CALM-AF10 Fusion Research Articles

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 role of the PZP domain of AF10 in acute leukemia driven by AF10 translocations

Chromosomal translocations of the AF10 (or MLLT10) gene are frequently found in acute leukemias. Here, we show that the PZP domain of AF10 (AF10PZP), which is consistently impaired or deleted in leukemogenic AF10 translocations, plays a critical role in blocking malignant transformation. Incorporation of functional AF10PZP into the leukemogenic CALM-AF10 fusion prevents the transforming activity of the fusion in bone marrow-derived hematopoietic stem and progenitor cells in vitro and in vivo and abrogates CALM-AF10-mediated leukemogenesis in vivo. Crystallographic, biochemical and mutagenesis studies reveal that AF10PZP binds to the nucleosome core particle through multivalent contacts with the histone H3 tail and DNA and associates with chromatin in cells, colocalizing with active methylation marks and discriminating against the repressive H3K27me3 mark. AF10PZP promotes nuclear localization of CALM-AF10 and is required for association with chromatin. Our data indicate that the disruption of AF10PZP function in the CALM-AF10 fusion directly leads to transformation, whereas the inclusion of AF10PZP downregulates Hoxa genes and reverses cellular transformation. Our findings highlight the molecular mechanism by which AF10 targets chromatin and suggest a model for the AF10PZP-dependent CALM-AF10-mediated leukemogenesis.

A Role for the CRM1 Nuclear Export Receptor in HOXA Gene Deregulation and CALM-AF10 Leukemogenesis

Background:CALM-AF10 translocations are seen in 5-10% of T-cell acute lymphoblastic leukemia (T-ALL) and are characterized by HOXA gene overexpression, a feature shared with leukemias associated with translocations of the Mixed-Lineage Leukemia (MLL) gene. The ability of CALM-AF10 to aberrantly activate HOXA gene transcription is mediated by interaction of the AF10 moiety with the H3K79 methyltransferase DOT1L. We previously demonstrated that a nuclear export signal (NES) within CALM is essential for CALM-AF10's ability to upregulate HOXA genes and cause leukemia in mice. The CRM1 (XPO1) nuclear export receptor specifically interacts with NES motifs, including the CALM NES. Fusing an 18 amino acid NES motif to AF10 recapitulates the oncogenic properties of CALM-AF10. Interfering with the CRM1/CALM-AF10 interaction by either genetic (mutation of the CALM NES motif) or pharmacologic (Leptomycin B - LMB) inhibition abolishes CALM-AF10's ability to bind to HOXA genes and activate their expression. We discovered that CRM1 itself is able to bind to HOXA loci, suggesting that CRM1 may recruit CALM-AF10 to its target genes. The NES binding cleft within CRM1 is maintained in a closed conformation by the C-terminal helix (CRM1 aa 1029-1071), and its truncation greatly enhances the NES-binding affinity of CRM1.Hypothesis: Since the NES motif of CALM is necessary and sufficient for the leukemogenic properties of CALM-AF10, we hypothesized that a CRM1-AF10 fusion protein would also be leukemogenic. To determine the importance of the NES binding ability of CRM1 in leukemogenesis, we investigated a deletion mutant of CRM1-AF10 that lacks the inhibitory C-terminal 43 amino acids of CRM1 (CRM1Δ-AF10).Methods: Confocal immunofluorescence microscopy of transfected HEK293 cells was used to study the intracellular localization of CRM1-AF10 and CRM1Δ-AF10 fusion proteins, and their transcriptional activity wasmeasured with a HOXA7 -luciferase reporter. We used LMB to study the impact of blocking the CRM1/NES interaction on HOXA7 transcriptional activation and cellular localization. Retroviral transduction and transplantation of murine bone marrow were performed to determine the ability of CRM1-AF10 and CRM1Δ-AF10 to induce leukemias.Results: CALM-AF10, CRM1-AF10 and CRM1Δ-AF10 fusion proteins localize to both the cytoplasm and nucleus (in a speckled pattern). While all three fusion proteins induced HOXA gene transcription compared with empty vector, CALM-AF10 (7.3 fold increase) and CRM1Δ-AF10 (7.8 fold) were significantly more potent than CRM1-AF10 (5.1 fold). LMB treatment (4 nM, 25 h) repressed HOXA7 transcription induced by CALM-AF10 (68% repressed), CRM1-AF10 (31% repressed), and CRM1Δ-AF10 (60% repressed). LMB exposure (2 nM, 2 h) induced nuclear retention of all three fusion proteins. In vivo, while all three fusions induced murine myeloid leukemias characterized by overexpression of Hoxa genes, CRM1-AF10 leukemias displayed incomplete penetrance (50%) with prolonged latency (250-300 days) compared with both CALM-AF10 and CRM1Δ-AF10, which demonstrated complete penetrance and similar latencies (100-130 days).Conclusions: We have shown that CRM1 can substitute for CALM to induce HOXA gene expression and cause leukemias when fused to AF10, suggesting that the contribution of the CALM moiety in the CALM-AF10 oncoprotein is to recruit CRM1. Deletion of the C-terminal inhibitory helix of CRM1 potentiates the oncogenic properties of CRM1-AF10. Moreover, pharmacologic inhibition of the NES/CRM1 interaction with LMB counteracts the ability of CRM1-AF10 and CRM1Δ-AF10 to activate the transcription of HOXA genes. These results suggest that binding to an as yet unidentified, NES-containing partner protein plays an important role in CRM1-AF10 leukemogenesis. Of note, the CRM1 C-terminal helix is missing in a CRM1-AF10 translocation described in a T-ALL patient (Bond, BLOOD, 2014). These studies shed light on the importance of CRM1 in leukemias associated with HOXA deregulation, and warrant further studies of the mechanisms underlying the therapeutic efficacy of CRM1 inhibitors (selective inhibitors of nuclear export - SINEs) that target the CRM1/NES interaction. DisclosuresNo 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.

