Journal articles: 'Leukemia initiation and transformation'

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He, Jin, Anh Tram Nguyen, and Yi Zhang. "KDM2b/JHDM1b, an H3K36me2-specific demethylase, is required for initiation and maintenance of acute myeloid leukemia." Blood 117, no. 14 (April 7, 2011): 3869–80. http://dx.doi.org/10.1182/blood-2010-10-312736.

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Abstract The histone H3 lysine 36 dimethyl–specific demethylase KDM2b/JHDM1b, which is highly expressed in various human leukemias, was previously found to be important in regulating cell proliferation and cellular senescence. However, its functions in leukemia development and maintenance are unclear. Here, we demonstrate that ectopic expression of Kdm2b/Jhdm1b is sufficient to transform hematopoietic progenitors. Conversely, depletion of Kdm2b/Jhdm1b in hematopoietic progenitors significantly impairs Hoxa9/Meis1-induced leukemic transformation. In leukemic stem cells, knockdown of Kdm2b/Jhdm1b impairs their self-renewing capability in vitro and in vivo. The functions of Kdm2b/Jhdm1b are mediated by its silencing of p15Ink4b expression through active demethylation of histone H3 lysine 36 dimethyl. Thus, our study suggests that Kdm2b/Jhdm1b functions as an oncogene and plays a critical role in leukemia development and maintenance.

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Hurtz, Christian, Katerina Hatzi, Leandro Cerchietti, Melanie Braig, Eugene Park, Yong-mi Kim, Sebastian Herzog, et al. "BCL6-mediated repression of p53 is critical for leukemia stem cell survival in chronic myeloid leukemia." Journal of Experimental Medicine 208, no. 11 (September 12, 2011): 2163–74. http://dx.doi.org/10.1084/jem.20110304.

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Chronic myeloid leukemia (CML) is induced by the oncogenic BCR-ABL1 tyrosine kinase and can be effectively treated for many years with tyrosine kinase inhibitors (TKIs). However, unless CML patients receive life-long TKI treatment, leukemia will eventually recur; this is attributed to the failure of TKI treatment to eradicate leukemia-initiating cells (LICs). Recent work demonstrated that FoxO factors are critical for maintenance of CML-initiating cells; however, the mechanism of FoxO-dependent leukemia initiation remained elusive. Here, we identified the BCL6 protooncogene as a critical effector downstream of FoxO in self-renewal signaling of CML-initiating cells. BCL6 represses Arf and p53 in CML cells and is required for colony formation and initiation of leukemia. Importantly, peptide inhibition of BCL6 in human CML cells compromises colony formation and leukemia initiation in transplant recipients and selectively eradicates CD34+ CD38− LICs in patient-derived CML samples. These findings suggest that pharmacological inhibition of BCL6 may represent a novel strategy to eradicate LICs in CML. Clinical validation of this concept could limit the duration of TKI treatment in CML patients, which is currently life-long, and substantially decrease the risk of blast crisis transformation.

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Mendoza-Castrejon, Jonny, Emily B. Casey, Riddhi M. Patel, Elisabeth Denby, and Jeffrey Magee. "Fetal MLL-ENL Initiation Induces Developmental Stage-Specific Programs That Restrict Transformation and Depend on MLL3." Blood 142, Supplement 1 (November 28, 2023): 950. http://dx.doi.org/10.1182/blood-2023-178865.

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Infant leukemias present as either acute lymphoblastic leukemia (ALL) or acute myeloid leukemia (AML) and are commonly driven by KMT2A/ MLL rearrangements (MLLr). MLLr account for ~70% of infant ALL and ~50% of infant AML. Most of the MLLr that drive infant leukemias occur before birth and require very few cooperating mutations. These observations suggest that MLLr transform fetal/neonatal progenitors with great efficiency. However, this model introduces important paradoxes. First, the incidence of MLLr infant leukemia is ~20-fold lower than the incidence of adult MLLr leukemias. Second, fetal leukemias are exceedingly rare despite the fact that MLLr can arise before birth and require a low secondary mutation threshold for transformation. Third, even in mouse models that demonstrate efficient leukemogenesis, the fraction of MLLr progenitors that actually give rise to AML is quite small. Fourth, in our prior studies, we developed a doxycycline (DOX) inducible MLL-ENL mouse model (Tet-ME) to activate MLL-ENL and found that transformation efficiency is higher when MLL-ENL is induced shortly after birth rather than before birth (PMID: 31405949). These observations raise the question of whether fetal/neonatal progenitors carry intrinsic protection against transformation. To test whether fetal identity conveys protection against MLLr leukemia initiation, we activated MLL-ENL at embryonic day 10.5 (E10.5) or postnatal day 14 (P14), by removing DOX. We then assessed the leukemogenic potential of postnatal progenitors in transplantation assays. Strikingly, progenitors that expressed MLL-ENL from E10.5 onward could engraft efficiently but did not give rise to AML in recipient mice. In contrast, progenitors that expressed MLL-ENL induced from P14 onward gave rise to rapid, fully penetrant AML in recipient mice (Figure 1A). Our data show that MLL-ENL induces heritable epigenetic changes in fetal progenitors, that limit transformation potential after transplantation. To assess the unique response of fetal progenitors to MLL-ENL activation, we used our Tet-ME model and induced MLL-ENL before or after birth. We isolated P28 progenitors, repeated transplantation/survival assays and performed Cellular Indexing of Transcriptomes and Epitopes (CITE-seq), in parallel, to test whether MLL-ENL differentially reprograms pre- and post-natal progenitors (i.e., do P28 progenitors have distinct expression programs depending on when MLL-ENL is induced?). Hematopoietic progenitors had similar differentiation trajectories by pseudotime analysis, but distinct gene expression profiles after fetal and postnatal MLL-ENL induction. Some known MLL-ENL targets, including Hoxa9 and Pim1, were maintained at similar levels following fetal and postnatal induction. Other targets, including Meis1, Pbx1 and Sox4, were only expressed in the HSC/MPP cluster following postnatal induction. Metabolic and differentiation states also differed. As in the initial survival assays, MLL-ENL expressing progenitors only gave rise to AML following postnatal induction. We next sought to identify epigenetic regulators that enforce non-malignant cell fates following fetal MLL-ENL induction. We focused our screen on epigenetic factors with known tumor suppressor roles and identified the SET methyltransferase MLL3 as a candidate effector of fetal protection. Specifically, heterozygous Kmt2c deletions greatly accelerated AML initiation after fetal MLL-ENL induction, but not postnatal induction (Figure 1B). Notably, the MLL3/4 COMPASS-like complex was recently shown to antagonize the KMT2A-MENIN interaction and promote differentiation in AML cells. Our data reinforce the notion that there are ontological windows that permit or repress leukemic transformation. Quite surprisingly, fetal progenitors appear to be more resistant to transformation than neonatal progenitors, potentially accounting for the paucity of fetal MLLr leukemias. Infant leukemias that do occur must bypass heritable protective mechanisms, either via mutations or epigenetic reprogramming.

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Ugale, Amol Sanjay, Gudmundur Logi Norddahl, Martin Wahlestedt, Petter Säwén, Pekka Jaako, Cornelis J. H. Pronk, Shamit Soneji, Jorg Cammenga, and David Bryder. "Hematopoietic Stem Cells Are Intrinsically Protected Against MLL-ENL Mediated Transformation." Blood 124, no. 21 (December 6, 2014): 839. http://dx.doi.org/10.1182/blood.v124.21.839.839.

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Abstract Studies on the developmental pathways of hematopoietic stem cells (HSCs) have led to roadmaps of differentiation and resulted in key information concerning lineage relationships and restriction points in the blood system. This knowledge is also central to understand the etiology of acute myeloid leukemia (AML), where recent work has proposed that the heterogeneity and aggressiveness of AML can associate with the developmental stage of transformation. Balanced chromosomal translocations that result in fusion proteins with aberrant transcriptional regulatory activities are frequent initiating events in acute myeloid leukemia, and a prototype family of such chimeric transcription factors is represented by fusions involving the mixed lineage leukemia-1 (MLL1) gene. Previous work using mouse models have suggested that at some stage of normal differentiation there is a loss of competence to induce AML. However discrepancies exists between these mouse models concerning the target cells of MLL fusion genes. While it is clear that cells can lose competence for leukemic transformation as part of their normal differentiation, the question remains whether the most primitive HSCs are always imbued with leukemogenic competency as part of their normal biology. To address this, we developed a Doxycycline inducible transgenic mouse model of the human chimeric transcription factor Mixed Lineage Leukemia-Eleven Nineteen Leukemia (MLL-ENL). Prospective isolations of candidate leukemia-initiating cells followed by adoptive transfers allowed us to detail leukemia-initiation and competence throughout the hematopoietic hierarchy. We show that AML can origin from multiple HPC subsets with intrinsic granulocytic/monocytic potential. Closely related myeloid progenitors displayed distinct leukemic- and functional capacity in response to physiological levels of MLL-ENL, highlighting the importance of a careful prospective isolation of progenitor populations. AML could also develop efficiently from common lymphoid progenitors, supporting a latent myeloid potential of these cells. By contrast, early commitment to the megakaryocytic/erythroid lineages was incompatible with leukemic development. By contrast, disease failed to arise from the most primitive progenitor subsets, including HSCs. Investigations of the immediate transcriptional responses to MLL-ENL showed evidence for a block in differentiation in both myeloid progenitors and HSCs, while MLL-ENL restricted cell cycle progression uniquely in HSCs. Our study highlights how an oncogene can exert unique functions depending on the developmental position of its cellular targets and demonstrate the existence of a mechanism, operational at the level of immature HSCs/progenitors, which act to prevent leukemic development. Figure 1 Graphical abstract Figure 1. Graphical abstract Disclosures No relevant conflicts of interest to declare.

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Okeyo-Owuor, Theresa, Yanan Li, Riddhi M. Patel, Wei Yang, Emily B. Casey, Andrew S. Cluster, Shaina N. Porter, David Bryder, and Jeffrey A. Magee. "The efficiency of murine MLL-ENL–driven leukemia initiation changes with age and peaks during neonatal development." Blood Advances 3, no. 15 (August 12, 2019): 2388–99. http://dx.doi.org/10.1182/bloodadvances.2019000554.

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Abstract MLL rearrangements are translocation mutations that cause both acute lymphoblastic leukemia and acute myeloid leukemia (AML). These translocations can occur as sole clonal driver mutations in infant leukemias, suggesting that fetal or neonatal hematopoietic progenitors may be exquisitely sensitive to transformation by MLL fusion proteins. To test this possibility, we used transgenic mice to induce one translocation product, MLL-ENL, during fetal, neonatal, juvenile and adult stages of life. When MLL-ENL was induced in fetal or neonatal mice, almost all died of AML. In contrast, when MLL-ENL was induced in adult mice, most survived for >1 year despite sustained transgene expression. AML initiation was most efficient when MLL-ENL was induced in neonates, and even transient suppression of MLL-ENL in neonates could prevent AML in most mice. MLL-ENL target genes were induced more efficiently in neonatal progenitors than in adult progenitors, consistent with the distinct AML initiation efficiencies. Interestingly, transplantation stress mitigated the developmental barrier to leukemogenesis. Since fetal/neonatal progenitors were highly competent to initiate MLL-ENL–driven AML, we tested whether Lin28b, a fetal master regulator, could accelerate leukemogenesis. Surprisingly, Lin28b suppressed AML initiation rather than accelerating it. This may explain why MLL rearrangements often occur before birth in human infant leukemia patients, but transformation usually does not occur until after birth, when Lin28b levels decline. Our findings show that the efficiency of MLL-ENL–driven AML initiation changes through the course of pre- and postnatal development, and developmental programs can be manipulated to impede transformation.

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Bunworasate, Udomsak, Hilal Arnouk, Hans Minderman, Kieran L. O'Loughlin, Sheila N. J. Sait, Maurice Barcos, Carleton C. Stewart, and Maria R. Baer. "Erythropoietin-dependent transformation of myelodysplastic syndrome to acute monoblastic leukemia." Blood 98, no. 12 (December 1, 2001): 3492–94. http://dx.doi.org/10.1182/blood.v98.12.3492.

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Abstract Acute monoblastic leukemia (acute myeloid leukemia [AML], French-American-British type M5a) with leukemia cutis developed in a patient 6 weeks after the initiation of erythropoietin (EPO) therapy for refractory anemia with ringed sideroblasts. AML disappeared from both marrow and skin after the discontinuation of EPO. Multiparameter flow cytometric analysis of bone marrow cells demonstrated coexpression of the EPO receptor with CD45 and CD13 on the surface of blasts. The incubation of marrow cells with EPO, compared to without, resulted in 1.3- and 1.6-fold increases, respectively, in tritiated thymidine incorporation and bromodeoxyuridine incorporation into CD13+ cells. Clinical and laboratory findings were consistent with the EPO-dependent transformation of myelodysplastic syndrome (MDS) to AML. It is concluded that leukemic transformation in patients with MDS treated with EPO may be EPO-dependent and that management should consist of the discontinuation of EPO followed by observation, if clinically feasible.

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Tsuruta-Kishino, Takako, Keisuke Kataoka, Hiroshi Kobayashi, Junji Koya, Kensuke Narukawa, Tomohiko Sato, and Mineo Kurokawa. "Loss Of p53 Induces Leukemic Transformation In a Murine Model Of JAK2V617F-Induced Polycythemia Vera." Blood 122, no. 21 (November 15, 2013): 269. http://dx.doi.org/10.1182/blood.v122.21.269.269.

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Abstract Myeloproliferative neoplasms (MPN) have an inherent tendency toward leukemic transformation, but its mechanisms remain largely unknown. Recently, TP53 mutation is reported to be frequently found in cases with post-MPN leukemia. Here, to address the contribution of p53 loss to leukemic transformation from MPN in vivo, we retrovirally transduced c-kit+ bone marrow (BM) cells from p53 knockout (p53-/-) and littermate mice (p53+/+) with either wild-type Jak2 (Jak2WT) or Jak2V617F respectively, and transplanted them into lethally irradiated mice. At 3 weeks after transplantation, both recipients of Jak2V617F/p53-/- and Jak2V617F/p53+/+ cells developed a polycythemia vera-like disease characterized by high WBC count and elevated hemoglobin (Hb) level. Jak2V617F/p53+/+ mice survived and continued to have elevated Hb level, whereas 5 weeks after transplantation, Jak2V617F/p53-/- recipients developed cachexia, and their Hb level declined. Eventually, these mice developed fatal leukemia with a median survival of 46.5 days after transplantation, suggesting loss of p53 cooperates with Jak2V617F mutation to promote leukemic transformation from MPN. To characterize these leukemias, we analyzed leukemic tissues from moribund Jak2V617F/p53-/- mice. Peripheral blood smears and BM specimen from Jak2V617F/p53-/- recipients showed a marked increase of erythroid precursors with dysplastic features, leading to suppression of normal hematopoiesis. Notably, Jak2V617F/p53-/- mice displayed marked hepatosplenomegaly and extensive pulmonary hemorrhage. Consistent with the histopathologic findings, Jak2V617F/p53-/- animals exhibited a remarkable accumulation of erythroid precursors (CD71+), and especially more immature progenitors (Ter119-/CD71+) in the BM and spleen, compared with Jak2V617F/p53+/+ animals. These data suggest Jak2V617F/p53-/- recipients developed infiltrative disease with accumulation of immature erythroid cells, fulfilling the Bethesda Criteria of erythroleukemia in mice. To assess the transplantability of Jak2V617F/p53-/- leukemia, we injected unfractionated BM cells from Jak2V617F/p53-/- mice into lethally irradiated mice. In all cases, lethal leukemia developed earlier than in primary recipients. Moreover, there was a significant increase in erythroid progenitors in secondary recipients, suggesting the erythroid component is the predominant lineage involved in this leukemia model. As Jak2V617F/p53-/- leukemic tissues contained three major populations: CD71+ erythroid progenitors, Mac1+ mature myeloid cells, and lineage-negative (CD71-/Mac1-) primitive leukemic cells, we purified and transplanted these subfractions into secondary recipients to evaluate their leukemia-initiating potential. As a result, both lineage-negative (CD71-/Mac1-) cells and CD71+ erythroid progenitors possessed leukemia- initiating capacity, but Mac1+ myeloid cells could not reconstitute the disease. In addition, these two fractions had different capacities to induce leukemias; recipients of CD71+ cells rapidly developed erythroleukemia, whereas lineage-negative cells caused lethal leukemia after the polycythemic state. Moreover, hematopoietic tissues in recipients transplanted with CD71+ cells mainly consisted of erythroid lineages, whereas lineage-negative cells produced both erythroid and myeloid lineages, suggesting lineage-negative cells are more immature than CD71+ erythroid precursors. Furthermore, subsequent fractionation of lineage-negative cells revealed leukemia-initiating cells were enriched in Lin-/Sca-1+/c-kit+ (LSK) cells. To further characterize two types of leukemia-initiating cells in Jak2V617F/p53-/- leukemia, we assessed their sensitivity to a JAK2 inhibitor, INCB18424, in vitro. Interestingly, INCB18424 treatment significantly reduced CD71+ cell proliferation, whereas LSK cells were able to expand in the presence of INCB18424, indicating different leukemia-initiating cells existing in post-MPN leukemia have different responsiveness to JAK2 inhibiton. In summary, these results demonstrate p53 loss is sufficient for inducing leukemic transformation in JAK2V617F-postive MPN and offers an in vivo model to assess novel therapeutic approaches for post-MPN leukemia. In addition, we revealed leukemia-initiating cells at different differentiation stages could exist in post-MPN leukemia. Disclosures: Kurokawa: Novartis: Consultancy, Research Funding; Bristol-Myers Squibb: Research Funding; Celgene: Consultancy, Research Funding.

