AML Progenitor Cells Research Articles

Combining Allogeneic Gamma Delta T Cells with a Phosphoantigen Prodrug Promotes Apoptosis of Acute Myeloid Leukemia Stem and Progenitor Cells

Despite recent therapeutic advances, patients with relapsed/refractory acute myeloid leukemia (r/r AML) have limited treatment options and poor prognosis. Leukemic stem cells (LSCs) are considered as relapse-initiating cells and the “culprit” of treatment resistance. Allogeneic and autologous gamma delta (γδ) T cell-based therapies have been explored for a long time and proven to be safe in patients through multiple clinical trials. However, quite a few AML blasts obtained from r/r AML patients show resistance to γδ T cell-mediated cytotoxicity. Novel strategies targeted LSCs may improve efficacy of γδ T cell-mediated r/r AML therapy. Given that γδ T cells exert antitumor capacity via multiple pathways, to identify the way matters most in the resistance of γδ T cell-mediated cytotoxicity, we examined the median fluorescence intensity (MFI) of the ligands expressed in tumor cells by flow cytometry, including butyrophilin 3A (BTN3A), butyrophilin 2A1 (BTN2A1), the human MHC class I chain-related genes (MICA and MICB), the unique-long 16 binding proteins (ULBP1, ULBP2/5/6, ULBP3), Fas, the receptor of TNF-related apoptosis-inducing ligand (TRAIL) DR5, CD112 and CD155. The cytotoxicity activity of γδ T cells was measured by flow cytometry-based killing assay or bright-lite luciferase assay system after coculturing with AML cell lines and blasts at an appropriate effector to target (E:T) ratio. Then we employed a Support Vector Machine model to predict the killing efficiency and identified that BTN3A and BTN2A1 play the predominant role in γδ T cell-mediated cytotoxicity based on the Shapley additive explanation. BTN3A and BTN2A1 are the key ligands in the γδ TCR-phosphoantigen (pAg) recognition activity. PAgs, which are produced in tumor cells function as “molecular glues” to promote heteromeric association of BTN3A1 and BTN2A1 (Yuan L et al., BioRxiv 2022). To promote the γδ T cell-mediated cytotoxicity targeting r/r AML, here we synthesized a pAg prodrug, which show great plasma stability and membrane permeability. We anticipate this small molecule would lead pAg accumulation in tumor cells and cause increased γδ T cell activation. To identify the killing efficacy of pAg prodrug-induced γδ T cell activation, we select KG-1α (a human leukemia stem cell-like cell) as target cell. Zoledronate (ZOL), which is employed to stimulate γδ T cells in multiple γδ T cell-based clinical trials, is used in our killing assay as compared group. As expected, combining γδ T cells with pAg prodrug or ZOL both significantly improved the percent of specific killing when the E:T ratio is 1:1. The pAg prodrug showed more potent activity than ZOL group with EC50 value of 0.1 nM vs 17.93μM. And the specific killing percent of pAg prodrug group is 83.26±2.1 % (1 nM), comparing with negative control 15.17±3.4 % (only γδT cells). Futhermore, at relatively low E:T ratio (1:4), γδ T cells with pAg prodrug remain excellent cytotoxicity when targeting MV411 cell and OCI-AML3 cell, which are widely used in r/r AML research. After coculturing with primary leukemia stem and progenitor cells (CD34 +) obtained from r/r AML patients for 6~8 h, the apoptosis percentage of CD34 + leukemia cells was 42.34% comparing with negative control (only γδT cells) 2.53%. Then we evaluated the in vivo efficacy of combining γδT cells and pAg prodrug in NSG mice bearing MV411 cells. Compared with only γδT cells-treated group, combination with pAg prodrug presented better tumor clearing. To elucidate the pathway of pAg prodrug-mediated killing, we tested mitochondria apoptosis related events. After treated by γδ T cells with pAg prodrug, KG-1α cells showed loss of mitochondrial outer membrane potential and increasingly actived caspase 3/7. Meanwhile, pAg prodrug directly activates effector γδ T cells with increased secretion of CD107a and IFN-γ. Considering the possibility of toxicity towards normal hematopoietic stem and progenitor cells, we conducted colony-forming units (CFUs) assay. After treatment with γδT cells and pAg prodrug, the number of CFUs of CD34 + cells from cord blood was not effected compared with the CD34 + cells only group. Overall, we explored the predominant factor in γδ T cell-mediated killing and synthesized a pAg prodrug to improve the killing efficacy. We demonstrated that combining γδ T cells with pAg prodrug has effectively promoted apoptosis of AML stem and progenitor cells sparing normal hematopoietic stem and progenitor cells.

PRMT5 Inhibition Enhances Elimination of FLT3-ITD AML Stem Cells in Combination with TKI Treatment

Introduction: The FMS-like tyrosine kinase-3 (FLT3) internal tandem duplication (ITD) is a commonly observed molecular abnormality in AML and is associated with increased risk of relapse and poor survival. FLT3 tyrosine kinase inhibitors (TKIs) have shown benefit in the treatment of FLT3-ITD AML but do not eliminate primitive leukemia stem cells and are limited by frequent relapse and resistance. There is considerable interest in understanding mechanisms of FLT3-ITD AML stem cell persistence to develop strategies to enhance their elimination. Here we evaluated the role of epigenetic regulators in maintenance of human FLT3-ITD AML cells. Methods: We performed an epigenetic screen using a well-curated collection of epigenetic probes (Epigenetics Probes Collection, Structural Genomics Consortium, n=41). FLT3-ITD (MOLM-13 and MV4-11), and FLT3-WT AML cell lines (OCI-AML3) were exposed to inhibitors for 7 days and CellTiter-Glo and Annexin V labeling were used to evaluate response. We further studied the effects of the PRMT5 inhibitors, GSK-591and LLY-283, with and without FLT3 TKIs, Quizartinib and Giltertinib, on FLT3-ITD+ and FLT3-WT AML cell lines. Synergy between PRMT5 inhibitors and TKIs was analyzed with Calcusyn software using the median effects method of Chou and Talalay. We performed in vivo analysis of the effects of PRMT5 and FLT3 kinase inhibition in a FLT3-ITD Tet2 deleted genetic mouse model of FLT3-ITD AML. Finally, we studied in vitro and in vivo effects of PRMT5 and FLT3 inhibition on primary CD34+ cells from FLT3-ITD AML patients. Results: Screening with epigenetic probes identified PRMT5 arginine methyl transferase inhibitors, GSK591 and LLY-283, amongst the probes with the greatest inhibitory effects on growth and survival of FLT3-ITD MOLM-13 and MV4-11 AML cells. In contrast, inhibitors of PRMT1, PRMT3. PRMT4 and PRMT7 were associated with significantly less growth inhibition of FLT3-ITD AML cell lines. FLT3-WT OCI-AML3 AML cells were also sensitive to PRMT5 inhibition. However, ectopic FLT3-ITD expression in FLT3-WT THP1 cells led to enhanced sensitivity to PRMT5 inhibition. The combination of GSK-591 or LLY-283 with either of the FLT3 TKIs Quizartinib or Giltertinib resulted in synergistically enhanced inhibition of FLT3-ITD+ MOLM-13 and MV4-11 AML cells, compared to TKI or PRMT5 inhibitor alone. On the other hand, FLT3-WT cells were insensitive to TKI alone, and the combination of PRMT5 inhibitor and TKI did not lead to synergistic enhancement of inhibition. We next evaluated the effect of PRMT5 inhibition in a genetic mouse model of FLT3-ITD AML. AML mice were treated with vehicle (control), Giltertinib, LLY283 or the combination of Giltertinib and LLY283 for 2 weeks. AML cells from LLY-283 treated mice showed significantly reduced symmetric dimethylarginine in SmB protein. Significant inhibition of mature AML cells and of AML stem and progenitor cells was seen in the bone marrow and spleen of mice treated with LLY283, and significantly enhanced inhibition was seen with combination of LLY283 and Giltertinib (Figure 1a). Finally, we evaluated the effect of TKI (Giltertinib or Quizartinib), LLY283 or the combination on primary FLT3-ITD AML CD34+ cells with diverse co-mutations (n=8). LLY283 significantly inhibited FLT3-ITD AML CD34+ cell growth and survival in vitro by itself, and the combination of TKI and LLY283 resulted in significantly increased inhibition compared to TKI alone, including both CD34+CD38- and CD34+CD38+ cell populations (Figure 1b). In addition, in vivo treatment of immunodeficient NRGS mice xenografted with human FLT3-ITD AML cells with LLY283 or the combination of Giltertinib and LLY283 led to significant depletion of AML cells in BM and spleen. Conclusion: An epigenetic probe screen identified PRMT5 as a key regulator of FLT3-ITD AML cell viability. We show an important role for PRMT5 in maintenance of murine and human FLT3-ITD AML stem cells and in their persistence following FLT3 TKI treatment. Treatment with a PRMT5 inhibitor in combination with a FLT3 TKI could be a promising approach to enhance elimination of FLT3-ITD AML stem cells. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Inhibition of ERK1/2 Reverses Venetoclax-Induced Autophagy to Overcome Resistance in Acute Myeloid Leukemia

