Primary AML Blasts Research Articles

The Allo-HLA-DP Restricted T Cell Repertoire Contains a Variety of Tissue-Restricted Specificities with Therapeutic Value

T cell-depleted allogeneic stem cell transplantation (alloSCT) is applied in patients with hematological malignancies to reduce the risk of graft versus host disease (GvHD), but the associated increased risk of infections and disease relapse makes scheduled donor-lymphocyte-infusion (DLI) necessary. Since HLA-DP is not taken into account in the matching procedure, stem cell grafts from unrelated donors are often only 10/10 matched, and mismatched for HLA-DP. Under non-inflammatory conditions, expression of HLA-DP is restricted to hematopoietic cells. Therefore, treatment with HLA-DP mismatched donor CD4 T cells can induce a specific graft versus leukemia (GvL) effect. However, in some cases, patients receiving HLA-DP mismatched CD4 T cells suffer from GvHD mediated by a profound allo-HLA-DP specific immune response. Adoptive transfer of in-vitro selected allo-HLA-DP restricted donor T cells directed against antigens specifically expressed on hematopoietic cells may be an elegant strategy to induce a specific GvL effect without coinciding GvHD. To investigate the feasibility of this approach, the allo-HLA-DP restricted T cell repertoire was dissected to unravel potential tissue specificities and to investigate the presence of hematopoiesis-specific CD4+ T cells with therapeutic value within this compartment.To induce allo-HLA-DP directed T cell responses, HLA-DP mismatched (10/10 matched) donor/patient pairs were selected. Donor PBMC were co-cultured with patient mature monocyte-derived dendritic cells (DC) for 14 days. The donor cells were first re-stimulated with autologous DC and depleted of activated auto-reactive CD137+ T cells using magnet cell separation (MACS). The negative fraction was then stimulated with the HLA-DP mismatched patient DC to induce activation of allo-reactive T cells. These allo-reactive CD137+ CD4+ T cells were clonally isolated using flowcytometric cell sorting, and expanded. Allo-HLA-DP restricted recognition of different hematopoietic and non-hematopoietic stimulator cells by the T cell clones was assessed using IFNγ and IL-4 ELISA. The T cells were tested against a large panel of hematopoietic cells (monocytes, DC, EBV-LCL and PHA blasts) of donor and patient origin, leukemic cell samples (AML) and non-hematopoietic cells (IFNγ pretreated, HLA-class-II expressing, skin-derived fibroblasts, and target-HLA-DPB1-transduced HELA, lung, kidney, and colon carcinoma cell lines).After re-stimulation with patient DC, flowcytometry showed frequencies of 0.5-10% of allo-DC activated (CD137+) CD4+ T cells. After cell sorting 1521 T cell clones from 4 different HLA-DP mismatched patient/donor pairs were tested against donor EBV-LCL, donor DC, patient EBV-LCL and patient DC as initial screening for allo-reactivity. 80% of the T cell clones showed allo-reactivity, as defined by recognition of patient, but not donor-derived EBV-LCL and/or DC. 14% of the tested clones showed no reactivity and 6% were auto-reactive. The HLA-DP restriction was analyzed of 400 selected allo-reactive T cell clones, using a panel consisting of donor, patient and 3rd party EBV-LCL or DC, 3rd party fibroblasts and target HLA-DPB1 transduced HELA cells. Of these 400 T cell clones, 65% were confirmed to be HLA-DP restricted. From these allo-HLA-DP restricted T cell clones 80% recognized both hematopoietic and non-hematopoietic cells expressing the target allo-HLA-DPB1. The other 20% of the allo-HLA-DP restricted T cell clones only produced cytokines when stimulated with hematopoietic cells (EBV-LCL and/or DC), and not when stimulated with non-hematopoietic cells (Fibroblasts, HELA, carcinoma cell lines). Moreover, 40% of these T cell clones showing hematopoiesis-restricted recognition only recognized DC, but not EBV-LCL expressing the target HLA-DPB1 allele. These clones also recognized primary AML blasts and proliferating CD34+ progenitor cells, illustrating a myeloid lineage restricted recognition pattern.These results illustrate that reactivity of allo-HLA-DP restricted T cells is not only determined by the expression of the target HLA-DPB1 allele, but is also dictated by cell lineage-specific gene expression causing differential peptide expression in HLA-DPB1. As a result, a significant portion of the allo-HLA-DP specific T cell repertoire harbors a specific GvL recognition pattern with high therapeutic value. DisclosuresNo relevant conflicts of interest to declare.

MicroRNA-126 Orchestrates a Stem Cell-like Program in AML and B-ALL and Can be Exploited to Dissect Disease Heterogeneity

MicroRNA-126 reinforces hematopoietic stem cell (HSC) quiescence by dampening PI3K-AKT signaling. We recently reported key functions of miR-126 in acute leukemia: in human AML, it was required to maintain leukemic stem cell (LSC) quiescence (Lechman et al, Cancer Cell 2016), while its ectopic expression in mouse HSC induced leukemia (75% B-ALL, 25% AML) that fully regressed when switching a tetracycline-repressible miR-126 cassette off (Nucera et al, Cancer Cell 2016). RNA sequencing showed that miR-126 targeted cell cycle, apoptosis and p53 response genes, prevented differentiation and sustained oncogenic/pro-survival pathways typically associated with stem and progenitor cells (Kit, Wnt, Thy1, Jak/Stat, Bcl2). We quantified miR-126 expression levels in a cohort of 45 newly diagnosed AML patients (n=38 de novo, of which 37% favorable, 34% int-1, 16% int-2, 13% adverse according to the ELN classification) presenting at the San Raffaele Hospital between 2010 and 2015 using a robust digital droplet PCR assay. In addition to core-binding factor (CBF) mutated AML, we found that the group of AML with chromosomal aneuploidy showed significantly elevated miR-126 levels, suggesting that this subgroup is characterized by high LSC frequencies and/or a specific need to suppress p53 responses, a hypothesis supported by the data obtained in our mouse model. To measure miR-126 at single cell resolution, we stably transduced primary AML blasts (n=5 diseases) with a lentiviral miR-126 reporter vector, xenotransplanted them into NSG mice and quantified miR-126 activity by FACS in the engrafted cells recovered from the mice's bone marrow. Across different genetic subgroups including CBF leukemia, cells with the highest miR-126 activity were enriched in the CD34+ or CD34+CD38- fractions, consistent with LSC. To facilitate prospective LSC isolation based on miRNA activity, we screened a series of combination miRNA reporters incorporating response elements for LSC-enriched and LSC-depleted miRNAs and have now identified an optimized construct that highlights easily sortable, distinct subpopulations that are currently undergoing functional validation. Applying this tool to AML with complex and monosomal karyotype, we are addressing whether high miR-126 expression refers to elevated LSC frequency or is a specific feature of this clinically relevant AML entity. We next applied the miR-126 reporter to n=15 primary, human B-ALL measuring miR-126 activity in the xenograft. Surprisingly, we identified well-separated blast subpopulations that differed in miR-126 activity within single diseases. Heterogeneity for miR-126 appeared to be a general feature of B-ALL as we detected 2-3 subpopulations in most of the diseases studied. We purified miR-126(high) and miR-126(low) B-ALL subpopulations from the primografts and verified up to 1log differences in miR-126 expression, while we detected equal levels of the BCR-ABL fusion transcript in all subpopulations from Philadelphia+ B-ALL confirming their neoplastic nature. When transplanting miR-126(high) and miR-126(low) subpopulations into secondary or tertiary recipients, post-sorting miR-126 levels were tightly maintained indicating that miR-126 levels were static rather than dynamically regulated in distinct B-ALL subpopulations, as expected from a subclonal architecture. RNAseq performed on miR-126(high) and miR-126(low) human B-ALL fractions evidenced a miR-126 signature reminiscent of the one obtained in the B-ALL mouse model and cord blood CD34+ cells, uncovering physiological miR-126 activity in human primary B-ALL. Taken together, these data support a broad, pathogenetically important role for miR-126 in human AML and B-ALL that goes beyond LSC and open up opportunities to better understand leukemia disease biology, dissect intratumoral heterogeneity and therapeutically target resistant and refractory disease, considering that miR-126 knockdown expands normal HSC while depleting the leukemia. DisclosuresCiceri:MolMed SpA: Consultancy.

