ALBERT

Description: Abstract 224 During hematopoiesis, all-trans-retinoic acid (ATRA), a natural derivative of vitamin A, has been shown to induce both myelomonocytic progenitor/stem cell differentiation and self-renewal. Although these opposing effects are likely to be partly due to developmental differences, it has been shown that pro- and anti-differentiation effects of ATRA are mediated by distinct retinoic acid receptor isotypes (RARα and RARγ, respectively). With the exception of acute promyelocytic leukemia (APL), ATRA treatment as a single agent has not been successful in other types of acute myeloid leukemia (AML). We have previously hypothesized that one of the underlying reasons for poor response of non-APL AML to ATRA (pan-RAR agonist) is aberrant expression and/or activities of RAR isotypes favoring RARγ and cell growth versus differentiation. Consistently, we have reported that expression of RARα isoforms, particularly ATRA-inducible RARα2, are down-regulated in AML (Blood. 2008; 111:2374). Epigenetic analysis of patient samples revealed that relative to normal CD33+ cells, the loss of RARα2 in AML is associated with a diminution in levels of histone histone H3 lysine 4 dimethylation (H3K4me2) on the ATRA-responsive RARA2 promoter (a modification associated with transcriptional activation). Interestingly, the H3K4me1/me2 demethylase LSD1/KDM1 (AOF2) is highly expressed in AML patients (www.proteinatlas.org). A number of small molecules that target this enzyme (LSD1i) are in development and, collectively, these data predict that the use of LSD1i will facilitate induction of expression of genes that are required for differentiation of AML cells. In this study we used tranylcypromine (TCP, a monoamine oxidase used as an antidepressant and anxiolytic agent in the clinical treatment of mood and anxiety disorders, respectively), which functions a time-dependent, mechanism-based inhibitor of LSD1. Here we show that TCP unlocked the ATRA-driven therapeutic differentiation response in non-APL AML cell lines including the TEX cell line, which is derived from primitive human cord blood cells immortalized by expression of the TLS-ERG oncogene. TEX cells are 〉90% CD34+, respond poorly to ATRA and mimic features of primary human AML and leukemia initiating cells (Leukemia. 2005; 19:1794). Consistent with this, ATRA/TCP treatment increased differentiation in primary patient samples. ATRA alone had in general only small effects in primary AML samples and TCP showed minimal activity in most cases. Furthermore, shRNA-mediated knockdown of LSD1 confirmed a critical role for this enzyme in blocking the ATRA response in AML cells. The effects of ATRA/TCP on AML cell maturation were paralleled by enhanced induction of genes associated with myelomonocytic differentiation, including direct ATRA targets. LSD1i treatment did not lead to an increase in genome-wide H3K4me2, but did increase H3K4 dimethylation of myelomonocytic differentiation-associated genes. Importantly, treatment with ATRA/TCP dramatically diminished the clonogenic capacity of AML cells in vitro and engraftment of cells derived from AML patients in vivo, suggesting that ATRA/TCP may also target leukemic stem cells. These data strongly suggest that LSD1 may, at least in part, contribute to AML pathogenesis by inhibiting the normal function of ATRA in myelomonocytic development and pave the way for effective differentiation therapy of AML. Disclosures: No relevant conflicts of interest to declare.

