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Adipocytes are the prevalent stromal cell type in adult bone marrows (BM). With increasing age, BM stroma-resident mesenchymal stem cells (MSCs), increase their capacity to differentiate into adipocytes, which leads to the progressive accumulation of fat in the BM space. It is conceivable that the increased BM adipocyte content promotes leukemogenesis and negatively affects responsiveness to chemotherapy. We previously reported that free fatty acids (FFAs) promote the metabolic shift from pyruvate oxidation to fatty acid oxidation (FAO), which causes uncoupling of mitochondrial oxidative phosphorylation and promotes leukemia cell survival (Samudio, J Clin Invest. 2010). We further demonstrated the prominent antiapoptotic effects of BM-derived adipocytes co-cultured with cells from acute monocytic leukemia (AMoL), a poor-prognosis subtype of AML (Tabe ASH. 2013). Proteomic analysis with isobaric tags for relative and absolute quantification (iTRAQ) showed upregulation of protein folding pathways which increases the expression of antiapoptotic chaperone proteins HSP70 and HSP90, of integrin-mediated cell adhesion and migration pathways and downregulation of oxidative phosphorylation along with repression of cytochrome c.Metacore gene ontology (GO) analysis identified NF-kB, c-Jun, SP1, AP-1, and HMG as the potent relevant transcription factors that closely interact with and activate chaperone proteins, chromatin, and gene transcription. In this study, we characterized a gene signature linking transcription with metabolic changes that contribute to AMoL cell survival under conditions mimicking aging BM with prevalent adipocytes. We confirmed the antiapoptotic role of FFAs produced by the primary BM MSC-derived adipocytes via pharmacologic inhibition of FAO by etomoxir (EX), which inhibits fatty acid entry into the mitochondria. EX (50mM) treatment reversed the prosurvival effects of adipocytes on serum-starved U937 monoblast cells (% Annexin V, -/+ EX: mono-culture, 30.1±9.0 / 31.5±4.6; co-culture with adipocytes, 8.9±2.1 / 29.6±9.2; P=0.02). To assess the molecular links between metabolic pathways and gene expression triggered by BM adipocytes, we performed RNA-seq transcriptome analysis with a next generation sequencer system HiSeq1500 (Illumina) using TopHat software for alignment and Cufflinks software for identifying differential gene expression. RNA-Seq detected upregulation of 21 genes in U937 cells after co-culture with BM-derived adipocytes (false discovery rate, DNA array (GeneSQUARE) analysis and quantitative RT-PCR detected upregulation of fatty acid binding protein 4 (FABP4), scavenger receptor CD36, nuclear receptor PPARG, and antiapoptotic Bcl-2 in U937 cells co-cultured with adipocytes. It is known that FFAs, the ligand of nuclear receptor PPARγ, activate PPARγ and promote FFA uptake through transcriptional induction of CD36 and FABP4 in monocytic cells. EX treatment, which blocks the entry of fatty acids into the mitochondria, induced prominent elevation of FABP4 in U937 cells co-cultured with adipocytes. These results indicate that the FABP4-mediated internalization and ligation of fatty acids to PPARγ facilitates transcriptional activation. From the transcriptome analysis and the mitochondrial uncoupling metabolic changes, we conclude that the survival of AMoL cells depends on the cooperative interactions between lipid metabolism and transcriptional activation of factors associated with chaperones, chemokines, and integrins. Strategies targeting FAO warrant further exploration in patients with monocytic leukemia, which is highly dependent on altered lipid metabolism. Disclosures No relevant conflicts of interest to declare.