Decitabine enhances anti-CD33 monoclonal antibody BI 836858–mediated natural killer ADCC against AML blasts

AbstractAcute myeloid leukemia (AML) is the most common type of acute leukemia, affecting older individuals at a median age of 67 years. Resistance to intensive induction chemotherapy is the major cause of death in elderly AML; hence, novel treatment strategies are warranted. CD33-directed antibody-drug conjugates (gemtuzumab ozogamicin) have been shown to improve overall survival, validating CD33 as a target for antibody-based therapy of AML. Here, we report the in vitro efficacy of BI 836858, a fully human, Fc-engineered, anti-CD33 antibody using AML cell lines and primary AML blasts as targets. BI 836858–opsonized AML cells significantly induced both autologous and allogeneic natural killer (NK)-cell degranulation and NK-cell–mediated antibody-dependent cellular cytotoxicity (ADCC). In vitro treatment of AML blasts with decitabine (DAC) or 5-azacytidine, 2 hypomethylating agents that show efficacy in older patients, did not compromise BI 836858–induced NK-cell–mediated ADCC. Evaluation of BI 836858–mediated ADCC in serial marrow AML aspirates in patients who received a 10-day course of DAC (pre-DAC, days 4, 11, and 28 post-DAC) revealed significantly higher ADCC in samples at day 28 post-DAC when compared with pre-DAC treatment. Analysis of ligands to activating receptors (NKG2D) showed significantly increased NKG2D ligand [NKG2DL] expression in day 28 post-DAC samples compared with pre-DAC samples; when NKG2DL receptor was blocked using antibodies, BI 836858–mediated ADCC was significantly decreased, suggesting that DAC enhances AML blast susceptibility to BI 836858 by upregulating NKG2DL. These data provide a rationale for combination therapy of Fc-engineered antibodies such as BI 836858 with azanucleosides in elderly patients with AML.

399. Evaluation of CD123 Targeting CART Cells in Non-Human Primates

The CAR-T platform has provided an exceptionally potent means to treat cancers that have proven resistant to standard treatments. This potency, however, can work against normal tissue as well. Some clinical trials have identified adverse consequences as a result of CAR-T cell targeting of critical normal tissue. To enable as many targets as possible using the CAR-T cell approach, it is important to understand the potential liabilities of those targets in normal tissues prior to initiating clinical trials. CD123, the IL-3 receptor alpha subunit, is a viable target for treatment of acute myeloid leukemia, as it is expressed highly on primary AML blasts. We have previously shown that, in mice transplanted with human hematopoietic stem cells, CD123 targeting CAR T-cells eradicated these precursors and, in turn, normal hematopoiesis (Gill et al 2014xPreclinical targeting of human acute myeloid leukemia and myeloablation using chimeric antigen receptor-modified T cells. Gill, Saar, Tasian, Sarah K., Ruella, Marco, Shestova, Olga, Li, Yong, Porter, David L., Carroll, Martin, Danet-Desnoyers, Gwenn, Scholler, John, Grupp, Stephan A., June, Carl H., and Kalos, Michael. Blood. 2014; 123: 2343–2354Crossref | PubMed | Scopus (120)See all ReferencesGill et al 2014). Here, we describe an animal model developed to address the potential effects of targeting CD123 on non-hematopoietic tissue, namely endothelial cells that are found to express significant levels of CD123. An scFv that bound cynomolgus monkey CD123 was identified and a chimeric lentivirus that efficiently transduced monkey PBMCs was developed. Cells were demonstrated to be active in vitro and dosed into monkeys, after which cellular expansion was observed. In vivo safety assessments and histopathology results will be discussed.

Human cytokine-induced memory-like NK cells expand in patients with AML and display enhanced anti-leukemia responses.

Abstract NK cells are innate lymphoid cells that mediate anti-tumor responses. Relapsed/refractory AML patients have a poor prognosis and new therapy approaches are needed. Recently, memory-like properties of NK cells have been discovered, including cytokine-induced memory-like NK cells. Human NK cells briefly pre-activated with IL-12, IL-15, and IL-18 and then allowed to differentiate in vitro have greater re-stimulation responses compared to control cells. We hypothesized that such human memory-like NK cells possess enhanced anti-leukemia functionality. Indeed, memory-like NK cells produced increased IFN-γ in response to primary AML blasts in vitro, compared to controls. Multi-dimensional mass cytometry analyses revealed minimal alterations of memory-like NK cell inhibitory KIR repertoire, and both KIR-ligand matched and mismatched memory-like NK cells responded to primary AML blasts. In the context of a first-in-human clinical trial, allogeneic memory-like NK cells expanded to become the majority of the cells in the blood and bone marrow from AML patients 7–14 days after infusion. Furthermore, donor memory-like NK cells displayed enhanced leukemia-triggered IFN-γ production compared to recipient NK cells in the same samples, and clinical responses have been observed in 3 of 6 evaluable patients. In one patient, mass cytometry revealed expansion of CD3− CD56+ CD8+ NK cells with restricted KIR expression and multiple activating receptors. Deep NK cell immunophenotyping of in vivo expanded memory-like NK cells in additional patients is ongoing. Since memory-like NK cells expand in vivo and display enhanced anti-leukemia functionality, they represent a promising immunotherapy approach for patients with AML.

Novel evidence that pituitary gonadotropins directly stimulate human leukemic cells-studies of myeloid cell lines and primary patient AML and CML cells.

We recently reported that normal hematopoietic stem cells express functional pituitary sex hormone (SexH) receptors. Here we report for the first time that pituitary-secreted gonadotrophins stimulate migration, adhesion, and proliferation of several human myeloid and lymphoid leukemia cell lines. Similar effects were observed after stimulation of human leukemic cell lines by gonadal SexHs. This effect seems to be direct, as the SexH receptors expressed by leukemic cells responded to stimulation by phosphorylation of MAPKp42/44 and AKTser473. Furthermore, in parallel studies we confirmed that human primary patient-derived AML and CML blasts also express several functional SexH receptors. These results shed more light on the potential role of SexHs in leukemogenesis and, in addition, provide further evidence suggesting a developmental link between hematopoiesis and the germline.