Description: Acute myeloid leukemia (AML) is the most common form of adult acute leukemia that is associated with a low long-term survival rate. While chemotherapy achieves remission in more than 70% of the treated AML patients, most relapse due to residual chemotherapy-resistant AML populations. Allogeneic hematopoietic stem cell transplantation reduces the relapse rate and demonstrates the efficacy of a cell-mediated treatment for the chemotherapy-resistant disease. However, its wide application is limited by donor availability and associated toxicity such as graft-versus-host disease (GvHD). Hence, there is a need for a new treatment approach that targets chemotherapy-resistant AML blasts with minimal side effects. The goals of this study were to characterize allogeneic CD3+ CD4-CD8- double negative T cells (DNTs) as a potential new therapy for AML patients, and to dissect its underlying mechanisms. Using a flow cytometry-based in vitro killing assay, we demonstrated that the allogeneic DNTs expanded from healthy volunteers were cytotoxic against 23/29 primary AML patient blasts in a dose-dependent manner. Thirteen blast samples were obtained from chemotherapy refractory or relapsing AML patients and, of those, 9 were susceptible to DNTs. Further, the average level of specific killing mediated by DNTs against chemotherapy-susceptible and -resistant blasts were comparable (19.30% ± 3.34% and 15.91% ± 3.63%, respectively). The anti-leukemia activity of DNTs was further validated in AML-NSG xenograft models. A single infusion of DNTs into mice pre-engrafted with primary AML blasts from chemoresponsive, chemorefractory, and relapsed patients significantly reduced the leukemia burden. While a single injection of DNTs resulted in a significant reduction of leukemia burden from 30% to 12.8%, it was further reduced to 2.6% after three injections of DNTs. Furthermore, the survival of NSG mice administered with a lethal dose of AML cell line, MV4-11, in DNT-treated group was significantly superior to the untreated group (57% vs. 0% survival on day 34, respectively). Although residual blasts were observed from the DNT -treated group, their susceptibility to DNT cell-mediated cytotoxicity remained comparable to blasts obtained from the PBS-treated group and primary AML blasts initially used for engraftment, suggesting that AMLs do not develop resistance to DNT-mediated cytotoxicity. In contrast to its potent cytolytic activity against leukemic cells, DNTs did not target allogeneic peripheral blood mononuclear cells (PBMC) and hematopoietic stem/progenitor cells (HSPC) in vitro. Administration of allogeneic DNTs into NSG mice engrafted with human HSPC had no effect on the engraftment level of human hematopoietic cells and their differentiation into different lineages. Further, in contrast to human PBMC, infusion of human DNTs did not cause xenogeneic GvHD in mice, collectively demonstrating the selective cytotoxic activity of allogeneic DNTs against leukemic cells. Using blocking assays, we showed that HLA-class I, NKG2D, and DNAM-1 were involved in DNT cell-mediated cytotoxicity against AML, whereas HLA-class II and T cell receptor did not play a significant role. We detected high levels of IFNγ release by DNTs upon encounter of susceptible AML targets. While IFNγ treatment alone did not induce AML cell death, neutralizing IFNγ reduced and pretreating AML cells with recombinant IFNγ increased their susceptibility to DNT cell killing. IFNγ treatment induced higher expressions of NKG2D and DNAM-1 ligands and blocking of NKG2D and DNAM-1 partially abrogated the effect of IFNγ on the AML-DNT interaction. Collectively, these studies demonstrated the safety and efficacy of allogeneic DNT therapy as a potential treatment for AML patients, including those with chemotherapy-resistant leukemia, and revealed the important molecules for the anti-leukemic activity of DNTs. Disclosures No relevant conflicts of interest to declare.

Description: 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. Disclosures Etchin: 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.

Description: The NOD.SCID xenotransplantation assay is a key model system for interrogating the biology of leukemic stem cells (LSCs) in human acute myeloid leukemia (AML). Approximately 50% of AMLs can generate human grafts in immunodeficient mice that recapitulate the features of the parent sample. However, some AML samples generate non-leukemic grafts upon xenotransplantation. We recently reported that multilineage (ML) grafts, comprised of both B-lymphoid and myeloid cells, are generated by preleukemic hematopoietic stem cells (preL-HSCs; Shlush et al. Nature 2014). PreL-HSCs contain a subset of the mutations present in leukemic blasts and have a competitive growth advantage over wildtype HSCs leading to clonal expansion in vivo, yet retain multilineage differentiation capacity. To investigate the relationship between patient outcomes and the biological properties of LSCs and preL-HSCs as reflected by different engraftment patterns, we transplanted 272 diagnostic patient samples, representing a broad cross-section of adult AML, into sublethally irradiated NOD.SCID mice by intrafemoral injection. Human chimerism was assessed 8-10 weeks post-transplant by flow cytometry. 41% of samples generated AML xenografts, defined as a human graft containing 〉90% myeloid (CD33+CD19-CD45+) cells. Three patterns of engraftment were seen with the remaining samples: no human graft, defined as 10% CD19+CD33-CD45+ B-cells (22%). Among patients whose samples generated ML grafts compared to AML grafts or TC/no graft, secondary AML was less common (10% vs. 27% vs. 26%, respectively; P=0.03). Associations between engraftment pattern and other baseline clinical characteristics, including age, white blood cell (WBC) count and cytogenetics, did not reach statistical significance in this cohort. However, there was a strong correlation between AML engraftment capacity and response to standard induction chemotherapy. AML engrafters had lower complete remission (CR) rates compared to all other patients as a group (51% vs. 81%; P