Background: Activation of the RAS/MAPK pathway confers inherent/acquired resistance to the Bcl-2 inhibitor, venetoclax, in AML (Zhang et al., Signal Transduct Target Ther. 2022). Pro-survival Bcl-2 inhibits apoptosis as well as autophagy. Phosphorylation of Bcl-2 by ERK1/2 leads to dissociation of the autophagic protein Beclin 1 from Bcl-2 and promotes autophagy (Pattingre et al., Cell. 2005). Thus, while Bcl-2 inhibition can induce apoptosis, its inhibition leads to induction of autophagy (Pattingre et al., Cell. 2005) that can protect tumor cells from organelle damage and nutrient deprivation. Objective: We hypothesized that terminal MAPK pathway inhibition using an ERK1/2 inhibitor would reverse venetoclax-induced autophagy and synergistically lead to cell death/apoptosis. Methods: We inhibited ERK1/2 using Compound 27 (Heightman et al., J Med Chem. 2018), a close analog of ASTX029, which is a dual-mechanism ERK1/2 inhibitor currently under clinical investigation in solid tumors (NCT03520075). We studied the potential synergy of ERK1/2 and Bcl-2 inhibition in decreasing cell proliferation and inducing apoptosis and in overcoming venetoclax resistance in AML preclinical models and primary patient samples (n=8). We performed simultaneous analysis of cell proliferation, stress response and DNA damage using single cell proteomics analysis to understand the mechanism of synergy. Autophagy was measured by lipidated LC3 quantification (flow cytometry and immunoblotting). Colony formation assay using NRAS-mutant PDX was performed to analyze the effect on progenitor cells. For insights into the mechanisms of synergy, we conducted large-scale proteomics analyses by single cell mass cytometry (CyTOF) and simultaneously measured cell proliferation, stress response, DNA damage and Bcl-2 family proteins. Results: Inhibition of ERK1/2 sensitized OCI-AML3 (NRAS-mutant) cells (Combination Index [CI]: 0.008) and venetoclax-resistant OCI-AML2 cells (CI: 0.05) to venetoclax and shifted the cytostatic effect of the single agents to a cytotoxic effect. Combination treatment induced apoptosis in leukemia initiating progenitor cells (LICs, CD34+CD38-) in primary AML samples with 54% apoptosis in combination versus 10% with venetoclax alone (CI = 0.03-0.23), while sparing normal CD34+CD38- cells. Mechanistically, basal ERK1/2 phosphorylation and autophagy (LC3 lipidation), were higher in venetoclax-resistant OCI-AML2 cells versus parental cells (p=0.0008) and both were inhibited with ERK1/2 inhibition alone and in combination with venetoclax. Simultaneous analysis of cell proliferation, stress response and DNA damage showed downregulation of proliferation (Ki-67) and an increase in ATF4 in response to ERK1/2 inhibition. The combination of Compound 27 and venetoclax inhibited autophagy (lipidated LC3) and increased apoptosis (increased cleaved PARP and cleaved caspase 3, Fig 1a). Venetoclax-induced autophagy was also confirmed in OCI-AML3 cells at 24 h post treatment (p=0.03). ERK1/2 inhibition reversed venetoclax-induced autophagy in OCI-AML3 (p=0.0003) and OCI-AML2-venetoclax-resistant cells (p=0.0007). CyTOF analysis showed decreased p-AMPK in response to ERK1/2 inhibition in OCI-AML3 cells. As AMPK/FIS1-mediated mitochondrial autophagy is required for self-renewal of AML stem cells (Pie et al., Cell Stem Cell. 2018), we confirmed decreased clonogenic potential of AML progenitor cells by reduced colony formation in response to ERK1/2 inhibition using an NRAS-mutant AML PDX model (p=0.0002). Single cell proteomics (CyTOF) showed decreased p-AMPK in response to single agent ERK1/2 inhibition or the combination with venetoclax in LIC populations (CD34+CD38-) in the NRAS-mutant AML PDX model. In addition to p-AMPK the expression of cMyc and CD44 was also reduced in the combination treatment, indicating reduced stemness (Fig 1b). Conclusion: Mechanistically, our results indicate that pharmacological inhibition of ERK1/2 impairs autophagy and sensitizes venetoclax-resistant AML cells to apoptosis, thus overcoming primary and secondary resistance to venetoclax. In addition, combination treatment induced apoptosis of LICs and reduced clonogenic potential and markers of stem cell function. These data provide a strong rationale for the combination of ERK1/2 and Bcl-2 inhibitors in treatment of AML and warrant further investigation in the clinic. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The metabolic enzyme hexokinase 2 localizes to the nucleus in AML and normal haematopoietic stem and progenitor cells to maintain stemness

Mitochondrial metabolites regulate leukaemic and normal stem cells by affecting epigenetic marks. How mitochondrial enzymes localize to the nucleus to control stem cell function is less understood. We discovered that the mitochondrial metabolic enzyme hexokinase 2 (HK2) localizes to the nucleus in leukaemic and normal haematopoietic stem cells. Overexpression of nuclear HK2 increases leukaemic stem cell properties and decreases differentiation, whereas selective nuclear HK2 knockdown promotes differentiation and decreases stem cell function. Nuclear HK2 localization is phosphorylation-dependent, requires active import and export, and regulates differentiation independently of its enzymatic activity. HK2 interacts with nuclear proteins regulating chromatin openness, increasing chromatin accessibilities at leukaemic stem cell-positive signature and DNA-repair sites. Nuclear HK2 overexpression decreases double-strand breaks and confers chemoresistance, which may contribute to the mechanism by which leukaemic stem cells resist DNA-damaging agents. Thus, we describe a non-canonical mechanism by which mitochondrial enzymes influence stem cell function independently of their metabolic function.

