Search

Macrophages are known to mediate anti-helminth responses, but it remains uncertain which subsets are involved or how macrophages actually kill helminths. Here we show rapid monocyte recruitment to the lung after infection with the nematode parasite,Nippostrongylus brasiliensis. In this inflamed tissue microenvironment these monocytes differentiate into an alveolar-like macrophage (AM) phenotype, expressing both Siglec-F and CD11c, surround invading parasitic larvae and preferentially kill parasites in vitro. Monocyte-derived AMs (Mo-AMs) express type 2-associated markers and show distinct remodeling of the chromatin landscape relative to tissue-derived AMs. In particular, they express high amounts of Arg1 (arginase-1), which we demonstrate mediates helminth killing through L-arginine depletion. These studies indicate that recruited monocytes are selectively programmed in the pulmonary environment to express AM markers and an anti-helminth phenotype.

We examined the activation and effector functions of lung macrophages in mediatingimmunity against helminth parasites. Using fate mapping mice, we showed thatmonocytes recruited to the lung assume an alveolar macrophage phenotype andcomprise as many as 10% of alveolar macrophages by 7 days after inoculation.Monocyte-derived alveolar macrophages assume distinct transcriptional and metabolicprofiles that include high levels of arginase expression and preferentially mediatehelminth damage, both of which are dependent on neutrophil help. In further studies,arginase was shown to mediate helminth killing through localized depletion of arginine.

Currently, applications of graphene and its derivatives have been reported in many fields. In particular, graphene oxide (GO) has been studied for biomedical applications, such as cell imaging, biosensors and drug delivery systems, because of its good biocompatibility and excellent physicochemical properties. Recently, much research has been conducted to control cell behavior, including attachment, proliferation and differentiation by using GO. On the other hand, the effects of GO on myogenic differentiation have not been explored. In the present study, the influence of GO on myogenic differentiation was investigated. This study assessed the cytotoxicity of GO and evaluated the myogenic differentiation of C2C12 murine myoblasts in growth media containing 10 μg/mL of GO. The physicochemical properties of GO were characterized by using atomic force microscopy (AFM) and Raman spectroscopy. The cytotoxicity was evaluated by using a WST-8 assay. An analysis of myogenic differentiation was performed by using immunofluorescence staining for the myosin heavy chain (MHC; myogenic differentiation marker) and was determined by calculating the amount of multinucleate myotube formation, the fusion index and the maturation index. GO did not exhibit cytotoxicity at low concentrations and effectively stimulated the myogenic differentiation of C2C12 murine myoblasts. Therefore, GO has potential skeletal tissue engineering applications.

In this study, hyaluronic acid (HA)/poly(lactic-co-glycolic acid, PLGA) core/shell fiber meshes loaded with epigallocatechin-3-O-gallate (EGCG) (HA/PLGA-E) for application to tissue engineering scaffolds for skin regeneration were prepared via coaxial electrospinning. Physicochemical properties of HA/PLGA-E core/shell fiber meshes were characterized by SEM, Raman spectroscopy, contact angle, EGCG release profiling and in vitro degradation. Biomechanical properties of HA/PLGA-E meshes were also investigated by a tensile strength test. SEM images showed that HA/PLGA-E fiber meshes had a three-dimensional interconnected pore structure with an average fiber diameter of about 1270 nm. Raman spectra revealed that EGCG was uniformly dispersed in the PLGA shell of meshes. HA/PLGA-E meshes showed sustained EGCG release patterns by controlled diffusion and PLGA degradation over 4 weeks. EGCG loading did not adversely affect the tensile strength and elastic modulus of HA/PLGA meshes, while increased their hydrophilicity and surface energy. Attachment of human dermal fibroblasts on HA/PLGA-E meshes was appreciably increased and their proliferation was steadily retained during the culture period. These results suggest that HA/PLGA-E core/shell fiber meshes can be potentially used as scaffolds supporting skin regeneration.

Background/Aims: Constitutive activation of the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin signaling pathway preferentially occurs in aggressive blastoid variants of mantle cell lymphoma (MCL) and is implicated in the pathogenesis of this disease. In this study, we investigated the role of PI3K isoforms on proliferation of aggressive MCL cells. Methods: The changes in cell viability, cell cycle distribution and apoptosis induction by the PI3K isoform-selective inhibitors were evaluated. The molecular basis underlying the effects of the specific inhibition of PI3K isoforms was investigated by Western blot analysis. Results: Our results demonstrated that a class IA PI3K isoform is most commonly involved in the constitutive activation of Akt in aggressive MCL. Treatment with a p110α isoform-specific inhibitor induced prominent cell cycle arrest followed by apoptosis through complete abolishment of phosphorylated (p)-Akt and its downstream targets. An inhibitor of isoform p110δ induced moderate cell cycle arrest with downregulation of p-Akt and p-S6K. A dual inhibitor of p110α and p110δ GDC-0941 caused more prominent cell growth inhibition compared to selective p110α or p110δ inhibitors. Inhibition of the class IB PI3K isoform p110γ did not cause cell cycle arrest or induce apoptosis in MCL cells. Conclusion: These findings suggest that the therapeutic ablation of class IA PI3K may be a promising strategy for the treatment of refractory, aggressive MCL.

Both phosphatidylinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin signaling and antiapoptotic Bcl-2 family members are critical for survival of acute myeloid leukemia (AML) cells. Here, we demonstrate the antileukemic effects of simultaneous inhibition of PI3K by the selective class I PI3K inhibitor GDC-0941 and of Bcl-2 family members by the BH3 mimetic ABT-737 in the context of the bone marrow microenvironment, where hypoxia and interactions with bone marrow stromal cells promote AML cell survival and chemoresistance. The combination of GDC-0941 and ABT-737 profoundly downregulated antiapoptotic Mcl-1 expression levels, activated BAX, and induced mitochondrial apoptosis in AML cells co-cultured with bone marrow stromal cells under hypoxic conditions. Hypoxia caused degradation of Mcl-1 and rendered Mcl-1-overexpressing OCI-AML3 cells sensitive to ABT-737. Our findings suggest that pharmacologic PI3K inhibition by GDC-0941 enhances ABT-737-induced leukemia cell death even under the protective conditions afforded by the bone marrow microenvironment.Combined blockade of PI3K and Bcl-2 pathways down-regulates anti-apoptotic Mcl-1 expression PI3K and Bcl-2 induced Mcl-1 down-regulation activates BAX PI3K and Bcl-2 blockage induces apoptosis in AML under hypoxic BM microenvironment.

Recently, there has been considerable effort to develop suitable scaffolds for tissue engineering applications. Cell adhesion is a prerequisite for cells to survive. In nature, the extracellular matrix (ECM) plays this role. Therefore, an ideal scaffold should be structurally similar to the natural ECM and have biocompatibility and biodegradability. In addition, the scaffold should have biofunctionality, which provides the potent ability to enhance the cellular behaviors, such as adhesion, proliferation and differentiation. This study concentrates on fabricating cell-adhesive matrices composed of RGD peptide-displaying M13 bacteriophage (RGD-M13 phage) and poly(lactic-co-glycolic acid, PLGA) nanofibers. Long rod-shaped M13 bacteriophages are non-toxic and can express many desired proteins on their surface. A genetically engineered M13 phage was constructed to display RGD peptides on its surface. PLGA is a biodegradable polymer with excellent biocompatibility and suitable physicochemical property for adhesive matrices. In this study, RGD-M13 phage/PLGA hybrid nanofiber matrices were fabricated by electrospinning. The physicochemical properties of these matrices were characterized by scanning electron microscopy, atomic force microscopy, Raman spectroscopy, and contact angle measurement. In addition, the cellular behaviors, such as the initial attachment, proliferation and differentiation, were analyzed by a CCK-8 assay and immunofluorescence staining to evaluate the potential application of these matrices to tissue engineering scaffolds. The RGD-M13 phage/PLGA nanofiber matrices could enhance the cellular behaviors and promote the differentiation of C2C12 myoblasts. These results suggest that the RGD-M13 phage/PLGA nanofiber matrices are beneficial to myoblast differentiation and can serve as effective tissue engineering scaffolds.

Osteoprogenitor cells play a significant role in the growth or repair of bones, and have great potential as cell sources for regenerative medicine and bone tissue engineering, but control of their specific differentiation into bone cells remains a challenge. Graphene-based nanomaterials are attractive candidates for biomedical applications as substrates for stem cell (SC) differentiation, scaffolds in tissue engineering, and components of implant devices owing to their biocompatible, transferable and implantable properties. This study examined the enhanced osteogenic differentiation of human mesenchymal stem cells (hMSCs) by reduced graphene oxide (rGO) nanoparticles (NPs), and rGO NPs was prepared by reducing graphene oxide (GO) with a hydrazine treatment followed by annealing in argon and hydrogen. The cytotoxicity profile of each particle was examined using a water-soluble tetrazolium-8 (WST-8) assay. At different time-points, a WST-8 assay, alkaline phosphatase (ALP) activity assay and alizarin red S (ARS) staining were used to determine the effects of rGO NPs on proliferation, differentiation and mineralization, respectively. The results suggest that graphene-based materials have potential as a platform for stem cells culture and biomedicalapplications.