Donor-Derived B Cells Produce Potent AML-Specific Antibodies That Recognize Novel Tumor-Specific Antigens and Mediate Graft-Versus-Leukemia Immunity

Unleashing the tumor-specific immune response by immunotherapies such as checkpoint inhibitors or allogeneic stem cell transplantation can result in long lasting tumor regression. While the role of T cells in such graft versus leukemia (GvL) immune responses has been established, the contribution of B cells to GvL responses is less clear. Using SEREX and other techniques, the presence of antibodies directed against established tumor antigens following allogeneic hematopoietic stem cell transplantation (HSCT) has been demonstrated. The function of these antibodies remains to be established though. Here we tested the hypothesis that B cells contribute to GvL.We selected three patients with high-risk AML who mounted potent GvL responses after allogeneic HSCT. Of these patients we established antibody-producing clonal B cell lines following transduction of memory B cells from the peripheral blood of these patients with Bcl-6 and Bcl-xL and screened those for producing antibodies specifically binding to surface antigens on AML cell lines and AML blasts. A number of antibodies were identified that recognized primary AML blasts isolated from newly diagnosed AML patients, but did not bind to healthy bone marrow, peripheral blood mononuclear cells or tissues such as liver, skin and colon. Target identification analyses revealed two novel, AML-specific surface proteins as primary targets of these antibodies. These newly identified AML-specific antigens are widely expressed on different types of AML, including blasts of the last 17 AML patients that entered our clinic of whom diagnosis material was made available for this project.Strikingly, antibody binding to one of these targets induced direct cell death of cultured AML cell lines and of primary AML blasts. Cytotoxicity of the antibodies was rapid, occurred both at 37o C and 4o C and could be prevented by Cytochalasin D, an actin polymerization inhibitor that stabilizes the cytoskeleton. This, in combination with the observation that cell death could not be prevented by apoptosis inhibitors indicated that the tumor cells were killed through a necrotic pathway like oncosis. Other antibodies induced antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent-cytotoxicity (CDC).Together these data indicate that tumor selective antibodies are elicited following allogeneic HSCT in AML patients with a strong GvL response. The direct and indirect cytotoxic activities against tumor cells of these antibodies suggest that they contribute to the GvL response. DisclosuresNo relevant conflicts of interest to declare.

Nuclear Export Inhibitor KPT-8602 Is Highly Active Against Leukemic Blasts and Leukemia-Initiating Cells in Patient-Derived Xenograft Models of AML

Currently available combination chemotherapy for AML induces complete remission in about 75% of younger and 45% of older patients, but often fails to induce long-term disease-free survival and is toxic to normal hematopoietic cells. The frequent disease relapse observed in chemotherapy treated AML patients is thought to occur through the inability of the existing drugs to specifically target the self-renewing leukemia-initiating cells (LICs). These cells have the functional capacity to replenish AML blasts. An attractive target for AML therapy is the nuclear export protein exportin 1 (XPO1), which is functionally required for continuous nuclear export of a subset of proteins and mRNAs. Small molecule Selective Inhibitor of Nuclear Export (SINE) compounds covalently bind to Cysteine528 in the cargo-binding groove of XPO1 and inhibit nuclear export. The orally bioavailable clinical SINE compound, selinexor (KPT-330), is currently in Phase 1 and 2 clinical trials in adult patients with AML (NCT01607892, NCT02088541, NCT02249091, NCT02403310, NCT02093403, NCT02485535, NCT02299518, NCT02416908, NCT02212561, and NCT01607892) and in a Phase 1 trial for relapsed childhood ALL and AML (NCT02091245). The results of these trials are encouraging, as they have demonstrated that selinexor alone or in combination is active in inducing remission in patients with relapsed or refractory AML. Here, we explore the anti-leukemia activity of a next generation SINE compound, KPT-8602.In preclinical toxicology studies as well as clinical trials, noted adverse events for selinexor are anorexia with weight loss, fatigue and thrombocytopenia, which limit the frequency of dosing to no more than every other day, three times a week. KPT-8602 is orally bioavailable and has similar pharmacokinetic properties to selinexor, with reduced brain penetration. Preliminary toxicology studies in rats and monkeys suggest that KPT-8602 has a substantially better tolerability profile, with reduced fatigue, thrombocytopenia, and anorexia compared to selinexor. Thus, KPT-8602 can be dosed orally on a daily basis for 5 days each week. The increased drug exposure with daily dosing and better tolerability of KPT-8602 leads directly to superior efficacy in pre-clinical models compared with selinexor.To define the anti-leukemia activity of KPT-8602 against primary AML blasts and LICs in a relevant pre-clinical setting, we established patient-derived xenograft (PDX) models, in which leukemic blasts from AML patients were transplanted into immunodeficient NOD-SCID-IL2Rcgnull (NSG) mice. Mice engrafted with leukemic blasts were treated with vehicle, selinexor (20 mg/kg three times per week), or KPT-8602 (15 mg/kg daily for 4 weeks). KPT-8602 was highly active against blast cells from three patients with poor-prognosis disease (cytogenetically normal AML with FLT3-ITD (AML-CN), AML with complex karyotype (AML-CK), and MDS-derived AML (MDS/AML)), as evidenced by a reduction in leukemic engraftment in primary mice after treatment (Fig. 1A-C). At these doses, KPT-8602 demonstrated higher anti-leukemic activity than selinexor, with no leukemic cells detected in the bone marrow of two of the eight AML-CN PDX treated mice (Fig. 1A-C). In secondary transplantation assays, KPT-8602 greatly reduced the frequency of LICs in the PDX model derived from AML-CN cells, indicating that this agent not only targets the bulk leukemic cells, but also eliminates LICs (Fig. 1D). In mice engrafted with normal cord blood human CD34+ cells, KPT-8602 reduced bone marrow human CD45+ cell numbers (Fig. 1E), but the impact on normal hematopoietic stem and progenitor cells, as determined by secondary transplantation assays (Fig. 1E), was modest when compared to the LIC reduction after KPT-8602 treatment. These findings demonstrate that KPT-8602 has better tolerability, perhaps through decreased CNS penetration, and is better at eliminating both AML LIC and blast cells when compared to selinexor. Therefore, KPT-8602 should be tested for efficacy and tolerability in patients with relapsed and refractory AML, with the goal to overcome a difficult obstacle in curing AML; destroying the LIC compartment while sparing normal hematopoietic cells. [Display omitted] DisclosuresEtchin:Karyopharm: Research Funding. Baloglu:Karyopharm Therapeutics Inc.: Employment, Equity Ownership. Landesman:Karyopharm: Employment. Senapedis:Karyopharm Therapeutics, Inc.: Employment, Patents & Royalties. Ellis:Karyopharm Therapeutics Inc: Employment. McCauley:Karyopharm Therapeutics, Inc: Employment. Stone:Agios: Consultancy; AROG: Consultancy; Abbvie: Consultancy; Sunesis: Consultancy, Other: DSMB for clinical trial; Karyopharm: Consultancy; Celator: Consultancy; Novartis: Research Funding; Merck: Consultancy; Juno: Consultancy; Pfizer: Consultancy; Amgen: Consultancy; Roche/Genetech: Consultancy; Celgene: Consultancy. DeAngelo:Bristol Myers Squibb: Consultancy; Incyte: Consultancy; Novartis: Consultancy; Pfizer: Consultancy; Agios: Consultancy; Ariad: Consultancy; Amgen: Consultancy; Celgene: Consultancy. Kauffman:Karyopharm: Employment, Equity Ownership. Shacham:Karyopharm: Employment, Equity Ownership. Look:Karyopharm Therapeutics, Inc: Research Funding.