Acute myeloid leukemia driven by the CALM-AF10 fusion gene is dependent on BMI1

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 often ineffective 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. Here we demonstrate that the Polycomb Repressive Complex 1 geneBMI1is consistently overexpressed in adult and pediatric CALM-AF10-positive leukemias. We demonstrate that genetic Bmi1 depletion abrogates CALM-AF10-mediated transformation of murine hematopoietic stem and progenitor cells (HSPCs). Furthermore, CALM-AF10-positive murine and human AML cells are sensitive to the small-molecule BMI1 inhibitor PTC-209 as well as to PTC-596, a compound in clinical development that has been shown to result in downstream degradation of BMI1 protein. PTC-596 significantly prolongs survival of mice injected with a human CALM-AF10 cell line in a xenograft assay. In summary, these results validate BMI1 as a bona fide candidate for therapeutic targeting in AML with CALM-AF10 rearrangements.

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.

Transfer of Ex Vivo Expanded NK and γδT Cells from Untouched Posttransplant PBMCs to Clear Minimal Residual Disease in Acute Myeloid Leukemia

GMP-grade NK cell expansion for clinical purpose has been demonstrated feasible and safe. Here we share our pilot data on posttransplant immunotherapy with ex vivo expanded NK cells to treat minimal residual disease in a pediatric patient with posttransplant relapsed myeloid leukemia. Our patient, a 13 year old boy who underwent 2nd allogeneic stem cell transplantation (haploidentical stem cell transplantation from his mother) due to posttransplant relapsed acute myeloid leukemia. After the 2nd haploidentical stem cell transplantation (SCT) minimal residual disease (MRD) was detected by multiparameter flow cytometry and by two molecular markers CALM-AF10 fusion transcript and a NRAS-mutation. For posttransplant compassionate use immunotherapy by NK cell transfer, NK cells were expanded from untouched isolated PBMCs of the patient post 2nd haploidentical SCT. GMP-grade expansion of the NK cells was done under static conditions in our GMP-facility. Isolated PBMCs were pooled with 100Gy irradiated K562mb15 4-1BBL feeder cells (kindly provided by Dario Campana) in a proportion of 1:20 (NK to K562mb15 4-1BBL). PBMCs and K562mb15 4-1BBL were seeded in conventional cell culture flasks (175cm2) at a density of 1.1E6 cells/ml. Cell culture media contained RPMI1640 supplemented with 10% AB-human serum, 1% L-glutamine and 100IU Proleukine® IL2/ml. Cell culture was monitored daily for cell number, white blood cell differentiation, pH of the cell culture, glucose metabolism, lactate production and microbial sterility testing at the beginning and the end of the expansion period. The cell product was harvested on day 15-17. Fresh isolated PBMCs and the expanded NK cell product were characterized by flow cytometry. NK cells were expanded >1000 fold (3.1 and 3.4 log-fold) in 14-17 days. The product contained a total number of 9.8E9 and 19.9E9 cells, which was 328 and 665E6/kgBW. The expansion protocol supports NK and γδ T cell expansion whereas the number of αβ T cells stays stable. Cytotoxicity assay against various targets revealed excellent cellular cytotoxicity and antibody dependent cellular cytotoxicity. To prevent relapse in our patient with posttransplant MRD positivity, NK cells from the patient post 2nd haploidentical SCT were expanded for cellular immunotherapy. 