Treatment using Fms-like tyrosine kinase-3 (FLT3) inhibitors is a promising approach to overcome the dismal prognosis of acute myeloid leukemia (AML) with activating FLT3 mutations. Current trials are combining FLT3 inhibitors with p53-activating conventional chemotherapy. The mechanisms of cytotoxicity of FLT3 inhibitors are poorly understood. We investigated the interaction of FLT3 and p53 pathways after their simultaneous blockade using the selective FLT3 inhibitor FI-700 and the MDM2 inhibitor Nutlin-3 in AML. We found that FI-700 immediately reduced antiapoptotic Mcl-1 levels and enhanced Nutlin-induced p53-mediated mitochondrial apoptosis in FLT3/internal tandem duplication cells through the Mcl-1/Noxa axis. FI-700 induced proteasome-mediated degradation of Mcl-1, resulting in the reduced ability of Mcl-1 to sequester proapoptotic Bim. Nutlin-3 induced Noxa, which displaced Bim from Mcl-1. The FI-700/Nutlin-3 combination profoundly activated Bax and induced apoptosis. Our findings suggest that FI-700 actively enhances p53 signaling toward mitochondrial apoptosis and that a combination strategy aimed at inhibiting FLT3 and activating p53 signaling could potentially be effective in AML.

Transforming growth factor beta1 (TGF-beta1) is an essential regulator of cell proliferation, survival and apoptosis, depending on the cellular context. TGF-beta1 is also known to affect cell-to-cell interactions between tumour cells and stromal cells. We investigated the role of TGF-beta1 in the survival of myelo-monocytic leukaemia cell lines co-cultured with bone marrow (BM)-derived mesenchymal stem cells (MSC). Treatment with recombinant human (rh)TGF-beta1 inhibited spontaneous and cytarabine-induced apoptosis in U937 cells, most prominently in U937 cells directly attached to MSCs. Conversely, the pro-survival effects of TGF-beta1 were inhibited by LY2109761 or TGF-beta1 neutralizing antibody. rhTGF-beta1 increased pro-survival phosphorylation of Akt, which was inhibited by LY2109761. The combination of rhTGF-beta1 and MSC co-culture induced significant upregulation of C/EBPbeta gene (CEBPB) and protein expression along with increased C/EBPbeta liver-enriched activating protein: liver-enriched inhibitory protein ratio, suggesting the novel role of C/EBPbeta in TGF-beta1-mediated U937 cell survival in the context of stromal cell support. In summary, these results indicate that TGF-beta1 produced by BM stromal cells promotes the survival and chemoresistance of leukaemia cells under the direct cell-to-cell interactions. The blockade of TGF-beta signalling by LY2109761, which effectively inhibited the pro-survival signalling, may enhance the efficacy of chemotherapy against myelo-monocytic leukaemic cells in the BM microenvironment.

Chronic myelogenous leukemia (CML) is driven by constitutively activated Bcr-Abl tyrosine kinase, which causes the defective adhesion of CML cells to bone marrow stroma. The overexpression of p210Bcr-Abl was reported to down-regulate CXCR4 expression, and this is associated with the cell migration defects in CML. We proposed that tyrosine kinase inhibitors, imatinib or INNO-406, may restore CXCR4 expression and cause the migration of CML cells to bone marrow microenvironment niches, which in turn results in acquisition of stroma-mediated chemoresistance of CML progenitor cells. In KBM5 and K562 cells, imatinib, INNO-406, or IFN-α increased CXCR4 expression and migration. This increase in CXCR4 levels on CML progenitor cells was likewise found in samples from CML patients treated with imatinib or IFN-α. Imatinib induced G0-G1 cell cycle block in CML cells, which was further enhanced in a mesenchymal stem cell (MSC) coculture system. MSC coculture protected KBM-5 cells from imatinib-induced cell death. These antiapoptotic effects were abrogated by the CXCR4 antagonist AMD3465 or by inhibitor of integrin-linked kinase QLT0267. Altogether, these findings suggest that the up-regulation of CXCR4 by imatinib promotes migration of CML cells to bone marrow stroma, causing the G0-G1 cell cycle arrest and hence ensuring the survival of quiescent CML progenitor cells. [Mol Cancer Ther 2008;7(1):48–58]

In this study, hyaluronic acid (HA)/poly(lactic-co-glycolic acid, PLGA) core/shell fiber meshes loaded with epigallocatechin-3-O-gallate (EGCG) (HA/PLGA-E) for application to tissue engineering scaffolds for skin regeneration were prepared via coaxial electrospinning. Physicochemical properties of HA/PLGA-E core/shell fiber meshes were characterized by SEM, Raman spectroscopy, contact angle, EGCG release profiling and in vitro degradation. Biomechanical properties of HA/PLGA-E meshes were also investigated by a tensile strength test. SEM images showed that HA/PLGA-E fiber meshes had a three-dimensional interconnected pore structure with an average fiber diameter of about 1270 nm. Raman spectra revealed that EGCG was uniformly dispersed in the PLGA shell of meshes. HA/PLGA-E meshes showed sustained EGCG release patterns by controlled diffusion and PLGA degradation over 4 weeks. EGCG loading did not adversely affect the tensile strength and elastic modulus of HA/PLGA meshes, while increased their hydrophilicity and surface energy. Attachment of human dermal fibroblasts on HA/PLGA-E meshes was appreciably increased and their proliferation was steadily retained during the culture period. These results suggest that HA/PLGA-E core/shell fiber meshes can be potentially used as scaffolds supporting skin regeneration.

The effect of pH on layer formation onto clean Cu surfaces in benzotriazole (BTA) aqueous solutions was studied by in situ spectroscopic ellipsometry. The effect of H2O2 addition was also investigated. Time changes in the ellipsometric parameters Ψ and Δ, which correspond to the structural changes of the layers on Cu, were discussed. In acidic solutions, a BTA or a Cu–BTA complex layer grows directly on Cu. The out-diffusion of Cu is suppressed at the Cu layer interface. When H2O2 was mixed, the Cu surface is eroded in acidic solutions. In alkaline solutions, the BTA layer grows on the oxidized Cu layer, or no growth occurs, depending on the composition of the solutions. In neutral solutions, the Cu–BTA complex layer forms on Cu, and the uncovered part is oxidized in the presence of H2O2.

Background Electrospinning is a simple and effective method for fabricating micro- and nanofiber matrices. Electrospun fibre matrices have numerous advantages for use as tissue engineering scaffolds, such as high surface area-to-volume ratio, mass production capability and structural similarity to the natural extracellular matrix (ECM). Therefore, electrospun matrices, which are composed of biocompatible polymers and various biomaterials, have been developed as biomimetic scaffolds for the tissue engineering applications. In particular, graphene oxide (GO) has recently been considered as a novel biomaterial for skeletal muscle regeneration because it can promote the growth and differentiation of myoblasts. Therefore, the aim of the present study was to fabricate the hybrid fibre matrices that stimulate myoblasts differentiation for skeletal muscle regeneration. Results Hybrid fibre matrices composed of poly(lactic-co-glycolic acid, PLGA) and collagen (Col) impregnated with GO (GO-PLGA-Col) were successfully fabricated using an electrospinning process. Our results indicated that the GO-PLGA-Col hybrid matrices were comprised of randomly-oriented continuous fibres with a three-dimensional non-woven porous structure. Compositional analysis showed that GO was dispersed uniformly throughout the GO-PLGA-Col matrices. In addition, the hydrophilicity of the fabricated matrices was significantly increased by blending with a small amount of Col and GO. The attachment and proliferation of the C2C12 skeletal myoblasts were significantly enhanced on the GO-PLGA-Col hybrid matrices. Furthermore, the GO-PLGA-Col matrices stimulated the myogenic differentiation of C2C12 skeletal myoblasts, which was enhanced further under the culture conditions of the differentiation media. Conclusions Taking our findings into consideration, it is suggested that the GO-PLGA-Col hybrid fibre matrices can be exploited as potential biomimetic scaffolds for skeletal tissue engineering and regeneration because these GO-impregnated hybrid matrices have potent effects on the induction of spontaneous myogenesis and exhibit superior bioactivity and biocompatibility.

