CALM-AF10 Leukemias Research Articles

Genetic and Pharmacological Interruption of the SIX1/EYA2 Axis Impairs Leukemia Proliferation

Background: The CALM-AF10 translocation is found in 5-10% of T-cell acute lymphoblastic leukemias (T-ALL), and a subset of acute myeloid leukemias (AML). CALM-AF10 leukemias are characterized by elevated expression of proleukemic HOXA genes. Since HOXA genes are difficult to target, we sought to identify non- HOXA effector genes through RNA sequencing and Microarray analyses, and showed that SIX1 expression is increased in CALM-AF10 leukemia cells. Like HOXA genes, SIX1 is a homeobox gene involved in cell proliferation, embryogenesis, and apoptosis. SIX1 interacts with the cofactor Eyes Absent 2 (EYA2), a protein phosphatase, to transcriptionally activate developmental genes, the TGFβ pathway, and the epithelial-mesenchymal transition (EMT). SIX1 expression is elevated during development but decreases with maturation. Intriguingly, overexpression of SIX1 has been observed in numerous malignancies including breast cancer, ovarian cancer, gliomas and esophageal cancer, and SIX1 is involved in accelerating the epithelial mesenchymal transition (EMT) and metastasis. Knockdown of either SIX1 or EYA2 has been shown to reverse these effects, and two novel inhibitors - one of the SIX1/EYA2 complex (Compound 8430, “8430”) and one of the EYA2 phosphatase (Compound 9987, “9987”) - have replicated the effects of SIX1 or EYA2 knockdown. Methods: SIX1 gene and protein expression were assessed in leukemia cell lines via RT-qPCR and Western Blot. SIX1 expression vectors were transduced into fetal liver hematopoietic stem cells (HSC) and immortalization was assessed using colony assays. CALM-AF10 leukemia cells lines were derived from irradiated mice transplanted with HSC retrovirally transfected with CALM-AF10 plasmids created in our lab. The established leukemia cell lines Jurkat, MOLT16, OCI-M2, TF-1, and SHI-1 were obtained from ATCC or DSMZ. shRNAs targeting SIX1 were lentivirally transduced into CALM-AF10 and SHI1leukemia cell lines. Cell-Titer-Glo assays and liquid culture were used to assess the effects of shRNAs, 8430, or 9987 on cell proliferation and cell growth. SIX1 gene expression in leukemias was determined from The Cancer Dependency Map from The Broad Institute using R. Results: We first validated RNAseq and Microarray studies, showing that CALM-AF10 induces SIX1 expression in murine fibroblasts and murine HSC, and localizes to the SIX1 locus. Furthermore, SIX1 overexpression was sufficient to immortalize murine fetal liver HSC (FL-mHSCs), implying a role in leukemic transformation. Multiple murine derived CALM-AF10 cell lines displayed increased SIX1 expression compared to fresh murine bone marrow and isolated HSCs. shRNA knockdown of SIX1 decreased viability of murine CALM-AF10 cells in culture. In addition, both the SIX1/EYA2 complex inhibitor (8430) and the EYA2 inhibitor (9987) decreased CALM-AF10 leukemia cell proliferation in vitro. To expand the relevance of SIX1 to other leukemias, we then queried The Cancer Dependency Map from the Broad Institute and identified other leukemia cell lines with increased SIX1 expression, including Jurkat and Molt16 (T-ALL), and SHI-1, OCIM2, and TF-1 (AML). SIX1 expression in these cells was validated by immunoblot and shRNA knockdown of SIX1 slowed the proliferation of SHI-1 cells. Finally, the 8430 and 9987 inhibitors reduced the proliferation of Jurkat, OCI-M2, and SHI-1 cells at doses similar to those used in CALM-AF10 leukemias. Conclusions: We have identified involvement of the SIX1/EYA2 axis in several different leukemias, including T-ALL and AML. Furthermore, we have shown that inhibitors of the SIX1/EYA2 interaction and of EYA2 reduce the proliferation of SIX1 expressing leukemias, suggesting a potential new therapeutic target in these leukemias.

CXCR4 Mediates Enhanced Cell Migration in CALM-AF10 Leukemia.

Leukemia transformed by the CALM-AF10 chromosomal translocation is characterized by a high incidence of extramedullary disease, central nervous system (CNS) relapse, and a poor prognosis. Invasion of the extramedullary compartment and CNS requires leukemia cell migration out of the marrow and adherence to the cells of the local tissue. Cell adhesion and migration are increasingly recognized as contributors to leukemia development and therapeutic response. These processes are mediated by a variety of cytokines, chemokines, and their receptors, forming networks of both secreted and cell surface factors. The cytokines and cytokine receptors that play key roles in CALM-AF10 driven leukemia are unknown. We find high cell surface expression of the cytokine receptor CXCR4 on leukemia cells expressing the CALM-AF10 oncogenic protein, contributing to the migratory nature of this leukemia. Our discovery of altered cytokine receptor expression and function provides valuable insight into the propagation and persistence of CALM-AF10 driven leukemia.

A SIX1/EYA2 Inhibitor Impairs CALM-AF10 and Jurkat Leukemia Cell Proliferation

Background: The CALM-AF10 translocation is found in 5-10% of T-cell acute lymphoblastic leukemias (T-ALL), and a subset of acute myeloid leukemias (AML). CALM-AF10 leukemias are characterized by elevated expression of proleukemic HOXA genes. Since HOXA genes are difficult to target, we hypothesized that identification of non-HOXA CALM-AF10 effector genes could potentially yield novel therapeutic targets. To discover novel CALM-AF10-regulated genes, we took advantage of our prior observation that the nuclear export factor CRM1/XPO1 tethers CALM-AF10 to HOXA genes by interacting with a nuclear export signal within CALM. Using microarrays, we identified a set of genes that showed decreased expression in response to the CRM1 inhibitor Leptomycin B (LMB), similar to Hoxa genes, in murine CALM-AF10 leukemia cells. Then using RNA-sequencing, we discovered a set of genes increased in murine hematopoietic stem cells transduced with CALM-AF10. There were 11 genes that were both decreased in response to LMB and increased in response to CALM-AF10, which included the Hoxa gene cluster, as well as Six1. We demonstrated that CALM-AF10 increases Six1 expression and localizes to the Six1 locus, as it does the Hoxa genes. SIX1, like the Hoxa genes, is a homeobox gene that is associated with embryogenesis and is quiescent post-embryologically. In addition, SIX1 and its cofactor EYA2 are overexpressed in numerous solid tumors, and an inhibitor of the SIX1/EYA2 complex (Compound 8430) has recently been described. While there is evidence of a role for SIX1 in solid tumors, its role in leukemias has not been explored.Objective: Evaluate the effect of a SIX1/EYA2 complex inhibitor on leukemia cell proliferation.Design/Methods: SIX1 gene and protein expression were assessed in CALM-AF10, Jurkat (T-ALL) and NOMO1 (AML) leukemia cell lines via Western Blot and RT-qPCR. CALM-AF10 leukemias were derived from murine models in our lab, Jurkat and NOMO1 cell lines were obtained from ATCC. The effect of compound 8430 - an inhibitor of the Six1/Eya2 interaction - on cell proliferation was evaluated using Cell-Titer-Glo Assays and liquid culture proliferation assays. In addition, we used the the CRM1 Nuclear Export Inhibitor KPT-330 alone and in combination with 8430 in these cell lines. SynergyFinder2 (https://synergyfinder.fimm.fi/) was used to assess synergy of 8430 and KPT-330. δ-score is a calculated value that indicates synergistic drug interaction, with a higher δ-score indicative of a synergistic effect of the drugs.Results: SIX1 gene and protein expression are increased in CALM-AF10 leukemia cell lines and Jurkat T-ALL cells, but not NOMO1 cells. Compound 8430 decreases cell proliferation in CALM-AF10 leukemias and Jurkat leukemia cell lines, however it did not affect the AML line NOMO1. Correspondingly, liquid cultures showed that 8430 alone slowed the proliferation of CALM-AF10 leukemia and the Jurkat cells, but not NOMO1 cells. The addition of KPT-330 to 8430 was synergistic in CALM-AF10 leukemia cells with a KPT-330 dose of 60 nM and multiple dose levels of 8430 (δ-scores 17-19) while in the Jurkat leukemia cells a dose of 30 nM of KPT-330 was synergistic at multiple dose levels of 8430 (δ-score 6-8) (Figure 1).Conclusions: The SIX1 homeobox gene is highly expressed during development, and its expression is silenced post-embryogenesis. Through an initial unbiased screen, we discovered that Six1 may play a role in CALM-AF10 leukemogenesis. We have determined that Six1 expression is upregulated in the presence of CALM-AF10. A role for Six1 in CALM-AF10 leukemogenesis is further supported by the ability of a SIX1/EYA2 inhibitor to slow the proliferation of CALM-AF10 leukemia cells. Importantly, based on our observation that 8430 slows proliferation of Jurkat cells, SIX1 inhibition may be relevant in other leukemias. Finally, our demonstration that 8430 synergizes with KPT-330, a Nuclear Export Inhibitor, suggests the possibility of a novel therapeutic approach for CALM-AF10 and other leukemias. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Establishing the Role of EPS15, DVL2 and Cortactin in CALM-AF10 Leukemogenesis

