ALBERT

Description: Despite advances in understanding of the biology of acute myeloid leukemia (AML), cure remains elusive for the majority of patients. Pro-survival molecules of BCL-2 family play critical roles in leukemia transformation and chemoresistance. The anti-leukemia potency of selective BCL-2 inhibitor venetoclax (ABT-199/GDC-0199) has been demonstrated in AML models (Pan et al. Cancer Discovery 2014). However, venetoclax is often associated with resistance due to its poor inhibition of MCL-1. RAF/MEK/ERK (MAPK) pathway is commonly activated in AML, and can stabilize anti-apoptotic MCL-1 and inactivate the pro-apoptotic BIM. In this study, we evaluated the anti-leukemia effects of concomitant BCL-2 and MAPK blockade by venetoclax in combination with MEK1/2 inhibitor GDC-0973 (cobimetinib). First, anti-leukemia activity of cobimetinib and venetoclax was examined in 18 primary AML samples with diverse genetic alterations. The combination significantly enhanced cell death, as compared to the single agent treatment (Fig 1A). Cobimetinib inhibited cell proliferation in the majority of AML cases (34.2 ± 23.7%) and the cell growth suppression was more profound in the combination group (60.2 ± 28.8%, p

Description: Acute myeloid leukemia (AML) cells highly depend on oxidative phosphorylation (OxPhos) to satisfy their heightened demands for energy, and the complex I OxPhos inhibitor IACS-010759 (Molina, Nat. Med. 2018) is currently in Phase 1 clinical trial in AML. In this study, we investigated how the bone marrow (BM) microenvironment affects the response to OxPhos inhibition in AML. To characterize the molecular mechanisms of sensitivity to OxPhos inhibition, we performed Cap Analysis of Gene Expression analysis (CAGE) on 31 genetically diverse primary AML samples (20 were defined as sensitive and 11 as resistant to IACS-010759; cut off 〉3.0 fold annexin V(+) by 100 nM IACS-010759/DMSO at 72 hours). CAGE identified higher expression of transcription start sites (TSS) for 17 genes in IACS-010759 resistant AML samples compared to sensitive (fold change 〉2.0, FDR 〈 0.05, EdgeR), which were related to cell adhesion, integrin and/or Rho GTPase family genes that modulate intracellular actin dynamics. We next investigated the interactions between IACS-010759 sensitive OCI-AML3 cells and BM-derived mesenchymal stem cells (MSC). Under conditions mimicking the BM microenvironment, IACS-010759 upregulated the pathways of focal adhesion and ECM-receptor interaction in OCI-AML3 cells (KEGG analysis based on CAGE). In turn, MSC co-culture increased oxygen consumption by AML, induced generation of mitochondrial ROS (control 4.4% vs IACS 44.4%), increased mtDNA (2-fold by q-PCR) and upregulation of mitochondrial proteins VDAC and cytochrome C, translating into dampened growth-inhibitory effects of IACS-010759. We further demonstrated that OCI-AML3 cells adhering to MSCs were fully protected from IACS-010759 induced apoptosis (IACS-induced specific apoptosis: non-adherent cells 16.2% ± 1.6% vs adherent cells 1.6% ± 0.7%, p=0.008, 30nM, 72hours). Similarly, adherent cells were fully protected from apoptosis induced by combination of IACS and AraC. These findings indicate that direct interactions with MSC trigger compensatory activation of mitochondrial respiration, increase in mitochondrial mass and resistance to OxPhos inhibition in AML. We next hypothesized that the trafficking of mitochondria from BM stroma cells to AML cells could represent a putative mechanism of an acquired resistance to OxPhos inhibition. To visualize mitochondria, OCI-AML3 and MSC were stably transfected with mitochondria-targeted PDHA1-GFP and -dsRed, respectively. We discovered that IACS-010759 induced transfer of MSC-derived mitochondria to OCI-AML3 cells (% of GFP/dsRed double-positive OCI-AML, control 4.1 ± 1.7 vs IACS 26.2 ± 13.4, p=0.002) via tunneling nanotubes (TNTs) detected by confocal and electron microscopy (Fig.1). Mitochondria transfer was only observed in the direct contact but not in the transwell co-cultures, and was abrogated by ICAM-1 neutralizing antibody and TNT blockade with Cytochalasin B. Likewise, combination of IACS with AraC increased mitochondrial transfer. We further found that IACS-010759 induced autophagy in OCI-AML3 cells co-cultured with MSC, as noted by increased conversion of LC3-I to LC3-II, which was further enhanced by the lysosome inhibitor Bafilomycin. Additionally, we observed autophagosome formation enwrapping MSC-derived mitochondria (Fig.1F), along with the degradation of an outer mitochondrial membrane protein Tom20. Finally, IACS-010759-induced transfer of mtDNA in BM-resident AML cells was confirmed in vivo in humanized AML PDX models (n=2). Daily