Mantle cell lymphoma (MCL) is an aggressive B-cell lymphoma that frequently demonstrates chemoresistance. Since a number of signaling pathways are dysregulated in MCL, novel strategies for restoring multiple tumor suppressors and pathways are of considerable interest. Exportin 1 (XPO1/CRM1) mediates nuclear export of numerous molecules, including oncogenic transcription factors, ribosomal subunits, and RNAs, and is critical for cancer survival and proliferation. We previously reported that single-agent XPO1 antagonist KPT-185 exhibited antiproliferative and proapoptotic activities against MCL cells via inhibiting synthesis of proteins, such as chaperone proteins (HSP70), through ribosomal biogenesis and via nuclear export of transcription factors and oncogenic mRNAs, including cyclin D1, c-Myc, and PIM1 (Tabe et al. ASH 2013). Intriguingly, proteomic analysis detected significant upregulation of glycolysis and gluconeogenesis pathways in KPT-185–treated MCL cells. Aerobic glycolysis plays an important role in sustaining tumor metabolism and may negatively affect the antitumor activity of KPT-185. We therefore assessed the efficacy of combining this regulator of nucleocytoplasmic shuttling with an inhibitor of mTOR signaling, which is a central regulator of cell metabolism integrating nutrients, with KPT-185 targeting the altered metabolism. We first investigated the antitumor effects and molecular mechanisms of simultaneous treatment with KPT-185 and the ATP-competitive second-generation mTOR kinase inhibitor AZD-2014 in three MCL cell lines: JVM2, Jeko-1, and MINO (KPT-185 IC50 values: 92, 103 and 96 nM, respectively, at 48 h by MTT). AZD-2014 treatment resulted in downregulation of p-S6K and c-Myc and upregulation of p27KIP and cleaved caspase-9, which translated into concentration-dependent reduction of cell proliferation (IC50: JVM2, 247 nM; Jeko-1, 86 nM; MINO, 370 nM, at 48 h by MTT). The KPT-185/AZD-2014 combination inhibited cell growth (% of control absorbance; values given are for KPT-185 [100nM], AZD-2014 [100nM for JVM2 and MINO, 50nM for Jeko-1], and KPT-185/AZD-2014: JVM2 49.4±2.0, 61.6±3.7, 25.2±0.2; Jeko-1 46.7±4.8, 66.9±3.3, 28.6±3.4; MINO 55.0±5.6, 79.6±0.6, 21.6±2.5, at 48 h by MTT). We next investigated changes of protein expression and signaling pathways induced by AZD-2014 or the KPT-185/AZD-2014 combination (24 h) in Jeko-1 cells (KPT-100nM, AZD-2014 200nM). The proteomic technology of isobaric tags for relative and absolute quantitation (iTRAQ) demonstrated that AZD-2014 affected expression of 68 proteins (42 upregulated / 26 downregulated) and caused downregulation of fatty acid synthase expression (P We then performed comprehensive and quantitative analysis of charged metabolites by capillary electrophoresis mass spectrometry in Jeko-1 cells after treatment with KPT-185 or KPT-185/AZD-2014 (24 h) to detect differences in 52 polar metabolites (P Taken together, our findings suggest that inhibition of mTOR kinase enhances the antitumor effects of the XPO1 antagonist KPT-185 with effective repression of XPO1 blockage–induced glycolysis/gluconeogenesis upregulation and of fatty acid synthesis and with possible disruption of bidirectional nucleocytoplasmic shuttling in MCL cells. These findings suggest a novel, rationally designed combinatorial strategy targeting pro-survival metabolism in MCL. Figure 1 Figure 1. Disclosures Andreeff: Karyopharm: Research Funding.

Adult T-cell acute lymphoblastic leukemia (T-ALL) has a poor prognosis associated with resistance and rapid relapses, necessitating novel therapeutic strategies. Malignant T cells require high concentrations of nutrients to sustain their increased rates of proliferation, and frequent activation of mTOR signaling, a central regulator of cell metabolism, has been shown in a high-risk subgroup of T-ALL with constitutive Notch activation (Chan, Blood, 2007;110,278). In this study, we analyzed the metabolic response mechanisms of Notch-activated aggressive T-ALL to the ATP-competitive mTOR inhibitor AZD2014 and to the combination of AZD2014 with a conventional chemotherapeutic agent, L-asparaginase (ASNase). To investigate the molecular modifications of key cellular metabolic processes induced by these agents, we undertook a comprehensive and quantitative analysis of charged metabolites by capillary electrophoresis mass spectrometry (CE-MS, Agilent Technologies) for metabolic profiling. We first assessed the anti-proliferative effects of the combination of AZD2014 and ASNase in CUTLL1, a T-ALL cell line with a Notch gain-of-function mutation. The medianinhibitory concentrations of AZD-2014 and ASNase were 250 nM and 0.005 U/mL, respectively (MTT assay, 48 hours). The combination of AZD2014 (100 nM) and ASNase (0.001 U/mL) caused apoptosis induction with increase in the % of sub-G1 fraction (Control: 5.5±1.8, AZD-2014: 9.5±2.1, ASNase: 10.9 ±1.5, AZD-2014/ASNase: 17.9 ±4.9; p We next investigated the metabolic changes in CUTLL1 cells after AZD2014 (100 nM) and/or ASNase (0.001 U/mL) treatment (24 hours). CE-MS analysis demonstrated differences in 66 polar metabolites (p These results indicate that AZD2014 effectively facilitates the ASNase-induced blockade of glycolysis/gluconeogenesis and fatty acid synthesis and represses asparate and glutamate metabolism in Notch-activated T-ALL cells. The multiple molecular alterations resulting from mTOR blockade by AZD2014, such as repression of translation initiation through potent inhibition of S6K and 4EBP1, induction of autophagy, and inhibition of c-Myc, may further impair the cellular metabolism altered by ASNase in aggressive T-ALL. In conclusion, metabolic re-programming by the combination of AZD2014 and L-asparaginase in Notch-activated T-ALL provides avenues for novel rationally designed combination regimens in ALL. Disclosures No relevant conflicts of interest to declare.