Background M13 bacteriophages can be readily fabricated as nanofibers due to non-toxic bacterial virus with a nanofiber-like shape. In the present study, we prepared hybrid nanofiber matrices composed of poly(lactic-co-glycolic acid, PLGA) and M13 bacteriophages which were genetically modified to display the RGD peptide on their surface (RGD-M13 phage). Results The surface morphology and chemical composition of hybrid nanofiber matrices were characterized by scanning electron microscopy (SEM) and Raman spectroscopy, respectively. Immunofluorescence staining was conducted to investigate the existence of M13 bacteriophages in RGD-M13 phage/PLGA hybrid nanofibers. In addition, the attachment and proliferation of three different types of fibroblasts on RGD-M13 phage/PLGA nanofiber matrices were evaluated to explore how fibroblasts interact with these matrices. SEM images showed that RGD-M13 phage/PLGA hybrid matrices had the non-woven porous structure, quite similar to that of natural extracellular matrices, having an average fiber diameter of about 190 nm. Immunofluorescence images and Raman spectra revealed that RGD-M13 phages were homogeneously distributed in entire matrices. Moreover, the attachment and proliferation of fibroblasts cultured on RGD-M13 phage/PLGA matrices were significantly enhanced due to enriched RGD moieties on hybrid matrices. Conclusions These results suggest that RGD-M13 phage/PLGA matrices can be efficiently used as biomimetic scaffolds for tissue engineering applications.

LaVO4 thin films have been deposited on Pt / Ti /SiO2 /Si substrates by pulsed laser deposition in order to explore the resistive switching (RS) behavior of the Au/LaVO4/Pt devices. It is found that the as-prepared Au/LaVO4/Pt devices stay in the low resistance state (LRS) and no electroforming process (forming-free) is needed to trigger fresh devices for the subsequent RS. Furthermore, the devices show excellent switching parameters such as a reproducible switching effect, a high resistance ratio of ~104 between the LRS and high resistance state (HRS), good endurance and retention characteristics, and non-overlapping switching voltages. The dominant conduction mechanisms are Ohmic conduction in the LRS and the lower voltage region of the HRS, and Poole–Frenkel emission in the higher voltage region of the HRS. The combination of temperature dependence of resistance, x-ray photoelectron spectroscopy, and model analysis suggests that the forming-free unipolar RS behavior can be explained by the formation and rupture of conical conducting filaments formed out of oxygen vacancies. These results may be important for practical application in nonvolatile resistive switching memory.

Constitutive activation of the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin signaling pathway preferentially occurs in aggressive blastoid variants of mantle cell lymphoma (MCL) and is implicated in the pathogenesis of this disease. In this study, we investigated the role of PI3K isoforms on proliferation of aggressive MCL cells.The changes in cell viability, cell cycle distribution and apoptosis induction by the PI3K isoform-selective inhibitors were evaluated. The molecular basis underlying the effects of the specific inhibition of PI3K isoforms was investigated by Western blot analysis.Our results demonstrated that a class IA PI3K isoform is most commonly involved in the constitutive activation of Akt in aggressive MCL. Treatment with a p110α isoform-specific inhibitor induced prominent cell cycle arrest followed by apoptosis through complete abolishment of phosphorylated (p)-Akt and its downstream targets. An inhibitor of isoform p110δ induced moderate cell cycle arrest with downregulation of p-Akt and p-S6K. A dual inhibitor of p110α and p110δ GDC-0941 caused more prominent cell growth inhibition compared to selective p110α or p110δ inhibitors. Inhibition of the class IB PI3K isoform p110γ did not cause cell cycle arrest or induce apoptosis in MCL cells.These findings suggest that the therapeutic ablation of class IA PI3K may be a promising strategy for the treatment of refractory, aggressive MCL.

We and others have previously demonstrated that p210 Bcr-Abl tyrosine kinase inhibits stromal cell-derived factor-1α/CXCR4 chemokine receptor signaling, contributing to the deficient adhesion of chronic myeloid leukemia (CML) cells to bone marrow stroma. Conversely, exposure of CML cells to a tyrosine kinase inhibitor (TKI) enhances migration of CML cells towards stromal cell layers and promotes non-pharmacological resistance to imatinib. Src-related kinase Lyn is known to interact with CXCL12/CXCR4 signaling and is directly activated by p210 Bcr-Abl. In this study, we demonstrate that TKI treatment promoted CXCR4 redistribution into the lipid raft fraction, in which it co-localized with active phosphorylated form of Lyn (LynTyr396) in CML cells. Lyn inhibition or cholesterol depletion abrogated imatinib-induced migration, and dual Src/Abl kinase inhibitor dasatinib induced fewer CML cells to migrate to the stroma. These findings demonstrate the novel mechanism of microenvironment-mediated resistance through lipid raft modulation, which involves compartmental changes of the multivalent CXCR4 and Lyn complex. We propose that pharmacological targeting of lipid rafts may eliminate bone marrow-resident CML cells through interference with microenvironment-mediated resistance.

Abstract 3920 Mantle cell lymphoma (MCL) is a subtype of B-cell lymphoma and frequently resistant to standard chemotherapeutic agents, and the aggressive blastoid MCL cases often possess mutant (mt-) TP53. Although a proteasome inhibitor bortezomib demonstrates single agent efficacy in MCL, more than 50% of the relapsed or refractory MCL are not enough sensitive to bortezomib. Therefore, development of rationally designed combinations of bortezomib and other antineoplastic agents is needed. We previously reported a dose- and time- dependent inhibition of cellular proliferation in wt-TP53 MCL cells by an MDM2 inhibitor nutlin-3, and its synergistic effects when combined with bortezomib in both mt-TP53- and wt-TP53- bearing MCL cells (Tabe et al., Clin Cancer Res. 2009). In this study, we investigated the molecular mechanism of the synergistical cytotoxic effects of nutlin-3/bortezomib in the mt-TP53 MCL cells which are intrinsically resistant to bortezomib. The MCL cell lines with wild-type and mutant TP53 (wt-TP53: Z-138, Granta-519, mt-TP53: MINO) have been utilized. First, we established the IC50 of nutlin-3 and bortezomib (nutlin-3: 19.1 μM for MINO, 8.2 μM for Granta 519, 1.0 μM for Z-138, bortezomib: 21.8 nM for MINO, 3.7 nM for Granta, 5.0 nM for Z138, MTS assay at 48 hours). The combination of nutlin-3 and bortezomib synergistically induced cytotoxicity more prominently in the bortezomib resistant and mt-TP53 bearing MINO cells (combination index: 0.05 for MINO, 0.71 for Granta 519, 0.12 for Z-138, Calcusyn software). The combination of nutlin-3/bortezomib caused G0/G1 cell cycle arrest in the bortezomib sensitive wt-TP53 Granta 519 and Z138, but not in MINO. In contrast, nutlin-3/bortezomib induced marked increase in the sub-G1 fraction in MINO, but only minimal or no further increase in Granta 519 and Z138, which was confirmed by annexin V/PI staining. BH3-only protein NOXA expression was increased by bortezomib in all tested cells, and the nutlin-3/bortezomib enhanced NOXA accumulation in MINO but not in Granta 519 and Z138 (western blot). On the other hand, anti-apoptotic Mcl-1 has been upregulated by bortezomib in Granta 519 and Z138. In MINO cells, Mcl-1 upregulation was induced only by nutlin-3/bortezomib, which was coimmunoprecipitated with NOXA along with the induction of caspase-3 and -9 cleavage products. We then, assess the subsequent conformational change of proapoptotic BAX/BAK by the nutlin-3/bortezmib combination in MINO (flow cytometric analysis). Taken together, our findings indicate that mitochondrial apoptotic pathway may be an important mechanism contributing to synergistic apoptosis induction by the nutlin-3/bortezomib combination in MCL cells expressing mt-TP53 and intrinsically resistant to bortezomib. The nutlin-3/bortezomib combination may be a potential efficient therapeutic strategy for the chemorefractory MCL. Disclosures: No relevant conflicts of interest to declare.