Background:Leukemia is the most common type of childhood cancer. Although the prognosis for many pediatric leukemias has improved, leukemias associated with the t(10;11) CALM-AF10 translocation remain difficult to treat. CALM-AF10 leukemias account for ~5-10% of childhood T-cell acute lymphoblastic leukemia (T-ALL)as well as a subset of acute myeloid leukemia (AML). CALM-AF10 leukemias exhibit increased expression of proleukemic HOXA genes, but relatively little is known about the cellular mechanisms that drive CALM-AF10 leukemogenesis. Our laboratory has demonstrated that the CALM protein contains a nuclear export signal (NES) that is critical for CALM-AF10-dependent leukemogenesis. The NES interacts with the CRM1/XPO1 nuclear export receptor, which shuttles proteins from the nucleus to the cytoplasm through the nuclear pore complex. We have shown that transcriptional activation of HOXA genes by CALM-AF10 is dependent on its interaction with CRM1. Importantly, CRM1 does not contain a recognized DNA binding domain, and it is not currently understood how the CALM-AF10/CRM1 complex interacts with regulatory regions of HOXA genes. To identify proteins that mediate the interaction between the CALM-AF10/CRM1 complex and DNA, we took advantage of a proximity-based labeling approach using BioID2, a second-generation biotin ligase. When fused to a protein of interest and in the presence of biotin, BioID2 biotinylates proteins in close proximity to the ligase. These biotinylated proteins can then be identified by mass spectrometry (MS).Methods:We prepared an expression plasmid in which BioID2 was cloned in-frame with CALM-AF10. Human Embryonic Kidney 293 (HEK293) cells were transiently transfected with BioID2-CALM-AF10 and grown in the presence or absence of biotin. MS was performed to identify candidate interacting proteins. We validated direct interactions of candidate proteins with CALM-AF10 using co-immunoprecipitation experiments in HEK293 cells transfected with a CALM-AF10 plasmid. We confirmed that candidate proteins are present in murine CALM-AF10 leukemia cells via Western blotting. In order to efficiently knockout (KO) candidate proteins, we have generated a human U937 cell line (which harbors a t(10;11) CALM-AF10 translocation) with a stable incorporated Cas9. To assess whether KO of EPS15, DVL2 or CTTN affects HOXA5 expression, we performed RT-qPCR in U937-Cas9 cells lines with confirmed KO.Results:We carried out three independent transfections/MS experiments, which identified 71, 95 and 61 proteins, respectively. Of the proteins identified, 12 candidates were common to all three experiments . Importantly, we identified Disruptor Of Telomeric silencing 1-Like (DOT1L), a protein known to interact with AF10, and Nuclear pore complex protein 214 (NUP214), a protein that interacts with CRM1 and that is involved in leukemogenic translocations. We chose EPS15, DVL2 and CTTN for further study, as each of these proteins plays a role in leukemogenesis. We performed initial validation of direct interactions via co-immunoprecipitation and found that all three proteins co-precipitate with CALM-AF10. Western blotting showed that all three proteins are expressed in a murine CALM-AF10 leukemia cell line. We effectively knocked out EPS15 protein expression in U937 cells, and showed that HOXA5 expression is reduced in the setting of EPS15 knockout.Conclusion:We used biotin ligase-dependent proximity-based labeling to identify candidate proteins that potentially interact with the CALM-AF10 fusion protein. Our identification of DOT1L validates the approach, since DOT1L is known to interact with CALM-AF10. We have started to investigate three candidate proteins - EPS15, DVL2 and CTTN - all of which are involved in leukemogenic transformation. We have shown that EPS15, DVL2 and CTTN are expressed in murine CALM-AF10 leukemia cells and directly interact with the CALM-AF10 fusion protein. Knockout of EPS15 in U937 cells results in decreased HOXA5 expression, suggesting the importance of EPS15 in CALM-AF10 leukemogenesis. Evaluation of the roles of these proteins in leukemogenesis may lead to identification of novel pathways involved in CALM-AF10 leukemogenesis. DisclosuresNo relevant conflicts of interest to declare.