The STAT3-MYC axis promotes survival of leukemia stem cells by regulating SLC1A5 and oxidative phosphorylation

AbstractAcute myeloid leukemia (AML) is characterized by the presence of leukemia stem cells (LSCs), and failure to fully eradicate this population contributes to disease persistence/relapse. Prior studies have characterized metabolic vulnerabilities of LSCs, which demonstrate preferential reliance on oxidative phosphorylation (OXPHOS) for energy metabolism and survival. In the present study, using both genetic and pharmacologic strategies in primary human AML specimens, we show that signal transducer and activator of transcription 3 (STAT3) mediates OXPHOS in LSCs. STAT3 regulates AML-specific expression of MYC, which in turn controls transcription of the neutral amino acid transporter gene SLC1A5. We show that genetic inhibition of MYC or SLC1A5 acts to phenocopy the impairment of OXPHOS observed with STAT3 inhibition, thereby establishing this axis as a regulatory mechanism linking STAT3 to energy metabolism. Inhibition of SLC1A5 reduces intracellular levels of glutamine, glutathione, and multiple tricarboxylic acid (TCA) cycle metabolites, leading to reduced TCA cycle activity and inhibition of OXPHOS. Based on these findings, we used a novel small molecule STAT3 inhibitor, which binds STAT3 and disrupts STAT3-DNA, to evaluate the biological role of STAT3. We show that STAT3 inhibition selectively leads to cell death in AML stem and progenitor cells derived from newly diagnosed patients and patients who have experienced relapse while sparing normal hematopoietic cells. Together, these findings establish a STAT3-mediated mechanism that controls energy metabolism and survival in primitive AML cells.

Chimeric Antigen Receptor (CAR) T Cell Therapy for CD7-Positive Acute Myeloid Leukemia

Expression of CD7 is an adverse prognostic factor in both AML and myelodysplastic syndromes. Because CD7 is not constitutively expressed by mature myeloid cells or early myeloid progenitor cells, we explored the feasibility of targeting these malignancies using T cells expressing a CD7-directed chimeric antigen receptor (CAR7). Due to high expression of CD7 on normal T cells, expression of CAR7 induced substantial fratricide of T cells, preventing their expansion and long term survival. We therefore disrupted the CD7 gene in T cells using CRISPR/Cas9 prior to transduction with the CAR. Electroporation of the Cas9 protein with CD7-specific gRNA produced little toxicity to T cells, and the resulting CD7KOCAR7 T cells (henceforth CAR7 T cells) had no evident fratricide, expanding >300-fold within two weeks.Upon coculture with primary AML blasts, CAR7 T cells eliminated a mean 85% of malignant cells within 48h (Fig. A) and expanded 5-fold. Furthermore, CAR7 T cells effectively targeted primitive AML progenitor cells resulting in a mean >25-fold decrease in the number of leukemic colonies after a 5h coculture (Fig. B). CAR7 T cells demonstrated robust cytotoxicity against CD7+ AML cell lines eliminating >95% of malignant cells after 72h of coculture at a 1:40 effector-to-target ratio but had no activity against CD7-negative tumor cells. To test the protective effect in vivo, we established a mouse xenograft model of disseminated AML by intravenous injection of the CD7+ AML cell line KG-1a. A single injection of 2x10^6 CAR7 T cells produced a >2-log reduction in tumor burden and significantly extended median survival (65 days in control group; median survival not reached at 88 days for the CAR7 T treated animals, P=0.0025). In a second, more aggressive AML model using Kasumi-3 cells, we observed a 2-log reduction in tumor volume following administration of CAR7 T cells.CD7 may also be transiently expressed in normal early myeloid progenitors. To assess the off-tumor hematotoxicity of CAR7 T cells, we cocultured them with cord blood-derived hematopoietic progenitor cells and measured colony formation on a methylcellulose medium. We observed no differences in the numbers of granulocytic, monocytic and erythrocytic colonies after exposure to CAR7 T or control non-transduced T cells indicating a lack of toxicity to normal myeloid and erythroid progenitor cells (Fig. C). Similarly, we observed no reactivity of CAR7 T cells against peripheral monocytes. However, CAR7 T cells were cytotoxic to normal peripheral T- and NK-cells suggesting potential aplasia of these lymphocyte subsets subsequent to in vivo administration of the CAR7 T cells. We may be able to circumvent this toxicity, by infusing pathogen-directed CD7KO T cells from the same donor to repopulate the host, since CD7KO cells appear to retain TCR-mediated activity against a range of pathogens. Hence, CD7KO T cells expressing a CD7-directed CAR can expand without fratricide and have high activity against AML while sparing normal myeloid cells. These data support the feasibility of this approach for the targeted therapy of AML. [Display omitted] DisclosuresLulla:ASH Scholar Award: Research Funding; Leukemia Texas Research grant: Research Funding; Junior Faculty sees funding-Baylor College of Medicine: Research Funding; Lymphoma SPORE: Research Funding; ASBMT Young Investigator Award: Research Funding; Leukemia Texas: Membership on an entity's Board of Directors or advisory committees.

Pre-Clinical Efficacy of Co-Targeting GFI1/KDM1A and BRD4 or JAK1/2 Against AML and Post-MPN Secondary AML Blast Progenitor Cells