Exportin 1 (XPO1/CRM1) mediates transport of a number of cargo molecules including transcription factors and ribosomal subunits from the nucleus to cytoplasm. XPO1 is critical for cancer cell survival and proliferation, and we reported that high XPO1 expression correlates with poor prognosis in AML (Kojima, Blood, 2013). Mantle cell lymphoma (MCL) is an aggressive B-cell lymphoma that frequently shows chemoresistance. The overexpression of cyclin D1 due to the specific translocation t(l1 ;14)(q13;q32) in MCL cells is believed to be associated with oncogenesis, and additional genetic events such as mutation/overexpression of p53 are adverse prognostic indicators. Since a number of signaling pathways are dysregulated in MCL, novel strategies aimed at restoring multiple anti-oncogenic pathways, are of considerable interest. We have previously reported anti-proliferative effects of the small molecule SINE XPO1 antagonist KPT-185 in MCL cells, in which KPT-185 abrogates MCL-related cyclin Dl overexpression and upregulates pro-apoptotic PUMA in a p53-independent manner (Tabe, ASH. 2012). In this study, we identified pro-survival pathways involved in XPO1-dependent nuclear export in MCL cells, using the isobaric tags for relative and absolute quantification (iTRAQ) with two-dimensional-liquid chromatography-tandem mass spectrometry. Two MCL cell lines with known p53 status and sensitivities to KPT-185 were analyzed; wt-p53 Zl38 (IC50 35 nM, ED50 62 nM) and mt-p53 Jeko-1 (IC50 103 nM, ED50 618 nM). iTRAQ proteomics identified a total of 2,255 unique proteins in Zl38 and of 2,179 in Jeko-1 cells (KPT-185 of 50 nM for Zl38 and 100 nM for Jeko-1, 18 h), including 75 proteins (62 downregulated and 13 upregulated proteins) consistently altered after KPT-185 treatment in both cells lines. Notably, 81% of the downregulated proteins (50/62) were ribosomal proteins, and iTRAQ further detected the significant repressions of EIF4A1/PIM2 (eukaryotic translation initiation factor 4A1) and EEF2 (eukaryotic elongation factor 2), suggesting that KPT-185 inhibited the XPO1-dependent nuclear export of ribosomal subunits, which led to a defect of ribosomal biogenesis. Very recently, the coordination between the net translational activity of ribosomal biogenesis and the transcriptional regulation via the multifaceted transcription factor HSF1 (heat shock factor 1) has been reported (Santagata, Science, 2013) and HSF1 was identified as a prime transducer that regulates a transcriptional network of genes driving heat-shock proteins, protein synthesis, and energy metabolism. In our study, iTRAQ consistently detected the KPT-185 induced decreased protein levels of HSF1 target HSP70 (Heat shock protein 70), FASN (Fatty acid synthase), phospho-HSP90 (Heat shock protein 90) and EEF1A1 (Eukaryotic translation elongation factor 1 alpha 1), and increased levels of phospho-HNRNPD (Heterogeneous nuclear ribonucleoprotein D, a nucleic acid binding protein which contributes pre-mRNA processing in nucleus). These results indicate that XPO1 may also be affecting transcriptional processes critical for cellular metabolism and survival. Translation initiation factor EIF4Al/PlM2 is known to be associated with an aggressive clinical course in B-cell lymphomas (Gomez-Abad, Blood. 2011), and downregulation of PIM1 kinase via ribosomal protein deficiency induces cell-cycle inhibitor p27KIP (Morishita, Cancer Res. 2008) and inhibits oncogenic transcription factor c-Myc (Iadevaia, Oncogene. 2010). Of note, iTRAQ detected the KPT-185 induced depletion of ribosomal proteins RPS19 and RPL11, which interact with PIM1 kinase and c-Myc, respectively. We confirmed KPT-185 induced downregulation of PIM1 and c-Myc and upregulation of p27KIP by Western blot. KPT-185 further reduced phospho-S6K, a substrate of mTORC1 and a major negative regulatory axis of autophagy, and induced a shift from LC3-I to LC3-II, suggesting that CRM1 inhibition by KPT-185 causes autophagy through suppression of mTOR signaling. In summary, this is the first investigation of XPO1 inhibition in MCL cells using the iTRAQ proteomics approach. The results suggest that XPO1 inhibition targets ribosomal biogenesis, in addition to its nuclear retention of numerous client proteins including p53. This finding elucidates a novel mechanism and target of KPT-185 and warrants further investigations. Disclosures: Andreeff: Karyopharm Therapeutics: Research Funding.