oral treatment of mice harboring human AML with IACS-010759 (5.0 mg/kg/day, 21 days) increased the ratio of murine/human mtDNA in human AML cells isolated from BM, in 5 days on/2 days off PDX models tested (2.1 ± 0.3 fold, n=2). In conclusion, the findings of this study indicate an important role of mitochondria trafficking from BM stromal cells to AML cells in a compensatory adaptation to OxPhos inhibition in BM microenvironment. We propose that blocking of mitochondrial transfer could enhance the anti-AML efficacy of OxPhos targeting agents. Disclosures Zhang: The University of Texas M.D.Anderson Cancer Center: Employment. Kuruvilla:The University of Texas M.D.Anderson Cancer Center: Employment. Andreeff:BiolineRx: Membership on an entity's Board of Directors or advisory committees; Breast Cancer Research Foundation: Research Funding; Oncolyze: Equity Ownership; Oncoceutics: Equity Ownership; Senti Bio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Eutropics: Equity Ownership; Reata: Equity Ownership; Aptose: Equity Ownership; 6 Dimensions Capital: Consultancy; Daiichi Sankyo, Inc.: Consultancy, Patents & Royalties: Patents licensed, royalty bearing, Research Funding; Jazz Pharmaceuticals: Consultancy; Celgene: Consultancy; Amgen: Consultancy; AstaZeneca: Consultancy; CPRIT: Research Funding; NIH/NCI: Research Funding; Center for Drug Research & Development: Membership on an entity's Board of Directors or advisory committees; Cancer UK: Membership on an entity's Board of Directors or advisory committees; NCI-CTEP: Membership on an entity's Board of Directors or advisory committees; German Research Council: Membership on an entity's Board of Directors or advisory committees; Leukemia Lymphoma Society: Membership on an entity's Board of Directors or advisory committees; NCI-RDCRN (Rare Disease Cliln Network): Membership on an entity's Board of Directors or advisory committees; CLL Foundation: Membership on an entity's Board of Directors or advisory committees. Konopleva:Astra Zeneca: Research Funding; Agios: Research Funding; Eli Lilly: Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Cellectis: Research Funding; Amgen: Consultancy, Honoraria; F. Hoffman La-Roche: Consultancy, Honoraria, Research Funding; Genentech: Honoraria, Research Funding; Ascentage: Research Funding; Kisoji: Consultancy, Honoraria; Reata Pharmaceuticals: Equity Ownership, Patents & Royalties; Ablynx: Research Funding; Forty-Seven: Consultancy, Honoraria; Calithera: Research Funding; Stemline Therapeutics: Consultancy, Honoraria, Research Funding.

Description: Annamycin (Ann) is an anti-tumoral anthracycline whose anti-leukemia activity is relatively unaffected by P-glycoprotein-related multidrug resistance. Unlike for the related doxorubicin (DOX), Ann accumulates in multidrug resistant cell lines, which is accompanied by DNA damage and apoptosis. In preclinical toxicology studies, in contrast to DOX, free Ann displayed a greatly reduced cardiotoxicity, while L-Ann appeared to be non-cardiotoxic. A liposomal formulation of Ann, termed L-Annamycin (L-Ann), is currently evaluated in patients with acute myeloid leukemia (AML). Anti-leukemia activity of Ann was demonstrated in several leukemia models as judged by circulating blast cytoreduction and extension of overall survival. However, the efficacy of L-Ann in the microenvironment of the bone marrow and other organ tissues remains unclear. In the current study, we assessed the anti-AML efficacy of Ann in a novel AML model that allows visualizing the dynamics of individual AML cells in vivo by two-photon microscopy. In this model, mouse AML cells bearing the MLL/ENL-FLT3/ITD[p53-/-] mutations co-express high levels of the cyan fluorescent protein mTurquoise2. Upon intravenous infusion of several tens of thousands cells into syngeneic immunocompetent C57BL6 mice, lethal AML disease reliably develops within 2 weeks. Using host mice expressing appropriate fluorescence reporter genes, the bright cyan fluorescence enables sensitive intravital imaging of individual AML cells in the context of organ architecture. Using this model in Thy1-RFP reporter mice expressing red fluorescence in all organ tissues with the blood flow marked by BSA-AF647 fluorescence, we evaluated AML cellularity reduction in the bone marrow and other organs after a single dose of L-Ann as well as in response to chronic treatment. In addition, we assessed the localization of the surviving AML cells at a high spatial resolution. We evaluated the in vivo organ biodistribution of intravenously infused L-Ann in C57BL6 mice by flow cytometry and two-photon microscopy based on the intrinsic fluorescence of the drug. In addition, we visualized the intracellular