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1
Gross, Alec W., Xiaowu Zhang, and Ruibao Ren. "Bcr-Abl with an SH3 Deletion Retains the Ability To Induce a Myeloproliferative Disease in Mice, yet c-Abl Activated by an SH3 Deletion Induces Only Lymphoid Malignancy." Molecular and Cellular Biology 19, no. 10 (October 1, 1999): 6918–28. http://dx.doi.org/10.1128/mcb.19.10.6918.
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ABSTRACT The bcr-abl oncogene plays a critical role in the pathogenesis of chronic myelogenous leukemia (CML). The fusion of Bcr sequences to Abl constitutively activates the Abl protein tyrosine kinase. We have recently shown that expression of Bcr-Abl in bone marrow cells by retroviral transduction efficiently induces in mice a myeloproliferative disease resembling human CML and that Abl kinase activity is essential for Bcr-Abl to induce a CML-like myeloproliferative disease. However, it is not known if activation of the Abl kinase alone is sufficient to induce a myeloproliferative disease. In this study, we examined the role of the Abl SH3 domain of Bcr-Abl in induction of myeloproliferative disease and tested whether c-Abl activated by SH3 deletion can induce a CML-like disease. We found that Bcr-Abl with an Abl SH3 deletion still induced a CML-like disease in mice. In contrast, c-Abl activated by SH3 deletion induced only lymphoid malignancies in mice and did not stimulate the growth of myeloid colonies from 5-fluorouracil-treated bone marrow cells in vitro. These results indicate that Bcr sequences in Bcr-Abl play additional roles in inducing myeloproliferative disease beyond simply activating the Abl kinase domain and that functions of the Abl SH3 domain are either not required or redundant in Bcr-Abl-induced myeloproliferative disease. The results also suggest that the type of hematological neoplasm induced by an abl oncogene is influenced not only by what type of hematopoietic cells the oncogene is targeted into but also by the intrinsic oncogenic properties of the particular abl oncogene. In addition, we found that ΔSH3 c-Abl induced less activation of Akt and STAT5 than did Bcr-Abl, suggesting that activation of these pathways plays a critical role in inducing a CML-like disease.
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Cross, Nick. "BCR-ABL Negative CML-Like Disorder." Clinical Lymphoma Myeloma and Leukemia 17 (September 2017): S107—S108. http://dx.doi.org/10.1016/j.clml.2017.08.052.
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He, Yiping, Jason A. Wertheim, Lanwei Xu, Juli P. Miller, Fredrick G. Karnell, John K. Choi, Ruibao Ren, and Warren S. Pear. "The coiled-coil domain and Tyr177 of bcr are required to induce a murine chronic myelogenous leukemia–like disease by bcr/abl." Blood 99, no. 8 (April 15, 2002): 2957–68. http://dx.doi.org/10.1182/blood.v99.8.2957.
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Abstract The bcr/abl fusion in chronic myelogenous leukemia (CML) creates a chimeric tyrosine kinase with dramatically different properties than intact c-abl. In P210 bcr/abl, the bcr portion includes a coiled-coil oligomerization domain (amino acids 1-63) and a grb2-binding site at tyrosine 177 (Tyr177) that are critical for fibroblast transformation, but give variable results in other cell lines. To investigate the role of the coiled-coil domain and Tyr177 in promoting CML, 4 P210 bcr/abl-derived mutants containing different bcr domains fused to abl were constructed. All 4 mutants, Δ(1-63) bcr/abl, (1-63) bcr/abl, Tyr177Phe bcr/abl, and (1-210) bcr/abl exhibited elevated tyrosine kinase activity and conferred factor-independent growth in cell lines. In contrast, differences in the transforming potential of the 4 mutants occurred in our mouse model, in which all mice receiving P210 bcr/abl-expressing bone marrow cells exclusively develop a myeloproliferative disease (MPD) resembling human CML. Of the 4 mutants assayed, only 1-210 bcr/abl, containing both the coiled-coil domain and Tyr177, induced MPD. Unlike full-length P210, this mutant also caused a simultaneous B-cell acute lymphocytic leukemia (ALL). The other 3 mutants, (1-63) bcr/abl, Tyr177Phe bcr/abl, and Δ(1-63) bcr/abl, failed to induce an MPD but instead caused T-cell ALL. These results show that both the bcr coiled-coil domain and Tyr177 are required for MPD induction by bcr/abl and provide the basis for investigating downstream signaling pathways that lead to CML.
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Hao, Sheryl X., and Ruibao Ren. "Expression of Interferon Consensus Sequence Binding Protein (ICSBP) Is Downregulated in Bcr-Abl-Induced Murine Chronic Myelogenous Leukemia-Like Disease, and Forced Coexpression of ICSBP Inhibits Bcr-Abl-Induced Myeloproliferative Disorder." Molecular and Cellular Biology 20, no. 4 (February 15, 2000): 1149–61. http://dx.doi.org/10.1128/mcb.20.4.1149-1161.2000.
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ABSTRACT Chronic myelogenous leukemia (CML) is a clonal myeloproliferative disorder resulting from the neoplastic transformation of a hematopoietic stem cell. The majority of cases of CML are associated with the (9;22) chromosome translocation that generates thebcr-abl chimeric gene. Alpha interferon (IFN-α) treatment induces hematological remission and prolongs life in 75% of CML patients in the chronic phase. It has been shown that mice deficient in interferon consensus sequence binding protein (ICSBP), a member of the interferon regulatory factor family, manifest a CML-like syndrome. We have shown that expression of Bcr-Abl in bone marrow (BM) cells from 5-fluorouracil (5-FU)-treated mice by retroviral transduction efficiently induces a myeloproliferative disease in mice resembling human CML. To directly test whether icsbp can function as a tumor suppressor gene, we examined the effect of ICSBP on Bcr-Abl-induced CML-like disease using this murine model for CML. We found that expression of the ICSBP protein was significantly decreased in Bcr-Abl-induced CML-like disease. Forced coexpression of ICSBP inhibited the Bcr-Abl-induced colony formation of BM cells from 5-FU-treated mice in vitro and Bcr-Abl-induced CML-like disease in vivo. Interestingly, coexpression of ICSBP and Bcr-Abl induced a transient B-lymphoproliferative disorder in the murine model of Bcr-Abl-induced CML-like disease. Overexpression of ICSBP consistently promotes rather than inhibits Bcr-Abl-induced B lymphoproliferation in a murine model where BM cells from non-5-FU-treated donors were used, indicating that ICSBP has a specific antitumor activity toward myeloid neoplasms. We also found that overexpression of ICSBP negatively regulated normal hematopoiesis. These data provide direct evidence that ICSBP can act as a tumor suppressor that regulates normal and neoplastic proliferation of hematopoietic cells.
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Zheng, Xiaomin, Saskia Güller, Gesine Bug, Bithia Grace, Roxana Bistrian, Dieter Hoelzer, Oliver G. Ottmann, Reinhard Henschler, and Martin Ruthardt. "The Reciprocal t(9;22)-Translocation Products ABL/BCR Have Leukemogenic Potential Independently from BCR/ABL." Blood 104, no. 11 (November 16, 2004): 214. http://dx.doi.org/10.1182/blood.v104.11.214.214.
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Abstract In 95% of chronic myeloid leukemia (CML) and in 25% of acute lymphatic leukemia (ALL) the t(9;22) translocation fuses the bcr gene on chromosome 22 to the abl gene on chromosome 9 and vice versa. On 22+ the different breakpoints leads to the formation of two different major fusion genes: the major breakpoint (M-bcr) related to CML and the minor (m-bcr) related to ALL. The chimaeric fusion gene on 22+ (Philadelphia-chromosome) encodes for the BCR/ABL protein, the p210(BCR/ABL) in CML and the p185(BCR/ABL) in Ph+ALL. The fusion gene on 9+ encodes for the reciprocal ABL/BCR proteins, the p40(ABL/BCR) in CML and the p96(ABL/BCR) in Ph+ALL. The respective ABL/BCR transcripts are detectable in 65% of CML and 100% of Ph+ ALL patients. The ABL/BCRs are BCR mutants and thus N-terminally truncated Rho-guanine-nucleotide exchange factors (Rho-GEF’s). It is known that the N-terminal truncation can confer transformation potential to Rho-GEFs, such as NET-1. In addition, both ABL/BCRs, like wt BCR, contain a C-terminal Rac-GTPase activating protein (GAP)-domain. CML-associated ABL/BCR (p96(ABL/BCR)) differs from the Ph+ ALL-associated p40(ABL/BCR) in that that it misses the ‘dbl homology domain’(DH domains) of potential oncogenic function. Hence it seems that Ph+ALL blasts, in contrast to CML-blasts, express, as a consequence of t(9;22) translocation, two oncogenic fusion proteins, the p185(BCR-ABL) as well as the p40(ABL/BCR) protein. Actually nothing is known about the contribution of the reciprocal t(9;22) translocation products to the CML- and the ALL-phenotype. Thus we studied the phenotype induced by the ABL/BCRs in hemopoietic progenitors. Here we report that both ABL/BCRs i) lost the capacity of wt BCR to suppress the activation of RAC by its Rac-GAP domain, but did not influence the activation status of Rho or cdc42; ii) as a consequence of the deregulation of Rac the cytoskeleton modelling by BCR (Filopodia - cdc42-like phenotype) was altered in p40(ABL/BCR)- and p96(ABL/BCR)-expressing fibroblasts (stress fibers - Rho-like phenotype and “microspikes”, respectively); iii) the increase of migration of BCR-expressing 32D cells into a stroma cell-spheroid model was reverted in p40(ABL/BCR)- and p96(ABL/BCR)-expressing 32D cells; iv) adhesion to TNFalpha activated endothelial cell layer in the “flow chamber” was increased in BCR-positive 32D cells but not in p40(ABL/BCR)- and p96(ABL/BCR)-positive cells. Regarding their leukemogenic potential we showed that i) both ABL/BCRs, in contrast to wt BCR, activated RAS; ii) both ABL/BCRs were unable to transform fibroblasts and to render Ba/F3 cells factor-independent. iii) p96(ABL/BCR) increased the replating efficiency of Sca1+/lin- hemopoietic stem cells (HSC) by selecting a population of immature HSC exclusively expressing c-kit and Sca-1 more strongly than p40(ABL/BCR); iv.) both ABL/BCR blocked the myeloid differentiation of HSC v) the inoculation of p96(ABL/BCR)- or p40(ABL/BCR)-expressing HSC into lethally irradiated recipient mice led in the 40% and 60% of the cases, respectively, to a clinical picture of either acute leukemia or myeloproliferative syndrome within 2–9 months. These data show for the first time that the t(9;22) leads to two leukemogenic fusion proteins - the BCR/ABL and the ABL/BCR - in CML as well as in Ph+ALL, which might represent an additional target for molecular therapy approaches.
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Million, Ryan P., and Richard A. Van Etten. "The Grb2 binding site is required for the induction of chronic myeloid leukemia-like disease in mice by the Bcr/Abl tyrosine kinase." Blood 96, no. 2 (July 15, 2000): 664–70. http://dx.doi.org/10.1182/blood.v96.2.664.014k52_664_670.
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The BCR/ABL oncogene results from a balanced translocation between chromosomes 9 and 22 and is found in patients with chronic myeloid leukemia (CML) and in some patients with acute B-lymphoid leukemia. The Bcr/Abl fusion protein is a constitutively active tyrosine kinase that stimulates several intracellular signaling pathways, including activation of Ras through direct binding of the SH2-containing adapter protein Grb2 to Bcr tyrosine 177. A tyrosine-to-phenylalanine mutation (Y177F) at this site blocks the co-association of Bcr/Abl and Grb2 in vivo and impairs focus formation by Bcr/Abl in fibroblasts. However, the Bcr/Abl Y177F mutant can transform hematopoietic cell lines and primary bone marrow cells in vitro, so the importance of the Bcr/Abl–Grb2 interaction to myeloid and lymphoid leukemogenesis in vivo is unclear. We have recently demonstrated the efficient induction of CML-like myeloproliferative disease by BCR/ABL in a murine bone marrow transduction/transplantation model system. The Y177F mutation greatly attenuates the myeloproliferative disease induced by BCR/ABL, with mice developing B- and T-lymphoid leukemias of longer latency. In addition, the v-abl oncogene of Abelson murine leukemia virus, whose protein product lacks interaction with Grb2, is completely defective for the induction of CML-like disease. These results suggest that direct binding of Grb2 is required for the efficient induction of CML-like myeloproliferative disease by oncogenic Abl proteins.
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Million, Ryan P., and Richard A. Van Etten. "The Grb2 binding site is required for the induction of chronic myeloid leukemia-like disease in mice by the Bcr/Abl tyrosine kinase." Blood 96, no. 2 (July 15, 2000): 664–70. http://dx.doi.org/10.1182/blood.v96.2.664.
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Abstract The BCR/ABL oncogene results from a balanced translocation between chromosomes 9 and 22 and is found in patients with chronic myeloid leukemia (CML) and in some patients with acute B-lymphoid leukemia. The Bcr/Abl fusion protein is a constitutively active tyrosine kinase that stimulates several intracellular signaling pathways, including activation of Ras through direct binding of the SH2-containing adapter protein Grb2 to Bcr tyrosine 177. A tyrosine-to-phenylalanine mutation (Y177F) at this site blocks the co-association of Bcr/Abl and Grb2 in vivo and impairs focus formation by Bcr/Abl in fibroblasts. However, the Bcr/Abl Y177F mutant can transform hematopoietic cell lines and primary bone marrow cells in vitro, so the importance of the Bcr/Abl–Grb2 interaction to myeloid and lymphoid leukemogenesis in vivo is unclear. We have recently demonstrated the efficient induction of CML-like myeloproliferative disease by BCR/ABL in a murine bone marrow transduction/transplantation model system. The Y177F mutation greatly attenuates the myeloproliferative disease induced by BCR/ABL, with mice developing B- and T-lymphoid leukemias of longer latency. In addition, the v-abl oncogene of Abelson murine leukemia virus, whose protein product lacks interaction with Grb2, is completely defective for the induction of CML-like disease. These results suggest that direct binding of Grb2 is required for the efficient induction of CML-like myeloproliferative disease by oncogenic Abl proteins.
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Albers, Corinna, Anna L. Illert, Cornelius Miething, Christian Peschel, and Justus Duyster. "Grb10 Mediated Akt Activation Is Required for Induction of CML Like Myeloproliferative Disease in Mice by BCR-ABL." Blood 110, no. 11 (November 16, 2007): 1012. http://dx.doi.org/10.1182/blood.v110.11.1012.1012.
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Abstract Chronic myelogenous leukaemia (CML) results from the neoplastic transformation of hematopoietic stem cells (HSC) and is characterized by a chromosomal translocation t(9;22)(q34;q11). This aberration leads to the expression of the oncogenic tyrosine kinase BCR-ABL, which mediates signals for proliferation, transformation and anti-apoptosis via various signalling pathways. Grb10, a member of the growth factor bound proteins, is known to bind activated tyrosine kinases like BCR-ABL and might be involved in the activation of the Akt signalling pathway. Here we report the impact of Grb10 for BCR-ABL mediated transformation. We exerted a siRNA based approach in combination with a murine bone marrow transplantation model. To this end we designed a MSCV based retrovirus encoding both a Grb10 microRNA and the BCR-ABL oncogene on a single construct. This approach ensured knockdowns of more than 90% in every BCR-ABL transformed cell. Methylcellulose assays demonstrated that bone marrow coexpressing Grb10 microRNA and BCR-ABL had a 4-fold decreased colony forming ability compared to control cells. We then transduced bone marrow (BM) with retrovirus coexpressing Grb10 microRNA and p185 BCR-ABL and transplanted lethally irradiated recipient Balb/C mice. The onset and progression of leukaemia was significantly delayed in mice transplanted with Grb10 microRNA and BCR-ABL compared with the BCR-ABL transduced control microRNA group. However, we were not able to completely avoid the development of leukaemia by Grb10 knockdown. Mice transplanted with the Grb10 knockdown construct showed a delayed lymphoblastic disease, positive for B220, whereas the control group developed a rapid myeloproliferative disease, characterized by CD11b and Gr-1. In vitro analysis of BaF/3 and 32D cells showed that Grb10 knockdown in combination with BCR-ABL expression leads to a reduced phosphorylation of Akt. Taken together our data demonstrate that Grb10 is required for the development of a myeloproliferative disease by BCR-ABL in mice. Hereby, Grb10 seems to be critical for the BCR-ABL induced activation of the Akt pathway. In addition, this study describes a novel approach to express an oncogene and a microRNA using a single retroviral construct. This tool can be used to systematically screen for drugable signalling targets involved in oncogenesis.