Identification of CKMT1B As a New Target in EVI1-Positive AML

Recent investigations into the relationship between cancer and cellular metabolism have revealed the strong dependency of different cancers on a diverse array of metabolic pathways. For instance, a variety of cancers depend on the PI3K/AKT pathway for a wide range of glucose-related mechanisms, including AKT-mediated membrane translocation of glucose transporters, activation of the glycolytic enzymes hexokinase and phosphofructokinase, and up-regulation of de novo fatty acid synthesis. A major area of therapeutic and biological interest in the study of cancer metabolism is the process by which these metabolic pathways become deregulated in the first place en route to, or as a result of, the development of cancer. The deregulation of metabolic pathways can occur through alterations in the cellular landscape brought about by mutations in metabolic enzymes (e.g., IDH1/IDH2 and SDH), aberrant expression of transcription factors, such as the proto-oncogene MYC, or the loss of tumor suppressors, such as p53.Through gene expression and metabolic profiling analyses, we found that the transcription factor EVI-1, whose overexpression in acute and chronic myeloid leukemia (AML and CML) is correlated with poor patient outcome, induced key metabolic perturbations in hematopoietic progenitor cells. These result in a decrease of the mitochondrial oxygen consumption rate, a blockade of the de novo purine and pyrimidine synthesis, and an increase in glycolysis. Using a library of pooled shRNAs targeting genes involved in each of these pathways, we then established a direct link between EVI-1 expression and the development of a heretofore undescribed cellular dependency on the overexpression of the ATP-buffering mitochondrial creatine kinase protein CKMT1B. We showed that EVI-1 directly promotes CKMT1B expression through repression of the master regulator of myeloid differentiation RUNX1. Alteration of the CKMT1B-dependent pathway, either with shRNA or with the small molecule cyclocreatine, impairs production of intracellular phospho-creatine, which in turn alters cell viability specifically in EVI-1-positive (n = 8) versus EVI-1-negative AML cell lines (n = 8), and in EVI-1-positive (n = 5) versus EVI-1-negative (n = 17) primary AML blasts. This decrease in cell viability is associated with the activation of an erythroid differentiation program concomitant with a downregulation of the immature lineage marker c-KIT, both induced by GSK3A/B inhibition. Overexpression of a constitutively activated form of GSK3B (S9A) or GSK3A (S21A) impairs the cell differentiation induced by CKMT1B inhibition. Finally, suppression of CKMT1B alleviates leukemic burden in vivo in two AML mouse models: i) an orthotopic model of transplanted human EVI1-positive AML cells and ii) a syngeneic model of transplanted murine NrasG12D + Evi1 AML cells.This new interplay between EVI-1 and the creatine pathway uncovers CKMT1B as a new target of interest in EVI-1-positive AML, a high-risk subtype of AML for which current treatment regimens remain inadequate. DisclosuresDeAngelo:Novartis: Consultancy; Ariad: Consultancy; Bristol Myers Squibb: Consultancy; Pfizer: Consultancy; Amgen: Consultancy; Incyte: Consultancy; Agios: Consultancy; Celgene: Consultancy. Stone:Celgene: Consultancy; Agios: Consultancy; Karyopharm: Consultancy; Sunesis: Consultancy, Other: DSMB for clinical trial; Novartis: Research Funding; Celator: Consultancy; Merck: Consultancy; Roche/Genetech: Consultancy; Abbvie: Consultancy; Amgen: Consultancy; Pfizer: Consultancy; AROG: Consultancy; Juno: Consultancy. Stegmaier:Novartis Pharmaceuticals: Consultancy.

CDC25A governs proliferation and differentiation of FLT3-ITD acute myeloid leukemia.

We investigated cell cycle regulation in acute myeloid leukemia cells expressing the FLT3-ITD mutated tyrosine kinase receptor, an underexplored field in this disease. Upon FLT3 inhibition, CDC25A mRNA and protein were rapidly down-regulated, while levels of other cell cycle proteins remained unchanged. This regulation was dependent on STAT5, arguing for FLT3-ITD-dependent transcriptional regulation of CDC25A. CDC25 inhibitors triggered proliferation arrest and cell death of FLT3-ITD as well as FLT3-ITD/TKD AC-220 resistant cells, but not of FLT3-wt cells. Consistently, RNA interference-mediated knock-down of CDC25A reduced the proliferation of FLT3-ITD cell lines. Finally, the clonogenic capacity of primary FLT3-ITD AML cells was reduced by the CDC25 inhibitor IRC-083864, while FLT3-wt AML and normal CD34+ myeloid cells were unaffected. In good agreement, in a cohort of 100 samples from AML patients with intermediate-risk cytogenetics, high levels of CDC25A mRNA were predictive of higher clonogenic potential in FLT3-ITD+ samples, not in FLT3-wt ones.Importantly, pharmacological inhibition as well as RNA interference-mediated knock-down of CDC25A also induced monocytic differentiation of FLT3-ITD positive cells, as judged by cell surface markers expression, morphological modifications, and C/EBPα phosphorylation. CDC25 inhibition also re-induced monocytic differentiation in primary AML blasts carrying the FLT3-ITD mutation, but not in blasts expressing wild type FLT3. Altogether, these data identify CDC25A as an early cell cycle transducer of FLT3-ITD oncogenic signaling, and as a promising target to inhibit proliferation and re-induce differentiation of FLT3-ITD AML cells.

Abstract 2623: Molecular mechanism of synergy between BET-protein bromodomain antagonist (BA) and pTEFb kinase inhibitor against human AML blast progenitor cells

Abstract We have previously reported that treatment with BET-protein bromodomain antagonist (BA) reduces the enhancer/promoter occupancy of BRD4 and induces the expression of HEXIM1, which sequesters and inhibits pTEFb (a complex of CDK9 and cyclin T). pTEFb phosphorylates serine 2 in the C-terminal heptad repeats in the stalled RNA pol II (RNAP2), causing its pause-release and the mRNA transcript elongation. While relatively sparing normal bone marrow progenitor cells, BA treatment attenuates the expression of cMYC, BCL2 and CDK4/6 and induces apoptosis of cultured (OCI-AML3, MOLM13 and MV4-11) and primary AML blast progenitor cells (BPCs), including those expressing FLT3-ITD. Because of its ability to inhibit CDK9 and pTEFb, we determined whether co-treatment with flavopiridol (FP), a known inhibitor of CDK9, would enhance the activity of BA against the cultured and primary AML BPCs. Our findings demonstrate that co-treatment with BA and FP synergistically induced apoptosis of the cultured and primary AML BPCs (CI of < 1.0). Combination of BA and FP, versus each drug alone, was associated with greater attenuation of the levels of p-RNAP2, cMYC, BCL-2 and CDK4/6 but greater induction of HEXIM1 and p21 levels. Notably, co-treatment with FP and BA markedly reduced the levels of the anti-apoptotic protein MCL1, which is known to be transcriptionally regulated by pTEFb-mediated phosphorylation of p-RNAP2. However, treatment with BA and FP did not affect the levels of cyclin T or CDK9. Co-treatment with BA and FP also synergistically induced apoptosis of MOLM13-TKIR cells (expressing FLT3-ITD) that exhibit > 100-fold in vitro resistance to FLT3 tyrosine kinase inhibitors. We next determined whether HEXIM1 levels regulate the activity of BA and FP in the AML BPCs. While shRNA-mediated stable knockdown of HEXIM1 significantly reduced the lethal activity of BA in OCI-AML3/HKD and MOLM13/HKD cells, tetracycline-inducible ectopic overexpression of HEXIM1 significantly enhanced BA-induced apoptosis of MOLM13 and OCI-AML3 cells. Notably, the significant improvement in the median survival of NOD/SCID mice treated with JQ1 (50 mg/kg/5 days per week/for 3 weeks) was not observed in the similarly treated mice engrafted with MOLM13/HKD cells (p < 0.01). The lethality induced by co-treatment with JQ1 and FP was significantly reduced in MOLM13/HKD versus MOLM13 cells; conversely, it was significantly increased in MOLM13 cells with ectopic overexpression of HEXIM1 (p < 0.01). These findings indicate that co-treatment with BA and FP exerts synergistic lethality, and BA-mediated induction of HEXIM1 is mechanistically involved in mediating the lethal effects of BA and FP against human AML BPCs. Citation Format: Santhana G. T. Devaraj, Bhavin Shah, Warren Fiskus, Baohua Sun, Saikat Saha, Sai Ravi Pingali, Swaminathan P. Iyer, Sunil Sharma, James E. Bradner, Kapil N. Bhalla. Molecular mechanism of synergy between BET-protein bromodomain antagonist (BA) and pTEFb kinase inhibitor against human AML blast progenitor cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2623. doi:10.1158/1538-7445.AM2015-2623

