ALBERT

Description: Abstract 3415 The advent of imatinib, a Bcr-Abl tyrosine kinase inhibitor (TKI) has revolutionized the treatment of patients with CML. Development of resistance and limited activity in blast crisis (BC) CML are evolving problems facing this therapy. We found that XIAP, a potent caspase inhibitor, is highly expressed in CML cells, in both, cell lines and patient samples. Treatment with imatinib deceased XIAP levels in imatinib-sensitive KBM5 but much less so in imatinib-resistant KBM5STI571 cells (harboring T315I mutation) suggesting that XIAP expression in CML is regulated at least in part via Bcr-Abl and that targeting XIAP may promote cell death in CML cells by circumventing imatinib resistance. To test this, we treated BC CML cells with XIAP antisense oligonucleotide (ASO) and with SMAC mimetic ABT-10 and found that inhibition of XIAP induced apoptotic cell death with similar efficacy in KBM5 cells and KBM5STI517 cells (EC50=6.3±0.3 μM and 8.4±0.4 μM at 48 hours, respectively for ABT-10). However, we noted that inhibition of XIAP by ASO induced the expression, in both KBM5 and KBM5STI571 cells, of apoptosis repressor with caspase recruitment domain (ARC) in both mRNA and protein levels but not the expression of Bcl-2 protein. ARC is a unique antiapoptotic protein. It acts through inhibiting caspases and antagonizing the activity and function of p53 and Bax. Therefore, its induction may antagonize the effect of XIAP downregulation. Indeed, inhibition of both XIAP and ARC by ASO induced significantly more cell death than inhibiting either protein alone in both KBM5 and KBM5STI cells. Furthermore, we demonstrated that XIAP inhibition induced-apoptosis was enhanced by imatinib in KBM5, but not in KBM5STI cells. Interestingly, inhibition of Bcr-Abl tyrosine kinase by imatinib not only decreased XIAP, but also suppressed ARC levels in KBM5 but had minimal effects on the levels of these proteins in KBM5STI571 cells and enforced expression of the Bcr-Abl p185 fusion protein (in HL-60 cells) greatly increased both XIAP and ARC levels. This induction was inhibited by imatinib suggesting that ARC is also a downstream target of Bcr-Abl tyrosine kinase. Therefore, imatinib enhancing XIAP inhibition induced-apoptosis in KBM5, not KBM5STI cells can be explained at least in part by its ability to decrease XIAP and ARC levels. In conclusion, XIAP is highly expressed in CML cells and upregulated by Bcr-Abl. Targeting XIAP promotes death of BC and TKI resistant CML cells. Results suggest that XIAP is a potential target in BC and TKI resistant CML cells and that XIAP inhibition-induced apoptosis is enhanced by imatinib in TKI sensitive cells and by ARC inhibition independent of cellular responses to TKIs. Inhibition of XIAP and ARC as a novel therapeutic strategy in CML warrants further investigation. Disclosures: Koller: Isis Pharmaceuticals: Employment.