Purpose: Mantle cell lymphoma (MCL) has one of the poorest prognoses of the non-Hodgkin's lymphomas, and novel therapeutic approaches are needed. We wished to determine whether Nutlin-3, a novel small-molecule murine double minute 2 (MDM2) antagonist that efficiently activates TP53, might be effective in inducing cell death in MCL. Experimental Design: MCL cell lines with known TP53 status were treated with Nutlin-3, and biological and biochemical consequences were studied. Synergies with the prototypic genotoxic agent doxorubicin and the novel proteasome inhibitor bortezomib were assessed. Results: Nutlin-3 resulted in a reduction in cell proliferation/viability (IC50 < 10 μmol/L), an increase in the apoptotic fraction, and cell cycle arrest in wild-type (wt) TP53 Z-138 and Granta 519 cells. These effects were accompanied by TP53 accumulation and induction of TP53-dependent proteins p21, MDM2, Puma, and Noxa. Cell cycle arrest was characterized by suppression of S phase and an increase in the G0-G1 and G2-M fractions and accompanied by suppression of total and phosphorylated retinoblastoma protein and a decrease in G2-M-associated proteins cyclin B and CDC2. The combination of Nutlin-3 with doxorubicin or bortezomib was synergistic in wt-TP53 MCL cells. Nutlin-3 also induced cell cycle arrest and reduced cell viability in the mutant TP53 MINO cells but at a significantly higher IC50 (22.5 μmol/L). These effects were associated with induction of the TP53 homologue p73, slight increases in p21 and Noxa, and caspase activation. Nutlin-3 and bortezomib synergistically inhibited cell growth of MINO. Conclusion: These findings suggest that the MDM2 antagonist Nutlin-3 may be an effective agent in the treatment of MCL with or without wt-TP53.

Integrin-linked kinase (ILK) directly interacts with β integrins and phosphorylates Akt in a phosphatidylinositol 3-kinase (PI3K)–dependent manner. In this study, we examined the functional role of ILK activation in leukemic and bone marrow stromal cells on their direct contact. Coculture of leukemic NB4 cells with bone marrow–derived stromal mesenchymal stem cells (MSC) resulted in robust activation of multiple signaling pathways, including ILK/Akt, extracellular signal-regulated kinase 1/2 (ERK1/2), signal transducers and activators of transcription 3 (STAT3), and Notch1/Hes. Blockade of PI3K or ILK signaling with pharmacologic inhibitors LY294002 or QLT0267 specifically inhibited stroma-induced phosphorylation of Akt and glycogen synthase kinase 3β, suppressed STAT3 and ERK1/2 activation, and decreased Notch1 and Hes1 expression in leukemic cells. This resulted in induction of apoptosis in both leukemic cell lines and in primary acute myelogenous leukemia samples that was not abrogated by MSC coculture. In turn, leukemic cells growing in direct contact with bone marrow stromal elements induce activation of Akt, ERK1/2, and STAT3 signaling in MSC, accompanied by significant increase in Hes1 and Bcl-2 proteins, which were all suppressed by QLT0267 and LY294002. In summary, our results indicate reciprocal activation of ILK/Akt in both leukemic and bone marrow stromal cells. We propose that ILK/Akt is a proximal signaling pathway critical for survival of leukemic cells within the bone marrow microenvironment. Hence, disruption of these interactions by ILK inhibitors represents a potential novel therapeutic strategy to eradicate leukemia in the bone marrow microenvironment by simultaneous targeting of both leukemic cells and activated bone marrow stromal cells. [Cancer Res 2007;67(2):684–94]

The goal of this study was to elucidate the functional roles of PI3K/AKT and MEK/ERK signaling on the proliferation and apoptosis of STI571-sensitive and -resistant CML cell lines in a co-culture system with human marrow stromal cells (MSCs), mimicking the bone marrow microenvironment. The phosphorylation of AKT and ERK was enhanced by co-culture with MSCs in both STI571-sensitive KBM-5 and STI571-resistant KBM-5/STI cells. In KBM-5 cells, the STI571 and PI3K inhibitor LY294002 combination was effective on apoptosis induction in the MSC co-culture system. In KBM-5/STI cells, treatment with LY294002 or PD98059 alone resulted in massive apoptosis, which was enhanced by co-culture with MSCs. These results provide a rationale for multi-molecular target therapy approaches based on a combination of signal transduction inhibitors with STI571 in CML.

The acute promyelocytic leukemia-specific PML-RARalpha fusion protein is a dominant-negative transcriptional repressor of retinoic acid receptor (RAR) target genes, which recruits HDAC and corepressor proteins and inhibits coactivators. Another oncogenic transcription factor, AML1-ETO, was proposed to cause an HDAC-dependent repression of RAR target genes. The RAR target RARbeta2 gene has been reported to be frequently silenced by hypermethylation in many types of cancer cells. We examined the methylation status of the RARbeta2 and asked if demethylation could reverse ATRA resistance in ATRA-resistant PML-RARalpha and AML1-ETO-positive cells. PML-RARalpha positive NB4 and its ATRA-resistant subvariant MR2 and AML1-ETO expressing Kasumi-1 cells had heterozygous methylation of RARbeta2. Although DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine partially reversed RARbeta2 CpG methylation in these cells, it did not significantly enhance ATRA-induced RARbeta2 mRNA expression and induction of maturation. However, the histone acetylase inhibitor SAHA combined with ATRA significantly reactivated RARbeta2 mRNA both in NB4 and MR2 cells with degradation of PML-RARalpha, which was associated with maturation. In contrast, SAHA did not affect AML1-ETO levels and failed to induce RARbeta2 expression and maturation in Kasumi-1 cells. In primary AML samples, RARbeta2 expression was uniformly low; however, no specific correlation was observed between the methylation of the RARbeta2 gene and expression of the fusion proteins, PML-RARalpha, and AML1-ETO. These results demonstrate that oncogenic PML-RARalpha and AML1-ETO translocations are rarely associated with RARbeta2 promoter methylation in primary AML samples.

The multidrug resistance 1 (MDR1) gene product P-glycoprotein (P-gp) is frequently implicated in cross-resistance of tumors to chemotherapeutic drugs. In contrast, acute promyelocytic leukemia (APL) cells do not express MDR1 and are highly sensitive to anthracyclines. The combination of ATRA and the novel histone deacetylase inhibitor (HDACI) depsipeptide (FK228) induced P-gp expression and prevented growth inhibition and apoptosis in NB4 APL cells subsequently exposed to doxorubicin (DOX). ATRA/FK228 treatment after exposure to DOX, however, enhanced apoptosis. Both agents, ATRA or FK228, induced MDR1 mRNA. This effect was significantly enhanced by ATRA/FK228 administered in combination, due in part to increased H4 and H3-Lys9 acetylation of the MDR1 promoter and recruitment of the nuclear transcription factor Y alpha (NFYA) transcription activator to the CCAAT box. Cotreatment with specific P-gp inhibitor PSC833 reversed cytoprotective effects of ATRA/FK228. G1 cell-cycle arrest and p21 mRNA induction were also observed in response to ATRA/FK228, which may restrict DOX-induced apoptosis of cells in G2 phase. These results indicate that epigenetic mechanisms involving NF-YA transcription factor recruitment and histone acetylation are activated by ATRA and HDACI, induce MDR1 in APL cells, and point to the critical importance of mechanism-based sequential therapy in future clinical trials that combine HDAC inhibitors, ATRA, and anthracyclines.

In the title molecule, C14H13NO2, the dihedral angle between the planes of the benzene rings is 65.18 (4)°. The central amide group has about the same degree of twist with respect to both ring planes, as indicated by the dihedral angles of 34.70 (8) and 30.62 (8)° between its plane and that of the phenyl and 4-methoxybenzene rings, respectively. The C atom of the methoxy group is close to being coplanar with its attached ring [deviation = −0.112 (2) Å]. In the crystal, molecules are linked by inter-amide N—H...O hydrogen bonds, which generateC(4) chains propagating in the [100] direction. Adajcent molecules in the chain are related by translational symmetry.

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.

Hypoxia and interactions with bone marrow (BM) stromal cells have emerged as essential components of the leukemic BM microenvironment in promoting leukemia cell survival and chemoresistance. High levels of transforming growth factor beta 1 (TGFβ1) produced by BM stromal cells in the BM niche regulate cell proliferation, survival, and apoptosis, depending on the cellular context. Exogenous TGFβ1 induced accumulation of acute myeloid leukemia (AML) cells in a quiescent G0 state, which was further facilitated by the co-culture with BM-derived mesenchymal stem cells (MSCs). In turn, TGFβ-neutralizing antibody 1D11 abrogated rhTGFβ1 induced cell cycle arrest. Blocking TGFβ with 1D11 further enhanced cytarabine (Ara-C)-induced apoptosis of AML cells in hypoxic and in normoxic conditions. Additional constituents of BM niche, the stroma-secreted chemokine CXCL12 and its receptor CXCR4 play crucial roles in cell migration and stroma/leukemia cell interactions. Treatment with 1D11 combined with CXCR4 antagonist plerixafor and Ara-C decreased leukemia burden and prolonged survival in an in vivo leukemia model. These results indicate that blockade of TGFβ by 1D11 and abrogation of CXCL12/CXCR4 signaling may enhance the efficacy of chemotherapy against AML cells in the hypoxic BM microenvironment.

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.