Description: miRNA expression is deregulated in human acute myeloid leukemia (AML), however the impact of altered post-transcriptional programs on the genesis and maintenance of leukemia stem cells (LSC) remains undefined. In order to elucidate the functional role of miRNA in LSC and identify relevant miRNA candidates, we performed global miRNA profiling on sorted cell subpopulations from 16 AML patient and 3 umbilical cord blood samples (Eppert et al, Nature Medicine 2011). Supervised analysis guided by the ability of each sub-population to initiate leukemic engraftment after xenotransplantation into immune-deficient mice generated a unique miRNA signature. miR-126, a miRNA that we previously demonstrated to have a conserved role in maintaining hematopoietic stem cell (HSC) quiescence (Lechman et al. Cell Stem Cell, 2012), was more highly expressed in LSC-enriched fractions and chosen for further validation. To confirm that miR-126 is a bona fide LSC determinant, we utilized a bidirectional lentiviral reporter vector specific for miR-126 (Gentner et al. Science Translational Medicine, 2010) to sort cells from AML patient samples based on miR-126 bioactivity, and demonstrated that all in vivo leukemia-initiating capacity was confined to cells with elevated miR-126 bioactivity. Lentiviral enforced expression of miR-126 in primary AML patient samples significantly increased LSC frequency (3.5-52.3 fold) as assessed by limiting dilution transplantation assays, while diminishing cell cycle entry, differentiation marker expression (CD14,CD15) and colony forming potential. Sponge-mediated knockdown of miR-126 expression resulted in the opposite effects. These findings suggest that high levels of miR-126 bioactivity support self-renewal/maintenance of primitive AML cells at the cost of aberrant differentiation. Moreover, by preserving LSC quiescence miR-126 promoted chemotherapy resistance, in part through suppression of CDK3, a gatekeeper of G0 to G1 cell cycle transit. Enforced expression of CDK3 partially rescued the functional consequences of supra-physiological levels of miR-126 bioactivity, rendering previously resistant LSC susceptible to killing by AraC/Daunorubicin combination chemotherapy. Our human LSC miRNA signature, optimized by regression analysis on a cytogenetically normal AML patient cohort, was prognostic for survival in a large independent AML patient cohort (Ley et. al N Engl. J Med, 2013) further validating the clinical significance of miRNA as stem cell determinants. Furthermore, miRNA-126 alone was prognostic for survival in two independent cohorts of AML patients with normal cytogenetics. These data demonstrate a mechanistic role for miR-126 in governing intrinsic LSC properties and establish miR-126 as a critical biomarker for clinical outcome. Disclosures: Wang: Trillium Therapeutics/Stem Cell Therapeutics: Research Funding.

Description: The NOD.SCID xenotransplantation assay has emerged as a key model system for interrogating the biology of human acute myeloid leukemia (AML). Previous work has established that approximately 50% of AMLs can generate grafts resembling the patient’s leukemia in immunodeficient mice. An understanding of the biologic properties of AML that enable engraftment in xenotransplant assays may aid in the development of prognostic tools and individualized therapy. To investigate these properties in a broad cross-section of human AML, we transplanted bone marrow (BM) or peripheral blood (PB) cells from 307 AML patients intra-femorally into sublethally irradiated NOD.SCID mice pre-treated with an anti-CD122 antibody. Human engraftment in the recipient BM was assessed 8-10 weeks post-transplant via flow cytometry. AML xenografts, defined as a human graft composed of 〉90% myeloid (CD33+CD19-CD45+) cells, were generated by 134 samples (44%). The remainder (hereafter referred to as non-engrafting samples) generated 3 patterns of non-leukemic engraftment: no human graft, defined as