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Wagenblast, Elvin, Joana Araújo, Olga I. Gan, Sarah K. Cutting, Alex Murison, Jessica L. McLeod, Gabriela Krivdova, et al. "A Human Model of Down Syndrome Associated Leukemia Reveals Different Cell of Origins for Initiation and Progression." Blood 136, Supplement 1 (November 5, 2020): 11–12. http://dx.doi.org/10.1182/blood-2020-135867.

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Introduction: Leukemia is the most common cancer in children and sequencing data suggest that the first genetic alterations often occur in utero. Children with Down syndrome (Trisomy 21, T21) have a 150-fold increased risk of childhood leukemia. In 30% of newborns with Down syndrome, a transient myeloproliferative disorder (pre-leukemia) occurs, which is characterized by a clonal proliferation of immature megakaryoblasts carrying somatic mutations in the GATA1 transcription factor (GATA1s) and resolves spontaneously in most cases. In 20% of the cases, acute megakaryoblastic leukemia (AMKL) evolves from the pre-leukemic clone by acquisition of additional mutations, such as in the cohesin subunit STAG2. It is hypothesized that this represents a multi-step process of leukemogenesis with three distinct genetic events: T21, GATA1s and STAG2. Yet, it remains unclear how an extra copy of chromosome 21 predisposes towards leukemia, the interplay between each genetic event and the cellular origin of transformation. Methods: Human long-term hematopoietic stem cells (LT-HSCs) were sorted from normal karyotype and T21 fetal livers (N-FL and T21-FL) and subsequently CRISPR/Cas9 edited to try to establish a humanized model of Down Syndrome associated pre-leukemia and AMKL. To model the initiation of the pre-leukemic state, GATA1s mutations were introduced, while additional STAG2- mutations were overlaid to model the progression to fully transformed AMKL. CRISPR/Cas9-edited control, GATA1s, STAG2- and GATA1s/STAG2- LT-HSCs were functionally interrogated in near-clonal xenograft assays, along with transcriptional and epigenetic profiling. Results: T21 status in combination with GATA1s had a profound synergistic effect on megakaryocytic lineage output in vivo compared to normal karyotype with GATA1s. Moreover, a high percentage of blasts were found in xenografts of GATA1s edited T21-FL LT-HSCs (>30%) but not in xenografts of GATA1s edited N-FL LT-HSCs. Conversely, GATA1s/STAG2- edited LT-HSCs generated grafts with >50% of blasts, regardless of T21 status. The immunophenotype of these blasts recapitulated those observed in patients diagnosed with Down Syndrome pre-leukemia and AMKL (CD117+CD34+CD41+CD71+CD33+CD4+CD7+). Thus, T21 is required for pre-leukemia development, but seems dispensable for AMKL as both N- and T21-FL LT-HSCs underwent leukemic transformation upon GATA1s/STAG2-. Serial xenotransplantation assays from primary engrafted mice were carried out to assess self-renewal properties of GATA1s-induced pre-leukemia and GATA1s/STAG2- induced AMKL. Only GATA1s/STAG2- edited N- and T21-FL grafts were able to propagate the leukemic phenotype with a high stem cell frequency, which was endowed by the additional STAG2- knock-out. ATACseq and RNAseq profiling of blast populations revealed an enrichment of GATA-binding sites with concomitant up-regulation of genes implicated in translation. To assess the role of progenitor cells in pre-leukemic initiation and leukemic progression, we CRISPR/Cas9 edited short-term HSCs, common myeloid progenitors and myelo-erythroid progenitors with GATA1s and/or STAG2- and subjected them to xenotransplantation. Strikingly, all progenitor subsets with combined GATA1s/STAG2- editing were able to drive leukemic transformation, while single GATA1s editing in the same subsets did not initiate pre-leukemia. This data strongly suggests that the initial GATA1s mutation must occur in T21 LT-HSCs, but subsequent STAG2 mutations can occur further downstream in progenitors. Lastly, to gain insight into how chromosome 21 predisposes towards pre-leukemia, three chromosome 21 miRNAs (miR-99a, -125b-2 and -155) were identified to be up-regulated in T21-FL LT-HSCs compared to N-FL LT-HSCs. Over-expression of these miRNAs in N-FL LT-HSCs induced a T21-like state with increased myeloid and megakaryocytic skewing. Dramatically, CRISPR/Cas9-edited knock-out of these miRNAs in GATA1s edited T21-FL LT-HSCs resulted in a block of pre-leukemia initiation. Conclusion: Our findings demonstrate that T21 is required for pre-leukemia initiation, which is mediated by over-expression of chromosome 21 miRNAs in LT-HSCs. Further, this data demonstrates different cell of origins between pre-leukemia initiation and AMKL progression. Ongoing studies focus on preventing the progression of pre-leukemia to AMKL by pharmacological targeting. Figure Disclosures Dick: Bristol-Myers Squibb/Celgene: Research Funding.

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Wang, Pin-Yi, Fay Young, Chun-Yu Chen, Brett M. Stevens, Sarah J. Neering, Randall M. Rossi, Timothy Bushnell, et al. "The biologic properties of leukemias arising from BCR/ABL-mediated transformation vary as a function of developmental origin and activity of the p19ARF gene." Blood 112, no. 10 (November 15, 2008): 4184–92. http://dx.doi.org/10.1182/blood-2008-02-142190.

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Abstract Recent reports have shown that upon expression of appropriate oncogenes, both stem cells and more differentiated progenitor populations can serve as leukemia-initiating cells. These studies suggest that oncogenic mutations subvert normal development and induce reacquisition of stem-like features. However, no study has described how specific mutations influence the ability of differentiating cell subsets to serve as leukemia-initiating cells and if varying such cellular origins confers a functional difference. We have examined the role of the tumor suppressor gene p19ARF in a murine model of acute lymphoblastic leukemia and found that loss of p19ARF changes the spectrum of cells capable of tumor initiation. With intact p19ARF, only hematopoietic stem cells (HSCs) can be directly transformed by BCR/ABL expression. In a p19ARF-null genetic background expression of the BCR/ABL fusion protein renders functionally defined HSCs, common lymphoid progenitors (CLP), and precursor B-lymphocytes competent to generate leukemia stem cells. Furthermore, we show that leukemias arising from p19ARF-null HSC versus pro-B cells differ biologically, including relative response to drug insult. Our observations elucidate a unique mechanism by which heterogeneity arises in tumor populations harboring identical genetic lesions and show that activity of p19ARF profoundly influences the nature of tumor-initiating cells during BCR/ABL-mediated leukemogenesis.

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Sharma, Ritul, Chunfen Zhang, and Aru Narendran. "The Small-Molecule E26-Transformation-Specific Inhibitor TK216 Attenuates the Oncogenic Properties of Pediatric Leukemia." Genes 14, no. 10 (October 8, 2023): 1916. http://dx.doi.org/10.3390/genes14101916.

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The E26-transformation-specific (ETS) transcription factors regulate multiple aspects of the normal hematopoietic system. There is an increasing body of evidence suggesting aberrant ETS activity and its contribution to leukemia initiation and progression. In this study, we evaluated the small-molecule ETS inhibitor TK216 and demonstrated its anti-tumor activity in pediatric leukemia. We found TK216 induced growth inhibition, cell cycle arrest and apoptosis and inhibited the migratory capability of leukemic cells, without significantly inhibiting the cell viability of normal blood mononuclear cells. Priming the leukemic cells with 5-Azacitidine enhanced the cytotoxic effects of TK216 on pediatric leukemia cells. Importantly, we found purine-rich box1 (PU.1) to be a potential target of TK216 in myeloid and B-lymphoid leukemic cells. In addition, TK216 sharply decreased Mcl-1 protein levels in a dose-dependent manner. Consistent with this, TK216 also potentiated the cytotoxic effects of Bcl-2 inhibition in venetoclax-resistant cells. The sustained survival benefit provided to leukemic cells in the presence of bone-marrow-derived conditioned media is also found to be modulated by TK216. Taken together, our data indicates that TK216 could be a promising targeted therapeutic agent for the treatment of acute myeloid and B-lymphoid leukemia.

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Ohlsson, Ewa, Marie Sigurd Hasemann, Anton Willer, Felicia Kathrine Bratt Lauridsen, Nicolas Rapin, Johan Jendholm, and Bo Torben Porse. "Initiation of MLL-rearranged AML is dependent on C/EBPα." Journal of Experimental Medicine 211, no. 1 (December 23, 2013): 5–13. http://dx.doi.org/10.1084/jem.20130932.

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MLL-fusion proteins are potent inducers of oncogenic transformation, and their expression is considered to be the main oncogenic driving force in ∼10% of human acute myeloid leukemia (AML) patients. These oncogenic fusion proteins are responsible for the initiation of a downstream transcriptional program leading to the expression of factors such as MEIS1 and HOXA9, which in turn can replace MLL-fusion proteins in overexpression experiments. To what extent MLL fusion proteins act on their own during tumor initiation, or if they collaborate with other transcriptional regulators, is unclear. Here, we have compared gene expression profiles from human MLL-rearranged AML to normal progenitors and identified the myeloid tumor suppressor C/EBPα as a putative collaborator in MLL-rearranged AML. Interestingly, we find that deletion of Cebpa rendered murine hematopoietic progenitors completely resistant to MLL-ENL–induced leukemic transformation, whereas C/EBPα was dispensable in already established AMLs. Furthermore, we show that Cebpa-deficient granulocytic-monocytic progenitors were equally resistant to transformation and that C/EBPα collaborates with MLL-ENL in the induction of a transcriptional program, which is also apparent in human AML. Thus, our studies demonstrate a key role of C/EBPα in MLL fusion–driven transformation and find that it sharply demarcates tumor initiation and maintenance.

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Yokoyama, Akihiko, and Hiroshi Okuda. "The Molecular Mechanism of Transcriptional Activation By MLL-AEP Fusion Proteins." Blood 126, no. 23 (December 3, 2015): 2435. http://dx.doi.org/10.1182/blood.v126.23.2435.2435.

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Abstract Chromosomal translocations generate a variety of mixed lineage leukemia (MLL) fusion genes, which cause aggressive leukemia. Although >70 different fusion partners have been identified, the majority of the cases are caused by the chimeric genes of MLL and a component of the AEP co-activator complex (hereafter referred to as AEP), which comprises of AF4 family proteins (e.g. AF4, AF5Q31), ENL family proteins (e.g. ENL, AF9), and the P-TEFb elongation factor. MLL-AEP fusion proteins constitutively activate their target genes by recruiting AEP components to their target chromatin, whereas wild-type MLL recruits AEP in a context-dependent manner. In the hematopoietic lineage, MLL fusion proteins aberrantly activate a subset of genes implicated in the hematopoietic stem cell (HSC) program, such as HOXA9 and MEIS1. Constitutive expression of these HSC program genes in hematopoietic progenitors has been shown to induce leukemia in a mouse model. It has been speculated that MLL-AEP activates transcription of those HSC program genes by aberrantly activating transcription elongation. However, it is largely unclear how AEP activates transcription. Using an extensive structure/function analysis, we revealed that a serine-rich domain of the AF4 family proteins, termed pSER, is an essential functional component of MLL-AEP-dependent gene activation and leukemic transformation. Through biochemical purification, we have identified Selectivity Factor 1 (SL1) as a novel factor associated with the pSER domain. SL1 comprises TBP and four TBP-associated factors (TAF1A, TAF1B, TAF1C, TAF1D), and is known as a core component of the pre-initiation complex (PIC) of RNA polymerase I (RNAP1). In the presence of UBF, SL1 forms a PIC on the promoters of ribosomal RNA genes, to drive RNAP1-dependent transcription. However, its role in RNAP2-dependent transcription was unknown. The initiation of RNAP2-dependent transcription in eukaryotes occurs through the loading of TBP to the promoter, via a direct association with the TATA element or through as-yet-unidentified mechanisms. Our results demonstrate that AEP facilitates the initiation of RNAP2-dependent transcription via the loading of TBP onto the TATA element, through SL1 activity. MLL-AEP fusion proteins utilize this TBP-loading function to activate transcription initiation in leukemic transformation. The wild-type AEP complex activates gene expression in the same manner in the physiological conditions. Taken together, our results unveil a novel role of SL1 as a TBP-loading factor in RNAP2-dependent gene activation, and a previously unknown transcription initiation mechanism involving AEP, which is more important than its transcription elongation activities for leukemic transformation. These findings greatly advance our understanding of the molecular mechanism of MLL fusion-dependent leukemic transformation, which was previously interpreted simply as mis-regulated transcription elongation. Disclosures Yokoyama: Dainipon Sumitomo Pharma Co., Ltd.: Research Funding.

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Horton, Sarah J., Vanessa Walf-Vorderwülbecke, Steve J. Chatters, Neil J. Sebire, Jasper de Boer, and Owen Williams. "Acute myeloid leukemia induced by MLL-ENL is cured by oncogene ablation despite acquisition of complex genetic abnormalities." Blood 113, no. 20 (May 14, 2009): 4922–29. http://dx.doi.org/10.1182/blood-2008-07-170480.

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Abstract Chromosomal translocations involving 11q23 are frequent in infant acute leukemia and give rise to the formation of MLL fusion genes. The mechanism of leukemic transformation by these fusions has been the subject of numerous investigations. However, the dependence of acute leukemia on MLL fusion activity in vivo and the efficacy of targeting this activity to eliminate disease have not been established. We have developed a model for conditional expression of MLL-ENL in hematopoietic progenitor cells, in which expression of the fusion oncogene is turned off by doxycycline. Conditionally immortalized myeloblast cells derived from these progenitors were found to induce leukemia in vivo. Leukemic cells isolated from primary recipient mice were shown to have acquired additional genetic abnormalities and, when transplanted into secondary recipients, induced leukemia with shortened latencies. However, the leukemic cells remained dependent on MLL-ENL expression in vitro and in vivo, and its ablation resulted in regression of established leukemias. This study demonstrates that even genetically complex leukemias can be reversed on inactivation of the initiating MLL fusion and has important implications for the design of novel leukemia therapies.