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Mohi, M. Golam, Wayne W. Chan, Shaoguang Li, Benjamin Neel, and Richard A. Van Etten. "Distinct Gab2-Mediated Signaling Pathways Are Essential for Myeloid or Lymphoid Transformation and Leukemogenesis by BCR-ABL." Blood 112, no. 11 (November 16, 2008): 570. http://dx.doi.org/10.1182/blood.v112.11.570.570.
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Abstract The BCR-ABL oncogene encodes an activated fusion tyrosine kinase that causes chronic myelogenous leukemia (CML) and B-lymphoid acute lymphoblastic leukemia (B-ALL) in humans. An autophosphorylation site at Tyr 177 of BCR-ABL recruits Grb2 via its SH2 domain, and is required for efficient induction of CML-like myeloproliferative disease by BCR-ABL in a mouse BM retroviral transduction/transplantation model. We showed previously (Sattler et al., Cancer Cell2002;1:479) that the scaffolding/adapter protein Gab2 is recruited to Y177 of BCR-ABL via a Grb2/Gab2 complex, and in vitro transformation of primary myeloid and lymphoid progenitors by BCR-ABL was impaired in bone marrow from mice with homozygous null mutations in the Gab2 gene (Gab2−/− mice), coincident with decreased activation of the ERK and Akt signaling pathways. Here, we demonstrate an essential requirement for Gab2 in myeloid and lymphoid leukemogenesis by BCRABL. Whereas recipients of BCR-ABL-transduced Gab2+/+ BM develop fatal CML-like myeloproliferative disease within 4 weeks of transplantation, recipients of BCR-ABLtransduced Gab2−/− BM fail to develop CML but succumb after a long latent period to T-cell acute lymphoblastic leukemia, phenocopying the disease induced by the BCR-ABL Y177F mutant. These results suggest that the Y177F and Gab2 mutations have an epistatic relationship, and that the critical transforming signals from Tyr177 of BCR-ABL are transmitted through Gab2. Co-expression of Gab2 with BCR-ABL in Gab2−/− BM restored efficient induction of CML-like leukemia, but mutants of Gab2 that lacked either the pleckstrin homology domain or Tyr binding sites for the SH2 domains of the downstream Gab2 effector molecules SHP2 or p85 PI3K failed to rescue myeloid leukemogenesis by BCR-ABL, although the mutant Gab2 proteins were expressed in circulating myeloid cells. Gab2 deficiency attenuated B-lymphoid transformation by BCR-ABL in vitro, and significantly prolonged the latency of B-ALL induced by BCR-ABL in mice. In contrast to CML, induction of B-ALL in Gab2−/− BM was rescued by either WT Gab2 or the Gab2 mutant defective in p85 binding. These results demonstrate that BCR-ABL absolutely requires signaling via Gab2 to both SHP2 and PI3K to cause CML, while a Gab2-SHP2 signaling pathway contributes to the pathogenesis of BCR-ABL+ B-ALL.
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Albers, Corinna, Anna Lena Illert, Cornelius Miething, Christian Peschel, and Justus Duyster. "Raf1 Is Required for Induction of a Bcr-Abl Positive CML Like Myeloproliferative Disease in Mice." Blood 112, no. 11 (November 16, 2008): 3207. http://dx.doi.org/10.1182/blood.v112.11.3207.3207.
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Abstract Introduction: Chronic myelogenous leukemia (CML) results from neoplastic transformation of hematopoietic stem cells (HSC), characterized by a chromosomal translocation t(9;22)(q34;q11). This aberration leads to the expression of the oncogenic tyrosine kinase Bcr-Abl, which mediates signals for proliferation, transformation and anti-apoptosis via various different pathways including the Raf/MEK/ERK cascade. The cytoplasmic protein Raf1 is a key molecule within this cascade. Recent studies have revealed an additional function of the Raf-1 kinase that is independent of the activation of the MAPK cascade and whose effect is to increase resistance to apoptosis. Therefore Raf1 is an interesting target for molecular therapies and more effective Raf1 inhibitors have recently been developed by the pharmaceutical industry. Here we report the impact of Raf1 signalling for Bcr-Abl mediated transformation. Methods: We exerted a siRNA based approach in combination with a murine bone marrow transplantation model. To this end we designed a MSCV based retrovirus encoding both the Raf1 microRNA and the Bcr-Abl oncogene on a single construct. This approach ensured knockdowns of more than 90% of Raf1 in every Bcr-Abl transformed cell. Results: Methylcellulose assays demonstrated that bone marrow coexpressing Raf1 microRNA and Bcr-Abl had a 2 fold decreased colony forming ability compared to control cells. We then transduced bone marrow (BM) with retrovirus coexpressing Raf1 microRNA and p185 Bcr-Abl and transplanted lethally irradiated recipient Balb/C mice. The onset and progression of leukemia was significantly delayed in mice transplanted with Raf1 microRNA and Bcr-Abl compared with the Bcr- Abl transduced control microRNA group. Raf1 knockdown mice showed only a moderate rise of white blood cell (WBC) counts and prolonged overall survival (median survival 39 ± 7.1 days) in comparison to control mice (23.3 ± 2.4 days). However, we were not able to completely avoid the development of leukemia by Raf1 knockdown. Conclusion: Taken together our data demonstrate that Raf1 is important for the development of a myeloproliferative disease by Bcr-Abl in mice. Therefore Raf1 inhibition in combination with Bcr-Abl kinase inhibition depicts an interesting approach towards eradication of Bcr- Abl positive leukemia. In addition, this study describes a novel and versatile approach to express an oncogene and a microRNA using a single retroviral construct. Thus this powerful tool can be used to systematically screen drugable signalling targets involved in oncogenesis.
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Pelletier, Shawn D., Daniel S. Hong, Yiguo Hu, Yuhua Liu, and Shaoguang Li. "Lack of the adhesion molecules P-selectin and intercellular adhesion molecule-1 accelerate the development of BCR/ABL-induced chronic myeloid leukemia-like myeloproliferative disease in mice." Blood 104, no. 7 (October 1, 2004): 2163–71. http://dx.doi.org/10.1182/blood-2003-09-3033.
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Abstract In vitro studies show that BCR/ABL-expressing hematopoietic cells exhibit altered adhesion properties. No in vivo studies show whether the altered adhesion properties affect BCR/ABL leukemo-genesis. Using mice with homozygous inactivation of genes encoding the 2 adhesion molecules P-selectin and intercellular adhesion molecule-1 (ICAM1), we show that the mutant mice develop BCR/ABL-induced chronic myeloid leukemia (CML)-like leukemia at a significantly faster rate than do wild-type (WT) mice. Lack of P-selectin and ICAM1 did not have a significant effect on the development of B-cell acute lymphoblastic leukemia (BALL) induced by BCR/ABL. Using mice deficient for P-selectin or ICAM1 alone, we show that P-selectin plays a major role in the acceleration of CML-like leukemia. Lack of P-selectin resulted in early release of BCR/ABL-expressing myeloid progenitors from bone marrow, appearing to alter the biologic properties of leukemic cells rather than their growth rate by increasing their homing to the lungs, causing fatal lung hemorrhages. These results indicate that adhesion of BCR/ABL-expressing myeloid progenitors to marrow stroma through P-selectin and ICAM1 play an inhibitory role in the development of CML-like disease, suggesting that improvement of adhesion between BCR/ABL-expressing myeloid progenitor cells and bone marrow stroma may be of therapeutic value for human CML. (Blood. 2004;104:2163-2171)
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Fredericks, Jessica, Chaitali Parikh, Ramesh Subrahmanyam, and Ruibao Ren. "The RALGEF Pathway Contributes to the Pathogenesis of Chronic Myelogenous Leukemia by BCR/ABL." Blood 112, no. 11 (November 16, 2008): 3208. http://dx.doi.org/10.1182/blood.v112.11.3208.3208.
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Abstract Selected inhibitors of the BCR/ABL tyrosine kinase have shown a remarkable clinical activity in patients with chronic myelogenous leukemia (CML). However, these drugs do not completely eradicate leukemic cells and drug resistance emerges. Identification of additional contributors to the pathogenesis of CML remains to be important for developing strategies for overcoming resistance to BCR/ABL kinase inhibitors and for eradicating leukemic cells. We and others have previously shown that expression of BCR/ABL in mouse bone marrow cells by retroviral transduction and transplantation (BMT) efficiently induces a CML-like myeloproliferative disorder (MPD). Using this BMT model for CML, we have found that the Grb2 SH2 binding site Y177 of BCR/ABL, which is critical for BCR/ABL to activate the RAS pathway, is required for the induction of CML-like disease by BCR/ABL. We have further shown that oncogenic NRAS (NRASG12D) can rescue the defect of the Y177F mutant BCR/ABL in the induction of CML-like disease. To further elucidate the pathways downstream of NRAS responsible for this “rescue” effect, we utilized three well-characterized NRAS effector mutants, G12D/T35S (activating RAF but is impaired in binding PI3K and RALGEF), G12D/E37G (activating RALGEF but not RAF or PI3K), and G12D/Y40C (activating PI3K but not RAF or RALGEF). None of the three NRAS effector mutants could induce hematological malignancies by themselves, but all were capable of “rescuing” the CML defect of BCR/ABLY177F to some extent. Interestingly, the NRAS G12D/E37G effector mutant was the most efficient. We further examined the role of RALGEF pathway by introducing an activated RALGDSCAAX into our BCR/ABLY177F mouse model. RALGDS-CAAX was able to cooperate with BCR/ABLY177F to induce a CML-like MPD in nearly half of mice whereas RALGDSCAAX alone did not induce diseases in any mice. As a complement to this study, we also introduced dominant negative RAL A or RAL B in the background of BCR/ABL. This resulted in a significant delay in disease onset. Taken together, this data sheds light on the importance of the RALGEF pathway in BCR/ABL-mediated CML induction and supports the notion of inhibition of this pathway as a potential therapeutic strategy.
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Million, Ryan P., Jon Aster, D. Gary Gilliland, and Richard A. Van Etten. "The Tel-Abl (ETV6-Abl) tyrosine kinase, product of complex (9;12) translocations in human leukemia, induces distinct myeloproliferative disease in mice." Blood 99, no. 12 (June 15, 2002): 4568–77. http://dx.doi.org/10.1182/blood-2001-12-0244.
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Several patients with clinical features of chronic myeloid leukemia (CML) have fusion of the TEL (ETV6) gene on 12p13 with ABL on 9q34 and express a chimeric Tel-Abl protein that contains the same portion of the Abl tyrosine kinase fused to Tel, an Ets family transcription factor, rather than Bcr. In a murine retroviral bone marrow transduction-transplantation model, a Tel (exon 1-5)–Abl fusion protein induced 2 distinct illnesses: a CML-like myeloproliferative disease very similar to that induced by Bcr-Abl but with increased latency and a novel syndrome characterized by small-bowel myeloid cell infiltration and necrosis, increased circulating endotoxin and tumor necrosis factor α levels, and fulminant hepatic and renal failure. Induction of both diseases required the Tel pointed homology oligomerization domain and Abl tyrosine kinase activity. Myeloid cells from mice with both diseases expressed Tel-Abl protein. CML-like disease induced by Tel-Abl and Bcr-Abl was polyclonal and originated from cells with multilineage (myeloid, erythroid, and B- and T-lymphoid) repopulating ability and the capacity to generate day-12 spleen colonies in secondary transplantations. In contrast to findings with Bcr-Abl, however, neither Tel-Abl–induced disease could be adoptively transferred to irradiated secondary recipient syngeneic mice. These results show that Tel-Abl has leukemogenic properties from distinct from those of Bcr-Abl and may act in a different bone marrow progenitor.
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Mian, Afsar, Isabella Haberbosch, Oliver G. Ottmann, and Martin Ruthardt. "Transphosphorylation of Endogenous BCR Mediates the Effect of T315I on the Transformation Potential of BCR/ABL." Blood 124, no. 21 (December 6, 2014): 4523. http://dx.doi.org/10.1182/blood.v124.21.4523.4523.
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Abstract Targeting of BCR/ABL by ABL-kinase inhibitors (AKI) such as Imatinib, Nilotinib, or Dasatinib is a proven concept in Philadelphia chromosome positive (Ph+ ) leukemia. In the majority of cases the acquisition of resistance is related to point mutations in BCR/ABL, such as the E255K, Y253F/H (P-loop), H396R (activation loop) or the T315I (gatekeeper). Noteworthy, Ph+ leukemias, both CML and Ph+ ALL, never emerge at diagnosis with a BCR/ABL harboring a resistance mutation even if the clone with the mutation is already existing and detectable by very sensitive methods. This indicates that the mutations confer biological features to the clones unveiled by the treatment. One can hypothesize that the presence of mutations such as the T315I confers a growth disadvantage with respect to native BCR/ABL. We and others have previously shown that the resistance mutations may influence the biology of BCR/ABL regarding its transformation potential. The aim of the study was to determine whether the ‘‘gatekeeper’’ mutation T315I is able not only to induce biochemical modification of BCR/ABL responsible for the to resistance of patients against first and second generation AKI, but also to confer biological features to BCR/ABL influencing its leukemogenic potential. We recently showed that T315I is able to fully restore factor independent growth in Ba/F3 cells of loss of function mutants (LOFM) of BCR/ABL such as that lacking the Y177 (Y177F) or the N-terminal coiled coil oligomerization interface (ΔCC), which was accompanied by a transphosphorylation of endogenous BCR. Based on these findings we systematically investigated the influence of T315I on the biology of BCR/ABL and the role of BCR in this process. As models we used a syngeneic mouse model of BCR/ABL induced CML-like disease, factor dependent 32D and Ba/F3 cells, and Ph+ ALL patient derived long term culture (PD-LTCs). These models allowed the direct comparison of BCR/ABL with BCR/ABL-T315I. Furthermore we took advantage of LOFM such as ΔCC-BCR/ABL and Y177F-BCR/ABL which we studied for their capacity to mediate either transformation potential in Rat-1 fibroblasts (contact inhibition and anchorage-independent growth) and/or factor independent growth in 32D cells in the presence of T315I. The role of the transphosphorylation of BCR was assessed by RNAi against BCR in 32D cells and the Rat-1 cells. Here we show that proliferation of 32D or Ba/F3 cells or the PD-LTCs expressing BCR/ABL-T315I was significantly slower than that of correspondent cells expressing native BCR/ABL. Also the induction of a CML-like disease in syngeneic mice was significantly delayed in the presence of T315I (median: BCR/ABL - 27 days; BCR/ABL-T315I - 61 days). On the other hand T315I was able to restore both transformation potential and factor independent growth of LOFM of BCR/ABL in Rat-1 and 32D cells, respectively. This was accompanied by a transphosphorylation of endogenous BCR at Y177, which led to an activation of Ras/Erk1/2 pathway. This effect of T315I on factor independent growth and transformation mediated by the LOFM and related activation of Ras/Erk1/2 was reverted by targeting the BCR with RNAi. Taken together these data suggest that T315I confer biological features to BCR/ABL which are unveiled only upon treatment or in the presence of LOFM. How these features may influence the destiny of the BCR/ABL-T315I clone in the patient and the role of Ras/Erk1/2 pathway in this process has to be further investigated. Disclosures No relevant conflicts of interest to declare.
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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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Zafar, Usva, Mohammed Yusuf, Rikhia Chakraborty, El-Nasir M. A. Lalani, and Afsar Ali Mian. "The ''Gatekeeper'' Mutation T315I in BCR/ABL Confers Additional Oncogenic Activities to Philadelphia Chromosome Positive Leukemia." Blood 134, Supplement_1 (November 13, 2019): 5196. http://dx.doi.org/10.1182/blood-2019-131694.