Ionizing radiation is known to induce remodeling of stromal microenvironment and enhance cancer progression. In this study, we investigated the molecular alterations of low-dose ionizing radiation (LDIR) induced non-targeted/bystander responses which affect a complex interplay of stromal cells and pre-leukemic cells in the bone marrow (BM) microenvironment. As a model of BM stromal cells and pre-leukemic cells, we utilized primary BM-derived stromal cells (MSCs) and the Epstein- Barr virus (EBV) infected and immortalized pre-leukemic B-lymphocyte cell line (EBV-B). LDIR (100 mGy, 4MV X ray from a LINAC) caused cell growth inhibition and moderate apoptosis induction in MSCs (viable cells % of control; 75.8 ± 2.4, specific SubG1 % 7.1 ± 0.8 at 24 h) but not in EBV-B cells. We further observed persistent upregulation of p21 mRNA (p< 0.001, RQ-PCR) after acute low-dose irradiation in MSCs but not in EBV-B cells, suggesting radiation induced senescence-like changes in MSCs. In EBV-B cells co-cultured with MSCs, low-dose irradiation induced moderate cell growth inhibition (viable cells % of control; 81.3 ± 6.5) without significant apoptosis induction. To gain insights into the molecular changes induced by LDIR in both, MSC and EBV-B cells we utilized genomic and proteomic analyses. To exclude possible contamination of MSCs, we confirmed negative expression of CD90 mRNA in the tested EBV-B samples. We first screened up to 28,869 genes by cDNA microarray (Affymetrix) and performed functional network analysis by MetaCore (GeneGo). LDIR induced upregulation of 48 genes and the downregulation of 45 (i.e., > 1.3-fold regulation) with prominent stimulation of cell adhesion pathways in MSCs. Of note, 31 of 48 up-regulated genes were small nucleolar RNAs. In EBV-B cells, LDIR upregulated 69 genes/downregulated 130 genes with significant stimulation of the TGFbeta dependent induction of epithelial-mesenchymal transition (EMT) pathways. In EBV-B cells co-cultured with MSCs, LDIR induced immune response signaling along with integrin-mediated cell-matrix adhesion pathway with 42 genes upregulation / 34 genes downregulation. Up-regulation of inflammatory IL8 mRNA (2.0±0.03 fold, p In summary, we have demonstrated the LDIR effects on BM-derived stromal cells and pre-leukemic cells under MSC co-culture conditions mimicking the BM microenvironment. We conclude that LDIR may support pre-malignant cells survival via direct activation of TGFbeta dependent EMT pathways, which contribute to antiapoptosis, and via interactions with irradiated neighboring BM stromal cells which promote quiescence and survival of pre-leukemic cells. Disclosures: No relevant conflicts of interest to declare.

Acute monocytic leukemia (AMoL) is a distinct subtype of AML with an average 3-year overall survival of 31% (Tallman, J Clin Oncol 2004), and the majority of patients die from disease progression after relapse. Adipocytes represent an essential component of the adults bone marrow (BM) microenvironment (Battula, Blood 2013, Tabe, Blood 2004, Konopleva, Blood 1999), and promote survival of monoblastic leukemia cells (Tabe, ASH, 2012). In this study, we employed a proteomic approach based on isobaric tags for relative and absolute quantification (iTRAQ, Applied Biosystems) to examine the molecular mechanism of pro-survival properties of BM adipocytes in co-culture with AMoL. The specific pathway alterations were identified by Metacore (GeneGo, St. Joseph, MI). We first confirmed the more prominent protective role of BM-derived adipocytes as compared to BM stromal cells (MSC) on AMoL cells. Co-culture with adipocytes derived from differentiated MSCs significantly protected U937 and primary AMoL cells (n = 5) from serum starvation-induced apoptosis compared to MSCs (U937 p = 0.029, primary samples p = 0.034). Gene expression analysis demonstrated striking upregulation of mRNA expression levels of CD36 (4.1+0.4-fold),FABP4 (569.3+40.1-fold), and PPARG (2.1+0.2-fold) in U937 cells co-cultured with adipocytes compared to the MSC co-cultured cells. Notably, Bcl-2 mRNA expression in U937 cells was significantly upregulated after adipocyte co-culture compared to MSCs (5.4+0.3-fold). We previously reported that fatty acids (FAs) promote leukemic cell survival via leukemia cells metabolic shifting from pyruvate oxidation to fatty acid oxidation for glycolysis, which links to the Bcl-2 anti-apoptotic machinery (Samudio, J Clin Invest. 