compartmentalization of L-Ann using confocal microscopy. Consistent with in vitro findings, we observed a rapid and deep reduction of AML blasts in the peripheral blood after a single dose of L-Ann in a dose-dependent manner (1-4 mg/kg). This reduction was strongly correlated with prolongation of animal survival from 14 days (vehicle) to 37 days (L-Ann 4 mg/kg once weekly started on day 10). In vitro and intravital microscopy revealed a distinct pattern of L-Ann distribution in organ tissues, which correlated in part with the local index of AML cellularity reduction and residual disease localization. Interestingly, in addition to the expected uptake of Ann in the cell's nucleus, Ann was also accumulated in the cytosol of the cells. This bi-compartmental intracellular distribution pattern contrasted with the nuclear-only localization of DOX. Administration of L-Ann early in the course of AML resulted in occasional complete responses some of which associated with resistance to AML re-challenge, suggesting capacity for anti-AML immune memory induction. This study confirms the efficacy of the drug in the model setting of syngeneic, immune-competent AML. Besides reinforcing the rationale for further development of Annamycin in AML, this study demonstrates a highly advantageous AML mouse model that is highly informative in studies of AML pharmacology, minimum residual disease (MRD), microenvironment and immunology. Disclosures Fokt: Moleculin Biotech, Inc.: Equity Ownership, Research Funding. Andreeff:Oncoceutics: Equity Ownership; Senti Bio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo, Inc.: Consultancy, Patents & Royalties: Patents licensed, royalty bearing, Research Funding; Jazz Pharmaceuticals: Consultancy; Celgene: Consultancy; Amgen: Consultancy; AstaZeneca: Consultancy; 6 Dimensions Capital: Consultancy; Reata: Equity Ownership; Aptose: Equity Ownership; Eutropics: Equity Ownership; Leukemia Lymphoma Society: Membership on an entity's Board of Directors or advisory committees; NCI-RDCRN (Rare Disease Cliln Network): Membership on an entity's Board of Directors or advisory committees; CLL Foundation: Membership on an entity's Board of Directors or advisory committees; BiolineRx: Membership on an entity's Board of Directors or advisory committees; German Research Council: Membership on an entity's Board of Directors or advisory committees; NCI-CTEP: Membership on an entity's Board of Directors or advisory committees; Cancer UK: Membership on an entity's Board of Directors or advisory committees; Oncolyze: Equity Ownership; Breast Cancer Research Foundation: Research Funding; CPRIT: Research Funding; NIH/NCI: Research Funding; Center for Drug Research & Development: Membership on an entity's Board of Directors or advisory committees. Priebe:Moleculin Biotech, Inc.: Consultancy, Equity Ownership, Research Funding. Zal:VueBio.com: Equity Ownership; BioLineRx: Research Funding; Daiichi-Sankyo: Research Funding; Moleculin Biotech, Inc.: Research Funding; NIH-CTEP: Research Funding; CPRIT: Research Funding; NIH/NCI: Research Funding.

Description: Abstract 3505 Activating mutations of fms-like tyrosine kinase 3 (FLT3), including Internal tandem duplications (ITD) mutations and point mutations of tyrosine kinase domain (TKD), can be detected in 1/3 of acute myeloid leukemia (AML) patients, and are associated with higher risk of relapse. (1) We reported that sorafenib, by directly targeting mutant FLT3-ITD, exerted anti-leukemia effects in AML with FLT3-ITD mutations in Phase I/II clinical trial and increased the complete remission rate to 100% (CR and CRp) in combination with Idarubicin/cytarabine.(2, 3) However, resistance to sorafenib develops in most AML patients with ITD mutations during prolonged therapy and leads to relapse.(3) Therefore, understanding the mechanisms of sorafenib resistance remains a challenge that may aid approaches to overcoming drug resistance in this dismal prognostic group of AML patients. The present study found that acquired point mutations in the TKD1 and TKD2 domains of the FLT3 gene play a crucial role by elevating levels of phosphorylated FLT3 and its downstream signaling proteins. cDNA-based mutation analysis of the FLT3 gene was performed in blood/bone marrow samples from AML patients who showed sensitivity to sorafenib during the first cycles of therapy in Phase I/II trials and then became resistant. Results showed that 50% (3 of 6 cases) developed single or multiple acquired point mutations of the FLT3 gene, which included mutations of D651G(H), G619C and I687F in TKD1 and/or E858K in TKD2. Further, a murine sorafenib-resistant AML cell line (Ba/F3-ITD-Res) was developed by long-term/low-dose exposure to sorafenib in vitro. Sequence