Description: p53, a key regulator of apoptosis, functions primarily upstream in the apoptotic cascade by directly and indirectly modulating Bcl-2 family of proteins. XIAP, a potent antiapoptotic protein, functions primarily downstream by suppressing caspases. Activation of p53 by MDM2 antagonist nutlin3a or inhibition of XIAP by small molecule inhibitors such as phenylurea 1396-11 was found to induce apoptosis in AML cells. Since the functions of XIAP and p53 are mediated and their activities controlled by a network of numerous components, some of which cross-regulate each other, we hypothesized that simultaneous activation of p53 and inhibition of XIAP would be a more effective at activating apoptotic signaling in AML cells. To test this idea, we treated AML cells with nutlin3a and 1396-11 and found that the combination synergistically induced cell death at 24 hours in OCIAML3 cells (combination index CI=0.200±0.047) and Molm13 cells (CI=0.565±0.082), two cell lines harboring wild type p53. Knocking down p53 expression by shRNA blunted the synergistic effect and downregulation of XIAP by antisense oligonucleotide (ASO) enhanced nutlin3a-induced apoptosis in OCI-AML3 cells, suggesting that the synergy was mediated by both p53 activation and XIAP inhibition. The specificity was further supported by data showing that inhibition of MDM2 and XIAP by their respective ASOs induced significantly more cell death than either ASO alone. Although nutlin3a alone induced apoptosis in OCI-AML3 cells, the cell death was not robust and caspase-3 activation was minimal by itself even at 48 hours with 10 μM of nutlin3a. Immunoblot analysis showed increased expression of p53 and its downstream target p21. Of note, because p21 not only induces G1 cell cycle block, it additionally exhibits antiapoptotic activity that diminishes the effects of p53 activation, we also studied effects of these agents on p21 levels. When nutlin3a and 1396–11 were combined, caspase-3 activation was greatly increased and nutlin3a-induced p21 expression was significantly diminished. Moreover, in these experiments, caspase inhibition restored p21 levels and diminished apoptosis enhanced by 1396-11, suggesting that XIAP inhibition-mediated caspase activation eliminates p21, enhancing nutlin3a-induced apoptosis. Furthermore, activation of p53 by nutlin3a increased caspase-6 protein levels and induced mitochondrial release of SMAC, an antagonist of XIAP, suggesting that p53 activation shifts the balance toward apoptosis, promoting the effect of XIAP inhibition. Most importantly, p53 activation and XIAP inhibition greatly enhanced apoptosis in primary blasts from AML patients. Five out of six samples treated showed synergistic killing at 24 hours (CI=0.73±0.13), even when the cells were protected from drug-induced and spontaneous apoptosis by MS-5 stroma cells (CI=0.45±0.06). In conclusion, results demonstrate that simultaneous activation of p53 by antagonizing MDM2 and inhibition of XIAP synergistically activate apoptotic signaling pathways and promote death of AML cells, in part by modulating p21, caspases, and cytosolic SMAC levels. Since both, XIAP and p53, are presently being targeted by ongoing clinical trials in leukemia patients, the combination strategy holds promise for expedited translation into the clinic.

Description: Recent studies suggest that the Bcl-2 and mitogen-activated protein kinase (MAPK) pathways together confer an aggressive, apoptosis-resistant phenotype on acute myelogenous leukemia (AML) cells. In this study, we analyzed the effects of simultaneous inhibition of these 2 pathways. In AML cell lines with constitutively activated MAPK, MAPK kinase (MEK) blockade by PD184352 strikingly potentiated the apoptosis induced by the small-molecule Bcl-2 inhibitor HA14-1 or by Bcl-2 antisense oligonucleotides. Isobologram analysis confirmed the synergistic nature of this interaction. Moreover, MEK blockade overcame Bcl-2 overexpression-mediated resistance to the proapoptotic effects of HA14-1. Most importantly, simultaneous exposure to PD184352 significantly (P = .01) potentiated HA14-1–mediated inhibition of clonogenic growth in all primary AML samples tested. These findings show that the Bcl-2 and MAPK pathways are relevant molecular targets in AML and that their concurrent inhibition could be developed into a new therapeutic strategy for this disease.

Description: We tested the effects of small-molecule XIAP antagonists based on a polyphenylurea pharmacophore on cultured acute myelogenous leukemia (AML) cell lines and primary patient samples. X-linked inhibitor of apoptosis protein (XIAP) antagonist N-[(5R)-6-[(anilinocarbonyl)amino]-5-((anilinocarbonyl){[(2R)-1-(4-cyclohexylbutyl)pyrrolidin-2-yl]methyl}amino)hexyl]-N-methyl-N′-phenylurea (1396-12), but not a structurally related control compound, induced apoptosis of primary leukemia samples with a lethal dose (LD50) of less than 10 μM in 16 of 27 (60%) samples. In contrast, XIAP antagonist 1396-12 was not lethal to the normal hematopoietic cells in short-term cytotoxicity assays. Response of primary AML specimens to XIAP inhibitor correlated with XIAP protein levels, with higher levels of XIAP associated with sensitivity. The XIAP antagonist 1396-12 induced activation of downstream caspases 3 and 7 prior to the activation of upstream caspase 8 and caspase 9. Apoptosis induction was also independent of B-cell lymphoma protein-2 (Bcl-2) or caspase 8, indicative of a downstream effect on apoptotic pathways. Thus, polyphenylurea-based XIAP antagonsists directly induce apoptosis of leukemia cells and AML patient samples at low micromolar concentrations through a mechanism of action distinct from conventional chemotherapeutic agents.

