Journal articles on the topic 'Blast Crisis, BCR, BCR-ABL, bone marrow precursors'
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1
Eiring, Anna M., Paolo Neviani, Ramasamy Santhanam, Joshua J. Oaks, Ji Suk Chang, Carlo Gambacorti-Passerini, Stefano Volinia, et al. "Requirement of the E2F3 Transcription Factor for BCR/ABL Leukemogenesis." Blood 110, no. 11 (November 16, 2007): 33. http://dx.doi.org/10.1182/blood.v110.11.33.33.
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Abstract Several RNA binding proteins (RBPs) have been implicated in the progression of chronic myelogenous leukemia (CML) from the indolent chronic phase to the aggressively fatal blast crisis. In the latter phase, expression and function of specific RBPs are altered at transcriptional or post-translational levels by the increased constitutive kinase activity of the BCR/ABL oncoprotein, resulting in enhanced resistance to apoptotic stimuli, growth advantage and differentiation arrest of CD34+ CML blast crisis (CML-BC) progenitors. In the current study, we identified by RIP (RNA immunoprecipitation)-mediated microarray analysis that mRNA encoding the E2F3 transcription factor associates to the BCR/ABL-regulated RBP hnRNP A1. Moreover, RNA electrophoretic mobility shift and UV-crosslinking assays revealed that hnRNP A1 interacts with E2F3 mRNA through a binding site located in the 3’UTR of both human and mouse E2F3 mRNA. Accordingly, E2F3 protein levels were upregulated in BCR/ABL-transformed myeloid precursor cell lines compared to parental cells in a BCR/ABL-kinase- and hnRNP A1 shuttling-dependent manner. In fact, treatment of BCR/ABL-expressing myeloid precursors with the kinase inhibitor Imatinib (2mM, 24 hr) or introduction of a dominant-negative shuttling-deficient hnRNP A1 protein (NLS-A1) markedly reduced E2F3 protein and mRNA levels. Similarly, upregulation of BCR/ABL expression/activity in the doxycycline inducible TonB2.10 cell line resulted in increased E2F3 protein expression. BCR/ABL kinase-dependent induction of E2F3 protein levels was also detected in CML-BCCD34+ compared to CML-CPCD34+ progenitors from paired patient samples and to normal CD34+ bone marrow samples. Importantly, the in vitro clonogenic potential of primary mouse BCR/ABL+ lineage negative (Lin−) progenitors was markedly impaired in BCR/ABL+ E2F3−/− compared to BCR/ABL-transduced E2F3+/+ myeloid progenitors and upon shRNA-mediated downregulation of E2F3 expression (90% inhibition, P<0.001). Furthermore, subcutaneous injection of shE2F3-expressing BCR/ABL+ cells into SCID mice markedly impaired in vivo tumorigenesis (>80% reduction in tumor burden, P<0.01). Accordingly, BCR/ABL leukemogenesis was strongly inhibited in SCID mice intravenously injected with E2F3 shRNA-expressing 32D-BCR/ABL cells and in mice transplanted with BCR/ABL-transduced Lin− bone marrow cells from E2F3−/− mice. Specifically, we demonstrate that reduced or absent levels of E2F3 resulted in dramatically decreased numbers of circulating BCR/ABL+ cells as determined by nested RT-PCR at 4 weeks post-injection (P=0.0001), normal splenic architecture and bone marrow cellularity and the absence of infiltrating myeloid blasts into non-hematopoietic compartments (i.e. liver). By contrast, SCID mice transplanted with vector-transduced 32D-BCR/ABL cells or BCR/ABL+ E2F3+/+ Lin− BM progenitors showed signs of an overt acute leukemia-like process with blast infiltration of hematopoietic and non-hematopoietic organs. Altogether, these data outline the importance of E2F3 expression for BCR/ABL leukemogenesis and characterize a new potential therapeutic target for the treatment of patients with advanced phase CML.
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Chang, Ji Suk, Ramasamy Santhanam, Rossana Trotta, Paolo Neviani, Anna M. Eiring, Edward Briercheck, Mattia Ronchetti, et al. "High levels of the BCR/ABL oncoprotein are required for the MAPK-hnRNP-E2–dependent suppression of C/EBPα-driven myeloid differentiation." Blood 110, no. 3 (August 1, 2007): 994–1003. http://dx.doi.org/10.1182/blood-2007-03-078303.
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Abstract The inability of myeloid chronic myelogenous leukemia blast crisis (CML-BC) progenitors to undergo neutrophil differentiation depends on suppression of C/EBPα expression through the translation inhibitory activity of the RNA-binding protein hnRNP-E2. Here we show that “oncogene dosage” is a determinant factor for suppression of differentiation in CML-BC. In fact, high levels of p210-BCR/ABL are required for enhanced hnRNP-E2 expression, which depends on phosphorylation of hnRNP-E2 serines 173, 189, and 272 and threonine 213 by the BCR/ABL-activated MAPKERK1/2. Serine/threonine to alanine substitution abolishes hnRNP-E2 phosphorylation and markedly decreases its stability in BCR/ABL-expressing myeloid precursors. Similarly, pharmacologic inhibition of MAPKERK1/2 activity decreases hnRNP-E2 binding to the 5′UTR of C/EBPα mRNA by impairing hnRNP-E2 phosphorylation and stability. This, in turn, restores in vitro and/or in vivo C/EBPα expression and G-CSF–driven neutrophilic maturation of differentiation-arrested BCR/ABL+ cell lines, primary CML-BCCD34+ patient cells and lineage-negative mouse bone marrow cells expressing high levels of p210-BCR/ABL. Thus, increased BCR/ABL oncogenic tyrosine kinase activity is essential for suppression of myeloid differentiation of CML-BC progenitors as it is required for sustained activation of the MAPKERK1/2-hnRNP-E2-C/EBPα differentiation-inhibitory pathway. Furthermore, these findings suggest the inclusion of clinically relevant MAPK inhibitors in the therapy of CML-BC.
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Ciccarelli, Bryan T., Tinghui Hu, Qing Wang, Julia J. Kim, and Ian P. Whitehead. "Examination of Clinically-Derived p210 BCR/ABL Rhogef Mutations in a Murine Bone Marrow Transplantation Model of CML." Blood 136, Supplement 1 (November 5, 2020): 24. http://dx.doi.org/10.1182/blood-2020-141685.
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The Philadelphia chromosome is formed by a balanced, reciprocal translocation that pairs sequences from BCR on Chromosome 22 with sequences from ABL on Chromosome 9, resulting in the production of the constitutively active tyrosine kinase (TK) BCR-ABL. Depending on the location of the breakpoint within BCR, three different sizes of BCR-ABL may form, each associated with distinct clinical sequelae. Previous studies identified a functional domain within the BCR sequences preserved by the more-indolent forms of BCR-ABL (i.e., p210 and p230) that demonstrates a constitutive Rho guanine nucleotide exchange factor (RhoGEF) activity. Using the structurally derived S509A mutation, which does not affect TK activity but abrogates RhoGEF signaling, we, subsequently, showed that this feature regulates leukemia progression in mice. The RhoGEF domain was recently reported to contain two missense mutations (F547L and T654K) in a chronic myelogenous leukemia (CML) blast crisis (BC) patient, suggesting that it may play a role in human disease as well. To evaluate the clinical significance of this region, we, therefore, examined p210 BCR-ABL constructs containing these clinically derived RhoGEF mutations (CDRMs), both in isolation and in combination, using a murine bone marrow transplant (mBMT) model of CML. The mutations did not destabilize p210 BCR-ABL expression or TK activity but decreased RhoGEF signaling. Relative to mice transplanted with wild-type (WT) p210 BCR-ABL, those that received the CDRMs exhibited an earlier onset of disease, frequently developing previously unseen dermatologic involvement or myeloid sarcomas, but demonstrated significantly increased survival in an additive manner [Fig. 1]. While mice transplanted with WT p210 BCR-ABL exhibited neutrophilia that progresses to a less-differentiated phenotype at death, disease in the CDRM mice was characterized by eosinophilia and monocytosis with no maturation arrest. The most likely cause of death in mice transplanted with WT p210 BCR-ABL is widespread hematogenous involvement of the lungs resulting in acute respiratory distress. In contrast, mice receiving the CDRMs demonstrated pulmonary involvement which was limited to the bronchovascular bundles or subpleural space. This is consistent with a switch to lymphatogenous spread, likely secondary to skewed differentiation, and it implies an alternate cause(s) of death. To help determine the mechanism responsible for the observed differences in differentiation and maturation arrest, we next studied the CDRMs in vitro using the 32Dcl3 murine myeloid precursor cell line. A growing body of evidence suggests that the DNA damage response can affect lineage restriction in myeloid cells; it is also well-established that the accumulation of DNA damage is necessary for progression to BC. Accordingly, the CDRMs were associated with the restoration of p53 activation and the G1/S cell cycle checkpoint relative to WT p210 BCR-ABL. This suggests that the RhoGEF domain may be responsible for the suppression of the DNA damage response. Because the mBMT phenotype associated with the S509A mutation was distinct from that of the CDRMs, it may indicate that this capability is mediated by some functionality yet to be described. Importantly, the CDRMs were also associated with increased Src family kinase activity, which is thought to be a major mechanism of BCR-ABL-independent TK inhibitor resistance and minimal residual disease. Collectively, these results suggest that the BCR-ABL RhoGEF domain can influence disease progression and response to treatment in human leukemia. Disclosures No relevant conflicts of interest to declare.
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Wei, E., V. Bellamkonda, and J. Polski. "Primary Chronic Myelogenous Leukemia Blast Crisis with Precursor B Lymphoblastic Leukemia, a Case Report." American Journal of Clinical Pathology 158, Supplement_1 (November 1, 2022): S108. http://dx.doi.org/10.1093/ajcp/aqac126.228.
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Abstract Introduction/Objective Primary chronic myelogenous leukemia (CML) blast crisis at the initial disease presentation is rare. Most CML blast crisis cases present with increased myeloblasts with a minority of patients showing lymphoblastic leukemia. Differentiating primary CML lymphoblast crisis from de novo acute lymphoblastic leukemia may represent a diagnostic challenge to both pathologists and treating clinicians. This distinction is important as it has significant implications on patient management. Methods/Case Report A 15-year-old male patient was admitted to our University Hospital for hyperleukocytosis. Patient reportedly had weight loss with occasional sweats and cervical lymphadenopathy. She was found to have massive splenomegaly. Peripheral blood showed hyperleukocytosis with predominance of granulocytes at all maturation stages ranging from blast to segmented neutrophils with increased blasts. Subsequent bone marrow findings were consistent with extensive involvement by B-lymphoblastic leukemia. Ten-color Flow cytometry showed approximately 30% blasts with rare lymphocytes and monocytes. The blasts revealed precursor B-lymphoblastic immunophenotypic expression of CD45, CD10, CD19, CD20, CD22, CD34, CD38, CD200, HLA-DR and TdT expression. The results were similar to that of peripheral blood. Granulocytes showed abnormal maturation pattern with increased immature precursors and partial expression of CD4 and CD56 with no abnormalities detected in lymphocytes. In this case, while the bone marrow findings are consistent with B-lymphoblastic leukemia, the peripheral blood findings are consistent with blast phase of chronic myeloid leukemia. Further evaluation by cytogenetic and molecular studies confirmed the presence of Philadelphia chromosome, p210 transcripts, and rearrangement of BCR-ABL1, which supported the impression of precursor B-lymphoblastic leukemia in primary blast phase of CML. The patient was treated with tyrosine kinase inhibitor combined chemotherapy and went into remission. She has been followed up without significant complications for a year. Results (if a Case Study enter NA) NA. Conclusion The diagnosis of CML in primary lymphoblastic crisis is rare and needs to be systemically excluded before giving the diagnosis of de novo BCR-ABL1-positive acute lymphoblastic leukemia. If the patient does not have splenomegaly or previous leukocytosis, it needs cytological examination and extensive molecular analyses.
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Chen, Ying-Yu, Mobeen Malik, Brian E. Tomkowicz, Ronald G. Collman, and Andrzej Ptasznik. "BCR-ABL1 Disrupts SDF-1-Dependent Hematopoietic Cell Migration and Adhesion through the LFA-1 Integrin-Mediated Mechanism." Blood 110, no. 11 (November 16, 2007): 1011. http://dx.doi.org/10.1182/blood.v110.11.1011.1011.
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Abstract Stromal-derived factor-1 (SDF-1) and its receptor, CXCR4, are essential for normal stem/progenitor cell movement, adherence, and retention within the bone marrow environment. Two mechanisms through which BCR-ABL1 are thought to disrupt CXCR4-mediated chemotactic responses have been described in leukemia: the inhibition of CXCR4 expression (Geay et al. 2005, Cancer Res.) and intra-cellular signaling defects without modification of CXCR4 expression (Salgia et al. 1999, Blood; Ptasznik et al. 2002, J. Exp. Med.). These opposing mechanisms suggest that the actual situation is more complex and that new signaling paradigms are needed. To address this, we studied the effects of BCR-ABL1 on SDF-1-dependent, integrin-mediated, migration and adhesion of hematopoietic precursors. Stimulation of BCR-ABL1(−) hematopoietic cells with SDF-1 showed reduced cell adherence to surfaces coated with ICAM-1 (a ligand for the LFA-1 integrin), which was associated with down-regulated expression of activation-dependent epitopes of the β2 integrin, LFA-1, on hematopoietic cells. Inhibition of Lyn expression with siRNA prevented the SDF-1-triggered down-regulation of LFA-1 and cell adherence, indicating that CXCR4 inhibited the function of LFA-1 through Lyn. Expression of BCR-ABL1 in these cells resulted in increased expression of activation-dependent epitopes of LFA-1 and prevented SDF-1-dependent regulatory effects on both LFA-1 affinity and ICAM-1 adherence. Also, expression of BCR-ABL1 prevented Lyn-mediated regulation of cell adhesion to ICAM-1 as well as Lyn-mediated regulation of LFA-1 affinity. These results indicate that BCR-ABL1 constitutively increases the affinity of the LFA-1 integrin to its ligand ICAM-1, locking the integrin into an “active” conformation. The net result is the loss of responsiveness of LFA-1 to SDF-1-induced ‘inside-out’ signaling involving CXCR4 and Lyn kinase. Because in our experiments BCR-ABL1 had no significant effect on the expression of CXCR4 in Mo7e cells, transfected with low and high amounts of p210-BCR-ABL, or in primary BCR-ABL(+) cells from CML blast crisis patients (n=3), we conclude that BCR-ABL1 inhibits CXCR4-triggered ‘inside-out’ integrin signaling rather than CXCR4 expression. Taken together, we propose that BCR-ABL1 disrupts the signaling link between the chemokine receptor, CXCR4 and the β2 integrin LFA-1 so as to inhibit normal SDF-1-mediated chemotaxis and adhesion in hematopoietic cells.
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Harb, Jason G., Paolo Neviani, Claudia S. Huettner, Guido Marcucci, and Danilo Perrotti. "Combined Pharmacologic Inhibition of Bcl-Xl/Bcl-2 and mTORC1/2 Survival Signals Trigger Apoptosis in BCR-ABL1+in Vitro Models of Blast Crisis Chronic Myelogenous Leukemia (CML-BC), and Primary CD34+/CD38− Stem and CD34+ progenitor Cells From CML-BC Patients." Blood 118, no. 21 (November 18, 2011): 2738. http://dx.doi.org/10.1182/blood.v118.21.2738.2738.
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Abstract Abstract 2738 Tyrosine kinase inhibitors (TKIs) have become frontline therapy for CML; however, alternative therapies are required, as TKIs do not induce long-term response in CML patients undergoing blastic transformation and are ineffective against Philadelphia-positive (Ph+) quiescent stem cells, which show innate resistance to BCR-ABL1 kinase inhibitors. Therapeutic targets of interest are survival factors conferring resistance to TKI-induced apoptosis and/or those increasing proliferation of leukemic progenitors. We previously reported (Harb JG et al., ASH 2007) that genetic inactivation of Bcl-x did not inhibit BCR-ABL1 leukemogenesis in an inducible mouse model of CML. Thus, we hypothesize that BCR/ABL mediated post-translational modification and inactivation of pro-apoptotic BAD negates the requirement for the anti-apoptotic function of Bcl-xL in stem/progenitor cells from SCLtTA-BCR/ABL1/Bcl-x−/− mice. Following this rationale, we tested if simultaneous pharmacologic BAD activation and Bcl-xL inhibition may be an efficient way of killing CML stem/progenitor cells. To test this, loss of Bcl-xL function with increased levels of active BAD was achieved by expressing Bcl-x shRNA in 32D-BCR/ABL mouse myeloid precursors that were then treated with LY294002 (LY), which suppresses the inhibitory effects of PI-3K/Akt activation on BAD. Flow cytometric analysis of Annexin V+ cells revealed that levels of apoptosis were three times higher in BCR-ABL1+ cells expressing the Bcl-x shRNA when compared with vector-transduced BCR-ABL1+ cells. As expected, increased levels of dephosphorylated (active) BAD at the mitochondrial membrane were found in LY-treated BCR-ABL+ cells. Interestingly, co-treatment of Bcl-x shRNA-expressing BCR-ABL1+ cells with LY and the Bcl-xL/Bcl-2 antagonist ABT-263 (ABT) did not further promote apoptosis, suggesting that decreased survival of BCR-ABL1+ cells was dependent on downregulation of Bcl-xL and not Bcl-2. To determine efficacy of combined pharmacologic Bcl-xL inhibition and BAD activation, 32D-BCR/ABL and K562 cells were treated with compounds expected to activate BAD upon inhibition of PI-3K/Akt/mTOR-generated signals, used alone or in combination with ABT. Individually, at suboptimal doses, LY, Rapamycin (RAP), mTORC1/2 inhibitor PP242, and ABT were tolerated with apoptosis levels lower than 20%. Notably, when combined with ABT, all three efficiently induced apoptosis (∼90% Annexin V+) of BCR-ABL1+ cells. As with LY, increased levels of active BAD were found at the mitochondrial membrane of RAP- and PP242-treated BCR-ABL1+ cells. We found that PP242 downregulated p-Akt (92%), Mcl-1 (67%) and Bcl-xL (51%) more efficiently than RAP or LY. It has been shown that PP242 impairs the clonogenic potential of TKI-resistant mononuclear BM CML-BC cells; however, its effects when used alone or in combination with ABT on survival of normal and leukemic hematopoietic stem (HSCs) and progenitor cells is still unknown. Thus, HSC-enriched (CD34+/CD38-) and progenitor (CD34+) CML-BC cell fractions were isolated from bone marrow and peripheral blood and used in colony forming (CFC) assays with ABT, PP242 or ABT/PP242. ABT alone did not suppress colony formation of Ph+ CD34+/CD38− cells, while PP242 reduced it by nearly 50%. Conversely, ABT/PP242 combination decreased Ph+ stem and progenitor colony formation by ∼80%. Furthermore, the self-renewal of Ph+ CD34+/CD38− cells was markedly impaired by ABT/PP242 as demonstrated by the 80% decrease in replating efficiency. To assess if non-leukemic stem cells would tolerate ABT/PP242, colony assays were performed with LSK from wild type mice treated with ABT, PP242, RAP and ABT/PP242. We did not find a significant effect of ABT or PP242 on clonogenic potential when given as single agents. More importantly, combined treatment decreased CFC output by only 35% while RAP, which has an acceptable toxicity profile as it has been used in clinical trials for patients unresponsive to TKIs, decreased LSK colony forming potential by 50%. In summary, our data showing that combined treatment with the mTORC1/2 inhibitor/BAD activator PP242 and the BCl-xL/Bcl-2 antagonist ABT-263 markedly induces apoptosis of BCR-ABL+ cell lines, in HSCs and in progenitors from CML-BC patients. This approach warrants further pre-clinical investigation aimed at inclusion in clinical protocols for treating blast crisis CML. Disclosures: No relevant conflicts of interest to declare.
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Falchi, Lorenzo, Giorgia Desantis, Fabrizio Liberati, Roberta Pace, Maria G. Morandi, Patrizia Scaravaglio, Giuseppe Saglio, and Anna M. Liberati. "Ph’-Positive (Ph’+) Chronic Myeloid Leukemia (CML) Presenting as Ph’+ T-Lymphoblastic Lymphoma (LL) Resistant to High-Dose Chemotherapy and Gleevec." Blood 106, no. 11 (November 16, 2005): 4869. http://dx.doi.org/10.1182/blood.v106.11.4869.4869.