Full textAbstract:
Chronic myeloid leukemia (CML) and 30% of adult acute lymphatic leukemia (ALL) are characterized by the Philadelphia chromosome (Ph+), having a (9;22) chromosomal translocation. The BCR/ABL fusion protein is the hallmark of Ph+ leukemia. BCR/ABL is characterized by deregulated and constitutively activated ABL tyrosine kinase activity that determines its transformation potential. Tyrosine kinase inhibitors (TKI) have greatly improved the overall prognosis of these diseases, particularly by altering the natural history of chronic phase (CP) CML and preventing the previously inexorable progression to terminal blast crisis (BC). However, unsatisfactory responses in advanced disease stages, resistance and long-term tolerability of BCR/ABL inhibitors represent major clinical problems. The most important mechanism of resistance against TKIs is the selection of leukemic clones driven by BCR/ABL harboring point mutations, such as the E255K, Y253F/H (P-loop), H396R (activation loop) or the T315I (gatekeeper). The "gatekeeper" mutation T315I confers resistance against all approved TKIs, with the only exception of Ponatinib, a multi-target kinase inhibitor. CML and Ph+ ALL, rarely present at diagnosis with a BCR/ABL harboring a resistance mutation to TKI. Resistant clones may be present and only detectable by highly sensitive methods. We have previously shown that the resistance mutations may influence the biology of BCR/ABL and its transformation potential. We therefore hypothesized that the presence of mutations such as the T315I select for a "dormant cell population" which manifests following initial treatments with TKI inhibitors and treatment failure. The aim of this study was to determine whether the ''gatekeeper'' mutation T315I is able to confer biological features to BCR/ABL influencing its leukemogenic potential. We investigated the influence of T315I on the biology of BCR/ABL in CML and Ph+ ALL. We used Ph+ ALL patient derived long term culture (PDLTCs), factor dependent Ba/F3 cells and syngeneic mouse model of BCR/ABL induced CML-like disease. These models allowed the direct comparison of BCR/ABL with BCR/ABL-T315I. We observed significantly slower proliferation of Ba/F3 cells and PDLTCs expressing BCR/ABL-T315I compared to the native BCR/ABL. This was further confirmed by undertaking mitotic index calculations and colony formation assays on both cell types. Furthermore, the induction of a CML-like disease in syngeneic mice was significantly delayed in the presence of T315I (median: BCR/ABL - 27 days; BCR/ABL-T315I - 61 days). We undertook functional studies to determine the putative signaling pathway and found that Ras/Erk1/2 pathway was activated inT315I positive cells. This study may assist towards therapy decisions in patients with CML/Ph+ ALL with a T315I mutation. Disclosures No relevant conflicts of interest to declare.
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Hantschel, Oliver D., Eva Eckelhart, Ines Kaupe, Florian Grebien, Kay-Uwe Wagner, Veronika Sexl, and Giulio Superti-Furga. "Bcr-Abl Directly Activates Stat5 Independent of Jak2." Blood 116, no. 21 (November 19, 2010): 511. http://dx.doi.org/10.1182/blood.v116.21.511.511.
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Abstract Abstract 511 Persistent activation of the transcription factor Stat5 is a signaling hallmark of Chronic Myelogenous Leukemia (CML). In mouse models, Stat5 was required for initial myeloid and lymphoid transformation (by Bcr-Abl p210 or p185 and v-Abl). Most importantly, we and others recently showed that Stat5 was also required for maintenance of Bcr-Abl-dependent leukemia in vivo and for engraftment and reconstitution of Bcr-Abl p210-positive leukemia in secondary recipients. Therefore, Stat5 is of central functional importance in the Bcr-Abl signaling network and represents a possible critical vulnerable node in CML. In contrast to the well-studied functional role of Stat5 in disease models, the molecular mechanism of Bcr-Abl dependent Stat5 activation, including the tyrosine kinase(s) that phosphorylate and activate Stat5, remain only partially understood. In particular, conflicting views on the involvement of the Jak2 kinase and its potential role as a drug target in CML exist. We used conditional Jak2 knock-out mice to study the contribution of Jak2 in Bcr-Abl-dependent transformation and leukemogenicity. Jak2 ablation did not compromise the Bcr-Abl p210-mediated transforming capability in primary murine bone marrow- or fetal liver-derived hematopoietic cells in vitro. In contrast, initial lymphoid transformation by v-abl and Bcr-Abl p185 was abolished in Jak2 knock-out mice. Jak2 deletion did not have an effect on maintenance of lymphoid leukemia cells in vitro, whereas deletion of Stat5 induced a G1 arrest and subsequent apoptosis. In line with this, ablation of Jak2 expression after leukemia induction did not alter disease latency or disease phenotype. Consistently, we did not observe a decrease in Stat5 activation upon siRNA-mediated knock-down of Jak2 alone or all four Jak kinases (Jak1, Jak2, Jak3 and Tyk2) in CML cell lines. Using a panel of pharmacological inhibitors, we found that neither Jak2-selective, nor pan-Jak kinase inhibitors or Src family kinase-selective inhibitors led to a decrease in Stat5 phosphorylation, while the highly selective Bcr-Abl inhibitor nilotinib completely abrogated Stat5 phosphorylation. To study possible contributions of other tyrosine kinases in the Bcr-Abl dependent activation of Stat5, we used Ba/F3 cells expressing the TKI-resistant Bcr-Abl mutant T315I in combination with different broad-specificity tyrosine kinase inhibitors, like dasatinib. At dasatinib concentrations that inhibited several dozens of tyrosine kinases, Stat5 phosphorylation in Ba/F3 Bcr-Abl T315I cells was unaffected, excluding a role for most tyrosine kinases other than Bcr-Abl in Stat5 activation and pointing towards a direct phosphorylation of Stat5 by Bcr-Abl. Together, this data excludes a role of Jak and Src kinases in the activation of Stat5 in Bcr-Abl positive cell lines. Finally, in comprehensive enzyme kinetic analysis experiments using recombinant kinase, Stat5 had a similar KM value for Bcr-Abl as the canonical direct Bcr-Abl substrate CrkL and displayed only mildly lower kinase substrate parameters (vmax, kcat) than CrkL, fully compatible with direct phosphorylation of Stat5 by Bcr-Abl. Together with our earlier data on the pivotal role of Stat5 in the transcriptional and signaling network of Bcr-Abl, we propose a hypersensitive switch-like behavior of the Bcr-Abl-Stat5 kinase substrate pair that mechanistically rationalizes the central functional role of Stat5 in the signaling of CML cells. In summary, we provide compelling evidence that activation of Stat5 by Bcr-Abl is likely to be direct and that targeting of Jak2 in CML may not be of therapeutic benefit, as Jak2 is not required for CML initiation or maintenance. Disclosures: Hantschel: Novartis: Honoraria; Bristol-Myers Squibb: Honoraria.
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Crawford, Lisa, Philip Windrum, Laura Magill, Junia V. Melo, Lynn McCallum, MaryFrances McMullin, Huib Ovaa, Brian Walker, and Alexandra Irvine. "Bcr-Abl Positive Cells Display Increased Proteasome Activity and Greater Sensitivity to Proteasome Inhibition." Blood 112, no. 11 (November 16, 2008): 3192. http://dx.doi.org/10.1182/blood.v112.11.3192.3192.
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Abstract Chronic myeloid leukemia (CML) is a malignant disorder of the hematopoietic stem cell, characterised by the constitutively active tyrosine kinase BCR-ABL. The current first-line therapy for CML is the tyrosine kinase inhibitor imatinib. Although imatinib induces durable responses, a number of patients develop resistance to this treatment, highlighting the need to identify new molecular targets in this disease. Proteasome inhibition has recently emerged as a novel anti-cancer therapy. There is evidence to suggest that the proteasome is a valid target in CML. We have previously reported that proteasome activity is higher in bone marrow from patients with CML than normal controls. Furthermore, we demonstrated using a cell line model that Bcr-Abl positive cells were more sensitive to induction of apoptosis by proteasome inhibition than Bcr-Abl negative cells. The present study investigates the relationship between Bcr-Abl expression and proteasome activity and the effect of proteasome inhibition on primary human CML cells. Conventional fluorogenic substrate assays for all three catalytic activities of the proteasome [chymotrypsin-like (CT-L), trypsin-like (T-L), post glutamyl peptide hydrolysing (PGPH)] and proteasome activesite label DansylAhx3L3VS were used to profile proteasome activity levels in ts-Bcr-Abl FDCP-Mix cells and mock transfected FDCP-Mix cells. Both methods confirmed that Bcr-Abl positive cells have higher levels of proteasome activity than Bcr-Abl negative cells (p ≤ 0.04; Figure 1a). Conversely, downregulation of BCR-ABL using si-RNA was associated with a significant decrease in proteasome activity (p < 0.05; Figure 1b). The ability of the proteasome inhibitor BzLLLCOCHO to induce apoptosis in primary human CML cells was evaluated using Mitosensor™ and Hoescht/Propidium Iodide staining. Treatment with BzLLLCOCHO (1μM), selectively induced apoptosis in primary CML cells compared to normal mononuclear cells (39 ± 9.6 % vs 18.1 ± 4.02 %, 72 hrs, p = 0.01). Drug combination experiments were performed with BzLLLCOCHO (1 μM) and imatinib (1 μM) in ts-Bcr-Abl FDCP-mix cells and primary CML cells. Using Calcusyn software to generate the median effect of Chou-Talalay, the sequential addition of imatinib followed by BzLLLCOCHO was found to synergistically enhance the induction of apoptosis in ts-Bcr-Abl FDCP-Mix cells and resulted in additive effects in primary CML cells (n=4). The effect of the compounds on Bcr-Abl kinase activity was assessed by immunoblotting for phosphorylated Crkl. No effect on Bcr-Abl activity was seen following treatment of ts-Bcr-Abl FDCP-Mix cells and primary CML cells with BzLLLCOCHO alone, however, the combination of BzLLLCOCHO and imatinib resulted in a greater reduction of Bcr- Abl activity (59.8 ± 9.9 %) than imatinib alone (34.1 ± 9.6 %). Finally, we investigated the effect of BzLLLCOCHO on two human CML cell lines which are resistant to imatinib (KCL22-r, LAMA84-r). Imatinib resistant cells were found to be equally as sensitive to induction of apoptosis by BzLLLCOCHO as their imatinib sensitive counterparts (KCL22-s 30.7 ± 5.7 % vs KCL22-r 32 ± 1.7 %; LAMA84-s 56.3 ± 3.2 % vs Lama84-r 57.2 ± 7.9 %; 72 hrs). The present findings suggest that higher proteasome activity in Bcr- Abl positive cells may render these cells more susceptible to induction of apoptosis by proteasome inhibition and provide a rational basis to examine the potential of proteasome inhibitors as a therapeutic target in CML, particularly in imatinib resistant disease. Figure 1a. Bcr-Abl+ cells contain significantly greater levels of proteasome activity than Bcr-Abl cells siRNA directed against BCR-ABL decreases proteasome activity. Figure 1a. Bcr-Abl+ cells contain significantly greater levels of proteasome activity than Bcr-Abl cells . / siRNA directed against BCR-ABL decreases proteasome activity.
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Sha, Xiaojin, Dan Liebermann, and Barbara Hoffman. "Loss of Gadd45b Accelerates BCR-ABL-Driven CML." Blood 132, Supplement 1 (November 29, 2018): 5138. http://dx.doi.org/10.1182/blood-2018-99-114890.
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Abstract Gadd45b is a member of Gadd45 stress sensor protein family that also includes Gadd45a & Gadd45g. To investigate the effect of Gadd45b in bcr-abl oncogene driven chronic myeloid leukemia (CML) development, syngeneic wild type lethally irradiated mice were reconstituted with either wild type or Gadd45b null myeloid progenitors transduced with a retroviral vector expressing BCR-ABL. Loss of Gadd45b was observed to accelerate BCR-ABL driven CML development with shortened median mouse survival time. BCR-ABL Gadd45b deficient CML progenitors exhibited increased proliferation and decreased apoptosis, associated with hyper-activation of c-Jun NH2-terminal kinase and Stat5. These results provide novel evidence that gadd45b, like gadd45a, functions as a suppressor of BCR-ABL driven leukemia, albeit via a different mechanism. Disclosures No relevant conflicts of interest to declare.
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Zhang, Bin, Yin Wei Ho, Sung-UK Lee, Takahiro Maeda, Claudia Huettner, and Ravi Bhatia. "Characterization of Leukemia-Initiating Cells in a Transgenic Model of Chronic Phase Chronic Myelogenous Leukemia (CML)." Blood 114, no. 22 (November 20, 2009): 858. http://dx.doi.org/10.1182/blood.v114.22.858.858.
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Abstract Abstract 858 In normal hematopoiesis, only a small population of lin-Sca-1+c-kit+ (LSK) cells with Flt3-CD150+48− immunophenotype has long-term hematopoietic stem cell (LT-HSC) capacity, whereas Flt3-CD150+CD48+ and Flt3-CD150-CD48+ LSK cells represent more differentiated multipotent progenitors (MPP1 and MPP2) without long-term engrafting capacity. Despite extensive investigation into BCR-ABL induced leukemogenesis, the impact of BCR-ABL expression on LSK subpopulations and the specific subpopulation with leukemia-initiating capacity remain unknown. Targeted expression of the BCR-ABL gene in murine hematopoietic stem and progenitor cells (HSPC), using a Tet-regulated SCL promoter, results in development of a chronic phase CML-like disorder (Blood 105:.324, 2005). Mice consistently develop leukocytosis, splenomegaly and expansion of bone marrow (BM) myeloid progenitors and primitive LSK cells following induction of BCR-ABL expression. Here we employed the SCL-tTA-BCR/ABL mouse model to investigate the effect of BCR-ABL expression on HSPC populations. BCR/ABL expression resulted in a 3-fold increase in granulocyte-macrophage progenitors (GMP) and a 1.5-fold increase in LSK cell numbers compared with non-induced controls, whereas numbers of common myeloid progenitors (CMP) and megakaryocyte-erythrocyte progenitors (MEP) were reduced. Despite expansion of total LSK cells, the number of LT-HSC was markedly reduced in the BM of BCR-ABL expressing mice (610±246 vs. 4,038±982). In contrast, an increase in MPP1 (6,150±1,813 vs. 3,185±1,247) and MPP2 (39,580±14,079 vs. 25,115±7,090) was seen. BCR-ABL mRNA expression was confirmed in each population by RT-PCR, with highest levels of expression seen in MPP cells. In vivo EdU labeling demonstrated increased cycling of LSK cells from BCR/ABL expressing mice compared to controls. We observed a vast increase in the number of GMP (11-fold), CMP (10-fold), MPP (4.5 fold) and LT-HSC (2.5 fold) in the spleen of BCR/ABL mice compared to controls. Since the functional potential of HSPC cannot be determined solely on the basis of cell surface markers, we also studied the ability of transplanted populations to generate leukemia in recipient mice. SCL-tTA/BCR-ABL transgenic mice were crossed with GFP transgenic mice to facilitate tracking of transplanted cells. Only LSK cells, but not CMP or GMP, from BM and spleen of BCR-ABL expressing mice were capable of generating CML-like disease and long term engraftment (>16 weeks) in recipient mice. The leukemic phenotype of donors was recapitulated in recipient mice; and leukemia could be transplanted to secondary and tertiary recipients. Further analysis revealed that the subpopulation of cells with a LT-HSC phenotype (Flt3-CD150+CD48-) within the LSK population was capable of generating CML-like disease and long term engraftment in recipient mice. Consistent with the diminished LT-HSC numbers demonstrated by flow cytometry, the frequency of functional HSC within the LSK population, as measured by competitive repopulation limiting dilution assays, was reduced in BCR-ABL expressing mice (1 in 234) compared to control mice (1 in 14). Interestingly, not all transplanted mice with long-term engrafted BCR-ABL-expressing cells developed leukemia. We determined that 1 in 6 Flt3-CD150+CD48- LSK cells possessed repopulation activity, whereas only 1 in 80 cells was capable of initiating leukemia in transplanted mice within 20 weeks, indicating that only a subset of BCR-ABL+ cells with long-term repopulating potential has leukemia-initiating capacity. In summary, BCR-ABL expression is associated with significant reduction in LT-HSC and expansion of MPP and GMP in the BM, and a marked increase in LT-HSC, MPP, CMP and GMP in the spleen of transgenic mice. Reduced LT-HSC numbers in BM may be explained by increased proliferation of BCR-ABL-expressing HSC and their enhanced egress from the BM to extramedullary locations such as the spleen. BCR-ABL expressing LT-HSC demonstrated long term engraftment and secondary transplantation capacity. However, only a fraction of BCR-ABL-expressing long-term repopulating cells has leukemia-initiating capacity, suggesting that additional cell intrinsic or extrinsic factors besides BCR-ABL expression may play a role in determining their leukemogenic potential. Disclosures: No relevant conflicts of interest to declare.