Description: Imatinib, a Bcr-Abl tyrosine kinase inhibitor has revolutionized the treatment of patients with CML. However, resistance develops due to Bcr-Abl gene mutations and various other mechanisms. Although second generation Bcr-Abl inhibitors can overcome most of the mutation driven resistance, they cannot overcome other resistance mechanisms. Furthermore, Imatinib has limited effectiveness in patients with blast crisis (BC) CML. We have previously shown that triptolide, an anti-cancer agent isolated from a Chinese herb, potently induces apoptosis in AML cells in part by decreasing the levels of XIAP and Mcl-1, two potent antiapoptotic proteins. Here we investigated its effect on Philadelphia chromosome positive (Ph+) cells and found that at low nM concentrations, triptolide induced significant cell death in K562 (IC50=113.4±3.9 nM) and KBM5 (IC50=30.0±2.1 nM) cells, two cell lines derived from BC CML patients, as well as in ALL-1 cells (IC50=113.8±1.4 nM), a cell line derived from Ph+ ALL. Interestingly, KBM5-STI571 cells, an Imatinib resistant KBM5 subline bearing the T315I mutation which is resistant to most available Bcr-Abl tyrosine kinase inhibitors, were as sensitive as KBM5 cells to triptolide. Likewise, triptolide killed Ba/F3 cells harboring BCR-ABL mutants (E255K and T315I) with similar efficacy as Ba/F3 cells carrying the wild type BCR-ABL gene. We then treated 8 samples from 7 CML patients with blasts ranging from 10–91% with triptolide (up to 100 nM) in vitro. Triptolide induced cell death in all samples tested. Importantly, 6/7 samples were from patients resistant/relapsed after Imatinib. Three were also nonresponsive to Nilotinib and one to neither Nilotinib nor Dasatinib. Next we tried to elucidate the possible apoptosis regulators involved in triptolide-induced cell death. Triptolide decreased antiapoptotic XIAP, Mcl-1 and Bcr-Abl protein levels in K562 cells and in blast cells from CML patients. Based on this observation, we treated CML cells with both triptolide and Imatinib. The combination synergistically induced cell death in K562 cells (CI=0.50±0.14). In KBM5 cells, Imatinib antagonized rather than enhanced triptolide when administrated simultaneously: Triptolide alone induced cell death with IC50=24.3±2.8 nM at 48 hours, while in combination with 1 μM Imatinib, the IC50 increased to 82.9±4.1 nM. This is probably due to the fact that Imatinib primarily blocks KBM5 cells in G0/G1 and that resting cells were less sensitive to triptolide. We therefore pretreated KBM5 cells with triptolide for 24 hrs followed by 1 μM Imatinib for 24 hrs. This sequential treatment was more effective to induce cell death in KBM5 cells (IC50=15.4±0.6 nM). Triptolide did not sensitize Imatinib resistant KBM5-STI571 cells. Conclusion: Results suggest that triptolide potently induces cell death in BC CML cells and that the cell death induced by triptolide is independent of response to Imatinib or other second generation Bcr-Abl kinase inhibitors. Triptolide could be of potential benefit to CML patients in blast crisis and CML patients failing Bcr-Abl tyrosine kinase inhibitors.