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Abstract BACKGROUND Extramedullary blast crisis (BC) of Ph’+ CML is infrequent and commonly affects bone, lymphoid tissue, skin and soft tissues and central or peripheral nervous system. Most of nodal lymphoid tumors occuring in the setting of CML derive from T-cell precursors and represent the evolution of CML to a lymphoid nodal BC. CASE HISTORY In February 2004, a 52 year-old patient underwent diagnostic wide biopsy of a nasopharingeal mass that caused severe acute respiratory symptoms. At the histological examination, the nasopharyngeal mucosa exhibited a diffuse pattern of infiltration by neoplastic cells with a characteristic single-file arrangement. The cells showed typical convoluted nuclei with one or two nucleoli and abundant cytoplasm (L2, lymphoblasts). The malignant cell population expressed a preT-cell immunophenotype: cytoplasmic CD3(+), CD43 (+), TdT (+/−), CD34(+), CD4(−), CD8(−). Thus, the diagnosis of T-cell LL was formulated. Whole-body CT scan revealed nasopharingeal mass, retropharyngeal, laterocervical, axillary, inguinal enlarged nodes and splenomegaly. Laboratory tests indicated leukocytosis (58,000/μl) with a differential WBC count typical of CML in chronic phase (myeloblasts <1%). This diagnosis was confirmed by a low alkaline phosphatase value (score: 1), histological features of bone marrow (BM) biopsy, classical cytogenetics (presence of the Ph’ in 100% of 25 metaphases analysed and absence of other cytogenetic abnormalities) and FISH evaluation (D-FISH bcr-abl in interphase and metaphase, Oncor probe). The nested RT-PCR (JQ Guo et al., Leukemia;2002,15:2447) disclosed the presence of the hybrid protein p210 (b2,a2), but not that of p190. A laterocervical node was excised to perform cytogenetic and molecular analyses in order to determine whether the T-cell LL was an unrelated disease or the expression of an extramedullary BC. The histology confirmed the presence of a uniform population of T-lymphoblasts in which classical cytogenetic analysis disclosed the following kariotype: 49,XY,t(9;22)(q34;q11),+9,+19,+der(22)t(9;22)(q34;q11) and 50–52,XY t(9;22)(q34;q11),+6+9,+9q+,+19,+20,+22q−. The FISH analysis showed the presence of bcr-abl gene in all cells analyzed and the presence of multiple copies of this gene as well as of double Ph’ chromosome. Nested RT-PCR showed the presence of both p210 and p190 transcripts. These findings indicated that the T-cell LL was an extramedullary BC of a CML simultaneously diagnosed in chronic phase in BM and peripheral blood. The patient was initially treated with vincristine, daunomicine, asparaginase and prednisone combined with Gleevec (800mg/die). Consolidation therapy, consisting of high-dose Ara-C (4g/m2 for 4 days) and mithoxantrone (10mg/m2 for 2 days), followed by a mieloablative course with mithoxantrone (60 mg/m2) and melphalan (180 mg/m2) and autologous stem cell support (ASCT) was administered after an initial clinical response. Gleevec was given during the entire treatment period. A fugacious complete clinical-hematological and a partial cytogenetic (FISH: 12% of cells bcr-abl+) and molecular (number of bcr-abl/104 ABL copies= 184, real-time quantitative RT-PCR: J Gabert et al., Leukemia;2003:1) remission was documented after ASCT. The patient refused further consolidation treatment with Gleevec and died, due to progressive disease, in December 2004.
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Walker, Christopher, Joshua J. Oaks, Paolo Neviani, Jason G. Harb, Ramasamy Santhanam, Y. Ma, Guido Marcucci, et al. "PP2A Activating Drugs (PAD): Anti-Leukemic and Non-Toxic Activity of Two Novel and Non-Immunosuppressive FTY720 Derivatives." Blood 116, no. 21 (November 19, 2010): 2901. http://dx.doi.org/10.1182/blood.v116.21.2901.2901.
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Abstract Abstract 2901 FTY720 is a sphingosine analog proposed by the FDA for treating Multiple Sclerosis patients because of its immunosuppressive activity, which depends on its ability to prevent lymphocyte egress into the peripheral blood. To act as an immunosuppressive drug, FTY720 undergoes sphingosine kinase 2 (SPHK2) phosphorylation and internalization upon interaction with the sphingosine-1-phosphate receptor 1 (S1PR1). FTY720 also acts as a potent activator of protein phosphatase 2A (PP2A), a tumor suppressor found inactivated in chronic and blast crisis CML with wild type or imatinib/dasatinib-resistant BCR-ABL1, Ph+ B-ALL, KitD816V AML, Jak2V617F+ MPDs and other leukemias/lymphomas. FTY720 treatment of cell lines and primary progenitors isolated from bone marrow of patients with these malignancies, markedly suppressed leukemic cell proliferation/survival and induced apoptosis in a PP2A-dependent manner. Notably, long-term treatment with FTY720 of mice carrying these hematopoietic malignancies significantly prolonged survival and restored normal myelopoiesis without exerting any toxic effect in hematopoietic and non-hematopoietic organs. However, in vivo administration of FTY720 strongly, albeit reversibly, decreases the number of circulating B and T lymphocytes. Here we report that a synthetically phosphorylated FTY720 (FTY720-P) is unable to induce neither PP2A activation nor apoptosis of BCR-ABL1-, Jak2V617F-, or KitD816V-expressing myeloid precursors, indicating that FTY720 phosphorylation is dispensable for its anti-leukemic activity. Thus, we functionally characterize two FTY720 derivatives, QC-FTYSM and OSU-2S, which were synthesized as molecules unable to undergo SPHK2 phosphorylation. Treatment (2.5 uM; 24h) of FTY720-sensitive 32D-BCR/ABL cells with QC-FTYSM and OSU2S results in ∼80% and 40%, respectively, more efficient suppression of BCR-ABL1 expression and kinase activity than that observed with FTY720. Moreover, QC-FTYSM, OSU-2S and FTY720 (2.5uM; 0–60h) induce a progressive block of proliferation and marked induction of apoptosis of 32D-BCR/ABL cells. In fact, a 96%, 98%, and 79% decrease in viability is observed after treatment with QC-FTYSM, OSU-2S and FTY720, respectively. Notably, viability of non-transformed myeloid 32Dcl3 cells is not significantly affected by treatment with FTY720 or its derivatives. Consistent with the ability of FTY720 to induce apoptosis through rescue of PP2A activity, phosphatase assays show identical ability of FTY720, QC-FTYSM and OSU-2S to restore PP2A functionality. In fact, comparable and marked decrease in the amount of inactive Y307-phosphorylated PP2Ac was detected in 32D-BCR/ABL cells treated with FTY720 or its derivatives. To formally demonstrate that QC-FTYSM and OSU-2S lack immunosuppressive activity, we first assessed their ability to be internalized upon interaction/association with the S1PR1 receptor. Thus, cells were transduced with a GFP-tagged S1PR1 and treated with either QC-FTYSM, OSU-2S, or, as positive control, FTY720-P. Confocal microscopy revealed that treatment FTY720-P resulted in a strong S1PR1 internalization. Conversely, exposure of the cells to QC-FTYSM and OSU-2S did not alter the S1PR1 membrane localization, indicating that these molecules did not undergo SHPK2 phosphorylation. Further demonstration of the inability of these compounds to act as immunosuppressive molecules was gained upon in vivo administration of a single dose of FTY720, QC-FTYSM or OSU-2C (10 mg/kg) to wild type FVB/N mice. As expected, percentage of B220+/CD19+ circulating B-cells decreased of ∼90% in FTY720-treated animals. Conversely, the percentage of B-cells after exposure to QC-FTYSM and OSU-2S remained unchanged (≤ 1% decrease). Likewise, the number or CD4+ and CD8+ cells also was not affect by treatment with the QC-FTYSM compound. Note that effect of OSU-2S on T-cells and the toxicity profile and anti-leukemic activity of these drugs in healthy animals and mouse models of deadly leukemias (e.g. T315I+ and blast crisis CML and Ph+ ALL) as well as Ph− MPDs are currently being assessed. Altogether our data indicate that QC-FTYSM and OSU-2S represent two potentially powerful and safe drugs which could be introduced in the current therapeutic protocols for different types of hematopoietic and non-hematopoietic malignancies characterized by functional inactivation of the PP2A tumor suppressor. Disclosures: No relevant conflicts of interest to declare.
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Frisch, Benjamin, John M. Ashton, Adam Olm-shipman, Lianping Xing, Craig T. Jordan, and Laura Calvi. "Reciprocal Synergistic Interactions of Leukemic Cells with Osteoclast Progenitors in the Bone Microenvironment." Blood 112, no. 11 (November 16, 2008): 322. http://dx.doi.org/10.1182/blood.v112.11.322.322.
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Abstract The bone marrow provides an essential regulatory microenvironment for adult hematopoiesis, however the relationship between the bone marrow microenvironment and malignant hematopoiesis remains poorly understood. To investigate the interactions between leukemia and the bone marrow microenvironment we utilized a mouse model of blast-crisis chronic myelogenous leukemia (BC-CML), in which primitive normal murine hematopoietic cells are modified to leukemic cells by expressing the translocation products BCR/ABL and Nup98/HoxA9. The presence of each translocation was confirmed by their co-expression of Green Fluorescent Protein (GFP) and Yellow Fluorescent Protein (YFP) respectively. Ten days after injection of GFP+/YFP+ leukemic cells into strain-matched immunocompetent, non-myeloablated recipient mice, 50% of the bone marrow was composed of leukemic cells as determined by flow cytometric analysis. Histologic analysis of the contralateral tibiae and femora demonstrated not only progressive replacement of the bone marrow by leukemic cells, but also a significant bone loss. Histomorphometric analysis confirmed 50% decreased trabecular bone volume in leukemic mice compared to control mice that were not injected with leukemic cells (bone volume/total volume (%): 12±2 vs 26±2 p=0.01). Interestingly, numerous multi-nucleated osteoclasts were observed in the bone marrow of leukemic mice and were localized adjacent to leukemic cells, suggesting that leukemic cells may affect osteoclastogenesis and result in massive bone loss. To test this hypothesis, we first measured the expression of known regulators of osteoclastogenesis, including RANKL, in our leukemic cells by quantitative RT-PCR analysis. Compared to GFP−/YFP− cells, GFP+/YFP+ cells have 3-fold increased expression of RANKL, a major osteoclastogenic cytokine. We then examined if leukemic cells can give rise to osteoclasts in the presence of RANKL and M-CSF in vitro and found that these cells were unable to differentiate into osteoclasts themselves. To determine if leukemic cells can induce osteoclastogenesis of normal osteoclast progenitors, we cocultured spleen-derived osteoclast precursors from wild-type mice with GFP+/YFP+ leukemic cells or GFP−/YFP− non-leukemic cells in osteoclastic differentiating media containing optimal concentrations of M-CSF and RANKL. As expected, there was abundant formation of mature osteoclasts, identified as TRAP+ multinucleated cells, in control cultures containing non-leukemic cells and osteoclast precursors. Leukemic cells significantly increased TRAP+ mono-nucleated osteoclast precursors (No. TRAP+ mononucleated cells/well: 34±3.3 vs 20±6.0 in non-leukemic cells, p=0.0136). Under this culture condition, we did not observe increased mature osteoclast formation by leukemic cells. Surprisingly, we found that osteoclast precursors strongly prolonged the survival of leukemia cells. In control cultures without a feeder layer of osteoclast precursors there were no viable leukemia cells present after 6 days in culture while in the co-culture system viable leukemia cells were still abundant after 6 days in culture, identifiable by their expression of GFP/YFP (No. GFP+/YFP+/high power field: 0 vs 142±6.4, p<0.01). In summary, in a murine model of BC-CML, there was a rapid loss of trabecular bone and an increase in the number of osteoclasts. Expression of osteoclast-regulating cytokines in leukemic cells favored osteoclastogenesis, however modified cells did not themselves give rise to osteoclasts, while increasing the population of normal immature osteoclasts. In turn, osteoclast progenitors prolonged survival of leukemic cells in vitro. Taken together, these data strongly suggest reciprocal synergistic interactions of leukemic cells with osteoclast progenitors in the bone microenvironment. These previously unrecognized interactions could be exploited to modify disease progression, providing a novel approach for leukemia treatment.
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Zipeto, Maria Anna, Angela Court Recart, Nathaniel Delos Santos, Qingfei Jiang, Leslie A. Crews, and Catriona HM Jamieson. "Inflammatory Cytokine-Responsive ADAR1 Impairs Let-7 Biogenesis and Promotes Leukemia Stem Cell Generation." Blood 126, no. 23 (December 3, 2015): 4014. http://dx.doi.org/10.1182/blood.v126.23.4014.4014.
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Abstract Background In advanced human malignancies, RNA sequencing (RNA-seq) has uncovered deregulation of adenosine deaminase acting on RNA (ADAR) editases that promote therapeutic resistance and leukemia stem cell (LSC) generation. Chronic myeloid leukemia (CML), an important paradigm for understanding LSC evolution, is initiated by BCR-ABL1 oncogene expression in hematopoietic stem cells (HSCs) but undergoes blast crisis (BC) transformation following aberrant self-renewal acquisition by myeloid progenitors harboring cytokine-responsive ADAR1 p150 overexpression. Emerging evidence suggests that adenosine to inosine editing at the level of primary (pri) or precursor (pre)-microRNA (miRNA), alters miRNA biogenesis and impairs biogenesis. However, relatively little is known about the role of inflammatory niche-driven ADAR1 miRNA editing in malignant reprogramming of progenitors into self-renewing LSCs. Methods Primary normal and CML progenitors were FACS-purified and RNA-Seq analysis as well as qRT-PCR validation were performed according to published methods (Jiang, 2013). MiRNAs were extracted from purified CD34+ cells derived from CP, BC CML and cord blood by RNeasy microKit (QIAGEN) and let-7 expression was evaluated by qRT-PCR using miScript Primer assay (QIAGEN). CD34+ cord blood (n=3) were transduced with lentiviral human JAK2, let-7a, wt-ADAR1 and mutant ADAR1, which lacks a functional deaminase domain. Because STAT signaling triggers ADAR1 transcriptional activation and both BCR-ABL1 and JAK2 activate STAT5a, nanoproteomics analysis of STAT5a levels was performed. Engrafted immunocompromised RAG2-/-γc-/- mice were treated with a JAK2 inhibitor, SAR302503, alone or in combination with a potent BCR-ABL1 TKI Dasatinib, for two weeks followed by FACS analysis of human progenitor engraftment in hematopoietic tissues and serial transplantation. Results RNA-seq and qRT-PCR analysis in FACS purified BC CML progenitors revealed an over-representation of inflammatory pathway activation and higher levels of JAK2-dependent inflammatory cytokine receptors, when compared to normal and chronic phase (CP) progenitors. Moreover, RNA-seq and qRT-PCR analysis showed decreased levels of mature let-7 family of stem cell regulatory miRNA in BC compared to normal and CP progenitors. Lentiviral human JAK2 transduction of CD34+ progenitors led to an increase of ADAR1 transcript levels and to a reduction in let-7 family members. Interestingly, lentiviral human JAK2 transduction of normal progenitors enhanced ADAR1 activity, as revealed by RNA editing-specific qRT-PCR and RNA-seq analysis. Moreover, qRT-PCR analysis of CD34+ progenitors transduced with wt-ADAR1, but not mutant ADAR1 lacking functional deaminase activity, reduced let-7 miRNA levels. These data suggested that ADAR1 impairs let-7 family biogenesis in a RNA editing dependent manner. Interestingly, RNA-seq analysis confirmed higher frequency of A-to-I editing events in pri- and pre-let-7 family members in CD34+ BC compared to CP progenitors, as well as normal progenitors transduced with human JAK2 and ADAR1-wt, but not mutant ADAR1. Lentiviral ADAR1 overexpression enhanced CP CML progenitor self-renewal and decreased levels of some members of the let-7 family. In contrast, lentiviral transduction of human let-7a significantly reduced self-renewal of progenitors. In vivo treatments with Dasatinib in combination with a JAK2 inhibitor, significantly reduced self-renewal of BCR-ABL1 expressing BC progenitors in the bone marrow thereby prolonging survival of serially transplanted mice. Finally, a reduction in ADAR1 p150 transcripts was also noted following combination treatment only suggesting a role for ADAR1 in CSC propagation. Conclusion This is the first demonstration that intrinsic BCR-ABL oncogenic signaling and extrinsic cytokines signaling through JAK2 converge on activation of ADAR1 that drives LSC generation by impairing let-7 miRNA biogenesis. Targeted reversal of ADAR1-mediated miRNA editing may enhance eradication of inflammatory niche resident cancer stem cells in a broad array of malignancies, including JAK2-driven myeloproliferative neoplasms. Disclosures Jamieson: J&J: Research Funding; GSK: Research Funding.
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Buesche, Guntram, Oliver Bock, Kais Hussein, Nils von Neuhoff, Brigitte Schlegelberger, and Hans H. Kreipe. "Coincidence of bcr/abl-Positive Chronic Myeloid Leukemia and bcr/abl-Negative Chronic Myeloproliferative Disorder and Its Relevance to the Course of Disease." Blood 110, no. 11 (November 16, 2007): 2950. http://dx.doi.org/10.1182/blood.v110.11.2950.2950.
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Abstract Recently, several case reports have been published reporting the occurrence of a JAK2-V617F-mutated clone in the bone marrow from patients with bcr/abl-positive chronic myeloid leukemia (CML) treated with imatinib mesylate. Representative data on the probability and prognostic significance of the coincidence of CML and bcr/abl-negative chronic myeloproliferative disorder (CMPD) do not exist as yet. The bone marrow biopsies from a total of 600 patients with CML treated with interferon-alpha (n = 200) or imatinib mesylate (n = 400) and a total of 1600 patients with CMPD were evaluated for the coincidence of bcr/abl-positive CML and bcr/abl-negative CMPD, and in all suspect cases, the bone marrow cells were analyzed for the JAK2-V617F-mutation. During a median follow-up period of three years, a total of six patients evolved morphologic features of CML as well as of bcr/abl-negative CMPD: One of 200 CML patients treated with interferon-alpha (.5%), four of 400 CML patients treated with imatinib (1.0%), and one of 1600 patients with CMPD (.06%). All patients were bcr/abl-positive with a bcr/abl-negative JAK2-V617F-mutated clone besides the bcr/abl-positive clone. In one patient, bcr/abl-positive CML occurred 15 years after diagnosis of polycythemia vera. Four patients showed morphologic features of bcr/abl-positive CML and bcr/abl-negative CMPD in the diagnostic bone marrow biopsy. One patient evolved features of bcr/abl-negative CMPD two years after imatinib treatment. However, in the diagnostic bone marrow biopsy of this patient, 5% JAK2-V617F-mutated cells were detected besides the bcr/abl-positive clone. During treatment with imatinib (n = 4) or interferon-alpha (n = 1), the proportion of JAK2-V617F-mutated alleles increased to 21 – 90% (median: 23%); in one patient, the follow-up was too short for a statistical analysis. One of five patients evolved essential thrombocythemia, one showed a relapse of polycythemia vera, and three patients suffered from chronic idiopathic myelofibrosis. The patient with essential thrombocythemia did not show a progression of disease. The patient with polycythemia vera died from JAK2-V617F-positive myeloid blast crisis, and all three patients with chronic idiopathic myelofibrosis suffered from full-blown marrow fibrosis one to four years after detection of JAK2-V617F-mutated CMPD. Conclusion: In a minority of patients with CML, a JAK2-V617F-mutated clone exists besides the bcr/abl-positive clone, and rarely, bcr/abl-positive CML may evolve during the course of a bcr/abl-negative JAK2-V617F-mutated CMPD. Without cytoreductive treatment, the bcr/abl-positive clone overgrows the bcr/abl-negative JAK2-V617F-mutated clone. However, in the case of a significant remission of bcr/abl-positive CML during treatment with imatinib or interferon-alpha, the bcr/abl-negative clone gets the chance to expand within bone marrow with development of morphologic and clinical signs of bcr/abl-negative CMPD. Depending on the type of CMPD, these patients show a significantly increased risk of imatinib-resistant marrow fibrosis or blast crisis.
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Pear, Warren S., Juli P. Miller, Lanwei Xu, John C. Pui, Benny Soffer, Robert C. Quackenbush, Ann Marie Pendergast, et al. "Efficient and Rapid Induction of a Chronic Myelogenous Leukemia-Like Myeloproliferative Disease in Mice Receiving P210 bcr/abl-Transduced Bone Marrow." Blood 92, no. 10 (November 15, 1998): 3780–92. http://dx.doi.org/10.1182/blood.v92.10.3780.