Abstract 2438 CRM1, a member of the importin b super family of nuclear transport receptors, functions as a major nuclear export factor by shuttling transcription factors including p53, p21, I-kB, and FOXO3a from nucleus to cytoplasm, thereby preventing their activity. CRM1 is also involved in the transport of rRNA and a certain subset of mRNAs including Cyclin D1. Upregulated CRM1 expression has been reported to correlate with poor prognosis in various hematopoietic malignancies. MCL is a subtype of B-cell lymphoma which is frequently resistant to standard chemotherapy. The t(11,14)(q13;32) translocation of MCL juxtaposes the cyclin D1 gene, and constitutively overexpressed cyclin D1 is believed to be associated with oncogenesis. Additional genetic events such as mutation/overexpression of TP53 have been reported as adverse prognostic indicators. TP53 mutations are rare in typical MCL, although about 30% of aggressive blastoid MCL have mt-TP53. Because of the multiple signaling pathways that are dysregulated in MCL, a novel strategy aimed at restoring multiple anti-oncogenetic pathways, especially targeting p53-independent signaling pathways, is of considerable interest. In this study, we investigated the antitumor effects and molecular mechanisms of the SINE CRM1 antagonist KPT-185 (Karyopharm Therapeutics) in 4 MCL cells with known TP53 mutation status (wt-TP53: JVM2, Z138; mt-TP53: MINO, Jeko-1). Treatment with KPT-185 resulted in reduction of cell proliferation in a concentration-dependent manner without significant differences between wt- and mt-TP53 cells (IC50 at 72hrs by trypan blue exclusion method; 35nM for Z138, 92 nM for JVM2, 96 nM for MINO, 103 nM for Jeko-1). KPT-185 exhibited limited pro- apoptotic activity in the tested MCL cells except Z138 (ED50 at 48hrs by Annexin V positivity; 62 nM for Z138, 910 nM for JVM2, 665 nM for MINO, 618 nM for Jeko-1). We then investigated KPT-185-induced TP53 target gene expression changes (24 genes) by TaqMan low density arrays (TLDA) (Applied Biosystems). In wt-TP53 JVM2 and Z138 cells, KPT-185 (100nM for Z138, MINO, and 500nM for JVM2, Jeko-1) upregulated classical p53 targets such as p21 and MDM2 mRNA (>2.0 fold), while there was no increase in mt-TP53 MINO and Jeko-1 cells. Of note, in both wt- and mt-TP53 cells, KPT-185 upregulated gene expression of PUMA which is a target of FOXO3a, p73 and p53 (3.3 fold for JVM2, 2.5 fold for Z138, 3.3 fold for MINO, 4.8 fold for Jeko-1). Recently, CRM1 has been reported to positively modulate the nuclear export of Cyclin D1 mRNA in a eIF4E-dependent manner. We therefore examined Cyclin D1 protein levels by western blot analysis, and observed significantly high baseline expression of Cyclin D1 in Z-138 cells which are highly sensitive to KPT-185, as compared to less sensitive MCL cells. KPT-185 treatment decreased Cyclin D1 expression in a dose-dependent manner (50nM and 100nM) after 12hrs of treatment accompanied by p21 induction and decreased p-Rb. These findings demonstrate that KPT-185 successfully inhibits CRM1 activity in MCL resulting in inhibition of Cyclin D1 and cell proliferation, and in the p-53-independent upregulation of pro-apoptotic PUMA. In conclusion, CRM1 inhibition by KPT-185 results in cell growth inhibition and in moderate cell death in a TP-53 independent manner. Results also suggest that the sensitivity to KPT-185 in MCL may be dependent on Cyclin D1 expression. Therefore, KPT-185may be an effective agent for the treatment of MCL. Disclosures: Shacham: Karyopharm Therapeutics: Employment. Kauffman:Karyopharm Therapeutics Inc: Employment.

Abstract 2582 Survival of adult patients with acute monocytic leukemia remains dismal. We proposed the hypothesis that bone marrow (BM) adipocytes abundant in aging marrow microenvironment play a role in chemoresistance of AML cells. We have reported that BM adipocytes promote monocytic differentiation, activation, and survival of monoblastic leukemia cells (ASH, 2012).In this study, we examined the molecular mechanism of BM adipocytes causing survival and differentiation of monoblastic leukemia cells. Monoblastic leukemia cell line U937 and primary samples from acute monoblastic leukemia patients (n=6) were co-cultured with BM-derived marrow stromal cells (MSC) and MSC-differentiated adipocytes. Under limiting serum conditions, MSCs and adipocytes inhibited spontaneous cell death of U937 cells with decrease in subG1 fractions, more prominently in co-cultures with adipocytes than with MSCs (% subG1 fraction; cultured alone 23.9+9.0, co-cultured with MSC 15.7+1.9%, co-cultured with adipocytes 11.9+1.2%, p=0.04). Adipocytes but not MSC moderately reduced spontaneous apoptosis in primary leukemic blasts (n=6, p=0.02). We next focused on adipocytokines, lepin and plasminogen activator inhibitor 1(PAI-1), both implicated in cancer progression, as potential mediators of the protective effects by adipocytes. mRNA levels of leptin, leptin receptor and PAI-1 were up-regulated in MSC-derived adipocytes during adipocytic differentiation (relative expression to GAPDH, leptin: MSC 0, adipocyte 123.3+35.0; leptin receptor: MSC 0.16+0.05, adipocyte 4.47+0.37; PAI-1: MSC 314.9+46.5, adipocyte 3766.1+656.2). High level of secreted leptin was confirmed by ELISA in adipocyte conditioning medium (MSC 23.1+2.9, adipocyte 110.0+4.6 pg/mL). In serum-starved conditions, human recombinant leptin (5ng/mL) stimulated cell growth of monoblastic U937 cells known to express leptin receptor (35.8+7.9% increase of viable cell number at 48 hours, p=0.02) and inhibited apoptosis (%subG1 fraction; control 26.2+3.9, leptin16.4+4.1%, p=0.04).Human recombinant PAI-1 inhibited spontaneous and ara-C-induced apoptosis in U937 cells (11.2+1.1% and 15.1+1.7% decrease of Annexin V posivitity p=0.01). These results suggest that BM adipocytes produced leptin and PAI-1contribute to support myeloid leukemia cells survival within BM microenvironment. We next focused on molecular changes in U937 cells co-cultured with adipocytes. We found up-regulation of monocyte/macrophage differentiation gene PPARg and of PPARg target genes, CD36 and FABP4, by quantitative RT-PCR (relative expression to GAPDH, control vs co-culture with adipocytes: PPARg: 8.8+0.9 v.s.18.8+2.6, CD36; 105.0+11.8v.s.425.7+35.6; FABP4: 0.27+0.03v.s.153.7+12.8). The purity of U937 cells separated from adipocytes was confirmed by lack of CD90 mRNA expression by PCR. PPARγ is known to promote monocytic differentiation and uptake of oxidized cholesterol through the induction of scavenger receptor CD36, a protein marker of mature monocytes/macrophages. The up-regulation of cell surface CD36 expression by co-culture with adipocytes was indeed found in U937 cells and in 4 of the 6 primary AML samples by flow cytometory (increase in CD36 MFI: U937; 230.28, Pt samples; 46.4+20.2). These data suggest that BM adipocytes promote monocytic differentiation of monoblastic leukemia cells. In summary, our findings indicate that secretion of leptin and PAI-1 by BM adipocytes supports leukemic cell survival, while PAI-1 primarily and directly inhibits apoptosis of AML cells. It is conceivable that increased adipocyte content of BM in elderly AML patients may negatively affect the responsiveness of monocytic leukemia cells to chemotherapy. Disclosures: No relevant conflicts of interest to declare.