413. Pre-Clinical Preparation and Validation of Tumor Cell-Based IL-12 Immunotherapy for Acute Myeloid Leukemia

Interleukin(IL)-12 is a potent pro-inflammatory cytokine that stimulates a variety of effector cells involved in anti-tumor immunity. Systemic administration of IL-12 has associated toxicities, however. Various strategies are being developed to reduce such toxicities by restricting IL-12 distribution. Options here include generating fusions with tumor-targeting molecules and directing gene delivery to specific cells. First – we used lentivirus vectors (LVs) to engineer expression of murine IL-12 in tumor cells ex vivo and subsequently infused such modified cells into recipient mice. This strategy restricts IL-12 to the local tumor microenvironment thereby promoting immune activation in the context of tumor-associated antigens (TAAs). Using mouse models of both leukemia and solid tumors, we found that this cell-based approach generated effective anti-tumor protection when as little as 1% of the tumor burden expressed IL-12 as long as threshold expression levels on a per cell basis were reached. Second – our groups showed that anti-tumor mechanisms here involve CD4+ killer T cells, dendritic cells, and direct cell-cell contact with effectors. Clinical translation of this cell-based IL-12 therapy is in progress in Toronto for AML using a novel LV to modify patients’ own blast cells. Patient AML cells collected to date (n = 21) were stratified based on in vivo growth kinetics and transduced with a near-GMP-grade bicistronic LV that encodes the human IL-12 cDNA as a p40-p70 fusion, as well as a mutant thymidylate kinase (tmpK) fused to the ectodomain of LNGFR (trLNGFR) as a suicide (cell-fate control) cassette. The trLNGFR/tmpK element allows selection and also selective ablation of transduced cells by administration of AZT. Furthermore, it also allows tracking of transduced cells and quantification of transduction frequencies/transgene expression levels. With our current protocol, functional transduction efficiencies of primary patient AML blasts ranged from 20% to 70% (n=17). Transduced AML cells displayed a strong correlation between vector copy number and trLNGFR/tmpK + IL-12 levels and dose-dependent sensitivity to AZT. In vitro immortalization (IVIM) assays determined that the near-GMP LV/IL-12 vector displayed minimal genotoxic risk in transduced lin- cells; insertion site analyses carried out on expanded clones displayed poly-to oligo-clonality patterns. Pre-clinical data on toxicity and scale-up considerations are being accumulated in preparation for a Clinical Trial Application to Health Canada targeting AML. This LV/IL-12 immunotherapy platform targeting tumor cells themselves thus holds potential to be effective against a wide variety of cancers.

NKG2D Signaling Leads to NK Cell Mediated Lysis of Childhood AML.

Natural killer cells have been shown to be relevant in the recognition and lysis of acute myeloid leukemia. In childhood acute lymphoblastic leukemia, it was shown that HLA I expression and KIR receptor-ligand mismatch significantly impact ALL cytolysis. We characterized 14 different primary childhood AML blasts by flow cytometry including NKG2D ligands. Further HLA I typing of blasts was performed and HLA I on the AML blasts was quantified. In two healthy volunteer NK cell donors HLA I typing and KIR genotyping were done. Blasts with high NKG2D ligand expression had significantly higher lysis by isolated NK cells. Grouping the blasts by NKG2D ligand expression led to a significant inverse correlation of HLA I expression and cytolysis in NKG2D low blasts. Furthermore, a significant positive correlation of NKG2D ligand expression and blast cytolysis was shown. No impact of KIR ligand-ligand mismatch was found but a significantly increased lysis of homozygous C2 blasts by KIR2DL1 negative NK cells (donor B) was revealed. In conclusion, NKG2D signaling leads to NK cell mediated lysis of childhood AML despite high HLA I expression.

Bone Marrow Stromal Cells Protect Both FLT3-ITD and FLT3-WT Primary AML Cells from the Anti-Leukemic Activity of the FLT3 Inhibitor AC220

FTL3 mutations are found in about 30% of AML patients, conferring a leukemic blast growth advantage, drug therapy resistance in the bone marrow (BM) and poor outcome. Mesenchymal stem/stromal cells (MSCs) are essential components of the bone marrow microenvironment, and growing evidence suggest that MSCs play a critical role in AML chemo-resistance, although the molecular mechanisms involved are poorly understood. The purpose of the study was to (1) establish an novel in vitro co-culture system between primary AML blasts and healthy donor BM-MSCs (HD-MSCs) or AML patient-derived MSCs (AML-MSCs), (2) evaluate the impact of culture with BM-MSCs on the sensitivity of AML cells to AC220 using patients samples with FLT3-ITD (n=4) or FLT3-WT (n=3). We first cultured HD-MSCs (n=5) and AML-MSC (n=3) and observed no phenotypical differences (CD14- CD34- CD45- CD73+ CD90+ CD105+), although HD-MSCs grew faster. We evaluated the effect of co-culturing AML samples (n=6) with HD-MSCs or AML-MSCs for 5 and 12 days on leukemic cell growth and found that both types of MSCs significantly and equally enhanced AML cell proliferation while maintaining blast phenotype. Using clonogenic assays on 4 AML specimens cultured alone or with either HD- or AML-MSCs for 5 and 12 days, we found that co-culture with either source of BM-MSCs drastically increased colony-forming cells number at day 5 and day 12 while CFC number decreased in the absence on BM-MSCs (no colonies at day 12 for the 4 samples), indicating that AML co-culture with HD/AML-MSCs supports the survival and/or proliferation of AML stem/progenitor cells. We next assessed the effect of increasing doses of AC220 (1, 10, 50, 100 and 500nM) on the apoptosis of FLT3-ITD (n=3) and FLT3-WT (n=4) AML cells cultured alone or with HD-MSCs. Exposure to AC220 for 72 hours significantly, and in a dose-dependent manner, increased the apoptosis of AML FLT3-ITD cells in monoculture (n=3, 21±1% of Annexin V positive cells for control, AC220 1nM 29±3.7%, 10nM 31±2.5%, 50nM 32±1.5%, 100nM 34±1.7% and 500nM 38±3.6%). In contrast, AML FLT3-ITD cells co-cultured with HD-MSCs were resistant to the drug (n=3, 21±2.6% of Annexin V positive cells for control, AC220 1nM 23±3%, 10nM 22±3%, 50nM 25±5.7%, 100nM 30±8.3% and 500nM 33±9.5%). Interestingly, we found that AML FLT3-WT are much less sensitive to increasing doses of AC220 compared to ITD samples (n=4, 27±3.9% of Annexin V positive cells for control, AC220 1nM 30±6.5%, 10nM 35±14%, 50nM 37±11%, 100nM 39±13% and 500nM 43±11%), and co-culture with BM-MSCs further decreased the sensitivity of AML FLT3-WT cells to AC220-induced apoptosis (n=4, 19±3.2% of Annexin V positive cells for control, AC220 1nM 17±3.9%, 10nM 20±3.4%, 50nM 19±3.7%, 100nM 21±4.5% and 500nM 26±1%). AC220 treatment for 3 days significantly, and in a dose-dependent manner, inhibited CFCs in AML FLT3-ITD (n=4, with 26±8%, 46±6%, 60±9%, 69±10% and 86±3% inhibition with 1, 10, 50, 100 and 500nM of AC220 respectively) while AML FLT3-ITD co-culture with HD-MSCs were less sensitive (n=4, with 9±10%, 30±6%, 42±9%, 57±11% and 72±7% inhibition with 1, 10, 50, 100 and 500nM of AC220, respectively). Similarly to the AC220-induced apoptosis, we observed that AML FLT3-WT CFCs are less sensitive to AC220-induced growth inhibition compared to ITD samples, although a 3 days exposure to AC220 significantly, and in a dose-dependent manner, inhibited AML FLT3-WT CFCs (n=3, with 38±16%, 44±14%, 58±12%, 70±21% and 81±19% inhibition with 1, 10, 50, 100 and 500nM of AC220, respectively). Interestingly, we observed that co-culture of AML FLT3-WT with stromal cells were significantly more resistant to increasing doses of AC220 (n=3, with 22±7%, 36±5%, 43±8%, 46±8% and 57±6% inhibition with 1, 10, 50, 100 and 500nM of AC220, respectively). Altogether, these results suggest that AML FLT3-ITD cells in monoculture are more sensitive to AC220 treatment compared to AML FLT3-WT primary cells, but more importantly, upon interaction with primary HD-MSCs, both WT and FLT3-ITD primary samples are protected from apoptosis and growth inhibition induced by AC220, indicating a critical role for the BM microenvironment in AC220 resistance. We are currently testing the impact of BM-MSCs co-culture on leukemic stem cell sensitivity to AC220 using transplantation in NSG mice. We will also evaluate if this co-culture model can be predictive of the response to in vivo treatment with AC220 in a patient-derived xenograft model. DisclosuresDos Santos:Janssen R&D: Research Funding. Danet-Desnoyers:Janssen R&D: Research Funding.