Transcriptional regulators (TFs) involved in cell-growth, differentiation and survival of AML stem/progenitor cells (LSCs) include RUNX1, PU.1, CEBPα, c-Myb and c-Myc. LSD1 (KDM1A) is an FAD-dependent amine-oxidase that demethylates mono and dimethyl histone H3 lysine 4 (H3K4Me1 and H3K4Me2). LSD1 is part of the repressor complexes involving GFI1, CoREST and HDAC1/2, that regulate active super-enhancers/enhancers (SEs/Es) and their target genes, mediating transcriptional repression and differentiation block in LSCs. GFI1 is a zinc-finger transcriptional repressor involved in AML development and differentiation. GFI1 contains an N-terminal domain through which it binds to the CoREST/LSD1/HDAC1/2 complex to regulate differentiation in LSCs. CRISPR-suppressor scanning revealed that enzymatic activity of LSD1 was not required for LSC differentiation, instead disruption of binding of LSD1 to GFI1 and CoREST induced differentiation in LSCs. LSD1 and GFI1 expression correlates with worse prognosis in MDS/AML. In present studies, we demonstrate first-time ever that knockout (KO) or degradation of LSD1 utilizing CRISPR-Cas9 or LSD1-FKBP12(F36V) and dTAG-13, respectively, disrupted LSD1-binding to GFI1/1B and CoREST, inhibiting colony growth and inducing differentiation markers (CD86 and CD11b) and morphologic differentiation of AML and post-MPN sAML blast progenitor cells (BPCs). CRISPR-mediated knockout of LSD1 in the AML OCI-AML5 and sAML SET2 cells significantly increased the permissive H3K4Me2/3-marked chromatin, reduced H3K27Ac occupancy at SEs/Es (by ChIP-Seq), especially of c-Myc and CDK6, as well as repressed DNMT1, CoREST, c-Myc, CDK6, and c-KIT, while inducing GFI1, PU.1, CEBPα, p21, CD11b, and CD86 levels (log2 -fold change by RNA-Seq and by Western analyses). This correlated with growth inhibition, % differentiation and apoptosis of AML and sAML cells. CRISPR-mediated GFI1-KO ± the irreversible LSD1 inhibitor (LSD1i) (INCB059872, INCB), repressed GFI1 levels, yet enhanced expressions of PU.1, p21 and CD11b and significantly increased % morphologic differentiation. Treatment with INCB (0.25 to 1.0 µM) also disrupted binding of LSD1 to GFI1 and to CoREST, increased GFI1/1B and PU.1 and repressed c-Myc protein levels, while significantly inhibiting colony growth, inducing differentiation and loss of viability of AML and post-MPN sAML (SET2 and HEL92.1.7) cells, as well as patient-derived AML and post-MPN sAML blasts (p < 0.01). Following INCB treatment, ATAC-Seq analysis demonstrated gained peaks in GFI1 and PU.1-target genes. Following H3K27Ac ChIP-seq analysis rank-ordering of SEs (ROSE) plot highlighted active SEs of RUNX1, GFI1, BCL2, PU.1, IRF8 and SMYD3, accompanied by increased H3K27Ac occupancy at the chromatin of GFI1 and PU.1 targets. Notably, INCB treatment also increased BRD4 occupancy, especially at the GFI1 and PU.1-target genes. RNA-Seq analysis showed that INCB treatment perturbed mRNA expressions, with positive normalized enrichment scores (NES) for interferon α, inflammatory-response, GFI1-targets and E2F-target gene-sets, and negative NES for c-Myc-targets and oxidative-phosphorylation gene-sets. RNA-Seq analyses of INCB-treated compared to untreated OCI-AML5 and SET-2 cells also demonstrated log2 fold-increase in the mRNA expressions of GFI1, PU.1 and CEBPα target-genes. Utilizing a protein domain-scanning CRISPR-Cas9 sgRNA screen followed by LSD1i treatment, present studies also demonstrate co-dependencies, including BRD4, in AML cells. BET inhibitor (BETi) treatment also depleted LSD1 protein levels, and co-treatment with the BETi OTX015 and INCB induced synergistic lethality in AML and post-MPN sAML blasts (Combination Indices < 1.0). Pre-treatment with INCB re-sensitized JAKi-resistant sAML cells to ruxolitinib-induced apoptosis and BETi-resistant post-MPN sAML cells to BETi-induced apoptosis. Notably, co-treatment with INCB (1.5 mg/kg) and ruxolitinib (20 mg/kg) or OTX015 (50 mg/kg), administered orally for 21 days, compared to ruxolitinib alone or vehicle control, significantly reduced the sAML burden and improved survival of immune-depleted mice engrafted with luciferized sAML HEL92.1.7 xenografts (p < 0.01). Collectively, these findings support further pre-clinical development of LSD1i-based combinations with ruxolitinib and BETi against post-MPN sAML. Disclosures Bose: CTI BioPharma: Honoraria, Research Funding; NS Pharma: Research Funding; Celgene Corporation: Honoraria, Research Funding; Pfizer, Inc.: Research Funding; Constellation Pharmaceuticals: Research Funding; Astellas Pharmaceuticals: Research Funding; Blueprint Medicines Corporation: Honoraria, Research Funding; Promedior, Inc.: Research Funding; Incyte Corporation: Consultancy, Honoraria, Research Funding, Speakers Bureau; Kartos Therapeutics: Honoraria, Research Funding. Kadia:Incyte: Research Funding; Pulmotec: Research Funding; Cellenkos: Research Funding; Celgene: Research Funding; Amgen: Research Funding; Genentech: Honoraria, Research Funding; JAZZ: Honoraria, Research Funding; Cyclacel: Research Funding; Novartis: Honoraria; Ascentage: Research Funding; Astellas: Research Funding; Pfizer: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding; Astra Zeneca: Research Funding; BMS: Honoraria, Research Funding. Verstovsek:CTI Biopharma Corp: Research Funding; AstraZeneca: Research Funding; Sierra Oncology: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Incyte Corporation: Consultancy, Research Funding; PharmaEssentia: Research Funding; Blueprint Medicines Corp: Research Funding; NS Pharma: Research Funding; Roche: Research Funding; Gilead: Research Funding; Protagonist Therapeutics: Research Funding; Promedior: Research Funding; Genentech: Research Funding; Celgene: Consultancy, Research Funding; ItalPharma: Research Funding.

A single-cell survey of cellular hierarchy in acute myeloid leukemia

BackgroundAcute myeloid leukemia (AML) is a fatal hematopoietic malignancy and has a prognosis that varies with its genetic complexity. However, there has been no appropriate integrative analysis on the hierarchy of different AML subtypes.MethodsUsing Microwell-seq, a high-throughput single-cell mRNA sequencing platform, we analyzed the cellular hierarchy of bone marrow samples from 40 patients and 3 healthy donors. We also used single-cell single-molecule real-time (SMRT) sequencing to investigate the clonal heterogeneity of AML cells.ResultsFrom the integrative analysis of 191727 AML cells, we established a single-cell AML landscape and identified an AML progenitor cell cluster with novel AML markers. Patients with ribosomal protein high progenitor cells had a low remission rate. We deduced two types of AML with diverse clinical outcomes. We traced mitochondrial mutations in the AML landscape by combining Microwell-seq with SMRT sequencing. We propose the existence of a phenotypic “cancer attractor” that might help to define a common phenotype for AML progenitor cells. Finally, we explored the potential drug targets by making comparisons between the AML landscape and the Human Cell Landscape.ConclusionsWe identified a key AML progenitor cell cluster. A high ribosomal protein gene level indicates the poor prognosis. We deduced two types of AML and explored the potential drug targets. Our results suggest the existence of a cancer attractor.

Abstract IA17: T cell receptor-like antibody 8F4 targets leukemia and non-hematopoietic cancer

Abstract Adaptive anti-tumor immune surveillance of leukemia, called the graft-versus-leukemia (GVL) effect in the setting of allogeneic stem cell transplantation (alloSCT), is responsible for inducing and maintaining long term remission. However, although donor T cells mediate GVL, they also mediate graft-versus-host disease (GVHD), which results in significant morbidity and can be fatal, and is thus a substantial barrier to the more widespread application of alloSCT for many patients with hematological malignancies. CD8 T cells are activated by cognate peptide/MHC-I target cell antigens through binding of clonally unique αβ-heterodimer T cell receptors (TCRs) on the cell surface. Thus, because GVL and GVHD target antigens can be unique, immunotherapy strategies that target GVL antigens such as adoptively transferred T cells could be developed to mediate GVL without GVHD. Likewise, TCR-like monoclonal antibodies (mAbs) that similarly bind to peptide/MHC-I also could be useful as novel immunotherapeutic agents to mediate GVL in the absence of cellular therapies. Moreover, if potent anti-tumor activity was mediated by such TCR-like mAbs, they could have advantages over adoptively transferred donor T cells, including easier standardized manufacturing, ease of dose and scheduled administration, and they could be given to patients in the absence of an alloSCT. We identified PR1, an HLA-A2-restricted 9-mer peptide a peptide derived from aberrantly expressed proteinase 3 (P3) and neutrophil elastase (NE) in myeloid leukemia, as a leukemia-associated target antigen for CD8 T cells that mediate GVL. PR1-specific CD8 T cells target HLA-A2+ AML, CML and MDS but not normal hematopoietic cells and they contribute to cytogenetic and molecular remission of myeloid leukemia. We developed a murine TCR-like mAb that binds to PR1/HLA-A2 (8F4) and showed that 8F4 mediated complement-dependent cytotoxicity (CDC) of AML, CML and MDS progenitor cells and blasts, but not normal bone marrow cells. Because 8F4 did not eliminate normal human hematopoietic cells in a xenograft model, we humanized 8F4 to an IgG1 isotype (h8F4). In AML PDX models, 8F4 and h8F4 eliminated HLA-A2+ AML and leukemia stem cells (LSCs). In preclinical safety models, only mild reversible blood cytopenia was observed after multiple-dose IV administration of high-dose 8F4. Based on these findings, h8F4 is being developed for a first-in-human phase I safety study in HLA-A2+ patients with high-risk AML. In addition, 8F4 also mediated lysis of many non-hematopoietic cancers due to PR1 cross-presentation on surface HLA-A2, which increased cancer cells susceptibility to 8F4-mediated lysis. Therefore, 8F4 also could be tested for the treatment of non-hematopoietic HLA-A2+ cancers such as breast cancer, non-small cell lung cancer, and colon cancer. Citation Format: Jeffrey J. Molldrem, Anna Sergeeva, Hong He, Amanda C. Herrmann, Tian-Hui Yang, Celine Kerros, Haley Peters, Jin Seon Im, Sapna Parshottam, Sijie Lu, Qing Ma, Karen C. Dwyer, Elizabeth Mittendorf, Gheath Alatrash. T cell receptor-like antibody 8F4 targets leukemia and non-hematopoietic cancer. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr IA17.