Abstract 4999 Natural killer (NK) cell neoplasms, including extranodal NK/T-cell lymphoma, nasal type (ENKL) and aggressive NK cell leukemia (ANKL), show a highly aggressive clinical course with poor response to chemotherapy, and new treatment approaches are urgently needed to improve cure rates. Patients with NK cell neoplasms cluster in Asia and Latin American countries, and the frequency of p53 mutations has been reported to be various by district. We have demonstrated that MDM2 protein was overexpressed in aggressive subclasses of NK cell neoplasms (Sugimoto et al. Jap J Cancer Res. 2002), which suggests that wild-type p53 expressing malignant NK cells may be a good candidate for biologic therapies that abrogate MDM2-p53 interactions and lead to cell death. Nutlin-3 is a small-molecule antagonist of MDM2 that efficiently blocks the MDM2-p53 interaction. In this study, we investigated the effects of nutlin-3 in 3 cell lines of ENKL and ANKL with known p53 mutation status (wt-p53: NK-YS, HANK-1; mt-p53: KHYG-1). Since aggressive NK-cell neoplasms arise in hypoxic environments and usually show an angiodestructive-infiltration pattern resulting in the tissue necrosis, we tried to assess the anti-proliferative effects and molecular mechanisms of nutlin-3 in the hypoxic condition. For hypoxia experiments, cells were cultured under 1.0% O2 for at least 14 days to assure their continuous proliferation and survival. Under hypoxia, more cells were positive to Annexin V than in normoxia, indicating that hypoxic conditions promote apoptosis in NK cell neoplasms. Nutlin-3 treatment in normoxia resulted in a reduction of cell proliferation with G0/G1 cell cycle arrest in a time and concentration-dependent manner in wt-p53 cells (IC50 at 48 hrs; 3.2 μM for NK-YS and 5.0 μM for HANK-1, MTT test). In hypoxia, nutlin-3 further enhanced cell growth inhibition and G0/G1 cell cycle arrest. An increase in the specific apoptosis (sub G1 and annexin V positivity) by nutlin-3 was observed with similar level between normoxia and hypoxia. The mt-p53 KHYG-1 cells demonstrated neither cell cycle arrest nor increase in the apoptotic cell fraction after nutlin-3 treatment. In the wt-p53 NK-YS and HANK-1 cells, nutlin-3 treatment increased the cellular levels of p53, and p53 dependent proteins including p21, MDM2 itself and the proapoptotic BH3-only proteins Noxa and Puma followed by the activation of caspase-9 and caspase-3 regardless of foxygen level. We observed no significant increase in the p53 targets in the mt-p53 overexpressing KHYG-1 cells. L-asparaginase has been demonstrated to induce apoptosis in aggressive NK cell neopplasms. To determine if inhibition of the TP53-MDM2 interaction by nutlin-3 in NK cell neoplasms might potentiate the effects of L-asparaginase, we assessed the effect of combining the two drugs. However, L-asparaginase induced apoptosis only in NK-YS cells, and no synergistic anti-proliferative effect was observed in any of the cell lines analyzed. These findings demonstrate that nutlin-3 successfully activates wt-p53 in NK cell neoplasms leading to the upregulation of traditional targets such as p21 and proapoptotic proteins including Noxa and Puma, and result in apoptotic cell death regardless of oxygen concentration. The data suggest that p53 activators such as nutlin-3 may be considerable for selected patients with wt-p53 NK cell neoplasms. Disclosures: No relevant conflicts of interest to declare.

Abstract 1501 Acute monoblastic leukemia occurs most commonly in young individuals, whereas acute monocytic leukemia is common in adults (WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues, 2008). Adipocytes are the prevalent stromal cell type in adult bone marrow (BM) and play an important role in the leukemic bone marrow microenvironment (Tabe et al., Blood 2004 103: 1815–22). BM stromal cells (MSCs) from elderly subjects have a reduced capacity to differentiate into osteoblasts and an increased capacity to differentiate into adipocytes, which leads to progressive accumulation of fat in the BM space with increasing age. In this study, we examined whether BM-derived adipocytes participate in the molecular events associated with proliferation, differentiation and apoptosis of monoblastic leukemia cells. To this end, we performed gene expression microarray analysis of 339 genes associated with lipid metabolism (GeneSQUARE, Kurabo, Japan) using RNA from monoblastic leukemia cell line U937 co-cultured with MSC or MSC-derived adipocytes. Five genes were upregulated (>2.0 fold) and 2 genes down-regulated in U937 cells co-cultured with adipocytes compared to U937 cultured alone. The up-regulated genes included monocyte/macrophage differentiation specific genes, such as scavenger receptor CD36, adipsin or fatty acid binding protein 4 (FABP4). CD36 and FABP4 are classical target genes of PPARγ which was also upregulated in U937 co-cultured with adipocytes. Upregulation of the inflammation-related haptoglobin gene was observed in U937 cells co-cultured with either adipocytes or MSC. These findings were confirmed by quantitative RT-PCR assay with concordant results. Down-regulated genes included monocyte chemoattractant protein-1 (MCP-1) (0.1 fold) and glycolytic enzyme hexokinase 2 (0.4 fold). In turn, U937 cells co-cultured with premature or mature adipocytes accumulated in G2 cell cycle phase compared to U937 cultured alone or co-cultured with MSC (% G2/M-phase fraction; U937 cultured alone, 14.6, co-culture with MSC 25.1, co-culture with preadipocyte 32.0, with mature adipocyte 31.9) and were protected from spontaneous cell death under serum-starved conditions (% subG1 fraction: U937 cultured alone 23.9%, co-cultured with MSC 15.7%, with preadipocytes 3.9% and with mature adipocytes 11.6%). In summary, results suggest that BM adipocytes, abundant in adult BM space, promote monocytic differentiation, proliferation and survival of monoblastic leukemia cells. We propose that these cells represent an essential component of the BM microenvironment that contributes to malignant phenotypes of adults with monocytic leukemia. Disclosures: No relevant conflicts of interest to declare.

Abstract 1424 Hypoxia confers pro-survival signals to tumor cells via multiple mechanisms including activation of phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR pathway. Constitutive activation of PI3K/Akt signaling pathway is documented in aggressive blastoid variants of mantle cell lymphoma (MCL) and is implicated in the pathogenesis of MCL. mTOR signaling, one of the important downstream targets of Akt, positively regulates protein translation by phosphorylating p70 ribosomal S6 kinase (S6K) or eukaryotic initiation factor 4E-binding protein-1 (4E-BP1). We have reported that the Class IA PI3K isoforms, mainly p110α and partially p110δ, are responsible for PI3K/Akt/mTOR signaling activation in blastoid MCL cells (Zhou et al., ASH abstract 2010). In this study, the activity and molecular mechanisms of action of a selective pan-isoform class I PI3K inhibitor GDC-0941 (Genentech) were investigated in the context of hypoxic microenvironment in MCL cells. For hypoxia experiments, MCL cells were cultured under 1% O2 for at least 14 days to assure their sustained proliferation and survival. Under hypoxia, more cells accumulated in G0/G1 phase, indicating that hypoxic conditions promote cell cycle quiescence in MCL cells. GDC-0941 treatment in normoxia resulted in a reduction of cell proliferation in a dose-dependent manner (IC50 at 48 hrs; 0.7 μM for Granta519, 0.5 μM for JVM2, 1.0 μM for MINO, 0.7 μM for Jeko-1, MTT test). In hypoxia, higher doses of GDC-0941 were required to cause cell growth inhibition compared to normoxic conditions in Granta519 (IC50; 1.7 μM), JVM2 (3.5 μM) and MINO (1.2 μM). Further, GDC-0941 failed to inhibit growth of Jeko-1 cells under hypoxia. GDC-0941 (0.5 μM) induced G0/1 cell cycle arrest under both normoxic and hypoxic conditions in Granta519, JVM2 and MINO cells. No significant induction of apoptosis by GDC-0941 (0.5 μM) was observed in any of the tested MCL cells. GDC-0941 completely inhibited phosphorylated (p-Ser473) Akt in all cell lines analyzed under both normoxic and hypoxic conditions, which resulted in decreased expression of p-GSK-3β and its downstream target cyclin D1. GDC-0941 further downregulated the mTOR downstream targets p-S6K and p-4EBP1 in Granta519, JVM2 and MINO cells under both, normoxia and hypoxia. On the contrary, GDC-0941 failed to affect hypoxia-induced p-S6K and p-4EBP1 expression levels in Jeko-1 cells, whereas potently suppressed these proteins at ambient oxygen level. These results suggest that in Jeko-1 cells hypoxia activates mTOR through PI3K-independent pathways. To test this, we examined consequences of mTOR blockade in Jeko-1 cells. mTOR inhibitor rapamycin (100 nM) moderately diminished cell growth under normoxic conditions causing accumulation of cells in G0/1 cell cycle phase but no apoptosis. In turn, under hypoxia rapamycin profoundly inhibited cell growth, inducing >80% of MCL cells to undergo cell death. These findings indicate that in certain MCL cells hypoxia activates mTOR through alternative, PI3K-independent pathways, which facilitate cell survival under hypoxia. Hence, blockade of class I PI3K may eliminate circulating MCL cells but not tissue-resident cells in hypoxic areas of the bone marrow or lymph nodes. In turn, simultaneous therapeutic ablation of PI3K/Akt and mTOR pathway may represent a promising strategy to target the aggressive blastoid variants of MCL cells in the hypoxic microenvironments. Disclosures: No relevant conflicts of interest to declare.