2010). Mature adipocytes are capable of releasing abundant free fatty acids (Fas), the essential ligands of a nuclear receptor PPARgamma (PPARG), which are internalized by leukemic cells via scavenger receptor CD36 and ligate PPARG through fatty acid binding protein 4 (FABP4). Enhanced transcriptional activity of FA-ligated PPARG may in turn stimulate glucose metabolism, regulate fatty acids storage, maintain mitochondrial membrane potential, and prevents apoptosis by upregulating anti-apoptotic Bcl-2 resulting in further increase of its downstream target genes including CD36 and FABP4. To gain further insights into metabolic alterations induced in adipocyte-leukemia co-cultures, we performed iTRAQ proteomic analysis of U937 cells cultured in the presence of MSCs or adipocytes. A total of 1,610 proteins were detected by this technology, with 106 proteins differentially expressed between co-culture conditions; 50 genes were up-regulated and 56 genes were down-regulated by adipocytes co-culture compared to MSCs. Among the upregulated proteins, FA synthase (p = 0.007) ATP-citrate synthase responsible for cytosolic acetyl-CoA synthesis (p = 0.019); and glyceraldehyde-3-phosphate dehydrogenase (p = 0.028) involved in the activation of glycolysis and gluconeogenesis pathways (p< 0.0001) were identified. Of note, 20% of the upregulated proteins (10/50) were ribosomal proteins, accompanied by an increase in six DNA elongation or replication related proteins. Anti-apoptotic chaperone proteins known to assist ribosome biogenesis, HSP70, HSP90alpha and HSP90beta were also increased by adipocyte co-culture (p Summary our results indicate that abundant FAs produced by BM adipocytes support AMoL cell survival via stimulation of PPARG transcription which directly increases the FA metabolism through uptake and translocation of FAs by CD36 and FABP4 in AMoL cells. FA metabolism in turn facilitates supply of Acetyl-CoA via oxidative phosphorylation, which drives the TCA cycle; and enhances ribosomal biogenesis of AMoL cells, resulting in pro-survival signaling in AMoL cells. It is conceivable that increased adipocyte content of the BM in adult AML patients may promote leukemogenesis and negatively affect the responsiveness to chemotherapy. Disclosures: No relevant conflicts of interest to declare.

Despite impressive outcomes achieved with combination chemotherapy regimens in acute lymphoblastic leukemia (ALL), resistance and relapses occur frequently necessitating a quest for novel therapeutic targets. PI3K/AKT/mTOR signalling is a central regulator of cell metabolism, growth, proliferation and survival and has been shown to be constitutively activated in the aggressive T-ALL with Notch gain-of-function mutations (Palomero, Leukemia, 2006;20:1279). In a high-risk subgroup of B-ALL harbouring mutations in the CRLF2 gene, the frequent activation of mTOR and JAK/STAT pathways was recently reported (Roberts, Cancer Cell, 2012;22:153). In this study, we tested the pre-clinical efficacy and mechanisms of action of the novel ATP-competitive mTOR inhibitor AZD2014 which effectively inhibits both TORC1 and TORC2 complexes. Pharmacological interactions with chemotherapeutic agent L-asparaginase (L-Asp) or JAK2 inhibitor TG101348 were further evaluated in high-risk immature T-ALL (PF-382, CCRF-CEM, RPMI-8402 cells, Notch1-mutated CUTLL1) and CRLF2-mutated B-ALL cells (MUTZ-5). Inhibition of mTOR kinase activity by AZD2014 used at sub-micromolar concentrations resulted in downregulation of mTORC1 substrates phospho-(p-)S6K (Ser240-244), p-4EBP1 (Thr37/46), mTORC2 target p-Akt (Ser473), and of the oncogenic transcription factor c-Myc. This translated into reduction of cell proliferation with G0/G1 cell cycle arrest in all tested ALL cells, with IC50s ranging from 70 nM to 1,000 nM. In Notch-activated CUTLL1 T-ALL cells, L-Asp enhanced the cell growth inhibition by AZD2014 and further decreased p-S6K levels. In CRLF2-rearranged MUTZ-5 B-ALL cells, the AZD2014/TG-101348 combination enhanced the anti-proliferative effects of AZD2014 with further decrease of p-4EBP1 and down-regulation of Pim-1, a downstream target of Akt and JAK/STAT signaling, more prominently than in other tested pre-B-ALL cells, Nalm6 and REH. AZD2014 induced autophagy as shown by the conversion of the autophagosomal marker LC3-I to LC-II in CUTLL1 and MUTZ-5 cells. To investigate the molecular modifications of the key cellular metabolic processes in response to mTOR blockade, we employed the proteomic technology of isobaric tags for relative and absolute quantitation (iTRAQ) with two-dimensional-liquid chromatography-tandem mass spectrometry. Using iTRAQ platform, more than 1,770 unique proteins were identified in each of the four cell lines tested (T-ALL, PF-382 and CUTLL1; B-ALL, Nalm6 and MUTZ-5). AZD2014 frequently and significantly downregulated anti-apoptotic chaperone heat shock proteins HSP70 and HSP90 (p < 0.05) along with upregulation of the