2 weeks post 1st NK cell transfer (day +170) the patient achieved complete MRD response in the bone marrow. Unfortunately the patient showed detectable MRD one month later. Therefore another NK cell expansion and transfer was done. 2 weeks post 2nd NK cell transfer (day +232) the patient again achieved complete MRD response in the bone marrow and is in complete molecular remission ever since (day +340). The NK cell products were tolerated well. Transient coughing and temporary increase of temperature were registered. Both, in vitro and in vivo effect of the NK cell product were documented. Clinical use of expanded and activated NK cells and γδ T cells can induce molecular remission in posttransplant MRD positive acute myeloid leukemia. DisclosuresNo relevant conflicts of interest to declare.

Abstract 1569: The nuclear export signal (NES) within CALM is necessary for CALM-AF10-induced leukemia

Abstract The CALM-AF10 fusion gene, which results from a t(10;11) translocation, is found in a variety of hematopoietic malignancies. Certain HOXA cluster genes and MEIS1 genes are upregulated in patients and mouse models that express CALM-AF10. Wild-type CALM primarily localizes in a diffuse pattern within the cytoplasm, whereas AF10 localizes in the nucleus; however, it is not clear where CALM-AF10 acts to induce leukemia. To investigate the influence of localization on leukemogenesis involving CALM-AF10, we determined the nuclear export signal (NES) within CALM that is necessary and sufficient for cytoplasmic localization of CALM-AF10. Mutations in the NES eliminated the capacity of CALM-AF10 to immortalize murine bone-marrow cells in vitro and to promote development of acute myeloid leukemia in mouse models. Furthermore, a fusion of AF10 with the minimal NES could immortalize bone-marrow cells and induce leukemia in mice. These results suggest that during leukemogenesis, CALM-AF10 plays its critical roles in the cytoplasm. Citation Format: Mai Suzuki, Kazutsune Yamagata, Yukiko Aikawa, Toshio Watanabe, Issay Kitabayashi. The nuclear export signal (NES) within CALM is necessary for CALM-AF10-induced leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1569. doi:10.1158/1538-7445.AM2014-1569

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.

Nuclear export signal within CALM is necessary for CALM‐AF10‐induced leukemia

The CALM–AF10 fusion gene, which results from a t(10;11) translocation, is found in a variety of hematopoietic malignancies. Certain HOXA cluster genes and MEIS1 genes are upregulated in patients and mouse models that express CALM-AF10. Wild-type clathrin assembly lymphoid myeloid leukemia protein (CALM) primarily localizes in a diffuse pattern within the cytoplasm, whereas AF10 localizes in the nucleus; however, it is not clear where CALM-AF10 acts to induce leukemia. To investigate the influence of localization on leukemogenesis involving CALM-AF10, we determined the nuclear export signal (NES) within CALM that is necessary and sufficient for cytoplasmic localization of CALM-AF10. Mutations in the NES eliminated the capacity of CALM-AF10 to immortalize murine bone-marrow cells in vitro and to promote development of acute myeloid leukemia in mouse models. Furthermore, a fusion of AF10 with the minimal NES can immortalize bone-marrow cells and induce leukemia in mice. These results suggest that during leukemogenesis, CALM-AF10 plays its critical roles in the cytoplasm.