Unique and Potent Tumor Specific Antibodies in Graft Versus Leukemia Responses

IntroductionAcute myeloid leukemia (AML) is a high-risk malignancy with a poor prognosis. Allogeneic hematopoietic stem cell transplantation (HSCT) can be curative if it induces a potent graft versus leukemia (GvL) response. GvL responses and graft versus host disease (GvHD) are typically considered T cell mediated, because T cell depletion from hematopoietic stem cell grafts reduces the risk of GvHD at the cost of leukemia relapse. In addition, depletion of B-lymphocytes with rituximab has led to amelioration of GvHD in a number of studies suggesting that B cells are also important in the pathophysiology of GvHD and, in analogy, in GvL responses. However, the characteristics of the antibodies produced by these leukemic specific B cells have not yet been studied.MethodsWe selected three patients with high-risk myelomonocytic leukemia who remained disease free years after allogeneic HSCT, from whom we established clonal human B cell lines, using a unique and innovative technology that was developed in our laboratory (Kwakkenbos ea, Nat Med 2010). These B cell lines, that concomitantly express immunoglobulin on their membranes and secrete antibodies, were used to select antibodies specific for cell surface antigens on AML cell lines with similar morphologic and immunophenotypic characteristics as the patients’ leukemic blasts, using a FACS based assay.ResultsFrom each patient, several AML specific B cell clones were retrieved. Their antibodies recognized surface antigens on primary AML blasts derived from multiple patients and on AML cell lines, but not on healthy bone marrow, peripheral blood mononuclear cells or tissues such as liver, skin and colon. The majority of the antibodies were of the IgG3 isotype. Approximately 40% of the AML-specific antibodies induced direct death of cultured AML cell lines and of primary AML blasts. The cell death pathway induced by these cytotoxic antibodies was oncotic rather than apoptotic.ConclusionOur data demonstrate that high-risk AML patients with a potent GvL response mount robust antibody responses against surface antigens that are specifically expressed on tumor cells and binding of these antibodies induces direct cell death. The targets recognized by the recovered antibodies are also expressed on leukemic blast from other patients suggesting evidence for a common immune mechanism responsible for AML clearance. Our findings suggest that antibody responses are important in GvL and open up new ventures for specific antibody treatment of AML patients. DisclosuresNo relevant conflicts of interest to declare.

Human Cytokine-Induced Memory-like (CIML) NK Cells Are Active Against Myeloid Leukemia in Vitro and in Vivo

NK cells are innate lymphoid cells that mediate anti-leukemia responses. The ability of MHC-haploidentical NK cells to recognize and eliminate AML blasts have been established in the setting of stem cell transplantation and early phase adoptive NK cell immunotherapy trials. However, the optimal approach to prepare human NK cells for maximal anti-leukemia capacity is unclear. As one form of innate NK cell memory, cytokine-induced memory-like (CIML) NK cells are induced by a brief (16 hour) pre-activation of human NK cells with the combination of IL-12, IL-15, and IL-18, while control NK cells from the same donor are activated by IL-15 only. In published work, this combined IL-12, IL-15, and IL-18 pre-activation results in enhanced proliferation and augmented IFN-gamma responses to cytokine or activating receptor-based re-stimulation following a rest period of 1 – 6 weeks. We hypothesized that CIML NK cells exhibit improved anti-leukemia properties compared to control NK cells from the same individual. Purified primary human CIML NK cells [both CD56bright and CD56dim subsets] produce more IFN-gamma, compared to control NK cells, upon re-stimulation with K562 cells or primary AML blasts after 7 days of rest (p<0.05 and p<0.001, N=5). CIML NK cells also exhibit higher granzyme B protein expression (p<0.01; N=8), and increased cytotoxicity against K562 leukemia targets in vitro (p<0.001, 2.5:1 and 5:1 E:T ratios). We next established a NOD-SCID-gamma-c-/- (NSG) xenograft model to investigate primary human CIML NK cell responses in vivo, with survival supported by low dose IL-2 administered every other day. Seven days following injection of 4 million NK cells / mouse, human CIML NK cells traffic to the bone marrow, spleen, liver and blood, and exhibited better in vivo expansion and persistence, compared to control NK cells (p=0.05 in the blood and bone marrow). Further, the characteristic enhanced functionality of CIML compared to control NK cells when restimulated with K562 targets was retained when assessed ex vivo 7 days post-transfer (p<0.05). Next, we investigated the ability of CIML versus control NK cells from the same donor to clear K562 AML cells in vivo. First, luciferase expressing K562 cells (1 million / mouse) were engrafted into sub-lethally irradiated (250 cGy) NSG mice. On day 3 after K562 challenge, primary human CIML or control NK cells from the same donor (4 million / mouse) were injected, which were supported in vivo using low dose IL-2. CIML NK cells exhibited significantly improved in vivo leukemia clearance as evidenced by whole mouse bioluminescence imaging (see Figure, P=0.03, N=7 mice per group). Thus, human CIML NK cells exhibit enhanced in vitro and in vivo anti-leukemia effects, compared to control NK cells. Based on these findings, a first-in-human phase 1 study of CIML NK cells in relapsed/refractory AML is currently underway. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Inclusion of RBC-Transfusion Dependency Can Improve the Prognostic Value of Revised-IPSS in MDS Patients