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Hantschel, Oliver D., Florian Grebien, Ines Kaupe, Boris Kovacic, John Wojcik, Gerald D. Gish, Shohei Koide, Hartmut Beug, Tony Pawson, and Giulio Superti-Furga. "The Bcr-Abl SH2-Kinase Domain Interface Is Critical for Leukemogenesis and An Additional Therapeutic Target in CML." Blood 114, no. 22 (November 20, 2009): 37. http://dx.doi.org/10.1182/blood.v114.22.37.37.
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Abstract Abstract 37 We previously showed that the Abl SH2 domain is an allosteric activator of c-Abl tyrosine kinase activity and substrate phosphorylation (Filippakopoulos et al. (2008) Cell 134(5), 793-803). This effect is exerted directly by docking of the SH2 domain onto the N-lobe of the kinase domain in the active conformation of c-Abl. We also showed that the same structural mechanism is a critical factor for full activation of the oncogenic fusion kinase Bcr-Abl. Disruption of binding of the SH2 domain to the kinase domain in Bcr-Abl by the Ile164Glu mutation in the SH2 domain, led to a strong reduction in in vitro tyrosine kinase activity and Bcr-Abl autophosphorylation. Unexpectedly, we observed a differential attenuation of downstream signaling pathways upon disruption of the SH2-kinase domain interface, indicating different activation thresholds of Bcr-Abl downstream signaling pathways. Here, we show that disrupting the SH2-kinase domain interface abrogates the transforming capacity of Bcr-Abl. Cells expressing the Bcr-Abl Ile164Glu mutant were unable to generate cytokine-independent colonies in vitro. Furthermore, mice transplanted with Bcr-Abl Ile164Glu expressing bone marrow cells did not develop the characteristic MPD-like disease that is caused by wild-type Bcr-Abl. Mice that received Bcr-Abl Ile164Glu cells showed normal survival, blood counts and histology after more than 100 days post-transplant, despite the presence of Bcr-Abl Ile164Glu-expressing cells in all blood lineages. This shows that the formation of the SH2-kinase domain interface is strictly necessary for Bcr-Abl to cause CML. Together with our data that show sensitization to imatinib inhibition of Bcr-Abl Ile164Glu as compared to Bcr-Abl wild-type, this argues for the SH2-kinase domain interface as an additional drug target on Bcr-Abl that may synergize with tyrosine kinase inhibitors and may be useful to inhibit tyrosine kinase inhibitor resistant Bcr-Abl clones. To address possibilities to interfere with the SH2-kinase domain interface, we are using an engineered binding protein that binds to the Abl SH2 domain with high-affinity and specificity and supposedly disrupts the interface with the kinase domain, resulting in a decrease in Bcr-Abl kinase activity. In conclusion, we provide strong evidence that the structural positioning of the SH2 domain is a crucial factor for constitutive activity, signal transduction and leukemogenicity of Bcr-Abl. Besides oligomerization via the N-terminal coiled-coiled domain and loss of the auto-inhibitory N-terminal myristoyl group, the proper positioning of the SH2 domain appears to be another critical factor that is required for constitutive activation of Bcr-Abl. Inhibitors of the SH2-kinase domain interface of Bcr-Abl may comprise alternative or additional points of pharmacological intervention for the treatment of imatinib-sensitive or -resistant CML or Ph+ acute lymphocytic leukemia. Disclosures: No relevant conflicts of interest to declare.
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Moore, James C., Chi Ly, Halbur Luke, and S. Tiong Ong. "Enhanced Killing of Chronic Myelogenous Leukemia Cells by Rapamycin and Imatinib Is Associated with Differential Inhibition of 4E-BP1 and eIF4E Phosphorylation and Decreased Protein Expression by Non-Overlapping Mechanisms." Blood 104, no. 11 (November 16, 2004): 1993. http://dx.doi.org/10.1182/blood.v104.11.1993.1993.
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Abstract The Bcr-abl tyrosine kinase is known to promote transformation by dysregulating gene transcription, but its role in dysregulating translation is less well documented. Our recent work has implicated the mammalian target of rapamycin (mTOR) signaling as a downstream target of Bcr-Abl, since we find that the mTOR effectors, 4E-BP1 and S6, are phosphorylated in a Bcr-Abl kinase-dependent manner (Ly et al., Cancer Research, 2003). Because mTOR is a central regulator of eukaryotic translation, and inhibitors of mTOR act synergistically with imatinib mesylate (imatinib) to kill CML cells, these results suggest that, like transcription, translation may be a general cellular process dysregulated by Bcr-Abl activity. If this were so, then components of the cellular apparatus co-opted by Bcr-Abl to increase translation would constitute rational therapeutic targets. These would include signaling pathways mediating increased translation, components of the cap-binding complex (eIF4E, eIF4GI, and eIF4A) that regulate cap-dependent mRNA translation, as well as proteins whose translation is increased by Bcr-Abl kinase activity. Here we identify eIF4E as well as cyclin D3 as potential therapeutic targets in CML. Since eIF4E is essential for cap-dependent translation, and increased translation parallels eIF4E phosphorylation at Ser209, we determined the status of eIF4E phosphorylation in murine hematopoietic Ba/F3 cells expressing Bcr-Abl (Ba/F3-Bcr-Abl), and its dependence on Bcr-Abl kinase activity. Using phosphospecific antibodies to eIF4E, we found that Bcr-Abl kinase activity was essential for phosphorylation of eIF4E at Ser209, but had no effects on total levels of the protein. In contrast, rapamycin had no effect on the degree of eIF4E phosphorylation, although it was able to inhibit phosphorylation of 4E-BP1 completely (unlike imatinib). By examining total mRNA and protein levels of known targets of Bcr-Abl, we determined that cyclin D3, but not cyclin D2, was post-transcriptionally regulated by Bcr-Abl. Metabolic labeling studies were also conducted in Ba/F3-Bcr-Abl cells treated with media alone, imatinib, rapamycin, or both. Our results demonstrated that translation of cyclin D3 protein is regulated by the mTOR kinase in Bcr-Abl-expressing cells, and that combined inhibition of mTOR and Bcr-Abl resulted in an additional decrease in protein levels. Together, these results demonstrate that Bcr-Abl promotes protein translation of specific genes via mTOR, and that the activity of both Bcr-Abl and mTOR kinases contribute to dysregulated protein expression via non-overlapping mechanisms in CML cells. Ongoing studies are being conducted to determine the role of both eIF4E and cyclin D3 in the pathogenesis of CML.
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Burgess, Gem S., Elizabeth A. Williamson, Larry D. Cripe, Sara Litz-Jackson, Jay A. Bhatt, Kurt Stanley, Mark J. Stewart, Andrew S. Kraft, Harikrishna Nakshatri, and H. Scott Boswell. "Regulation of the c-jun Gene in p210 BCR-ABL Transformed Cells Corresponds With Activity of JNK, the c-jun N-Terminal Kinase." Blood 92, no. 7 (October 1, 1998): 2450–60. http://dx.doi.org/10.1182/blood.v92.7.2450.
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Abstract Activity of the c-jun N-terminal kinase (JNK) has been shown in hematopoietic cells transformed by p210 BCR-ABL. However, analysis has not been reported for hematopoietic cells on the consequences of this activity for c-jun promoter regulation within its distinctive proximal 8-base consensus CRE-like element, an element linked to JNK-mediated increase in c-jun transcription. In the present study, regulation of the proximal c-jun promoter was studied in murine myeloid cells transformed by p210 BCR-ABL. Promoter regulation in p210 BCR-ABL transformed cells was compared with regulation of the promoter in nontransformed interleukin-3 (IL-3)–dependent parental cells. The composition of nuclear AP-1 proteins contained within cells with p210 BCR-ABL, and their binding to the c-jun promoter proximal CRE-like element, was compared with the composition and binding of AP-1 proteins in IL-3–treated parental cells without p210 BCR-ABL. The present analysis found fivefold increased c-jun transcription occurring in p210 BCR-ABL transformed murine myeloid cells possessing a corresponding magnitude of increased kinase activity of JNK, compared with IL-3–stimulated parental cells. Augmented JNK activity was accompanied by increased nuclear abundance of c-jun and c-fos proteins that bound specifically to the proximal c-jun promoter CRE element. Also, representative human leukemic cell lines expressing p210 BCR-ABL and possessing abundant kinase activity of JNK, when compared with parental cells that were deficient in JNK activity, had increased c-jun and c-fosproteins. Finally, to show the relevance of these observations in model systems, we studied blast cells from patients with Philadelphia chromosome–positive acute leukemic transformation, and observed comparable activities of JNK catalysis and c-jun/AP-1 protein relative to the cell lines that possessed p210 BCR-ABL and JNK activity. These studies provide a basis for investigating the set of downstream genes which augmented c-jun/AP-1 activity enlists in the process of transformation by p210 BCR-ABL.
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Burgess, Gem S., Elizabeth A. Williamson, Larry D. Cripe, Sara Litz-Jackson, Jay A. Bhatt, Kurt Stanley, Mark J. Stewart, Andrew S. Kraft, Harikrishna Nakshatri, and H. Scott Boswell. "Regulation of the c-jun Gene in p210 BCR-ABL Transformed Cells Corresponds With Activity of JNK, the c-jun N-Terminal Kinase." Blood 92, no. 7 (October 1, 1998): 2450–60. http://dx.doi.org/10.1182/blood.v92.7.2450.2450_2450_2460.
Full textAbstract:
Activity of the c-jun N-terminal kinase (JNK) has been shown in hematopoietic cells transformed by p210 BCR-ABL. However, analysis has not been reported for hematopoietic cells on the consequences of this activity for c-jun promoter regulation within its distinctive proximal 8-base consensus CRE-like element, an element linked to JNK-mediated increase in c-jun transcription. In the present study, regulation of the proximal c-jun promoter was studied in murine myeloid cells transformed by p210 BCR-ABL. Promoter regulation in p210 BCR-ABL transformed cells was compared with regulation of the promoter in nontransformed interleukin-3 (IL-3)–dependent parental cells. The composition of nuclear AP-1 proteins contained within cells with p210 BCR-ABL, and their binding to the c-jun promoter proximal CRE-like element, was compared with the composition and binding of AP-1 proteins in IL-3–treated parental cells without p210 BCR-ABL. The present analysis found fivefold increased c-jun transcription occurring in p210 BCR-ABL transformed murine myeloid cells possessing a corresponding magnitude of increased kinase activity of JNK, compared with IL-3–stimulated parental cells. Augmented JNK activity was accompanied by increased nuclear abundance of c-jun and c-fos proteins that bound specifically to the proximal c-jun promoter CRE element. Also, representative human leukemic cell lines expressing p210 BCR-ABL and possessing abundant kinase activity of JNK, when compared with parental cells that were deficient in JNK activity, had increased c-jun and c-fosproteins. Finally, to show the relevance of these observations in model systems, we studied blast cells from patients with Philadelphia chromosome–positive acute leukemic transformation, and observed comparable activities of JNK catalysis and c-jun/AP-1 protein relative to the cell lines that possessed p210 BCR-ABL and JNK activity. These studies provide a basis for investigating the set of downstream genes which augmented c-jun/AP-1 activity enlists in the process of transformation by p210 BCR-ABL.
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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.
Full textAbstract:
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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Illert, Anna Lena, Cornelius Miething, Rebekka Grundler, Manuel Schmidt, Andreas Burchert, Andreas Neubauer, Claudia Mugler, Christian Peschel, and Justus Duyster. "Interferon Regulatory Factor 4 Is Not Required for Induction of Chronic Myeloid Leukaemia-Like Myeloproliferative Disease by Bcr/Abl in Mice." Blood 106, no. 11 (November 16, 2005): 2866. http://dx.doi.org/10.1182/blood.v106.11.2866.2866.
Full textAbstract:
Abstract Interferon regulatory factors (IRF) are activating and/or repressing transcription factors induced by treatment with type I and II Interferon (IFN), other cytokines, receptor cross-linking and viral infection. In contrast to IRF-1 and IRF-2, which are widely expressed, IRF-4 and IRF-8 are tissue-restricted factors. IRF-8 is expressed mainly in cells of haematopoietic origin and has recently been shown to inhibit mitogenic activity of p210 Bcr/Abl-transformed myeloid progenitor cells by activating several genes that interfere with the c-Myc pathway. IRF-4 is most homologous with IRF-8 (approximately 70% overall homology) and its expression is highly restricted to lymphocytes of the B-cell type (pre-B, B, and plasma cells), mature T-cells and macrophages. Furthermore IRF-4 expression is significantly impaired in CML and AML patient samples predominately in T-cells. To examine a potential role of IRF-4 in Bcr/Abl mediated transformation we used a bone marrow transplant model (BMT). We transduced IRF-4 knockout (KO) bone marrow with retrovirus expressing p210 Bcr/Abl and transplanted it into lethally irradiated recipient C57/bl6 mice. For proper control we transplanted also wildtype (WT) bone marrow transduced with Bcr/Abl and mock transfected IRF-4 KO bone marrow (BM). All recipients transplanted with Bcr/Abl transduced BM (regardless of which IRF-4 KO or WT) developed rapidly a myeloproliferative disorder characterized by leukocytosis and expression of the myeloid lineage markers CD11b and Gr1. Surprisingly, IRF-4 KO Bcr/Abl infected BM recipient mice survived slightly longer than the control group transplanted with WT p210 BM (12 vs. 19 days). Histopathologic studies of the affected organs (spleen/lung) revealed extramedullary haematopoiesis in the spleens of both groups and a distinct infiltration of the tumor cells in the lung of WT Bcr/Abl transduced BM recipient mice, resulting in massive punctuated bleedings. Interestingly, preliminary analysis suggest a significantly reduced lung infiltration with almost no pulmonary bleedings in IRF-4 KO Bcr/Abl infected BM recipient mice, which we assume to be the reason for the differences in the overall survival. Taken together our data demonstrate that IRF-4 is not required for the induction of a myeloproliferative disorder by Bcr/Abl in vivo and for its ability to transform BM cells in vitro, but IRF-4 deficiency seems to have an impact on the fulminant pulmonary haemorrhage occurring in the murine CML-like disease.
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Zhao, Xingwang, Hengyi Xie, Meng Zhao, Asma Ahsan, Xinxin Li, Fei Wang, Junyang Yi, et al. "Fc receptor–like 1 intrinsically recruits c-Abl to enhance B cell activation and function." Science Advances 5, no. 7 (July 2019): eaaw0315. http://dx.doi.org/10.1126/sciadv.aaw0315.
Full textAbstract:
B cell activation is regulated by the stimulatory or inhibitory co-receptors of B cell receptors (BCRs). Here, we investigated the signaling mechanism of Fc receptor-like 1 (FcRL1), a newly identified BCR co-receptor. FcRL1 was passively recruited into B cell immunological synapses upon BCR engagement in the absence of FcRL1 cross-linking, suggesting that FcRL1 may intrinsically regulate B cell activation and function. BCR cross-linking alone led to the phosphorylation of the intracellular Y281ENV motif of FcRL1 to provide a docking site for c-Abl, an SH2 domain-containing kinase. The FcRL1 and c-Abl signaling module, in turn, potently augmented B cell activation and proliferation. FcRL1-deficient mice exhibited markedly impaired formation of extrafollicular plasmablasts and germinal centers, along with decreased antibody production upon antigen stimulation. These findings reveal a critical BCR signal-enhancing function of FcRL1 through its intrinsic recruitment to B cell immunological synapses and subsequent recruitment of c-Abl upon BCR cross-linking.