The prognosis of CALM-AF10-positive adult T-cell acute lymphoblastic leukemias depends on the stage of maturation arrest

CALM-AF10 (also known as PICALM-MLLT10) is the commonest fusion protein in T-cell acute lymphoblastic leukemia, but its prognostic impact remains unclear. Molecular screening at diagnosis identified CALM-AF10 in 30/431 (7%) patients with T-cell acute lymphoblastic leukemia aged 16 years and over and in 15/234 (6%) of those aged up to 15 years. Adult CALM-AF10-positive patients were predominantly (72%) negative for surface (s)CD3/T-cell receptor, whereas children were predominantly (67%) positive for T-cell receptor. Among 22 adult CALM-AF10-positive patients treated according to the LALA94/GRAALL03-05 protocols, the poor prognosis for event-free survival (P=0.0017) and overall survival (P=0.0014) was restricted to the 15 T-cell receptor-negative cases. Among CALM-AF10-positive, T-cell receptor-negative patients, 82% had an early T-cell precursor phenotype, reported to be of poor prognosis in pediatric T-cell acute lymphoblastic leukemia. Early T-cell precursor acute lymphoblastic leukemia corresponded to 22% of adult LALA94/GRAALL03-05 T-cell acute lymphoblastic leukemias, but had no prognostic impact per se. CALM-AF10 fusion within early T-cell precursor acute lymphoblastic leukemia (21%) did, however, identify a group with a poor prognosis with regards to event-free survival (P=0.04). CALM-AF10 therefore identifies a poor prognostic group within sCD3/T-cell receptor negative adult T-cell acute lymphoblastic leukemias and is over-represented within early T-cell precursor acute lymphoblastic leukemias, in which it identifies patients in whom treatment is likely to fail. Its prognosis and overlap with early T-cell precursor acute lymphoblastic leukemia in pediatric T-cell acute lymphoblastic leukemia merits analysis. The clinical trial GRAALL was registered at Clinical Trials.gov number NCT00327678.

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.

Abrogation of MLL-AF10 and CALM-AF10 Mediated Transformation Through Genetic Inactivation or Pharmacological Inhibition of the H3K79 Methyltransferase DOT1L.

AbstractAbstract 2384The t(10;11)(p12;q23) and the t(10;11)(p12;q14), which encode the MLL-AF10 and CALM-AF10 fusion oncoproteins respectively, are two recurrent chromosomal rearrangements observed in patients with acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). Patients with AML harboring either MLL-AF10 or CALM-AF10 rearrangements have a particularly poor outcome compared to patients whose leukemia cells do not harbor these translocations. Thus new therapeutic approaches are clearly needed for patients with leukemias bearing rearrangements of the AF10 gene. Previous studies have suggested that the histone H3 lysine 79 (H3K79) methyltransferase DOT1L is recruited by a part of the AF10 protein retained in leukemic MLL-AF10 and CALM-AF10 fusions, suggesting a role of this methyltransferase in transformation mediated by these fusion oncoproteins. The general amenability of enzymes to pharmacological inhibition makes DOT1L an attractive therapeutic target in leukemias bearing AF10-fusions. We decided to test whether transformation mediated by the MLL-AF10 or CALM-AF10 fusions can be impaired by Dot1l inhibition. We transformed bone marrow cells with either the MLL-AF10 or the CALM (400–648)-AF10 (677–758) fusion proteins that have been previously shown to immortalize hematopoietic progenitors in vitro and in vivo. In contrast to the large, highly clonogenic compact colonies observed in methylcellulose culture of wildtype transformed cells, Dot1l excision from the MLL-AF10 or CALM-AF10 immortalized progenitors led to the formation of small, diffuse colonies that had lost their replating capability. In MLL-AF10 or CALM-AF10 transformed cells, Dot1l- inactivation significantly reduced global H3K79 dimethylation as assessed by Western blotting, and Wright Giemsa staining confirmed morphological features consistent with myeloid differentiation. Moreover, the expression of MLL-AF10 and CALM-AF10 targets, such as Hoxa cluster genes and Meis1, decreased significantly after Dot1l inactivation as assessed by quantitative PCR. We then assessed the impact of Dot1l deletion on the in vivo leukemogenic activity of MLL-AF10 and CALM-AF10 transformed bone marrow cells. Ongoing experiments clearly demonstrate that Dot1l excision strongly impairs the initiation and maintenance of both MLL-AF10 as well as CALM-AF10 mediated murine leukemias.Having established that genetic inactivation of Dot1l inhibits H3K79 methylation and consequently transforming potential of the AF10-fusions, we investigated the efficacy of Dot1l inhibitors against MLL-AF10 or CALM-AF10 transformed murine bone marrow cells. Treatment of MLL-AF10 or CALM-AF10 transformed cells with EPZ004777, the first specific small-molecule inhibitor of Dot1l, suppressed expression of downstream oncogenic targets such as Hoxa cluster genes and Meis1, and selectively impaired the proliferation of MLL-AF10 and CALM-AF10 transformed, but not Hoxa9/Meis1 transformed cells. MLL-AF10 and CALM-AF10 transformed cells underwent differentiation and cell cycle arrest after EPZ004777 treatment. Results from in vivo colony-forming units-spleen (CFU-S) assays showed that pre-treatment of MLL-AF10 or CALM-AF10 transformed cells with the EPZ004777 inhibitor profoundly impaired their spleen-colony forming ability, suggesting that EPZ004777 may show in vivo efficacy against AF10-fusion transformed cells. Taken together, our results demonstrate that Dot1l inhibition impairs the in vitro and in vivo oncogenic activity of the MLL-AF10 and CALM-AF10 fusion oncogenes. These results indicate that patients with leukemias bearing chromosomal rearrangements of the AF10 gene may benefit from small molecule inhibition of H3K79 methylation. Disclosures:Olhava:Epizyme: Employment. Daigle:Epizyme, Inc.: Employment. Richon:Epizyme, Inc.: Employment, Equity Ownership. Pollock:Epizyme Inc.: Employment, Equity Ownership. Armstrong:Epizyme: Consultancy.