Description: Leukemia in humans arises from the multistep accumulation of mutations. However, the identity of the cell of origin, the nature of the first genetic lesion and the order of subsequent mutations remain poorly understood, as most cases of de novo acute myeloid leukemia (AML) are diagnosed without prior observation of a pre-leukemic clonal expansion. As part of studies to examine intra-tumoral genetic heterogeneity in AML, we carried out deep targeted sequencing (read depth 250×) of 101 commonly mutated leukemia genes on samples from 12 patients at diagnosis. Normal T-cells from each sample were expanded in vitro to provide a non-leukemic hematopoietic tissue for comparison. In 3 of 4 patients, we unexpectedly identified DNMT3a mutation not only in AML cells but also in T-cells at a low allele frequency (1-20%). Other genetic alterations such as NPM1 mutation (mutNPM1) were found only in AML cells and not in T-cells, ruling out contaminating AML cells as the source of the DNMT3a signal in cultured T-cells. To investigate the prevalence of T cell involvement, an additional 71 samples from AML patients at diagnosis were screened by Sanger sequencing for DNMT3a mutations. 17 of 71 AML samples (24%) carried R882 codon mutations (mutDNMT3a), and 15 of 17 (88%) also carried mutNPM1. Mutant allele frequency in freshly isolated T-cells was measured by droplet digital PCR (ddPCR). mutDNMT3a with no evidence of mutNPM1 was detected in T-cells of 12 of 17 patients (70.5%), suggesting that DNMT3a mutation occurs before NPM1 mutation in an ancestral stem/progenitor cell that gives rise to both T-cells and the dominant AML clone present at diagnosis. To directly determine whether phenotypic stem/progenitor cells that carried the mutDNMT3a allele could be identified within the non-leukemic hematopoietic compartment of AML blood and bone marrow samples, we undertook genetic analysis of highly-resolved phenotypically-defined normal stem, progenitor and mature lymphoid cell fractions from 10 patient samples. mutDNMT3a without detectable mutNPM1was present in stem cells and all downstream progenitors, with mean allele frequency among multipotent progenitor (MPP), multilymphoid progenitor (MLP) and common myeloid progenitor (CMP) of 31.7%. mutDNMT3a and mutNPM1 were found together only in granulocyte monocyte progenitor (GMP) and CD33+ blasts. Importantly, even for patients in whom mutDNMT3a was not detected in mature lymphoid populations, mutDNMT3a without mutNPM1 was found in MPP, MLP, CMP, providing strong evidence that mutDNMT3a precedes mutNPM1 during leukemogenesis. Analysis of diagnostic and remission samples revealed similar or higher proportion of cells with mutDNMT3a alone at remission compared to diagnosis. Xenotransplantation of cells from the diagnostic samples of 2 patients with mutDNMT3a and mutNPM1generated predominantly non-leukemic multilineage grafts (18 of 19 mice) with predominance of cells bearing mutDNMT3a without mutNPM1 (mean allele frequency 57%), confirming that mutDNMT3a was present in HSC. Kinetic analysis at 8 and 16 weeks revealed increasing mutDNMT3a allele frequency in multilineage xenografts over time, suggesting that mutDNMT3a confers a competitive growth advantage over non-mutated HSC. Collectively, our results are consistent with the clonal expansion in AML patients of mutDNMT3a HSC that survive chemotherapy. These cells may therefore represent a reservoir for further genomic progression leading to relapse. Our findings now offer the possibility of therapeutic intervention during remission to eliminate these surviving pre-leukemic clones and prevent relapse in a large proportion of AML patients carrying mutDNMT3a. As well, our work provides a framework for the identification of other early events in leukemogenesis and examination of how these changes disrupt normal HSC function and lead to leukemia. Disclosures: Wang: Trillium Therapeutics/Stem Cell Therapeutics: Research Funding.