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Fishman, Hila, Shreyas Madiwale, Ifat Geron, Vase Bari, Wouter Van Loocke, Yael Kirschenbaum, Itamar Ganmore, et al. "ETV6-NCOA2 fusion induces T/myeloid mixed-phenotype leukemia through transformation of nonthymic hematopoietic progenitor cells." Blood 139, no. 3 (January 20, 2022): 399–412. http://dx.doi.org/10.1182/blood.2020010405.

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Abstract Mixed-phenotype acute leukemia is a rare subtype of leukemia in which both myeloid and lymphoid markers are co-expressed on the same malignant cells. The pathogenesis is largely unknown, and the treatment is challenging. We previously reported the specific association of the recurrent t(8;12)(q13;p13) chromosomal translocation that creates the ETV6-NCOA2 fusion with T/myeloid leukemias. Here we report that ETV6-NCOA2 initiates T/myeloid leukemia in preclinical models; ectopic expression of ETV6-NCOA2 in mouse bone marrow hematopoietic progenitors induced T/myeloid lymphoma accompanied by spontaneous Notch1-activating mutations. Similarly, cotransduction of human cord blood CD34+ progenitors with ETV6-NCOA2 and a nontransforming NOTCH1 mutant induced T/myeloid leukemia in immunodeficient mice; the immunophenotype and gene expression pattern were similar to those of patient-derived ETV6-NCOA2 leukemias. Mechanistically, we show that ETV6-NCOA2 forms a transcriptional complex with ETV6 and the histone acetyltransferase p300, leading to derepression of ETV6 target genes. The expression of ETV6-NCOA2 in human and mouse nonthymic hematopoietic progenitor cells induces transcriptional dysregulation, which activates a lymphoid program while failing to repress the expression of myeloid genes such as CSF1 and MEF2C. The ETV6-NCOA2 induced arrest at an early immature T-cell developmental stage. The additional acquisition of activating NOTCH1 mutations transforms the early immature ETV6-NCOA2 cells into T/myeloid leukemias. Here, we describe the first preclinical model to depict the initiation of T/myeloid leukemia by a specific somatic genetic aberration.

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Cai, Xiongwei, Yoshihiro Hayashi, Mark Wunderlich, Nancy A. Speck, James C. Mulloy, Gang Huang, and Yi Zheng. "Loss of Function RUNX1 Mutations Restrict Protein Biosynthesis during Pre-Leukemia and MDS Transition but Not after Leukemic Transformation." Blood 128, no. 22 (December 2, 2016): 3860. http://dx.doi.org/10.1182/blood.v128.22.3860.3860.

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Abstract Runx1, a DNA binding subunit of core binding factors, is found frequently mutated in hematological malignancies. Runx1 mutation can be an early event in leukemogenesis endowing pre-leukemic stem cells with a selective advantage in the bone marrow, and is associated with an unfavorable outcome. In mouse models, loss of function (LOF) Runx1 mutations cause a broad decrease of ribosome biogenesis in hematopoietic stem and progenitor cells (HSPCs) by directly binding to ribosomal related genes essential for protein synthesis, and confers resistance to genotoxic stress (Cai et al. 2015 Cell Stem Cell 17(2):165-77). Paradoxially, leukemia cells generally require higher biosynthetic activity, and AML patients with LOF Runx1 mutations show upregulated ribosome signatures compared with those without Runx1 mutations (Silva et al., 2009 Blood 114:3001-3007). It remains unclear whether RUNX1 plays a role in regulating protein synthesis in leukemogenesis as in normal HSPCs, and if LOF Runx1 mutations are important for leukemia initiation, transformation and/or maintenance. To examine such mechanistic roles of RUNX1 in AML progression, we have used a previously reported MLL-PTD; Mx Cre; Runx1 Flox/Flox (Double mutant -DM) mouse model (Hayashi et al., 2015 Blood 126:303 ) that allows experimental tracking of the step-wise transition of HSPCs from pre-disease stage to a MDS-like stage, prior to full blown AML. Various subpopulations of the HSPCs, including genotypic HSCs, MPP, GMP, CMP, MEP, were isolated from the mice at pre-disease, MDS-like, and AML full-blown stages, and were assayed for protein synthesis rates by O-propargyl puromycin incorporation, DNA synthesis rates by BrdU labeling, and FACS analysis. At the pre-disease state, DM HSCs, as well as all the progenitor populations, had lower protein biosynthesis activity compared with similar populations of wild-type control or MLL-PTD mutant mice, consistent with LOF Runx1 mutations providing stress-resistance and survival advantage. As disease progressed, the DM mice developed MDS-like phenotypes including severe anemia and bone marrow fibrosis, with the HSCs (LSK CD34-Flt3- cells) showing increased protein synthesis rate compared with the pre-disease DM mice. Upon the onset of full-blown leukemia, the protein translation rates in all subpopulations of DM HSPCs were significantly faster than the control non-leukemic cells, regardless of the Runx1 mutant status. Importantly, preliminary analyses of two human AML samples found that CD34+ cells with LOF Runx1 mutations displayed a similarly enhanced protein synthesis rates than CD34+ leukemia bone marrow cells carrying wild type Runx1, as seen in the mouse model. Our results show that at early initiation, LOF Runx1 mutation supresses protein biosynthesis; during transition to MDS, the inhibitory regulation was bypassed in LT-HSCs (LSK CD34-Flt3-), suggesting that Runx1-controlled protein translation is involved in the early clonal selection of disease progression. In full-blown leukemia cells including the primitive subpopulations, however the protein synthesis rate appears to become uncoupled from Runx1 regulation possibly due to an activation of compensatory machineries. This study of the role of Runx1 mutation in pre-leukemia cell progression to full blown leukemia raises the question that while some tumor initiating mutations such as LOF Runx1 mutations contribute to the tumor initiation and transformation process, they may not be essential for maintaining certain crucial leukemia cell phenotypes such as protein biosynthesis. Disclosures No relevant conflicts of interest to declare.

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Gasic, Vladimir, Teodora Karan-Djurasevic, Djordje Pavlovic, Branka Zukic, Sonja Pavlovic, and Natasa Tosic. "Diagnostic and Therapeutic Implications of Long Non-Coding RNAs in Leukemia." Life 12, no. 11 (November 2, 2022): 1770. http://dx.doi.org/10.3390/life12111770.

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Leukemia is a heterogenous group of hematological malignancies categorized in four main types (acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML) and chronic lymphocytic leukemia (CLL). Several cytogenetic and molecular markers have become a part of routine analysis for leukemia patients. These markers have been used in diagnosis, risk-stratification and targeted therapy application. Recent studies have indicated that numerous regulatory RNAs, such as long non-coding RNAs (lncRNAs), have a role in tumor initiation and progression. When it comes to leukemia, data for lncRNA involvement in its etiology, progression, diagnosis, treatment and prognosis is limited. The aim of this review is to summarize research data on lncRNAs in different types of leukemia, on their expression pattern, their role in leukemic transformation and disease progression. The usefulness of this information in the clinical setting, i.e., for diagnostic and prognostic purposes, will be emphasized. Finally, how particular lncRNAs could be used as potential targets for the application of targeted therapy will be considered.

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Melo, Junia V., and Michael W. N. Deininger. "Biology of chronic myelogenous leukemia—signaling pathways of initiation and transformation." Hematology/Oncology Clinics of North America 18, no. 3 (June 2004): 545–68. http://dx.doi.org/10.1016/j.hoc.2004.03.008.

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Joffre, Carine, Charlotte Ducau, Laura Poillet-Perez, Charly Courdy, and Véronique Mansat-De Mansat-De Mas. "Autophagy a Close Relative of AML Biology." Biology 10, no. 6 (June 18, 2021): 552. http://dx.doi.org/10.3390/biology10060552.

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Autophagy, which literally means “eat yourself”, is more than just a lysosomal degradation pathway. It is a well-known regulator of cellular metabolism and a mechanism implicated in tumor initiation/progression and therapeutic resistance in many cancers. However, whether autophagy acts as a tumor suppressor or promoter is still a matter of debate. In acute myeloid leukemia (AML), it is now proven that autophagy supports cell proliferation in vitro and leukemic progression in vivo. Mitophagy, the specific degradation of mitochondria through autophagy, was recently shown to be required for leukemic stem cell functions and survival, highlighting the prominent role of this selective autophagy in leukemia initiation and progression. Moreover, autophagy in AML sustains fatty acid oxidation through lipophagy to support mitochondrial oxidative phosphorylation (OxPHOS), a hallmark of chemotherapy-resistant cells. Nevertheless, in the context of therapy, in AML, as well as in other cancers, autophagy could be either cytoprotective or cytotoxic, depending on the drugs used. This review summarizes the recent findings that mechanistically show how autophagy favors leukemic transformation of normal hematopoietic stem cells, as well as AML progression and also recapitulates its ambivalent role in resistance to chemotherapies and targeted therapies.

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Jamrog, Laura, Guillaume Chemin, Vincent Fregona, Lucie Coster, Marlène Pasquet, Chloé Oudinet, Nelly Rouquié, et al. "PAX5-ELN oncoprotein promotes multistep B-cell acute lymphoblastic leukemia in mice." Proceedings of the National Academy of Sciences 115, no. 41 (September 26, 2018): 10357–62. http://dx.doi.org/10.1073/pnas.1721678115.

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PAX5 is a well-known haploinsufficient tumor suppressor gene in human B-cell precursor acute lymphoblastic leukemia (B-ALL) and is involved in various chromosomal translocations that fuse a part of PAX5 with other partners. However, the role of PAX5 fusion proteins in B-ALL initiation and transformation is ill-known. We previously reported a new recurrent t(7;9)(q11;p13) chromosomal translocation in human B-ALL that juxtaposed PAX5 to the coding sequence of elastin (ELN). To study the function of the resulting PAX5-ELN fusion protein in B-ALL development, we generated a knockin mouse model in which the PAX5-ELN transgene is expressed specifically in B cells. PAX5-ELN–expressing mice efficiently developed B-ALL with an incidence of 80%. Leukemic transformation was associated with recurrent secondary mutations on Ptpn11, Kras, Pax5, and Jak3 genes affecting key signaling pathways required for cell proliferation. Our functional studies demonstrate that PAX5-ELN affected B-cell development in vitro and in vivo featuring an aberrant expansion of the pro-B cell compartment at the preleukemic stage. Finally, our molecular and computational approaches identified PAX5-ELN–regulated gene candidates that establish the molecular bases of the preleukemic state to drive B-ALL initiation. Hence, our study provides a new in vivo model of human B-ALL and strongly implicates PAX5 fusion proteins as potent oncoproteins in leukemia development.

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Horton, Sarah J., Vanessa Walf-Vorderwülbecke, Steve J. Chatters, Neil J. Sebire, Jasper de Boer, and Owen Williams. "Acute Myeloid Leukemia Induced by MLL-ENL Is Cured by Oncogene Ablation despite Acquisition of Complex Genetic Abnormalities." Blood 112, no. 11 (November 16, 2008): 3109. http://dx.doi.org/10.1182/blood.v112.11.3109.3109.

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Abstract Chromosomal translocations involving the Mixed-Lineage-Leukemia (MLL) gene on chromosome 11q23 are frequent in infant acute leukemia and give rise to the formation of MLL-fusion genes. Several studies have addressed the importance of MLL-fusion activity for the initiation and maintenance of hematopoietic transformation. However, the dependence of established leukemias on MLL-fusion activity has not been previously addressed. We have developed a model for conditional expression of MLL-ENL in hematopoietic progenitor cells, in which expression of the fusion oncogene is turned off by doxycycline. In this study, immortalized myeloid cells conditionally or constitutively expressing the MLL-ENL fusion gene were used to induce acute myeloid leukemia (AML) in vivo. Primary recipients developed AML with a mean latency of 81.4 (±4.8) days. Secondary recipients developed AML with much shorter latencies than primary recipients regardless of whether the leukemic cells were freshly transplanted (26.8 (±6.8) days) or cultured in vitro for one month prior to transplantation (18 (±3.9) days). Genetic analysis revealed that some leukemic cells had acquired gross chromosomal abnormalities such as trisomy 6 or gains and losses of chromosome regions, which were not detected in the immortalised cells from which they were derived. Despite the acquisition of additional genetic abnormalities, the leukemic cells remained dependent upon MLL-ENL expression in vitro and in vivo. The leukemic cells terminally differentiated into neutrophils upon doxycycline treatment in vitro and established leukemias regressed following administration of doxycycline to recipient mice in their drinking water. Leukemic regression was accompanied by the complete loss of leukemic cells from the peripheral blood and differentiation of leukemic cells in the spleen. In 7 out of 34 doxycycline treated mice, remission was not sustained and the leukemias relapsed. However, most of these were shown to have acquired constitutive expression of MLL-ENL. This study demonstrates that leukemic cells are addicted to MLL-ENL expression and suggests that targeting the transcriptional/signalling networks established by MLL-fusion oncogenes in patients with 11q23 rearrangements would be a major therapeutic advance.

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Quéré, Ronan, Göran Karlsson, Falk Hertwig, Marianne Rissler, Beata Lindqvist, Thoas Fioretos, Peter Vandenberghe, Marilyn L. Slovak, Jörg Cammenga, and Stefan Karlsson. "Smad4 binds Hoxa9 in the cytoplasm and protects primitive hematopoietic cells against nuclear activation by Hoxa9 and leukemia transformation." Blood 117, no. 22 (June 2, 2011): 5918–30. http://dx.doi.org/10.1182/blood-2010-08-301879.

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Abstract We studied leukemic stem cells (LSCs) in a Smad4−/− mouse model of acute myelogenous leukemia (AML) induced either by the HOXA9 gene or by the fusion oncogene NUP98-HOXA9. Although Hoxa9-Smad4 complexes accumulate in the cytoplasm of normal hematopoietic stem cells and progenitor cells (HSPCs) transduced with these oncogenes, there is no cytoplasmic stabilization of HOXA9 in Smad4−/− HSPCs, and as a consequence increased levels of Hoxa9 is observed in the nucleus leading to increased immortalization in vitro. Loss of Smad4 accelerates the development of leukemia in vivo because of an increase in transformation of HSPCs. Therefore, the cytoplasmic binding of Hoxa9 by Smad4 is a mechanism to protect Hoxa9-induced transformation of normal HSPCs. Because Smad4 is a potent tumor suppressor involved in growth control, we developed a strategy to modify the subcellular distribution of Smad4. We successfully disrupted the interaction between Hoxa9 and Smad4 to activate the TGF-β pathway and apoptosis, leading to a loss of LSCs. Together, these findings reveal a major role for Smad4 in the negative regulation of leukemia initiation and maintenance induced by HOXA9/NUP98-HOXA9 and provide strong evidence that antagonizing Smad4 stabilization by these oncoproteins might be a promising novel therapeutic approach in leukemia.

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Cai, Qi, Robin Jeannet, Hongjun Liu, and ya-Huei Kuo. "CBFβ-SMMHC Impairs Erythroid Differentiation and Induces Expansion of Aberrant Megakaryocytic/Erythroid Progenitors Capable of Leukemia Initiation." Blood 124, no. 21 (December 6, 2014): 2149. http://dx.doi.org/10.1182/blood.v124.21.2149.2149.