Disrupting Wnt/ß-Catenin Signaling By a ß-Catenin/CBP Modulator C-82 Suppresses Cell Growth and Induces Apoptosis in AML Blasts and CD34+38- AML Progenitor Cells

Wnt/ß-catenin signaling has been reported to be active in AML and essential for AML stem cells but not absolutely required for self-renewal of adult hematopoietic stem cells. Targeting ß-catenin may represent a novel therapeutic opportunity for AML that should be further investigated in clinical studies.Using reverse-phase protein array, we determined ß-catenin expression in samples obtained from 511 newly diagnosed AML patients and found that high ß-catenin level is associated with unfavorable cytogenetics (p = 0.01). Expression of ß-catenin is correlated with its target gene survivin in these samples (n= 511, R=0.24, p < 0.0001). In paired samples, ß-catenin expression is significantly higher in bone marrow (BM) than peripheral blood samples (n=140, p < 0.001) and in relapsed compared to newly diagnosed samples (n=47, p = 0.04).C-82, like ICG-001 is a ß-catenin/cAMP-response element-binding protein (CBP) modulator which binds to CBP and inhibits the interaction between ß-catenin and CBP thus disrupts Wnt/ß-catenin/CPB mediated cell proliferation/self-renewal signaling. In the present study, we investigated the effects of inhibition of ß-catenin/CBP by C-82 in AML cells on cell growth, cell survival, the expression of the downstream targets, and the therapeutic potential of C-82 in AML.Treatment with C-82 suppressed cell growth in AML cell lines. Cell cycle analysis showed that the treatment inhibited Edu incorporation and greatly decreased S-phase cells accompanied with increased G1 and subG1 cells in OCI-AML3 and Molm13 cells. Significant cell death was observed in C-82 treated OCI-AML3 and Molm13 cells (EC50=0.82±0.04 μM and 0.79±0.02 μM, IC50=0.42±0.06 μM and 0.39±0.02 μM, respectively) at 48 hours and cells from AML patients (n=7, EC50=1.90±0.28 μM and IC50=1.17±0.26 μM) including CD34+38- AML stem/progenitor cells (n=6, EC50=4.25±0.69 μM and IC50=2.11± 0.04 μM) at 72 hours, while minimal cell death was found in CD34+ cells from normal BM (n=3, <10% with 2.5 μM C-82 at 48 hours). Decreased expression of survivin, c-Myc, and CD44, all downstream of ß-catenin/CBP signaling were found in cell lines, bulk primary AML and CD34+38-cells (n=7) by western blot or flow cytometry analysis.Co-cultures of AML cells with BM-derived mesenchymal stromal cells (MSCs) induced ß-catenin and the adhesion protein CD44, a target of ß-catenin in AML cells suggesting that the BM microenvironment regulates ß-catenin expression in AML cells. This finding is supported by the observed higher ß-catenin levels in BM compared to peripheral blood cells. C-82 suppressed MSC-induced CD44, inhibited the adhesion of OCI-AML3 cells to MSCs, and induced apoptosis of AML cell lines and blasts (n=5) and CD34+38-cells (n=4) from AML patients co-cultured with MSCs. Furthermore, combination of C-82 with Ara C further enhanced death of AML cells (combination index < 1).The data demonstrate that disrupting Wnt/ß-catenin signaling by a ß-catenin/CBP modulator C-82 suppresses cell growth and induces apoptosis in AML cells including CD34+38- AML stem/progenitor cells while it has minimal toxicity in normal CD34+ cells supporting the clinical development of the strategy to test the therapeutic potential of targeting wnt/ß-catenin signaling in AML. PRI-724, a C-82 pro-drug is currently in clinical trial as a single agent and in combination with Ara C in patients with advanced AML. DisclosuresKouji:PRISM Pharma Co.: Employment. Cortes:PRISM Pharma Co.: clinic trial Other. Carter:PRISM Pharma Co.: Research Funding.

Blocking TNF and IL-1 Sensitizes AML Stem Cells to NF-κB Inhibitor Treatment

Background: NF-κB inhibition selectively eliminates leukemia stem cells (LSCs) but has less effect on healthy hematopoietic stem cells (HSCs), suggesting an advantageous target for leukemia therapy. However, NF-κB inhibition alone does not have a significant effect on in vivo models of leukemia. We have reported that many types of AML cells produce tumor necrosis factor-α (TNF) which appears to protect LSCs from NF-κB inhibition by stimulating JNK-AP1 survival/proliferative signaling parallel to NF-κB. Complete inactivation of TNF signaling cannot fully reproduce the effects of JNK-AP1 inhibition, suggesting that some other inflammatory cytokine(s) might also compensate for NF-κB inhibition through activation of JNK-AP1. We found that IL-1α and β (IL-1) is such a cytokine.Methods: TNF and IL-1 expression was examined in 430 AML samples by microarray and verified by qRT-PCR. IL-1α and β protein levels in the sera of AML patients and medium from cultured AML cells were examined by ELISA. MLL-AF9-transduced murine AML cells, human AML cell lines and primary patient samples were used in this study. TNF signal was blocked by genetic deletion of receptors Tnfr1 and 2, or treatment with a TNF-specific monoclonal antibody. IL-1 signal was blocked by shRNA knockdown of IL-1R or treatment with an IL-1R antagonist. The effects of IL-1 and TNF signal co-inactivation on growth and response to NF-κB inhibition in AML cells were evaluated by in vitro culture and colony-forming assay, and in vivo transplantation/leukemogenesis assays.Results: We found that most types of human AML cells both express and produce TNF and IL-1. Treatment with these cytokines stimulated the growth and colony-forming ability of AML cells but repressed the growth of normal HSPC in in vitro culture. Inactivation of either the TNF or IL-1 signaling axes suppressed the growth of and significantly sensitized AML stem cells to NF-κB inhibition both in vitro by targeting clonogenic AML progenitor cells, and in vivo through compromising leukemogenesis. Simultaneous inactivation of both TNF and IL-1 resulted in an enhanced repressive effect on AML cells compared to inactivation of either individual cytokine’s receptors alone. AML cells with inactivation of both TNF and IL-1 signals showed significantly lower clonogenic ability in vitro, further reduced leukemogenic capacity in vivo and enhanced sensitivity to NF-kB inhibitor treatment both in vitro and in vivo. Mechanistically, we found that both TNF and IL-1 stimulate JNK-AP1 and NF-κB, two parallel survival signaling pathways in AML cells, but induce JNK-mediated death signaling in normal HSPCs.Conclusion: Both TNF and IL-1 provide protection to LSCs from the effects of NF-κB inhibitor treatment in an autocrine manner and contribute to repression of normal hematopoiesis in a paracrine fashion. This protection is mediated by JNK-AP1. Our studies show inhibiting both TNF and IL-1 inflammatory signals can block this effect, and may be a better strategy to successfully treat AML when combined with NF-κB inhibitors. DisclosuresNo relevant conflicts of interest to declare.

Highly active combination of BRD4 antagonist and histone deacetylase inhibitor against human AML.