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Abstract Expression of the 210-kD bcr/abl fusion oncoprotein can cause a chronic myelogenous leukemia (CML)-like disease in mice receiving bone marrow cells transduced by bcr/abl-encoding retroviruses. However, previous methods failed to yield this disease at a frequency sufficient enough to allow for its use in the study of CML pathogenesis. To overcome this limitation, we have developed an efficient and reproducible method for inducing a CML-like disease in mice receiving P210 bcr/abl-transduced bone marrow cells. All mice receiving P210 bcr/abl-transduced bone marrow cells succumb to a myeloproliferative disease between 3 and 5 weeks after bone marrow transplantation. The myeloproliferative disease recapitulates many of the hallmarks of human CML and is characterized by high white blood cell counts and extensive extramedullary hematopoiesis in the spleen, liver, bone marrow, and lungs. Use of a retroviral vector coexpressing P210 bcr/abl and green fluorescent protein shows that the vast majority of bcr/abl-expressing cells are myeloid. Analysis of the proviral integration pattern shows that, in some mice, the myeloproliferative disease is clonal. In multiple mice, the CML-like disease has been transplantable, inducing a similar myeloproliferative syndrome within 1 month of transfer to sublethally irradiated syngeneic recipients. The disease in many of these mice has progressed to the development of acute lymphoma/leukemia resembling blast crisis. These results demonstrate that murine CML recapitulates important features of human CML. As such, it should be an excellent model for addressing specific issues relating to the pathogenesis and treatment of this disease.
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Pear, Warren S., Juli P. Miller, Lanwei Xu, John C. Pui, Benny Soffer, Robert C. Quackenbush, Ann Marie Pendergast, et al. "Efficient and Rapid Induction of a Chronic Myelogenous Leukemia-Like Myeloproliferative Disease in Mice Receiving P210 bcr/abl-Transduced Bone Marrow." Blood 92, no. 10 (November 15, 1998): 3780–92. http://dx.doi.org/10.1182/blood.v92.10.3780.422k15_3780_3792.
Full textAbstract:
Expression of the 210-kD bcr/abl fusion oncoprotein can cause a chronic myelogenous leukemia (CML)-like disease in mice receiving bone marrow cells transduced by bcr/abl-encoding retroviruses. However, previous methods failed to yield this disease at a frequency sufficient enough to allow for its use in the study of CML pathogenesis. To overcome this limitation, we have developed an efficient and reproducible method for inducing a CML-like disease in mice receiving P210 bcr/abl-transduced bone marrow cells. All mice receiving P210 bcr/abl-transduced bone marrow cells succumb to a myeloproliferative disease between 3 and 5 weeks after bone marrow transplantation. The myeloproliferative disease recapitulates many of the hallmarks of human CML and is characterized by high white blood cell counts and extensive extramedullary hematopoiesis in the spleen, liver, bone marrow, and lungs. Use of a retroviral vector coexpressing P210 bcr/abl and green fluorescent protein shows that the vast majority of bcr/abl-expressing cells are myeloid. Analysis of the proviral integration pattern shows that, in some mice, the myeloproliferative disease is clonal. In multiple mice, the CML-like disease has been transplantable, inducing a similar myeloproliferative syndrome within 1 month of transfer to sublethally irradiated syngeneic recipients. The disease in many of these mice has progressed to the development of acute lymphoma/leukemia resembling blast crisis. These results demonstrate that murine CML recapitulates important features of human CML. As such, it should be an excellent model for addressing specific issues relating to the pathogenesis and treatment of this disease.
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Koschmieder, Steffen, Berthold Göttgens, Pu Zhang, Junko Iwasaki-Arai, Koichi Akashi, Jeffery L. Kutok, Tajhal Dayaram, et al. "Inducible chronic phase of myeloid leukemia with expansion of hematopoietic stem cells in a transgenic model of BCR-ABL leukemogenesis." Blood 105, no. 1 (January 1, 2005): 324–34. http://dx.doi.org/10.1182/blood-2003-12-4369.
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Abstract To develop murine models of leukemogenesis, a series of transgenic mice expressing BCR-ABL in different hematopoietic cell subsets was generated. Here we describe targeted expression of P210 BCR-ABL in stem and progenitor cells of murine bone marrow using the tet-off system. The transactivator protein tTA was placed under the control of the murine stem cell leukemia (SCL) gene 3′ enhancer. Induction of BCR-ABL resulted in neutrophilia and leukocytosis, and the mice became moribund within 29 to 122 days. Autopsy of sick mice demonstrated splenomegaly, myeloid bone marrow hyperplasia, and extramedullary myeloid cell infiltration of multiple organs. BCR-ABL mRNA and protein were detectable in the affected organs. Fluorescence-activated cell sorter (FACS) analysis demonstrated a significant increase in mature and immature myeloid cells in bone marrow and spleen, together with increased bilineal B220+/Mac-1+ cells in the bone marrow. tTA mRNA was expressed in FACS-sorted hematopoietic stem cells expanded 26-fold after BCR-ABL induction. Thirty-one percent of the animals demonstrated a biphasic phenotype, consisting of neutrophilia and subsequent B-cell lymphoblastic disease, reminiscent of blast crisis. In summary, this mouse model recapitulates many characteristics of human chronic myeloid leukemia (CML) and may help elucidate basic leukemogenic mechanisms in CML stem cells during disease initiation and progression. (Blood. 2005;105:324-334)
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Minami, Yosuke, Scott Stuart, Tomokatsu Ikawa, Asoka Banno, Irina Hunton, Karl Willert, Cornelis Murre, Catriona Jamieson, and Jean Y. J. Wang. "Imatinib-Resistant Activation of β-Catenin by BCR-ABL in a Murine Model of Chronic Myelogenous Leukemia Stem Cells." Blood 108, no. 11 (November 1, 2006): 2126. http://dx.doi.org/10.1182/blood.v108.11.2126.2126.
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Abstract [INTRODUCTION] Chronic myelogenous leukemia (CML) is effectively treated with imatinib, a small molecule inhibitor of the BCR-ABL tyrosine kinase that is expressed in the hematopoietic compartment including stem and progenitor cells in CML patients. While imatinib induces disease remission, it does not eradicate BCR-ABL-positive stem cells. Recently, granulocyte-macrophage progenitors (GMP) with nuclear β-catenin and an aberrant potential for self-renewal were detected in CML blast crisis (Jamieson et al, NEJM, 2004). We have established a murine model that generates BCR-ABL-positive GMP and a transplantable CML. In this model, BCR-ABL activates β-catenin through an imatinib-resistant pathway. [METHODS] Retroviral mediated gene transfer was employed to express BCR-ABL (p210) and/or GFP in a pluripotent murine hematopoietic progenitor cell line (mHPC) (Ikawa et al, Immunity, 2004). Infected cells were isolated using GFP-sorting and characterized ex vivo for leukemogenic phenotypes and gene expression profiles. The GFP-sorted cells were transplanted into lethally irradiated congenic mice. The distribution of GFP-positive cells in the hematopoietic compartments was determined by FACS. [RESULTS] BCR-ABL expression induced leukemogenic traits in mHPC, i.e., stroma- and cytokine-independent survival and proliferation. BCR-ABL also induced the expression of myeloid lineage markers, a process that was inhibited by imatinib. By contrast, imatinib did not abolish the stabilization of β-catenin in BCR-ABL transduced mHPC. Transplantation of BCR-ABL-transduced mHPC, but not GFP-transduced mHPC, induced in mice a CML-like disease presented with increased white blood cell counts and splenomegaly. The bone marrow cells from these mice reproducibly transferred CML phenotypes to secondary recipient mice. The diseased bone marrow contains an expanded population of GFP-positive GMP. The effect of imatinib on the leukemogenic potential of these BCR-ABL-positive GMP in vivo is under investigation. [CONCLUSIONS] Expression of BCR-ABL in the context of mHPC induces leukemogenic traits, including the stabilization of β-catenin, without requirement for additional genetic events. The BCR-ABL-transduced mHPC generates CML-like disease in mice accompanied by the expansion of a BCR-ABL-positive GMP population, similar to that observed in blast crisis CML patients. The stabilization of β-catenin by BCR-ABL is not inhibited by imatinib; consistent with the notion that imatinib cannot eradicate CML stem cells. This murine model can be used to identify therapeutics that targets the CML stem cells.
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Skorski, T., P. Kanakaraj, M. Nieborowska-Skorska, MZ Ratajczak, SC Wen, G. Zon, AM Gewirtz, B. Perussia, and B. Calabretta. "Phosphatidylinositol-3 kinase activity is regulated by BCR/ABL and is required for the growth of Philadelphia chromosome-positive cells." Blood 86, no. 2 (July 15, 1995): 726–36. http://dx.doi.org/10.1182/blood.v86.2.726.bloodjournal862726.
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The BCR/ABL oncogenic tyrosine kinase is responsible for initiating and maintaining the leukemic phenotype of Philadelphia chromosome (Ph1)- positive cells. Phosphatidylinositol-3 (PI-3) kinase is known to interact with and be activated by receptor and nonreceptor tyrosine kinases. We investigated whether PI-3 kinase associates with and/or is regulated by BCR/ABL, whether this interaction is functionally significant for Ph1 cell proliferation, and, if so, whether inhibition of PI-3 kinase activity can be exploited to eliminate Ph1-positive cells from bone marrow. We show that the p85 alpha subunit of PI-3 kinase associates with BCR/ABL and that transient expression of BCR/ABL in fibroblasts and down-regulation of BCR/ABL expression using antisense oligodeoxynucleotides (ODNs) in Ph1 cells activates and inhibits, respectively, PI-3 kinase enzymatic activity. The use of specific ODNs or antisense constructs to downregulate p85 alpha expression showed a requirement for p85 alpha subunit in the proliferation of BCR/ABL-dependent cell lines and chronic myelogenous leukemia (CML) primary cells. Similarly, wortmannin, a specific inhibitor of the enzymatic activity of the p110 subunit of PI-3 kinase, inhibited growth of these cells. The growth of normal bone marrow and erythromyeloid, but not megakaryocyte, progenitors was inhibited by p85 alpha antisense [S]ODNs, but wortmannin, at the concentrations tested, did not affect normal hematopoiesis. The proliferation of two BCR/ABL- and growth factor-independent cell lines was not affected by downregulation of the expression of the p85 alpha subunit or inhibition of p110 enzymatic activity, confirming the specificity of the observed effects on Ph1 cells. Thus, PI-3 kinase is one of the downstream effectors of BCR/ABL tyrosine kinase in CML cells. Moreover, reverse transcriptase-polymerase chain reaction performed on single colonies to detect BCR-ABL transcripts showed that wortmannin was able to eliminate selectively CML-blast crisis cells from a mixture of normal bone marrow and Ph1 cells.
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Ma, Cheng-Cheng, Ye Chai, Hui ling Chen, Xin Wang, Ying Gao, Wan li Hu, and Xue Xiang. "Clonal evolution of AML1-ETO coexisting with BCR-ABL and additional chromosome abnormalities in a blastic transformation of chronic myeloid leukemia." Journal of International Medical Research 48, no. 5 (May 2020): 030006052091923. http://dx.doi.org/10.1177/0300060520919237.
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Blast crisis develops in a minority of patients with chronic myeloid leukemia even in the era of tyrosine kinase inhibitor (TKI) therapy. Reports suggest that we know little about the mechanism of BCR-ABL and AML1-ETO co-expression in blast crisis of chronic myeloid leukemia, and that other chromosomal abnormalities also coexist. Here, we document an unusual and interesting case of a 51-year-old female diagnosed in the chronic phase of chronic myeloid leukemia. After undergoing TKI treatment for 3 months, her bone marrow aspirates in the chronic phase had transformed to blast crisis. Molecular genetic testing indicated she was positive for p210 form of BCR-ABL (copy number decreased from 108.91% to 56.96%) and AML1-ETO fusion (copy number, 5.65%) genes and had additional chromosomal abnormalities of t(8; 21)(q22; q22)/t(9; 22)(q34; q11), t(2; 5)(p24; q13) and an additional +8 chromosome.
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Skorski, T., M. Nieborowska-Skorska, P. Wlodarski, G. Zon, RV Iozzo, and B. Calabretta. "Antisense oligodeoxynucleotide combination therapy of primary chronic myelogenous leukemia blast crisis in SCID mice." Blood 88, no. 3 (August 1, 1996): 1005–12. http://dx.doi.org/10.1182/blood.v88.3.1005.1005.
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Abstract The proliferation of chronic myelogenous leukemia (CML) cells and the transformation of normal hematopoietic cells by BCR-ABL appear to require the expression of a functional MYC protein, suggesting an approach to treatment of Philadelphia leukemias based on simultaneous targeting of BCR-ABL and c-MYC. To test this hypothesis, CML-blast crisis (CML-BC) primary cells were treated in vitro with bcr-abl and c- myc antisense phosphorothioate oligodeoxynucleotides ([S]ODNs), individually or in combination. Compared with antisense ODNs targeting of individual oncogenes, downregulation of both BCR-ABL and c-MYC by specific antisense [S]ODNs resulted in a synergistic antiproliferative effect. Colony formation of normal bone marrow cells was not affected by either treatment. To assess the therapeutic potential of multiple oncogene downregulation, SCID mice injected with CML-BC primary cells were treated systematically with equal doses of bcr-abl or c-myc antisense [S]ODNs or with a combination of both antisense [S]ODNs. Compared with mice treated with individual compounds, the disease process was significantly retarded in the group treated with both [S]ODNs as revealed by flow cytometry, clonogenic assay, and RT-PCR analysis to detect leukemic cells in mouse tissue cell suspensions. These effects correlated with a markedly increased survival of leukemic mice treated with both antisense [S]ODNs. Leukemic cells harvested from antisense [S]ODN-treated mice were sensitive to the effects of antisense [S]ODNs in vitro, suggesting that the treatment can be successfully repeated. These data demonstrate the therapeutic potential of targeting multiple cooperating oncogenes.
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Skorski, T., M. Nieborowska-Skorska, P. Wlodarski, G. Zon, RV Iozzo, and B. Calabretta. "Antisense oligodeoxynucleotide combination therapy of primary chronic myelogenous leukemia blast crisis in SCID mice." Blood 88, no. 3 (August 1, 1996): 1005–12. http://dx.doi.org/10.1182/blood.v88.3.1005.bloodjournal8831005.
Full textAbstract:
The proliferation of chronic myelogenous leukemia (CML) cells and the transformation of normal hematopoietic cells by BCR-ABL appear to require the expression of a functional MYC protein, suggesting an approach to treatment of Philadelphia leukemias based on simultaneous targeting of BCR-ABL and c-MYC. To test this hypothesis, CML-blast crisis (CML-BC) primary cells were treated in vitro with bcr-abl and c- myc antisense phosphorothioate oligodeoxynucleotides ([S]ODNs), individually or in combination. Compared with antisense ODNs targeting of individual oncogenes, downregulation of both BCR-ABL and c-MYC by specific antisense [S]ODNs resulted in a synergistic antiproliferative effect. Colony formation of normal bone marrow cells was not affected by either treatment. To assess the therapeutic potential of multiple oncogene downregulation, SCID mice injected with CML-BC primary cells were treated systematically with equal doses of bcr-abl or c-myc antisense [S]ODNs or with a combination of both antisense [S]ODNs. Compared with mice treated with individual compounds, the disease process was significantly retarded in the group treated with both [S]ODNs as revealed by flow cytometry, clonogenic assay, and RT-PCR analysis to detect leukemic cells in mouse tissue cell suspensions. These effects correlated with a markedly increased survival of leukemic mice treated with both antisense [S]ODNs. Leukemic cells harvested from antisense [S]ODN-treated mice were sensitive to the effects of antisense [S]ODNs in vitro, suggesting that the treatment can be successfully repeated. These data demonstrate the therapeutic potential of targeting multiple cooperating oncogenes.
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Eiring, Anna M., Paolo Neviani, Ramasamy Santhanam, Joshua J. Oaks, Ji Suk Chang, Mario Notari, William Willis, et al. "Identification of novel posttranscriptional targets of the BCR/ABL oncoprotein by ribonomics: requirement of E2F3 for BCR/ABL leukemogenesis." Blood 111, no. 2 (January 15, 2008): 816–28. http://dx.doi.org/10.1182/blood-2007-05-090472.
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Several RNA binding proteins (RBPs) have been implicated in the progression of chronic myelogenous leukemia (CML) from the indolent chronic phase to the aggressively fatal blast crisis. In the latter phase, expression and function of specific RBPs are aberrantly regulated at transcriptional or posttranslational levels by the constitutive kinase activity of the BCR/ABL oncoprotein. As a result, altered expression/function of RBPs leads to increased resistance to apoptotic stimuli, enhanced survival, growth advantage, and differentiation arrest of CD34+ progenitors from patients in CML blast crisis. Here, we identify the mRNAs bound to the hnRNP-A1, hnRNP-E2, hnRNP-K, and La/SSB RBPs in BCR/ABLtransformed myeloid cells. Interestingly, we found that the mRNA encoding the transcription factor E2F3 associates to hnRNP-A1 through a conserved binding site located in the E2F3 3′ untranslated region (UTR). E2F3 levels were up-regulated in CML-BCCD34+ in a BCR/ABL kinase– and hnRNP-A1 shuttling–dependent manner. Moreover, by using shRNA-mediated E2F3 knock-down and BCR/ABL-transduced lineage-negative bone marrow cells from E2F3+/+ and E2F3−/− mice, we show that E2F3 expression is important for BCR/ABL clonogenic activity and in vivo leukemogenic potential. Thus, the complexity of the mRNA/RBP network, together with the discovery of E2F3 as an hnRNP-A1–regulated factor, outlines the relevant role played by RBPs in posttranscriptional regulation of CML development and progression.
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Zhang, Bin Amber, Dandan Zhao, Huafeng Wang, Chen Liang, Le Xuan Truong Nguyen, Junjing Qiao, Shanshan Suo, et al. "Microrna-142 Deficiency Promotes Chronic Myeloid Leukemia (CML) Transformation from Chronic Phase (CP) to Blast Crisis (BC)." Blood 136, Supplement 1 (November 5, 2020): 4. http://dx.doi.org/10.1182/blood-2020-143202.
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Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm resulting from the BCR-ABL1 fusion gene that encodes a constitutively activated tyrosine kinase (TK). Although TK inhibitors (TKIs) induce disease remission and prolonged survival in CML patients, a subset are resistant and progress from chronic phase (CP) to blast crisis (BC) with poor prognosis. Understanding the molecular mechanisms of transformation from CP to BC is necessary in the development of effective treatments. Here, we used the inducible SCLtTA/BCR-ABL transgenic CP CML model to study the molecular mechanism of disease evolution. Upon tetracycline withdrawal to induce BCR-ABL expression, both the SCLtTA/BCR-ABL homozygous (homo, i.e., SCLtTA+/+BCR-ABL+/+, hereafter called BCR-ABL) and heterozygous (het, i.e., SCLtTA+/-BCR-ABL+/-) mice developed and died of CP CML without developing BC CML, implying that BCR-ABL dosage is insufficient to induce transformation. MicroRNA (miR)-142 is highly expressed in hematopoietic cells with a critical role in normal hematopoiesis. In miR-142 knockout (KO)(miR-142−/−) mice, hematopoietic stem and progenitor cells expanded with a decrease of hematopoietic output. Loss of miR-142 function has been reported in lymphoma, acute lymphocytic leukemia and acute myeloid leukemia. Of note, we also observed lower levels of miR-142 in CD34+CD38- cells from patients with BC CML versus (vs) patients with CP CML. Thus, we hypothesized that miR-142 insufficiency may promote CML transformation from CP to BC. To test our hypothesis, we generated miR-142 KO BCR-ABL (i.e., miR-142−/−BCR-ABL) mice and observed increasing leukemic blasts over time after BCR-ABL induction in the blood and bone marrow (BM), but not in miR-142 wt (miR-142+/+)BCR-ABL controls even when the latter became moribund. MiR-142−/−BCR-ABL mice had larger spleens and significantly shorter survival [median: 26 vs 54 days (d); p<0.0001] than miR-142+/+BCR-ABL controls. Of note, while both homo (miR-142−/−) and het (miR-142+/−) miR-142 KO BCR-ABL mice eventually developed BC CML, the former had a significantly faster progression to BC and shorter survival (median: 26 vs 45 d; p=0.003) than the latter, suggesting miR-142 deficiency alone is sufficient to initiate BC transformation in the CP CML model in a dose-dependent manner. Importantly, all these features were recapitulated in congenic recipient mice transplanted with BM Lin-Sca-1+c-Kit+ cells (LSKs, 2000/mouse) from diseased miR-142−/−BCR-ABL mice, suggesting LSKs were enriched in leukemic stem cells and able to reproduce BC. Of note, in an RNA-seq analysis comparing LSKs from diseased miR-142−/−BCR-ABL (BC) and miR-142+/+ BCR-ABL(CP) mice, 504 genes were found differentially expressed. Gene set enrichment analysis (GSEA) showed only four pathways differentially expressed (upregulated); three [i.e., oxidative phosphorylation, glycolysis and adipogenesis] regulating cell metabolism and the fourth regulating protein secretion. Next, we developed a novel CpG-miR-142 mimic oligonucleotide, hereafter called CpG-M-miR-142, to restore miR-142 levels. Treatment with CpG-M-miR-142 (20mg/kg/day, iv, 4 weeks) on day 2 after BCR-ABL induction significantly prolonged survival of miR-142−/−BCR-ABL mice compared with CpG-scramble (SCR) (75% vs 33% survival rate at day 40 after BCR-ABL induction; median survival: not reached vs 25 d; p=0.03). Since we observed lower miR-142 levels in TKI-resistant vs TKI-sensitive CML patients (p=0.02), we selected LSKs from diseased miR-142−/−BCR-ABL and miR-142+/+BCR-ABL mice and exposed them to TKI nilotinib (NIL; 2µM) or vehicle for 72 hours to evaluate if downregulation of miR-142 was associated with TKI resistance. We observed lower apoptosis and higher cell growth in NIL-treated miR-142−/−BCR-ABL LSKs vs NIL-treated miR-142+/+BCR-ABL LSKs. The decreased sensitivity of miR-142−/−BCR-ABL LSKs to TKI was rescued by treatment with CpG-M-miR-142. CpG-M-miR-142 (2µM) plus NIL significantly increased apoptosis and reduced cell growth in miR-142−/−BCR-ABL LSKs compared with SCR+ NIL. We showed a key role of miR-142 deficiency in the transformation of CP CML to BC CML associated with deregulation of metabolic pathways. Restoring miR-142 expression in vivo with CpG-M-miR-142 significantly decreased the BC transformation rate, prolonged survival of miR-142−/−BCR-ABL mice and may increase sensitivity to TKIs. Disclosures Marcucci: Iaso Bio: Membership on an entity's Board of Directors or advisory committees; Abbvie: Speakers Bureau; Novartis: Speakers Bureau; Pfizer: Other: Research Support (Investigation Initiated Clinical Trial); Takeda: Other: Research Support (Investigation Initiated Clinical Trial); Merck: Other: Research Support (Investigation Initiated Clinical Trial).