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Li, Shaoguang, Robert L. Ilaria, Ryan P. Million, George Q. Daley, and Richard A. Van Etten. "The P190, P210, and P230 Forms of the BCR/ABL Oncogene Induce a Similar Chronic Myeloid Leukemia–like Syndrome in Mice but Have Different Lymphoid Leukemogenic Activity." Journal of Experimental Medicine 189, no. 9 (May 3, 1999): 1399–412. http://dx.doi.org/10.1084/jem.189.9.1399.
Full textAbstract:
The product of the Philadelphia chromosome (Ph) translocation, the BCR/ABL oncogene, exists in three principal forms (P190, P210, and P230 BCR/ABL) that are found in distinct forms of Ph-positive leukemia, suggesting the three proteins have different leukemogenic activity. We have directly compared the tyrosine kinase activity, in vitro transformation properties, and in vivo leukemogenic activity of the P190, P210, and P230 forms of BCR/ABL. P230 exhibited lower intrinsic tyrosine kinase activity than P210 and P190. Although all three oncogenes transformed both myeloid (32D cl3) and lymphoid (Ba/F3) interleukin (IL)-3–dependent cell lines to become independent of IL-3 for survival and growth, their ability to stimulate proliferation of Ba/F3 lymphoid cells differed and correlated directly with tyrosine kinase activity. In a murine bone marrow transduction/transplantation model, the three forms of BCR/ABL were equally potent in the induction of a chronic myeloid leukemia (CML)–like myeloproliferative syndrome in recipient mice when 5-fluorouracil (5-FU)–treated donors were used. Analysis of proviral integration showed the CML-like disease to be polyclonal and to involve multiple myeloid and B lymphoid lineages, implicating a primitive multipotential target cell. Secondary transplantation revealed that only certain minor clones gave rise to day 12 spleen colonies and induced disease in secondary recipients, suggesting heterogeneity among the target cell population. In contrast, when marrow from non– 5-FU–treated donors was used, a mixture of CML-like disease, B lymphoid acute leukemia, and macrophage tumors was observed in recipients. P190 BCR/ABL induced lymphoid leukemia with shorter latency than P210 or P230. The lymphoid leukemias and macrophage tumors had provirus integration patterns that were oligo- or monoclonal and limited to the tumor cells, suggesting a lineage-restricted target cell with a requirement for additional events in addition to BCR/ABL transduction for full malignant transformation. These results do not support the hypothesis that P230 BCR/ABL induces a distinct and less aggressive form of CML in humans, and suggest that the rarity of P190 BCR/ABL in human CML may reflect infrequent BCR intron 1 breakpoints during the genesis of the Ph chromosome in stem cells, rather than intrinsic differences in myeloid leukemogenicity between P190 and P210.
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Krause, Daniela S., Ulrich H. von Andrian, and Richard A. Van Etten. "Selectins and Their Ligands Are Required for Homing and Engraftment of BCR-ABL+ Leukemia-Initiating Cells." Blood 106, no. 11 (November 16, 2005): 697. http://dx.doi.org/10.1182/blood.v106.11.697.697.
Full textAbstract:
Abstract Autologous hematopoietic stem cell (HSC) transplantation is a feasible form of treatment for many types of leukemias and lymphomas, including chronic myeloid leukemia (CML). Malignant cells contaminating the graft, however, can engraft and lead to relapse of the original disease. Previous studies have demonstrated that BCR-ABL+ leukemic progenitors have defects in the adhesive function of beta-1 integrins and in their response to the chemokine SDF-1alpha, pathways that are critical for homing and engraftment of normal HSC. We hypothesized that BCR-ABL-expressing leukemic stem cells differ from normal HSC in their homing and engraftment properties. Using a retroviral transduction/transplantation model of CML and donor/recipient mice with mutations in adhesion molecules, we investigated the role of specific adhesion pathways in the engraftment of CML-like leukemia. We found no difference in the expression levels of integrins, LFA-1, and CXCR4 between normal and BCR-ABL+ c-Kit+ Lin- cells, but lower expression levels of P-selectin glycoprotein ligand-1 (PSGL-1) and of L-selectin. In transplantation experiments, VCAM-1, the principal bone marrow ligand for beta-1 integrin, was not required in the bone marrow endothelium of the recipient for efficient engraftment of CML-like disease, confirming that progenitors capable of initiating CML-like leukemia upon transplantation are independent of the beta-1 integrin pathway for engraftment. Likewise, recipient P-selectin was also not required for the engraftment of CML-like leukemia. By contrast, deficiency of PSGL-1 in the leukemic cells or of E-selectin in the recipient significantly reduced engraftment by BCR-ABL-expressing stem cells, as assessed by Southern blot quantitation of proviral clone frequency. The requirement for recipient E-selectin could be bypassed by direct intrafemoral injection of BCR-ABL-expressing cells, leading to polyclonal leukemia. BCR-ABL-expressing cells that were deficient for the selectin ligand-synthesizing enzymes Core-2 or Fucosyltransferases IV and VII also exhibited decreased engraftment and increased disease latency. Treatment of BCR-ABL-transduced cells with neuraminidase, which destroys selectin binding sites, completely blocked leukemic engraftment. Whereas L-selectin has no role in homing and engraftment of normal HSC, BCR-ABL-expressing L-selectin-deficient progenitors were profoundly defective for engraftment, with decreased disease clonality, increased disease latency, and frequent death of recipients from graft failure. Importantly, efficient engraftment and leukemogenesis of BCR-ABL-expressing L-selectin-deficient cells was restored by co-expression of a chimeric E/L-selectin molecule that is resistant to cell surface shedding. These results establish that BCR-ABL-expressing leukemic stem cells rely to a greater extent on selectins and their ligands for homing and engraftment than normal HSC. Specific blocking of selectin-ligand interactions is a novel clinical strategy to exploit the differences in normal and Ph+ stem cells that may be beneficial in an autologous transplantation setting.
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le Coutre, Philipp, Elena Tassi, Marileila Varella-Garcia, Rossella Barni, Luca Mologni, Gonçalo Cabrita, Edoardo Marchesi, Rosanna Supino, and Carlo Gambacorti-Passerini. "Induction of resistance to the Abelson inhibitor STI571 in human leukemic cells through gene amplification." Blood 95, no. 5 (March 1, 2000): 1758–66. http://dx.doi.org/10.1182/blood.v95.5.1758.005a41_1758_1766.
Full textAbstract:
The 2-phenylaminopyrimidine derivative STI571 has been shown to selectively inhibit the tyrosine kinase domain of the oncogenicbcr/abl fusion protein. The activity of this inhibitor has been demonstrated so far both in vitro with bcr/abl expressing cells derived from leukemic patients, and in vivo on nude mice inoculated with bcr/abl positive cells. Yet, no information is available on whether leukemic cells can develop resistance to bcr/ablinhibition. The human bcr/abl expressing cell line LAMA84 was cultured with increasing concentrations of STI571. After approximately 6 months of culture, a new cell line was obtained and named LAMA84R. This newly selected cell line showed an IC50 for the STI571 (1.0 μM) 10-fold higher than the IC50 (0.1 μM) of the parental sensitive cell line. Treatment with STI571 was shown to increase both the early and late apoptotic fraction in LAMA84 but not in LAMA84R. The induction of apoptosis in LAMA84 was associated with the activation of caspase 3–like activity, which did not develop in the resistant LAMA84R cell line. LAMA84R cells showed increased levels of bcr/abl protein and mRNA when compared to LAMA84 cells. FISH analysis with BCR- and ABL-specific probes in LAMA84R cells revealed the presence of a marker chromosome containing approximately 13 to 14 copies of the BCR/ABL gene. Thus, overexpression of the Bcr/Abl protein mediated through gene amplification is associated with and probably determines resistance of human leukemic cells to STI571 in vitro.
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Mian, Afsar, Anahita Rafiei, Claudia Oancea, Oliver G. Ottmann, and Martin Ruthardt. "The Functional Interplay Between the t(9;22)-Associated Fusion Proteins BCR/ABL and ABL/BCR in Philadelphia Chromosome Positive Acute Lymphatic Leukemia." Blood 124, no. 21 (December 6, 2014): 2402. http://dx.doi.org/10.1182/blood.v124.21.2402.2402.
Full textAbstract:
Abstract The successful targeting of BCR/ABL by selective ABL-kinase inhibitors (AKI) such as Imatinib, Nilotinib, or Dasatinib alone is unable to eradicate the leukemic clone in Philadelphia chromosome positive (Ph+ ) leukemia. The t(9;22)(q34;q11) is a balanced translocation. Der22 involves the BCR (breakpoint cluster region) gene locus with two principal breaks: the M-bcr, encoding for the p210BCR/ABL and the m-bcr, encoding for the 185BCR/ABL fusion proteins, respectively. The constitutively activated BCR/ABL kinase is responsible for the leukemic transformation through an aberrant activation of multiple signaling pathways, such as Stat, Pi3K and Ras/Erk. The der9 encodes for the reciprocal ABL/BCR fusion proteins the p40ABL/BCR, present in 65% of patients with chronic myeloid leukemia (CML) and the p96ABL/BCR, detectable in 100% of patients with Ph+ acute lymphatic leukemia (ALL). ABL/BCRs are oncogenes able to influence the lineage commitment of hematopoietic progenitors. Aim of this study was to further disclose the role of p96ABL/BCR for the pathogenesis of Ph+ ALL. We co-expressed p96ABL/BCRand p185BCR/ ABL from a p2A peptide-linked multi-cistronic retroviral vector, which allows the expression of multiple proteins from a single open reading frame (ORF) to identical levels. The co- expression of p96ABL/BCR enhanced the kinase activity and, as a consequence, the transformation potential of p185BCR/ABL in factor dependent progenitor cells and untransformed fibroblasts. Targeting p96ABL/BCR by RNAi inhibited growth of Ph+ ALL cell lines and primary Ph+ ALL patient-derived long-term cultures (PD-LTCs). Furthermore p96ABL/BCR negatively influenced the response to AKI in these models as shown by an increased response to AKI when p96ABL/BCR was down-regulated. Our in vitro and in vivo stem cell studies on murine fetal liver cells and adult HSCs revealed a functional hierarchy between p96ABL/BCR and p185BCR/ABL. In fact, p96ABL/BCR strongly increased stem cell capacity in replating efficiency and colony forming unit-spleen day 12 (CFU-S12) assays, whereas p185BCR/ABL showed no effect. In contrast co-expression of p96ABL/BCR and p185BCR/ABL increased significantly both serial replating potential and CFU-S12 colony formation as compared to p96ABL/BCR alone. In a syngeneic mouse model co-expression of p96ABL/BCR abolished the capacity of p185BCR/ABL to induce a CML-like disease and led to the induction of ALL. Taken together our here presented data reveal an important role of p96ABL/BCR for the pathogenesis of Ph + ALL. Disclosures No relevant conflicts of interest to declare.
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Demirel, Özlem, Olivier Balló, Hubert Serve, and Christian H. Brandts. "The Role Of SOCS1 In BCR-ABL Mediated Transformation and Leukemogenesis." Blood 122, no. 21 (November 15, 2013): 2507. http://dx.doi.org/10.1182/blood.v122.21.2507.2507.
Full textAbstract:
Abstract The BCR-ABL oncogene activates several signaling pathways, most notably by constitutive phosphorylation of the signal transducer and activator of transcription protein 5 (STAT5). After phosphorylation and nuclear translocation, STAT5 transcriptionally activates numerous genes responsible for proliferation, survival and differentiation of hematopoietic stem and progenitor cells. Among the STAT5 target genes are suppressor of cytokine signaling (SOCS) proteins. SOCS proteins inhibit JAK kinases by multiple mechanisms, thereby terminating cytokine signaling in a classical negative feedback loop. The SOCS family of proteins comprises eight members: cytokine-inducible SRC homology 2 (SH2) domain protein (CIS) and SOCS1–SOCS7. SOCS1 is frequently silenced by hypermethylation in multiple myeloma and inactivating mutations have been found in Hodgkin lymphoma with consecutive increase in JAK2 kinase activity. More recently, we identified SOCS1 as a “conditional oncogene” in the context of the FLT3-ITD oncogene (Reddy et al, Blood 2012): SOCS1 significantly enhanced FLT3-ITD-mediated myeloid transformation, both in vitro and in vivo. We hypothesized that this may be a more general mechanism of transformation and therefore analyzed the role of SOCS proteins in BCR-ABL mediated transformation and leukemogenesis. First, we investigated gene expression levels of SOCS proteins in BCR-ABL positive (versus BCR-ABL negative) cell lines and primary ALL long term-cultured cells. Upon treatment with the BCR-ABL inhibitor imatinib, mRNA expression levels of CIS and SOCS1-4 were reduced. SOCS5-7 did not exhibit any changes and were non-responsive to ABL-kinase inhibition. In lineage-depleted primary murine bone marrow retrovirally transduced with BCR-ABL, high induction of CIS and SOCS1-3 mRNA was detected, while SOCS4-7 showed only minor changes. When overexpressed in IL-3 dependent cell lines, SOCS1 led to a very rapid cell death within few days. Similar effects were demonstrated for CIS and SOCS2 overexpression, however, with a slower kinetics. In contrast, BCR-ABL transduced cells were insensitive to SOCS1 overexpression. In colony formation assays performed with primary hematopoietic cells, expression of SOCS1 led to a significant decrease of colony numbers. Interestingly, co-expression of SOCS1 and BCR-ABL (hereafter abbreviated as SOCS1/BCR-ABL) also lowered colony numbers compared to cells expressing BCR-ABL alone. However, when cells were subjected to interferon alpha or interferon gamma treatment, SOCS1/BCR-ABL positive cells displayed higher colony numbers and gained a growth advantage over BCR-ABL expressing cells, since anti-proliferative effects of the cytokines were inhibited by the presence of SOCS1. A careful analysis of the downstream signaling cascade of BCR-ABL and SOCS1/BCR-ABL expressing cells did not demonstrate any differences in the phosphorylation of AKT, ERK1/2 and STAT5. However, when BCR-ABL was inhibited by imatinib, STAT5 phosphorylation was significantly decreased in SOCS1/BCR-ABL transduced cells. Finally, the influence of SOCS1 in BCR-ABL mediated leukemia was investigated in a murine bone marrow transplantation model. BCR-ABL or SOCS1/BCR-ABL expressing cells led to disease formation with a chronic myeloid leukemia-like phenotype. Interestingly, the co-expression of SOCS1 and BCR-ABL prolonged disease latency, as opposed to the phenotype observed with FLT3-ITD (where SOCS1 co-expression shortened latency). In this setting SOCS1 acts as a tumor suppressor, protecting BCR-ABL transformed cells from rapid disease development, and a molecular analysis will be presented. Disclosures: No relevant conflicts of interest to declare.
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Sha, Xiaojin, Dan A. Liebermann, and Barbara Hoffman. "Loss of Growth Arrest DNA Damage 45a,b (GADD45a,b) Enhances Oncogenicity in BCR/ABL-Driven Chronic Myelogenous Leukemia." Blood 114, no. 22 (November 20, 2009): 3264. http://dx.doi.org/10.1182/blood.v114.22.3264.3264.