voltage-dependent anion channels (VDAC) (p < 0.01) and histone H1 (p < 0.05), both functioning as dynamic initiators of mitochondria-mediated apoptosis. In T-ALL cells, L-Asp alone or in combination with AZD2014 further upregulated VDAC, histone H1, and apoptosis related mitochondrial chaperone HSP10 (p < 0.05), and repressed translation initiation factors and proteasome-related proteins (p < 0.05). Interestingly, in Notch-mutant CUTLL1 cells L-Asp downregulated DNA methyltransferase Dnmt1 (p < 0.05), and the combination with AZD2014 further decreased histone-binding protein RBBP4 (p < 0.05) which interacts with histone deacetylase, suggesting previously unrecognized epigenetic anti-tumor modifications by AZD2014/L-Asp treatment. In B-ALL cells, the AZD2014/TG101348 combination induced significant repression of ribosomal and spliceosomal pathways (p < 0.001) along with marked repression of HSP70 and ubiquitin related protein E3 ubiquitin-protein ligase (p < 0.05), and increased expression of the apoptosis regulators VDAC (p < 0.01), histone H1 (p < 0.05) and chaperone HSP60 (p < 0.05). In summary, these results suggest that mTOR blockade by AZD2014 inhibits ALL cell growth through multiple mechanisms, which include repression of translation initiation through potent inhibition of S6K and 4EBP1, induction of autophagy, modulation of HSP chaperone proteins and epigenetic modifications. These cellular alterations are further impaired by the concomitant inhibition of asparagine metabolism with L-Asp in T-ALL or through blockade of JAK2 signaling in B-ALL, providing avenues for novel rationally designed combinatorial regimens in ALL. Disclosures: No relevant conflicts of interest to declare.

Hypoxia is one of the essential components of the leukemia bone marrow (BM) microenvironment that promotes leukemia cell homing, survival and chemoresistance (Benito, PlosOne 2011). Tyrosine kinase inhibitors (TKIs) do not eradicate the total mass of chronic myeloid leukemia (CML) cells including primitive and quiescent cells. Persistence of CML cells in the hypoxic BM niche after cessation of TKI therapy may result in disease relapse. We have reported that hypoxia adapted CML cells acquire TKI resistance associated with higher glyoxalase-1 (Glo-1) enzyme activity which detoxifies methylglyoxal, a cytotoxic by-product of glycolysis (Takeuchi, Cell Death Differ 2010). In this study, we employed a proteomic approach based on isobaric tags for relative and absolute quantification (iTRAQ, Applied Biosystems) to investigate additional molecular mechanisms of CML adaptation to hypoxia and acquired resistance to dasatinib under hypoxic conditions. The specific pathway alterations were identified by KEGG(Kyoto University) and MetaCore (GeneGo). We utilized two hypoxia-adapted (HA) subclones of CML cell lines, KCL22-HA and KBM5-HA cells, which were selected over a month under 1.0 % oxygen culture conditions. The growth rate of both HA-CML cell lines was slower than that of the corresponding parental cells (ratio of incremental increase in cell numbers, 0.39 for KCL22-HA/KCL22, 0.54 for KBM5-HA/KBM5 at 48hrs). Although parental KCL22 cells were sensitive to dasatinib, KCL22-HA cultured under hypoxia acquired resistance to dasatinib (IC50: KCL22 0.1 nM, KCL22-HA >20nM, at 48hrs by MTT). In contrast, dasatinib induced more prominent cell growth inhibition in KBM5-HA cells cultured under hypoxia compared to KBM5 parental cells (IC50: KBM5 1.3 nM, KBM-5/HA 0.3 nM). Dasatinib effectively downregulated the phosphorylation levels of Stat5 and ERK in parental KCL22 and KBM5 cells. Notably, the baseline levels of p-Stat5 and p-ERK were markedly diminished in both KCL22-HA and KBM5-HA cells. We next performed iTRAQ proteomic analysis and detected more than 1,300 proteins in each cell type. Comparison of the basal proteome of KCL22 vs KCL22-HA and KBM5 vs KBM5-HA cells showed differential expression of 54 proteins in KCL22 isogenic cells (37 upregulated, 17 downregulated) and of 159 proteins in KBM5 cells (56 upregulated, 103 downregulated). These alterations included consistent activation of glycolysis and gluconeogenesis pathway (p In summary, these findings demonstrate the dramatic metabolic changes in hypoxia adjusted CML cells and indicate that hypoxia adapted / dasatinib resistant CML cells repress their stimulated glycolic metabolic state and oxidative phosphorylation along with spliceosomal mRNA splicing after dasatinib exposure, which allow them to enter quiescent stage and escape from apoptosis. Altogether these data indicate novel mechanisms of acquired bone marrow microenvironment-mediated resistance to TKI in CML. Disclosures: Tabe: Bristol-Myers Squibb: Research Funding.