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Santhanam, Ramasamy, Paolo Neviani, Anna Eiring, Joshua Oaks, Mario Notari, Bradley W. Blaser, Shujun Liu, et al. "FTY720, a New and Alternative Strategy for Treating Blast Crisis CML and Ph1 ALL Patients." Blood 108, no. 11 (November 16, 2006): 288. http://dx.doi.org/10.1182/blood.v108.11.288.288.
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Abstract Blast crisis chronic myelogenous leukemia (CML-BC) and Philadelphia chromosome positive (Ph1) acute lymphoblastic leukemia (ALL) are two fatal BCR/ABL-driven leukemias against which the current therapy with Abl kinase inhibitors fails to induce a long-term response, as the majority of patients are either refractory or relapse after a few months of treatment. We recently reported that functional loss of the PP2A tumor suppressor occurs during CML disease progression and that restoration of PP2A activity impairs in vitro and in vivo BCR/ABL leukemogenesis. Here we assessed the therapeutic potential of the PP2A activator FTY720 in CML-BC and Ph1 ALL patient cells and in in vitro and in vivo models of these BCR/ABL+ leukemias. FTY720 (500 nM-2.5 mM) induces caspase-dependent apoptosis (70–98% annexin V+) and impairs the clonogenic potential (70–95% inhibition) of imatinib/dasatinib-sensitive and -resistant (T315I) p210 and p190 BCR/ABL-expressing myeloid and lymphoid progenitor cell lines (Ph1 K562, 32D-p210BCR/ABL, 32D-p210(T315I)BCR/ABL and BaF3-p190BCR/ABL), respectively, and of primary bone marrow CML-BCCD34+ (n=11) and Ph1 ALLCD34+/CD19+ (n=12) patients cells. Interestingly the cytokine (IL-3 or IL-7)-dependent growth and differentiation of normal CD34+ myeloid and CD34+/CD19+ lymphoid progenitors (n=8) is not affected by FTY720 treatment. Furthermore, pharmacologic doses of FTY720 markedly suppress leukemogenesis in SCID mice (n=13 per group) transplanted with myeloid and lymphoid progenitor cells transformed with p210BCR/ABL and p190BCR/ABL, respectively. In fact, the median survival has not yet been reached in FTY720-treated (10 mg/kg/day) BCR/ABL+ cell-injected mice. Conversely, all of untreated 32D-p210BCR/ABL, 32D-p210BCR/ABL(T315I) and BaF3-p190BCR/ABL leukemic mice died of an overt acute leukemia-like process with a median survival of 4.3, 4.8 and 4.1 weeks, respectively (P<0.001). After 11 weeks of FTY720 treatment, 80% and 90% of p210 and p190 mice, respectively, were alive and in molecular remission. Moreover, long-term (189 days) FTY720 daily administration (10 mg/kg/day) did not induce any adverse effect, and achieved sustained absence of BCR/ABL+ cells (assessed by nested RT-PCR) in 50% of mice transplanted with myeloid progenitors expressing the imatinib/dasatinib-resistant T315I p210BCR/ABL mutant. Mechanistically, the anti-leukemic effects of FTY720 are sphingosine 1-phosphate receptor 1 (SIP1)-mediated and dependent on the ability of FTY720 to activate PP2A phosphatase. That, in turn, inhibits the activity and expression of wild type and mutant p210 and p190 BCR/ABL oncoproteins and important regulators (e.g. Akt) of malignant cell survival and proliferation. Altogether, these results not only reinforce the importance of the PP2A tumor suppressor in the biology of Ph1 leukemias but, because FTY720 has been shown to be feasible in Phase I-III clinical trials for multiple sclerosis or solid organ transplant patients, they strongly support the use of this PP2A activator as a novel therapeutic approach for CML-BC and Ph1 ALL.
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Milojkovic, Dragana, Satyajit Sahu, Jeffery Henderson, Barbara H. Czepulkowski, John R. Salisbury, William T. Bellamy, Stephen Devereux, Antonio Pagliuca, Aloysius Y. L. Ho, and Ghulam J. Mufti. "Immunohistochemical Characterisation of Vascular Endothelial Growth Factor (VEGF) and its Receptors Flt-1 and KDR in Chronic Myeloid Leukaemia (CML) Patients Treated with Imatinib Mesylate." Blood 104, no. 11 (November 16, 2004): 1999. http://dx.doi.org/10.1182/blood.v104.11.1999.1999.
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Abstract VEGF is a potent angiogenic regulator implicated in increased angiogenesis, which is characteristic of CML. VEGF interacts with its two tyrosine kinase (TK) receptors, Flt-1 (VEGFR-1) and KDR (Flk-1/VEGFR-2). CML therapy with imatinib mesylate, which targets BCR-ABL TK activity, induces haematological remission in 95% and complete cytogenetic remission (CCR)/ major response (MR) in 60% of cases. We describe the serial evaluation of patient bone marrow (BM) trephines and the analysis of the impact of imatinib on VEGF in all stages of CML during therapy. Consecutive CML (n=38) patients (25 males, 13 females, median age of 56 years (19 to 81) were sequentially analysed during the course of imatinib therapy. Chronic phase (CP) n=24, accelerated phase (AP) n=11 and blast crisis (BC) n=3 prior to imatinib therapy. BM examination was performed at diagnosis, at 1 month, then 3 monthly following imatinib therapy. Immunohistochemical analysis determined expression of VEGF, VEGFR1/Flt-1 or VEGFR2/KDR. Results were correlated with cytogenetic response. In normal BM cellular VEGF and its receptors were expressed in megakaryocytes and macrophages, but rarely in myeloid cells. VEGF was not expressed in erythroblasts, lymphocytes or plasma cells. A weak VEGFR1 signal was observed in monocytes/histiocytes and VEGFR2 in histiocytes only. VEGF expression in CML was detected as a diffuse cytoplasmic pattern in myeloid and monocyte precursors, classified as 4+ (very intense staining), 3+ (strong), 2+ (moderate), 1+ (weak), or 0 (completely negative) throughout. The intensity of staining varied and did not correlate with the stage of disease, but VEGF was strongly expressed in the megakaryocytes. Both VEGFR1/Flt-1 and VEGFR2/KDR were expressed in monocytic/myeloid population and in megakaryocytes. The staining intensity varied from borderline detectable to a very strong staining intensity. 14/38 (37%) of patients showed a reduction in VEGF expression, which was particularly marked with respect to VEGFR2 receptor staining. Of these, 10/14 patients (71%) achieved CCR, one MR (major response) and 3 minimal/NR (no response). All patients in this group had a reduction in BM cellularity, median 30% (15–70%). No change in VEGF receptor expression was detected in 10/38 (26%). Only 1 patient achieved CCR, 3 MR, with a minor/NR in the rest (60%), and a median BM cellularity of 30% (10–100%). Increasing VEGF expression was observed in 14/38 patients (37%). Here 6/14 (43%) patients showed NR (2 minor/minimal responses) with CCR in only 4 of the evaluable patients. The median BM cellularity was 25% (10–95%). 3/14 patients went on to develop progressive disease In summary, reduced expression of VEGF and its receptors, particularly KDR, can predict a favourable response to imatinib and correlates with a reduced BM cellularity and cytogenetic response. In patients with an increase or no change in VEGF expression there is a greater incidence of a poor cytogenetic response and a higher tendency to relapse despite a reduction in BM trephine cellularity, but a longer follow-up may be necessary.
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Li, Shaoguang, Yiguo Hu, Sarah Swerdlow, Theodore M. Duffy, Roberto Weinmann, and Francis Y. Lee. "Targeting BCR-ABL Kinase Activity-Independent Signaling Pathways and Leukemia Stem Cells Is Essential for Curative Therapy of Philadelphia Chromosome Positive (Ph+) Leukemia." Blood 106, no. 11 (November 16, 2005): 1990. http://dx.doi.org/10.1182/blood.v106.11.1990.1990.
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Abstract Therapeutic efforts for Philadelphia chromosome positive (Ph+) leukemia have focused on targeting mainly BCR-ABL kinase activity with kinase inhibitors, since it has generally been believed that shutting down BCR-ABL kinase activity will completely inhibit its functions, leading to inactivation of downstream signaling pathways. Inhibition of BCR-ABL kinase activity by imatinib mesylate (Gleevec) is highly effective in treating human Ph+ chronic myeloid leukemia (CML) in chronic phase, but not Ph+ B-cell acute lymphoblastic leukemia (B-ALL) and CML blast crisis. The reasons for this are not well understood, but the fact that imatinib is a strong inhibitor of BCR-ABL kinase activity suggests that BCR-ABL kinase activity-independent pathways also play a critical role in the development of both forms of Ph+ leukemia. We have previously shown that the SRC family kinases LYN, HCK, and FGR are activated by BCR-ABL in pre-B leukemic cells and are required for the development of B-ALL (Hu et al, Nat Genet36:453, 2004). Others have shown that cells from imatinib-resistant patients imatinib expressed an activated form of LYN (Donato et al, Blood101:690, 2003), and that a BCR-ABL mutant with no kinase activity was still able to activate HCK (Warmuth et al, J Biol Chem272:33260, 1997). Based on these observations, we hypothesized that inhibition of BCR-ABL kinase by imatinib might not inactivate SRC kinases activated by BCR-ABL in pre-B leukemic cells, which may explain the relatively poor activity of imatinib against Ph+ B-ALL and lymphoid blast crisis CML. We find that SRC kinases activated by BCR-ABL remain fully active in imatinib-treated mouse leukemic cells and this BCR-ABL kinase activity-independent pathway is essential for leukemic cell survival and proliferation. Blockade of this pathway also prevents CML transition to lymphoid blast crisis. In mice with B-ALL, inhibition solely of BCR-ABL kinase activity by imatinib is not curative, but inhibition of both SRC and BCR-ABL kinase activities by the novel, oral, multi-targeted kinase inhibitor dasatinib (BMS-354825), while not killing leukemic stem cells, affords complete remission, maintained as long as treatment is continued. In these mice, we identified the B-ALL leukemic stem cells as B220+CD43+ pro-B cells. CML mice treated with dasatinib lived significantly longer than those treated with imatinib, which correlated with significantly lower numbers of BCR-ABL-expressing leukemic cells in bone marrow, peripheral blood, and spleens of dasatinib-treated CML mice versus placebo- or imatinib-treated mice. However, neither dasatinib nor imatinib were curative in these mice, which was attributed to an inability of both drugs to completely kill Lin-c-kit+CD34-Hoe- CML stem cells. Our studies indicate that complete eradication of leukemic cells in B-ALL and CML mice requires not only targeting BCR-ABL kinase activity-dependent and SRC-dependent pathways, but also killing BCR-ABL-expressing stem cells insensitive to both imatinib and dasatinib. However, the rapid and striking hematologic response of B-ALL mice to dasatinib suggests that the pro-B progenitors with acquired self-renewal capacity are the major source of highly proliferating B-lymphoid leukemic cells in B-ALL mice, and that complete inhibition of growth of this leukemic population with dasatinib could achieve long-term survival in B-ALL.
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Patel, Beena, Pina Trivedi, Manisha Brahmbhatt, Sarju Gajjar, Ramesh Iyer, Esha Dalal, Shilin Shukla, Pankaj Shah, and Sonal Bakshi. "Detection of derivative 9 deletion by BCR-ABL fluorescence in-situ hybridization signal pattern to evaluate treatment response in CML patients." Archive of Oncology 17, no. 1-2 (2009): 13–18. http://dx.doi.org/10.2298/aoo0902013p.
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Background: To evaluate prognostic effect of submicroscopic deletions involving breakage and fusion points of the derivative chromosome 9 and 22 in chronic myeloid leukemia in untreated patients and their follow up samples to correlate with disease outcome. Methods: The study included 78 pretreatment (PT) samples from CML patients and 90 follow-up samples, classified as complete responders (CR, n=33), nonresponders (NR, n =54), and partial responder (PR, n=3) depending on the treatment status of the follow-up samples. Karyotype analysis was performed on metaphases obtained through short term cultures of bone marrow and blood. Detection of BCR-ABL fusion gene was performed using dual color dual fusion (D-FISH) translocation probes. Results: BCR-ABL fusion gene detection by D-FISH showed ABL-BCR deletion on derivative 9 in 47.8% of nonresponders which was higher as compared to pretreatment (11%). Mix D-FISH signal pattern was found in around 20% of pretreatment and non-responder samples. Average interval from chronic phase to blast crisis and accelerated phase was respectively 3.5 and 18 months and accelerated to blast crisis was 16.5 months from the time of diagnosis. The follow-up duration of 31 patients responded to therapy was significantly higher (p=0.0001) as compared to 45 patients who did not respond to therapy. Variant D-FISH signal pattern was seen at the time of diagnosis in patient who responded to therapy as well as those patients who did not respond to therapy. Conclusion: This is the first study from India reporting deletion in ABL, BCR, or ABL-BCR on derivative 9 did not correlate with response to therapy.
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Liu, Xiaoli, Song Zhang, Qingfeng Du, Wei Fan, Yu Wang, and Feng Yao. "ATP Binding Domain Mutants of BCR-ABL in Patients with Chronic Myeloid Leukemia Treated with Imatinib." Blood 106, no. 11 (November 16, 2005): 4859. http://dx.doi.org/10.1182/blood.v106.11.4859.4859.
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Abstract Objective:Imatinib (also called STI571 or Gleevec), a competitive inhibitor at the ATP binding site of BCR-ABL, has been shown remarkable clinical activity in patients with chronic myeloid leukemia (CML). However, a significant proportion of Imatinib-treated CML patients with advanced stage disease develop resistance. In this study, we try to detect ATP binding domain mutants of BCR-ABL in Imatinib-treated CML patients and explore the possibility that ATP binding domain mutants might confer resistance to imatinib. Patients and Methods:We analyzed bone marrow samples from 16 Imatinib-treated CML patients, including 6 chronic phase, 2 accelerated phase (AP) and 8 blast crisis (BC) patients. 8 patients were sensitive to imatinib and 8 patients resistant. A polymerase chain reaction strategy was used to amplify and sequence the ATP binding domain of BCR-ABL. Results:Point mutations were found in the ATP binding domain of BCR-ABL in 3 of 16 patients. In 2 of 8 imatinib-resistance patients, a single nucleotide change was detected and resulted in a threonine to isoleucine substitution at position 315(Thr315Ile) of ABL kinase. This point mutation has proved to interfere with drug binding and cause resistance to imatinib. In addition, we found a mutation (Lys357Arg) remain sensitive to imatinib. Discussion:Chronic myeloid leukemia (CML) is a hematopoietic disorder characterized by the malignant expansion of bone marrow stem cells. Its malignant clonal marker is Philadelphia chromosome (Ph) which harbors the BCR-ABL fusion gene. The latter encodes a chimeric BCR-ABL protein, identified as having a central role in the pathogenesis of CML. As a competitor for ATP binding of BCR-ABL, Imatinib selectively induces apoptosis and blocks proliferation in BCR-ABL positive cells and has been shown to have high activity in CML. Clinical studies demonstrate that Many Imatinib-treated CML patients with advanced stage disease respond initially but then relapse. Till now, the resistant mechanisms of imatinib have not all been known. In this study, we find that ATP binding domain mutants of BCR-ABL is one of the potential mechanisms of resistance to imatinib, but not all mutants associate with imatinib resistance and some mutants remain sensitive to imatinib.
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Papadopoulou, Vasiliki, Korina Peste, Dafni Koumpi, Vasiliki Stavropoulou, Panagiotis Theodorou Diamantopoulos, Dimitrios Dimitroulis, Konstantinos Zervakis, et al. "A Pilot Study Of Bone Marrow and Adipose Tissue – Derived Mesenchymal Stem Cells In Chronic Myeloid Leukemia (CML); Mesenchymal Stem Cells Of The Bone Marrow Harbor The Bcr-Abl Fusion Gene In a CML Patient." Blood 122, no. 21 (November 15, 2013): 1480. http://dx.doi.org/10.1182/blood.v122.21.1480.1480.
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Abstract Background Chronic myeloid leukemia (CML) is a disease of the haemopoietic stem cell, hence its hallmark chromosomal rearrangement is detected in different haemopoietic lineages. Due to reported altered adhesion of haemopoietic cells to bone marrow stroma in CML, the cytogenetic profile and properties of bone marrow and adipose tissue mesenchymal cells have been the interest of our current study. Previous research has shown that the BCR-ABL fusion gene is not present in bone marrow mesenchymal stem cells; however, it has been detected in endothelial cells and in Flk1+ hemangioblastic progenitors. Aims To study the properties of mesenchymal stem cells in CML, we initially investigated the presence of the BCR-ABL fusion gene in bone marrow and adipose tissue mesenchymal stem cells (MSCs) of patients with CML at diagnosis. Although previous studies have yielded negative results, the numbers of patients studied have been limited. Moreover, the presence of BCR-ABL in hemangioblastic progenitors in some cases of CML patients highlights the possibility that the lesion can sometimes be detected in a progenitor cell much more primitive than the haemopoietic stem cell. Adipose tissue MSCs were studied concomitantly with those of bone marrow in order to establish possible cell motility between the two compartments during disease evolution. Methods We isolated mesenchymal stem cells from bone marrow and adipose tissue mononuclear cells of four CML patients via cell culture. MSCs were isolated from the mononuclear cell fraction via serial medium changes removing non-adherent cells, followed by at least three passages. Patient samples were collected before the onset of therapy; three of the patients were diagnosed in chronic phase and one in blast crisis. High purity of MSCs from haemopoietic cell contaminants (>94% in all cases) was confirmed by flow cytometry; mesenchymal stem cells were positive for CD90, CD73 and CD105 and negative for CD34 and CD45. FISH analysis was performed to detect the BCR-ABL gene (probe LSI BCR/ABL1, ES Dual Color Translocation, Vysis). For each sample, 500 nuclei were examined. Results The BCR-ABL fusion gene was detected in 15% of the bone marrow MSCs’ nuclei of one patient, who had chronic phase CML. The CD34+/CD45+ cell population percentage in the final MSC population isolated from this patient was only 1,5% on flow cytometry analysis, which means that this result cannot be accounted for by haemopoietic cell contamination of MSCs. The other three patients did not carry the translocation in their bone marrow MSCs upon FISH analysis. Adipose tissue MSCs of all four patients were negative for BCR-ABL. Conclusions We have shown the presence of the BCR-ABL fusion gene in bone marrow mesenchymal stem cells of one CML patient. This result implies an occurrence of the t (9;22) (q11;134) translocation at a more primitive level in some cases of the disease, so that the bone marrow stroma can be involved in the malignant clone. Because there is still limited knowledge about the role of MSCs, whether BCR-ABL–positive or negative, in the development of the malignant haemopoietic clone in CML, our study will be extended to study the cytogenetic and gene expression profile of mesenchymal stem cells in a larger number of CML patients. Disclosures: No relevant conflicts of interest to declare.