Abstract 2480 Hypoxia plays an important role in survival and chemoresistance of AML cells in bone marrow (BM) microenvironment. In AML, constitutively activating ITD mutations of FLT3 are associated with poor prognosis by activating downstream targets involved in the PI3K/AKT, RAS/MAPK or STAT5 signaling which confers oncogenic properties such as dysregulated proliferation, resistance to apoptosis, and blocked differentiation. We and others have demonstrated that the antileukemic effects of FLT3 inhibitors was less prominent in BM-resident leukemic FLT3-ITD blasts compared to circulating blasts (Zhang et al. J Natl Cancer Inst. 2008). <>The chaperone molecule 90-kDa heat shock proteins (Hsp90) is known to be highly expressed in malignant cells, and its activity is enhanced under hypoxic conditions. We have previously reported the cytotoxic efficacy of a tricyclic coumarin GUT-70 (5-methoxy-2,2-dimethyl-6-(2-methyl-1-oxo-2-butenyl)-10-propyl-2H,8H-benzo[1,2-b;3,4-b] dipyran-8-one (C23H26O5) (synthesized at Nippon Shinyaku, Kyoto, Japan) that induced degradation of Hsp90 client proteins including key components of multiple signaling pathways involved in cell proliferation and/or survival (Jin et al. Br.J. Cacer 2011). In this study, the anti-proliferative effects and molecular mechanisms of action of GUT-70 and another HSP90 inhibitor 17-DMAG (geldanamycin analog) were investigated in AML cells with/without FLT3-ITD mutation in hypoxic condition. Cells were adapted to hypoxia by culturing under 1.0% O2 for at least 14 days prior to experiments. The anti-proliferative effects of GUT-70 or 17-DMAG treatment were examined in 3 AML cell lines with known FLT mutation status (wt-FLT3: OCI-AML3; FLT3-ITD mutated: MOLM13, MV4;11) and paired isogenic Ba/F3 cells transfected with wt-FLT3 (Ba/F3-FLT3) or FLT3-ITD (Ba/F3-ITD) (kindly provided by Dr. D. Small). GUT-70 or 17-DMAG induced cell growth inhibition in a concentration-dependent manner with stronger effects in hypoxia than in normoxia (Table 1). In normoxic condition, GUT-70 or 17-DMAG caused more pronounced increase in the apoptotic fraction and G0/G1 accumulation in FLT3/ITD harboring cells than in wt-FLT3 cells. In hypoxia, however, wt-type FLT3 expressing cells were more sensitive to GUT-70 or 17-DMAG compared to the FLT3-ITD cells. Mutation status of p53 did not affect the anti-proliferative effects of GUT-70 or 17-DMAG, as determined by comparing anti-leukemia efficacy in OCI-AML3 and MOLM13 cells infected with p53 shRNA; and in wt- vs mt-p53 bearing MV4;11 (MV4;11R) cells. <>The mechanisms of action of GUT-70 or 17-DMAG were next investigated in FLT3-ITD bearing MOLM13 and Baf3/ITD, and in wt-FLT3 expressing OCI-AML3 and Baf3/FLT cells. Both GUT-70 and 17-DMAG induced Hsp70, a marker of Hsp90 inhibition, along with the degradation of Hsp90 client proteins, c-Raf, p-ERK, and p-Akt, without a significant difference between FLT3/ITD (+/−) cells irrespective to oxygen concentrations. The downregulation of Akt downstream targets p-GSK3b and p-4EBP1 as well as FLT3 target p-STAT5 were observed.The accumulation of cell cycle inhibitors p27 and p21 or proapoptotic BH3-only protein Noxa along with caspase-9 and -3 cleavage was more pronounced in hypoxia than in normoxia.Table 1.IC50 of GUT-70 and 17-DMAG in normoxia and hypoxia (MTT assay).IC50 ofGUT-70(μM)IC50 of17-DMAG(nM)NormoxiaHypoxiaaerobic IC50/hypoxic IC50NormoxiaHypoxiaaerobic IC50/hypoxic IC50OCI-AML37.74.61.750.518.92.7MOLM133.02.11.420.610.12.0MV4;115.94.31.418.12.09.1Baf3/FLT39.24.91.933.919.81.7Baf3/ITD7.33.91.923.215.21.5 In summary, our findings indicate that HSP-90 inhibitors successfully repress the pro-survival kinase pathways and stimulate cell cycle inhibitory and pro-apoptotic factors in FLT3-ITD as well as FLT3-wt AML cells under hypoxic condition. Results suggest that HSP-90 inhibitors may be useful in the eradication of chemoresistant AML cells sequestered in hypoxic BM niches. Disclosures: No relevant conflicts of interest to declare.

Abstract 926 We have previously reported pro-survival effects of TGF-β1 in myelo-monocytic leukemia cells (Xu et al.,Br J Haematol.2008). Hypoxia and interactions with bone marrow (BM) stromal cells have emerged as essential components of leukemic BM microenvironment that promote leukemia cell survival and chemoresistance. Our preliminary data indicate that TGF-β neutralizing antibody 1D11 (Genzyme) prevents accumulation of AML cells in a quiescent G0 state under co-culture condition with BM-derived mesenchymal stromal cells (MSC) (Jin et al., ASH abstract 2010). In turn, the chemokine CXCL12 and its receptor CXCR4 play crucial roles in cell migration and stroma/leukemia cell interactions. In this study, we investigated the anti-leukemic effects and molecular mechanisms of action of TGF-β neutralizing antibody 1D11 under hypoxic conditions. We further investigated the anti-leukemic efficacy of 1D11 combined with CXCR4 antagonist plerixafor in the in vivo leukemia models. AML cells (MV4;11 and U937) were propagated under 1% O2 for at least 14 days to assure their sustained proliferation and survival. Isotype control antibody 13C4 combined with ara-C induced no significant change in apoptosis or cell cycle progression. In MV4;11 cells cultured with 2ng/mL rhTGF-β1, 1D11 (10 μg/mL) induced only minimal apoptosis by itself, yet enhanced low-dose cytarabine (AraC, 0.5 μM) induced apoptosis. This effect was more prominent under hypoxia compared to normoxia (% of subG1 fraction, 21% O2: ara-C, 2.6 ± 0.2%, ara-C + 1D11, 10.8 ± 2.5%, p=0.03; 1% O2: ara-C, 11.3 ± 2.7%, AraC + 1D11, 21.4 ± 0.5%, p=0.001). 1D11 with ara-C abrogated rhTGFβ1-induced accumulation of cells in G0/G1 phase (21% O2; cont, 73.8 ± 4.1, rhTGFβ, 82.2 ± 3.2, rhTGFβ + AraC, 65.4 ± 2.5, rhTGFβ + AraC + 1D11, 50.3 ± 1.9, p=0.001: in 1% O2; cont, 71.8 ± 1.3, rhTGFβ, 85.4 ± 1.4, rhTGFβ + AraC, 79.3 ± 5.1, rhTGFβ + AraC + 1D11, 67.1 ± 4.0, p = 0.03). The anti-leukemic efficacy of 1D11 was next examined in an in vivo leukemia model. 1D11 administered at 5 mg/kg IP every other day in combination with ara-C (50 mg/kg IP weekly) decreased leukemia burden of nude mice injected with Baf3/ITD-luciferase leukemia cells (p=0.002). Administration of small molecule CXCR4 inhibitor plerixafor, which successfully diminished cell migration to CXCL12 in vitro, in combination with 1D11 decreased leukemia burden in vivo (p=0.05), and co-administration of ara-C, plerixafor and 1D11 was most effective (bioluminescence intensity, ×107 photons/sec) control, 1.2 ± 0.2; ara-C, 0.94 ± 0.3; plerixafor + 1D11, 0.56 ± 0.1; plerixafor + 1D11 + ara-C, 0.23 ± 0.09, p=0.003). We next examined the molecular mechanisms responsible for chemosensitization through blockade of TGFβ with 1D11. Treatment with rhTGF-β1 induced upregulation of p21 expression as well as pro-survival phosphorylation of Stat3 in MV4;11 and U937 cells, and these effects were abrogated by 1D11. Knock-down of Stat3 by siRNA increased apoptosis induction in U937 cells cultured in the presence of rhTGFβ1. Notably, 4-fold upregulation of the established TGFβ target, leukemia inhibitory factor (LIF) gene mRNA, was observed after rhTGF-β1 treatment and this was reversed by 1D11. These results indicate that 1D11 inhibits rhTGF-β1-induced autocrine stimulation of pro-survival LIF-JAK-Stat3 signal transduction pathway in AML cells. In summary, blockade of TGF-β by 1D11, and abrogation of CXCL12/CXCR4 signaling may enhance the efficacy of chemotherapy against AML cells in the hypoxic BM microenvironment. These findings warrant further investigations in human clinical trials. Disclosures: Konopleva: Genzyme: Research Funding.