The SIX1 homeobox Gene Is a Novel Therapeutic Target in CALM-AF10 Leukemogenesis

Background : The CALM-AF10 translocation is found 5-10% of T-cell acute lymphoblastic leukemias (T-ALL), and a subset of acute myeloid leukemias (AML). CALM-AF10 leukemias are characterized by elevated expression of proleukemic HOXA genes. Since HOXA genes are difficult to target, we hypothesized that identification of non-HOXA CALM-AF10 effector genes could potentially yield novel therapeutic targets. To discover novel CALM-AF10-regulated genes, we took advantage of our prior observation that the nuclear export factor CRM1/XPO1 tethers CALM-AF10 to HOXA genes by interacting with a nuclear export signal within CALM. Using microarrays, we identified a set of genes that showed decreased expression in response to the CRM1 inhibitor, Leptomycin B (LMB), similar to Hoxa genes, in murine CALM-AF10 leukemia cells. Then using RNA-sequencing, we discovered a set of genes increased in murine hematopoietic stem cells transduced with CALM-AF10. There were 11 genes that were both decreased in response to LMB and increased in response to CALM-AF10, which included the Hoxa gene cluster, as well as Six1. Similar to HOXA genes, SIX1 is a homeobox gene that is associated with embryogenesis and is quiescent post-embryologically. Additionally, SIX1 and its cofactor EYA2 have been found to be overexpressed in numerous solid tumors, and inhibitor of the SIX1/EYA2 complex has recently been described. While there is evidence of a role for SIX1 in solid tumors, its role in leukemias has not been explored. Objective: To evaluate the role of SIX1 in CALM-AF10 leukemias. Design/Methods: RT-qPCR and Chromatin Immunoprecipitation (ChIP) were performed using bone marrow progenitors transduced with CALM-AF10 or an empty vector, with and without LMB. Methylcellulose colony assays assessed the ability of SIX1 to enhance self-renewal of hematopoietic progenitors. An inhibitor of the Six1/Eya2 interaction (compound 8430) was used to evaluate cell proliferation. Downstream targets of Six1 were evaluated using RT-qPCR in CALM-AF10 cells treated with Six1/Eya2 inhibitor (8430). Results: RT-qPCR confirmed overexpression of SIX1 in CALM-AF10 leukemia cells, and showed decreased SIX1 expression in the presence of LMB. Furthermore, ChIP revealed that CALM-AF10 binds to the SIX1 gene locus. Overexpression of SIX1 in fetal liver progenitors was sufficient to increase self-renewal potential. The 8430 Six1/Eya2 inhibitor slowed cell growth in CALM-AF10 cells compared to cells treated with DMSO alone. Finally, downstream targets such as Slc2a1, Cdk2, and Cyclina2 were decreased in 8430-treated CALM-AF10 leukemia cells. Conclusions: The SIX1 homeobox gene is highly expressed during embryogenesis, and its expression is silenced post-embryogenesis. Through an initial unbiased screen, we discovered that Six1 may play a role in CALM-AF10 leukemogenesis. We have determined that Six1 expression is upregulated in the presence of CALM-AF10. Further, we have shown a potential oncogenic role for Six1, as it was able to increase the self-renewal potential of hematopoietic progenitors. The role of Six1 in CALM-AF10 leukemia is further supported by the ability of a SIX1/EYA2 inhibitor to slow the growth of CALM-AF10 leukemia cells and decrease the expression of downstream targets of SIX1. These observations suggest that Six1 plays a pathogenic role in leukemogenesis, and may be a novel therapeutic target in CALM-AF10 leukemias. Disclosures No relevant conflicts of interest to declare.

Characterizing Proteins That Mediate CALM-AF10 Leukemogenesis

Background: Leukemia is the most common type of childhood cancer. Although the prognosis for many pediatric leukemias has improved, leukemias associated with the t(10;11) CALM-AF10 translocation remain difficult to treat. CALM-AF10 leukemias account for ~5-10% of childhood T-cell acute lymphoid leukemia (T-ALL) as well as a subset of acute myeloid leukemia (AML). CALM-AF10 leukemias exhibit increased expression of proleukemic HOXA genes, but relatively little is known about the cellular mechanisms that drive CALM-AF10 leukemogenesis. Our laboratory has demonstrated that the CALM protein contains a nuclear export signal (NES) that is critical for CALM-AF10-dependent leukemogenesis. The NES interacts with the CRM1/XPO1 nuclear export receptor, which shuttles proteins from the nucleus to the cytoplasm through the nuclear pore complex. We have shown that transcriptional activation of HOXA genes by CALM-AF10 is critically dependent on its interaction with CRM1. Importantly, CRM1 does not contain a recognized DNA binding domain, and it is not currently understood how the CALM-AF10/CRM1 complex interacts with regulatory regions of HOXAgenes. In order to identify proteins that mediate the interaction between the CALM-AF10/CRM1 complex and DNA, we took advantage of a proximity-based labeling approach using BioID2, a second-generation biotin ligase. When fused to a protein of interest and in the presence of biotin, BioID2 biotinylates proteins in close proximity to the ligase. These biotinylated proteins can then be identified by mass spectrometry (MS). Methods: We prepared an expression plasmid in which BioID2 was cloned in-frame with CALM-AF10. We then transiently transfected Human Embryonic Kidney 293 (HEK293) cells with the BioID2-CALM-AF10 plasmid, grew them in the presence or absence of biotin, and performed streptavidin-pulldown followed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) to identify candidate interacting proteins. Proteins were considered candidates if they had a peptide spectrum match (PSM) score > 10 and at least a two-fold greater PSM score versus negative control. We validated direct interactions of candidate proteins with CALM-AF10 by performing co-immunoprecipitation experiments. Results: We first confirmed that the addition of BioID2 to CALM-AF10 does not affect the transcriptional activation of HOXA genes or CALM-AF10 mediated immortalization of hematopoietic stem cells. We carried out three independent transfections/LC-MS/MS experiments, which identified 71, 95 and 61 proteins, respectively. Of the proteins identified, 11 candidates were common to all three experiments.Of particular interest, we identified Disruptor Of Telomeric silencing 1-Like (DOT1L), a protein known to interact with AF10, and Nuclear pore complex protein 214 (NUP214), a protein that has been identified in leukemogenic translocations. The nine additional candidate proteins included: EPS15, DVL2, DVL3, and DDX3X -all known to play a role in leukemogenesis. We performed initial validation of direct interactions via co-immunoprecipitation and found that Epidermal Growth Factor Receptor Substrate 15(EPS15) co-precipitates with CALM-AF10. Conclusion: We used biotin ligase-dependent proximity-based labeling to identify candidate proteins that potentially interact with the CALM-AF10 fusion protein. Our identification of DOT1L validates the approach, since DOT1L is known to interact with CALM-AF10. We have started to investigate other candidate proteins, focusing on known translocation partners in various leukemias. Our screen identified EPS15, a protein involved in receptor-mediated endocytosis of epidermal growth factor and a known translocation partner for MLL/KMT2A. KMT2A-EPS15 translocations (t(1;11)(p32;q23)) have been identified in both AML and ALL, and KMT2A-EPS15 is among the eight most common KMT2A rearrangements. We have shown that EPS15 co-immunoprecipitates with CALM-AF10, suggesting that EPS15 may also play a role in CALM-AF10 leukemogenesis. Further evaluation of this interaction is underway, and may lead to identification of novel pathways involved in CALM-AF10 leukemogenesis. Disclosures No relevant conflicts of interest to declare.