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Moujalled, Donia M., Diane T. Hanna, Giovanna Pomilio, Veronique Litalien, Shaun Fleming, Marie Chanrion, Ana-Leticia Maragno, et al. "Identification of Potent BH3-Mimetic Combinations Targeting Pro-Survival Pathways in Human B-Cell Acute Lymphoblastic Leukemia." Blood 132, Supplement 1 (November 29, 2018): 567. http://dx.doi.org/10.1182/blood-2018-99-114051.
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Abstract Background Precursor-B acute lymphoblastic leukemia (B-ALL) is an aggressive hematological malignancy. Relapsed disease has a poor prognosis, despite improved outcomes with tyrosine kinase inhibitors for Ph+ cases and immunotherapeutic approaches, such as blinotumomab and CAR-T cells. Targeting cell survival with novel small molecule BH3-mimetic inhibitors of BCL-2 (e.g. Souers et al Nat Med 2013, Roberts et al, NEJM 2016 and Casara et al, Oncotarget 2018), BCL-XL (Lessene et al, Nat Chem Biol, 2013) or MCL1 (Kotschy et al, Nature 2016) is an emerging therapeutic option. BCL-2 is reported to have a pro-survival role in BCR-ABL1, JAK2 fusion, ETV6-RUNX1 and MLL-r driven ALL (Brown et al., Journal Biological Chemistry 2017). BH3-mimetics targeting BCL-2 and BCL-XL has efficacy in paediatric ALL xenografts (Khaw et al., Blood 2016), while ruxolitinib combined with ABT-737 is synergistic in JAK2-mutant pre-B-ALL (Waibel et al., Cell Reports 2013). We now report that combined targeting of BCL-2 and MCL1 has broad pre-clinical efficacy in adult B-ALL samples with Ph+, Ph- and Ph-like characteristics. Methods S55746 and S63845 were obtained from Servier/Novartis, A1331852 from Guillaume Lessene (WEHI), venetoclax, daunorubicin, dexamethasone (DXM) and tyrosine kinase inhibitors (TKIs) from Selleckchem. Bliss synergy scores were determined using a checkerboard approach to evaluate combinations (previously described Bliss, Ann Appl Biol 1939). Primary ALL cells were obtained from 14 patients (4 Ph+ and 10 Ph-) providing informed consent. Ex vivo cell viability (sytox blue exclusion) at 48h was determined over a 5-log dilution range (1nM-10uM) using drugs alone or in equimolar combinations. For in vivo studies, adult B-ALL patient derived xenografts were performed in NSG; NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice. Results Dual BH3-mimetic targeting of BCL-2 and MCL1 was strongly synergistic (Bliss sum >1000) in SUPB15 (Ph+ BCR-ABL1), BV173 (lymphoid blast crisis BCR-ABL1), MUTZ5 (Ph-like) and MHHCALL4 (Ph-like) B-ALL cell lines. This was more effective than single BH3-mimetic combinations with DXM or TKIs (dasatinib or ruxolitinib) (Fig. A, B). In B-ALL patient samples, combined BCL-2 and MCL1 targeting lowered the LC50 by 10-1000 fold (to LC50<10nM) in 4/4 Ph+ ALL cases and 8/10 Ph- cases. Similarly, combined MCL1 and BCL-XL targeting demonstrated synergy in 3/4 Ph+ cases and 7/10 Ph- cases (to LC50<10nM), confirming remarkable anti-leukemic activity compared to BH3-mimetics alone or chemotherapy (daunorubicin) (Fig. C). BH3-mimetic combination therapy (S55746/S63845) compared favourably in Ph+ ALL cases to S55746 (figure D) or S63845 (Figure E) in combination with dasatinib. Preliminary data using patient-derived xenografts in NSG mice revealed in vivo efficacy of combined S55746 and S63845 therapy against 3 adult B-ALL cases (1 Ph+ and 2 Ph-). Reduction of established ALL in the bone marrow was observed in mice receiving combined S55746/S63845 after one week of treatment (p=<0.05) (Fig. F-H). Conclusions Dual BH3-mimetic targeting of BCL-2 and MCL1 induces synergistic killing of human B-ALL cell lines and primary ALL samples in vitro and rapid cytoreduction in vivo. Simultaneous inhibition of BCL-2 and MCL1 represents a novel and effective approach for targeting Ph+, Ph- and Ph-like B-ALL without need for additional DNA-damaging chemotherapy or kinase inhibition. Our results support the translational investigation of dual BH3-mimetic targeting of BCL-2 and MCL1 in the clinic. Figure legend: BLISS synergy scores for A. Ph+ and B. Ph-like ALL cell lines for drug combinations targeting BCL-2, MCL1, BCR-ABL, JAK1/2 and DXM. C. LC50 activity in primary ALL after 48hr of treatment with BH3 mimetics and combinations targeting BCL-2, MCL1, BCL-XL, compared to daunorubicin (LC50< 10nM red; ~ 100nM yellow; >1uM green). D. Comparison of BH3-mimetics targeting D.BCL-2 or E. MCL1 in combination with dasatinib in Ph+ vs Ph- primary B-ALL samples. Activity expressed as LC50 activity after 48h, with median values shown. Irradiated NSG mice were transplanted with 106 primary B-ALL cells. Engraftment of F. Ph+ and G-H. Ph- B-ALL cells was confirmed at 10 weeks by detection of hCD45 in PB. Mice were then treated with i) vehicle (d1-5), ii) S55746 100mg/kg days 1-5 by gavage, iii) S63845 25 mg/kg IV on days 2 and 4 or iv) S55746+S63845. Mice were euthanized on day 8 and hCD45+ from flushed femurs quantified. Disclosures Chanrion: Servier: Employment. Maragno:servier: Employment. Kraus-Berthier:servier: Employment. Lessene:servier: Research Funding. Roberts:Janssen: Research Funding; AbbVie: Research Funding; Genentech: Research Funding; Walter and Eliza Hall: Employment, Patents & Royalties: Employee of Walter and Eliza Hall Institute of Medical Research which receives milestone and royalty payments related to venetoclax. Geneste:servier: Employment. Wei:Pfizer: Honoraria, Other: Advisory committee; Celgene: Honoraria, Other: Advisory committee, Research Funding; Amgen: Honoraria, Other: Advisory committee, Research Funding; Servier: Consultancy, Honoraria, Other: Advisory committee, Research Funding; Novartis: Honoraria, Other: Advisory committee, Research Funding, Speakers Bureau; Abbvie: Honoraria, Other: Advisory board, Research Funding, Speakers Bureau.
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Chang, Ji-Suk, Ramasamy Santhanam, Edward Briercheck, Anna Eiring, Rossana Trotta, Mario Notari, Denis-Claude Roy, Michael A. Caligiuri, and Danilo Perrotti. "Molecular and Pharmacologic Suppression of MAPK Activity Rescues Differentiation of BCR/ABL+ Myeloid Progenitors by Releasing C/EBPa from the Inhibitory Effect of hnRNP E2." Blood 108, no. 11 (November 1, 2006): 2240. http://dx.doi.org/10.1182/blood.v108.11.2240.2240.
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Abstract Impaired differentiation is a common feature of many hematological malignancies including blast crisis chronic myelogenous leukemia (CML-BC). We previously reported that the inability of CML-BC myeloid progenitors to undergo terminal neutrophil differentiation depends on suppression of C/EBPα expression through the translation inhibitory activity of the BCR/ABL-regulated RNA binding protein hnRNP E2. Here we show that p210-BCR/ABL oncoprotein post-translationally enhances hnRNP E2 expression and translation-regulatory function. In fact, hnRNP E2 protein but not mRNA expression directly correlates with BCR/ABL levels in myeloid progenitors expressing different BCR/ABL levels, in Ph1 K562 and EM3 and BCR/ABL-inducible TonB.210 cells. Likewise, graded BCR/ABL expression in primary Lin− mouse bone marrow cells results in increasing hnRNP E2 levels. This, in turn, inhibits the expression of C/EBPα and that of the C/EBPα-regulated G-CSF (granulocytic-colony stimulating factor) receptor. In these models of CML-BC, increased hnRNP E2 expression results from the BCR/ABL- and ERK1/2-dependent enhancement of hnRNP E2 protein stability, as hnRNP E2 levels are downregulated by treatment with imatinib or the MEK inhibitors PD098059 and U0126, and after expression of dominant negative ERK1/2 mutants. Accordingly, hnRNP E2 protein levels rapidly decrease and are barely detectable in parental 32Dcl3 cells treated for 30 min. with the protein synthesis inhibitor cycloheximide (CHX). By contrast, they remain unchanged in 32D-BCR/ABL cells CHX-treated for 4 hours. Accordingly, hnRNP E2 protein expression is markedly reduced in IL-3-deprived parental but not 32D-BCR/ABL cells through a proteasome-dependent mechanism. Mechanistically, we have evidence that the BCR/ABL-activated ERK1/2 kinases phosphorylate hnRNP E2 on four different sites (S173, S189, T213, and S272), and that serine to alanine substitution of these phosphorylation sites abolishes hnRNP E2 phosphorylation in living cells and in vitro kinase assay. Moreover, the non-phosphatable hnRNP E2S173A, S189A, T213A, S272A mutant is less stable than wild-type and phosphomimetic hnRNP E2. Functionally, suppression of hnRNP E2 phosphorylation/expression by chemical inhibition of ERK1/2 activity results in decreased hnRNP E2 binding to the C-rich element contained in the uORF-spacer region of C/EBPα mRNA. This, in turn, restores C/EBPα expression and allows G-CSF-driven neutrophilic maturation of differentiation-arrested 32D-BCR/ABL cells and Lin− mouse bone marrow cells expressing high levels of p210-BCR/ABL oncoprotein. Likewise, treatment of G-CSF-cultured (48 hours) CML-BCCD34+ bone marrow patient cells with the MEK1 inhibitor U0126 (25 mM, 32 h) destabilizes hnRNP E2, thus restoring C/EBPα expression. Altogether our data not only indicate that the BCR/ABL-induced ERK1/2 activity is essential for the hnRNP E2 stability and suppression of C/EBPα-dependent granulocytic differentiation of CML-BC myeloid progenitors, but they also suggest the potential use of clinically-relevant MAPK inhibitors in the therapy of CML-BC.
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Neviani, Paolo, Ramasamy Santhanam, Yihui Ma, Guido Marcucci, John C. Byrd, Ching-Shih Chen, Jorge Cortes, et al. "Activation of PP2A by FTY720 Inhibits Survival and Self-Renewal of the Ph(+) Chronic Myelogenous Leukemia (CML) CD34+/CD38− Stem Cell through the Simultaneous Suppression of BCR/ABL and BCR/ABL– independent Signals." Blood 112, no. 11 (November 16, 2008): 189. http://dx.doi.org/10.1182/blood.v112.11.189.189.
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Abstract CML is a clonal disorder of the pluripotent hematopoietic stem cell characterized by the sustained kinase activity of the BCR/ABL oncoprotein. We reported that the BCR/ABL-dependent and SET-mediated inhibition of protein phosphatase PP2A tumor suppressor activity is essential for the leukemogenic potential of CD34+ CML bone marrow progenitors, as molecular and pharmacologic restoration of PP2A inhibits the activity of BCR/ABL and that of several important regulators of cell survival/proliferation, thus resulting in marked apoptosis, impaired clonogenic potential and in vivo leukemogenesis of imatinib/dasatinib-sensitive and -resistant Ph(+), but not normal, CD34+ blasts and/or BCR/ABL+ mouse marrow progenitors. Here we show that SET-dependent suppression of PP2A activity is a common feature of Ph(+) progenitors (CMP and GMP) and imatinib/ dasatinib-insensitive CD34+/CD38- BCR/ABL+ (n=3) stem cells but not of the equivalent cell fractions from healthy individuals (n=3). To determine the biological importance and therapeutic implications of impaired PP2A activity in Ph(+) stem cells, we evaluated by clonogenic, CFC/replating, LTC-IC and CFSE-mediated cell division-tracking assays, the effects of FTY720 (2.5 mM), a PP2A activator currently in phase III trials for MS patients, and lentiviral-mediated ectopic PP2Ac expression on survival and self-renewal of BCR/ ABL+ stem/progenitor cells isolated from bone marrow of CML blast crisis patients (ntot=8; Ph1≥90%) and/or SCL-tTA-BCR/ABL transgenic animals (ntot=10). FTY720 treatment (2.5–5mM) severely suppressed the clonogenic potential of CD34+/CD38− and CD34+/CD38+/CD45RA−/+ CML stem/progenitor cells. Accordingly, self-renewal and long-term repopulating potential of CML leukemic stem cells was markedly impaired by pharmacologic PP2A reactivation. In fact, the CFC output of LTC-IC cultures (6 weeks) deriving from FTY720-treated (2.5 mM; 72h) Ph(+) CD34+ cells was more than 95% inhibited if compared to that of LTC-IC cultures from untreated CML cells. By contrast, imatinib (5 mM) and dasatinib (200 nM) treatment led to a 3.5 and 5-fold increase in CFC output, respectively. Consistent with the ability of FTY720 to impair self-renewal of CML stem cells, a 50–90% reduction of the CFSEMAX/quiescent cell population was observed in CFSE-stained CD34+ CML cells treated for 6–9 days with FTY720. Notably, FTY720 did not exert any significant effect on CFSE-stained CD34+ cells from healthy individuals whereas, as expected, imatinib (5 mM) and dasatinib (200 nM) treatment led to a 22% and 27% increase in CFSEMAX CML cells, respectively. Interestingly, only FTY720 triggered apoptosis of CFSEMAX CML cells (41% Annexin V+ cells) although BCR/ABL activity (phospho-ABL intracellular flow-cytometry staining) in CFSEMAX cells was efficiently inhibited by FTY720, Imatinib and dasatinib, suggesting that BCR/ABL-independent PP2A-regulated signals control the survival and self-renewal of CML stem cells. Indeed, lentiviral-driven PP2Ac-overexpression as well as treatment with FTY720, but not imatinib, significantly decreased (40–90% reduction) CFC/serial replating efficiency, colony size and percentage of CFSEMAX fraction (66–96% reduction) of Lin−/ Sca+/Kit+ (LSK) cells isolated from bone marrow and spleen of leukemic SCL-tTA-BCR/ ABL mice. Mechanistically, the detrimental effect of PP2A activation on survival and self-renewal of CML stem cells might depend on the ability of PP2A to inactivate b-catenin that, reportedly, is a PP2A target essential for the self-renewal of the CML blast crisis GMP progenitors. In fact, immunoblotting, direct immunofluorescence and LET/TCF luciferase assays showed that ectopic PP2Ac expression and/or FTY720, but not imatinib, treatment leads to inactivation/degradation of nuclear b-catenin in BCR/ABL+ primary mouse LSK and/or 32D-BCR/ABL cells. Altogether our data not only highlight the importance of PP2A inactivation for survival and self-renewal of CML stem cells but also suggest the existence of BCR/ABL-independent, PP2A-sensitive and b-catenin-mediated signals that may account for resistance of CML quiescent stem cells to tyrosine kinase inhibitor monotherapy. Thus, FTY720 treatment has the potential to eradicate CML by efficiently targeting both stem and progenitor Ph(+) cells regardless of their degree of sensitivity to imatinib and dasatinib.
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Mascarenhas, Cintia C., Anderson F. Cunha, Katia B. B. Pagnano, Rosana A. Silveira, Fernando F. Costa, Ricardo Pasquini, Nelma C. D. Clementino, and Carmino A. De Souza. "Screening of Mutations in BCR-ABL Kinase Domain in Chronic Myeloid Leukemia (CML) Patients Treated with Kinase Inhibitors by Denaturing High-Performance Liquid Chromatography (D-HPLC)." Blood 110, no. 11 (November 16, 2007): 4580. http://dx.doi.org/10.1182/blood.v110.11.4580.4580.
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Abstract Point mutations within the ABL kinase domain are the most frequent mechanism for reactivation of kinase activity of the BCR-ABL gene and have been associated with clinical resistance to tyrosine kinases (TK) inhibitors in CML patients conferring in some of them a poor prognosis. The T315I (Treonine → Isoleucine) is a mutation described in exon 6 of BCR-ABL gene that makes the protein resistant to all kinase inhibitors most currently used for treating CML (imatinib, nilotinib and dasatinib). D-HPLC allows for high throughput mutation screening. This technique is based on heteroduplex formation by PCR products amplified from wild type and mutant alleles. Under optimized denaturing conditions, these heteroduplexes can be distinguished from homoduplex. In this study we screened mutations in exon 6 of BCR-ABL gene in patients treated with kinase inhibitors, in different phases of the disease. We evaluated 85 patients: 9 at diagnosis, 81 in chronic phase, 3 in accelerated phase, one in blast crisis. Thirty four were resistant to imatinib, 10 of them to dasatinib and three had suboptimal response to imatinib. In 9 of 85 (10,5%) samples, D-HPLC showed an abnormal elution profile suggesting the presence of nucleotide changes. Automated sequencing confirmed the presence of two point mutations: T315I (two patients) and F359V (two patients). Five patients requires sequencing confirmation. Patients with T315I mutation failed to imatinib and dasatinib. One of them relapsed after bone marrow transplantation in blast crisis. Patients with F359V mutation were resistant to imatinib. One of them has partial hematological response with dasatinib and the other is in complete molecular response after bone marrow transplantation. D-HPLC seems to be a ship and practical method for routine clinical monitoring for emergence of kinase domain mutations and may be useful for optimizing therapy in CML. Early detection of emerging mutant clones may help in decision-making of alternative treatment.
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Agarwal, Anupriya, Ryan Mackenzie, Dorian LaTocha, Kavin Vasudevan, Eduardo Firpo, Marc Loriaux, Keiichi Nakayama, Brian J. Druker, James M. Roberts, and Michael Deininger. "The Oncogenic Role of Tumor Suppressor Protein p27 in Ph+ Chronic Myeloid Leukemia." Blood 114, no. 22 (November 20, 2009): 3276. http://dx.doi.org/10.1182/blood.v114.22.3276.3276.