analysis identified acquired point mutations of N676D and Y842C. To further investigate the correlation of these point mutations with drug resistance, selected single point mutations were individually introduced into Ba/F3-ITD cells by lentiviral infection. Apoptosis induction was measured after exposure to sorafenib. Results showed varying degrees of resistance in sublines with different point mutations and their EC50s for apoptosis induction were 0.69, 0.61 and 19 μM in D651G, N676D and Y842C mutation-containing cells, respectively. By comparison, EC50 for Ba/F3-ITD was 0.16 μM. However, cells with multiple concomitant point mutations (such as N676D plus Y842C) displayed impressive resistance (EC50 = 32μM). These results suggest that single mutations in either TKD1 or TKD2 are associated with resistance of sorafenib-induced apoptosis, and that structural alterations of TKD2 are more critical than those of TKD1. Further, concomitant mutations in both TKDs strongly impair response to sorafenib, suggesting a pivotal role for the structural integrity of both TKDs in maintaining sensitivity of FLT3-ITD AML cells to sorafenib. To better understand mechanisms of resistance, relevant biomarkers were investigated by Western blot in these cells with point mutations. The results showed that TKD1 mutations (D651G and/or N676D) increased basal levels of phospho–FLT 3 and its downstream targets phospho–ERK, –Stat5, –AKT and –S6K, suggesting that mutations of TKD1 result in increased FLT3 activation. However, one TKD2 mutation (Y842C) which does not result in increased basal levels of phospho-FLT3, was nevertheless resistant to sorafenib-induced suppression of FLT3 targets, implying that Y842C may interfere with the binding of sorafenib to FLT3 by a conformational shift of the adenosine triphosphate (ATP)–binding pocket, which reduces the accessibility for sorafenib and mediates resistance. Notably, cells with concomitant N676D and Y842C mutations in both TKDs displayed higher basal phosphorylation levels of FLT3 and its downstream targets, resulting in increased resistance to sorafenib-induced suppression of phosphorylation, suggesting that synergistic resistance to sorafenib may result from multiple mutations in by both, TKD1 and TKD2. Our findings provide an improved understanding of acquired FLT3-ITD/TDK point mutations associated with sorafenib resistance in FLT3-ITD-mutant AML, and might be useful in developing agents that have the potential of overcoming resistance of FLT3-ITD inhibitor in AML. Disclosures: Ravandi: Bayer: Research Funding; Onyx: Research Funding.

Description: The bone marrow microenvironment (BME) critically supports hematopoietic stem cells and protects leukemia cells from chemotherapy, immune surveillance, and related stresses. A critical component of the BME is the mesenchymal stem cell (MSC). Dan Link’s group demonstrated that MSC are essential for human hematopoiesis, particularly as a source of SDF-1, which regulates homing, proliferation, and differentiation of HSC. Moreover, studies from our group and others have demonstrated that MSC protect leukemia cells from chemotherapy. At present, very little is known about MSC derived from AML patients, and an understanding of the proteomic makeup of these cells in the leukemia microenvironment could help to elucidate mechanisms involved in supporting their pro-tumor function. We used reverse phase protein array analysis (RPPA) to compare the expression of 151 proteins in MSC derived from AML BMs (N = 106) with those from healthy donors (N = 71). The expression of 45 of these proteins was deemed significantly different (p 〈 0.01) between the two sets. AML MSC expressed higher levels of p53 and p21 (CDKN1A), and the expression of the latter was correlated with other proteins within each MSC set. Using beta-galactosidase staining, AML MSC were found to undergo senescence more frequently than normal MSC. Elevated p21 in AML MSC is consistent with this finding. While 15 proteins were positively, and 20 proteins negatively, correlated with p21 expression in normal MSC, there were only three proteins positively, and nine negatively, correlated in AML-derived MSC. In normal MSC, SMAD1 (a key component in MSC growth and differentiation involving multiple receptors like TGF beta and BMP) expression and AKT signaling were low when p21 is expressed. However, in AML MSC this association was not seen, albeit a negative correlation with ITGAL was observed. SMAD1 expression was higher in normal MSC. In normal MSC, the expression of SMAD1 was negatively correlated with PPARG and NPM1, and was positively correlated with the expression of phosphorylated ELK. The opposite relationship was seen in AML MSC (i.e., PPARG and NPM1 exhibited positive correlation with SMAD1 and phosphorylated ELK was negatively correlated with the protein). While the significance of these relationships remains to be determined it is interesting to note that PPARG is a key regulator of adipocyte differentiation in MSC, so perhaps this alteration of SMAD/PPARG in AML MSC could impede their differentiation potential. In an accompanying abstract from our group, we report that AML MSC are primed toward osteoblastic differentiation and do not differentiate into adipocytes (Battula VL et al, ASH 2014). The RPPA data on PPARG is consistent with this finding. SMAD1 also positively regulates miR-21. Since p21 is a miR-21 target, it seems possible that the differences in expression could be attributed to SMAD1 and miR-21 signaling. We analyzed miR-21 expression in normal and AML-derived MSC (N = 10, each) using qRT-PCR and found a statistically significant (p =0.014) increase in its expression in normal MSC relative to their disease counterparts. When anti-miR-21 was transduced into healthy donor MSC, which caused a 3-fold increase in p21 (but no difference in cyclin D1 expression, another miR-21 target whose expression was also increased in AML MSC). AML MSC also exhibited higher protein expression of the B55 alpha subunit (PPP2R2A) of protein phosphatase 2A (PP2A). This expression contrasted interestingly with that of leukemia cells, since we have previously reported low PPP2R2A levels in AML blasts associated with shorter remission durations (Ruvolo et al Leukemia 2011). Furthermore, AKT phosphorylation was negatively correlated with PPP2R2A expression in AML blasts, and normal MSC, but there was no correlation between PPP2R2A and phosphorylated AKT in AML MSC. Also, expression of PPP2R2A was positively correlated with the expression of the survival protein NOL3 (ARC) which may provide new clues to possible survival mechanisms in AML MSC. In summary, these findings represent insights into the proteomic profiling of normal and AML MSC. Results suggest that senescence (via p21), differentiation potential (involving SMAD/PPARG pathway), and survival signaling (including PP2A/AKT) are altered in AML MSC. Studies are underway to determine how these variations in MSC properties impact the AML microenvironment. Disclosures Carter: Tetralogic Pharmaceuticals: Research Funding.

Description: Background: A role for the Chemokine (C-C motif) ligand 2 (CCL2) in attracting tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSC) and infiltrating monocytes has been described for many solid tumors in which they play an essential role in modifying the adaptive immune response, ultimately favoring tumor progression. Unfortunately, little is known about the importance of this mechanism for the progression of AML. We recently identified CCL2 as the most prominent chemokine produced by bone marrow (BM) mesenchymal stromal cells (BM-MSC) in response to the interaction with myeloid leukemia cells (PMID: 24599548). In addition, elevated CCL2 plasma levels have been reported in patients of AML (PMID: 17822317), ALL (PMID: 21298741) and CLL (PMID: 22397722) when compared to normal controls. In this study we assessed the effects of blocking the CCR2-CCL2 axis on the migration and signaling of hematopoietic cells as well as on the infiltration of immune-suppressive cells in leukemia-bearing mice. Results: We first studied the efficacy and potency of agents at inhibiting CCL2-mediated migration, using the human monocytic leukemia cell line THP-1. Migration towards human recombinant CCL2 (5 ng/ml) was significantly inhibited by as little as 1 nM of NOX-E36, a human-specific CCL2 Spiegelmer (NOXXON Pharma, Berlin). Spiegelmers are RNA-like molecules built from L-ribose units that are able to bind molecules such as peptides and proteins with an affinity in the pico-to nanomolar range. Similar results were obtained with a CCR2 antagonist (100 ng/ml; Santa Cruz). In anticipation of in vivo studies in mice, we next confirmed the ability of a mouse-specific CCL2 Spiegelmer (mNOX-E36) to inhibit migration and signaling pathway activation in murine hematopoietic cells. For this purpose, we cloned and overexpressed via lentiviral transduction the murine CCL2 receptor (CCR2) in Ba/F3 cells (a murine pro-B cell line). Stimulation of Ba/F3-CCR2 cells with 5 ng/ml of mouse recombinant CCL2 induced a ~2000 fold increase in migration of Ba/F3-CCR2 cells and was successfully blocked with mNOX-E36 in a concentration-dependent manner. Western blot analysis of protein lysates from mCCL2-stimulated cells (30 minutes treatment) indicated activation of AKT, ERK and p38-MAPK. The CCL2-induced phosphorylation of these molecules was completely