Abstract 2773 Development of the second-generation ABL tyrosine kinase inhibitor (TKI) dasatinib in CML aimed at overcoming resistance to imatinib, to eliminate persistent residual disease and thus prevent recurrence of active leukemia after TKI discontinuation in chronic myeloid leukemia (CML). Hypoxia has recently been reported as an essential component of the leukemia BM microenvironment that promotes leukemia cell homing, survival and chemoresistance (Benito et al, PlosOne 2011). In this study, we investigated the anti-CML efficacy and molecular mechanisms of action of dasatinib under hypoxic conditions. We developed a hypoxia-adopted subclone of the KBM5 CML cell line (KMB5-HA), which was selected under 1.0% oxygen conditions, and an imatinib-resistant KBM5 subline bearing the T315I mutation (KBM5-T315I). KBM5-HA cells cultured under hypoxia accumulated in G0/G1 and exhibited moderate spontaneous apoptosis compared to KBM5 parental cells in normoxia (sub G1 %: KBM5 3.8±0.8, KBM5-HA 8.7±0.9; p=0.01; proportion of cells in G0/G1: KBM5 36.5±1.1%, KBM5-HA 52.4±6.6%; p=0.02, PI analysis). These cells displayed higher sensitivity to dasatinib than parental KBM cells (IC50; 1.3 nM for KBM5, 0.3 nM for KBM5-HA, at 48hrs by MTT). Low-dose dasatinib (0.5nM) which failed to cause inhibition of proliferation in parental KBM5 cells, caused significant apoptosis induction with cell cycle arrest in KBM-HA cells (sub G1 %: control 8.7±0.9, datatinib 49.1±16.9; p=0.02, G0/G1 %: control 52.4±6.4%, dasatinib 74.1±3.5%; p=0.01). In KBM5-T315I cells, dasatinib induced more prominent cell growth inhibition under hypoxia compared to normoxia (IC50 at 48hrs: normoxia 16.2 nM, hypoxia 3.7 nM). Treatment with 5nM dasatinib, which did not affect KBM5-T315I cell growth under normoxia, induced significant apoptotic effects under hypoxia (sub G1 %: control 6.9±0.9, dasatinib 20.3±3.3; p=0.05, G0/G1 %: control 57.3±5.8, dasatinib 67.4±8.9; p=0.41). We next investigated dasatinib-induced changes of Stat-5 and ERK activation in CML cells by immunoblotting Treatment with 0.5 nM dasatinib resulted in marked down-regulation of phosphorylated (p-) Stat-5 and p-ERK in both, KBM5 and KBM5-T315I cells. In KBM5-HA cells, however, no baseline expression of p-Stat-5 or p-ERK was detected. These results suggest that in KBM5-HA cells dasatinib induces apoptosis and cell cycle arrest via Stat-5 or ERK-independent pathways. To investigate the candidate signaling factors responsible for dasatinib effects on KBM-HA, we performed the proteomic analysis utilizing proteomic technology of isobaric tags for relative and absolute quantitation (iTRAQ, Applied Biosystems) coupled with two-dimensional-liquid chromatography-tandem mass spectrometry. A total of 1,234 proteins were detected, and 296 proteins were found to be significantly up-regulated in response to dasatinib treatment in KBM5-HA cells. Among the up-regulated proteins, we found 39 proteins involved in apoptosis induction including Cytochrome C and Cytochrome C oxydase subunits (p In summary, these findings unravel novel mechanisms of action of dasatinib under conditions mimicking the hypoxic BM microenvironment via induction of Cytochrome C oxydase and mitochondrial chaperones HSP10 and HSP60, which lead to loss of mitochondrial transmembrane potential and release of apoptogenic Cytochrome C in CML cells. Disclosures: Tabe: Bristol-Myers Squibb: Research Funding.