Description: Apoptosis inducing factor (AIF) is a mitochondrial flavoprotein that is released into the cytoplasm to form an active DNase and then translocates into the nucleus to induce DNA fragmentation when cells undergo apoptosis. AIF also acts as an essential NADPH oxidase, a function which is distinct from its proapoptotic role. We have shown previously that AIF expression level increased with increasing blast counts in primary acute myelogenous leukemia (AML) patient samples. Here we report that AIF expression levels are increased in primary CD34+ AML cells compared with CD34− cells. To investigate the role of AIF in primary AML, bone marrow or peripheral blood samples from AML patients (n=8) with CD34+ or CD34− cell populations were examined for changes in reactive oxygen species (ROS), mitochondrial membrane potential (ΔψM), apoptosis and chemo-sensitivity in the CD34+ and CD34− compartments after treatment with the mitochondrial complex 1 inhibitor rotenone. Similar experiments were performed following knockdown of AIF with lentiviral shRNA directed against AIF in K562 cells. Results demonstrate: significantly increased apoptosis rate (39.2% Annexin V+ cells in CD34+ vs. 14.1% in CD34− cells following 2 uM rotenone, p=0.05), and decreased ΔψM in CD34+ cells (44.5% ΔψM cells in CD34+ vs. 78.2% in CD34− cells, p=0.01). MCL-1 expression levels were significantly decreased in CD34+ but not in CD34− cells (p=0.007). The apoptotic effect of rotenone in CD34+ cells was potentiated by AraC (1uM). Profound inhibition of O2 consumption in AIF knockdown K562 cells (−5.2±0.3) compared with K562-neo (neo transfected K562, −13.1±0.3) cells (p=0.001). Rotenone treated AIF knockdown K562 cells and K562-neo cells revealed that decreased AIF expression led to higher ΔψM and higher superoxide generation (p=0.0002). In contrast, total mitochondrial mass (NAO) decreased slightly in K562KD cells. Data suggested that AIF contributes to leukemia cell survival by modulating mitochondrial metabolism, independent of its proapoptotic activity, which is consistent with recent reports demonstrating its importance in mitochondrial complex 1 activity. Decreased AIF levels are associated with increased generation of superoxide anion, suggesting that AIF critically regulates mitochondrial function in AML cells. Rotenone selectively decreases AIF and induces apoptosis in CD34+ cells in AML. AIF functions as a survival factor in AML strategies targeting AIF expression or function may be beneficial in the treatment of myeloid leukemia.

Description: Abstract 3311 Bcl-2 family proteins are key regulators of apoptosis. Aberrations in Bcl2 levels are known to promote tumorigenesis and chemoresistance. Thus strategies to target Bcl2 will likely provide effective therapies for malignancies such as acute myeloid leukemia (AML). ABT-737 is a small molecule BH3 mimetic that binds tightly to a hydrophobic cleft on Bcl-2 and Bcl-XL and exerts its proapoptotic function by preventing antiapoptotic Bcl-2 family members from sequestering activating BH3 proteins (Oltersdorf et al., Nature 2005). We have reported that ABT-737 effectively kills acute myeloid leukemia blast, progenitor, and stem cells without affecting normal hematopoietic cells. ABT-737 induces the disruption of the BCL-2/BAX complex and BAK-dependent but BIM-independent activation of the intrinsic apoptotic pathway (Konopleva et al., Cancer Cell 2006). The ABT-737 related clinical compound, ABT-263, is undergoing Phase I/II studies in chronic lymphocytic leukemia, with initial signs of clinical activity. However, the main side effect is thrombocytopenia resulting from inhibition of Bcl-XL. Hence, combinations of ABT-737 with non-cytotoxic agents are desirable to take full advantage of ABT236's unique spectrum of biophysical and preclinical activities. In this project, we studied pharmacologic interactions between ABT-737 and the DNA methyltransferase inhibitor 5-azacytidine (5-azaC). 