Full textAbstract:
Abstract 3264 Poster Board III-1 The bcr/abl oncogene causes chronic myelogenous leukemia (CML) in human. BCR/ABL induces the transformation of myeloid lineage through MAPK, JNK/SAPK, PI3K signaling pathways. Growth arrest DNA damage 45A (GADD45A) and GADD45B are upregulated during myeloid lineage terminal differentiation. They are involved in G2/M cell cycle arrest and apoptosis in response to exogenous stress stimuli through MAPK and JNK/SAPK pathways. To investigate the effect of GADD45A and GADD45B in the development of CML, syngeneic wild type lethally irradiated mice were reconstituted with wild type, gadd45a or gadd45b null myeloid progenitors transduced with a retrovirally expressed 210-kD BCR/ABL fusion oncoprotein. We found that loss of gadd45a or gadd45b accelerated the development of CML-like disease in wild type recipients. BCR/ABL transformed gadd45a or gadd45b deficient progenitor recipients exhibited a significantly accelerated kinetics of increase in the number of WBC and percentage of myeloid blasts in blood compared to mice reconstituted with the same number of wild type bone marrow cells transduced with BCR/ABL. There was also increase in the rate of accumulation of CD11b+Gr1+ cells in the bone marrow and spleen. Using in vitro and in vivo BrdU assays, enhanced proliferation capacity was observed for both BCR/ABL transduced gadd45a and gadd45b deficient myeloid progenitors. BCR/ABL transduced gadd45a and gadd45b deficient primary myeloid progenitors formed more and bigger colonies compared to BCR/ABL transformed wild type progenitors. Impaired apoptosis was showed in BCR/ABL transduced gadd45a deficient myeloid progenitors. These results indicate that both gadd45a and gadd45b function as suppressors of the development of BCR/ABL driven CML, where gadd45a appears to suppress CML via mechanism involving inhibition of cell proliferation enhancement of apoptosis, whereas gadd45b appears to only inhibit cellular proliferation. Dissecting the molecular nature of signaling paths involved in the suppressive function of gadd45a and gadd45b in BCR/ABL driven CML, as well as analysis of Gadd45 in CML patients, is underway. Disclosures: No relevant conflicts of interest to declare.
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Slupianek, Artur, Stanislaw Jozwiakowski, Ewa Gurdek, Michal O. Nowicki, and Tomasz Skorski. "BCR/ABL Regulates the Expression and Interacts with Werner Syndrome Helicase/Exonuclease To Modulate Its Biochemical Properties." Blood 106, no. 11 (November 16, 2005): 2873. http://dx.doi.org/10.1182/blood.v106.11.2873.2873.
Full textAbstract:
Abstract A genome-wide screen suggested that BCR/ABL kinase might stimulate WRN, a member of the RecQ-like DNA helicases family. The Werner syndrome protein (WRN) exerts DNA helicase and 3′-5′ exonuclease activities. Inactivating mutations in the WRN gene causes Werner syndrome, characterized by premature aging, genomic instability and cancer predisposition. The WRN helicase unwinds unusual DNA structures, which can occur physiologically, or can be accidentally generated during DNA repair (double-stranded DNA with mismatched tails, bimolecular G4 quartets and Holliday junctions). In addition, WRN physically interacts with components of two major systems for DNA double-strand breaks (DSBs) repair: non-homologous end-joining (NHEJ) and homologous recombination (HR). Here we demonstrated that BCR/ABL regulates the expression of WRN mRNA and protein in CML primary cells and BCR/ABL-transformed cell lines. BCR/ABL kinase-induced WRN expression is mediated by c-MYC, but not STAT5 - dependent transcription as well as by inhibition of caspases-dependent cleavage. In addition, immunoprecipitation and pull-down studies indicated that BCR/ABL interacts directly with WRN resulting in its tyrosine phosphorylation. Mutation analysis revealed that multiple domains/amino acid residues of BCR/ABL and WRN are involved in the interaction. BCR/ABL-positive leukemia cells exerted an enhanced WRN-dependent helicase activity. In addition, immunoprecipitation and double-immunofluorescence co-localization studies demonstrated an elevated interaction between WRN and RAD51 in BCR/ABL cells undergoing genotoxic stress in comparison to parental counterparts. Altogether, it is likely that WRN is involved in DSBs repair by HR in leukemia cells. More detailed studies are underway to pinpoint the role of WRN in DNA damage response in BCR/ABL-transformed cells.
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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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Cuiffo, Benjamin, and Ruibao Ren. "Dominant Negative Effect of Palmitoylation-Deficient NRAS In Suppression of BCR/ABL Leukemogenesis." Blood 116, no. 21 (November 19, 2010): 3157. http://dx.doi.org/10.1182/blood.v116.21.3157.3157.
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Abstract Abstract 3157 RAS mutations are found in ∼30% of all human cancers, with NRAS being the RAS isoform found most frequently mutated in hematological malignancies including acute myelogenous leukemia (AML) and chronic myelomonocytic leukemia (CMML). We have previously shown that expression of oncogenic NRAS efficiently induces a CMML- or AML-like disease in mice. Like all RAS proteins, NRAS must undergo a series of post-translational modifications for differential targeting to distinct cellular membrane microdomains. We have previously found that palmitoylation, in addition to prenylation, is an essential process for NRAS leukemogenesis. The results suggest that targeting RAS palmitoylation may be an effective therapy for hematological malignancies as well as other NRAS related cancers. In addition to the direct activation by mutations, RAS can also be functionally activated by other oncogenic mutations, including activated protein tyrosine kinases such as BCR/ABL. While significant advances have been made in the treatment of chronic myelogenous leukemia (CML) by targeting the kinase activity of BCR/ABL, these treatments do not constitute a cure, as BCR/ABL-positive cells persist and eventually develop mutations that cause resistance to the kinase inhibitors. In addition, although advances have also been achieved in the treatment of BCR/ABL+ B-acute lymphoblastic lymphoma (B-ALL) through the use of combination chemotherapy along with the ABL kinase inhibitor imatinib, BCR/ABL+ B-ALL continues to pose a huge challenge; they are highly refractory to imatinib, and are still the least treatable subtype of ALL. The identification of molecular targets that impede the progression of BCR/ABL+ leukemias not relying on targeting the kinase activity of BCR/ABL is likely beneficial. Previous studies have shown that RAS is a critical downstream transducer of the oncogenic signaling by BCR/ABL. We have shown that palmitoylation-deficient oncogenic NRAS is mislocalized away from the plasma membrane yet still capable of binding GTP in cells. This finding suggests that palmitoylation-deficient, activated RAS may have a dominant negative effect in RAS signaling. As a proof-of-concept study for treating cancers driven by RAS regulators through blocking RAS palmitoylation, we evaluated the effect of blocking RAS palmitoylation in BCR/ABL leukemogenesis. We found that expression of palmitoylation-deficient oncogenic NRAS or palmitoylation-deficient wild type NRAS significantly impeded progression of BCR/ABL induced B-ALL- and CML-like diseases in mice. The palmitoylation-deficient NRAS suppresses the activation of downstream effectors of RAS, possibly by sequestrating RAS effectors away from the plasma membrane. The results suggest that targeting RAS palmitoylation may also constitute an effective therapy in cancers driven by oncogenes upstream of RAS. Disclosures: No relevant conflicts of interest to declare.
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Krause, Daniela S., Katherine Lazarides, Ulrich H. von Andrian, and Richard A. Van Etten. "CD44 Is Selectively Required for the Homing and Engraftment of BCR-ABL-Expressing Leukemic Stem Cells." Blood 108, no. 11 (November 16, 2006): 743. http://dx.doi.org/10.1182/blood.v108.11.743.743.
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Abstract In chronic myeloid leukemia (CML) patients treated by autologous hematopoietic stem cell (HSC) transplantation, malignant progenitors in the graft can contribute to relapse of leukemia (Deisseroth et al., Blood1994; 83:3068), but the mechanisms of homing and engraftment of leukemic CML stem cells are unknown. Although the frequency of autografting in CML has decreased following the introduction of imatinib, most imatinib-responsive patients harbor residual BCR-ABL-expressing stem cells (Graham et al., Blood2002; 99:319) and some will develop progressive leukemia. Autografting with cells harvested at the time of minimal residual disease could be an important salvage therapy, but methods to selectively block the engraftment of leukemic stem cells are needed. In this study, we show that CD44 expression is increased on murine BCR-ABL-expressing stem/progenitor cells and contributes to the ability of these cells to bind to Selectins. In a retroviral transduction/transplantation model of CML, BCR-ABL-transduced progenitors from CD44-deficient donors were defective in homing to recipient marrow, resulting in defective engraftment and impaired induction of CML-like myeloproliferative disease. By contrast, CD44-deficient stem cells transduced with empty retrovirus engrafted as efficiently as wild-type HSC. In addition, CD44 was not required for induction of acute B-lymphoblastic leukemia (B-ALL) by BCR-ABL, indicating that the engraftment requirement for CD44 is specific to leukemic cells initiating CML, not B-ALL. The requirement for donor CD44 was bypassed by direct intrafemoral injection of BCR-ABL-transduced CD44-deficient stem cells, or by co-expression of human CD44 with BCR-ABL. Treatment of BCR-ABL-transduced stem/progenitor cells from wild-type donors with antibody to CD44 attenuated the induction of CML-like leukemia in recipients. These results demonstrate a major role for CD44 in homing and engraftment of BCR-ABL-expressing leukemic stem cells, possibly through adhesive interactions with Selectins and/or hyaluronan in the recipient bone marrow niche. They further argue that BCR-ABL-transduced stem/progenitor cells depend to a greater extent on CD44 for engraftment than do normal HSC, and suggest that CD44 blockade may be beneficial in autologous transplantation in CML.
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Hsieh, Mo-Ying, and Richard A. Van Etten. "Distinct Roles for the NF-κB Pathway In Myeloid and Lymphoid Transformation and Leukemogenesis by BCR-ABL." Blood 116, no. 21 (November 19, 2010): 1225. http://dx.doi.org/10.1182/blood.v116.21.1225.1225.
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Abstract Abstract 1225 The BCR-ABL tyrosine kinase, product of the t(9;22) Ph chromosome, activates multiple signaling pathways in leukemic cells from patients with chronic myeloid leukemia (CML) and Ph+ B-cell acute lymphoblastic leukemia (B-ALL). Previous studies have shown that NF-κB is activated in BCR-ABL-expressing cell lines and contributes to transformation of primary B-lymphoid cells by BCR-ABL (Reuther et al., Genes Dev. 1998;12:968), but the mechanism of activation has not been defined (Kirchner et al., Exp. Hematol. 2003;31:504), and importance of NF-kB to myeloid and lymphoid leukemogenesis by BCR-ABL is unknown. To interrogate the role of NF-κB in BCR-ABL-mediated transformation, we utilized a super-repressor mutant form of IκBα (IκBαSR), which has been used to block NF-κB nuclear localization and transactivation by constitutively sequestering NF-κB in the cytoplasm. Using retrovirus co-expressing BCR-ABL and IκBαSR, we found that IκBαSR blocked nuclear p65/RelA expression and inhibited the IL-3 independent growth of Ba/F3 cells and primary B-lymphoid cells transformed by BCR-ABL. The effect of NF-κB inhibition was primarily on proliferation rather than on cell survival, as there was no increase in apoptosis in cells expressing IκBαSR. When primary bone marrow cells were transduced and transplanted under conditions favoring induction of B-ALL or CML-like myeloproliferative neoplasm in recipient mice, co-expression of IκBαSR significantly attenuated disease development and prolonged survival of diseased mice. Molecular analysis of these leukemias demonstrated that NF-κB inhibition decreased the frequency of leukemia-initiating (“stem”) cells in the CML model, but not in the B-ALL model, and was associated with decreased expression of c-Myc, an NF-κB target. To clarify the mechanism of activation of NF-κB in BCR-ABL-expressing cells, we targeted two upstream kinases that negatively regulate IκBα, IKKα/IKK1 or IKKβ/IKK2. To accomplish this, we engineered retroviruses co-expressing BCR-ABL and kinase-inactive, dominant-negative mutants of IKK1 (IKK1KM) or IKK2 (IKK2KM). Co-expression of either IKK mutant inhibited both B-lymphoid transformation and leukemogenesis by BCR-ABL, as well as induction of CML-like MPN, with IKK1 inhibition more effective than IKK2. Together, these results demonstrate that NF-κB is activated in part through the canonical IKK pathway in BCR-ABL-expressing leukemia cells, and that NF-κB signaling plays distinct roles in the pathogenesis of myeloid and lymphoid leukemias induced by BCR-ABL. In CML, NF-κB may play a role for in generation and/or maintenance of leukemic stem cells. These results validate IKKs as targets for therapy in Ph+ leukemias, and motivate the evaluation of small molecule IKK inhibitors in these diseases. Disclosures: No relevant conflicts of interest to declare.
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Yu, Chuanjiang, Sivahari Prasad Gorantla, Tony Mueller, Lena Lippert, Zhenyu Yue, Robert Zeiser, Tobias Huber, Justus Duyster, and Anna Lena Illert. "Beclin-1 Phosphorylation By BCR-ABL Is Crucial for CML Leukemogenesis By Suppression of Autophagy." Blood 126, no. 23 (December 3, 2015): 16. http://dx.doi.org/10.1182/blood.v126.23.16.16.
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Abstract The constitutively activated chimeric Tyrosine kinase BCR-ABL is critical for initiation, progression and maintenance of chronic myelogenous leukemia (CML). Imatinib and second generation BCR-ABL tyrosine kinase inhibitors (TKIs) serve now as standard therapies for Ph+-patients. However, disease persistence occurs frequently and insensitivity of CML stem cells to TKI treatment is discussed as one major reason for this. Recent evidence accumulates, that autophagy, a genetically-regulated process of adaptation to metabolic stress, is involved in TKI-induced cell death. It is hypothesized, that TKI-induced autophagy could allow CML stem cells to become metabolically dormant enabling their survival under conditions that may mimic growth factor deprivation and thereby "antagonize" TKI-induced cell death. However, the molecular mechanism of BCR-ABL and TKI induced autophagy as well as its role as tumor suppressor or promoter is poorly understood. In our study, we aim to identify the precise role of autophagy and its´ effector molecules in a murine CML model. To test whether BCR-ABL regulates autophagy, we measured LC3 as a marker for autophagy in BCR-ABL+-K562 cell. Interestingly, inhibition of BCR-ABL activity by nilotinib led to increased LC3-II expression and punctual LC3 accumulation, indicating, that BCR-ABL activity can suppress autophagy. Consistent with this, Ba/F3 cells expressing BCR-ABL WT induce autophagy, whereas Ba/F3 cell expressing BCR-ABL-T315I fail to induce autophagy by nilotinib treatment, pointing to a BCR-ABL specific autophagy induction than an unspecific effect of TKI treatment. Next, we investigated the proteins involved in BCR-ABL mediated autophagosome formation. Recruitment of VPS34 and ATG14 to Beclin1 was increased in case of nilotinib treatment and could thereby positively regulate autophagosome formation, whereas Rubicon, a negative regulator was less recruited to the Beclin1-complex. To further identify the impact of Beclin1 as a key regulator of autophagy in BCR-ABL-driven leukemia, we used a targeted genetic approach in a CML mouse model. Interestingly, mice transplanted with Belin1 knockdown, BCR-ABL expressing bone marrow showed a less aggressive disease with significantly lower WBC-count, leukemic burden and prolonged overall survival of the mice. In contrast, deletion of ATG5, another central regulator of autophagy, was not able to change disease onset or progression in the CML model. To further clarify the function of Beclin1, we performed biochemical binding analyses and were able to show, that Beclin1 binds to BCR-ABL independent of BCR-ABL kinase activity and Beclin1 is phosphorylated by BCR-ABL. Interestingly, Beclin1 is an exclusive target of BCR-ABL and can not be phosphorylated by other aberrantly activated tyrosine kinases like Flt3-ITD, NPM-ALK and PDGFRA-D842V. In vitro kinase assay with active ABL-kinase confirm Beclin1 as a specific substrate of BCR-ABL. GST pulldown experiments mapped the N-terminal region of Beclin1 to interact with BCR-ABL. Cloning of different phospho-deficient mutants identified tyrosine residues Y233 and Y352 of Beclin1 as the crucial sites for specific BCR-ABL phosphorylation. To test the impact of BCR-ABL mediated Beclin1-phosphorylation on autophagy induction, we generated Beclin1 phospho-mimic (Y233E/Y352E) and phospho-deficient (Y233F/Y352F) mutations. Interestingly, nilotinib treatment fails to induce autophagy in cells expressing the Beclin1 phospho-mimic mutations, thereby highlighting the necessity of Beclin1 in BCR-ABL-mediated autophagy. Expression of Beclin1 mutations in Beclin1 knockout MEFs and K562 cells show decreased binding of UVRAG, ATG14 and VPS34 to Beclin1 Y233E/Y352E, suggesting an important role of Beclin1 phosphorylation for complex stabilization and autophagy suppression. Taken together our findings identify Beclin1 as a specific substrate of BCR-ABL. Downregulation of Beclin1 is associated with a prolonged overall survival of BCR-ABL transplanted animals; direct phosphorylation of Beclin1 on Tyrosine residues Y233 and Y352 lead to LC3 inhibition and suppression of autophagy. Our results thereby highlight the importance of Beclin1 in BCR-ABL-mediated leukemogenesis and show, that autophagy induction in CML cells may be rather a specific Beclin1-BCR-ABL interaction effect than a general microenvironmental stress phenomenon. Disclosures No relevant conflicts of interest to declare.