SIX1 Is a Novel Effector Gene in CALM-AF10 Leukemia

Background: The CALM-AF10 translocation is detected in ~10% of T-cell acute lymphoblastic leukemias (T-ALLs), and in some acute myeloid leukemias (AMLs). CALM-AF10 leukemias are characterized by high expression of proleukemic HOXA genes, which serve a critical role in hematopoiesis. We hypothesized that identification of novel CALM-AF10 effector genes may yield new therapeutic targets in this difficult to treat leukemia. We took advantage of our prior observation that the nuclear export factor CRM1/XPO1 tethers CALM-AF10 to HOXA genes by interacting with a nuclear export signal (NES) in CALM. Using next generation sequencing, we determined that, SIX1, similar to HOXA genes, is increased in CALM-AF10 leukemias and decreased in response to CRM1 inhibition with Leptomycin B (LMB). Design/Methods: RT-qPCR and Chromatin Immunoprecipitation were performed using both bone marrow progenitors and murine embryonic fibroblasts (MEFs) transduced with CALM-AF10 or an empty vector, with and without LMB. The ability of SIX1 to enhance self-renewal of hematopoietic progenitors was examined by measuring the colony-forming ability of transduced fetal liver hematopoietic progenitor cells. CRISPR-Cas9 was used to silence SIX1 in Human Embryonic Kidney 293 (HEK293) cells. Results: RT-qPCR confirmed overexpression of SIX1 in both CALM-AF10 transduced MEFs and CALM-AF10 leukemias, with decreased SIX1 expression observed in the presence of LMB. ChIP analysis showed that CALM-AF10 binds to the SIX1 gene locus. Overexpression of SIX1 in fetal liver cells was sufficient to increase the self-renewal potential of colony-forming progenitors. SIX1 was successfully knocked out in HEK293 cells without a significant effect on HEK293 proliferation. Conclusions: The SIX1 homeobox gene is highly expressed during development and its expression is silenced post-embryogenesis. Increased SIX1 expression has been reported in numerous solid tumors. We have determined that SIX1 is upregulated in CALM-AF10 leukemias, and increases the self-renewal potential of hematopoietic progenitors. Using CRISPR-Cas9 to silence SIX1, we have demonstrated that SIX1 is not essential for cell survival, and that its inhibition may impair CALM-AF10 leukemia cell proliferation. Thus, SIX1 may play a pathogenic role in leukemogenesis and is a potential therapeutic target in CALM-AF10 leukemias. Disclosures No relevant conflicts of interest to declare.

Genetic or Pharmacological Inhibition of the Polycomb Group Protein BMI1 Impairs Leukemic Transformation Mediated By the CALM-AF10 Fusion Oncoprotein

A subset of acute myeloid and lymphoid leukemia cases harbor a t(10;11)(p13;q14) translocation resulting in the CALM-AF10 fusion gene. Standard chemotherapeutic strategies are not very effective in treating patients with CALM-AF10 fusions. Hence, there is an urgent need to identify molecular pathways dysregulated in CALM-AF10 positive leukemias which may lay the foundation for novel targeted therapies. The polycomb repressive complex 1 gene BMI1 is consistently overexpressed in CALM-AF10 leukemias. Previous studies have shown that CALM-AF10 leukemias express high levels of BMI1, regardless of whether the leukemias are myeloid or lymphoid. Our analysis of TCGA acute myeloid leukemia (AML) data confirmed that AML cells with AF10-rearrangements displayed significantly higher expression of BMI1 transcripts compared to cells from non AF10-rearranged AML patients. These observations indicate that BMI1 may be directly activated by AF10-fusion oncogenes as suggested by our previous studies.We sought to investigate the role of BMI1 in CALM-AF10 mediated leukemogenesis using murine and human models of CALM-AF10-mediated AML. First, we tested whether BMI1 deficiency can affect CALM-AF10 mediated oncogenic transformation of hematopoietic stem and progenitor cells (HSPCs). Towards this end, we retrovirally transduced fetal liver cells from Bmi1 wild-type, heterozygous or homozygous null mice with the CALM-AF10 fusion oncogene. Upon plating these cells in colony forming unit (CFU) assays, we observed a significant decrease in the colony formation capacity of the CALM-AF10 fusion transduced cells on a Bmi1 deficient background. Next, we performed Cre-recombinase mediated excision of Bmi1 of already transformed CALM-AF10 myeloid leukemia cells (Bmi1 floxed background). Bmi1 deletion led to a significant reduction in the number of total CFUs compared to Bmi1 wild-type cells, with a particularly striking reduction in the number of blast-like colonies. These experiments, using Bmi1 constitutive or conditional knockout-mice, revealed that CALM-AF10 transformed AML cells are dependent on Bmi1.Recently, selective pharmacological BMI1 inhibitors have been developed. We tested the impact of pharmacologic BMI1 inhibition on a panel of CALM-AF10-driven mouse leukemias with the small molecule inhibitor PTC-209. PTC-209 treatment increased gene expression of the known BMI1-repressed targets Cdkn2a (p16) and Cdkn1a (p21) and led to a dose-dependent decrease in cell proliferation. We also observed a marked increase in Annexin V+ cells upon PTC-209 treatment. In addition, cell-cycle analysis using BrdU incorporation assays revealed a significant decrease in cells in the S-phase, demonstrating that PTC-209 treatment leads to growth arrest and apoptosis in CALM-AF10 AML cells.In order to confirm these findings in human AML with CALM-AF10 rearrangements, we treated human CALM-AF10 positive AML cell lines P31, U937 and KPMOTS with PTC-209. Consistent with our results in the murine AML model, we observed a time and dose-dependent decrease in proliferation of these human cell lines upon PTC-209 treatment. Drug treated human cells also showed concomitant cell-cycle arrest and apoptosis induction, coupled with an increase in expression of BMI1 repressed tumor suppressor genes such as CDKN2A and CDKN1A.In summary, our results demonstrate that BMI1 is a bonafide candidate for therapeutic targeting in AML with CALM-AF10 rearrangements and possibly other CALM-AF10 positive leukemias. We are now assessing clinical-grade BMI1 inhibitors for in vivo efficacy in mouse models of CALM-AF10-mediated AML. DisclosuresDeshpande:Salgomed Therapeutics: Membership on an entity's Board of Directors or advisory committees; A2A Pharma: Membership on an entity's Board of Directors or advisory committees.

Genomic and Proteomic Profiling of AF10-Fusion Oncoproteins Reveal Mechanisms of Leukemogenesis and Actionable Targets