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Abstract Approximately 12% of human acute myeloid leukemia (AMLs) harbor a recurrent chromosomal rearrangement inv(16)(p13q22). Inv(16) creates a fusion gene Cbfb-MYH11, encoding the fusion protein CBFß-SMMHC. Expressing CBFß-SMMHC in hematopoietic cells using a constitutive knock-in mouse model (Cbfb+/Cbfb-MYH11) or a conditional knock-in mouse model (Cbfb56M/+/Mx1-Cre; 129SvEv strain) causes defects in lymphoid and myeloid differentiation, and predisposes mice to AML. Previous studies with the constitutive knock-in mouse model showed impaired primitive erythropoiesis, however, Cbfb-MYH11 knocked-in cells were able to contribute to erythropoiesis in chimeric mice. To further delineate the effect of CBFß-SMMHC in adult erythropoiesis in the conditional knock-in mouse, we backcrossed Cbfb56M/+/Mx1-Cre into C57BL/6 and a Rosa26mT/mG Cre reporter strain. Induced expression of CBFß-SMMHC in adult mice leads to cell number dependent development of AML, consistent with previous studies in 129SvEv strain. Analysis of pre-leukemic bone marrow 2 weeks after induction revealed a 5.7-fold expansion of immunophenotypic pre-megakaryocyte/erythrocyte (Pre-Meg/E; Lin-cKit+Sca1-CD16-/loCD150+CD105-), and a 4.7 fold decrease of the erythroid progenitor (EP; Lin-cKit+Sca1-CD16-/loCD105hi) subset compared to similarly treated control mice. Both methylcellulose-based erythroid colony forming assay and in vitro erythroid differentiation culture showed that pre-leukemic Pre-Meg/Es expressing CBFß-SMMHC had an impaired differentiation potential for erythroid lineage. Using the Rosa26mT/mG Cre reporter allele, we tracked the proportions of CBFß-SMMHC- expressing cells (GFP+) in the Pre-Meg/E and EP subsets. We observed that the contribution of GFP+ cells sharply decreased in EPs but not in Pre-Meg/Es from primary pre-leukemic mice. Similar results were seen in transplant recipients engrafted with sorted GFP+ pre-leukemic Lin-cKit+Sca1+ cells. These results further confirmed that CBFß-SMMHC impairs cell-autonomous erythroid differentiation in vivo. Consistent with the impaired differentiation of Pre-Meg/Es, we observed altered expression pattern of erythroid regulatory genes, including Fog1, Gata2, and Gfi1b. The pre-leukemic Pre-Meg/Es exhibited increased colony forming and replating capacity in vitro and enhanced proliferation and survival in vivo. To determine whether these phenotypic Pre-Meg/E cells could be the cellular origin for leukemic transformation, we expressed a known cooperative onco-protein Mpl by retroviral transduction followed by transplantation. The majority of mice (83%) receiving 100,000 Pre-Meg/E cells developed leukemia with a medium onset of 92 days, suggesting that Pre-Meg/Es indeed are capable of leukemia initiation. In conclusion, the expression of CBFß-SMMHC impairs adult erythropoiesis at the transition of Pre-Meg/E to EPs, causing an expansion of Pre-Meg/E cells. These pre-leukemic Pre-Meg/Es could be the target cell of additional mutations contributing to leukemia transformation. Disclosures No relevant conflicts of interest to declare.

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El-Haj, Nura, Wilson I. Gonsalves, Vinay Gupta, Jacob P. Smeltzer, Sameer A. Parikh, Preet P. Singh, and Naseema Gangat. "Secondary Hemophagocytic Syndrome Associated with Richter’s Transformation in Chronic Lymphocytic Leukemia." Case Reports in Hematology 2014 (2014): 1–4. http://dx.doi.org/10.1155/2014/287479.

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Hemophagocytic syndrome (HPS) is an extremely rare condition arising from the overactivation of one’s own immune system. It results in excessive inflammation and tissue destruction. Prompt initiation of treatment is warranted in either scenario in order to decrease mortality. Most cases are triggered by infectious agents, malignancy, or drugs. We describe the first case of a CLL patient presenting with HPS due to acquisition of EBV-related large cell lymphoma in the setting of profound immunodeficiency.

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Cobaleda, C., N. Gutiérrez-Cianca, J. Pérez-Losada, T. Flores, R. Garcı́a-Sanz, M. González, and I. Sánchez-Garcı́a. "A primitive hematopoietic cell is the target for the leukemic transformation in human Philadelphia-positive acute lymphoblastic leukemia." Blood 95, no. 3 (February 1, 2000): 1007–13. http://dx.doi.org/10.1182/blood.v95.3.1007.003k35_1007_1013.

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BCR-ABL is a chimeric oncogene generated by translocation of sequences from the chromosomal counterpart (c-ABLgene) on chromosome 9 into the BCR gene on chromosome 22. Alternative chimeric proteins, BCR-ABLp190 and BCR-ABLp210, are produced that are characteristic of chronic myelogenous leukemia (CML) and Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph1-ALL). In CML, the transformation occurs at the level of pluripotent stem cells. However, Ph1-ALL is thought to affect progenitor cells with lymphoid differentiation. Here we demonstrate that the cell capable of initiating human Ph1-ALL in non-obese diabetic mice with severe combined immunodeficiency disease (NOD/SCID), termed SCID leukemia–initiating cell (SL-IC), possesses the differentiative and proliferative capacities and the potential for self-renewal expected of a leukemic stem cell. The SL-ICs from all Ph1-ALL analyzed, regardless of the heterogeneity in maturation characteristics of the leukemic blasts, were exclusively CD34+CD38−, which is similar to the cell-surface phenotype of normal SCID-repopulating cells. This indicates that normal primitive cells, rather than committed progenitor cells, are the target for leukemic transformation in Ph1-ALL.

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Quere, Ronan, Silja Andradottir, Ann Brun, Roman Zubarev, Göran Karlsson, Karin Olsson, Mattias Magnusson, Jörg Cammenga, and Stefan Karlsson. "High Levels of the Adhesion Molecule CD44 on Leukemic Cells Generate Acute Myeloid Leukemia Relapse After Withdrawal of the Initial Transforming Event." Blood 116, no. 21 (November 19, 2010): 3154. http://dx.doi.org/10.1182/blood.v116.21.3154.3154.

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Abstract Abstract 3154 Multiple genetic hits are detected in patients with acute myeloid leukemia (AML). To investigate this further, we developed a tetracycline inducible mouse model of AML, where the initial transforming event, overexpression of HOXA10, can be eliminated. Continuous overexpression of HOXA10 is required to generate AML in primary recipient mice, but is not essential for maintenance of the leukemia. Transplantation of AML to secondary recipients showed that in established leukemias, ∼80% of the leukemia-initiating cells (LICs) in bone marrow stopped proliferating upon withdrawal of HOXA10 overexpression. However, the population of LICs in primary recipients is heterogeneous since ∼20% of the LICs induce leukemia in secondary recipients despite elimination of HOXA10 induced overexpression (HOXA10OFF). Since the withdrawal of the initial transforming event can be made upon demand, we have been able to ask what co-operating events are essential to maintain growth of leukemic cells as overexpression of HOXA10 is removed. Intrinsic genetic activation of several proto-oncogenes was observed in leukemic cells resistant to inactivation of the initial transformation event. We have identified a frequent increase in the activation of the proto-oncogenes JUN, FOS and EGR1 in relapsed leukemia where overexpression of HOXA10 has been withdrawn (HOXA10OFF versus HOXA10ON conditions). In order to further investigate if another possible mechanism is involved in leukemia, upon withdrawal of the primary oncogenic event, we performed proteomic analysis using mass spectrometry. Interestingly, we observed that upon removal of the primary event, leukemia that continued to grow produced high levels of several proteins involved in cell-cell and cell-matrix interactions. Among these proteins, CD44 is expressed on the cell surface and participates in cell transmigration and is an important target, since this surface glycoprotein mediates cell adhesion, migration and homing of hematopoietic cancer cells. To determine whether an increase in CD44 is a key mechanism by which LICs are resistant, we performed a functional test by FACS sorting leukemic cells generated in primary donors and transplanted 20,000 cells expressing different levels of the CD44 surface marker (CD44low, CD44medium and CD44high) in the tail vein of lethally irradiated secondary recipient mice fed doxycycline or ciproxine. When we monitored mice for occurrence of leukemia, outgrowth of leukemic cells was not dependant on the CD44 protein level on the HOXA10ON (doxycycline) condition. Consistent with this, onset of leukemia was not delayed for mice transplanted with CD44low leukemic cells. When mice were fed with ciproxine to turn off HOXA10 overexpression, all mice injected with CD44high leukemic cells developed leukemia, whereas all mice injected with CD44low leukemic cells remained healthy. In conclusion, we confirmed that withdrawal of the initial HOXA10 oncogene promotes the outgrowth of LICs expressing high levels of CD44. This study suggests that extrinsic niche-dependent factors are also involved in the host-dependent outgrowth of leukemias after withdrawal of HOXA10 overexpression event that initiates the leukemia. Here we demonstrate the highly aggressive nature of LICs expressing high levels of CD44 and conversely, show the impaired outgrowth of LICs expressing low levels of this surface marker. In conclusion, our murine model of inducible HOXA10 expression recapitulates many of the features of human AML and is helpful to analyse the “oncogene addiction” and unravel the basic mechanisms involved in initiation and maintenance of leukemia, and to study whether adhesion molecules expressed on the surface of leukemic cells are important factors for leukemic relapse in the microenvironmental niches of the bone marrow. Our findings support the notion that cell intrinsic genetic events are not the only factors causing leukemic relapse, but suggest that host-dependant extrinsic factors in the bone marrow niche may also play a fundamental role in the mechanism mediating leukemic relapse. Disclosures: No relevant conflicts of interest to declare.

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Jeannet, Robin, Qi Cai, Hongjun Liu, Hieu Vu, and Ya-Huei Kuo. "Alcam Mediated Interaction Regulates Normal Hematopoietic Stem Cell Function and Cbfβ-SMMHC Induced Leukemogenesis." Blood 120, no. 21 (November 16, 2012): 4091. http://dx.doi.org/10.1182/blood.v120.21.4091.4091.

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Abstract Abstract 4091 The inv(16) acute myeloid leukemia (AML)-associated CBFβ-SMMHC fusion protein impairs hematopoietic differentiation and predisposes to leukemic transformation. Alcam, which encodes the activated leukocyte cell adhesion molecule (CD166), is a cell surface immunoglobulin superfamily member mediating homophilic adhesion as well as heterotypic interactions with CD6. We found that Alcam expression marks long-term repopulating HSCs (LT-HSC), multipotent progenitors (MPP), a subset of granulocyte-macrophage progenitors (GMP), and that Alcam expression is lost or reduced in subsets of pre-leukemic and leukemic progenitors expressing the Cbfβ-SMMHC fusion protein. We characterized the role of Alcam in HSC differentiation and self-renewal using an Alcam-null (Alcam−/−) mouse model (Weiner et al. 2004 Mol Cell Neurosci 27:1, 59–69). We show that Alcam is highly expressed in LT-HSCs where its level progressively increases with age. Young adult Alcam−/− mice had normal homeostatic hematopoiesis, and normal numbers of phenotypic HSCs. However, Alcam−/− HSCs had reduced long-term replating capacity in vitro and reduced long-term engraftment potential upon transplantation. We show that Alcam−/− BM contain a markedly lower frequency of long-term repopulating cells than wild type (WT). Further, the long-term repopulating potential and engraftment efficiency of Alcam−/− LT-HSCs was greatly compromised despite a progressive increase in phenotypic LT-HSC numbers during long-term serial transplantation. In addition, an age-associated increase in phenotypic LT-HSC cellularity was observed in Alcam−/− mice. This increase was predominately within the CD150hi fraction, and was accompanied by significantly reduced leukocyte output. Moreover, Alcam−/− LT-HSCs display premature elevation of Selp expression, a hallmark of HSC aging. To understand the role of Alcam in leukemic transformation, we generated conditional Cbfb-MYH11 knock-in (Cbfb56M/+/Mx1-Cre), Alcam-deficient (Alcam−/−) mice. Interestingly, we found that loss of Alcam drastically delayed or reduced leukemia incidence. Transplantation of Alcam−/−/Cbfb56M/+/Mx1-Cre pre-leukemic bone marrow cells into WT recipients also led to delayed and reduced incidence of leukemia development. These results suggest that Alcam contributes to leukemia transformation in a cell-intrinsic manner. Collectively, our study reveals that Alcam regulates the functional integrity and self-renewal of LT-HSCs, and contributes to leukemia initiation induced by CBFβ-SMMHC. Disclosures: No relevant conflicts of interest to declare.

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Chaubey, Aditya, Chinavenmeni Subramani Velu, Shane Horman, Anil Jegga, Monica L. Guzman, Nancy Zeleznik-Le, Craig T. Jordan, Martin Carroll, Brian Gebelein, and H. Leighton Grimes. "Intrinsic Requirement of MicroRNA In Hox-Based Leukemia Initiating Cell Maintenance." Blood 116, no. 21 (November 19, 2010): 4192. http://dx.doi.org/10.1182/blood.v116.21.4192.4192.

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Abstract Abstract 4192 Mouse models of MLL-Af9 leukemia have been exploited to roughly determine leukemia initiating cell (LIC) characteristics and biology. Each colony formed by MLL-Af9 leukemic splenocytes is capable of initiating leukemia upon transplantation. MLL-fusion oncoproteins signal through elevated expression of HoxA9, Meis and Pbx proteins. The HoxA9 transcription factor is of critical interest in human AML because it is; 1) directly affected by chromosomal translocations, 2) upregulated by leukemia oncoproteins, and 3) the level of HoxA9 in cytogenetically normal human AML predicts outcome. However, the direct transcriptional targets of endogenous HoxA9 that mediate transformation remain largely unknown. The Growth factor independent-1 (Gfi1) transcriptional repressor is known to induce granulopoiesis and inhibit myeloid progenitor proliferation. GFI1 is mutated in patients with severe congenital neutropenia (SCN). SCN patients are at increased risk for AML. We have shown that Hox and Gfi1 orthologs antagonize each other during Drosophila anterior posterior patterning, and that in mammalian myeloid progenitors Gfi1 directly represses the expression of Hoxa9, Pbx1 and Meis1. Moreover, Gfi1 regulates the expression of miR-21 and miR-196b, and forced expression of these miR blocks G-CSF instructed granulopoiesis. Here we demonstrate that microRNA genes are the targets of endogenous HoxA9 versus Gfi1 antagonism and that this antagonism is relevant in the context of human leukemia. Moreover, we also show that miR-196b and miR-21 are activated by Hox-signaling leukemia oncoproteins. Next, in both murine leukemia models and primary human AML samples, antagomir-mediated inhibition of microRNA function specifically disrupts colony replating potential of Lin- bone marrow cells transformed by HoxA9, Nup98-HoxA9 and MLL-Af9, as well as bona fide MLL-Af9 leukemias. In contrast, cells transformed by AML-ETO (which does not signal through HoxA9) are not affected. In vivo, antagomir treatment blocked MLL-Af9-initiated leukemia (but not AML-ETO leukemia) lethality. Finally, limiting dilution analyses demonstrate that antagomirs inhibit Hox-based transformation by targeting the LIC. Antagomir treatment induces the re-expression of a histone lysine demethylase, downregulation of gene expression associated with maintenance of MLL-Af9 leukemia and associated H3K4me3 marks on these genes. Our data establish microRNA genes as functional downstream targets of endogenous HoxA9, and implicate epigenetic signaling as critical client/mediators of Hox-based leukemia oncoproteins in LIC maintenance. Disclosures: Carroll: Sanofi Aventis Corporation: Research Funding; Kyowa Hakko Kirin Pharmaceuticals: Research Funding; Agios Pharmaceuticals: Research Funding.

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Ma, Haiqing, Saradhi Mallampati, Baohua Sun, Yun Gong, Véronique Lefebvre, and Xiaoping Sun. "Sox4 Is Essential for Initiation and Progression of Acute Lymphoblastic Leukemia Induced by P190-BCR-ABL Transformation." Blood 118, no. 21 (November 18, 2011): 564. http://dx.doi.org/10.1182/blood.v118.21.564.564.