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.

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.

Mutations of PHF6 are associated with mutations of NOTCH1, JAK1 and rearrangement of SET-NUP214 in T-cell acute lymphoblastic leukemia

Mutations in the PHF6 gene were recently described in patients with T-cell acute lymphoblastic leukemia and in those with acute myeloid leukemia. The present study was designed to determine the prevalence of PHF6 gene alterations in T-cell acute lymphoblastic leukemia. We analyzed the incidence and prognostic value of PHF6 mutations in 96 Chinese patients with T-cell acute lymphoblastic leukemia. PHF6 deletions were screened by real-time quantitative polymerase chain reaction and array-based comparative genomic hybridization. Patients were also investigated for NOTCH1, FBXW7, WT1, and JAK1 mutations together with CALM-AF10, SET-NUP214, and SIL-TAL1 gene rearrangements. PHF6 mutations were identified in 11/59 (18.6%) adult and 2/37 (5.4%) pediatric cases of T-cell acute lymphoblastic leukemia, these incidences being significantly lower than those recently reported. Although PHF6 is X-linked and mutations have been reported to occur almost exclusively in male patients, we found no sex difference in the incidences of PHF6 mutations in Chinese patients with T-cell acute lymphoblastic leukemia. PHF6 deletions were detected in 2/79 (2.5%) patients analyzed. NOTCH1 mutations, FBXW7 mutations, WT1 mutations, JAK1 mutations, SIL-TAL1 fusions, SET-NUP214 fusions and CALM-AF10 fusions were present in 44/96 (45.8%), 9/96 (9.4%), 4/96 (4.1%), 3/49 (6.1%), 9/48 (18.8%), 3/48 (6.3%) and 0/48 (0%) of patients, respectively. The molecular genetic markers most frequently associated with PHF6 mutations were NOTCH1 mutations (P=0.003), SET-NUP214 rearrangements (P=0.002), and JAK1 mutations (P=0.005). No differences in disease-free survival and overall survival between T-cell acute lymphoblastic leukemia patients with and without PHF6 mutations were observed in a short-term follow-up. Overall, these results indicate that, in T-cell acute lymphoblastic leukemia, PHF6 mutations are a recurrent genetic abnormality associated with mutations of NOTCH1, JAK1 and rearrangement of SET-NUP214.