The AF10/MLLT10 gene is recurrently involved in chromosomal rearrangements in human leukemia. AF10 rearrangements are linked to a poor prognosis in AML and T-ALL, underscoring the need to identify targeted therapies for AF10-fusion positive leukemia. Defining the molecular mechanisms of oncogenesis mediated by AF10-fusion proteins (AF10-FPs) may unravel novel actionable targets in leukemias with AF10-gene rearrangements. Towards this end, we established tetracycline (Tet)-inducible models of MLL-AF10 and CALM-AF10 AML and performed RNA-seq in AML cells treated with doxycycline (Dox) compared to vehicle treated counterparts. Since Dox treatment completely abrogates AF10-fusion gene expression from the Tet-regulated promoter, these models can be used to characterize the transcriptional landscape of potential AF10-FP target genes. We observed that among transcripts significantly downregulated upon Dox treatment, 168 genes were common in both the MLL-AF10 Tet-Off or CALM-AF10 Tet-Off conditions, indicating a high overlap between potential transcriptional targets of these distinct AF10-FPs. Expectedly, this list included genes previously implicated in leukemogenesis including Hoxa cluster genes, Meis1, Flt3, Mecom, Cd34, Gfi1b, Eya1 and Nkx2-3. Importantly, in addition to these well-characterized genes, we identified a number of novel pathways that were downregulated in the AF10-FP Tet-Off state. The most striking molecular signature of potential AF10-FP-regulated genes emerging from these analyses were factors involved in innate immunity and pro-inflammatory cytokine signaling. Prominent drivers of these molecular signatures included genes of the Jak/Stat and NFkB signaling pathways as well as Interferon response genes. We confirmed that AF10-FPs strongly activated Jak-Stat and NFkB signaling by performing Western blotting for key factors involved in these pathways. Since pro-inflammatory cytokines have been shown to play a role in AML cell survival, we tested the impact of cytokine depletion on murine AF10-FPs-driven AML cells. Proliferation assays demonstrated that AF10-FP-transformed cells could survive significantly better in cytokine-free medium compared to those transformed with other oncogenes such as MLL-AF9, which were completely dependent on cytokines for survival and proliferation in vitro. These results suggest that activation of cytokine signaling may contribute to increased survival of AF10-FP-driven AML cells.Next, we performed proteomic studies in which affinity-purified epitope-tagged AF10-FPs were evaluated for interacting proteins using Mass Spectrometry (MS). While studies on MLL-AF10 fusion are ongoing, our studies revealed that the strongest interactor of the CALM-AF10 fusion protein was the Janus kinase protein Jak1. We confirmed this finding by immunoprecipitation experiments in CALM-AF10 AML cells using a Jak1-specific antibody. Given the role of JAK1 in cytokine-mediated pro-inflammatory signaling, our findings indicate that CALM-AF10 may activate this pathway through direct recruitment of the Jak1 kinase. We sought to directly test the role of JAK1 in AF10-FP-mediated leukemogenesis. For this, we transformed bone marrow stem and progenitor cells from Jak1 floxed mice with the CALM-AF10 fusion. Deletion of Jak1 using Cre-recombinase in CALM-AF10 AML significantly reduced their proliferation in vitro. Furthermore, Jak1 deletion led to a highly significant reduction in the number of colony forming units (CFUs) from CALM-AF10 AML cells, with a particularly striking decrease in the number of blast-like colonies. We also observed a significant increase in differentiation of CALM-AF10 AML cells following Jak1 deletion, demonstrating that Jak1 activity is important for maintaining the CALM-AF10 leukemia cells in an undifferentiated state. Importantly, these results were recapitulated with two different small-molecule JAK1 inhibitors itacitinib and filgotinib that are being tested in clinical trials for a variety of human diseases. Treatment of CALM-AF10 AML cells with these selective JAK1 inhibitors led to a significant, dose-dependent decrease in proliferation accompanied by growth arrest and apoptosis. Taken together, our studies demonstrate that AF10 fusions activate pro-inflammatory signaling by co-opting the Jak-Stat pathway, presenting a potential therapeutic target in AF10-fusion-driven AML. DisclosuresLevine:Janssen: Consultancy, Honoraria; Celgene: Consultancy, Research Funding; Qiagen: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Prelude: Research Funding; Loxo: Consultancy, Equity Ownership; Imago: Equity Ownership; C4 Therapeutics: Equity Ownership; Novartis: Consultancy; Gilead: Honoraria; Isoplexis: Equity Ownership; Epizyme: Patents & Royalties; Roche: Consultancy, Research Funding. Deshpande:A2A Pharma: Membership on an entity's Board of Directors or advisory committees; Salgomed Therapeutics: Membership on an entity's Board of Directors or advisory committees.

CCI-007, a novel small molecule with cytotoxic activity against infant leukemia with MLL rearrangements

There is an urgent need for the development of less toxic, more selective and targeted therapies for infants with leukemia characterized by translocation of the mixed lineage leukemia (MLL) gene. In this study, we performed a cell-based small molecule library screen on an infant MLL-rearranged (MLL-r) cell line, PER-485, in order to identify selective inhibitors for MLL-r leukemia. After screening initial hits for a cytotoxic effect against a panel of 30 cell lines including MLL-r and MLL wild-type (MLL-wt) leukemia, solid tumours and control cells, small molecule CCI-007 was identified as a compound that selectively and significantly decreased the viability of a subset of MLL-r and related leukemia cell lines with CALM-AF10 and SET-NUP214 translocation. CCI-007 induced a rapid caspase-dependent apoptosis with mitochondrial depolarization within twenty-four hours of treatment. CCI-007 altered the characteristic MLL-r gene expression signature in sensitive cells with downregulation of the expression of HOXA9, MEIS1, CMYC and BCL2, important drivers in MLL-r leukemia, within a few hours of treatment. MLL-r leukemia cells that were resistant to the compound were characterised by significantly higher baseline gene expression levels of MEIS1 and BCL2 in comparison to CCI-007 sensitive MLL-r leukemia cells.In conclusion, we have identified CCI-007 as a novel small molecule that displays rapid toxicity towards a subset of MLL-r, CALM-AF10 and SET-NUP214 leukemia cell lines. Our findings suggest an important new avenue in the development of targeted therapies for these deadly diseases and indicate that different therapeutic strategies might be needed for different subtypes of MLL-r leukemia.

Abstract B23: Selective and rapid killing of mixed lineage leukemia and CALM-AF10 leukemia by SM7, a novel small molecule identified by chemical library screening

Abstract Translocations involving the Mixed Lineage Leukemia (MLL) gene define the major proportion of infant leukemia, a disease that is characterised by a dismal prognosis due to treatment toxicity and therapy resistance. There is an urgent need for the development of less toxic, more selective and targeted therapies for this disease. In this study, we performed a cell-based chemical library screen on an infant MLL cell line, PER-485, in order to identify selective inhibitors of MLL leukemia. After screening initial hits for viability against a panel of 25 cell lines including MLL rearranged and wild-type MLL leukemia, solid tumors and control cells, SM7 was identified as a compound that significantly and selectively decreased the viability of MLL-rearranged (four out of seven) and related CALM-AF10 (two out of two) leukemia cell lines, without killing cell lines derived from wild-type MLL leukemia, solid tumours or normal cells. In contrast to currently developed MLL-inhibitors, the effect of SM7 in suppressing the characteristic MLL-driven gene expression signature in MLL-rearranged cells, with downregulation of leukemogenic HoxA9 and Meis1 expression, occurred within an hour after treatment. The transcript levels of cMyc and Bcl2, important survival proteins regulated by HoxA9, were also rapidly decreased in response to SM7. SM7 induced a rapid caspase-dependent apoptosis, with mitochondrial depolarization within twenty four hours of treatment. This is distinct from the action of other previously described MLL small molecule inhibitors. The rapid and selective toxicity of SM7 towards MLL-rearranged and CALM-AF10 leukemia cell lines suggests an important new avenue in the development of targeted therapies for this disease. Citation Format: Daria Chudakova, Klaartje Somers, Shiloh Middlemiss, Victoria Wen, Alan Kwek, Bing Liu, Molly Clifton, Sukey Pan, Sarah Cruikshank, Marissa Scandlyn, Tim Failes, Wendy Hoang, Ursula Kees, Olga Chernova, Andrei V. Gudkov, Michelle Haber, Murray Norris, Michelle Henderson. Selective and rapid killing of mixed lineage leukemia and CALM-AF10 leukemia by SM7, a novel small molecule identified by chemical library screening. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B23.