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Abstract Abstract 564 Sox4 is an Sry-related HMG box transcription factor that plays critical roles in development. Sox4-null mice die at embryonic day 14 due to cardiac malformation. Recent studies have suggested that SOX4 may contribute to tumorigenesis, based on the fact that SOX4 is upregulated in many human tumor types, including brain, bladder, prostate, colon, and lung cancers. In addition, increased expression of Sox4 induced by retroviral insertional mutagenesis in mice has been shown to be associated with leukemia and lymphoma. We also found that SOX4 is highly expressed in human acute lymphoblastic leukemia (ALL) cells. We therefore set out in this study to determine the function of Sox4 and its underlying mechanisms in ALL. To overcome embryonic lethality caused by conventional Sox4 inactivation, we used Sox4 conditional null mice (Sox4fl/fl) to establish Sox4-null ALL cell lines. First, we obtained pure B220+ pro-B cells by co-culturing Sox4fl/fl fetal liver cells and OP-9 bone marrow stromal cells in the presence of IL-7. Subsequently, pro-B cells were transformed with p190-BCR-ABL and Sox4 inactivation was induced with Cre recombinase. We found that Sox4−/− pro-B transformed cells exhibited a significantly slower proliferation rate than Sox4+/− pro-B cells. Notably, cell cycle analysis indicated a lower percentage of S phase cells in Sox4−/− pro-B transformed cells and these cells yielded much fewer colonies than transformed Sox4+/− cells in colony formation assay. We further labeled the transformed cells with a luciferase reporter and injected them intravenously into sublethally irradiated NOD/SCID mice. Bioluminescence imaging showed that mice injected with Sox4−/− cells developed leukemia significantly more slowly and survive longer than mice injected with Sox4+/− cells. In conclusion, our data show that Sox4 significantly contributes to initiation and progression of leukemia from p190-BCR-ABL-transformed ALL cells in the mouse and thereby strongly support the notion that SOX4 overexpression critically contributes to acute lymphoblastic leukemia development in humans. Disclosures: No relevant conflicts of interest to declare.

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Cai, Qi, Robin Jeannet, Hongjun Liu, and Ya-Huei Kuo. "Aberrant Megakaryocytic/Erythroid Progenitors Contributes To Transformation Of Cbfb-SMMHC Induced Acute Myeloid Leukemia." Blood 122, no. 21 (November 15, 2013): 1652. http://dx.doi.org/10.1182/blood.v122.21.1652.1652.

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Abstract Inv(16)(p13q22) is a recurrent chromosomal rearrangement found in approximately 12% of human acute myeloid leukemia (AML) cases and creates a fusion gene between CBFb and MYH11. The fusion gene encodes a fusion protein CBFß-SMMHC which causes defects in lymphoid and myeloid differentiation. Previous studies also showed that primitive erythropoiesis is impaired by CBFß-SMMHC, however, CBFß-SMMHC knocked-in cells was able to contribute to adult erythropoiesis in chimeric mice. Expressing CBFß-SMMHC in the hematopoietic cells using a conditional knock-in mouse model (Cbfb56M/+/Mx1-Cre; 129SvEv strain) recapitulates inv(16)-associated AML. Previous studies in this model showed that CBFß-SMMHC expression leads to pre-leukemic hematopoietic alterations, and together with additional cooperative mutations, result in spontaneous myeloid leukemia in mice with a 3-6 month latency. We hypothesized that an expanded cell population at the pre-leukemic stage could be the target of additional mutations, and hence the cell of origin of leukemia initiating cells. To further delineate the pre-leukemic progenitors and leukemia initiating cells, we backcrossed Cbfb56M/+/Mx1-Cre into C57BL/6 for more than 10 generations. Similar to previous studies in the129SvEv strain, expressing CBFß-SMMHC in adult C57BL/6 mice leads to cell number dependent development of AML. Analysis of pre-leukemic bone marrow as early as 2 weeks after induction revealed a 5.7-fold expansion of Pre-Meg/E cells (Pre-Megakaryocyte/Erythrocyte: Lin-cKit+Sca1-CD16-/loCD150+CD105-) compared to similarly treated control mice. While there was a significant increase in Pre-Meg/E population, we did not find significant increase in their proliferation but observed a 4.7 fold decrease of the erythroid progenitor (EP; Lin-cKit+Sca1-CD16-/loCD105hi) subset. Methylcellulose-based colony forming assay showed that pre-leukemic Pre-Meg/E had an impaired differentiation potential for erythroid lineage. In vitro erythroid differentiation assay also showed a partial block of differentiation from pre-leukemic Pre-Meg/E progenitors. These Pre-Meg/E like progenitors were able to induce leukemia in the presence of a known cooperative oncoprotein MPL when transplanted in lethally irradiated congenic recipient mice. In summary, our Results suggest that expression of CBFß-SMMHC impairs adult erythropoiesis at the transition of Pre-Meg/E to EPs, causing an expansion of Pre-Meg/E cells, which can be the target cell of additional mutations contributing to leukemia transformation. Disclosures: No relevant conflicts of interest to declare.

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Wesolowski, Radoslaw, Elisabeth Kowenz-Leutz, Karin Zimmermann, Dorothea Dörr, Maria Hofstätter, Robert K. Slany, Alexander Mildner, and Achim Leutz. "Myeloid transformation by MLL-ENL depends strictly on C/EBP." Life Science Alliance 4, no. 1 (November 3, 2020): e202000709. http://dx.doi.org/10.26508/lsa.202000709.

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Chromosomal rearrangements of the mixed-lineage leukemia gene MLL1 are the hallmark of infant acute leukemia. The granulocyte-macrophage progenitor state forms the epigenetic basis for myelomonocytic leukemia stemness and transformation by MLL-type oncoproteins. Previously, it was shown that the establishment of murine myelomonocytic MLL-ENL transformation, but not its maintenance, depends on the transcription factor C/EBPα, suggesting an epigenetic hit-and-run mechanism of MLL-driven oncogenesis. Here, we demonstrate that compound deletion of Cebpa/Cebpb almost entirely abrogated the growth and survival of MLL-ENL–transformed cells. Rare, slow-growing, and apoptosis-prone MLL-ENL–transformed escapees were recovered from compound Cebpa/Cebpb deletions. The escapees were uniformly characterized by high expression of the resident Cebpe gene, suggesting inferior functional compensation of C/EBPα/C/EBPβ deficiency by C/EBPε. Complementation was augmented by ectopic C/EBPβ expression and downstream activation of IGF1 that enhanced growth. Cebpe gene inactivation was accomplished only in the presence of complementing C/EBPβ, but not in its absence, confirming the Cebpe dependency of the Cebpa/Cebpb double knockouts. Our data show that MLL-transformed myeloid cells are dependent on C/EBPs during the initiation and maintenance of transformation.

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Auberger, Patrick, and Alexandre Puissant. "Autophagy, a key mechanism of oncogenesis and resistance in leukemia." Blood 129, no. 5 (February 2, 2017): 547–52. http://dx.doi.org/10.1182/blood-2016-07-692707.

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AbstractAutophagy is a lysosomal pathway involved in degradation of intracellular material. It appears as an adaptation mechanism that is essential for cellular homeostasis in response to various stress conditions. Over the past decade, many studies have linked alteration of autophagy with cancer initiation and progression, autoimmune, inflammatory, metabolic, and degenerative diseases. This review highlights recent findings on the impact of autophagy on leukemic transformation of normal hematopoietic stem cells and summarizes its role on leukemic cell response to chemotherapy.

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Zhang, Jing, Jing Wang, Yangang Liu, Harwin Sidik, Ken H. Young, Harvey F. Lodish, and Mark D. Fleming. "Oncogenic Kras-induced leukemogeneis: hematopoietic stem cells as the initial target and lineage-specific progenitors as the potential targets for final leukemic transformation." Blood 113, no. 6 (February 5, 2009): 1304–14. http://dx.doi.org/10.1182/blood-2008-01-134262.

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Abstract KRAS is often mutated in human hematopoietic malignancies, including juvenile myelomonocytic leukemia (JMML) and T-cell lymphoblastic leukemia/lymphoma (TLL/L). However, the exact role and function of oncogenic KRAS mutations in the initiation and progression of JMML and TLL/L remain elusive. Here, we report the use of a mouse bone marrow transplantation model to study oncogenic Kras-induced leukemogenesis. We show that as the first genetic hit, oncogenic Kras mutations initiate both JMML and TLL/L, but with different efficiencies. Limiting dilution analyses indicated that an oncogenic Kras mutation alone is insufficient to produce frank malignancy. Instead, it cooperates with additional subsequent genetic event(s). Moreover, transplantation of highly purified hematopoietic stem cells (HSCs) and myeloid progenitors identified HSCs as the primary target for the oncogenic Kras mutation. Karyotypic analysis further indicated that secondary genetic hit(s) target lineage-specific progenitors rather than HSCs for terminal tumor transformation into leukemic stem cells. Thus, we propose the cellular mechanism underlying oncogenic Kras-induced leukemogenesis, with HSCs as the primary target by the oncogenic Kras mutations and lineage-committed progenitors as the final target for cancer stem cell transformation. Our model might be also applicable to other solid tumors harboring oncogenic Kras mutations.

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Kumar, Bijender, Lei Zhang, Yunan Miao, Gerald Wuenschell, Allen Lin, Vinod Pullarkat, Guido Marcucci, Robert Hickey, and Ching-Cheng Chen. "Proteomics Profiling of Leukemia Derived Exosomes: A Potential Role in Leukemic Transformation." Blood 126, no. 23 (December 3, 2015): 3857. http://dx.doi.org/10.1182/blood.v126.23.3857.3857.

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Abstract Exosomes are small sized vesicles (30-100nm) actively secreted by a wide variety of cells in body fluids. Exosomes are known carriers of mRNA, microRNA and proteins, and have been shown to transform the recipient cells. Exosomes are important in regulation of physiological processes in our body and depending on their content they can induce activation, proliferation, differentiation or apoptosis of the recipient cells. Increasingly, exosomes are studied for their potential as both indicators of disease initiation, progression, drug resistance and as a prospective new treatment approach. We hypothesize that identifying proteins that are differentially expressed on the exosomes among normal and leukemic cells will provide signature biomarkers and offer insight into the mechanism(s) contributing to the transformation of the leukemic niche. Exosomes were extracted from the serum samples of healthy and acute myeloid leukemia (AML) patients and were found to be more abundant in AML patients (p=0.049). Western blot analysis of the AML derived exosomes pellet confirmed the presence of CD63 and tumor susceptibility gene 101 (TSG101); two exosomal marker proteins indicating a good exosome preparation. Further, using ultracentrifugation, we prepared exosomes from the peripheral blood of two normal healthy individuals and MV411 (FLT3-ITD+) & KG1A cell lines after culturing in conditioned media in hypoxia for 48 hours. 2-Dimensional polyacrylamide gel electrophoresis (2D-PAGE) was performed from lysates of 100 µg of exosomes, and subsequently differently expressed spots were identified using 2D-PAGE analysissoftware. We identified 15 spots showing significantly up-regulated and 8 down-regulated patterns of expression in the exosomes from AML cell lines, relative to the pattern seen from the normal exosome lysates. In gel digestion with trypsin was performed, and proteins were identified in each differentially expressed spot using the Agilent 6510 QTOF Mass Spectrometer. The Scaffold Proteome Software was used to validate MS/MS based peptide and protein identifications. The Database for Annotation, Visualization and Integrated Discovery (DAVID ) v6.7 was used to interpret the biological functions of these differentially expressed proteins. In up-regulated datasets, the top enriched pathways and annotations (p<0.05) were proteins involved in: the core histone H2A/H2B/H3/H, Ras protein signal transduction, DNA packaging, telomere maintenance, translational elongation, the G1 to S phase transition, Aminoacyl-tRNA synthetase, negative regulation of programmed cell death, Transforming protein RhoA (multidrug resistance protein), lung cancer oncogene 7(HLC-7), Growth factor receptor-bound protein 2(GRB2), Nucleoside diphosphate kinase A (NME1),Transforming growth factor-beta-induced protein(TGFBI), regulation of DNA repair, and secretary granule associated Rab proteins. All these leukemia exosomal up-regulated proteins have already been known to be involved in tumor transformation. The down-regulated leukemia exosomes proteins were involved in negative regulation of cytoskeleton organization, histone methyltransferase complex, Phosphatidylinositol transfer protein, EHD1(tumor suppressor gene), Cysteine and methionine metabolism, and calcium-dependent phospholipid binding. More functional studies on the protein content of exosomes are needed to decipher their potential functions in leukemogenesis. Identification of unique exosomal protein markers from leukemia patients will allow for better identification of their role to stratify patients according to their disease status, and enable us to elucidate their specific role and dysfunctional status in hematopoietic stem cells. Disclosures No relevant conflicts of interest to declare.

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Dudenhöffer-Pfeifer, Monika, and David Bryder. "Immunoediting is not a primary transformation event in a murine model of MLL-ENL AML." Life Science Alliance 1, no. 4 (July 10, 2018): e201800079. http://dx.doi.org/10.26508/lsa.201800079.

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Although it is firmly established that endogenous immunity can prevent cancer outgrowth, with a range of immunomodulatory strategies reaching clinical use, most studies on the topic have been restricted to solid cancers. This applies in particular to cancer initiation, where model constraints have precluded investigations of immunosurveillance and immunoediting during the multistep progression into acute myeloid leukemia (AML). Here, we used a mouse model where the chimeric transcription factor MLL-ENL can be conditionally activated in vivo as a leukemic “first-hit,” which is followed by spontaneous transformation into AML. We observed similar disease kinetics regardless of whether AML developed in WT or immunocompromised hosts, despite more permissive preleukemic environments in the latter. When assessing transformed AML cells from either primary immunocompetent or immunocompromised hosts, AML cells from all sources could be targets of endogenous immunity. Our data argue against immunoediting in response to selective pressure from endogenous immunity as a universal primary transformation event in AML.

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Roe, Jae-Seok, and Christopher R. Vakoc. "C/EBPα: critical at the origin of leukemic transformation." Journal of Experimental Medicine 211, no. 1 (January 6, 2014): 1–4. http://dx.doi.org/10.1084/jem.20132530.

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Acute myeloid leukemia (AML) is a hematopoietic malignancy characterized by clonal expansion of myeloid progenitor cells. A major mechanistic theme in AML biology is the extensive collaboration among fusion oncoproteins, transcription factors, and chromatin regulators to initiate and sustain a transformed cellular state. A new study in this issue describes how the C/EBPα transcription factor is crucial for the initiation of AML induced by MLL fusion oncoproteins, but is entirely dispensable for the maintenance of established disease. These observations provide a unique glimpse into the pioneer round of regulatory events that are critical at the origin of AML formation. Furthermore, this study implies the existence of oncogene-induced positive feedback loops capable of bypassing the continuous need for certain regulators to propagate disease.

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Chen, Shuying, Qing Rao, Haiyan Xing, Jing Yu, Huan Li, Min Wang, and Jianxiang Wang. "Rac1 Gtpase Promotes Hematopoietic Stem Cell Migration, Self-Renewal and Participates in Leukemia Initiation and Maintenance." Blood 124, no. 21 (December 6, 2014): 2923. http://dx.doi.org/10.1182/blood.v124.21.2923.2923.