11107 Background: The bromodomain-containing protein 4 (BRD4) is a member of the BET (bromodomain and extraterminal) protein family, which regulate the assembly of chromatin complexes and transcriptional regulators at the target gene promoters, e.g., of Myc and BCL2. JQ1 competitively binds and inhibits the bromodomains of the BET proteins, including BRD4. Treatment with JQ1 attenuates Myc and BCL2 levels and promotes growth arrest, differentiation and apoptosis of AML cells. Methods: Here, we determined the growth inhibitory, differentiation and apoptotic effects of JQ1 (100 to 500 nM) alone and in combination with low concentrations of the deacetylase inhibitor panobinostat (PS) (5 to 20 nM) (gift from Novartis) in cultured (OCI-AML3, MV4-11 and MOLM13) and primary AML blast progenitor cells. We also determined the in vivo anti-AML activity of JQ1 (50 mg/kg/day IP) and/or PS (5 mg/kg/MWF IP) for 3 weeks against the OCI-AML3 xenograft in the NOD/SCID mice. Results: Treatment with JQ1 decreased the binding of BRD4 at the chromatin associated with Myc and BCL2 promoters (by ChIP-qPCR) and attenuated the mRNA and protein levels of Myc, BCL2 and cyclin D1, while increasing the levels of p21, CEBPα, BIM and cleaved PARP in the cultured and primary AML blasts. This was associated with an increase in the % of G1 phase cells, attenuation of the colony growth, and induction of apoptosis of the cultured (OCI-AML3 &gt;MV4-11 &gt;MOLM13) and primary AML cells. JQ1 treatment also dose-dependently inhibited the viability of CD34+ primary AML progenitor cells (n=10). Gene expression microarray analysis revealed a JQ1 treatment-associated signature of the most up- and down-regulated mRNAs that correlated with its anti-AML effects. Co-treatment with JQ1 and PS synergistically induced apoptosis of AML cells (by isobologram analysis; CI &lt; 1.0). As compared to the mice treated with vehicle alone, or with each agent alone, co-treatment with JQ1 and PS, without inducing toxicity, significantly improved the survival of the OCI-AML3 engrafted NOD/SCID mice (p &lt; 0.01). Conclusions: These pre-clinical findings warrant further in vivo evaluation of the efficacy of the combination of a BRD4 antagonist and PS, and its predictive biomarkers, against human AML.

Role of CXCR4 in the pathogenesis of acute myeloid leukemia.

The Chemokine receptor CXCR4 and its ligand stromal derived factor-1 (SDF-1/CXCL12) are important players involved in cross-talk between leukemia cells and the bone marrow (BM) microenvironment. CXCR4 expression is associated with poor prognosis in AML patients with and without the mutated FLT3 gene.CXCL12 which is constrictively secreted from the BM stroma and AML cells is critical for the survival and retention of AML cells within the BM. In vitro, CXCR4 antagonists were shown to inhibit the migration of AML cells in response to CXCL12. In addition, such antagonists were shown to inhibit the survival and colony forming potential of AML cells and abrogate the protective effects of stromal cells on chemotherapy-induced apoptosis in AML cells. In vivo, using immune deficient mouse models, CXCR4 antagonists were found to induce the mobilization of AML cells and progenitor cells into the circulation and enhance anti leukemic effects of chemotherapy. The hypothesis that CXCL12/CXCR4 interactions contribute to the resistance of AML cells to signal transduction inhibitor- and chemotherapy-induced apoptosis is currently being tested in a series of Phase I/II studies in humans.

Prognostic Impact and Targeting of CRM1 in Acute Myeloid Leukemia

Abstract 870p53 is a transcription factor that prevents abnormal cell growth. Cellular levels of p53 are critically regulated by MDM2, which is frequently over-expressed in AML. Nutlin-3a disrupts MDM2-p53 interaction, increases cellular levels of p53 in both nucleus and cytoplasm, and activates p53 signaling in cells. p53 status is the major determinant of response to MDM2 inhibitors. p53 is shuttled between the nucleus and the cytoplasm, and CRM1 mediates its nuclear export. Karyopharm Therapeutics has developed novel, potent and irreversible small molecule selective inhibitors of CRM1. We hypothesized that CRM1 inhibition would enhance the nuclear activity of p53, thereby enhancing p53-mediated transcription-dependent apoptotic signaling in AML. We measured CRM1 expression in primary AML samples and investigated if blockade of nuclear export of p53 by CRM1 inhibition would enhance MDM2 inhibitor-induced apoptosis in AML.CRM1 expression was investigated in 511 patient AML samples using a validated robust reverse-phase protein array. Higher levels of CRM1 were associated with higher marrow and peripheral blast percentages (P < 0.00001). Expression was lower in those with favorable cytogenetics compared to those with intermediate or unfavorable cytogenetics (P = 0.029). CRM1 levels were higher in patients with FLT3 mutations (P = 0.003). In 3-way correlation (using distance weighted least squares), there was a clear interaction with p53 levels being highest when CRM1 was high and MDM2 levels were low. Overall survival progressively worsened as CRM1 levels increased, with median survival of 66 weeks for those with CRM1 expression in the lowest third, 47 weeks for middle third and 37 weeks in the highest third (P = 0.007). CRM1 levels did not affect remission duration (P = 0.33).The CRM1 inhibitor KPT-185 exhibited dose-dependent anti-proliferative and cytotoxic activity in AML cell lines, as evidenced by low IC50 values and high Annexin V positivity (= low ED50 values). IC50 values for wild-type p53 cells ranged from 27 to 38 nM, and for mutant p53 cells from 48 to 112 nM, suggesting that KPT-185 potently inhibits AML cell growth largely independent of p53. In contrast, apoptosis induction by KPT-185 was much more prominent in p53 wild-type than in p53-defective cells: ED50 values for Annexin V induction were 150, 90 and 85 nM in p53 wild-type and > 1000 nM in 5 of 6 p53 mutant cell lines. Stable p53 knockdown (> 90% efficiency) rendered AML cells resistant to KPT-induced apoptosis. KPT-185 induced p53 target genes TP53I3, GDF15, MDM2 and ZMAT3 partially in a p53-dependent manner. Hence, p53 was identified as major determinant of CRM1 inhibition-induced apoptosis in AML.MDM2-inhibitor Nutlin-3a induced p53 in both nucleus and cytoplasm, while CRM1 inhibition accumulated p53 in the nucleus. Treatment with KPT-185 or Nutlin-3a caused time-dependent increase in cellular p53 levels. The KPT-185/Nutlin-3a combination induced p53 more efficiently than the individual agents by accumulating p53 exclusively in the nucleus, and synergistically induced apoptosis and cell death. p53 knockdown abrogated these synergistic effects.In primary AML cells, both KPT-185 (24.7 – 36.7% Annexin V) and Nutlin-3a (13.6 – 59.8%) induced apoptosis in a dose-dependent manner. Importantly, both KPT-185 and Nutlin-3a induced apoptosis in CD34+CD38- progenitor cell populations as effectively as they did in bulk AML cells, suggesting high sensitivity of CD34+CD38- cells to CRM1 inhibition and MDM2 inhibition. KPT-185 and Nutlin-3a synergized in the induction of apoptosis in both bulk and CD34+CD38- AML progenitor cells: combination index (CI) values were 0.26 (bulk) and 0.30 (CD34+CD38-) for ED50 and 0.93 (bulk) and 0.46 (CD34+CD38-) for ED75, indicating highly synergistic (CI < 1) efficacy in apoptosis induction. The relation between p53 status and sensitivity to Nutlin-induced apoptosis has been well established. Nutlin-resistant samples were much less sensitive to KPT-185 than Nutlin-sensitive cases (12.2 ± 0.06 % versus 30.9 ± 0.04 % Annexin V, P < 0.05). Synergistic induction of apoptosis was not observed in normal cord blood CD34+CD38- cells.Collectively, CRM1 inhibition offers a novel therapeutic strategy for AML that mostly retains wild-type p53. We propose to develop novel combinatorial approaches for the therapy of AML, aimed at maximal activation of p53 and apoptosis signaling by concomitant MDM2 and CRM1 inhibition. Disclosures:Shacham:Karyopharm Therapeutics: Employment. Kauffman:Karyopharm Therapeutics: Employment. Andreeff:Hoffmann-La Roche: Research Funding; Karyopharm Therapeutics: Unrestricted gift, Unrestricted gift Other.