abrogated by pre-treatment with mNOX-E36. Subsequently, we determined whether the expression of CCL2 by stromal cells in leukemia-resident organs triggers the infiltration of TAMs and possibly other immune-suppressive cells into those organs. We conducted preliminary in vivo studies in non-irradiated immunocompetent C57BL/6 mice (n=5 per group) injected with syngeneic AML1/ETO9a-expressing primary murine leukemia cells (PMID: 19339691). After confirmation of leukemia engraftment by IVIS imaging, mice were treated with mNOX-E36 (14.4 mg/kg, s.c., three times per week) or vehicle control for 3 weeks. At this point, all animals were sacrificed and their tissues (spleens and BM from femurs) were collected for analysis. Although we did not observe differences in leukemia burden by imaging between vehicle and mNOX-E36 treated groups, flow cytometry analysis revealed an increase in the frequency of CD11b+ Ly6Clow MHC IIlow macrophages (2 to 7 fold increase) in spleens of mice engrafted with leukemia (vehicle-treated group) when compared to spleens collected from healthy mice. These MHC IIlow macrophages were previously identified as immunosuppressive M2-like macrophages as opposed to MHC IIhi macrophages which show a pro-inflammatory M1-like phenotype (PMID: 20570887). Importantly, CCL2 inhibitor mNOX-E36 abrogated this macrophage infiltration within the leukemia microenvironment. Conclusions: Our results indicate that blockade of the CCR2-CCL2 axis not only affects migration and signaling of treated cells in vitro, but also interferes with the infiltration of M2-like macrophages into spleens of leukemia-bearing mice. Current in vivo experiments using a combination of standard chemotherapy with mNOX-E36 in AML immunocompetent models are undergoing. We expect that in vivo modulation of CCL2 will improve response to chemotherapy of AML by reducing the marrow infiltration of infiltrating monocytes and tumor-associated macrophages, which would facilitate translation of this novel concept into clinical trials in AML. Disclosures Zuber: Boehringer Ingelheim: Research Funding; Mirimus Inc.: Consultancy, Other: Stock holder. Eulberg:NOXXON Pharma AG: Employment. Kruschinski:NOXXON Pharma AG: Employment.

Description: The expression of Bcl-2 family proteins is perturbed in multiple types of cancers, including leukemias, and is associated with disease progression and resistance to chemotherapy. ABT-199 (GDC-0199) is a new BH3 mimetic that was developed to specifically target Bcl-2 while sparing Bcl-XL, hence avoiding thrombocytopenia intrinsic to 1st generation BH3 mimetics like ABT-737 (Souers et al., Nat Med, 2013). In this study, we report proteomic profiling of Bcl-2 family members in a large series of ALL patients (pts) and pre-clinical activity of ABT-199. Expression of 20 pro- and anti-apoptotic proteins was studied in 186 newly diagnosed ALL using reverse phase protein arrays (RPPA). Supervised clustering demonstrated distinct differences in 11 proteins in ALL with different cytogenetic and FAB characteristics (Fig. 1, p4µM). Next, the cytotoxic activity of ABT-199 was tested against a panel of 12 genetically diverse primary ALL samples, including 6 from pts with relapsed or refractory disease. Ten out of twelve samples (83%) were exquisitely sensitive to both agents, with IC50 values of 0.0001-0.14µM for ABT-199 and 0.0004-0.3µM for ABT-737. One of the four Ph+ samples was resistant to both agents, and one of the two T-ALL was less sensitive to ABT-199 compared to ABT-737. Two samples with t(4;11) were highly sensitive to ABT-199. All primary ALL samples tested (n=7) expressed high levels of Bcl-2, and no significant correlation between sensitivity and expression of Bcl-2 family members was found. Importantly, three human-derived xenografts from pediatric pre-B-ALL samples (1345, 1809, 0398) were very sensitive to ABT-199 (IC50 3nM, 0.1nM and 2.3nM, respectively). Finally, anti-leukemia activity of ABT-199 was tested in MLL-rearranged patient-derived xenograft NSG mice. Treatment with ABT-199 at 100mg/kg/d by oral gavage days 13-23 significantly reduced leukemia tumor burden as determined by bioluminescence imaging (average 70% reduction in BLI signal in 4 ABT-treated mice compared to 4 control animals at 9 weeks, p=0.03). In summary, proteomic profiling and patterns of sensitivity to Bcl-2 inhibition indicate that ALL, with exception of Burkitt's lymphoma, represents a Bcl-2 dependent disease. These results provide strong rationale for introducing ABT-199, which recently showed impressive efficacy in CLL trials, into the clinical armamentarium of ALL therapy. Disclosures: Konopleva: AbbVie, Inc: Research Funding. Leverson:AbbVie, Inc.: Employment, Equity Ownership.