Background: The Revised International Prognostic Scoring System (R-IPSS) stratifies MDS patients better than the original IPSS scoring system. Although RBC-transfusion dependency (RBC-TD) is associated with poor prognosis, it is not included in the R-IPSS. Another limitation of R-IPSS is that it is designed to assess the prognosis of patients only at the time of diagnosis; it does not provide prognostic guidance during the disease course. We hypothesise that the use of RBC-transfusion dependency status as a time-varying covariate improves R-IPSS.Aim: To assess the impact of RBC-TD as a time-varying covariate in addition to R-IPSS in predicting survival outcome of MDS patients.Materials and Methods: To match the patient selection criteria as in R-IPSS, primary MDS patients, AML (blast 20-30%) and CMML (WBC≤12x109/L) not treated with disease modifying agents or stem cell transplantation were included. RBC-TD was defined as RBC transfusion of at least 1 unit/8 weeks for at least 4 months (Malcovati et al; JCO 2007). For the statistical analysis of overall survival (OS) measured in months since diagnosis, the Akaike Information Criterion (AIC) was used to assess the goodness-of-fit of a model. Landmark analyses at 6, 12 and 24 months after the diagnosis were also conducted; individuals who experienced the event (i.e. death) before the landmark time point were excluded. The remaining patients were then classified into two groups – RBC-TD noted at or before the landmark time point and transfusion independent at the landmark time point. Results: In our study, 295 patients met the inclusion criteria for analysis, their median age was 75 years (21-97 years) and 66% patients were male. The majority of patients were RCMD, RAEB1 and RAEB2. R-IPSS improved the risk stratification of MDS patients, predominantly for the IPSS-intermediate group (Table I). The median OS in R-IPSS Very Low, Low, Intermediate, High and Very High risk group was 87, 62, 28, 13 and 12 months respectively (p<0.001). Median OS was significantly inferior in male patients (42.4 vs. 63.9 months; p<0.001) compared to females and elderly patients (129, 51 and 33 months in patients <60, 60-80 and >80 years respectively).Of the 295 patients, 22 patients died and 1 lost-to-follow-up within four months of diagnosis; they did not complete the four months qualifying period for RBC-TD and hence were excluded from further analysis. Of the models tested, the four factor model which included R-IPSS, RBC-TD as a time-varying covariate, age and sex was best supported. RBC-TD (HR 5.5; P<0.001) was associated with poor survival, independent of the R-IPSS category, sex and age at diagnosis (Table 2).At each land mark analysis RBC-TD was associated with poor survival, independent of R-IPSS, age and gender. The median survival of patients who became RBC-TD by 6 months was significantly poorer than patients who remained RBC-transfusion independent (18 vs.64 months; p<0.001; Fig 1A). Similarly median OS was significantly poorer in patients who became RBC-TD by 12 months (26 vs.71 months; p<0.001; Fig 1B) and 24 months (40 vs. 87 months; Fig.1C)Conclusions: Majority of MDS patients become progressively cytopaenic and require RBC transfusion during the disease course. RBC-transfusion dependency is always associated with poor prognosis irrespective of time to become RBC-TD. Assessing RBC-TD status along with R-IPSS provides more precise prognostic information during the disease course.Table 1:Refinement of IPSS patient risk categories by R-IPSS scoring systemR-IPSSIPSSVery LowLowIntermediateHighVery HighLow (n=126; 43%)6064200Intermediate1 (n=121; 41%)85445140Intermediate-2 (n=33; 11%)0071610High (n=15; 5%)000411Total=29568 23%118 40%54 18%34 11.5%21 7%Table 2:Factors Predicting Survival of Primary Untreated MDS PatientsFactorHazard RatioP value (Chi Sq)Age at diagnosis (Unit = 1 year)1.04<0.001Sex (Male)1.99<0.001R-IPSS Low (Ref Level = Very Low)1.460.106R-IPSS Int3.23<0.001R-IPSS High4.03<0.001R-IPSS Very High7.72<0.001Transfusion Dependency as a Time Varying Covariate5.50<0.001 [Display omitted] DisclosuresHiwase:Novartis pharmaceutical Ltd , Australia: Research Funding. Ross:Novartis: Honoraria, Research Funding; BMS: Honoraria. Reynolds:Novartis AG: Former Novartis AG employee and holder of Novartis AG stock Other.

Mutation in Wilms’ Tumor 1 Induces DNA Hypermethylation of PRC2 Targets, Blocks Myelomonocytic Differentiation, and Defines a Novel Subtype of AML Responsive to EZH2 Inhibition

Wilms’ Tumor 1 is a transcription factor found to be recurrently mutated (WT1mut) in 10% of normal karyotype acute myeloid leukemia (NK-AML), predominantly in young adults with intermediate-poor prognosis, often in association with FLT3-ITD and high white cell count. Mutations are usually heterozygous and consist of small insertions or deletions clustered around exons 7 and 9, which encode zinc-finger DNA binding domains. In general, mutations disrupt these DNA binding domains leading to a truncated protein that may act in a dominant negative fashion. Currently, the mechanism by which WT1mut contributes to leukemogenesis is unknown, and no lead drug targets linked to this mutation have been identified to date. Using a novel computational method based on Boolean implications that link the presence of a somatic mutation to CpG methylation on a site-by-site basis, we found that mutation in WT1 is strongly linked to DNA hypermethylation in AML patient samples. In order to validate these findings, we expressed mutant WT1 protein prematurely truncated at exon 7 in THP-1 AML cells (confirmed to be wildtype for WT1 at both alleles), and after 10 passages we measured DNA methylation by 450K bead-chip arrays. We found consistent upregulation of DNA methylation in mutant but not wildtype WT1-expressing cells when compared to parental THP-1 cells, validating WT1 mutation as an active driver of DNA hypermethylation. Additional methylome analysis of human hematopoietic stem and progenitor compartments (HSPC) including (HSC, MPP, L-MPP, CMP, and GMP) indicated that WT1mut induces predominantly de novo DNA methylation, as virtually all CpG sites induced by the mutant protein are unmethylated in normal HSPC. Strikingly, the pattern of methylation in both WT1mut patient samples and WT1mut-THP-1 cells was enriched for polycomb repressor complex 2 (PRC2) target genes (p<1.6E-87), implicating a role for this repressive chromatin-remodelling complex in WT1mut leukemogenesis. In keeping with this, gene expression analysis of WT1mut AMLs (but not other normal karyotype AMLs) showed marked repression of known hematopoietic PRC2 target genes (as defined by Chip-Seq), suggesting WT1mut may induce a differentiation block through deregulation and hypermethylation of PRC2 targets.To explore this possibility, we expressed WT1mut in purified normal cord blood CD34+ HSPC using lentiviral transduction and performed in vitro liquid culture differentiation assays in IL-3, SCF, FLT3L, and GM-CSF. We found that WT1mut (but not wildtype or empty vector) induced a myelomonocytic differentiation block with fewer cells expressing CD11b, CD11c, and CD14. Separately, we also showed that WT1mut induced a differentiation block in a TF-1 cell model of erythroid differentiation. These findings suggest a role for WT1mut in perturbing myeloid differentiation in early HSPC.To test the therapeutic implications of our findings, we asked whether inhibition of the major enzymatic histone trimethylase component of PRC2, EZH2, could reverse the differentiation block caused by WT1mut. Significantly, we found that treatment of primary WT1mut AML blasts with the selective EZH2 inhibitor GSK-126 induced upregulation of the mature myeloid markers CD11b, CD33, and CD14. In contrast, NK-AML without WT1mut or acute promyleocytic leukemia cells did not show a significant differentiation response.Our results indicate that mutation in WT1 defines a novel subgroup of DNA hypermethyated AML with de novo hypermethylation of PRC2 target genes that may clinically respond to selective EZH2 inhibitors through differentiation. Importantly, our methods show that genome-wide analysis of mutation-specific DNA methylation patterns may have a future role in determining epigenetic therapies for personalized medicine. DisclosuresNo relevant conflicts of interest to declare.