Rocaglamide Selectively Eradicates Human Leukemia Stem Cells and Synergizes with Multiple Agents to Target AML Cells

Abstract 1338Recent evidence suggests that myeloid leukemia is initiated and maintained by Leukemia Stem Cells (LSCs). Standard chemotherapy however, does not efficiently ablate LSCs. Consequently, even for leukemia patients who attain a clinical remission, LSCs are generally not destroyed and are thought to be responsible for subsequent relapse of the disease. Therefore, new treatment regimens are necessary to improve therapeutic outcomes. Nearly half of the agents used in cancer therapy today are either natural products or derivatives of natural products. The present studies demonstrate that rocaglamide, a compound derived from the traditional Chinese medicinal plants Aglaia induces robust apoptosis in primary human AML cells while sparing normal hematopoietic cells. Further analysis of progenitor cells using in vitro colony assays, as well as stem cells using the NOD Scid Gamma (NSG) xenograft model, show that rocaglamide also preferentially targets AML progenitor and stem cell populations.Methionine metabolic labeling experiments show that rocaglamide inhibits the translation of nascent protein synthesis within twenty-four hours and this inhibition results in the rapid loss of short-lived survival proteins such as c-myc, Mcl-1, and Bcl-xl. These results are consistent with previous work showing rocaglamide, and members of the rocaglamide family of compounds, inhibit translation. To investigate further the molecular mechanism of LSC-specific cell death induced by rocaglamide we performed next generation sequencing on 5 AML specimens treated with rocaglamide. Bioinformatic analysis and subsequent experiments showed that rocagalmide leads to P53 activation, NFkB inhibition, cell cycle inhibition as well as defects in mitochondrial integrity and energy metabolism. In addition to efficacy as a single agent, pre-treatment of leukemia cells with rocaglamide significantly sensitizes the cells to several anti-cancer compounds, including cytarabine and daunorubicin two of the front-line chemotherapuetic drugs for AML patients. Importantly, we show that many of the mechanistic features of rocaglamide as a single agent play a role in its ability to synergize. In comparison with translational inhibitors that are used clinically to treat AML patients, temsirolimus and ribovarin, rocaglamide is significantly more toxic to leukemia cells. Interestingly, this increased cytotoxicity does not directly correlate with ability of the compounds to inhibit translational inhibition. Temsirolimus, inhibits translation at levels equal to or greater than rocaglamide however it has a cytostatic effect on leukemia cells in contrast to the cytotoxic effects of rocaglamide. Temsirolimus also does not synergize with anti-cancer compounds to the same degree as rocaglamide. These results suggest that rocaglamide's ability to modulate several key pathways in addition to inhibiting translation are critical to the activity of rocagalmaide and may suggest ways to improve the efficacy of translational inhibitors currently used in the clinic. These studies along with preliminary in vivo pharmacodynamic and pharmacokinetic experiments indicate that rocaglamide may be a promising candidate for the development of a new class of compounds for the treatment of leukemia and for increasing the efficacy of treatments designed to specifically target AML cells. Disclosures:No relevant conflicts of interest to declare.

A novel nonradioactive CFDA assay to monitor the cellular immune response in myeloid leukemia

BackgroundDonor lymphocyte transfusion (DLT) may induce the graft-versus-leukemia (GVL) effect for patients with AML relapsed after transplant. However, AML is a highly diverse disease and the limited overall efficacy of DLT in clinical practice emphasizes the importance of identifying a specific subgroup of patients who might benefit from this treatment approach. ObjectiveTo monitor the cellular immune response after DLT, we developed an active specific immunization strategy using in vitro generated AML-trained T cells to induce a highly specific antileukemic T-cell response and thus established a novel nonradioactive assay system to assess the antileukemia immunity by flow cytometry, correlated with [3H]-thymidine uptake. MethodsThe myeloid blasts derived from five patients with AML relapsed post-allogeneic hematopoietic stem cell transplantation (allo-HSCT) were first labeled with CFDA (5,6-carboxyfluorescein diacetate succinimidyl ester). To analyze the growth inhibitory potential of the donor T cells trained by AML progenitor cells, the myeloid blasts were induced to proliferate by means of a cytokine cocktail (50ng/mL of SCF; 25ng/mL of IL-3; 100ng/mL of GM-CSF; 100ng/mL of G-CSF; 2U/mL of EPO; 0.47g/L of transferrin; and 5×10−5mmol/L of 2-ME). The T cell mediated growth inhibitory potential was detected after 5 days by flow cytometry and correlated with [3H]-thymidine uptake. The simultaneous use of TO-PRO-dye and calibrate beads allowed not only the cell viability to be known but also allowed quantification of the effector function. ResultsHere, we applied a CFDA dye to track the proliferation and expansion of AML blasts in response to the cytokine cocktail in vitro. AML-trained T cells, expressed high levels of the activation markers CD25 and CD69, and were generated to recognize the leukemic progenitor cells and inhibit cytokine-induced leukemic cell proliferation, which is an active specific immunization strategy circumventing the identification of leukemia-associated antigens. The capability of proliferation inhibition of AML-trained T cells evaluated with our nonradioactive, CFDA-based assay provided comparable results with the classic [3H]-thymidine assay with an even lower ratio of effector to target cells. ConclusionTaken together, the novel, nonradioactive, CFDA-based assay was a robust tool to monitor the antileukemic immune response after DLT in myeloid leukemias.

Anti-AML activity of a novel beta-catenin antagonist BC2059.

10605 Background: The canonical WNT-β-catenin pathway is essential for self-renewal, growth and survivalof AML stem and progenitor cells. Deregulated WNT signaling inhibits degradation of β-catenin, causing increased nuclear translocation and interaction of β-catenin with the TCF/LEF transcription factor, which up regulates cyclin D1, Myc and survivin expression in AML progenitor cells. BC2059 (β-Cat Pharmaceuticals) is a potent, small molecule, anthraquinone oxime-analog, which inhibits WNT-β catenin pathway by promoting the degradation and attenuation of β-catenin levels. Methods: We determined the in vitro anti-AML activity of BC2059 (BC) (250 to 1000 nM) against cultured and primary human AML blast progenitors, as well as evaluated the in vivo anti-AML efficacy of BC in NOD-SCID and NOD-SCID-IL2γ receptor deficient (NSG) mice. Results: BC induced cell cycle G1 phase accumulation and apoptosis (40%) of the cultured OCI-AML3, HL-60 and HEL92.1.7 (HEL) AML cells. BC dose-dependently also induced apoptosis of primary AML versus normal progenitors. Treatment with BC resulted in proteasomal degradation and decline in the nuclear levels of β-catenin, which led to decreased activity of the LEF1/TCF4 transcription factor highlighted by reduced TOP-FLASH luciferase activity in the AML cells. This was associated with reduced protein levels of cyclin D1, MYC and survivin. Co-treatment with BC and the histone deacetylase inhibitor panobinostat (PS) (10 to 20 nM) synergistically induced apoptosis of cultured and primary AML blasts. Following tail vein infusion and establishment of AML by OCI-AML3 or HEL cells in NOD-SCID mice, treatment with BC (5, 10 or 15 mg/kg b.i.w, IV) for three weeks demonstrated improved survival, as compared to the control mice (p &lt;0. 001). Survival was further improved upon co-treatment with BC and PS (5 mg/kg IP, MWF). BC treatment (5 or 10 mg/kg IV) also dramatically improved survival of NSG mice with established human AML following tail-vein injection of primary AML blasts expressing FLT3 ITD. Mice did not experience any toxicity or weight loss. Conclusions: These findings highlight the notable pre-clinical in vitro and in vivo activity and warrant further development and in vivo testing of BC against human AML.