TAL1 and MYB Abnormalities in Childhood T-Cell Acute Lymphoblastic Leukemia

T-cell acute lymphoblastic leukemia (T-ALL) accounts for 10% to 15% of newly diagnosed cases of childhood acute lymphoblastic leukemia (ALL). Although gene fusions generated through chromosomal translocations, deletions, and inversions are the most frequent genetic abnormalities detected in other types of leukemia, recurrent gene fusions except for SIL-TAL1 have been poorly defined in T-ALL. To discover driver mutations or fusion genes, which involved in the pathogenesis of childhood T-ALL and to identify novel prognostic markers of childhood T-ALL, we performed whole-exome sequencing (WES) and transcriptome sequencing (WTS) in 31 and 46 cases, respectively. We also performed SNP array karyotyping in 46 cases. To detect somatic mutations or fusion transcripts, we used our pipeline "Genomon-exome" and "Genomon-fusion" algorithm. Subsequently, somatic mutations were validated using deep amplicon sequencing. Candidate fusion transcripts were validated by reverse - transcription polymerase-chain-reaction and Sanger sequencing.Recurrent mutations observed in more than 3 cases were detected in NOTCH1 (n = 18, 58%), FBXW7 (n = 7, 23%), PHF6 (n = 5, 16%), GATA3 (n = 4, 13%), MYB (n = 3, 10%), and NRAS (n = 3, 10%), respectively. We identified previously known fusion genes, such as MLL-ENL (n = 2), CALM-AF10 (n = 2), NUP214-ABL1 (n = 1) and FGFROP1-FGFR1 (n = 1). MLL-ENL is one of the frequent translocation in infant multilineage leukemia, but also reported in non-infant B cell precursor ALL and T-ALL. FGFR1OP is ubiquitously expressed, and the predicted chimeric FGFR1OP-FGFR1 protein contains the catalytic domain of FGFR1. It is thought to promote hematopoietic stem cell proliferation and leukemogenesis through a constitutive phosphorylation and activation of the downstream pathway of FGFR1. CALM-AF10 leukemia is reported to increase HOXA cluster gene transcription, we could also confirm elevated HOXA genes expression by FPKM value. Four SIL-TAL1 fusions were detected in our cohort. Recently, a novel mutation in TAL1 enhancer region which introduce de novo MYB biding site has been reported. Since this abnormality lead high expression of TAL1, we also analyzed expression data obtained from WTS. Among 46 specimens, 19 samples showed high expression of TAL1 (FPKM value ≥5). In those cases, 4 cases had SIL-TAL1 fusions (8%), and 3 cases (6%) had insertions in enhancer region of TAL1. Subsequent analysis using Gene Set Enrich Analysis (GSEA) between TAL1 high and low expression samples revealed that "LEE_EARLY_T_LYMPHOCYTE_UP" was enriched in TAL1 high expression samples (Enrichment score = 0.73, FDR = 0.073). This gene set includes genes up-regulated at early stages of progenitor T lymphocyte maturation compared to the late stages, and MYB was included in this gene set. Intriguingly, MYB mutation samples were not represented TAL1 high expression. TAL1 related rearrangement or enhancer insertion was not detected in the rest of 12 cases with TAL1 high expression, suggesting that other mechanisms of TAL1 high expression might be exist. In conclusion, although NOTCH1 and FBXW7 mutations were relatively frequently detected in our series, we also found recurrent MYB mutations. SIL-TAL1 was known as most frequent rearrangement, TAL1 enhancer insertions were also frequent in TAL1 overexpressed samples. TAL1 enhancer insertion and MYB mutation was exclusive, suggesting that TAL1 and MYB have a key role in childhood T-ALL. Consistent with other reports, frequent translocations were not observed in T-ALL, suggesting the genetic differences between T-ALL and other hematological malignancies. Further studies will be necessary to unravel oncogenic mechanisms that implicated in new therapeutic strategy for childhood T-ALL. DisclosuresNo relevant conflicts of interest to declare.

The CRM1 Nuclear Export Receptor Activates HOXA Gene Expression in Leukemogenesis

HOXA genes are effectors of oncogenic transformation that are frequently upregulated in myeloid and T-cell acute leukemias. Chromosomal translocation-derived oncoproteins, including MLL fusions, NUP (NUP98 or NUP214) fusions or CALM-AF10, bind to HOXA genes and result in their overexpression. We have previously demonstrated that a CRM1-dependent Nuclear Export Signal (NES) within CALM is essential for CALM-AF10’s ability to upregulate HOXA genes and cause leukemia in mice. Interfering with the CRM1/CALM-AF10 interaction by either genetic or pharmacologic inhibition abolishes CALM-AF10’s ability to bind to and activate HOXA gene expression. Furthermore, we showed that CRM1 binds to HOXA loci, suggesting that CRM1 recruits CALM-AF10 to its target genes.To explore whether CRM1 is also involved in the upregulation of Hoxa genes associated with MLL- and NUP98-fusion genes, we measured Hoxa transcript levels in murine leukemia cells treated with the CRM1 inhibitor Leptomycin B (LMB). LMB is a small molecule that covalently binds to the NES binding domain of CRM1 and blocks its ability to interact with NES partner proteins. We found that treatment of MLL-AF10, MLL-ENL, NUP98-HOXA9 or NUP98-AF10 leukemia cells with LMB (1 nM, 2 hours) causes a 50% reduction of Hoxa7, Hoxa9, Hoxa10 and Hoxa11 levels, similar to what is observed in CALM-AF10 leukemia cells. This suggests that in addition to its ability to interact with CALM-AF10, CRM1 may also participate in the transcriptional activation of Hoxa genes caused by MLL- or NUP98-fusion proteins.To demonstrate the importance of the CRM1/CALM interaction in CALM-AF10-dependent oncogenesis, we studied the biological activity of an artificial CRM1-AF10 fusion protein. Using a murine bone marrow clonogenic progenitor replating assay, we found that while native CRM1 overexpression did not result in transformation, the CRM1-AF10 fusion significantly increased the self-renewal of clonogenic progenitors. This effect was even more pronounced when CRM1 was fused to the MLL partner ENL: transduction with a CRM1-ENL fusion gene caused the immortalization of clonogenic bone marrow progenitors. Both CRM1-AF10- and CRM1-ENL-transduced progenitors displayed overexpression of Hoxa genes. To investigate the leukemogenic potential of CRM1-AF10in vivo, we transplanted mice with retrovirally transduced bone marrow progenitors and found that CRM1-AF10 induces myeloid neoplasms with a low penetrance and long latency (after more than a year of observation, 5 of 15 mice developed myeloid neoplasms between 160 and 220 days). These primary CRM1-AF10 leukemias could be transplanted to secondary recipients and cause myeloid leukemias with a shorter latency. Experiments to determine the leukemogenic potential of CRM1-ENLin vivo are ongoing. In contrast to CRM1-AF10, CRM1-ENL-transduced progenitors displayed a marked proliferative advantage in all transplanted mice (assessed by the elevation in the percentage of GFP-expressing CRM1-ENL-transduced cells in the peripheral blood over time); mice transplanted 74 days ago will be followed to determine survival curves.In summary, our results demonstrate that CRM1 regulates the expression of Hoxa genes in mouse leukemia cells, and alteration of CRM1’s activity can drive murine leukemogenesis. This has implications for understanding the mechanisms of HOXA deregulation in human leukemias induced by various fusion oncoproteins. It is noteworthy that in addition to interacting directly with CALM-AF10 through the NES, CRM1 physiologically interacts with NUP98 and NUP214 to facilitate transport through the nuclear pore. Our data also suggest that the anti-tumor effects of CRM1 inhibitors (Selective Inhibitors of Nuclear Export, SINEs) currently undergoing clinical trials, could be mediated, at least in part, by their ability to block the transcriptional activation of tumor-promoting genes by CRM1. DisclosuresNo relevant conflicts of interest to declare.