5-azaC is a cytidine analog with clinical activity in myelodysplastic syndromes (MDS) and in AML. Since recent studies indicate that 5-azaC induce DNA damage in p53-dependent fashion, we tested the hypothesis that the pro-apoptotic effects of 5-azaC/ABT-737 combination are related to non-redundant activation of BH3-only proteins and mitochondrial apoptosis in AML cells with wt p53. In vitro, 5-azaC and ABT-737 in combination for 72 hrs induced growth inhibition and apoptosis in AML cell lines OCI-AML3, MOLM-13 and U937 in a highly synergistic, dose-dependent fashion, with combination indices (CIs) ranging from 0.1 to 0.22. These effects were observed even at low concentrations (5-azaC 100nM and ABT-737 10nM, at 10:1 ratio). In contrast, no synergistic apoptosis was seen in p53-null HL-60 cells. Likewise, ABT-737/5-azaC induced apoptosis in a synergistic fashion in OCI-AML3 cells infected with vector control shRNA (CI=0.04) but failed to induce cell death in OCI-AML3 p53 shRNA cells, indicating critical p53-dependent mechanisms of cell death. In primary AML samples sensitive to ABT-737 alone (n=3), synergistic and additive effects were seen. The combined effects of 5-azaC and ABT-737 were further investigated in NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ mice injected with cells from a patient with primary refractory AML. Seven days after leukemia transplantation, mice were treated with vehicle, ABT-737 (intraperitoneally (IP), 75mg/kg/day for 10 days), 5-azacytidine (IP, 4mg/kg/day for 5 days) or with the combination. Engraftment of patient leukemia cells was analyzed by the immunohistochemical detection of CD45-positive cells in bone marrow and spleen seven weeks after transplantation. Both, ABT-737 and 5azaC each exerted anti-leukemic effects, as evidenced by significant reduction in leukemia cells in bone marrow and spleen (10% and 3% CD45+ cells detectable in 1/3 mice in ABT-737 and 5-azaC groups, respectively), no CD45+ AML cells were detected in organs of 3/3 mice treated with the combination. No overt hemorrhage was detected in the animals. In summary, the combination of 5-azaC and ABT-737 induces synergistic cells death in AML cell lines and in a subset of primary AML samples in a p53-dependent fashion. The mechanisms of this pharmacologic interactions including the p53-dependent upregulation of BH3-only proteins, described by us for ABT-737/MDM2 inhibitor combinations, are currently under investigation. Results suggest that this therapeutic strategy can be successfully utilized in AML patients with low mutation rate and unimpaired signaling of p53. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2355 The interaction of bone marrow (BM) mesenchymal stem cells (MSC) and acute myeloid leukemia (AML) cells creates a microenvironment (McrEnv) that supports and regulates the survival and proliferation of leukemic cells. These same BM McrEnv interactions can also create a sanctuary that protects subpopulations of AML blasts from chemotherapy. The mechanisms by which the BM-MSC McrEnv effects these changes remain unclear and the heterogeneity of these effects across different subtypes of AML (FAB, WHO or cytogenetics) is unknown. Furthermore, the functional differences between normal BM-MSC and AML BM-MSC biology are undefined. To address this we set out to perform proteomic profiling comparing normal and AML BM derived-MSC and to ascertain how AML BM-MSC protein expression patterns correlated with AML blast protein expression. We cultured BM samples for 4 to 8 weeks in MEM-alpha media with 20% fetal calf serum to isolate AML-MSC (n=106), and NL-MSC (n=70). Cells defined as MSC were positive for CD90 and CD105 and negative for CD45 by flow cytometry. Whole cell protein lysates were prepared. Matched AML protein samples prepared from mononuclear cell fractions from the same BM collection were available for most cases (n=96). We generated a custom Reverse Phase Protein Array (RPPA) from these samples and probed the array with 151 validated antibodies. Statistical analysis was performed by two-way ANOVA using Tukey's test to identify differential expression of individual proteins between the samples and Ingenuity pathway analysis was performed to elucidate differential pathway utilization. Comparison of AML-BM-MSC to NL-BM-MSC demonstrated similar levels of expression for 66 proteins (43.7%) but 85, 67 and 28 were different at the p-value of 〈 0.05, 〈 0.01 and