Isocitrate Dehydrogenase Mutations Induce BCL-2 Dependence in Acute Myeloid Leukemia through Inhibition of Cytochrome C Oxidase Function

IDH1 and IDH2 are two of the most frequently mutated genes in acute myeloid leukemia at an overall frequency of about 15-20%. The genes encode enzymes in the citric acid cycle that normally catalyze the oxidative decarboxylation of isocitrate, producing α-ketoglutarate (α-KG). The mutant enzymes gain a neomorphic activity that catalyzes the conversion of α-KG to (R)-2-hydroxyglutarate (2-HG). The intracellular concentration of (R)-2-HG is over 100-fold higher in IDH-mutated cells than in wildtype cells. (R)-2-HG has been shown to be a competitive inhibitor of multiple α-KG dependent dioxygenases including TET2, and the Jumonji-C domain containing histone demethylases. These enzymes are thought to be the main targets through which (R)-2-HG exerts its effects on leukemogenesis.We previously reported that inhibition of the anti-apoptotic BCL-2 protein is synthetic lethal against mutant IDH which we discovered through a large-scale pooled lentiviral RNA interference screen. We confirmed that expression of mutant IDH1 or IDH2 strikingly sensitized AML cells to shRNA-mediated BCL-2 knockdown and pharmacologic BCL-2 inhibition with ABT-199, a highly specific BH3 mimetic. Importantly, we found that primary human AML blasts harboring IDH mutations were significantly more sensitive to ABT-199 than blasts with wildtype IDH ex vivo and in xenograft transplant models. Furthermore, we showed that ABT-199 was able to target the leukemic stem cell compartment in IDH-mutated samples.Here, we present our work to uncover the synthetic lethal mechanism. An important clue to the mechanism surfaced with the finding that treatment with a cell-permeable precursor of (R)-2-HG sensitized AML cells to BCL-2 inhibition, indicating that the intracellular accumulation of (R)-2-HG found in IDH-mutated cells is sufficient to mediate the phenotype. In addition, we found that (R)-2-HG was able to sensitize isolated mitochondria to ABT-199 with collapse of the mitochondrial transmembrane potential as a surrogate marker for commitment to apoptosis. This finding indicates that 1) the target mediating the synthetic lethal phenotype is localized to the mitochondria, and 2) changes in the epigenome and expression of nuclear-encoded genes are not required for the synthetic lethal phenotype.To identify the potential mitochondrial molecular target, we focused our analysis on the effect of (R)-2-HG on the enzymatic activity of individual complexes in the electron transport chain (ETC), given that ETC dysfunction can potentially alter the threshold for apoptosis. We found that (R)-2-HG at concentrations found in IDH mutated cells inhibited the in vitro enzymatic activity of complex IV (cytochrome C oxidase (COX)) in a dose-dependent manner, but had no effect on the remaining ETC complexes. This finding has in vivo significance as COX activity in intact IDH-mutated primary AML cells was found to be significantly decreased compared with non-mutated AML cells.Next, we investigated the possibility that COX inhibition is sufficient to induce BCL-2 dependence. We found that suppression of COX activity with chemical inhibitors or genetically through shRNA-mediated knockdown of a COX subunit (COX-IV) was sufficient to sensitize AML cells to ABT-199. Furthermore, treatment with tigecycline, a FDA-approved antibiotic that has previously been shown to disrupt ETC function through inhibition of mitochondrial translation resulting in decreased expression of mitochondrial-encoded proteins including the catalytic subunits of the COX complex, reproduced the sensitization effect in non-IDH mutated AML cells. Based on the above findings, we propose a mechanistic model in which (R)-2-HG accumulation in IDH mutant cells directly inhibits COX, thereby lowering the mitochondrial threshold for triggering apoptosis upon BCL-2 inhibition.In summary, we discovered that in addition to the previously described inhibition of α-KG dependent dioxygenases through (R)-2-HG production, IDH mutations also affect mitochondrial bioenergetics. This finding opens up the intriguing possibility that IDH mutations contribute to leukemogenesis not only through epigenetic changes but also through metabolic dysregulation. Lastly, our findings form the rational basis for combining agents that disrupt ETC function such as tigecycline with ABT-199 to target resistant cancer cells and maximize the clinical utility of this promising drug. DisclosuresMedeiros:Agios: Consulting - Ad board Other.

Targeting Aberrant Self-Renewal of Leukemic Cells with a Novel CBP/p300 Bromodomain Inhibitor

The CBP/p300 histone acetyltransferases are key transcriptional regulators of hematopoiesis that have been found to be involved in AML-associated recurrent chromosomal translocations and shown to function as co-activators of leukemogenic fusion oncogenes, suggesting that specific targeting of CBP/p300 may be beneficial for therapy. We characterized the anti-leukemic potential of I-CBP112, a novel chemical inhibitory probe targeting the CBP/p300 bromodomain (BRD). BRDs belong to a diverse family of evolutionary conserved protein-interaction modules recognizing acetylated lysine residues and thereby mediating recruitment of proteins to macromolecular complexes. I-CBP112 represents a new, potent and selective class of BRD inhibitors (oxazepines) binding to recombinant CBP/p300 BRDs with a KD of 151nM and 157nM respectively. Initial characterization by FRAP and BRET assays revealed that I-CBP112 displaced the isolated BRD construct from chromatin but not the full length CBP. I-CBP112 also impaired the interaction of CBP/p300 with p53, resulting in reduced p53-K382 acetylation, reduced p21 expression, and high sensitivity to Doxorubicin-induced DNA damage. We started to explore the effects of the compound on leukemic cells by exposing a series of murine cell lines immortalized by the MLL-CBP fusion and other potent leukemia-associated oncogenes including the MLL-AF9, MLL-ENL, or the NUP98-HOXA9 fusion to increasing doses of I-CBP112. Interestingly, no significant cytotoxicity was observed up to concentrations of 5μM. However, in all cell lines we observed a significant reduced number of colonies formed in methylcellulose, associated with morphological differentiation as observed in Giemsa stained cytospots. Similar to the murine leukemic cell lines we found that I-CBP112 did not cause immediate cytotoxic effects but impaired colony formation and induced cellular differentiation of a series of 18 human leukemic cell lines. Reduced colony formation upon I-CBP112 treatment was also observed of human primary AML blasts but not of CD34+ hematopoietic stem cells from two healthy donors. I-CBP112 effects were studied in more detail in three human leukemia cell lines: SEM (MLL-AF4+), MOLM13 (MLL-AF9+) and Kasumi-1 (AML1-ETO+). Long-term exposure of these cells to I-CBP112 in liquid medium, resulted in a dose-dependent G1 cell cycle arrest, with Kasumi-1 being the most sensitive to the inhibitor, demonstrating further morphological signs of differentiation and apoptotic cell death. Importantly, combination of I-CBP112 with the BET-BRD inhibitor JQ1 or Doxorubicin revealed a clear synergistic effect on cell survival of the AML cell lines except for the combination of I-CBP112 with Doxorubicin on MOLM13. Surprisingly only modest effects of I-CBP112 exposure on the transcriptional programs of SEM, MOLM13 and Kasumi-1 cells were found by microarray expression profiling. Genes found affected were mainly immune response regulators or NFkappaB targets suggesting that attenuation of NFkappaB downstream signals might impair the leukemia initiation capacity reflected by reduced colony formation. Extreme limited dilution assays (ELDA) in methylcellulose, as well as bone marrow transplantations in limiting dilutions using MLL-AF9-transformed murine leukemic blasts revealed that I-CBP112 significantly impaired self-renewal of the leukemic stem cell compartment in vitro and reduced the leukemia-initiating potential in vivo. Taken together, these data demonstrate that selective interference with the CBP/p300 BRD by I-CBP112 has the potential to selectively target leukemic stem cells and opens the way for novel combinatory “BRD inhibitor” therapies for AML and other human cancers. DisclosuresNo relevant conflicts of interest to declare.