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Abstract Acute myeloid leukemia (AML) is a hematological malignancy resulting from the transformation of normal hematopoietic stem cell (HSC). Except for the intrinsic factors, it is acceptable that some extrinsic events from microenvironment could be the important co-factors in the development of leukemia. In addition to the specific component, as an extrinsic factor, interaction between HSC and bone marrow niche regulates HSCs fate. Disruption on the interactions also influences hematopoiesis. It has become evident that Rac members of Rho GTPases family are important molecules regulating HSCs interactions with hematopoietic microenvironment and activation of Rac1 are observed in a serials of leukemia cells. We previously reported that Rac1 is highly expressed in leukemia cells and found that activation of Rac1 GTPase lead to an increase in leukemia cells migration, chemotherapy resistance, quiescence and trafficking to bone marrow niche. Furthermore, we showed that Rac1 mediated the localization in niche is further attributable to the maintenance of LSC quiescence. In this study, we investigated the effects of active Rac1 GTPase in the transformation of HSC and determined if the activation of Rac1GTPase could promote the interaction of HSC with osteoblastic niche and further contribute to the leukomogenesis. By forced expression of a constitutively active form of Rac1 GTPase (Rac1 V12）in c-Kit+ hematopoietic stem/progenitor cell, we show that activation of Rac1 GTPase promotes cell migration, adhesion and colony formation, and also lead to an increase in the frequency of cells in quiescent state. Gene expression analysis shows that activation of Rac1 up-regulates the expression of several molecules that mediated the interaction of LSC with osteoblastic niche, as well as the cell cycle inhibitors such as p21, p27, and p57. Furthermore, we established a mouse model of acute myeloid leukemia by transduction murine c-kit+HSPC with Rac1 V12 combined with AML1-ETO9a, followed by transplantation into lethally irradiated mice. To investigate the role of Rac1 activation in leukemogenesis in vivo, we treated the AML1-ETO-Rac1 leukemia cells with Rac1 GTPase inhibitor EHT1846 and then transplanted into recipient mice. After 40 μM EHT1846 treatment, no engraftment of AML cells in recipient mice was observed. Kaplan-Meier analyses indicate that treatment with EHT1846 significantly prolongs survival of the transplanted mice. 20μM dose of EHT1846 was less effective. These data indicated that active Rac1 might be an important contributing factor to leukemogenesis. In addition, short-term homing assays showed that EHT 1846 treatment causes a marked inhibition of AML cell homing into both bone marrow and spleen as compared with controls, indicating that Rac1 mediated homing could be an important step and participated in the leukemogensis. Altogether, our data suggest that activation of Rac1 GTPase is critical for the interaction between HSCs with BM niche and even be contributed to leukemia development. Disclosures Wang: Novartis: Consultancy; Bristol Myers Squibb: Consultancy.

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Kuchenbauer, Florian, Tobias Berg, Sarah M. Mah, Milijana mirkovic-Hosle, Anisa Salmi, Jens Ruschmann, Andrew Muranyi, et al. "Mir-223 Is Dispensable for the Onset of Acute Myeloid Leukemia." Blood 116, no. 21 (November 19, 2010): 501. http://dx.doi.org/10.1182/blood.v116.21.501.501.

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Abstract Abstract 501 The functional roles of microRNAs in the development of acute myeloid leukemia (AML) are not yet clear. Due to its myeloid-specific expression, miR-223 has been one of the most-investigated miRNAs in normal and malignant hematopoiesis. However, the role of miR-223 in myeloid differentiation is not completely understood, as contradicting data exists. Genetic depletion of miR-223 led to a significant increase of myeloid progenitor cells as well as circulating hyperreactive neutrophils. Here, we investigate the role of miR-223 in the development of AML in vivo, using retroviral overexpression models of Hoxa9 with Meis1 or MN1 as two potent models of leukemic transformation in a miR-223+/+ or miR-223−/− background. In contrast to the observed high level expression of miR-223 in human CD34- bulk AML cells (p=0.0106), we could show that miR-223 was dispensable for the development of AML and did not impact on either the leukemic stem cell frequency nor the AML cell phenotype in Hoxa9-Meis1 AML cells. While these findings reveal that miR-223 is not necessary for leukemic transformation in highly aggressive AML models, we became interested if miR-223 functions rather as modulator of disease progression, especially at the early development of AML. Therefore, we investigated the role of miR-223 with regards to differentiation and self-renewal in two preleukemic model systems by retrovirally infecting miR-223−/− and miR-223+/+ BM cells with AML1-ETO and Hoxa9 respectively. Characterization of these models demonstrated that miR-223 expression is a determinant of differentiation, as miR-223−/− preleukemic cells exhibit a significant lower Mac-1 expression (p=0.0003). However, in contrast to normal miR-223−/− BM cells, which show a significantly higher colony forming capacity in methylcellulose compared to miR-233+/+ BM cells, the colony forming capacity of miR-223−/− or miR-223+/+ preleukemic cells did not significantly change. These findings demonstrate that miRNA miR-223 is hierarchically expressed in AML cells, and functionally link miR-223 to impaired differentiation rather than increased self-renewal in the initiation of AML. This indicates that miR-223 is more likely a fine tuner of leukemic development than a potent tumor suppressor or oncogenes as suggested in the literature. However, it still remains to be shown if the presence of miR-223 influences the susceptibility of preleukemic cells to convert into leukemia initiating cells. Disclosures: No relevant conflicts of interest to declare.

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Wang, Yingzi, Andrei V. Krivtsov, Amit U. Sinha, Trista North, Wolfram Goessling, Leonard I. Zon, and Scott Armstrong. "β-Catenin Determines Developmental Stage Specific Transformation by Hox Genes." Blood 114, no. 22 (November 20, 2009): 385. http://dx.doi.org/10.1182/blood.v114.22.385.385.

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Abstract Abstract 385 Leukemia stem cells (LSC) possess extensive proliferative and self-renewal potential similar to normal hematopoietic stem cells (HSC). Therefore understanding the similarities and differences between HSC and LSC is critical if LSC specific therapies are to be developed. Hox genes represent a group of genes that can influence both normal HSC and LSC self-renewal, and are critical targets of leukemogenic MLL fusion proteins. Previous reports have described the ability of Hoxa9 and Meis1a (HoxA9/M) to induce leukemia when expressed in mouse bone marrow (BM). However, whether HoxA9/M can fully recapitulate the leukemogenic activity of MLL fusion proteins remains unclear. In this study, we show that HoxA9/M, unlike MLL-AF9, fails to induce leukemia from granulocyte-macrophage progenitors (GMP) but does so from HSC. Immunophenotypic analysis and in vivo limiting dilution transplantation of HSC-derived leukemias demonstrate heterogeneity with only a subset of cells possessing leukemia-propagating activity. The LSC in this model have an immunophenotype consistent with differentiating myeloid cells. Gene expression analysis of LSC induced by MLL-AF9 expression in GMP and HoxA9/M expression in HSC demonstrate an approximately 10-fold increase in prostaglandin-endoperoxide synthase 1 (PTGS1) (also known as Cycloxygenase-1 or Cox-1) and prostaglandin E receptor 1 (PTGER1) expression. As recent studies have highlighted a critical connection between prostaglandin synthesis and Wnt/ β-catenin signaling pathway, we hypothesized that β-catenin is aberrantly activated in LSC derived from either GMP expressing MLL-AF9 or HSC expressing HoxA9/M. Western blots and immunofluorescence using an antibody specific for dephosphorylated (activated) β-catenin identified active β-catenin in MLL-AF9-driven and HoxA9/M-driven LSC but not normal GMP. These data suggested that insufficient β-catenin activity might be a contributing factor to the inability of HoxA9/M to transform GMP and thus we sought to determine if activated β-catenin cooperated to induce leukemia from GMP. We found that co-expression of HoxA9/M and activated β-catenin efficiently induced leukemia from GMP whereas neither expressed alone had leukemogenic activity. Next, we assessed if β-catenin is required for HoxA9/M-mediated leukemogenesis initiated from HSC. Conditional β-catenin loss-of-function experiments demonstrated impaired in vivo expansion of cells derived from HoxA9/M transduced HSC, and β-cat-/- cells did not induce leukemia. This defect could be rescued by expression of a constitutively active form of β-catenin. Finally, we demonstrate that continued β-catenin activity is required for LSC maintenance by chemical suppression of the β-catenin pathway with indomethacin (a cox-1/cox-2 inhibitor), which shows remarkable selective elimination of the LSC fraction in mice transplanted with HoxA9/M transduced HSC. Our gain and loss-of-function studies demonstrate that β-catenin activity is required for leukemia initiation from HSC, and that constitutively active β-catenin can cooperate with HoxA9/M to efficiently transform GMP. Thus, Wnt/β-catenin activity makes cells permissive to transformation, which suggests that its restricted activation to stem cell populations in normal hematopoietic development limits the permissiveness of developmental cell types to transformation by specific oncogenes. These data have important implications for tumor development in other tissues/organs and for the development of β-catenin pathway antagonists in AML. Disclosures: No relevant conflicts of interest to declare.

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Ropa, James, Nirmalya SAHA, and Andrew G. Muntean. "SETDB1 Represses Hox Gene Expression and Suppresses Acute Myeloid Leukemia." Blood 132, Supplement 1 (November 29, 2018): 1320. http://dx.doi.org/10.1182/blood-2018-99-117690.

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Abstract Epigenetic regulators play an important role in normal and malignant hematopoiesis. Epigenetic deregulation of the HOXA gene cluster drives transformation of about 50% of acute myeloid leukemia (AML), including those harboring MLL rearrangements and NPM mutations, as well as others. Expression of Hoxa9 and its co-factor Meis1 is sufficient to transform bone marrow into a lethal AML in mouse models. We previously demonstrated that the pro-leukemic genes Hoxa9 and Meis1 are critically regulated by the histone H3 Lysine 9 (H3K9) methyltransferase SETDB1. Recent studies show that SETDB1 is required for normal hematopoiesis and MLL-AF9 mediated leukemia (Koide, et al. Blood 2016). Our lab recently demonstrated that SETDB1 negatively regulates the expression of HoxA9 and Meis1 through deposition of promoter H3K9 methylation in MLL-AF9 AML cells (Ropa et al. Oncotarget 2018). Consistent with these data, HOXA9 and MEIS1 expression negatively correlates with SETDB1 expression in AML patient samples. Therefore, we investigated the biological impact of SETDB1 on AML. We first noted that expression of SETDB1 in AML patient samples is significantly lower compared to normal hematopoietic cells. Further, higher SETDB1 expression correlated with a significantly better overall survival (p=0.003) and lower expected hazard (HR=0.9/100RSEM; p=0.009) in AML patients compared with lower SETDB1 expression. These data are consistent with SETDB1 negatively regulating pro-leukemic genes and suggests that SETDB1 expression may be correlated with AML patient prognosis. We tested this directly by expressing high levels of SETDB1 in AML cells. Ex vivo assays show that retroviral overexpression of SETDB1 in MLL-AF9 AML cells leads to cell differentiation, decreased leukemia colony formation, and decreased cell proliferation. Consistent with the AML patient data, overexpression of SETDB1 significantly delays MLL-AF9 mediated leukemogenesis in vivo (p=0.01). Further, we observed a strong selective pressure against exogenous SETDB1 expression in moribund mice. Transcriptome analyses demonstrate that SETDB1 globally represses Hox and pluripotency gene programs. Strikingly, we found that SETDB1 represses many of the same genes that exhibit reduced promoter H3K9me3 in AML patient samples relative to CD34+ cells. These data point to a role for SETDB1 in negatively regulating pro-leukemic target genes and suppressing AML. We also explored how chemical and genetic inhibition of H3K9 methylation and Setdb1 affects AML initiation and maintenance. We first confirmed the previously reported requirement for Setdb1 in AML cell lines by genetically deleting both alleles of Setdb1 in MLL-AF9 cells, which resulted in a complete arrest of proliferation (Koide, et al. Blood 2016). Combined with our data presented above, these results suggest a narrow window of SETDB1 expression is maintained in AML cells. To achieve reduced (but not complete loss of) activity, we investigated how small molecule inhibition of H3K9 methylation (UNC0638) or shRNA mediated knock down of Setdb1 affects AML initiation. We observed increased ex vivo colony formation of normal ckit+ bone marrow cells upon shRNA mediated knockdown of Setdb1 or upon UNC0638 treatment. We hypothesized that this expansion of colony forming unit potential of hematopoietic cells may translate to increased transformation potential by leukemic oncogenes. Indeed, cells pretreated with UNC0638 followed by retroviral transduction with MLL-AF9 exhibit significantly higher capacity for leukemic colony formation than vehicle treated cells. These data are consistent with H3K9 methylation repressing genes required for AML transformation. Our data identified a narrow window of expression of SETDB1 in AML patient samples. SETDB1 expression is reduced in AML patients relative to normal cells and chemical inhibition of H3K9 methylation expands the pool of cells amenable to MLL-AF9 mediated transformation ex vivo. While inhibition of SETDB1 and other H3K9 methyltransferases has been suggested as a possible therapeutic strategy, our data suggests this may also prime bone marrow cells for transformation by inhibiting epigenetic processes that repress pro-leukemic target genes. Further investigation of the roles of SETDB1 and H3K9 methylation levels is necessary to determine the value of these epigenetic modifiers as therapeutic targets in AML and is currently ongoing. Disclosures No relevant conflicts of interest to declare.

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Dong, Fang, Haitao Bai, Xiaofang Wang, Shanshan Zhang, Zhao Wang, Miner Xie, Sen Zhang, et al. "Mouse acute leukemia develops independent of self-renewal and differentiation potentials in hematopoietic stem and progenitor cells." Blood Advances 3, no. 3 (February 7, 2019): 419–31. http://dx.doi.org/10.1182/bloodadvances.2018022400.

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Abstract The cell of origin, defined as the normal cell in which the transformation event first occurs, is poorly identified in leukemia, despite its importance in understanding of leukemogenesis and improving leukemia therapy. Although hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) were used for leukemia models, whether their self-renewal and differentiation potentials influence the initiation and development of leukemia is largely unknown. In this study, the self-renewal and differentiation potentials in 2 distinct types of HSCs (HSC1 [CD150+CD41−CD34−Lineage−Sca-1+c-Kit+ cells] and HSC2 [CD150−CD41−CD34−Lineage−Sca-1+c-Kit+ cells]) and 3 distinct types of HPCs (HPC1 [CD150+CD41+CD34−Lineage−Sca-1+c-Kit+ cells], HPC2 [CD150+CD41+CD34+Lineage−Sca-1+c-Kit+ cells], and HPC3 [CD150−CD41−CD34+Lineage−Sca-1+c-Kit+ cells]) were isolated from adult mouse bone marrow, and examined by competitive repopulation assay. Then, cells from each population were retrovirally transduced to initiate MLL-AF9 acute myelogenous leukemia (AML) and the intracellular domain of NOTCH-1 T-cell acute lymphoblastic leukemia (T-ALL). AML and T-ALL similarly developed from all HSC and HPC populations, suggesting multiple cellular origins of leukemia. New leukemic stem cells (LSCs) were also identified in these AML and T-ALL models. Notably, switching between immunophenotypical immature and mature LSCs was observed, suggesting that heterogeneous LSCs play a role in the expansion and maintenance of leukemia. Based on this mouse model study, we propose that acute leukemia arises from multiple cells of origin independent of the self-renewal and differentiation potentials in hematopoietic stem and progenitor cells and is amplified by LSC switchover.

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Chan, Lai N., Mark A. Murakami, Rebecca Caesar, Christian Hurtz, Kohei Kume, Teresa Sadras, Seyedmehdi Shojaee, et al. "Signaling Input from Divergent Pathways Subverts Malignant B-Cell Transformation." Blood 134, Supplement_1 (November 13, 2019): 3944. http://dx.doi.org/10.1182/blood-2019-130774.