Description: Heat shock factor 1 (HSF1) is best known as a key sensor of proteotoxic stress, but accumulating evidence also supports a major role for this transcriptional regulator in cancer biology. In a variety of human solid tumor cells, downregulation of HSF1 inhibits growth, induces cell death and limits metastatic potential. In breast cancers, nuclear accumulation of HSF1 and a tumor-specific gene expression signature reflecting HSF1 activation were found to be strongly associated with poor outcome (Mendillo et al, Cell 2012). In addition, we have recently reported, as a counter-intuitive reversal of the central dogma, that inhibition of protein translation represses the constitutive activation of HSF1 in cancers, and that HSF1 inhibition induced by the potent eIF4a inhibitor rohinitib (RHT) exerts profound, far-ranging anti-tumor effects (Santagata et al, Science 2013). Review of public databases supports targeting of HSF1 and eIF4a in AML: mRNA levels of HSPA8, one of the primary HSF1 targets, are correlated with poor prognosis in AML (Prognoscan, data from Metzeler et al, Blood 2008) and eIF4a mRNA levels were highest in AML among 12 cancer types (Oncomine, data from Ramaswamy et al, PNAS 2001). Here, we demonstrate that inactivation of HSF1 in acute myeloid leukemias (AMLs) by RHT exerts pronounced apoptogeniceffects with preferential activity against FLT3-ITD mutant cells in cell culture and in mice. First, we confirmed our previous finding of inactivation of HSF1 by RHT in AML. In OCI-AML3, MOLM-13 and MV4;11 cells, mRNA levels of HSPA8 were reduced by 70% after RHT treatment compared to untreated controls. OCI-AML3 cells were then infected with lentivirus encoding a reporter GFP-luciferase fusion protein the expression of which is driven by promoter elements from either the HSPA1A or HSPA6 genes; an approximately 50% reduction of reporter induction by heat shock was observed after RHT treatment compared to untreated controls. Next, treatment of 7 human AML cell lines in culture showed that RHT induces marked anti-leukemia effects at low nanomolar concentrations (LD50s; 9.5 to 99.5 nM, IC50s; 4.7 to 8.8 nM, based on AnnexinV/PI-positivity as determined by flow cytometry at 72hr). The most pronounced cytotoxic effects were observed in FLT3-ITD+ cell lines (LD50s 〈 10 nM in MOLM13 and MV4;11 cells). Using two sets of isogenic cell lines (Ba/F3 and OCI-AML3 cells with FLT3-ITD or wild-type (wt) FLT3), we confirmed that RHT more potently kills FLT3-ITD cells (LD50s; 65.3 vs 20.1 nM in Ba/F3 cells). Furthermore, the combination of FLT3 inhibitor sorafenibwith RHT showed synergistic effects in cell culture (Combination Index: ED50 0.85, ED75 0.86, ED90 0.89). Immunoblot analysis showed higher phospho-HSF1 (Serine 326) in FLT3-ITD Ba/F3 cells than FLT3-wt cells, suggesting greater dependence of FLT3-ITD cells on HSF1 activation for survival. We also tested primary samples from 17 AML patients and bone marrow (BM) samples from 8 healthy donors. RHT potently induced apoptosis in AML cells, while relatively sparing normal BM cells (Figure 1A). Importantly, a similarly significant difference in sensitivity was also observed between AML and normal stem cells (CD45+CD34+CD38-). Moreover, the activity of RHT against the leukemic population was significantly higher in FLT3-ITD than in FLT3-wt cells (Figure 1B). We also evaluated the activity of RHT in a FLT3 mutant AML xenograft model using GFP-luciferase labeled MOLM-13 cells. Significantly decreased luciferase activity was detected by bioluminescence imaging and a dose-dependent reduction in GFP+ leukemic cells was seen in peripheral blood and BM by day 16 (Figure 2). Survival of the treatment groups was significantly prolonged (median; 18 vs 22.5 vs 24 days respectively, p 〈 0.0001). In conclusion, HSF1 function provides an attractive therapeutic target in AML. The eIF4a inhibitor RHT down-regulates HSF1 transcriptional function and exerts robust anti-leukemia activity in cell culture and in mice. Although the relative contributions of HSF1 inactivation and translation inhibition to the net anti-leukemic activity of RHT remain to be defined, promising features of this approach include its activity against AML stem cells, while sparing normal stem cells and its particularly potent cytotoxicity for poor-prognosis FLT3-ITD AMLs. Taken together, these preclinical findings strongly support further development of eIF4a inhibitors in the treatment of AML. Disclosures Ishizawa: Karyopharm: Research Funding. Konopleva:Novartis: Research Funding; AbbVie: Research Funding; Stemline: Research Funding; Calithera: Research Funding; Threshold: Research Funding.