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Abstract Abstract 3276 Poster Board III-1 Background: Several studies have indicated that BCR-ABL causes cell cycle defects by interfering with the cell cycle regulatory functions of p27, a Cyclin dependent kinase (Cdk) inhibitor and tumor suppressor. Studies in BCR-ABL positive cell lines have shown that BCR-ABL promotes proteasomal degradation of p27 in a pathway that involves the SCFSKP2 ubiquitin ligase, while cytoplasmic mislocalization has been described in primary CML cells. It has been suggested that the principal effect of this cytoplasmic mislocalization is to remove p27 from the nucleus, thereby relieving Cdks from p27 inhibition. However, recent studies have shown that a p27 mutant (p27CK-), that cannot bind to Cdks or Cyclins, actively contributes to oncogenesis. This raises the question as to whether cytoplasmic mislocalization of p27 in CML cells may in fact promote leukemogenesis rather than merely compromise Cdk inhibition. We therefore hypothesized that the net contribution of p27 in CML is to promote leukemogenesis due to the oncogenic activity of cytoplasmic p27. Experimental approach and results: We determined p27 localization in BCR-ABL positive cell lines and CD34+ progenitor cells from newly diagnosed chronic phase CML patients (N=7) and from CML patients in blast crisis (N=2) by immunoblotting of nuclear and cytoplasmic cellular fractions. We found that p27 is predominantly cytoplasmic in most CML cell lines and in CD34+ cells from 8/9 (89%) patient samples, including patients in blastic phase. Cytoplasmic localization of p27 in CD34+ cells from CML patients was also confirmed by immunofluorescence analysis. Further, we observed that inhibition of BCR-ABL kinase by imatinib, an Abl kinase inhibitor increased nuclear p27 in all cell lines tested and in 4/9 patient samples (3/7 chronic phase and 1/2 blastic phase samples). However, we did not observe a substantial change in the cytoplasmic p27 levels. Similar results were obtained in Ba/F3 and 32D murine hematopoietic cell lines expressing BCR-ABL when compared with the respective parental cells. Further, SKP2 was up-regulated in CD34+ cell from CML patients as compared to the normal patients consistent withSKP2 mediated down-regulation of nuclear p27. These data suggest that nuclear but not cytoplasmic p27 levels are predominantly regulated by BCR-ABL kinase activity. To test whether p27 is crucial for BCR-ABL-driven leukemia, we compared leukemogenesis between recipients of BCR-ABL transduced p27+/+ and p27-/- bone marrow. Mice transplanted with BCR-ABL infected p27-/- marrow had significantly longer median survival (70 days, range 48-150 days) compared to recipients of p27+/+ marrow (37 days, range 14-56 days) (p=0.0123). To exclude that this difference was related to the differences in homing and engraftment capabilities of p27+/+ and p27-/- bone marrow cells, we compared short term homing and long term engraftment of p27+/+ and p27-/- bone marrow cells transplanted into wild-type recipients and found no differences. These data suggest that the net contribution of p27 to BCR-ABL-mediated leukemogenesis is positive. Further, to investigate the contribution of nuclear p27 to leukemogenesis, we utilized marrow from p27S10A mice in the murine CML model. In p27S10A mice, p27 is nuclear to to abrogation of the phosphorylation site implicated in nuclear export. We injected BCR-ABL transduced bone marrow cells of p27S10A and p27+/+ mice into wild-type recipients and compared the disease progression. We observed that mice transplanted with BCR-ABL infected p27S10A marrow had significantly longer median survival (28 days, range 23-79 days) compared to the recipients of p27+/+ marrow (23 days, range 21-38 days) (p=0.0139). This data is consistent with nuclear tumor suppressor function of p27. Combined with the data above, this suggests that cytoplasmic p27 promotes BCR-ABL mediated leukemogenesis. Conclusions: Our data suggest that though nuclear p27 functions as a tumor suppressor, the net contribution of p27 in CML might be oncogenic due to an oncogenic role of the increased cytoplasmic p27. Restoring nuclear p27 or reducing cytoplasmic p27 may be therapeutically useful in malignancies with low nuclear and high cytoplasmic p27 expression. Disclosures: Druker: OHSU patent #843 - Mutate ABL Kinase Domains: Patents & Royalties; MolecularMD: Equity Ownership; Roche: Consultancy; Cylene Pharmaceuticals: Consultancy; Calistoga Pharmaceuticals: Consultancy; Avalon Pharmaceuticals: Consultancy; Ambit Biosciences: Consultancy; Millipore via Dana-Farber Cancer Institute: Patents & Royalties; Novartis, ARIAD, Bristol-Myers Squibb: Research Funding. Deininger:Genzyme: Research Funding; BMS: Consultancy; Novartis: Consultancy, Honoraria; Ariad : Research Funding.
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Iqbal, Tuba, Amber Younus, Uzma Zaidi, Jawad Hassan, and Tahir Sultan Shamsi. "Pediatric Chronic Myeloid Leukemia with Megakaryocytic Blast Crisis as Initial Presentation: Case Report and Review of Literature." National Journal of Health Sciences 5, no. 4 (July 12, 2021): 171–74. http://dx.doi.org/10.21089/njhs.54.0171.
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Abstract: Background: Pediatric Chronic Myeloid Leukemia (CML) is a rare entity accounting for 2-3% of pediatric malignancies. CML rarely presents as Blast Crisis (BC) at the time of diagnosis, and megakaryocytic blast crisis is even rarer. Case Presentation: We herein, report a case of a young female, 10-year-old who presented with anemia, leukocytosis and massive splenomegaly. Clinical features, peripheral film and bone marrow findings were consistent with CML in megakaryocytic blast crisis. Bone marrow cytogenetic analysis revealed karyotype of 46, XX, t(9:22)(q34;q11.2) in 20 metaphases and BCR-ABL P210 by PCR was detected with transcript level of 83%, which further confirmed our diagnosis. Conclusion: De novo presentation of chronic myeloid leukemia with megakaryocytic blast crisis is rarely observed in pediatric population with very few cases published till now. We are presenting this case because of its rarity, likelihood of misdiagnosis as AML (M7) and poor prognosis, if not treated precisely. Keywords: Chronic Myeloid Leukemia (CML), Acute Myeloid Leukemia (AML), Blast Crisis (BC), Acute Megakaryocytic Leukemia (AMKL), Chronic Phase (CP), Accelerated Phase (AP), Tyrosine Kinase Inhibitor (TKI).
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Sontakke, Pallavi, Richard W. J. Groen, Jennifer Jaques, Huipin Yuan, Anton Martens, Edo Vellenga, and Jan Jacob Schuringa. "Mouse Versus Human Extrinsic Cues Dictate Transformation Potential In BCR-ABL/BMI1-Induced Leukemia In Humanized Xenograft Models." Blood 122, no. 21 (November 15, 2013): 515. http://dx.doi.org/10.1182/blood.v122.21.515.515.
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Abstract The NSG [NOD/Lt-scid/IL2Rγnull] xenotransplantation mouse model is currently the model of choice to evaluate human hematopoietic engraftment and to study development of human leukemia. Indeed, we have previously shown that co-expression of BCR-ABL together with the polycomb repression complex 1 (PRC1) member BMI1 in human cord blood (CB) derived CD34+ cells was sufficient to induce a serially transplantable lymphoid leukemia (Rizo et al., Blood 2010). This leukemia was characterized by high levels of CD34+/CD19+/CD20-/IgM-/CD33-/CD15- lymphoid blasts in the bone marrow and a high degree of infiltration of blasts in spleen and liver. Clonal analysis revealed that similar clones gave rise to leukemia in primary and secondary recipients. Although in vivo no myeloid leukemias were observed, in vitro both lymphoid as well as myeloid immortalized long-term cultures could readily be established, in line with phenotypes observed in chronic myeloid leukemia patients whereby a chronic myeloid phase can egress into a myeloid or lymphoid blast crisis. It is very plausible that differences between murine and human hematopoietic stem cell niches underlie these observed differences. Human engraftment in NSG mice is typically lymphoid biased, and since many growth factors and cytokines are species-specific it is clear that the murine niche is not ideal to evaluate human hematopoietic engraftment and leukemic transformation potential. In our current study we have evaluated the in vivo leukemic transformation potential of human CB derived CD34+ cells expressing BCR-ABL and/or BMI1 in NSG mice in which scaffolds coated with culture-expanded human mesenchymal stromal cells (MSCs) were implanted subcutaneously 8 weeks prior to injection of transduced cells, to allow the development of a humanized niche containing mineralized bone-matrix, osteoblasts, stromal cells, as well as appropriate vascularization (Groen et al., Blood 2012). BCR-ABL/BMI1 transduced human CB derived CD34+ cells or primary blast crisis CML patient cells were injected either intravenously or directly into the humanized scaffolds, and leukemia development was evaluated. Our data indicate that in a humanized niche, in contrast to a murine niche, BCR-ABL was sufficient to induce leukemia as a single hit without overexpression of exogenous BMI1. Furthermore, both ALL as well as erythro/myeloid leukemias could be induced. The ALL could be transplanted to secondary recipients and besides the lymphoid marker CD19, the cells also expressed CD33 and CD15, but not CD11b or GPA. These data are in sharp contrast to results obtained in xenograft mouse models without human niches, where BCR-ABL expression alone in human cells was not sufficient to induce leukemia, and secondary hits such as BMI1 were essential. Efficient engraftment of a blast-crisis CML patient sample was also observed in the human niche model, whereby the immature blast-like phenotype was maintained in the human scaffold niche, while more differentiated cells were observed in the mouse bone marrow niche. In vitro, long-term self-renewing cultures could readily be established with cells retrieved from the human scaffold niche of these leukemic mice, while no long-term cultures could be initiated with cells retrieved from the murine bone marrow niche, from the same mouse. These data indicate that a human niche is required to maintain appropriate in vivo self-renewal of human BC CML cells. Interestingly, the endogenous BMI1 levels were significantly higher in cells retrieved from the human scaffold niche as compared to the mouse BM niche. In conclusion, our data indicate that BCR-ABL transformed cells needs secondary event such as over expression of oncogene like BMI1 for its full transformation potential, most likely to overcome or repress oncogene-induced senescence. The mouse environment is not able to provide these secondary events in human cells, whereas the human niche is able to provide signals that together with BCR-ABL are sufficient to fully transform human cells in xenograft models. Disclosures: No relevant conflicts of interest to declare.
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Wang, Pin-Yi, Fay Young, Randall M. Rossi, Timothy Bushnell, and Craig T. Jordan. "Modeling the Origins of Lymphoid Blast Crisis Via Expression of BCR/ABL in Hematopoietic Cells Mutated at the INK4a/ARF Locus." Blood 106, no. 11 (November 16, 2005): 464. http://dx.doi.org/10.1182/blood.v106.11.464.464.
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Abstract Homozygous deletion of exon 2 at the INK4a/ARF locus is found in approximately 50% of BCR/ABL-mediated lymphoid blast crisis specimens, indicating that dysfunction of the locus is a contributing factor in lymphoid leukemogenesis. The INK4a/ARF locus encodes tumor suppressors, p16INK4a and p14 (human)/p19 (mouse) ARF, which are involved in regulating the Rb-mediated G1 cell cycle check point and MDM2-mediated p53 degradation respectively. Here we report a novel murine acute lymphoblastic leukemia (ALL) model which creates a disease similar to human B-ALL. In this system, we used a retrovirus encoding BCR/ABL-p210 and green fluorescent protein (GFP) to transduce INK4a/ARF null hematopoietic cells (exon 2 deleted), followed by transplantation into wild-type C57Bl/6 recipient mice. As early as 10–12 days post-transplant, transduced GFP+ cells were detected in the peripheral circulation. By 14–16 days post-transplant, recipient mice had highly elevated white blood cell counts and signs of advanced disease (hunched posture, roughened coat, anorexia and inactivity). Flow cytometric studies indicated that peripheral GFP+ cells were almost entirely B lymphoid (B220 and CD19+), and cytological analysis showed a blast-like morphology. In addition, the disease was readily transferred to secondary and tertiary recipients by bone marrow transplantation. These findings indicated rapid and reproducible development of B-ALL in recipient animals. To characterize potential leukemic progenitor cells, in vitro colony assays were performed using marrow, spleen and peripheral blood cells. These experiments showed that almost all in vitro colony-forming ability resided in marrow and represented approximately 2% of the overall leukemic population. Further, the phenotype of colony-forming leukemic cells correlated with expression of VLA5 and VCAM-1. In order to explore potential target cells responsible for the disease phenotype, different subpopulations of donor INK4a/ARF null hematopoietic cells were sorted, transduced with the BCR/ABL-p210 retroviral vector, and transplanted into wild type recipients. These experiments demonstrated that B-ALL could arise from BCR/ABL expression in INK4a/ARF null hematopoietic stem cells (HSC) and Pro-B cells, but not from common myeloid progenitors (CMP) or IgM+B cells. Interestingly, while transplantation of infected Pro-B cells generated only B-ALL, the HSC-transduced recipient mice showed mixed lineage engraftment with contribution to both myeloid and lymphoid tissues. In these animals lymphoid GFP+ cells showed the same immature B-ALL phenotype as observed previously, while myeloid GFP+ cells showed heterogeneous myeloid subsets, consistent with a CML-like myeloproliferative disease. Over time, the B-ALL population in these mice progressed rapidly and the animals succumbed to ALL. Subsequent transplantation analysis of marrow from mixed lineage animals demonstrated that both the B-ALL and myeloid phenotypes could be transferred to secondary recipients. These findings indicate that leukemic stem cells (LSC) are detectable in this model of lymphoid blast crisis, but that the clinical presentation is highly dependent upon the developmental stage at which mutations occur. Given the typical myeloid phenotype observed upon expression of the BCR/ABL retroviral vector in wild type HSC, our findings indicate that INK4a/ARF function critically influences developmental fate choices in the progression of BCR/ABL-mediated leukemia from chronic to blast crisis phase.
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Harb, Jason, Paolo Neviani, Claudia Huettner, Guido Marcucci, and Danilo Perrotti. "BCR/ABL Dosage Hierarchically and Temporally Influences hnRNP A1, hnRNP K and hnRNP E2 Expression in Hematopoietic Stem and Progenitor Cells." Blood 114, no. 22 (November 20, 2009): 191. http://dx.doi.org/10.1182/blood.v114.22.191.191.
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Abstract Abstract 191 The molecular mechanism leading to the progression of chronic myelogenous leukemia (CML) from the indolent chronic phase (CML-CP) to the rapidly fatal blast crisis (CML-BC) are still unclear although a plausible assumption is that enhanced expression of BCR/ABL, as that observed in most of patients undergoing progression, represents the factor promoting clonal evolution of CML. Given that a) BCR/ABL levels are increased in the CML-BC stem/leukemia-initiating cell population; b) a causal relationship exists between levels and activity of the BCR/ABL oncoprotein and aberrant mRNA processing, nuclear export and/or translation; and c) molecular and/or pharmacologic interference with the expression and/or activity of the BCR/ABL-regulated RNA binding proteins (hnRNP A1, hnRNP K and hnRNP E2) antagonizes both in vitro and in vivo BCR/ABL leukemogenesis by impairing proliferation, inhibiting survival and/or restoring differentiation of BCR/ABL+ hematopoietic progenitors; we hypothesized that BCR/ABL initiates a hierarchical activation of signals leading to a temporally-organized increase in the expression/function of specific RNA binding proteins (RBPs), and that this represents an essential step for disease progression. To determine whether expression of hnRNP A1, K and E2 is hierarchically regulated by BCR/ABL and at which stage of the CML stem/progenitor cell development it occurs, we first transduced 32Dcl3 cells with the MigR1-BCR/ABL construct and allowed the clones from a 32D-BCR/ABLlow cell population to become BCR/ABLhigh/hnRNP E2high /C/EBPa− within 21 days of culture in the presence of IL-3. Western blots indicate that BCR/ABL-dependent full induction of hnRNP A1 expression precedes that of hnRNP K and E2 which occurs only after BCR/ABL levels and activity increase to levels capable of conferring cytokine-independent growth and differentiation arrest, suggesting that hnRNP A1 may have the role of a “gatekeeper”, as it allows the increase of BCR/ABL expression through inhibition of PP2A. This, in turn, will promote disease progression in part by inducing expression and activity of hnRNP K and E2 that, as we previously reported, regulate survival, proliferation and differentiation of CD34+ CML-BC progenitors. Notably, we observed a similar pattern of hnRNP induction in the lymphoid BCR/ABL-inducible TonB210157 cells. We also have evidence that differences in hnRNP A1, hnRNP K and hnRNP E2 expression exist between the stem and progenitor cell fractions of BCR/ABL+ primary cells and that they are differentially regulated in leukemic and normal cells. In fact, FACS analysis followed by intracellular protein staining performed on bone marrow- and spleen-derived LSK (Lin−/Sca-1+/c-kit+), common myeloid progenitors (CMPs) (Lin−/Sca-1−/c-kit+/CD34+, FCgRII/IIIdim) and granulocyte monocyte progenitors (GMPs) (Lin−/Sca-1−/c-kit+/CD34+/FCgRII/IIIbright) from leukemic SCLtTA-BCR/ABL double-transgenic mice showed that levels of hnRNP A1 were 3 to 5-fold higher in CMP/GMP than LSK cell fractions (LSK<CMP<GMP). By contrast, in non-induced animals, hnRNP A1 expression was overall markedly inhibited (10-fold lower) with respect to leukemic mice and progressively decreased during LSK maturation into GMPs (LSK>CMP>GMP). Likewise, hnRNP K and E2 levels were 2-fold increased in the progenitors compared to the LSK of leukemic animals, whereas an opposite trend in the expression of hnRNP K was observed in the LSK and CMPs/GMPs of non-induced animals. Moreover, hnRNP K levels in the leukemic CMPs/GMPs were 30 to 40-fold higher than those detected in the same cell fractions from non-induced animals. Interestingly, the highest levels of hnRNP A1 and K in the CMP/GMP fractions correlated with the development of a lymphoid blast crisis-like phenotype as determined by the 30% increase in splenic B220+/Mac-1+ cells. Altogether these data suggest that hierarchical and temporal changes in the expression of hnRNPs occur upon BCR/ABL transformation in stem and progenitor cells and during disease progression. Furthermore, these results are consistent with the reported role of the BCR/ABL-regulated hnRNP A1, K and E2 as a positive regulator of BCR/ABL stability through the SET-dependent inhibition of PP2A, a direct enhancer of Myc translation, and as an inhibitor of C/EBPa-dependent myeloid maturation of blast crisis CML progenitors, respectively. Disclosures: No relevant conflicts of interest to declare.
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Demehri, Shadmehr, Thoralf Lange, Peter Paschka, Susan Branford, Tamio Koizumi, Takeshi Sugimoto, Lay-Cheng Lim, et al. "CML with E8A2 BCR-ABL Fusion: The Fourth Breakpoint Cluster Region?." Blood 104, no. 11 (November 16, 2004): 1018. http://dx.doi.org/10.1182/blood.v104.11.1018.1018.
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Abstract In Ph+ leukemia, the breakpoints within BCR cluster in 3 distinct breakpoint cluster regions (bcr). Rearrangements in the minor (m−), major (M−) and micro (m−) breakpoint cluster region (bcr) give rise to e1a2, e13/14a2 and e19a2 BCR-ABL fusion proteins that are associated with Ph+ ALL, CML and chronic neutrophilic leukemia, respectively. Atypical BCR-ABL fusions have also been reported, mostly in isolated cases. Here, we describe eight patients with an unusual e8a2 BCR-ABL transcript and provide an overview of their common characteristics. The index case (#1) was a 56 year old man diagnosed with CML in 1993. Therapy with busulfan and hydrea induced a complete hematologic remission (CHR) that was maintained until March 2001, when accelerated disease developed. Treatment with imatinib and subsequently AML-type chemotherapy were only transiently effective and the patient died from myeloid blast crisis in March 2003. Cytogenetics demonstrated a standard t(9;22)(q34;q11). Unexpectedly, FISH was consistent with an m-bcr rearrangement and multiplex PCR showed an unusually large band. Sequencing identified a fusion between BCR exon e8 and ABL exon a2, with a 55 bp insert corresponding to an inverted segment of ABL intron Ib (corresponding to nt 29861–29915, Genbank U07561). Immunoblotting of bone marrow mononuclear cells with anti-ABL antibody identified a 200 kDa protein (p200BCR-ABL). Seven additional patients with an e8a2 BCR-ABL fusion have been identified (table 1). These patients tend to have relatively high platelet counts (median 569 x 109/L, range 216 – 1123) and relatively low white counts (median 42 x 109/L, range 4.7 – 146) but no other distinguishing characteristics at diagnosis. With a median follow-up of 36 months (range, 4 –97), two patients had progressed to blast crisis and died, one died from an unrelated cause, and five were alive, one after allogeneic BMT. Remarkably, none of the patients treated with interferon-a achieved even a minor cytogenetic remission, similar to patients with p190-positive CML. Two patients besides case #1 have received imatinib, one has achieved a CHR at four months, and information on the other is not available. Compared to P190 and P210, p200 lacks the pleckstrin homology (PH) domain but retains the Dbl-like and CDC24 homology domains of BCR. Deletion of the PH domain may disrupt the function of the Dbl-like domain to serve as a GTP exchange factor for Rac, Cdc42 and Rho. This may lead to disruption of the actin cytoskeleton and would be expected to cause a disease that is biologically closer to p190 than to p210.
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Di Stefano, Carla, Marco Tafani, Bruna Pucci, Elisabetta Abruzzese, Margherita Trawiska, Manuela Indelicato, Margherita Trawiska, et al. "A Distinctive Pattern of Different Gene Expression in Chronic Myelogenous Leukemia Patients." Blood 112, no. 11 (November 16, 2008): 4231. http://dx.doi.org/10.1182/blood.v112.11.4231.4231.