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Background and significance: Normal B-cells constantly exchange information with their environment and depend on external cues for proliferation and survival that engage multiple divergent pathways (e.g. cytokine receptors; B-cell receptor, BCR). The dependency of normal B-cells on signaling input from a diverse repertoire of surface receptors is in contrast to transforming oncogenes that engage one single pathway. Beyond the established concept that diverse signal input from multiple cell surface receptors becomes dispensable in transformed cells, we here provide evidence that inactivation of divergent pathways that are not aligned with the principal oncogenic driver represents a critical step during malignant transformation. Tracking early stages of leukemia-initiation, we identified convergence on one principal oncogenic driver and inactivation of diverging pathways as critical events during B-cell transformation. Our results support a scenario in which reactivation of divergent and potentially conflicting signaling pathways represents a powerful barrier to malignant transformation. Here we studied the interaction of STAT5- and ERK-signaling pathways during normal B-cell development and malignant B-cell transformation, and found that convergence on one principal oncogenic driver represents a critical event during B cell transformation and a previously unrecognized vulnerability. Results: Our analysis of 987 patient-derived B-cell acute lymphoblastic leukemia leukemia (B-ALL) samples revealed that individual mutations did not promote leukemogenesis unless they converged on one single oncogenic pathway. Mutations that were not aligned with the central oncogenic driver would activate divergent pathways and subvert malignant transformation. Oncogenic lesions in B-ALL frequently mimic survival and proliferation signals downstream of cytokine receptors (STAT5) or the B cell receptor (ERK). STAT5- (286 cases) and ERK- (386 cases) activating lesions were frequently found but rarely co-occurred in the same sample (35 cases; P=2.5E-16). Single-cell mutation and phosphoprotein analyses revealed that even in these rare cases, oncogenic STAT5- or ERK-activation were mutually exclusive and segregated to competing clones. STAT5 and ERK engaged opposing biochemical and transcriptional programs orchestrated by MYC and BCL6, respectively. Genetic reactivation of the divergent (suppressed) pathway came at the expense of the principal oncogenic driver and reversed malignant transformation. Conversely, Cre-mediated deletion of divergent pathway components triggered leukemia-initiation and accelerated development of fatal disease. Thus, persistence of divergent signaling pathways represents a powerful barrier to malignant transformation and convergence on one principal driver defines a key event during leukemia-initiation. Proof-of-concept studies in patient-derived B-ALL cells revealed that small molecule agonists of STAT5 or ERK to reactivate the suppressed divergent circuits subvert oncogenic signaling and strongly synergized with direct inhibition of the principal oncogenic driver. Hence, pharmacological reactivation of divergent pathways can be leveraged as a previously unrecognized strategy to deepen treatment responses and to overcome drug-resistance. Current treatment approaches for drug-resistant cancer are focused on drug-combinations to suppress the central oncogenic driver and multiple alternative pathways. Here, we introduce a concept based on inhibition of the principal driver combined with pharmacological reactivation of divergent pathways Conclusions: These results provide evidence that inactivation of divergent pathways that are not aligned with the principal oncogenic driver represents a critical step during malignant transformation. Unlike B-ALL, where reactivation of a divergent pathway suppresses the principal pathway and compounds toxicity, activation of an alternative pathway in solid tumors represents a route for survival and drug resistance. While current treatment approaches for drug-resistant cancer are focused on drug-combinations to inhibit multiple pathways, we introduce a scenario that is based on inhibition of the principal pathway combined with reactivation of divergent pathways. Figure Disclosures Wiita: UCSF: Patents & Royalties; Indapta Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Protocol Intelligence: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Izraeli:sightdx: Consultancy; novartis: Honoraria; prime oncology: Speakers Bureau. Weinstock:Celgene: Research Funding; Verastem Oncology: Research Funding.

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Aivalioti, Maria M., Tushar D. Bhagat, Aditi Paranjpe, Boris Bartholdy, Kith Pradhan, Mario Pujato, Amit Verma, and Britta Will. "PU.1-Dependent Enhancer Decommissioning Drives Transformation of Tet2 deficient Hematopoietic Stem and Progenitor Cells." Blood 136, Supplement 1 (November 5, 2020): 40. http://dx.doi.org/10.1182/blood-2020-142070.

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Acute myeloid leukemia (AML) is the most frequent leukemia in elderly individuals with a median age at diagnosis of 67 years (Juliusson et al., Blood 2009). It arises in a step-wise process and originates from hematopoietic stem cells (HSC) (Jan et al.,Sci Transl Med. 2012). Genetic and epigenetic alterations drive the formation of pre-leukemic HSC clones with altered function, which can gain dominance and eventually give rise to AML upon the acquisition of cooperating lesions (Jan et al.,Sci Transl Med. 2012). However, it is currently impossible to predict which healthy elderly individuals with clonal hematopoiesis will eventually develop myeloid malignancies, as the pathways to leukemia are unknown. Heterozygous inactivating mutations of the epigenetic regulator Ten-Eleven Translocation-2 (TET2) are commonly found in patients with AML, yet also in a remarkable fraction of healthy elderly individuals in whom it is associated with clonal hematopoiesis (Busque, et al Nat Genet. 2012). These observations and studies in Tet2-deficient mice strongly suggest that TET2 inactivation is an early event in the pathogenesis of myeloid malignancies, but is not sufficient to fully transform HSC (Moran-Crusio et al., Cancel Cell 2011). TET2 cooperates with several transcription factors to regulate hematopoiesis (Rasmussen et al., Genome Res 2019), one of which is PU.1 (de la Rica et al., Genome Biol. 2013), an essential transcription factor governing normal hematopoiesis (Iwasaki et al., Blood 2005). In humans, PU.1 activity or expression is only moderately impaired in the majority of AML patients, and remarkably, also in aged HSC (Will et al., Nat Med. 2015), underscoring the essentiality of PU.1. Importantly, PU.1 target genes are frequently found hypermethylated in AML (Sonnet et al., Genome Med. 2014, Kaasinen et al., Nat Commun. 2019), suggesting a profound epigenetic inactivation of the PU.1 network. We hypothesized that moderate impairment of PU.1 abundance, as found in AML, can cooperate with loss-of-function mutations of Tet2 to initiate malignancy. We developed a novel tissue-specific compound mutant mouse model carrying heterozygous deletion of an upstream regulatory element (URE) of Pu.1 along with Tet2 deletion (Vav-iCre+ PU.1URΕ∆/+Tet2+/flox; Vav-iCre+ PU.1URΕ∆/+Tet2flox/flox). While none of the single mutant mice developed AML, compound mutant mice developed aggressive myeloid leukemia whose penetrance and latency exhibited Tet2 dose dependency. The disease presented with leukocytosis, anemia and splenomegaly. By cell morphology analysis of the peripheral blood, bone marrow and spleen, the leukemic mice exhibited accumulation of differentiation-blocked myeloblasts, myelocytes and/or metamyelocytes, that was confirmed using detailed myeloid differentiation markers, distinguishing the disease in immature or mature AML. Furthermore, gold standard in vitro and in vivo assays, assessing both self-renewal and differentiation capacity of double mutant mice-derived cells, revealed that the expanded differentiation-blocked stem and progenitor cells bear aberrant self-renewal and disease-initiating capacities. Comprehensive molecular profiling by next generation sequencing of disease-initiating cells uncovered a substantial overlap with human AML, such as functional GF1b loss with concomitant overexpression of CD90/Thy1 (Thivakaran et al., Haematologica 2018). Importantly, our analyses also revealed transcriptional dysregulation, hypermethylation of PU.1 regulated enhancers with concomitant loss of enhancer activity and alterations in chromatin accessibility of particularly genes co-bound by PU.1 and TET2. Current efforts focus on identifying key effectors of the dysregulated PU.1/TET2 sub-network driving malignant transformation in clonal hematopoiesis. Our collected data provide proof of concept that moderate PU.1 dose impairment can functionally cooperate with the inactivation of Tet2 in the initiation of myeloid leukemia and uncovers a likely unifying AML pathomechansim. Disclosures Will: Novartis Pharmaceuticals: Other: Service on advisory boards, Research Funding.

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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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Hurtz, Christian, Cihangir Duy, Leandro Cerchietti, Eugene Park, Weimin Ci, Srividya Swaminathan, Soo-mi Kweon, et al. "BCL6 Is Required for Leukemia-Initiation and Self-Renewal Signaling in Chronic Myeloid Leukemia." Blood 114, no. 22 (November 20, 2009): 2167. http://dx.doi.org/10.1182/blood.v114.22.2167.2167.

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Abstract Abstract 2167 Poster Board II-144 The transcriptional repressor BCL6 was discovered as a potent protooncogene in diffuse large B-cell lymphoma (DLBCL) and is also expressed in normal germinal center (GC) B cells. BCL6 protects DLBCL and GC B cells against DNA damage-induced apoptosis by suppressing target genes including p53, p21, ATR and p27. In preliminary experiments for this study, we found that treatment of BCR-ABL1-driven Ph+ ALL cells with Imatinib results in a 60- to 90-fold up-regulation of BCL6, reaching protein levels as high as in DLBCL and GC B cells. Since the oncogenic BCR-ABL1 kinase also drives leukemic transformation in CML cells, we studied regulation of BCL6 in human CML cells upon TKI-treatment. Like in Ph+ ALL, TKI-treatment resulted in rapid BCL6 mRNA and protein upregulation in CML cells, albeit to lesser degree than in Ph+ ALL and DLBCL. We hypothesized that upregulation of BCL6 in CML cells in response to TKI-treatment serves the same function as in DLBCL and protects from p53-mediated apoptosis. Through transcriptional repression of p53, p21, ATR and p27 as in DLBCL, upregulation of BCL6 in response to TKI-treatment would represent a defense mechanism of CML cells to evade TKI-induced cell death. We tested the role of BCL6 in CML in a genetic loss-of-function experiment: To this end, myeloid progenitor cells from bone marrow of BCL6+/+ and BCL6-/- mice were transformed with BCR-ABL1. Compared to their BCL6+/+ counterparts, BCL6-/- mouse CML cells were highly sensitive to Imatinib-treatment. In a complementary approach, we treated human CML cells with either Imatinib alone or with a novel BCL6 peptide inhibitor (Retro-inverso BCL6-peptide inhibitor, RI-BPI). Consistent with findings in BCL6-/- mouse CML cells, also human CML cells were significantly sensitized to Imatinib by BCL6 inhibition (using 5 mmol/l RI-BPI). Of note, the BCL6 inhibitory peptide RI-BPI alone induced no significant toxicity in human CML cells but synergized with Imatinib-treatment. Since RI-BPI alone had no cytotoxic effect on CML cells, we tested whether loss of BCL6 function affects self-renewal capacity of CML cells. This possibility was explored in two complementary colony formation assays: In one set of experiments, we tested the ability of BCL6+/+ and BCL6-/- mouse CML cells to form colonies in semisolid methylcellulose agar. The comparison between BCL6+/+ (94 ± 11 colonies) and BCL6-/- (<1 ± 1 colonies; p<0.004) mouse CML cells revealed that BCL6 represents an absolute requirement for self-renewal signaling in CML cells. In line with these results, we observed that BCL6-/- mouse CML cells undergo replicative senescence and cell cycle arrest under cell culture conditions after ∼2 months, whereas leukemic growth proceeds undiminished in BCL6+/+ CML cells. These results were confirmed studying BCL6 function in human CML cells using RI-BPI. Despite lack of toxicity, 5 mmol/l RI-BPI drastically reduced the number of colonies that were formed by human CML cells (980 ± 79 vs 262 ± 174; p= 0.001). Based on these findings we conclude that inhibition of BCL6 using a novel peptide inhibitor effectively disrupts self-renewal signaling and may be developed as a novel therapeutic approach for leukemia stem cell eradication in CML. Disclosures: No relevant conflicts of interest to declare.

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Ozpolat, Bulent, Ugur Akar, Magaly Barria, and Gabriel Lopez-Berestein. "PKCδ Regulates Eukaryotic Initiation Factor eIF2α through PKR during Retinoic Acid-Induced Myeloid Cell Differentiation." Blood 108, no. 11 (November 16, 2006): 1928. http://dx.doi.org/10.1182/blood.v108.11.1928.1928.

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Abstract Dysregulation of mRNA translation can contribute to malignant transformation. Translation initiation is a rate limiting step of mRNA translation and protein synthesis and plays a critical role in regulation of cell growth, proliferation and differentiation. We previously reported that ATRA induces translational suppression through multiple posttranscriptional mechanisms during terminal cell differentiation detected by proteomic analysis (Harris et al, Blood, 104 (5) 2004). Here we investigated the regulation of translation initiation and the role of eIF2α during terminal differentiation of myeloid leukemia cells. We found that ATRA and other granulocytic differentiation inducing agents, such as dimethyl sulfoxide (DMSO), arsenic trioxide (ATO) induce phosphorylation of eIF2α on serine 51 in promyelocytic leukemia (NB4) cells, indicating the suppression of translation initiation. However, monocytic/macrophagic differentiation of NB4 cells by phorbol 12-myristate 13-acetate (phorbol ester, PMA), or by ATRA in U937 and THP-1 myelomonoblastic myeloid leukemia (AML) cells, was not accompanied with induction of eIF2α phosphorylation. ATRA, ATO or DMSO-induced granulocytic differentiation closely correlated with induction of expression and phosphorylation/activation of protein kinase C-delta (PKCδ) on threonin 505 and serine 643 in NB4 cells. The specific PKCδ inhibitor, rottlerin, markedly inhibited ATRA-induced expression and phosphorylation (serin 51) of eIF2a in NB4 cells. Rottlerin reduced phosphorylation of eIF2α expression not only in the leukemia cells but also in solid tumor cells such as breast (MCF7) and pancreatic (Panc28) cancer cells. Because protein kinase R (PKR) has been shown to inhibit mRNA translation by inducing phosphorylation of eIF2α, we also examined whether this pathway is involved in ATRA-induced phosphorylation of eIF2α and whether it is downstream of PKCδ. We observed that ATRA induces expression and phosphorylation/activation of PKR in NB4 cells. Rottlerin inhibited ATRA-induced expression and activity of PKR , suggesting that activity of PKR is regulated by PKCδ in response to ATRA in NB4 cells. Overall, our data suggest that retinoic acid suppresses translation initiation through PKCδ/PKR/eIF2α pathway during granulocytic but not monocytic differentiation of acute myeloid leukemia cells. These results revealed a novel role of ATRA in granulocytic cell differentiation of myeloid cells. Because malignant cells usually have hyperactivated mRNA translation, targeting translational factors/regulators of initiation may offer new strategies for the treatment of myeloid leukemia cells.

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Trinh, Long Xuan, Young Kwang Chae, Preetesh Jain, Susan Lerner, Ohad Benjamini, Xuemei Wang, and Michael J. Keating. "Second Cancers Affect the Clinical Outcomes of Patients with Chronic Lymphocytic Leukemia (CLL) Treated with Frontline FCR-Based Therapy." Blood 120, no. 21 (November 16, 2012): 2900. http://dx.doi.org/10.1182/blood.v120.21.2900.2900.