Effects of Iron Depletion on CALM-AF10 Leukemias

Iron, an essential nutrient for cellular growth and proliferation, enters cells via clathrin-mediated endocytosis (CME). The clathrin assembly lymphoid myeloid (CALM) protein plays an essential role in the cellular import of iron by CME. CALM-AF10 leukemias harbor a single copy of the normal CALM gene, and may therefore be more sensitive to the growth inhibitory effect of iron restriction compared with normal hematopoietic cells. We found that Calm heterozygous (CalmHET) murine fibroblasts exhibit signs of iron deficiency, with increased surface transferrin receptor (sTfR) levels and reduced growth rates. CalmHET hematopoietic cells are more sensitive in vitro to iron chelators than their wild type counterparts. Iron chelation also displayed toxicity towards cultured CalmHETCALM-AF10 leukemia cells and this effect was additive to that of chemotherapy. In mice transplanted with CalmHETCALM-AF10 leukemia, we found that dietary iron restriction reduces tumor burden in the spleen. However, dietary iron restriction, used alone or in conjunction with chemotherapy, did not increase survival of mice with CalmHETCALM-AF10 leukemia. In summary, while Calm heterozygosity results in iron deficiency and increased sensitivity to iron chelation in vitro, our data in mice do not suggest that iron depletion strategies would be beneficial for the therapy of CALM-AF10 leukemia patients.

A critical role for CRM1 in regulating HOXA gene transcription in CALM-AF10 leukemias

The leukemogenic CALM-AF10 fusion protein is found in patients with immature acute myeloid and T-lymphoid malignancies. CALM-AF10 leukemias display abnormal H3K79 methylation and increased HOXA cluster gene transcription. Elevated expression of HOXA genes is critical for leukemia maintenance and progression; however, the precise mechanism by which CALM-AF10 alters HOXA gene expression is unclear. We previously determined that CALM contains a CRM1-dependent nuclear export signal (NES), which is both necessary and sufficient for CALM-AF10-mediated leukemogenesis. Here, we find that interaction of CALM-AF10 with the nuclear export receptor CRM1 is necessary for activating HOXA gene expression. We show that CRM1 localizes to HOXA loci where it recruits CALM-AF10, leading to transcriptional and epigenetic activation of HOXA genes. Genetic and pharmacological inhibition of the CALM-CRM1 interaction prevents CALM-AF10 enrichment at HOXA chromatin, resulting in immediate loss of transcription. These results provide a comprehensive mechanism by which the CALM-AF10 translocation activates the critical HOXA cluster genes. Furthermore, this report identifies a novel function of CRM1: the ability to bind chromatin and recruit the NES-containing CALM-AF10 transcription factor.

A CALM-derived nuclear export signal is essential for CALM-AF10–mediated leukemogenesis

AbstractThe t(10;11) chromosomal translocation gives rise to the CALM-AF10 fusion gene and is found in patients with aggressive and difficult-to-treat hematopoietic malignancies. CALM-AF10–driven leukemias are characterized by HOXA gene up-regulation and a global reduction in H3K79 methylation. DOT1L, the H3K79 methyltransferase, interacts with the octapeptide/leucine zipper domain of AF10, and this region has been shown to be necessary and sufficient for CALM-AF10–mediated transformation. However, the precise role of CALM in leukemogenesis remains unclear. Here, we show that CALM contains a nuclear export signal (NES) that mediates cytoplasmic localization of CALM-AF10 and is necessary for CALM-AF10–dependent transformation. Fusions of the CALM NES (NESCALM-AF10) or NES motifs from heterologous proteins (ABL1, Rev, PKIA, APC) in-frame with AF10 are sufficient to immortalize murine hematopoietic progenitors in vitro. The CALM NES is essential for CALM-AF10–dependent Hoxa gene up-regulation and aberrant H3K79 methylation, possibly by mislocalization of DOT1L. Finally, we observed that CALM-AF10 leukemia cells are selectively sensitive to inhibition of nuclear export by Leptomycin B. These findings uncover a novel mechanism of leukemogenesis mediated by the nuclear export pathway and support further investigation of the utility of nuclear export inhibitors as therapeutic agents for patients with CALM-AF10 leukemias.

Iron Deprivation Impairs Proliferation of CALM-AF10 leukemia Cells in Vitro and in Vivo

Abstract 1386 Background:The CALM-AF10 fusion protein arises from the t(10;11) chromosomal translocation and is found in 8–10% of T-cell acute lymphoblastic leukemias and a smaller percentage of acute myeloid leukemias; these hematopoietic malignancies are associated with a poor clinical outcome. The precise contributions of the Clathrin Assembly Lymphoid Myeloid leukemia (CALM) protein to leukemogenesis remain uncertain. CALM plays a role in clathrin-dependent endocytosis, which mediates the entry of growth factor receptors and nutrients into cells and is essential for the internalization of iron-bound transferrin. We have previously shown that Calm-deficient (Calm−/−) mouse fibroblasts derived from fit1 mice are iron deficient, and are more sensitive to treatment with iron chelators. Objective:Since CALM-AF10 leukemia cells are haploinsufficient for CALM, we hypothesize that reduced levels of CALM in CALM-AF10 leukemia cells cause a relative iron deficiency, and result in enhanced sensitivity to iron deprivation. Design/Methods:Fibroblasts and fetal liver hematopoietic progenitors (HP) were derived from Calm−/−, Calm+/− or Calm+/+ E14 embryos. The proliferation rates of non-immortalized fibroblasts were compared in the presence and absence of supplemental iron (ferric ammonium citrate (FAC)) or treatment with an iron chelator (deferoxamine (DFO)). Surface transferrin receptor expression was quantified by flow cytometry. Primary HP cells were cultured in the presence or absence of DFO and viable cell numbers were determined. To examine the anti-leukemic effect of iron deprivation in vivo, C57BL/6J-Tyrc-2J mice were fed a low-iron diet, transplanted with CALM-AF10-transduced Calm+/− leukemia cells and leukemia latency was determined. Results:Heterozygous Calm+/− fibroblasts exhibit CALM protein levels that are intermediate between their wildtype (Calm+/+) and deficient (Calm−/−) counterparts. Calm+/− cells display a slower rate of proliferation in vitro (50% reduction of viable cells) compared to their wildtype fibroblasts, and this growth deficiency can be corrected by iron supplementation with 50 mM FAC. The presence of reduced intracellular iron levels in Calm+/− fibroblasts was manifested by increased transferrin receptor expression relative to wildtype cells. In vitro, Calm+/− HP cells showed greater sensitivity to iron chelation by DFO (5 mM) than Calm+/+ controls: the relative number of viable cells in the presence of DFO was 20% lower in Calm+/− compared to Calm+/− HP cells. To assess the impact of iron deprivation in vivo, bone marrow cells from Calm+/−CALM-AF10 leukemic mice were injected into secondary recipient mice preconditioned by having received a low-iron diet for 8 weeks (n=9). The onset of leukemia in transplanted mice maintained on the iron-deficient diet was considerably delayed (median survival 93 days versus 63 days, p=0.013) relative to age- and gender-matched control mice fed a normal diet (n=9). Conclusions:We have shown that reduced CALM expression impairs iron import and consequently limits the rate of cell proliferation. Both in vitro and in vivo results suggest that CALM haploinsufficient CALM-AF10 leukemias are particularly sensitive to iron deprivation. This raises the possibility that iron chelation may be a previously unappreciated treatment option for patients with aggressive CALM-rearranged leukemias. We are currently studying the impact of iron chelation in murine CALM-AF10 in vivo leukemia models. Disclosures:No relevant conflicts of interest to declare.