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Yamamoto, Kiyoko, Shinobu Tsuzuki, Tomoki Naoe, and Masao Seto. "Deregulated Activity of AML1/RUNX1 Cooperates with BCR-ABL to Immortalize Hematopoietic Progenitor Cells and Induces Blast Crisis-Like Disease of Chronic Myelogenous Leukemia in Mice,." Blood 118, no. 21 (November 18, 2011): 3749. http://dx.doi.org/10.1182/blood.v118.21.3749.3749.
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Abstract Abstract 3749 Since the introduction of imatinib treatment for chronic myeloid leukemia (CML), most patients in the chronic phase (CP) achieve long-lasting hematologic remission. However, some patients acquire resistance or intolerance to imatinib, which results in disease progression to blast crisis (BC). Although it is known that BCR-ABL induces genomic instability, which results in leukemia cells creating multiple gene defects, genes inducing the progression to BC in CML are largely unknown. Moreover, mutations in the AML1/RUNX1 gene and overrepresentation of AML1/RUNX1 in BC compared to CP have been revealed. We therefore hypothesized that deregulation of AML1/RUNX1 might be one of the mechanisms for disease progression of CML. We initially sought to establish a culture system in vitro that allows rapid identification of genes possibly contributing to the emergence of BC. Given that cells leading to the origin of BC are known to resemble committed progenitor rather than stem cells, we employed Sca-1(-)Lin(-) progenitors derived from mouse fetal liver for the culture. Thus, cells were retrovirally transduced with BCR-ABL alone or in combination with a gene of interest, and cultured in vitro without cytokines. BCR-ABL alone-transduced cells grew initially, but ceased to do so after a week. In contrast, cells co-transduced with BCR-ABL and either one of NUP98-HOXA9, Hes1 or Bmi1 (genes known to induce BC-like disease in concert with BCR-ABL) kept growing and were immortalized in the cytokine-free culture. Importantly, cells cultivated in vitro caused fetal disease when transplanted into mice. These findings suggest the utility of our in vitro culture system as an assay of the co-operativity of a given gene with BCR-ABL to induce BC. We therefore applied the culture system for the assay of AML1/RUNX1 mutants. We chose four AML1/RUNX1 mutants (K83Q, R139G, R80C, and D171N) for the analysis of their co-operativity with BCR-ABL. Wild-type AML1/RUNX1 and a vector-only control were also included. Results showed that K83Q and R139G mutants co-operated with BCR-ABL to immortalize progenitor cells in cytokine-free culture in vitro. In contrast, R80C and D171N mutants lacked such ability. The cells co-expressing BCR-ABL and either K83Q or R139G mutants cultivated in vitro caused fetal disease in mice following transplantation: all 5 mice transplanted with BCR-ABL/K83Q-transduced cells and 7 of 10 mice transplanted with BCR-ABL/R139G-transduced cells developed lethal hematologic diseases 7–79 days following transplantation. These findings are suggestive of variance among mutants of AML1/RUNX1 regarding the ability to induce BC. Unexpectedly, further analyses revealed that wild-type AML1/RUNX1 was as potent as K83Q and R139G in inducing BC: all 4 mice transplanted with BCR-ABL/wild-type AML1-transduced cells developed fetal disease. Examination of various lengths of C-terminally truncated versions of AML1/RUNX1 for their co-operativity with BCR-ABL revealed that AML1L419X and AML1H377X were able to co-operate with BCR-ABL to immortalize progenitor cells, while AML1I337X and AML1A297X did not. These findings suggest that transcriptional regulatory domains residing at the carboxyl-terminal of AML1/RUNX1 play important roles for wild-type AML1/RUNX1 to induce BC-like disease. Perhaps consistent with the increased expression of AML1/RUNX1 contributing to the occurrence of BC, shRNA-mediates silencing of AML1/RUNX1 inhibited growth of human CML cell lines (K562, MegA2, and MEG01). The overall findings of the present study suggest that (1) all mutants of AML1/RUNX1 do not contribute equally to the induction of BC-like disease, but there is a wide range of variance among mutants regarding their ability to induce BC in collaboration with BCR-ABL, and (2) increased expression of wild-type AML1/RUNX1 can induce BC-like disease in collaboration with BCR-ABL in mice. AML1/RUNX1 mutants tested here are known to dominant-negatively inhibit wild-type AML1/RUNX1 functions, but only two of 4 mutants exhibited BC-inducing activity. The over-expression of wild-type AML1/RUNX1 also exhibited such BC-inducing activity. We are currently conducting a detailed investigation of differences in cell lineage affected and growth properties caused by mutant and wild-type AML1/RUNX1s, which may reveal the dichotomic functions of AML1/RUNX1 in the development of BC. Disclosures: Naoe: Zenyaku-Kogyo: Research Funding; Novartis Pharma.: Honoraria, Speakers Bureau; Chugai Pharma.: Research Funding; Dainipponn-Sumitomo Pharma.: Research Funding; Kyowa-Hakko Kirin.: Research Funding; Otsuka Pharma.: Research Funding.
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Nardi, Valentina, Olaia Naveiras, Mohammad Azam, and George Q. Daley. "A Critical Role for CCL Chemokines in the Immuno-Protection Induced by Type I Interferons and IRF8/ICSBP Against Bcr/Abl-Induced Leukemia." Blood 110, no. 11 (November 16, 2007): 1001. http://dx.doi.org/10.1182/blood.v110.11.1001.1001.
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Abstract Until recently, the mainstay of Chronic Myelogenous Leukemia (CML) therapy was Interferon (IFN) alpha, which in a minority of patients induces long lasting cytogenetic remission. While the exact mechanism of action of IFN alpha in CML is still obscure, it is clear that the clinical response to IFN alpha correlates with immune system reactivity against leukemic clones. As minimal molecular disease often persists despite the use of imatinib and new Bcr-Abl inhibitors, immunotherapy remains an appealing adjunct to molecularly targeted inhibitors in CML therapy. We have shown that IRF8/ICSBP (Interferon Consensus Sequence Binding Protein) expression in Bcr-Abl transformed cells prevents their capacity to form a lethal leukemia when injected into mice, and that this protection is mediated by a long-lasting and potent CD8+ response against unknown epitopes on the leukemic cells. We hypothesized that the protection mediated by IRF8/ICSBP might be related to the anti-leukemic effects of IFN alpha. We now find that Type I IFNs like IFN alpha regulate IRF8/ICSBP expression in mouse and human cells and in Bcr-Abl transformed cells. Furthermore, type I IFNs can substitute for ICSBP in inducing the anti-leukemic immunity against Bcr-Abl transformed cells. Transcriptional profiling of cells expressing ICSBP, Bcr-Abl, or both ICSBP and Bcr-Abl identified two chemokines, CCL6 and CCL9, which were associated with the immune protection induced by IRF8/ICSBP expression. Type I IFNs and IRF8/ICSBP induce the expression of these chemokines in cells transformed with Bcr-Abl. RNAi-mediated inhibition experiments in our mouse model of CML show that these chemokines are required for the IRF8/ICSBP-mediated CD8+ anti-leukemic response to the Bcr-Abl transformed cells, suggesting that these chemokines could be exploited for immunotherapy in combination with existing Bcr-Abl peptide vaccines.
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Sanchez-Aguilera, Abel, Ami tava Sengupta, Joseph P. Mastin, Kyung H. Chang, David A. Williams, and Jose A. Cancelas. "Rac2 GTPase Activation Is Necessary for Development of p190-BCR-ABL-Induced B-Cell Acute Lymphoblastic Leukemia." Blood 112, no. 11 (November 16, 2008): 3790. http://dx.doi.org/10.1182/blood.v112.11.3790.3790.
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Abstract The fusion gene BCR-ABL, resulting from t(9;22) reciprocal chromosomal translocations, encodes a constitutively active tyrosine kinase. Two different isoforms of BCR-ABL, p190 and p210, are associated to two completely different diseases. In the tyrosine kinase inhibitor (TKI) era, while p210-BCR-ABL-induced CML is highly responsive to TKI, p190-BCR-ABL still induces a poor prognosis B-cell acute lymphoblastic leukemia (B-ALL). The only difference between these two forms of BCR-ABL is the existence of a DH/Cdc24/PH domain in p210-BCR-ABL, which acts as a guanine nucleotide exchange factor (GEF) able to activate Rho GTPases. Rac is a subfamily of Rho GTPases with regulatory activity on hematopoietic stem cell and progenitor (HSC/P) functions. We have previously shown that Rac2 and further the combination of Rac1 and Rac2 mediate downstream signals in p210 BCR-ABL-induced myeloproliferation (Thomas EK, et al., Cancer Cell, 2007). Interestingly, despite the absence of a GEF domain in p190-BCR-ABL, Rac is activated, suggesting the activation of other GEF(s). Here we have analyzed whether Vav and Rac family members are involved in p190-BCR-ABL-induced B-ALL. We have used a combination of in vitro (Ba/F3 pro-B cells transduced with p190 or p210 BCR-ABL) and in vivo (murine transduction-transplantation model of p190 BCR-ABL-induced B-ALL) approaches. In Ba/F3 cells, both p190 BCR-ABL and p210 BCR-ABL activated Rac and the Rac effector p21 activated kinase (PAK), and their proliferation and survival appeared severely decreased in response to the Rac activation inhibitor NSC23766. Stat3, Stat5 and Jnk, but not ERK, p38 or NF-kB, were constitutively hyperactivated in p190 BCRABL-expressing Ba/F3 cells and primary murine B-ALL cells. Intracellular flow cytometry analysis demonstrated that Stat5 was specifically activated in the pro/pre-B leukemic cell population, compared to normal B cells. In the murine model of B-ALL, loss of Rac2, but not Rac3, prolonged survival and impaired leukemia development. Like in Ba/F3 cells, primary B-CFU and outgrowth in Witte-Whitlock assays of leukemic primary cells from mice was severely decreased by the addition of NSC23766 to the culture. Although Vav was activated by both p190- and p210-BCR-ABL, since NSC23766 does not block the activation by Vav1, we hypothesized that other GEFs were involved. Indeed, the loss of Vav1 or even combined loss of Vav1 and Vav2 did not impair BCR-ABL-mediated lymphoid leukemogenesis in vivo. Vav3, another member in the Vav family which uses a different mechanism of activation of Rac GTPases was a likely candidate. In fact, loss of Vav3 alone was able to significantly prolong the survival and attenuate development of p190 BCR-ABL-driven B-ALL. In conclusion, the results of this study indicate that Rac activation is necessary for the development of B-ALL induced by p190-BCR-ABL in vitro and in vivo, and validate a new signaling pathway as a therapeutic target for BCR-ABL-induced B-ALL.
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Uchida, Naoya, Hideki Hanawa, Koiti Inokuchi, Kazuo Dan, and Takashi Shimada. "Leukemogenesis of the b2a2 Type p210 BCR/ABL in a Bone Marrow Transplantation Mouse Model Using a Lentivirus Vector." Blood 106, no. 11 (November 16, 2005): 2875. http://dx.doi.org/10.1182/blood.v106.11.2875.2875.
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Abstract [INTRODUCTION] BCR/ABL induces the chronic phase of chronic myeloid leukemia (CML). The three main principal forms (p190, p210 and p230 BCR/ABL) of the BCR/ABL gene are found in distinct forms of leukemia and have shown to be different leukemogenic activities in mice. The BCR breakpoint locations of p210 BCR/ABL falls either between the exons b2 and b3 (b2a2) or between the exons b3 and b4 (b3a2). Though the leukemogenic activity of the b3a2 type gene had been shown in mice, the leukemogenesis of the b2a2 type has not been tested yet. [PURPOSE] The purpose of this study is to evaluate the leukemogenesis of the b2a2 p210 BCR/ABL gene, for the first time, in a bone marrow (BM) transduction and transplantation (BMTT) mouse model, and to compare the leukemogenesis of the b2a2 and the b3a2 p210 BCR/ABL. [METHODS] The ecotropic envelope-pseudotyped self-inactivating lentivirus vectors carrying the b2a2 or the b3a2 p210 BCR/ABL cDNA driven by the murine stem cell virus (MSCV) U3 promoter was constructed. The BM cells were harvested from Balb/c mice without 5-fluorouracil pretreatment. The lineage-marker-negative (Lin−) BM cells were prepared by negative selections using a lineage antibodies cocktail (anti-mouse CD3e, CD11b, B220, Gr-1 and TER-119). The Lin− BM cells were prestimulated by cytokines (mIL3, mSCF, hTPO, hIL6) and then these were transduced for 12 hrs with the lentivirus vectors at a MOI (multiplicity of infection) 3 in the presence of the same cytokines on a RetroNectin (TAKARA)-coated 6-well plate. The initial transduction rates of the b2a2 and the b3a2 p210 BCR/ABL vectors were 0.38% and 0.16%, respectively, determined by real time PCR. The transduced BM cells (1 x 105) were transplanted by injection into the lateral tail vein of the lethally irradiated Balb/c mice. [RESULT] In our BMTT mouse model, both the b2a2 and the b3a2 p210 BCR/ABL genes developed a fatal CML-like myeloproliferative disease in 4 weeks after transplantation. The frequency of leukemia development with the b2a2 was 75% (6/8), while that with the b3a2 was 30% (3/10). The difference may depend on the initial transduction rate. The disease was characterized by expansion of mature myeloid cells in peripheral blood. The averaged copy-number of the vector in peripheral blood cells in leukemic mice (> 0.1 copy/diploid) was higher than that in leukemia-free mice (< 0.03 copy/diploid). There was no significant difference between the phenotypes of the b2a2 and the b3a2 p210 BCR/ABL genes, in white blood cell count (41.2±15.2 vs. 38.5±7.00 x103/mm3, p=.907), hemoglobin concentration (13.5±0.642 vs. 13.9±1.13 g/dl, p=.779) and platelet count (646±74.0 vs. 460±60.4 x103/mm3, p=.152). The survival time of each CML-like mice was also similar (57±6 vs. 62±15 days, p=.534). [DISCUSSION] Our BMTT model mice using lentivirus vectors survived longer (Mean: 58±5, Median: 48±2 days) than the previous BMTT model mice using retroviral vectors. Therefore our BMTT mouse model using the lentivirus vectors is more likely to mimic a human CML than using retroviral vectors. Using this model, the fatal CML-like myeloproliferative disease was developed with the b2a2 p210 BCR/ABL gene as well as the b3a2 gene. These data suggest the b2a2 p210 BCR/ABL had similar leukemogenic activities to the b3a2.
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Chen, Ying, Nicole Froehlich, and Stefan K. Bohlander. "Towards the In Vivo Identificaton of Leukemogenic Fusion Proteins." Blood 104, no. 11 (November 16, 2004): 2970. http://dx.doi.org/10.1182/blood.v104.11.2970.2970.