Description: Galectins are a family of b-galactoside binding proteins with effects on cell adhesion, apoptosis, cell cycle, and mRNA processing. Galectin-3 (LGALS3) is unique among galectins by having an N terminal region of roughly 130 amino acids that allows for multimerization and binding to other proteins independent of carbohydrate binding. In addition to promoting BCL2 gene expression and mitochondrial integrity, LGALS3 (along with LGALS1) positively regulates RAS signaling and thus stabilizes survival proteins dependent on ERK phosphorylation such as MCL-1. The pro-survival functions of LGALS3 and other galectins suggest that their targeting could be therapeutic for cancers including AML. Indeed, LGALS3 expression is a predictor of poor prognosis in acute myeloid leukemia (AML), as reported by Cheng and colleagues (Blood 2013) for patients with non-M3 AML and CN-AML. The modified pectin GCS-100 (La Jolla Pharmaceutical, San Diego, CA), now in a Phase II clinical trial for chronic kidney disease, binds and blocks the function of LGALS3. We report that GCS-100 suppresses the growth of AML cell lines OCI-AML3, THP-1, and HL60 in vitro as a single agent, at doses under the 250 ug/mL (i.e., within clinically-achievable concentrations). Short-term treatment of cells (i.e., 〈 6 hr) potently suppressed phosphorylation of AKT and ERK and reduced expression of BCL2 and MCL-1. Because LGALS3 positively regulates anti-apoptotic BCL2 family members, the Raz group has suggested targeting galectins to enhance efficacy of BH3 mimetic drugs (Harazano et al Cancer Metastasis Review 2013). We found that GCS-100 potently synergized with ABT-737 to kill OCI-AML3 cells: while 1 uM ABT-737 or 125 ug/mL GCS-100 reduced total viable cells by ~ 30% and induced apoptosis in 〈 20% of cells after 48 hr as single agents, their combination at those doses and time point reduced viable cells by ~ 94% and induced apoptosis in ~ 70% of cells. Suppression of LGALS3 by lentiviral shRNA reduced BCL2 gene expression as determined by qRT-PCR and augmented killing with ABT-737. Lentiviral suppression of LGALS3 protected cells from GCS-100 at doses of 250 ug/mL but reduction of the galectin failed to protect cells from higher doses of the drug (i.e., 500 ug/mL). This result suggests other galectins are likely inhibited at higher doses of the agent. We used gene expression profiling (GEP) on Illumina HT12v4 human whole-genome arrays to assess more broadly the molecular effects of inhibiting galectins in AML cell lines OCI-AML3 and THP-1 treated with 250 ug/mL or 500 ug/ml GCS-100 for 24 hr. Data were analyzed by Gene Set Enrichment Analysis (GSEA) using gene sets from the Molecular Signatures Database (www.broadinstitute.org/gsea/msigdb/). GSEA suggested that GCS-100 promotes differentiation and inhibits genes associated with proliferation. Multiple upregulated gene sets suggest that there may be a release of a differentiation block as a result of GCS-100 treatment. Furthermore, two gene sets suggest that GCS-100 behaves similar to a GSK3 inhibitor: Known pathways regulated by GSK3 in hematopoietic stem cells are mTOR and Wnt/beta Catenin. Inhibition of Wnt/beta Catenin can release a differentiation block. Consistent with GCS-100 promoting cell differentiation, lentiviral shRNA reduced LGALS3 protein 〉 90% in THP-1 cells and increased CD11b expression, suggesting increased differentiation, compared to cells with control shRNA. GCS-100 was tested in an in vitro model of the bone marrow microenvironment using BM-derived mesenchymal stromal cell (MSC). MSC can protect leukemia cells from a variety of clinically relevant chemotherapy drugs including AraC. GCS-100 was effective at killing AML cells despite the presence of MSC. Both THP-1 and OCI-AML3 cells exhibited 〉 80% and 〉 60% reduction of viable cells, respectively, despite the presence of MSC when treated with 250 ug/mL GCS-100 for 72 hours. In addition, GCS-100 was found to block adhesion of OCI-AML3 cells to MSC suggesting that GCS-100 could be effective in mobilizing AML cells. In summary, our findings suggest that GCS-100 can induce apoptosis in AML cells as a single agent or in combination with the BH3 mimetic ABT-737. The agent is effective even in the presence of MSC suggesting it could be efficacious in the leukemia niche. These findings suggest GCS-100 could be effective for AML therapy. Disclosures Rolke: La Jolla Pharmaceutical Company: Employment. Tidmarsh:La Jolla Pharmaceutical Company: Employment.