Abrogation of MLL–AF10 and CALM–AF10-mediated transformation through genetic inactivation or pharmacological inhibition of the H3K79 methyltransferase Dot1l

The t(10;11)(p12;q23) translocation and the t(10;11)(p12;q14) translocation, which encode the MLL (mixed lineage leukemia)-AF10 and CALM (clathrin assembly lymphoid myeloid leukemia)-AF10 fusion oncoproteins, respectively, are two recurrent chromosomal rearrangements observed in patients with acute myeloid leukemia and acute lymphoblastic leukemia. Here, we demonstrate that MLL-AF10 and CALM-AF10-mediated transformation is dependent on the H3K79 methyltransferase Dot1l using genetic and pharmacological approaches in mouse models. Targeted disruption of Dot1l using a conditional knockout mouse model abolished in vitro transformation of murine bone marrow cells and in vivo initiation and maintenance of MLL-AF10 or CALM-AF10 leukemia. The treatment of MLL-AF10 and CALM-AF10 transformed cells with EPZ004777, a specific small-molecule inhibitor of Dot1l, suppressed expression of leukemogenic genes such as Hoxa cluster genes and Meis1, and selectively impaired proliferation of MLL-AF10 and CALM-AF10 transformed cells. Pretreatment with EPZ004777 profoundly decreased the in vivo spleen-colony-forming ability of MLL-AF10 or CALM-AF10 transformed bone marrow cells. These results show that patients with leukemia-bearing chromosomal translocations that involve the AF10 gene may benefit from small-molecule therapeutics that inhibit H3K79 methylation.

Abstract LB-154: Nuclear export of CALM-AF10 is essential for leukemogenesis

Abstract Background: The t(10;11) chromosomal translocation gives rise to the CALM-AF10 fusion gene and is recurrently found in patients with aggressive and difficult-to-treat hematopoietic malignancies (AML and T-ALL). CALM-AF10 leukemias are characterized by upregulation of Hoxa cluster genes and a reduction in H3K79 methylation. The native AF10 protein localizes to the nucleus, while native CALM protein is found predominantly in the cytoplasm. It has been shown that the OM-LZ domain of AF10 interacts with the histone methyltransferase, Dot1L, and that this interaction is necessary for CALM-AF10-mediated transformation. Although it is known that CALM functions as an accessory endocytic protein, the precise role of CALM in leukemogenesis is unclear. Objective: To determine the role of CALM in CALM-AF10-mediated transformation in an effort to identify novel therapeutic targets. Results: Through structure-function analyses, we determined that a minimal region of CALM (aa 520-583) is critical for CALM-AF10-mediated transformation of murine hematopoietic progenitor cells (HPCs). Further analysis of this sequence identified a nuclear export signal (NES) encoded by CALM aa 544-553. The CALM NES mediates the cytoplasmic localization of CALM-AF10; point mutation of the CALM NES (NES*) results in exclusive CALM(NES*)-AF10 accumulation in the nucleus. In contrast to CALM-AF10, transduction of primary murine HPCs with CALM(NES*)-AF10 fails to cause in vitro transformation. Fusion of an 18 aa peptide that includes the CALM NES in frame with AF10 (NESCALM-AF10) is sufficient to induce transformation in vitro and leukemogenesis in vivo. Leukemias induced by NESCALM-AF10 recapitulated the phenotype of CALM-AF10 leukemias, including aberrant Hoxa cluster upregulation and decreased H3K79 methylation. Strikingly, fusion of the NES from different proteins, such as PKI-α and APC, to AF10 also conferred cytoplasmic localization and resulted in transformation in vitro. Finally, human CALM-AF10 leukemia cell lines (U937 and P31-FUJI) display increased sensitivity to a compound that inhibits nuclear export, Leptomycin B. Conclusions: We have determined that a CALM-derived nuclear export signal is both necessary and sufficient for CALM-AF10-mediated transformation. These findings reveal a novel mechanism by which nuclear export of CALM-AF10 mediates leukemogenesis. We postulate that nuclear export inhibitor (NEI) compounds may offer new therapeutic possibilities for patients with aggressive CALM-AF10 leukemias. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-154. doi:1538-7445.AM2012-LB-154

CALM Insufficiency Impairs Leukemia Cell Proliferation by Limiting Iron Uptake

Abstract 1478 Background:The CALM-AF10 fusion protein that arises from the t(10;11) chromosomal translocation is found in both acute myeloid leukemia and 8–10% of T-cell acute lymphoblastic leukemia and is associated with a poor clinical outcome. The interaction of AF10 with the DOT1L histone methyltransferase has been shown to be necessary for CALM-AF10 mediated transformation. However, the role of the Clathrin Assembly Lymphoid Myeloid leukemia (CALM) protein in leukemogenesis remains uncertain. CALM plays a role in clathrin-mediated endocytosis. This process mediates the entry of various growth factor receptors and nutrients into cells and is essential for the internalization of iron-bound transferrin. Objective:We hypothesize that loss of CALM function, as a result of CALM haploinsufficiency and/or a dominant negative CALM-AF10 effect, may contribute to transformation. Design/Methods:To study the effects of CALM deficiency, we used genetically modified fit1 mice that harbor a Calm gene mutation that results in a severely truncated and nonfunctional Calm protein. Fetal liver (FL) cells from Calm−/−, Calm+/− or CalmWT/WT E14 embryos were retrovirally transduced with MLL-ENL or CALM-AF10 and studied in vitro or transplanted into syngeneic mice. Interleukin-3 dependent cell lines were established from Calm−/− or CalmWT control FL cells immortalized by CALM-AF10 or MLL-ENL. Results:CALM-AF10 leukemias induced in Calm−/− or Calm+/− precursors were considerably delayed compared with those induced in CalmWT controls. Clathrin-mediated endocytosis of fluorescently labeled transferrin receptor (TfR) was impaired in Calm−/− cells. This was accompanied by a 2–4 fold increase in surface expression of TfR by flow cytometry and an increase in total TfR protein as measured by Western blot in Calm−/− cells. Calm -deficient cells also displayed lower levels of the iron storage protein ferritin, suggestive of an iron deficient state. Each of these features was reversed by re-expression of CALM via retroviral transduction of Calm−/− cells with a CALM expression vector. Paralleling the prolonged latency of Calm−/− leukemias, we found that Calm -deficient cells proliferated at a considerably slower rate than CALM -rescued cells. This cell proliferation defect could be rescued by supplementing the cells with iron (ferric ammonium citrate, 50 μM), indicating that reduced iron availability limits the expansion of Calm -deficient cells. Intriguingly, Calm−/− cells were significantly more sensitive to the growth inhibitory effect of iron chelation (deferoxamine, 5 μM) than CALM -rescued cells. Conclusions:These observations suggest that loss of CALM function impairs iron import and consequently limits the rate of cell proliferation. This raises the possibility that CALM haploinsufficiency present in CALM-AF10 leukemias might render cells particularly responsive to iron chelation therapy. Disclosures:No relevant conflicts of interest to declare.