Description: We have reported that “apoptosis repressor with caspase recruitment domain” (ARC), an antiapoptotic protein promotes leukemia-stromal interactions in part by increasing the expression of CXCR4 in AML cells and of CXCL12 (SDF-1) in mesenchymal stromal cells (MSCs) (Carter et al., ASH 2012). To further understand the mechanisms of action of ARC in mediating leukemia-stromal interactions, we examined the expression of multiple chemokines by quantitative cytokine PCR array in ARC knockdown (KD) and control MSCs. We found that among 6 C-X-C motif and 22 C-C motif chemokines, CXCL12, CCL2 and CCL4 are highly expressed and upregulated by ARC in MSCs. We then determined the protein levels of CCR2 and CCR5, the respective receptors for CCL2 and CCL4, in bone marrow samples from 8 AML patients and found that CCR2 was highly expressed in all samples and CCR5 was detectable in 75 % suggesting that the CCR2/CCL2 and CCR5/CCL4 axes may also contribute to leukemia-stromal interactions in AML. Trans-well migration assays showed that CCL2 and CCL4 induced the migration of AML cells which was antagonized by blocking antibodies or small molecule inhibitors. AML cells migrated less to ARC KD than to control MSCs. Primary leukemia cells from AML patient samples also migrated towards CCL2 and CCL4, and this migration correlated with the expression of the respective receptors in the leukemia cells. Co-culture of MSCs with leukemia cells greatly increased the levels of CXCL12, CCL2, and CCL4 in MSCs and the increase was diminished when co-cultured with ARC KD and increased when co-cultured with ARC overexpressing (OE) cells was compared to their respective controls, suggesting that chemokine expression in MSCs is in part regulated by ARC-mediated signaling from AML cells. Cytokine PCR array revealed that IL1β expression is increased in ARC OE and decreased in ARC KD cells. Conversely, the level of the IL1 receptor antagonist IL1RN is lower in ARC OE and higher in ARC KD. ARC OE cells also secreted more IL1β while ARC KD cells secreted less. Furthermore, co-cultures with MSCs increased the expression of IL1β in AML cells. IL1β greatly increased the levels of CCL2, CCL4, and CXCL12 in MSCs and the increase was largely diminished by co-treating the MSCs with IL1β antagonist IL1βRA. Similarly, treating MSCs with conditioned media from AML cells increased the expression of the chemokines in MSCs, and the increase was reduced with conditioned medium from ARC KD cells; this increase was diminished by IL1βRA. Furthermore, IL1βRA blocked the migration of these cells toward CCL2 and CCL4. Using a 3-D model in which cancellous bone was covered with MSCs, we found fewer OCI-AML3 cells attached to MSCs when ARC was knocked down in these cells further supporting the role of ARC in leukemia-stromal interactions. ARC is a member of caspase recruitment domain (CARD) containing proteins that have diverse functions such as antiapoptosis and regulation of NFκB activity. ARC participation in cell death suppression and leukemia-stromal interactions suggests that ARC may interact not only with apoptosis regulators but also with signaling proteins to regulate multiple cellular functions in AML. Conclusions Our findings suggest that multiple ARC-regulated receptor/ligand pairs play important roles in leukemia-stromal interactions, and that there is reciprocal crosstalk between malignant cells and microenvironment cells that is in part mediated through ARC-regulated inflammatory cytokine IL1β. ARC is therefore a novel target not only for direct apoptosis induction in leukemia cells but also for disruption of protective leukemia-stromal interactions to further sensitize leukemia cells to their elimination by chemotherapy. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2626 Hematopoietic cells express a wide range of adhesion molecules and bone marrow (BM) stroma cells produce their corresponding ligands. Through these ligand-receptor pairs, hematopoietic cells interact with their BM microenvironment. The same system is hijacked by AML and often adhesion molecules in leukemia cells and/or their ligands in stroma cells are upregulated, promote leukemia-stroma interactions, and protect leukemia cells from therapeutic agents. Understanding the underlying mechanisms is critical for therapeutic strategies aimed at disrupting leukemia-stroma interactions. For example, pharmacological blockage of the CXCR4-SDF1a axis has been shown to result in chemosensitization of AML cells in vitro, in vivo and in clinical trials (Zeng Z et al., Blood 2009; Uy GL et al., Blood 2012; Andreeff M et al., ASCO 2012). ARC (Apoptosis repressor with caspase recruitment domain) is an antiapoptotic protein. We recently determined ARC expression in samples from 511 newly diagnosed AML patients by reverse-phase protein array and reported that ARC is one of the strongest adverse prognostic markers in AML (Carter BZ et al., Blood 2011). In the same sample set, we also probed the expression of more than 200 additional proteins enabling us to correlate ARC expression with the expressions of other proteins. Surprisingly, ARC expression was correlated with multiple proteins involved in cell adhesion and migration. We generated stable ARC overexpressing (O/E) KG-1 cells, ARC knock down (K/D) OCI-AML3 and Molm13 cells, and ARC K/D BM derived mesenchymal stroma cells (MSCs) to investigate ARC's roles in leukemia-stroma interactions. Expression levels of FAK, integrinb3, fibronectin, and VLA4 were increased in ARC O/E and decreased in ARC K/D cells, compared to controls. CXCR4 mRNA and cell surface CXCR4 protein were higher in ARC O/E KG-1 (3.80- and 1.53-fold, P