The clathrin-binding domain of CALM and the OM-LZ domain of AF10 are sufficient to induce acute myeloid leukemia in mice

The t(10;11)(p13-14;q14-21) translocation, giving rise to the CALM-AF10 fusion gene, is a recurrent chromosomal rearrangement observed in patients with poor prognosis acute myeloid leukemia (AML). Although splicing of the CALM-AF10 fusion transcripts has been described in AML patients, the contribution of different CALM and AF10 domains to in vivo leukemogenesis remains to be defined. We therefore performed detailed structure-function studies of the CALM-AF10 fusion protein. We demonstrate that fusion of the C-terminal 248 amino acids of CALM, which include the clathrin-binding domain, to the octapeptide motif-leucine-zipper (OM-LZ) domain of AF10 generated a fusion protein (termed CALM-AF10 minimal fusion (MF)), with strikingly enhanced transformation capabilities in colony assays, providing an efficient system for the expeditious assessment of CALM-AF10-mediated transformation. Leukemias induced by the CALM-AF10 (MF) mutant recapitulated multiple aspects of full-length CALM-AF10-induced leukemia, including aberrant Hoxa cluster upregulation, a characteristic molecular lesion of CALM-AF10 leukemias. In summary, this study indicates that collaboration of the clathrin-binding and the OM-LZ domains of CALM-AF10 is sufficient to induce AML. These findings further suggest that future approaches to antagonize CALM-AF10-induced transformation should incorporate strategies, which aim at blocking these key domains.

Use of CALM-AF10 and NUP98-HOXD13 fusions to Identify Oncogenic Pathways That Collaborate with HOXA9 Overexpression

AbstractAbstract 2499Acute myeloid leukemias (AMLs) are thought to result from a series of complementary mutations that affect several vital pathways, including differentiation, self-renewal, apoptosis, and proliferation. Several mutations associated with AML (MLL fusions, NUP98 fusions, CALM-AF10 fusion, and NPM1 mutations) are thought to impair hematopoietic differentiation by dysregulation of target genes; one of the most consistently dysregulated target genes is HOXA9. Mice that express a CALM-AF10 (CA10) or NUP98-HOXD13 (NHD13) fusion gene overexpress HOXA9 and develop AML with a delayed onset and incomplete penetrance, suggesting the need for complementary mutations. We have used three techniques including candidate gene resequencing, retroviral insertional mutagenesis (RIM), and gene expression profiling to compare and contrast the complementary mutations that occur in these mice. The frequency of Ras pathway mutations is similar for both CA10 (20%) and NHD13 (32%) leukemic mice, and previously reported RIM studies identified common integration sites near Mn1 and Evi1 in both models, suggesting that dysregulation of similar collaborative pathways can lead to leukemia in both models. However, there were significant differences between these models. The aforementioned RIM studies identified Zeb2 as the most frequent insertion site for CA10 leukemias, suggesting that Zeb2 collaborated with CA10, whereas no Zeb2 insertions were found in the NHD13 RIM study. Another distinction between the NHD13 and CA10 leukemias is the expression of the HOXA9 co-regulatory factor Meis1. Meis1 is markedly upregulated in bone marrow from clinically healthy, pre-leukemic CA10 mice and CA10 leukemic tissues compared to wildtype bone marrow. Conversely, Meis1 expression was decreased in bone marrow from clinically healthy NHD13 mice compared to wildtype, and only occasionally overexpressed in NHD13 leukemias. Interestingly, RIM analysis of NHD13 mice identified Meis1 as the most frequent insertion site, suggesting that Meis1 overexpression can collaborate with NHD13 during leukemic transformation. Flt3, which is frequently mutated in patients with AML, has been shown to be transcriptionally activated by Meis1. Of note, Flt3 is consistently overexpressed in CA10 leukemias but is overexpressed only in those NHD13 leukemias that have upregulated Meis1. Intriguingly, the frequency of Flt3 activating mutations in CA10 leukemias (which overexpress Flt3) was 25%, but none were found in the NHD13 leukemias (which do not overexpress Flt3). Lastly, using gene expression arrays, N-myc was identified as one of the most differentially overexpressed mRNAs, suggesting that it may cooperate with CA10 during leukemic transformation. Furthermore, N-myc has recently been shown to promote AML in mice, and our preliminary experiments suggest that N-myc and CA10 collaborate and lead to increased cell number and replating potential in bone marrow colony formation assays. In conclusion, we have taken several genetic approaches to identify key similarities and differences in the biology of two leukemic models (CA10 and NHD13), both of which overexpress HOXA9. Disclosures:No relevant conflicts of interest to declare.