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Abstract Currently there are no methods available to identifiy leukemogenic fusion proteins in vivo. All available methods, like Southern blotting, PCR, FISH or Western blotting, require the destruction of the cells that are assayed. A method for the in vivo detection of leukemogenic fusion proteins would be highly desirable because it would open up new approaches to study leukemia and might lead to novel treatment strategies. We have developed a strategy for the in vivo detection of the BCR/ABL fusion protein. BCR/ABL is found in virtually all cases chronic myeloid leukemia (CML) and a large proportion of acute lymphoblastic leukemia (ALL). Animal model have shown that the BCR/ABL fusion protein is required for the induction and maintenance of leukemia. The fact that BCR/ABL fusion protein is crucial for the development of leukemia makes this fusion protein an attractive target for therapy development. Our BCR/ABL detection strategy is based on protein-protein interactions and a proof of principle for the strategy was implemented in the yeast system. Two detection proteins are expressed in the cells: 1) protein A, a Gal4-DNA binding domain/BCR interacting protein fusion protein and 2) protein B, a Gal4-activation domain/ABL interacting protein fusion protein. Only when BCR/ABL is present in the cell, do protein A, protein B, and BCR/ABL form a trimeric complex which activates the transcription of reporter genes under the control of Gal4-upstream activating sequence (UAS). Yeast cells (strain CG1945) transformed with a protein A expressing plasmid (pGBT9-BCR-interactor), a protein B expressing plasmid (pGAD424-ABL1-interactor), and a BCR/ABL expressing plasmid (pES1-BCR/ABL) showed expression of the reporter genes HIS3 and LACZ. The expression of the HIS3 reporter gene was assayed by growth of the yeast cells on medium lacking histidine. The expression of the LACZ gene was verified by a beta-galactosidase filter assay. Yeast cells that were transformed with the pES1 plasmid without the BCR-ABL coding region did not show activation of the reporter genes. Several other negative controls were also negative. Thus the method was able to clearly distinguish between BCR/ABL expressing cells and cells did not express BCR/ABL. We are presently adapting this system for use in mammalian cells. The flexibility of our strategy allows us to freely choose the reporter or effector genes. Therapeutically more useful effector genes are suicide genes, which encode pro-drug converting enzymes (e.g. HSV thymidine kinase), or markers that can easily be assayed (e.g. green fluorescent protein).
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Peng, Cong, Julia Brain, Yiguo Hu, Linghong Kong, David Grayzel, Roger Pak, Margaret Read, and Shaoguang Li. "IPI-504, a Novel, Orally Active HSP90 Inhibitor, Prolongs Survival of Mice with BCR-ABL T315I CML and B-ALL." Blood 108, no. 11 (November 1, 2006): 2183. http://dx.doi.org/10.1182/blood.v108.11.2183.2183.
Full textAbstract:
Abstract Development of mutations within the kinase domain is a major drug-resistance mechanism for tyrosine kinase inhibitors (TKIs) in cancer therapy. In CML (chronic myeloid leukemia), a disease driven by the constitutively active BCR-ABL oncoprotein, no available TKIs have been effective in treating patients with the BCR-ABL T315I mutation. Heat shock protein 90 (Hsp90) is a highly conserved, constitutively expressed molecular chaperone that facilitates folding of client proteins like BCR-ABL, and affects the stability of these proteins. Several labs have shown that Hsp90 inhibition in vitro results in the degradation of BCR-ABL T315I and induces potent killing of these cell lines. However, these results have not been demonstrated in animal models for BCR-ABL-induced CML and B-ALL (B-cell acute lymphoblastic leukemia, a disease that does not respond well to TKIs including imatinib and dasatinib). Thus, IPI-504, an orally administered Hsp90 inhibitor, was evaluated in murine models of CML and B-ALL. Treatment of mice with wild type (WT)- or T315I BCR-ABL-induced CML with IPI-504 resulted in BCR-ABL protein degradation and a decrease in circulating BCR-ABL positive cells. In response to treatment with vehicle the median survival time of WT and T315I CML mice is approximately 20 days. While the T315I CML mice were resistant to imatinib with a median survival of 21 days, IPI-504 (50 and 100 mg/kg, PO TIW) demonstrated dose-dependent prolonged survival of these mice by 30 and 70 days, respectively (p<0.001 for both doses). Both imatinib and IPI-504 similarly prolonged survival of mice with BCR-ABL-WT-induced CML. In the T315I CML mice prolonged survival of the IPI-504 treated cohort was associated with decreased peripheral blood BCR-ABL positive leukemia cells during treatment, less splenomegaly and improved pulmonary histopathlogy at necropsy. In CML mice receiving mixed BCR-ABL-WT- or T315I-transduced donor bone marrow cells, Hsp90 inhibition more potently suppressed T315I-expressing leukemia clones relative to the WT clones, consistent with in vitro studies where T315I BCR-ABL was more sensitive to IPI-504 induced degradation in cell lines than WT BCR-ABL. Combination treatment with IPI-504 and imatinib was more effective than either treatment alone in prolonging survival of mice bearing both WT and T315I leukemic cells. IPI-504 also significantly prolonged survival of B-ALL mice bearing the T315I mutation (p<0.001). These results provide a rationale for use of an Hsp90 inhibitor as a novel approach to overcoming resistance to TKIs as well as the potential for first line combination treatment in CML patients. The potential for IPI-504 to eliminate mutant kinases via Hsp90 inhibition provides a new therapeutic strategy for treating BCR-ABL-induced CML, ALL as well as other cancers resistant to treatment with TKIs.
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Gabert, Jean A., Christophe Picard, Sandrine Hayette, Christelle Bilhou-Nabera, Jean-Michel Cayuela, Elizabeth Macintyre, Nicole Frenoy, et al. "Prospective Multicentric Molecular Study for Poor Prognosis Fusion Transcripts at Diagnosis in Adult ALL Patients - The LALA94 Experience." Blood 106, no. 11 (November 16, 2005): 4476. http://dx.doi.org/10.1182/blood.v106.11.4476.4476.
Full textAbstract:
Abstract From 1994 to 2000, 984 adults aged from 15 to 55 years with newly diagnosed Acute Lymphoblastic Leukemia (ALL) were eligible for randomization in the multicentric LALA-94 clinical protocol. The t(9;22), t(1;19) and t(4;11) translocations corresponding to BCR-ABL, E2A-PBX1 and MLL-AF4 fusion gene transcripts respectively, were considered as independent poor prognostic factors. Standardized RT-PCR analysis of these fusion gene transcripts were performed by 17 laboratories in order to provide data before the second randomization (J35) on 787 patients. In this multicentric study, validated data were available for therapeutic stratification for 91% of these analysed patients. No false positive RT-PCR was reported. Secondarily to retrospective BCR-ABL FISH, few false BCR-ABL negative RT-PCR were identified, leading to the design of new BIOMED-1 primers for b3-a3 junctions detection. Moreover, the LALA-94 study allowed to define new guidelines for molecular analysis at diagnosis. Like in other studies, the BCR-ABL transcript was found to be the most frequent molecular abnormality in B-ALL (24%) whereas MLL-AF4 and E2A-PBX1 were detected in 5% and 3.5% of B-ALL, respectively. Epidemiological and clinical data of MLL-AF4 and E2A-PBX1 were concordant with previous publications. Interestingly, because of the large number of reviewed patients, the different BCR-ABL subtypes (M-BCR and m-BCR) were statistically characterized by few clinical data. M-BCR subgroups had a higher age than m-BCR (p= 0.016) and occurs especially during the second semester (p= 0.034). Moreover, the comparison of clinical data at diagnosis of M-BCR variants showed that median age of b3a2 was statistically younger than b2a2 (p= 0.04) and that b3a2 occurs more frequently in man (p= 0.02). For the first time, these data suggest that these BCR-ABL breakpoints: m-BCR and M-BCR and also b2a2 and b2a3, are secondary to different physio-oncologic mechanisms even if therapeutic regimens including the same targeted therapy (tyrosine kinase inhibitor) for all BCR-ABL variants is the rule today.
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Singh, Aditya, and Prateek Bhatia. "Effective Downregulation of BCR-ABL Tumorigenicity by RNA Targeted CRISPR-Cas13a." Current Gene Therapy 21, no. 3 (June 8, 2021): 270–77. http://dx.doi.org/10.2174/1566523221666210217155233.
Full textAbstract:
Aim: To induce BCR-ABL gene silencing using CRISPR Cas13a. Background: CML is a clonal myeloproliferative disorder of pluripotent stem cells driven by a reciprocal translocation between chromosomes 9 and 22 forming a BCR-ABL fusion gene. Tyrosine- kinase inhibitor drugs like imatinib are the mainstay of treatment and cases resistant to these drugs have a poor prognosis in the absence of a compatible stem-cell donor. However with rapid advancements in gene-editing technologies most studies are now focusing on developing a translational model targeting single-gene disorders with a prospective permanent cure. Objective: To explore the potential application of the RNA targeting CRISPR-Cas13a system for effective knockdown of BCR-ABL fusion transcript in a CML cell line K562. Methods: CRISPR Cas13a crRNA was designed specific to the chiCRISPR Cas13a crRNA was designed specific to the chimeric BCR-ABL gene and the system was transfected as a two-plasmid system into a CML cell line K562. The effects were enumerated by evaluating the expression levels of downstream genes dependent on the expression of the BCR-ABL gene. Also next-generation sequencing was used to ascertain the effects of CRISPR on the gene.meric BCR-ABL gene and the system was transfected as a two-plasmid system into a CML cell line, K562. The effects were enumerated by evaluating the expression levels of downstream genes dependent on the expression of the BCR-ABL gene. Also, next-generation sequencing was used to ascertain the effects of CRISPR on the gene. Results: The CRISPR system was successfully able to lower the expression of downstream genes [pCRKL and pCRK] dependent on the activated BCR-ABL kinase signal by up-to 4.3 folds. The viability of the CRISPR-treated cells was also significantly lowered by 373.83-fold [p-value= 0.000891196]. The time-dependent kinetics also highlighted the significant in-vitro suppressive activity to last up to 8 weeks [p-value: 0.025]. As per the cDNA sequencing data from the Oxford MinION next-generation sequencer the CRISPR treated cells show 62.37% suspected cleaved reads. Conclusion: These preliminary results highlight an excellent potential application of RNA targeting CRISPRs in Haematological neoplasms like CML and should pave the way for further research in this direction.
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Minami, Yosuke, Scott A. Stuart, Tomokatsu Ikawa, Akihiro Abe, Tomoki Naoe, Catriona H. M. Jamieson, and Jean Y. J. Wang. "Transformation of E2A-Deficient Pluripotent Progenitors by BCR-ABL Generates Imatinib-Resistant Leukemic Stem Cells." Blood 112, no. 11 (November 16, 2008): 1342. http://dx.doi.org/10.1182/blood.v112.11.1342.1342.
Full textAbstract:
Abstract Chronic myeloid leukemia (CML) is effectively treated with imatinib (imatinib mesylate, IM), a small molecule inhibitor of the BCR-ABL tyrosine kinase that is expressed in the entire hematopoietic compartment including stem cells (HSC) and progenitors. However, it is still unclear whether IM-therapy is able to eradicate BCR-ABL-positive HSC and progenitors. By transforming murine E2A-deficient pluripotent hematopoietic cells (Ikawa, et al., Immunity 04) with p210BCR-ABL, we determined that as few as 50 granulocyte macrophage progenitors (GMP)-like cells were sufficient to induce a transplantable CML-like disease in congenic mice, and that the leukemogenic GMP displayed higher levels of β-catenin activity than either the non-transformed GMP or the transformed nonGMP (most of which were myeloid differentiated cells), both in culture and in transplanted mouse bone marrow (Proc Natl Acad Sci USA 08, in press). The initiation of transformation required BCR-ABL kinase activity; however, whereas transformed nonGMP were sensitive to IM-treatment, expansion and survival of the leukemogenic progenitors in ex vivo-culture or in mice were not completely inhibited by IM-treatment. The drug resistance did not correlate with higher levels of BCR-ABL, mutations at ABL-kinase domain, induction of quiescence, stromal support or drug efflux. Additionally, the differential responses between the transformed progenitors and differentiated cells to IM-treatment were not affected by the restoration of E2A-function. These results imply that leukemic progenitors possess innate resistance to IM and that eradication of these cells with other drugs is required to cure CML. We are also investigating BCR-ABL-positive residual disease in HSC and progenitors of chronic phase CML patients on IM-therapy.
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Gleixner, Karoline V., Harald Herrmann, Barbara Peter, Katharina Blatt, Karina Schuch, Winfried F. Pickl, Christian Sillaber, and Peter Valent. "The Multi-Kinase/ABL Inhibitor R763/AS703569 Induces DNA Endoreduplication and Apoptosis In Imatinib-Resistant CML Cells and Synergizes with Nilotinib, Dasatinib, and the Plk-1 Inhibitor BI 2536, In Producing Growth Inhibition." Blood 116, no. 21 (November 19, 2010): 3394. http://dx.doi.org/10.1182/blood.v116.21.3394.3394.
Full textAbstract:
Abstract Abstract 3394 Resistance to imatinib is a major clinical problem and challenge in advanced chronic myeloid leukemia (CML). In most patients, drug-resistant mutants of BCR/ABL are detectable. Although most of these mutants still are responsive to second generation BCR/ABL kinase inhibitors (KI) such as nilotinib or dasatinib, drug responses are often short-lived. The BCR/ABL mutant T315I confers resistance against all available BCR/ABL KI, including nilotinib and dasatinib. More recent data suggest that several Aurora kinase (AuK) inhibitors block the kinase activity of BCR/ABL T315I. We have examined the growth-inhibitory effects of the AuK/ABL inhibitor R763/AS703569 (Merck-Serono, Darmstadt, Germany) on primary CML cells (chronic phase, n=12), the CML cell line K562, and Ba/F3 cells transfected with various imatinib-resistant mutants of BCR/ABL. As assessed by 3H-thymidine-uptake, R763/AS703569 was found to inhibit proliferation in imatinib-sensitive and imatinib-resistant primary CML cells in all donors tested, in imatinib-resistant and imatinib-responsive K562 cells, and in Ba/F3 cells harbouring various mutants of BCR/ABL (E255K, Y253F, H396P, T315I). The effects of R763/AS703569 on BCR/ABL-transformed cells were dose-dependent with IC50 values ranging between 0.001–0.1 μ M in K562 cells, <0.001-1 μ M in primary CML cells, and 0.001–0.1 μ M in BCR/ABL+ Ba/F3 cells. In all three patients in whom an imatinib-resistant BCR/ABL mutation was detected (one with V379I, one with F359V, and one with T315), R763/AS703569 was found to inhibit growth of primary CML cells at pharmacologic concentrations (IC50: 0.5, 0.005, and 0.05 μ M, respectively). As assessed by flow cytometry, the growth-inhibitory effects of R763/AS703569 were accompanied by DNA endoreduplication and consecutive apoptosis. Western blot experiments using anti-pCrkL antibody were performed and confirmed that R763/AS703569 blocks BCR/ABL activity at 1 μ M in K562 cells and BCR/ABL-transformed Ba/F3 cells. In addition, R763/AS703569 was found to block Aurora kinase A and Lyn phosphorylation in CML cells. By contrast, no effect of R763/AS703569 on phosphorylation or expression of Polo-like kinase-1 (Plk-1) was seen. In a next step, we explored the effects of drug combinations on growth of K562 cells and BCR/ABL-transformed Ba/F3 cells. In these experiments, R763/AS703569 was found to synergize with the ABL/multi-kinase inhibitors nilotinib (Novartis, Basel Switzerland) and dasatinib (Bristol-Myers Squibb, Princeton, NJ) in producing growth inhibition in CML cells (figure). Moreover, R763/AS703569 and the Plk-1 inhibitor BI 2536 (Boehringer Ingelheim, Vienna, Austria) were found to produce synergistic growth-inhibitory effects on CML cells in all samples tested. Synergistic effects of the KI applied were also seen in Ba/F3 cells exhibiting BCR/ABL T315I. In summary, our data show that the novel AuK/ABL inhibitor R763/AS703569 produces growth inhibition and apoptosis in BCR/ABL-transformed cells including those harbouring BCR/ABL T315I or other imatinib-resistant BCR/ABL mutants. Moreover, our data show that R763/AS703569 synergize with other multi-kinase/ABL inhibitors as well as with the Plk-1 inhibitor BI 2536 in producing growth inhibition in imatinib-resistant CML cells. Whether R763/AS703569, applied alone or in combination with other targeted drugs, can produces anti-leukemic effects in patients with advanced CML remains to be determined in clinical trials. Synergistic growth-inhibitory effects of R763/AS703569 and nilotinib in BCR/ABL T315I+ Ba/F3 cells (left), and R763/AS703569 and dasatinib in K562 cells (right). Disclosures: Valent: Novartis: Research Funding; Bristol-Myers Squibb: Research Funding; Merck-Serono: Research Funding.