Abstract 4315 A natural triterpene Avicin D induces apoptosis in various tumor cells and regulates cellular metabolism via Glucocorticoid receptor (GR) signalling, known to be involved in adipocyte differentiation (Tang, Annu Rev Biochem. 2012;81:715). Since adipocytes represent an essential component of the aging bone marrow (BM) microenvironment (Tabe, Blood 2004;103:1815), and promote monocytic differentiation and survival of monoblastic leukemia cells (Tabe, ASH, 2011), we examined the potential of Avicin D to modulate BM adipocytes and its effects on the survival of the leukemic cells. In mature adipocytes derived from BM mesenchymal stem cells (MSCs), Avicin D (1ƒÊM, 48 hrs) significantly increased the number of lipid vesicles without apoptosis induction or significant cell cycle arrest. This was associated with upregulation of adipose-specific genes, PPARG and fatty acid binding protein 4 (FABP4), and inflammation-related haptoglobin (HP) gene (p We next investigated the effects of MSCs and adipocytes pre-treated with 1mM Avicn D for 24 hours on co-cultured monoblastic leukemia cell line U937. Avicin D (1ƒÊM) moderately (26±5 %) inhibited cell proliferation of U937 cells cultured alone as detected by MTT assay. Serum-starvation induced cell death in U937 cells was inhibited by MCS or adipocyte co-culture, and further blocked by Avicin D-pretreated adipocytes, which was accompanied with cell cycle arrest (sub-G1 fraction; control U937 36.1±7.1%, co-culture with MSC 17.9±4.1%, MSC+Avicin D 20.9±3.9%, adipocyte 16.9±2.9, adipocyte+Avicin D 11.5±0.8%, p=0.03, G0/G1 phase; control 39.1±5.9%, with MSC 44.3±1.0%, MSC+Avicin D 40.3±1.3%, adipocyte 40.6±1.3, adipocyte+Avicin D 50.4±2.3%, p=0.03). Co-culture of U937 cells with Avicin D-pretreated adipocytes induced upregulation of PPARG and CD36, markers of monocytes/macrophages maturation in U937 cells (fold increase compared to control U937: PPARG: co-culture with adipocytes 2.1±0.4, adipocytes+Avicin D 13.8±6.2; CD36: adipocytes 2.2±0.4, adipocytes+Avicin D 4.7±2.9). Co-culture with Avicin D-pretreated adipocytes also caused 3.1 ±0.4 fold increase of BCL2 mRNA in U937 cells compared to the one of co-cultured with control adipocytes (p=0.02). The purity of U937 cells separated from adipocytes was confirmed by lack of CD90 mRNA expression by PCR. Utilization of the proteomic technology of isobaric tags for relative and absolute quantitation (iTRAQ) with two-dimensional-liquid chromatography-tandem mass spectrometry allowed the identification of 1,634 proteins. We found changes in 14 proteins in U937 cells co-cultured with Avicin D-pretreated adipocytes compared to untreated adipocytes. Among 13 down-regulated proteins, three are involved in pyruvate metabolism (Glyoxalase I; p=0.01, Lactate dehydrogenase B; p=0.02, Pyruvate kinase; p=0.03), and four proteins participate in cell cycle progression (DNA replication licensing factor MCM4, MCM5; p=0.02, p=0.03, DNA-dependent protein kinase; p=0.02, Interleukin enhancer-binding factor 3; p=0.005). Voltage-dependent anion-selective channel protein 2, which is involved in the mitochondrial apoptotic pathway via regulation of Bcl-2, and an enzymatic antioxidant Superoxide dismutase were up-regulated (p=0.04, p=0.03). These data indicate that U937 cells co-cultured with Avicin D pretreated adipocytes were induced to undergo cell cycle arrest, escape apoptosis with down-regulation of glucose metabolism. Altogether, these results suggest that Avicin D induces adipocytic differentiation of BM-derived stromal cells, promotes production of leptin and inflammatory cytokines, which in turn supports survival of monocytic leukemia cells. These findings suggest a contributory role of the aging “pro-inflammatory” bone marrow microenvironment in the reduced efficacy of cytotoxic chemotherapy which is prevalent in elderly AML patients. Disclosures: No relevant conflicts of interest to declare.

Abstract 4610 Low-dose irradiation (LDI) exposure is a form of environmental carcinogen, which is of significant interest after the nuclear accident in Japan. In this study, we investigated the LDI-induced molecular alterations of bone marrow (BM) –derived stromal cells (MSCs), and the biological response of pre-leukemic cells neighbor to LDI exposed MSCs. As the model of pre-leukemic cells, we utilized the Epstein- Barr virus (EBV) infected and immortalized B lymphocyte cell line (EBV-B). In MSCs, exposed to 100 mGy irradiation (4MVX rayfrom a LINAC) caused cell growth inhibition (75.8±2.4 % of control, p1.3 fold) in irradiated MSCs (at 24 hrs) compared to non-irradiated MSCs. The functional network analysis of cDNA microarray data by MetaCore (GeneGo) showed an enrichment of the adhesion/ECM remodeling pathways. TaqMan RT-PCR confirmed significant up-regulation of ECM scaffold Sulfatase1 mRNA (2.0±0.1 fold, p=0.004). Proteomic analysis utilized isobaric tags for relative and absolute quantitation (iTRAQ, Applied Biosystems), identified 32 up-regulated and 1 down-regulated proteins (p15.0 ng/mL, at 72hrs). We then investigated the effect of LDI exposed MSCs on EBV-B cell proliferation. Under low-serum growth condition (1% FBS), co-culture with pre-irradiated (100 mGy) MSC layers supported EBV-B cell proliferation more prominently than non-irradiated MSCs (118.3±4.0 % viable cell number, p Disclosures: No relevant conflicts of interest to declare.