Description: Background: Except for the ‘good risk’ groups identified by current cytogenetic and molecular criteria, patients with acute myelogenous leukemia (AML) largely relapse after attaining initial remissions, and outcomes of patients with early relapse is uniformly poor, highlighting the need for strategies to overcome resistance to agents used in frontline therapy. Autophagy is an ancestral adaptive mechanism to stress and can be triggered by exposure to chemotherapy. Autophagy is also believed to play an important role in tumor protection by the microenvironment. Autophagy inhibition increases chemosensitivity in several experimental models of solid tumors. The role of autophagy is not well studied in AML, but the hypoxic bone marrow niche is expected to induce autophagy in AML cells and render them chemo-resistant. Our reverse phase protein array (RPPA) analysis of over 500 newly diagnosed AML patient samples indicated that abnormal expression of several autophagy proteins in leukemic blasts (LKB1, BECLIN 1 and ATG7) is associated with poor prognosis in AML (Borthakur, G et al. ASH 2011# 2513), suggesting a therapeutic role for autophagy suppression. Atg7 is a key molecule in autophagy vesicle elongation with a role in two essential ubiquitin-like reactions (LC3 lipidation and Atg 5/12 conjugation), and its knockdown is expected to inhibit autophagy globally. Aim: To determine the effects of autophagy inhibition on chemosensitivity of AML cells and on stroma induced resistance. Results: We semi-quantitatively assessed ATG7 expression in AML cell lines (THP1, OCI-AML2 and 3, HL-60, MOLT-4, MOLM 13 and 16) by Western blot. HL-60 and MOLM13 cells had the lowest expression of Atg7 and were most sensitive to treatment with ara-C and idarubicin while OCI-AML3 and THP-1 had highest levels of Atg7 and were most resistant to treatment (p= .001-.004). In addition, high expression of Atg7 is associated with high levels of anti-apoptotic Mcl-1 in these lines. As available autophagy inhibitors are non-specific, we used lentiviral shRNA to knock down Atg7 and study the role of autophagy. Atg7 knockdown cells (ATG7-KD) were treated with ara-C and idarubicin for 48-96 hours. At all time points, apoptosis was significantly higher in ATG7-KD cells compared to cells transduced with a non-silencing scrambled control (ATG7-Scr) at 96 hrs: 39.6±2.4 vs 22.4±1.3, p=.002 for ara-C 2 µM and 47.9±2.6 vs 34.7±2.3, p=.0004 for idarubicin. We co-cultured OCI-AML3 (ATG7-KD and -Scr) cells with normal bone marrow derived mesenchymal stromal cells (MSCs) to mimic the bone marrow micro-environment. Sensitivity to ara-C and idarubicin was reduced by co-culture for both cell types, but ATG7-KD cells remained more sensitive than were ATG7-Scr cells. Western blot analysis confirmed increased p62 and decreased LC3 lipidation in ATG7-KD cells (LC3 II/I ratio= 0.15 vs 0.7 in ATG7-Scr), indicating a block in autophagy, and increased caspase 3 cleavage, indicating apoptosis. The higher expression of pro-apoptotic NOXA and BAX, and lower expression of anti-apoptotic MCL-1 and BCL-2, in ATG7-KD cells compare to ATG7-Scr indicating mitochondrial pathway apoptosis. Gene expression profiling on Illumina HT12 arrays was used to study OCI-AML3 ATG7-KD and -Scr cells at baseline and after 24 and 48 hr of araC 0.5 µM. Gene set enrichment analysis (GSEA) showed significantly lower baseline expression of interferon response genes and JAK-STAT pathway genes in OCI-AML3 ATG7-KD cells. Changes with araC treatment were largely similar, but the response to araC was different between ATG7-KD and –Scr cells for a small number of genes. In-vivo chemo-sensitivity experiments are in progress. Conclusion: Autophagy inhibition by genetic silencing of ATG7 increases chemosensitivity of AML cells, even in the presence of bone marrow stromal cells. ATG7-KD cells appear to be more primed for apoptosis compared to their controls. Concomitant inhibition of ATG7, a potentially drugable E1 ligase, appears to be a valid strategy to enhance sensitivity to front-line treatment agents in AML. Disclosures No relevant conflicts of interest to declare.