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Abstract Abstract 2900 Increasing use of chemo-immunotherapy in CLL has raised concerns over the carcinogenic effect of FCR regimen. Fludarabine based regimen in relapsed CLL has been reported to be predisposed to a higher risk of other cancers as compared to healthy individual. However, an association between the CLL treatment and the development of other cancers is not evident in the frontline FCR setting. We retrospectively studied the frequency, characteristics, and clinical outcomes of second malignancy in patients with CLL who were treated with frontline FCR-based therapy at University of Texas, MD Anderson Cancer Center from 2004 to 2010. Patients who developed other malignancies after the initiation of FCR-based therapy were considered as patients with second cancer post CLL treatment (n=39). Patients who had history of other malignancies before the initiation of FCR-based therapy were considered as patients with prior malignancy history (n=90). Patients who neither have history of other cancers nor patients developed second cancer during the study period were assigned as patients without second cancer group (n=82). There were 24 patients (n=24) who experienced Richter's transformation post FCR-based therapy. The overall survival (OS) was measured from the initiation of CLL treatment until death from any causes or last follow-up. Progression-free survival (PFS) was measured from the initiation of CLL treatment until disease progression, relapse or death. Kaplan-Meier method was used to estimate outcomes in four groups. Cox-proportional hazard regression model was used to assess the association between patient characteristics and survival outcomes. Pre-treatment characteristics were similar among these groups except for chromosome abnormalities. Ten patients (44%) out of 24 patients with Richter's transformation had TP53 gene mutation (p <0.05). Ninety patients (38%) of 235 patients had prior malignancy history includes non-melanoma skin cancer (n=32, 36%), melanoma (n=12, 13%), prostate cancer (n=15, 17%), colon cancer (n=5, 6%), renal cancer (n=2, 2%) and follicular lymphoma (n=1, 1%). Among 145 patients without prior malignancy history, there were 39 patients (27%) who developed second cancer including other leukemia, and 24 patients (17%) who developed Richter's transformation after the FCR-based therapy. The median time from initiation of FCR-based therapy to the development of Richter' transformation was 13.53 months (range, 1.63 to 40.87). There was no patient with prior history of other malignancies had recurrent cancers during the study. Thirty nine patients who developed second cancer includes non-melanoma skin (n=10, 26%), melanoma (n=2, 5%), head and neck (n=1, 2.5), Merkel cell (n=1, 2.5%), prostate (n=6, 15%), breast (n=1, 2.5%), lung (n=2, 5%) renal (n=1, 2.5%), gastric (n=2, 5%), liver (n=1, 2.5%), Hodgkin (n=1, 2.5%), and therapy-related MDS-AML (n=11, 28%). The estimated median PFS in the whole cohort was 4.57 years (95% CI: 3.678 to 5.462). PFS in patients without second cancer was not reached (NR) compared to 3.31 years (95% CI: 2.178 to 3.542) in patients with second cancer post CLL treatment (p<0.05). PFS in patients with Richter's transformation was 1.02 years (95% CI: .625 to 1.415). The median follow up duration for all 235 patients was 3.29 years (range, 0.08–8.51 years) with total of 61 deaths. The median overall survival in either group of patients with prior malignancy history or in patients without second cancers were not reached (NR) compared with 3.81 years (95% CI: 2.231 to 5.389) in patients with second cancer (p < 0.05). OS in patients with Richter's transformation was 3.84 years (95% CI, 1.434 to 6.246) There were 8 patients (10%) who died among 82 patients without second cancer as compared to 20 patients (49%) who died among 39 patients with second cancer (p <0.05). Second cancers in patients with CLL treated with FCR regimen are significantly associated with inferior clinical outcomes. We observed the high incidence of skin cancer, prostate and Richter's transformation in patients with CLL who underwent treatment with frontline FCR-based therapy. Further studies are warranted to determine the association between FCR regimen and the development of second cancer, especially Richter's transformation. Disclosures: No relevant conflicts of interest to declare.

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Xu, Dan, Xia Liu, Wen-Mei Yu, Howard J. Meyerson, Caiying Guo, Stanton L. Gerson, and Cheng-Kui Qu. "Non–lineage/stage-restricted effects of a gain-of-function mutation in tyrosine phosphatase Ptpn11 (Shp2) on malignant transformation of hematopoietic cells." Journal of Experimental Medicine 208, no. 10 (September 19, 2011): 1977–88. http://dx.doi.org/10.1084/jem.20110450.

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Activating mutations in protein tyrosine phosphatase 11 (Ptpn11) have been identified in childhood acute leukemias, in addition to juvenile myelomonocytic leukemia (JMML), which is a myeloproliferative disorder (MPD). It is not clear whether activating mutations of this phosphatase play a causal role in the pathogenesis of acute leukemias. If so, the cell origin of leukemia-initiating stem cells (LSCs) remains to be determined. Ptpn11E76K mutation is the most common and most active Ptpn11 mutation found in JMML and acute leukemias. However, the pathogenic effects of this mutation have not been well characterized. We have created Ptpn11E76K conditional knock-in mice. Global Ptpn11E76K/+ mutation results in early embryonic lethality. Induced knock-in of this mutation in pan hematopoietic cells leads to MPD as a result of aberrant activation of hematopoietic stem cells (HSCs) and myeloid progenitors. These animals subsequently progress to acute leukemias. Intriguingly, in addition to acute myeloid leukemia (AML), T cell acute lymphoblastic leukemia/lymphoma (T-ALL) and B-ALL are evolved. Moreover, tissue-specific knock-in of Ptpn11E76K/+ mutation in lineage-committed myeloid, T lymphoid, and B lymphoid progenitors also results in AML, T-ALL, and B-ALL, respectively. Further analyses have revealed that Shp2 (encoded by Ptpn11) is distributed to centrosomes and that Ptpn11E76K/+ mutation promotes LSC development, partly by causing centrosome amplification and genomic instability. Thus, Ptpn11E76K mutation has non–lineage-specific effects on malignant transformation of hematopoietic cells and initiates acute leukemias at various stages of hematopoiesis.

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Sengupta, Amitava, Ashley M. Ficker, Susan K. Dunn, Malav Madhu, and Jose A. Cancelas. "Bmi1 reprograms CML B-lymphoid progenitors to become B-ALL–initiating cells." Blood 119, no. 2 (January 12, 2012): 494–502. http://dx.doi.org/10.1182/blood-2011-06-359232.

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The characterization and targeting of Philadelphia chromosome positive (Ph+) acute lymphoblastic leukemia (ALL)–initiating cells remains unresolved. Expression of the polycomb protein Bmi1 is up-regulated in patients with advanced stages of chronic myelogenous leukemia (CML). We report that Bmi1 transforms and reprograms CML B-lymphoid progenitors into stem cell leukemia (Scl) promoter-driven, self-renewing, leukemia-initiating cells to result in B-lymphoid leukemia (B-ALL) in vivo. In vitro, highly proliferating and serially replatable myeloid and lymphoid colony-forming cultures could be established from BCR-ABL and Bmi1 coexpressing progenitors. However, unlike in vivo expanded CML B-lymphoid progenitors, hematopoietic stem cells, or multipotent progenitors, coexpressing BCR-ABL and Bmi1 did not initiate or propagate leukemia in a limiting dilution assay. Inducible genetic attenuation of BCR-ABL reversed Bmi1-driven B-ALL development, which was accompanied by induction of apoptosis of leukemic B-lymphoid progenitors and by long-term animal survival, suggesting that BCR-ABL is required to maintain B-ALL and that BCR-ABL and Bmi1 cooperate toward blast transformation in vivo. Our data indicate that BCR-ABL targeting itself is required to eradicate Ph+/Bmi1+ B-ALL–initiating cells and confirm their addiction to BCR-ABL signaling.

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Oancea, Claudia, Brigitte Rüster, Jessica Roos, Afsar Ali Mian, Tatjana Micheilis, Hannelore Held, Anjali Dubey, Hubert Serve, Reinhard Henschler, and Martin Ruthardt. "T(15;17)-PML/RAR-Induced Leukemogenesis: Long-Term Repopulating Hematopoietic Stem Cells as the Initial Target and More Mature Progenitors as the Potential Targets for Final Leukemic Transformation." Blood 114, no. 22 (November 20, 2009): 3980. http://dx.doi.org/10.1182/blood.v114.22.3980.3980.

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Abstract Abstract 3980 Poster Board III-916 Stem cells have been shown to play an important role in the pathogenesis and maintenance of a significant number of malignancies, including leukemias. Similar to normal hematopoiesis the AML cell population is thought to be hierarchically organized. According to this model, only a few stem cells (LSC) are able to initiate and maintain the disease. The inefficient targeting of the leukemic stem cells (LSC) is considered responsible for relapse after the induction of complete hematologic remission (CR) in AML. Acute promyelocytic leukemia (APL) is a subtype of AML characterized by the t(15;17) translocation and expression of the PML/RARα fusion protein. Treatment of APL with all-trans retinoic acid (t-RA) as monotherapy induces CR, but not molecular remission (CMR), followed by relapse within a few months. In contrast arsenic as monotherapy induces high rates of CR and CMR followed by a long relapse-free survival. We recently have shown that in contrast to t-RA, arsenic efficiently targets PML/RAR-positive stem cells, whereas t-RA increases their proliferation. For a better characterization of LSC in APL which has to be targeted for an efficient eradication of the disease we wanted to characterize the leukemia-initiating cell and the cell population able to maintain the disease in vivo. The model was based on a classical transduction/transplantation system of murine Sca1+/lin- HSC combined with a novel approach for the enrichment of transformed cells with long-term stem cell properties. We found that PML/RAR induced leukemia from the Sca1+/lin- HSC with a frequency of 40% and a long latency of 8-12 months independently of its capacity to increase dramatically replating efficiency and CFU-S12 potential as expression of the differentiation block and proliferation potential of derived committed progenitors. Based on the hypothesis that PML/RAR exerts its leukemogenic effects on only a small proportion of the Sca1+1/lin- population, we proceeded to select and to amplify rare PML/RAR-positive cells with the leukemia-initiating potential, by a negative selection of cell populations with proliferation potential without long term stem cell-capacity (LT). Therefore we expressed PML/RAR in Sca1+/lin- cells and enriched this population for LT- (lin-/Sca1+/c-Kit+/Flk2-) and ST-HSC (lin-/Sca1+/c-Kit+/Flk2+). After a passage first in semi-solid medium for 7 days and subsequent transplantation into lethally irradiated mice, cells from the ensuing CFU-S day12 were again transplanted into sublethally recipient mice. After 12 to 36 weeks, 6/6 mice developed acute myeloid leukemia without signs of differentiation in the group transplanted with the lin-/Sca1+/c-Kit+/Flk2- population but not from that transplanted with lin-/Sca1+/c-Kit+/Flk2+ cells. This leukemia was efficiently transplanted into secondary recipients. The primary leukemic cell population gave origin to 6 clearly distinct subpopulations defined by surface marker pattern as an expression of populations with distinct differentiation status, able - after sorting - to give leukemia in sublethally irradiated recipients: Sca1+/c-Kit+/CD34- (LT-HSC), Sca1+/c-Kit+/CD34+ (ST-HSC), Sca1-/c-Kit+, B220lo/GR1+/Mac1+, B220hi/GR1+/Mac1+, B220-/Gr1-/Mac1-. Interestingly, all leukemias from the different population presented an identical phenotype. These findings strongly suggest that there is a difference between a leukemia-initiating (L-IC) and leukemia-maintaining (L-MC) cell population in the murine PML/RAR leukemia model. In contrast to the L-IC, represented by a very rare subpopulation of primitive HSC, recalling a hierarchical stem cell model, the L-MC is represented by a larger cell population with a certain grade of phenotypical heterogeneity, but a high grade of functional homogeneity recalling a stochastic cancer induction model. Disclosures: No relevant conflicts of interest to declare.

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Wang, Xiaoli, Cing Siang Hu, Joseph Tripodi, Vesna Najfeld, Bruce Petersen, Raajit K. Rampal, Noushin Farnoud, Christopher Famulare, John Mascarenhas, and Ronald Hoffman. "Myeloproliferative Neoplasm (MPN) Blastic Transformation Occurs at the Level of Hematopoietic Stem Cells." Blood 132, Supplement 1 (November 29, 2018): 101. http://dx.doi.org/10.1182/blood-2018-99-117348.

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Abstract Myeloproliferative neoplasm-blast phase (MPN-BP) and de novo acute myeloid leukemia (AML) each have distinct mutational patterns and clinical courses. MPN-BP patients have a particularly dismal prognosis with a median survival of less than 6 months with currently available therapies. So far, the cellular hierarchy that characterizes MPN-BP and the evolution of various leukemia-initiating clones (LIC) in MPN-BP have not been well delineated. We therefore established an in vivo MPN-BP xenograft model to address these questions. Among the 22 patients with MPN-BP studied 11 were cytogenetically normal while the remainder had multiple chromosomal abnormalities including del(5), del(20q), del(14), +1q, del(17p). 86% of the patients had at least 2 myeloid malignancy gene mutations including JAK2, ASXL1,TET2, MPL, SF3B1, RUNX1, U2AF1, PTPN11, IDH1/2, SRSF2 and TP53. These findings indicate that MPN-BP is characterized by multiple mutational events and cytogenetic abnormalities. T cell-depleted mononuclear cells from 8 of 14 patients engrafted in NSG mice {>0.5% hCD45+ cells in bone marrow (BM)}. Among them, samples from 6 patients resulted in a high degree of hCD45+ cell chimerism (34.6±6.4% in BM) and recapitulated numerous aspects of MPN-BP within 4 months, including the presence of at least 20% hCD45dimCD33+ cells or hCD34+ cells, or at least 20% blasts as detected by morphological examination of the marrow and leukemia cell dissemination to the spleen and PB. These mice had a 2.8±0.6- fold increase in splenic weight as compared to mice receiving PBS alone. The leukemic mice were characterized by reduced blood counts, suggesting that MPN-BP cells suppressed normal murine hematopoiesis, or led to cytopenias due to hyper-splenism. Moreover, the greater degrees of blast cell chimerism and the higher frequency of leukemia initiating cells as determined by limiting dilution analyses correlated with a shorter time to leukemia initiation and an inferior clinical outcome of the transplanted NSG mice. Grafts from each of these 6 MPN-BP patients produced a large number of donor-derived myeloid cells and a smaller number of lymphoid cells (mostly CD3+ and few CD19+). Cells belonging to each of these lineages and leukemic cells in primary recipients produced from Pts 4, 5, 6 and 11 had an identical proportion of chromosomally abnormal and mutated cells as primary cells [Pt 4: JAK2V617F, TET2 and PHF6; Pt 5 and 11: Del (20q), +8; Pt 6: +1q, del(17p)], except that a small proportion of T cells from Pts 5 and 11 lacked chromosomal abnormalities. Furthermore, the degree of MPN-BP engraftment and leukemic cell burden increased with the subsequent 3 serial transplantations even when the recipients received progressively smaller numbers of MPN-BP cells from the prior recipient. Primary Pt 6 originally had a JAK2V617F+ PV but lost JAK2V617F at the time the MPN-BP occurred at which time there were two clonal cell populations, one with +1q (12%) and the other del(17p) (80%), the site of the TP53 gene, as well as normal cells (8%). In the primary recipient NSG the donor derived cells were JAK2V617F- but contained +1q (1%) and del(17p) (98.5%) and cytogenetically normal (0.5%). +1q and JAK2V617F were not observed, while cells containing the TP53 deletion alone were detected in donor derived leukemic cells, mature myeloid and T cells in the secondary and subsequent serial recipients. Furthermore, del(17p) was found in phenotypically isolated HSCs, MPPs, MLPs, CMPs, GMPs, MEPs, and mature T cells within the CD33- cell fraction as well as CD45dimCD33+ AML blasts selected from primary MPN-BP cells from Pt6. However, +1q was found exclusively in purified MLPs and MEP. These observations establish that cytogenetic and mutational events that lead to MPN-BP occur at different stages along the developmental HSC hierarchy and that a small population of normal HSCs persist. Furthermore, in JAK2V617F+ MPNs that develop MPN-BP and lose JAK2V617F, additional cytogenetic events occur at different stages along the JAK2V617F- MPN-BP-stem cell hierarchy. Our ability to serially transplant the LIC from these patients has allowed us to create the first MPN-BP PDX model that will not only extend our understanding of MPN-BP stem cell biology but might also prove useful for screening drugs to treat MPN-BP. Disclosures Rampal: Jazz: Consultancy, Honoraria; Incyte: Honoraria, Research Funding; Stemline: Research Funding; Constellation: Research Funding; Celgene: Honoraria. Mascarenhas:Incyte: Membership on an entity's Board of Directors or advisory committees, Research Funding; CTI Biopharma: Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Research Funding; Roche: Research Funding; Novartis: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Promedior: Research Funding; Janssen: Research Funding. Hoffman:Formation Biologics: Research Funding; Summer Road: Research Funding; Merus: Research Funding; Incyte: Research Funding; Janssen: Research Funding.

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Journal articles on the topic 'Leukemia initiation and transformation'

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