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Abstract Introduction: Molecular chaperones have many functions, such as protecting other proteins against aggregation, assisting in folding of nascent proteins/refolding of damaged proteins and targeting severely damaged proteins to degradation. As one of the molecular chaperones, Hsp90 functions to facilitate the folding of newly synthesized and denatured client proteins, including mutated p53, Bcr-Abl, p185ErbB2 and Raf-1. The Bcr-Abl fusion gene encodes for the p210Bcr-Abl tyrosine kinase (TK) implicated in the pathogenesis of chronic myelogenous leukemia (CML). Studies in cultured cells have identified many signal transduction pathways activated by Bcr-Abl, including activation of the Ras, MAPK, JNK/SAPK, phosphatidylinositol-3 kinase, nuclear factor-B and STAT pathways. Imatinib mesylate (imatinib IM) is a tyrosine kinase inhibitor that competitively inhibits ATP binding in the kinase domains of both the Bcr-Abl and c-Abl kinases. It has been suggested that resistance to imatinib stems from Bcr-Abl gene amplification, leading to overexpression of Bcr-Abl protein or point mutations in the Bcr-Abl gene However, several groups suggested that there might be other forms of Bcr-Abl-independent imatinib resistance Recently, it has been reported that changes in histone deacetylase (HDAC) expression in leukemic cells could be involved in mechanisms for abnormal cellular proliferation that operate through chromatin-independent pathways and thereby could lead to acquired drug resistance of the cells In the present study, we evaluated in primary leukemic blasts, obtained from chronic myelogenous leukemia patients at onset, patients in blast crisis and patients which were imatinib-resistant The espression the sirtuin members family and HSP70, HSP90 i-NOS and bcl-2 was evaluated by Nortern blot and Western blot analysis. Material and Methods: Primary leukaemia blasts We harvested primary blast rich mononuclear cells were obtained by gradient centrifugation on ficoll-hypaque of bone marrow and peripheral blood cells after obtaining appropriate informed consent. Northern blot Total RNAs from control or treated cells were isolated using Tri Reagent Aliquots of RNA were electrophoresed and blotted onto nylon membranes, that hybridized to 32P-labelled probe. Western Blot Cells were lysed and. then were centrifugated. Protein concentration was determined by the Bradford assay.. Equivalent amounts of protein loaded and electrophoresed and were transferred to nitrocellulose membranes, that were incubated with the different primary antibodies:, Result and Discussion:. In the present study, we evaluated a pattern of different gene expression by Northern Blot and Western Blot analysis in bone marrow and peripheral blood cells from 16 CML patients at onset, from 2 patients in blast crisis evolved under IM treatment, and 14 imatinib-resistant patients. Some RNAs were underexpressed in most or all samples tested and never overexpressed (eg SIRT2, SIRT3, SIRT4 and SIRT5), while others were overexpressed in the great majority of samples and rarely, if ever, underexpressed (eg SIRT1, SIRT7, HSP90, iNOS)Furthermore, we examined the level of heat-shock related proteins HSP90 and bcl-2 in 2 patients during treatment with IM. and one patient IM-resistant by western Blot analysis: HSP90 and BCl-2 increased one patient during treatment with IM, while both protein levels was very high in one one patient IM-resistant These results suggest that the difference of genes expression might contribute to patterns of clinical response.
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Ciccarelli, Bryan T., Ilona Tala, Tinghui Hu, Dan Li, Ru Chen, Qing Chen, and Ian P. Whitehead. "Investigation Of p210 Bcr-Abl Rhogef Activity In The Progression Of Chronic Myelogenous Leukemia." Blood 122, no. 21 (November 15, 2013): 3802. http://dx.doi.org/10.1182/blood.v122.21.3802.3802.
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Abstract The Philadelphia chromosome is formed by a balanced, reciprocal translocation that pairs sequences from BCR on Chromosome 22 with sequences from ABL on Chromosome 9 and results in the production of the constituitively active tyrosine kinase Bcr-Abl. Depending on the location of the breakpoint within BCR, three different sizes of Bcr-Abl can be produced (i.e., p190, p210 and p230) and they are associated with different clinical outcomes. The larger p210 form is observed in greater-than 95% of chronic myelogenous leukemia [CML], while the diminutive p190 is present in approximately 2/3 of Philadelphia-positive acute lymphoblastic leukemia [ALL]. Although both the p210 and p190 forms contain the same portion of Abl, importantly, they differ only in the amount of Bcr which is retained at the amino terminus. We previously identified a functional domain within the Bcr sequences preserved by p210, but not by p190, which demonstrates a constitutive Rho GTPase-specific guanine nucleotide exchange factor [RhoGEF] activity. To determine the contribution of this region to p210 Bcr-Abl-related disease progression in CML, we therefore introduced a single amino acid substitution [S509A] into this construct which abrogated its activity and then compared this mutant to the p210 and p190 variants in a murine bone marrow transplantation model. While all of the mice eventually developed myeloproliferative disease, those transplanted with either p210 Bcr-Abl S509A or p190 Bcr-Abl displayed a more rapid onset than the mice transplanted with p210 Bcr-Abl (within 12 vs. 23 days of transplantation, respectively). Interestingly, this reduced disease latency is associated with erythroid hyperplasia in the absence of anemia and expansion of megakaryocyte-erythrocyte progenitor, common myeloid progenitor and granulocyte-macrophage progenitor populations, which results in a phenotype that is similar to the M6 form of acute myeloid leukemia. This phenotype is also readily transplantable into secondary recipients, indicating that it is a true element of the malignancy and not a reactive process. Taken together, these results support a model wherein the RhoGEF activity of p210 Bcr-Abl actively regulates disease progression by downregulating the self-renewal of myeloid progenitors. While our animal studies indicate that the Bcr region plays a significant role in disease progression, to the best of our knowledge, this has yet to be evaluated using clinically derived mutations. Recently, the RhoGEF domain of p210 Bcr-Abl was reported to be mutated and/or partially deleted in tumors obtained from several CML blast crisis patients and a p210 Bcr-Abl-positive ALL patient. These findings suggest that the RhoGEF domain of p210 Bcr-Abl may in fact be actively involved in the aggressiveness of primary specimens as well. In order to determine the consequences of the reported mutations, we therefore examined their effects on disease progression using a murine bone marrow transplant model. Disclosures: No relevant conflicts of interest to declare.
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Wertheim, Jason A., Kevin Forsythe, Brian J. Druker, Daniel Hammer, David Boettiger, and Warren S. Pear. "BCR-ABL–induced adhesion defects are tyrosine kinase–independent." Blood 99, no. 11 (June 1, 2002): 4122–30. http://dx.doi.org/10.1182/blood.v99.11.4122.
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The t(9;22) chromosomal translocation results in expression of P210BCR-ABL, a fusion protein necessary for the development of chronic myelogenous leukemia (CML). The constitutive activation of the P210BCR-ABL tyrosine kinase results in phosphorylation of multiple signaling pathways leading to the transformed phenotype. Additionally, extracellular interactions between P210BCR-ABL–expressing progenitor cells and bone marrow stroma may provide external signals that facilitate CML development. In contrast to the intracellular signaling pathways involved in CML, little is known about how P210BCR-ABLexpression modifies cell-cell and cell-substratum interactions. To investigate the role of P210BCR-ABL in modulating cellular adhesion, we used a highly sensitive and quantitative cell detachment apparatus that measures the strength of association between a population of cells and an adhesive matrix. Our findings show that P210BCR-ABL expression increased adhesion nearly 2-fold between the myeloblastic cell line, 32D, and fibronectin compared to a control vector. We then investigated whether abnormal adhesion due to P210BCR-ABL expression was caused by its tyrosine kinase activity. A quantitative analysis of cell-fibronectin adhesion found that neither expression of a kinase-inactive P210BCR-ABL mutant in 32D cells or attenuation of kinase activity by STI571 (imatinib mesylate) in 32D cells transduced with wild-type P210BCR-ABL could correct the nearly 2-fold increase in cell-fibronectin adhesion. Similarly, STI571 treatment of Meg-01 cells, a P210BCR-ABL–expressing cell line derived from a patient in blast crisis, failed to inhibit adhesion to fibronectin. Together, our results indicate that changes in adhesion induced by P210BCR-ABL are independent of its tyrosine kinase activity.
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Benito, Juliana, Yuexi Shi, Barbara Szymanska, Hernan Carol, Ingrid Bohem, Hongbo Lu, Sergej Konoplev, et al. "Targeting the Leukemia-Associated Hypoxic Microenvironment with Hypoxia-Activated Prodrug PR-104." Blood 116, no. 21 (November 19, 2010): 868. http://dx.doi.org/10.1182/blood.v116.21.868.868.
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Abstract Abstract 868 Interactions between leukemia cells and the bone marrow (BM) microenvironment are known to promote leukemia cell survival and confer resistance to drugs commonly used in the management of this disease. We investigated the protective role of hypoxia in the BM microenvironment. We observed a marked expansion of hypoxic niches in the bone marrow of immunodeficient mice engrafted with the acute lymphoblastic leukemia (ALL) cell line Nalm6 and with primary ALL cells, as detected by the reductive 2-nitroimidazole compound pimonidazole (PIM), which forms stable adducts in hypoxic regions. We further demonstrated induction of the chemokine receptor CXCR4, and of the enzyme carbonic anhydrase 9 (CAIX), both targets of Hypoxia-Inducible Factor 1α (HIF-1a), in hypoxic areas of BM from mice harboring the Nalm6 xenografts. Furthermore, we used a CML blast crisis model to evaluate the time course of hypoxia expansion in the BM. In C57Bl6/J mice engrafted with murine HSCs co-expressing BCR/ABL and Nup98 we observed a time-dependent increase in PIM positive areas which coincided with the presence of GFP positive cells. In line with these findings, HIF-1α was highly expressed in BM biopsies from newly diagnosed ALL patients (n=15) but was significantly reduced when the patients achieved complete remission (CR). Culture under hypoxic conditions (1% O2) conferred resistance of pre-B ALL cells REH, Nalm-6, and of AML OCI-AML3 leukemic cells against several chemotherapeutic agents including vincristine, methotrexate and idarubicin (% of Annexin V(+) cells at 21% vs 1%O2; REH plus 1ng/ml vincristine:88.5+/−2.3 vs 18.1+/−10.3; REH plus 0.25uM etoposide: 86.4 +/−7 vs 14.4+/−8.6; Nalm6 plus 10ng/ml methotrexate: 33.4+/−2.7 vs 4.6+/−1; OCI-AML3 plus 50ng/ml idarubicin: 28.45+/−5 vs 12.76+/−1) . Taken together, these results provide rationale for examining the potential of hypoxia-activated pro-drugs to eliminate leukemia progenitor cells within hypoxic niches. To this end, we tested the hypoxia-activated prodrug PR104, a dinitrobenzamide nitrogen mustard that is reduced to its active metabolites under hypoxic conditions (Patterson et al., Clin Can Res 2007). In vitro, PR-104 induced cell death in three different leukemia cell lines (Nalm6, REH and the AML line Molm13) selectively under hypoxic (pO2 1%) conditions. The anti-leukemic efficacy of PR-104 as a single agent was next examined in several in vivo leukemia models. Administration of PR-104 prolonged survival and decreased leukemia burden of 1) NOD/Scid/IL2Rg-KO (NOG) mice injected with cells from primary refractory FLT3-mutated AML; 2) NOG mice injected with leukemic cells from an infant with MLL-rearranged B-lineage ALL; and 3) NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice injected with Nalm6-luciferase ALL cells. Next, we evaluated anti-tumor effects of PR-104 at clinically relevant doses (200, 100 and 50 mg/kg) and at maximal tolerated dose (550 mg/kg) in two ALL xenograft models (a T-lineage ALL and a B-cell precursor ALL) (Figure 1, studies supported by NCI NO1CM42216 and by PPTP contract NO1-CM91001-03). Compared to vehicle control, PR-104 significantly delayed progression of the T-ALL xenografts at all doses tested and at three of the four doses (550, 200 and 100 mg/kg) of B-cell pre-ALL, which resulted in significantly increased event-free survival (EFS) of mice in the treatment groups. Altogether, these findings strongly suggest that targeting hypoxia is feasible. If successful, this approach may significantly impact leukemia therapy and ultimately improve patient survival. This concept is currently being tested in an ongoing Phase I clinical trial of PR-104 in relapsed/refractory AML patients. Disclosures: Wilson: PROACTA: Equity Ownership. Konopleva:PROACTA: PI on clin trial funded by Proacta.
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Shiotsu, Yukimasa, Hitoshi Kiyoi, Ryohei Tanizaki, Yosuke Minami, Akihiro Abe, Yuichi Ishikawa, Kenichi Ishii, et al. "KW-2449, a Novel Multi-Kinase Inhibitor, Suppresses the Growth of Imatinib-Resistant Ph+ Leukemia Including BCR-ABL/T315I Both in Vitro and in Vivo." Blood 112, no. 11 (November 16, 2008): 1640. http://dx.doi.org/10.1182/blood.v112.11.1640.1640.
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Abstract Background: KW-2449 is a multi-kinase inhibitor against FLT3, ABL and ABL/T315I and Aurora kinases with IC50 values of 0.007, 0.014, 0.004 and 0.048 micro mol/L, respectively. We reported a possible mode of action of KW-2449 with respect to its anti-leukemic effects on FLT3-mutated and FLT3-wild type leukemia cells via FLT3 and Aurora inhibition, respectively (1). Currently KW-2449 is being investigated in a Phase 1/2 study in patients with acute myeloid leukemia. In this report, we investigated the activity of KW-2449 or imatinib in imatinib-resistant leukemia with the T315I mutation. Methods and results: We evaluated the effects of KW-2449 in vitro and in vivo on imatinib-resistant Ph+ leukemia. While imatinib suppressed the growth of K562 (Ph+CML with wild-type BCR-ABL) and TCC-Y (Ph+ALL with wild-type BCR-ABL) with GI50 values of 0.20 and 0.18 micro mol/L, it had little inhibitory effects on TCC-Y/sr (Ph+ALL with BCR-ABL/T315I) with a GI50 value of 24 micro mol/L. On the other hand, KW-2449 showed equivalent growth inhibitory activities against K562, TCC-Y and TCC-Y/sr giving the GI50 values of 0.2–0.6 micro mol/L. In addition, KW-2449 showed potent growth inhibitory activity against IL-3 dependent cells transfected with BCR-ABL and BCR-ABL/T315I with GI50 values below 0.50 micro mol/L, whereas imatinib had no growth inhibition in BCR-ABL/T315I cells. When we examined the ABL-signaling pathway, imatinib had no effects on the expression of phosphorylated BCR-ABL (P-BCR-ABL) and STAT5 (P-STAT5), a key downstream signal molecules of BCR-ABL in TCC-Y/sr cells. Furthermore, no obvious apoptosis or cell cycle effects were observed in BCR-ABL/T315I cells after imatinib treatment. In addition, the exposure to KW-2449 induced reduction of P-BCR-ABL and P-STAT5 at 0.25 micro mol/L and induced G2/M arrest and apoptosis over the GI50 value (0.50–1.0 micro mol/L). These data provide the evidence that BCR-ABL inhibition at a lower concentration of KW-2449 modulates its signaling pathway and that Aurora inhibition at a higher concentration may play a critical role in the anti-proliferative effects in imatinib-resistant CML and Ph+ALL. To assess the anti-leukemia activity of KW-2449 in vivo, the SCID mice intravenously inoculated with TCC-Y/sr leukemia were orally treated with KW-2449 or imatinib. While KW-2449 prolonged the survival, imatinib treatment had no effects in this model. Furthermore, anti-proliferative activity of KW-2449 was examined in primary samples from blast crisis CML patients who had BCR-ABL/T315I mutation. After inoculation of blast cells into NOG mice, KW-2449 or imatinib treatment started. In this model, oral treatments with KW-2449 decreased peripheral copy number of BCR-ABL mRNA and CD45+ blast cells in the bone marrow, though imatinib treatment showed limited activity. Conclusion: KW-2449 demonstrated anti-leukemia activity against imatinib resistant leukemia both in vitro and in vivo. These results suggest that KW-2449 would be effective against imatinib-resistant CML or Ph+ALL because of its potent and unique kinase inhibition profile.
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Sun, Kai, Xuejun Zhang, Zhen Wang, Yuqing Chen, Lei Zhang, Wei Cheng, Mingyue Shi, et al. "Allogeneic CAR-T Cell Therapy for Treatment of Relapse after Allo-HSCT in Patients with Refractory CML Lymphoid Blast Crisis: Significance of HLA Matched Donor/Patient Pair in the Safety/Efficacy of CAR-T Cell Therapy." Blood 132, Supplement 1 (November 29, 2018): 4275. http://dx.doi.org/10.1182/blood-2018-99-115768.
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Abstract Introduction: CD19-specific CAR-T cells have shown promise in the treatment of relapsed or refractory Ph+ ALL. It remains to be established whether allogeneic CAR-T cells have clinical activity in patients with relapsed CML lymphoid blast crisis with a history of allo-HSCT. Here we report our experience in two cases of allogeneic CAR-T cell therapy for treatment of relapse after allo-HSCT in patients with refractory CML lymphoid blast crisis. Methods: For manufacture of allogeneic CAR-T cells, peripheral blood mononuclear cells were collected from the same stem cell donor. Lentiviral construction and generation of CAR-T cells, clinical protocol design, assessment and management of cytokine release syndrome (CRS), were performed as described in our previous report (Leukemia. 2017;31:2587-2593). Fludarabine and cyclophosphamide had been administered for lymphocyte depletion before allogeneic CAR-T cells infusion. Patients: Patient 1 was a 52-year-old woman with refractory CML lymphoid blast crisis, who had a relapse after undergoing allo-HSCT from her daughter (HLA-10/10). Her initial examinations of peripheral blood and bone marrow were consistent with the diagnosis of CML lymphoid blast crisis. Cytogenetics and molecular analysis confirmed the presence of t(9;22)(q34;q11) and BCR-ABL1 210 fusion protein. In February 2017, examination of bone marrow revealed a further increase of lymphoblasts to 83.2%. In addition, ABL1 kinase mutations (Y253H and E255K/V) were identified. The patient underwent HLA 10/10-matched allo-HSCT without acute GVHD. A remission with a negative test for BCR-ABL1 210 and 99.62% donor chimerism had been achieved, then she had a lymphoblastic relapse occurred 2 months after allo-HSCT. Consistently, BCR-ABL1 210 turned positive, and chimerism analysis showed 67.4% donor chimerism. 3 weeks after relapse, allogeneic CAR-T cells were infused at the dose of 5×106 /kg CD19-specific CAR-T cells. Patient 2 was a 39-year-old male patient with relapsed CML lymphoid blast crisis with a history of allo-HSCT. He had received a diagnosis of CML chronic phase 7 years earlier. Bone marrow revealed a karyotype of 46, XY, t(3;9;22)(q27;q34;q11) and BCR-ABL mRNA transcript. From April 2011 to September 2012, the patient was treated with nilotinib. In September 2012, bone marrow examination revealed 78% lymphoblasts, thus the diagnosis of CML lymphoid blast crisis was established. In December 2012, the patient underwent HLA 7/10-matched sibling allo-HSCT (from his brother) without evidence of GVHD and maintained CR for 2 years. In December 2014, the patient developed bone marrow relapse (lymphoblast 9.5%) and extramedullary leukemia (testicular involvement) harboring the BCR-ABL-T315I mutation. During 2014 to 2018, the patient received multiple courses of CIKs, HDMTX and DLI, but failed to achieve CR. In March 2018, the patient received healthy donor derived allogeneic CAR19 T cells (2×105/kg) therapy. Result: Before CAR-T cells infusion, both patients with refractory CML lymphoid blast crisis had a relapse after successful allo-HSCT. Approximately 1 month after CAR-T cells infusion, a persistent morphologic remission, a recovering BM, and complete absence of BCR-ABL mRNA transcripts confirmed morphologic and molecular remission in both patients. Consistent with this, flow cytometry could not detect blasts or CD19+ B lineage cells. Patient 1 did not experience toxicities and allogeneic CAR-T cell therapy was well tolerated. Patient 2 developed severe CRS (Gr 4) including high-grade fevers (>40°C), hypotension, hypoxia, mental status changes, and seizures. These episodes ran for approximately 1 week before they were halted by treatment with steroids plus tocilizumab, and plasma exchange. The toxicity of allogeneic CAR-T cells is correlated with high levels of IL-6, IFN-γ, TNF-a, and CRP. Conclusion: The clinical outcomes from these 2 patients demonstrate the in vivo efficacy of allogeneic CD19-targeted T cells to induce clinical, morphology and molecular remissions as well as B cell aplasia in adults with relapsed CML lymphoid blast crisis with a history of allo-HSCT. The efficacy of allogeneic CAR-T cell therapy may not always be related to the risk of severe CRS. The degree of HLA matching may have a major impact on the prevention of CRS after allogeneic CAR-T cell therapy. Fully HLA-matched-pair may increase the safety and efficacy of the allogeneic CAR-T cell therapy. Disclosures No relevant conflicts of interest to declare.
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Koptyra, Mateusz, Scott Houghtaling, Marcus Grompe, and Tomasz Skorski. "Fanconi Anemia D2 Protein Contributes to BCR/ABL-Mediated Transformation of Hematopoietic Cells." Blood 106, no. 11 (November 16, 2005): 2878. http://dx.doi.org/10.1182/blood.v106.11.2878.2878.