Investigational NEDD8-Activating Enzyme (NAE) Inhibitor, MLN4924, Demonstrates Activity Against Primary AML Blast, Progenitor and Stem Cell Populations

Abstract 1414Acute myelogenous leukemia (AML) has a poor prognosis. Most patients die of either primary refractory or relapsed disease. It has been proposed that relapse may result from ineffective ablation of leukemia stem cells (LSCs) by chemotherapy. Therefore, in order to improve therapy, it is imperative to identify agents that eliminate leukemic progenitor and stem cell populations in addition to blasts. We have previously demonstrated that proteasome inhibition can result in selective ablation of LSCs, without harming normal hematopoietic stem cells (HSCs). Thus, we sought to investigate the activity of a more specific inhibitor of the ubiquitin-proteasome pathway, an investigational NEDD8-Activating Enzyme (NAE) inhibitor MLN4924. Treatment of primary human AML and blast crisis CML samples with MLN4924 resulted in a dose-dependent decrease in the total viability of the blast-cell populations, with an average LD50 of 0.367 μM (0.203–0.530 95% CI; N=17). The average LD50 for phenotypically described LSCs was 0.645μM (0.304–0.986; 95%CI; N=12). Interestingly, when performing progenitor/stem functional assays, we observed a 64% and 86% decrease in colony formation after treatment with 0.25μM and 1 μM of MLN4924 (p< 0.001; N=5; p< 0.001 N=8 respectively). Furthermore, MLN4924 had little to no effect on colony formation of normal hematopoietic cells. Thus, MLN4924 may impair the activity of AML stem/progenitor cells, but not normal cells, in vitro. Importantly, primary AML samples that were resistant to standard chemotherapeutics such as cytarabine in vitro were sensitive to MLN4924. Treatment of primary AML samples with MLN4924 showed increased γH2AX foci, indicating activation of stress responses. Together, these data suggest that MLN4924 can eliminate AML blasts, progenitor and stem cell populations in vitro. Disclosures:Smith:Millennium Pharmaceuticals: Employment.

Abstract C144: Treatment with β-catenin antagonist BC2059 exhibits single agent efficacy and exerts superior activity with tyrosine kinase inhibitor (TKI) or histone deacetylase (HDAC) inhibitor against human AML, CML, and myeloproliferative neoplasm (MPN) progenitor cells.

Abstract The canonical WNT-β-catenin signaling is essential to the cellular processes of self-renewal and growth, as well as required for self-renewal of leukemia stem. The multi-protein degradation complex for β-catenin is often inactivated in transformed hematopoietic progenitor cells. This results in the preservation, nuclear translocation and interaction of β-catenin with the T-cell factor (TCF)/lymphoid enhancer factor (LEF) transcription factor, which regulates the expression of genes such as cyclin D1, MYC and survivin. BC2059 (β-Cat Pharmaceuticals) is a potent small molecule, anthraquinone oxime-analog inhibitor of the WNT-β-catenin pathway. Treatment with BC2059 mediates the degradation and attenuated levels of β-catenin. In the present studies, we determined the activity of BC2059 in human cultured and primary AML, CML and advanced MPN versus normal cells. Exposure to 50 to 100 nM of BC2059 induced cell cycle G1 phase accumulation and apoptosis (40 to 80%) of the cultured AML cells (HL-60 and OCI-AML3), MPN HEL92.1.7 (HEL) and UKE1 cells expressing the mutant JAK2V617F, as well as of the CML K562 and LAMA-84 cells expressing BCR-ABL. BC2059 treatment also induced apoptosis of CD34+ primary MPN cells from patients with advanced MPN expressing mutant JAK2, as well as of primary CD34+ AML and CML progenitor cells. In contrast, BC2059 did not induce significant apoptosis of normal CD34+ progenitor cells. Exposure to BC2059 attenuated both β-catenin protein levels (significantly restored by co-treatment with the proteasome inhibitor) and the activity of the LEF1/TCF4 transcription factor, associated with reduced levels of cyclin D1, MYC and survivin in the cell lysates of BC2059-treated AML, CML and MPN cells. Following the tail vein infusion of HEL cells and establishment of MPN, NOD-SCID mice were treated with 15 or 20 mg/kg of BC2059 administered b.i.w for three weeks via the tail vein. As compared to the control, BC2059-treated mice demonstrated significantly improved survival (p &amp;lt;0.001). Next, we examined the effects of co-treatment with BC2059 (20 to 50 nM) and JAK2-targeted TKI TG101209 (TG) (200–1000 nM) or BCR-ABL-targeted TKI nilotinib (10–20 nM) against MPN or CML cells, respectively. As compared to treatment with each agent alone, co-treatment with BC2059 and TG synergistically induced apoptosis of HEL and primary CD34+ MPN cells. Additionally, co-treatment with BC2059 and nilotinib induced synergistic apoptosis of K562 and primary CML progenitor cells. Further, combined treatment with BC2059 and the HDAC inhibitor panobinostat (10 to 20 nM) also induced significantly more apoptosis of HL-60, HEL and K562 (p &amp;lt; 0.01), as well as of the primary CD34+ MPN, primary AML and CML progenitor cells. Notably, BC2059 (50 nM) also induced marked apoptosis of panobinostat-resistant cultured AML HL-60/LR cells (Blood 112: 2896, 2008). BC2059-based combinations were remarkably less toxic in normal CD34+ progenitor cells (p &amp;lt; 0.01). Thus, BC2059 exerts notable in vitro and in vivo activity against human myeloid malignant cells. Additionally, BC2059 exerts superior activity in combination with an HDAC inhibitor, as well as with JAK2 or BCR-ABL-targeted TKI against AML, MPN and CML progenitor cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C144.

Abstract 2145: AR-42, a novel histone deacetylase inhibitor with selectivity against leukemia stem cell via apoptosis induction, inhibition of chaperone function of heat shock protein 90 in acute myeloid leukemia

Abstract AML is a fatal disease where most patients relapse and die of their disease. Increasing evidence indicates that disease relapse is driven by leukemia stem cells (LSCs). Thus, it is imperative to identify new therapies that can ablate LSCs. We have previously reported that screening the NCBI Gene Expression Omnibus (GEO) can accelerate the identification of anti-LSC compounds by searching for those mimicking the gene expression pattern of a known anti-LSC compound, parthenolide (PTL). One of our “hits” was the HDACi, AR-42 (Arno Therapeutics, Inc). This compound represents a novel class of HDACis that are structurally similar to phenylbutyrate, but with improved HDACi activity at sub-micromolar concentrations. At first, we investigated effects of AR-42 on growth and colony forming ability by using AML cell lines and primary human AML samples. AR-42 exhibits low micromolar potency in biochemical and cellular assays. AR-42 efficiently suppressed AML cell growth at a low dosage (1 μM) after 24 hours of treatment. Just like PTL, AR-42 preferentially targets AML progenitor and stem cell populations. More than 50% cell death was induced by AR-42 in phenotypically defined AML stem / progenitor. Colony forming capability of LSCs treated with 1 μM AR-42 for 24 hours was significantly diminished. Furthermore, 1 μM AR-42 significantly impaired the potential of LSCs in a nonobese diabetic/severe combined immunodeficient (NOD/SCID) xenograft model. Surprisingly, even though AR-42 partially mimics the gene expression pattern of PTL, it does not activate Nrf2-controlled cytoprotective responses like PTL. AR- 42 inhibits NF-κB but, unlike PTL, does not activate HMOX-1. AR-42- mediated apoptosis is associated with activation of caspase 8, PARP cleavage. Further studies found that AR-42 induced inhibition of HSP90 in conjunction with degradation of FLT3 protein. Altogether, these findings indicate that AR-42 may be a promising LSC-targeted therapeutic agent. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2145. doi:10.1158/1538-7445.AM2011-2145