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Abstract Homologous recombination (HR), involving RAD51 protein, plays an important role in the response of BCR/ABL-positive leukemia cells to numerous DNA double-strand breaks (DSBs) induced by reactive oxygen species (ROS) or genotoxic treatment. Fanconi D2 protein (FANCD2), a member of the Fanconi protein family, is monoubiquitinated on K561 and phosphorylated by ATM on S222 in response to DSBs. The K561 monoubiquitinated form of FANCD2 interacts with RAD51 during HR, and phosphorylation of FANCD2 on S222 is important for activation of S phase checkpoint in response to DNA damage. Our studies detected an enhanced interaction between RAD51 and FANCD2 in BCR/ABL-positive leukemia cells in comparison to normal counterparts. In addition, although the expression of FANCD2 was stimulated by BCR/ABL and growth factors, higher levels of FANCD2 monoubiquitination was detectable in CML patient cells at chronic phase and in blast crisis, and in BCR/ABL-transformed cells in comparison to non-transformed cells. This effect was reversed after inhibition of BCR/ABL kinase with STI571. Therefore, monoubiquitination of FANCD2 may play a role in BCR/ABL-mediated leukemogenesis. BCR/ABL kinase displayed an impaired transformation potential in FANCD2-/- murine bone marrow cells in comparison to +/+ counterparts. In addition, expression of BCR/ABL kinase, but not the kinase-deficient K1172R mutant, inhibited the proliferation rate of FANCD2-/- human lymphoblast cell line. Growth ability of BCR/ABL-positive FANCD2-/- cells could be rescued by co-expression of the wild-type and S222A mutant of FANCD2, but not the K561R mutant. This observation suggested that K561 monoubiquitination, but not S222 phosphorylation might play an important role in BCR/ABL-mediated transformation. Since BCR/ABL cells employ RAD51-dependent HR to repair numerous DSBs induced by ROS, elevated expression of monoubiquitinated FANCD2 may facilitate this process. This hypothesis is supported by the observation that BCR/ABL-positive FANCD2-/- cells accumulate more DNA damage than +/+ counterparts as indicated by enzymatic assays converting oxidative DNA lesions into gaps detectable by comet assay. In addition, enhanced oxidative DNA damage in BCR/ABL-positive FANCD2-/- cells produced a variety of DNA lesions including abasic sites, and single- and double-strand breaks assessed by neutral comet assay. Moreover, BCR/ABL-positive FANCD2-/- cells accumulated higher numbers of DSBs detected by γ-H2AX immunostaining and displayed discrete apoptosis. In conclusion we hypothesize that monoubiquitination of FANCD2 may play a role in the initial steps of BCR/ABL dependent leukemogenesis, probably due to its ability to interact with RAD51 and facilitate HR repair of an excess of spontaneous DSBs induced by ROS.
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Geyer, C. Ronald, Michael Szeto, Ashton Craven, Marciano D. Reis, David P. Sheridan, and John F. DeCoteau. "Stromal Cell-Derived Secreted Factors Contribute to the Innate Imatinib Resistance of Leukemia Stem Cells In a Genetically Defined Murine Model of Chronic Myeloid Leukemia Blast Crisis." Blood 116, no. 21 (November 19, 2010): 204. http://dx.doi.org/10.1182/blood.v116.21.204.204.
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Abstract Abstract 204 Treatment of chronic myeloid leukemia (CML) patients with tyrosine kinase inhibitors (TKIs), such as imatinib, nilotinib, and dasatinib, results in a dramatic reduction in proliferating BCR-ABL expressing leukemia cells. However, these agents do not eliminate the CML stem cell population, indicating that inhibiting BCR-ABL kinase activity alone is not sufficient to eradicate the disease. In vitro studies of human CML cell lines and CD34+ cells isolated from CML patients, have shown that bone marrow stromal cell factor (BMSF) conditioned media can maintain important pro-survival and self-renewal activities downstream of BCR-ABL in the presence of TKIs, suggesting a role for secreted BMSFs in innate resistance to BCR-ABL kinase inhibition. However, the ability of BMSFs to maintain the leukemic potential of CML stem cells upon exposure to TKIs has not been reported. We used a standard murine retroviral transduction system to model CML blast crisis (BC-CML) and obtain cells highly enriched for leukemia initiating potential. Purified LIN-, Sca-1+, CD117+ cells (LSKs) were isolated from the bone marrow of C57BL6/J mice and retrovirally-transduced with BCR-ABL-GFP and Nup98/HoxA9-YFP then injected intravenously into recipient C57BL6/J mice. All animals developed leukemia within 21 days characterized by leukocytosis and extensive infiltration of bone marrow and spleen with leukemic blasts. LSKs expressing both BCR-ABL-GFP and Nup98/HoxA9-YFP (GFP+/YFP+ LSKs) were purified from the spleens or bone marrows of leukemic mice and cultured for 72 hrs in BMSF conditioned media across a range of concentrations (0% - 50%) in the presence and absence of imatinib (0 - 1000 nM). BMSF conditioned media reduced the cytotoxic effects of imatinib on GFP+/YFP+ LSKs as assessed by cell counts, trypan blue viability assays, and Annexin V expression by flow cytometry. Furthermore, BMSF conditioned media reduced the inhibitory effects of imatinib on GFP+/YFP+ LSK colony formation in methylcellulose, and beta-catenin expression as assessed by flow cytometry. These observations strongly suggest that signaling by stromal cell-derived soluble factors protects BC-CML stem cells from imatinib therapy by re-activating pro-survival and self-renewal pathways. The ability of BMSFs to reduce the inhibitory effect of imatinib on BC-CML stem cell self-renewal in vivo was assessed by performing secondary transplantation assays. GFP+/YFP+ LSKs were purified from primary CML mice and transplanted into secondary recipients following in vitro exposure to BMSF conditioned media in the presence and absence of 1000 nM imatinib. Survival after transplantation was compared in cohorts of 5 mice per experimental condition: Group 1 (0% BMSF, 0 nM imatinib), Group 2 (50% BMSF, 0 nM imatinib), Group 3 (50% BMSF, 1000 nM imatinib) and Group 4 (0% BMSF, 1000 nM imatinib). Survival was significantly prolonged in Group 4 mice treated with 1000 nM imatinib and this effect was abrogated by treatment with 50% BMSF conditioned media, indicating that cell-derived soluble factors contribute to maintaining BC-CML stem cell function in the presence of imatinib. Our findings strongly suggest that signaling by soluble BMSFs plays an important role in the innate imatinib resistance of CML stem cells, implicating these factors in disease relapse. Genetically defined murine models of CML provide a powerful in vivo system to identify and target soluble factors that contribute to stromal-mediated cytoprotection of CML stem cells from TKIs. Disclosures: No relevant conflicts of interest to declare.
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Park, Chun Shik, Koramit Suppipat, and H. Daniel Lacorazza. "The Transcription Factor ELF4 Promotes Survival of Myeloid Leukemic Stem Cells." Blood 116, no. 21 (November 19, 2010): 1209. http://dx.doi.org/10.1182/blood.v116.21.1209.1209.
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Abstract Abstract 1209 Chronic myeloid leukemia (CML) is a myeloproliferative disease that originate in hematopoietic stem cells (HSCs) as a result of the t(9;22) translocation, giving rise to the Ph (Philadelphia chromosome) and BCR-ABL oncoprotein. Although treatment of CML patients with tyrosine kinase inhibitor can efficiently eliminate most leukemic cells, chemoresistant leukemic stem cells (LSCs) can survive and drive recurrence of CML in these patients. A number of genes have been described to promote or inhibit proliferation of LSCs. Some of them have similar roles in normal HSCs. The transcription factor ELF4 promotes cell cycle entry of quiescent HSCs during homeostasis (Lacorazza et al., 2006). Thus, to investigate the function of ELF4 in CML initiation and maintenance, we developed a BCR-ABL-induced CML-like disease using retroviral transfer of BCR-ABL in Elf4-null bone marrow (BM) cells. We first investigated whether ELF4 is required for the induction of CML. Recipient mice of BCR-ABL-transduced WT BM cells developed CML and died with a latency 16–23 days, whereas recipient mice of BCR-ABL-transduced Elf4-/- BM cells showed longer latency of 45–47 days (n=20; p<0.0005). Progression of leukemia was monitored in peripheral blood, BM and spleen by flow cytometry. In mice transplanted with BCR-ABL-transduced Elf4-null BM cells, Gr-1+ leukemic cells expanded the first two weeks after BM transplantation followed by a decline at expense of a secondary expansion of B220+ cells. In contrast, Gr-1+ leukemic cells continuously expanded in mice receiving BCR-ABL-transduced WT BM cells. These results suggest that loss of ELF4 causes a profound abrogation in BCR-ABL-induced CML, while allowing progression of B-cell acute lymphocytic leukemia. Since loss of Elf4 led to impaired maintenance of myeloid leukemic cells, we postulated that ELF4 may affect survival of LSCs. Thus, we analyzed the frequency of Lin-c-Kit+Sca-1+ (LSK) cells that are BCR-ABL positive in BM and spleen. We found that BCR-ABL+ LSK cells were significantly reduced in recipients of BCR-ABL-transduced Elf4-/- BM cells. These studies indicate that ELF4 is essential to maintain the LSC pool in CML acting as a molecular switch between myeloid and lymphoid blast crisis. Disclosures: No relevant conflicts of interest to declare.
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Al-Bayati, Asmaa, Ayoub Al-Bayti, and Vian Husain. "A short review about chronic myeloid leukemia." Journal of Life and Bio Sciences Research 4, no. 01 (January 9, 2023): 15–19. http://dx.doi.org/10.38094/jlbsr40172.
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Chronic myeloid leukemia (CML) develops as a result of a clonal process in a pluripotent stem cell. Anemia, granulocytosis, basophilia, thrombocytosis and splenomegaly are some of the symptoms of the condition. According to clinical findings, the illness progresses through three stages, each of which is identified by a rise in number of the blast cells in peripheral blood or bone marrow: chronic (10%), accelerated (10-19%) and acute leukemia-like blast crisis (20%). Most CML cases could be preliminary diagnosed by the presence of splenomegaly in addition to mutation in the BCR-ABL gene as well as complete blood count (CBC) test. Moreover, a bone marrow biopsy can provide a major confirmation of the disease process and staging. Definitive diagnosis of the disease can be either achieved through fluorescence in situ hybridization (FISH) or polymerase chain reaction PCR technique.
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Koptyra, Mateusz, and Tomasz Skorski. "Fanconi Anemia D2 (FANCD2) Is Required To Overcome Reactive Oxygen Species (ROS) - Induced DNA Double-Strand Breaks in BCR/ABL-Positive Leukemias." Blood 108, no. 11 (November 1, 2006): 2119. http://dx.doi.org/10.1182/blood.v108.11.2119.2119.
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Abstract BCR/ABL-mediated transformation is associated with elevation of ROS which, in addition to enhancing the cytoplasmic signaling pathways, may increase the number of oxidative DNA lesions including DSBs. Homologous recombination (HR), involving RAD51 protein, plays an important role in the response of BCR/ABL-positive leukemia cells to numerous DSBs induced by ROS. Fanconi D2 protein (FANCD2), a member of the Fanconi protein family, is monoubiquitinated on K561 and phosphorylated by ATM on S222 in response to DSBs. The K561 monoubiquitinated form of FANCD2 interacts with RAD51 during HR, and phosphorylation of FANCD2 on S222 is important for activation of S phase checkpoint in response to DNA damage. Our studies detected an enhanced interaction between RAD51 and FANCD2 in BCR/ABL-positive leukemia cells in comparison to normal counterparts implicating the role in repair of ROS-dependent DSBs. In addition, higher levels of monoubiquitinated FANCD2 protein was detectable in CML patient cells at chronic phase and in blast crisis, and in BCR/ABL-transformed cells in comparison to non-transformed cells. This effect was reversed after inhibition of BCR/ABL kinase with STI571. Therefore, FANCD2 may play a role in BCR/ABL-mediated leukemogenesis. This speculation is supported by impaired transformation potential of the BCR/ABL kinase in FANCD2−/− murine bone marrow cells in comparison to +/+ counterparts. In addition, expression of BCR/ABL kinase, but not the kinase-deficient K1172R mutant, inhibited the proliferation rate of FANCD2−/− human lymphoblast cell line. The growth defect of BCR/ABL-positive FANCD2−/− cells was accompanied with delayed leukemogenesis in SCID mice. Growth ability of BCR/ABL-positive FANCD2−/− cells could be rescued by co-expression of the wild-type and S222A mutant of FANCD2, but not the K561R mutant. This observation suggested that K561 monoubiquitination, but not S222 phosphorylation might play an important role in BCR/ABL-mediated transformation. Since BCR/ABL cells employ RAD51-dependent HR to repair numerous DSBs induced by ROS, elevated expression of monoubiquitinated FANCD2 may facilitate this process. This hypothesis is supported by the observation that BCR/ABL-positive FANCD2−/− cells and +/+ counterparts display similar levels of ROS and oxidized DNA bases, however, the former cells accumulate more DSBs evaluated by neutral comet assay and detected by γ-H2AX foci immunostaining. This effect could be reversed by the expression of FANCD2 S222A, but not K561R mutant, again implicating HR in reparation of these DSBs. Elevated levels of ROS-mediated DSBs in BCR/ABL-positive FANCD2−/− cells did not cause any significant changes in cell cycle distribution, but resulted in discrete but persistent apoptosis. Scavenging of ROS by vitamin E and N-acetylcysteine reduced the number of DSBs and eliminated the growth defect in BCR/ABL-positive FANCD2−/− cells without affecting their +/+ counterparts. In conclusion we hypothesize that monoubiquitination of FANCD2 may play a role in BCR/ABL-dependent leukemogenesis, probably due to its ability to interact with RAD51 and facilitate HR repair of an excess of spontaneous DSBs induced by ROS.
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Hutchinson, Catherine, David P. Sheridan, John F. DeCoteau, and Ron Geyer. "Chaetocin Exhibits Anti-Leukemia Activity Against Chronic Myeloid Leukemia Stem Cells." Blood 120, no. 21 (November 16, 2012): 1667. http://dx.doi.org/10.1182/blood.v120.21.1667.1667.
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Abstract Abstract 1667 Treatment of chronic myeloid leukemia (CML) patients with tyrosine kinase inhibitors (TKIs) targeting BCR-ABL kinase results in a dramatic reduction in proliferating BCR-ABL expressing leukemia cells. However, these agents do not eliminate the CML stem cell population, indicating that inhibiting BCR-ABL kinase activity alone is not sufficient to eradicate the disease, and implicating TKI-insensitive CML stem cells in relapse. Thus, there is great interest in identifying new therapeutic agents that can be combined with TKIs to eliminate TKI-insensitive CML stem cells that are not fully addicted to BCR-ABL. Chaetocin, a mycotoxin that inhibits histone methyltransferase SUV39H1, exhibits anticancer properties against myeloma and AML cells. To investigate a potential role for chaetocin in CML treatment, we first compared its cytotoxic effects with those of imatinib on TonB210, a murine hematopoietic cell line with doxycycline-inducible BCR-ABL expression that is dependent on BCR-ABL for survival and proliferation in the absence of exogenous IL-3. As expected, imatinib treatment produced cytotoxic effects in TonB210 cells induced to express BCR-ABL, but not in uninduced TonB210 cells, as assessed by trypan blue viability assays and Annexin V expression by flow cytometry. In contrast, chaetocin, displayed significant cytotoxicity against both TonB210 cells induced to express BCR-ABL and uninduced TonB210 cells. We then used a standard murine retroviral transduction system that models CML blast crisis (BC-CML) to study chaetocin effects on BCR-ABL expressing cells highly enriched for leukemia initiating potential. Purified LIN-, Sca-1+, CD117+ cells (LSKs) were isolated from the bone marrow of C57BL6/J mice and retrovirally-transduced with BCR-ABL-GFP and Nup98/HoxA9-YFP then injected intravenously into recipient C57BL6/J mice. All animals developed leukemia within 21 days characterized by leukocytosis and extensive infiltration of bone marrow and spleen with leukemic blasts. LSKs expressing both BCR-ABL-GFP and Nup98/HoxA9-YFP (GFP+/YFP+ LSKs) were purified from the spleens or bone marrows of leukemic mice and cultured for 48 hrs in chaetocin across a range of concentrations (0 – 400 nM). Chaetocin exhibited cytotoxic effects against GFP+/YFP+ LSKs as assessed by trypan blue, Annexin V, and colony forming assays. Chaetocin effects on CML stem cell self-renewal in vivo were assessed by performing secondary transplantation assays and limiting dilution analysis (LDA). GFP+/YFP+ LSKs were purified from primary CML mice and transplanted into secondary recipients following in vitro exposure to IC50 concentrations of chaetocin and/or imatinib. Survival after transplantation and LSC frequency were compared in four treatment cohorts: Group 1 (untreated control), Group 2 (chaetocin), Group 3 (imatinib) and Group 4 (chaetocin + imatinib). Compared to untreated mice, survival was significantly prolonged in mice treated with imatinib alone and the survival benefit of imatinib was enhanced by co-treatment with chaetocin. LDA showed both chaetocin and imatinib treatment alone decreased LSC frequency (1 in 10,143 in chaetocin treated, and 1 in 95,782 in imatinib treated vs. 1 in 4,036 in untreated control) but that the combination of chaetocin and imatinib treatment resulted in a dramatic decrease in LSC frequency (1 in 293,628 in chaetocin + imatinib treatment vs. 1 in 4036 in untreated control). Chaetocin, imatinib, and chaetocin + imatinib treatment of normal murine hematopoietic stem cells resulted in only minimal cytotoxicity as assessed by trypan blue, Annexin V, and colony forming assays. Our findings suggest that chaetocin, or chaetocin-related compounds, might serve to complement TKI therapy in the treatment of CML. Disclosures: No relevant conflicts of interest to declare.
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Mukherjee, Kaushiki, Xiaojin Sha, Ravi Bhatia, Tomasz Skorski, Barbara Hoffman, and Dan Liebermann. "GADD45a Is a Tumor Suppressor In BCR-ABL-Driven Leukemogenesis." Blood 122, no. 21 (November 15, 2013): 1467. http://dx.doi.org/10.1182/blood.v122.21.1467.1467.
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Abstract The BCR-ABL fusion oncogene which encodes a fused deregulated tyrosine kinase causes chronic myelogenous leukemia (CML) in humans. Imatinib, a small molecule ABL kinase inhibitor has been highly effective in treating chronic phase (CP) CML patients. However, a substantial number of patients undergo relapse due to development of resistance to imatinib therapy that leads to blast crisis (BC-CML), which is invariably fatal within weeks to months. Additional genetic aberrations assist in progression and identification of key players that are responsible for transformation is of utmost importance from a therapeutic point of view. Growth arrest DNA damage 45a (Gadd45a) gene, a member in the gadd45 family of genes including Gadd45b & Gadd45g, was identified as a myeloid differentiation primary response gene. There is evidence consistent with it’s involvement in G2/M cell cycle arrest and apoptosis in response to multiple stressors, including genotoxic and oncogenic stress. To investigate the effect of Gadd45a in the development of CML, adaptive bone marrow transplantation experiments with either wild type or Gadd45a null myeloid progenitors expressing 210-kD BCR-ABL fusion oncoprotein revealed that loss of Gadd45a accelerated BCR-ABL driven CML resulting in the development of a more aggressive AML/BC like disease. Recent newly obtained data indicate that number of Gadd45a deficient Leukemic stem cells (LSC) harboring BCR-ABL increased as disease progressed confirming Gadd45a as a crucial tumor suppressor in CML. Recent data also indicate, that transformed Gadd45a deficient progenitors exhibit increased proliferation and decreased apoptosis, associated with enhanced PI3K-AKT-mTOR-4E-BP1 signaling and upregulated oncogenic p30C/EBPα. More importantly, newly obtained data indicate that Gadd45a transcript levels in peripheral blood of human blast crisis (BC-CML) samples was found to be reduced compared to accelerated phase (AP-CML), chronic phase (CP-CML) and normal controls, assessed by Quantitative real time PCR analysis. Collectivly these data strongly suggest that Gadd45a expression is a novel prognostic indicator of CML progression, implicating Gadd45a as a downregulated target of BCR-ABL associated with progression to more aggressive stages. To conclude, our findings provide novel evidence that Gadd45a functions as a suppressor of BCR/ABL driven myeloid leukemogenesis, & that suppresion of Gadd45a is associated with CML progression. These data provide the impetus to further elucidate the role Gadd45a plays in suppressing the development of CML, and explore how its loss contributes to the progression of CML to more aggressive leukemic phenotypes. Disclosures: No relevant conflicts of interest to declare.