Abstract 1814 Transforming growth factor β1 (TGF-β1) is an essential regulator of cell proliferation, survival, and apoptosis, depending on the cellular context. In the bone marrow (BM) microenvironment, high levels of TGF-β1 are known to affect cell-to-cell interactions between cancer and stromal cells and to stimulate cancer cells survival. We have previously reported pro-survival effects of TGF-β1 in myelo-monocytic leukemia cells (Xu et al., Br J Haematol. 2008). In turn, the chemokine CXCL12 and its receptor CXCR4 play crucial roles in cell migration and stroma/leukemia cell interactions. CXCR4 contains TGF-β-inducible element Smad-binding sites in the proximal enhancer region, and TGF-β has been reported to up-regulate CXCR4 expression in several cancer cells. In this study, we investigated the anti-proliferative effects of TGF-β neutralizing antibody in the context of BM microenvironment. Blocking TGF-β signaling with anti-TGF-β neutralizing antibody 1D11 (Genzyme) but not with isotype control antibody 13C4 decreased expression of the TGF-β downstream signaling targets plasminogen activator inhibitor-1 (PAI-1, mRNA) and Smad2 phosphorylation, in a dose-dependent manner (1-10 μg/mL) in three myelo-monocytic leukemia cell lines MV4;11, U937 and THP-1 cells. Treatment with rhTGF-β1 (2ng/mL, 72hrs) reduced cytarabine (0.5-2 μM) induced apoptosis (decrease in specific apoptosis measured by annexin flow cytometry: MV4;11, 9.2 +/− 2.3 %; U937, 14.5 +/− 0.7%; THP-1, 9.7 +/− 1.0 %, at 72 hrs). Further, rhTGF-β1 inhibited serum starvation-induced apoptosis and promoted G0/G1 cell cycle arrest (subG1, MV4;11: 17.8% vs 2.4%; U937, 45.9% vs 24.2%; G0/G1, MV4;11: 30.4% vs 50.6%, U937, 12.9% vs 35.6%, at 72 hrs), which was effectively reversed by anti-TGF-β antibody 1D11. Co-culture of leukemic cells with bone marrow (BM)-derived mesenchymal stromal cells (MSC) in the presence of rhTGF-β1 further increased the proportion of cells in G0/G1 phase (MV4;11: 75.5%, U937: 39.7%), which was partially reversed by 1D11. Under MSC co-culture condition, 1D11 treatment induced prominent G2/M accumulation (proportion of G2/M cells, 1D11 vs 13C4: MV4;11, 6.8% vs 27.3%, U937, 10.5% vs 15.0%) with minimal apoptosis induction. These results indicate that TGF-β inhibition prevents accumulation of AML cells in a quiescent Go state, but does not induce apoptosis in cells lodging in the BM microenvironment. We next evaluated effects of 1D11 antibody on CXCL12/CXCR4-mediated migration. While treatment with 1D11 only partially inhibited cell migration to CXCL12 in the presence of TGF-β1, CXCR4 antagonist AMD3100 completely diminished cell migration to CXCL12 with moderate increase of apoptosis in MSC co-cultures. Based on these findings, we are currently testing the hypothesis that the simultaneous blockade of TGF-β and CXCL12/CXCR4 signaling will inhibit AML cell migration and promote chemotherapy-induced cell killing of AML cells lodging in the BM microenvironment. The in vivo studies to test these hypotheses are ongoing and will be presented. Disclosures: Konopleva: Genzyme: Research Funding.

Abstract 1812 Deregulation of the phosphatidylinositol 3-kinase (PI3K)-mediated signaling plays an important role in the development of cell proliferation of mantle cell lymphoma (MCL). The PI3K pathway activation in MCL has been shown to result from constitutive B cell receptor (BCR) activation which is directly mediated by the Class IA PI3K p110 isoforms (a, β, and d). However, their relative contribution in MCL is not fully understood. In this study, the activity and molecular mechanisms of isoform-selective PI3K inhibitors which target different isoforms of the p110-kDa subunit has been investigated. We utilized the isoform-selective PI3K inhibitors; PI3-Ka inhibitor IV (p110a), TGX115 (p110b), IC87114 (p110d) and the non-specific PI3K inhibitor LY294002 (all inhibitors were purchased commercially). The p110a and p110d but not p110b isoform protein expression was detected in all tested MCL cell lines (Granta 519, JVM-2, Z138, Jeko-1, MINO). PI3-Ka inhibitor IV as well as non-specific PI3K inhibitor LY294002 induced cell growth inhibition with dose-dependent manner (IC50 at 48 hrs; PI3-Ka inhibitor IV: 17.5 μM for Granta 519, 14.3 μM for Jeko-1, 16.5 μM for Z138, LY294002: 14.8 μM for Granta 519, 19.4 μM for Jeko-1, 15.0 μM for Z138, MTT test). However, neither IC87114 nor TGX115 showed significant cell growth inhibition up to 40mM. Low dose of PI3-Ka inhibitor IV (5 μM) or LY294002 (5 μM) induced G0/G1 cell cycle arrest (increase of G0/G1 phase: PI3-Ka inhibitor IV 17.9 % for Granta 519, 28.2 % for Jeko-1, LY294002 19.3 % for Granta 519, 14.5 % for Jeko-1), and the higher dose (10 μM) increased apoptosis(specific apoptosis: PI3-Ka inhibitor IV 10.8 % for Granta 519, 15.3 % for Jeko-1, LY294002 13.6 % for Granta 519, 19.6 % for Jeko-1). No induction of cell cycle arrest/apoptosis by IC87114 or TGX115 treatment was observed. We then tried to assess the inhibition of PI3K/Akt signaling activation by p110a and p110d inhibitors. PI3-Ka inhibitor IV (10 μM) completely diminished phosphorylated (p-) Akt in all cell lines analyzed. Further investigation with 1–10 μM PI3-Ka inhibitor IV or IC87114 in Granta 519 and Jeko-1 cells declared that 1 μM PI3-Ka inhibitor IV almost diminished p-Akt and p-S6rp in both cells. The phosphorylation level of other PI3K/Akt signaling downstream substrates, GSK3-b and 4E-BP1, were down-regulated in dose dependent manner. Recently, GSK3-b kinase has been shown to negatively regulate cell cycle progression through Cyclin D1 repression in MCL. We observed that PI3-Ka inhibitor IV decreased Cyclin D1 expression and active pRb which are responsible for G0/G1 cell cycle arrest. The treatment with IC87114 (10 μM) performed moderate decrease of p-Akt, p-S6rp, and p-4E-BP, while no change in the levels of p-GSK3-b, Cyclin D1, or p-pRb was observed in both Granta 519 and Jeko-1 cells. We also tested whether the combination of PI3-Ka inhibitor IV or IC87114 with the proteasome inhibitor bortezomib induces synergistic cytotoxicity in MCL. No synergistic anti-proliferative effect was observed in any of the MCL cell lines analyzed. These findings demonstrate that p110a may be the responsible Class IA PI3K isoform for the development of MCL cell proliferation, and p110a isoform-selective PI3K inhibitor but not p110d or p110b inhibitors may provide a better therapeutic index relative to pan-PI3K inhibitors. Disclosures: No relevant conflicts of interest to declare.

Abstract 3977 Mantle cell lymphoma (MCL) is an aggressive B-cell lymphoma with poor prognosis, requiring novel anticancer strategies. Since p53 inactivating mutations occur primarily in the aggressive and refractory MCL variants, targeting p53-independent signaling pathways is of considerable interest. We previously reported the cytotoxic efficacy of a newly discovered tricyclic coumarin GUT-70 (5-methoxy-2,2-dimethyl-6-(2-methyl-1-oxo-2-butenyl) -10-propyl-2H,8H-benzo[1,2-b;3,4-b ]dipyran-8-one (C23H26O5) (synthesized at Nippon Shinyaku, Kyoto, Japan), originally derived from Calophyllum brasiliense, with more pronounced apoptotic effects in mutant-p53 MCL cells than in wild type-p53 cells (Jin et al., ASH abstract 2009). Some of the coumarin antibiotics are known to bind to chaperone molecule 90-kDa heat shock proteins (Hsp90) and induce degradation of Hsp90 client proteins including key components of multiple signaling pathways for cell proliferation and/or survival. In this study, the mechanisms of action of GUT-70 were investigated in MCL cell lines with known p53 mutation status (wt-p53: JVM-2, Granta-519, mt-p53: Jeko-1, MINO). GUT-70 demonstrated a dose-dependent binding affinity to Hsp90 (competitive binding assay using fluorescently labeled geldanamycin), increased ubiquitinated proteins accumulation, and further induced degradation of Hsp90 client proteins, including cyclin D1, Raf-1, Akt, and mt-p53, or increased Hsp70, a marker of Hsp90 inhibition. The downregulation of constitutively overexpressed cyclin D1 in MCL by GUT-70 was accompanied by p27 accumulation, decreased Rb phosphorylation, and impeded cell cycle progression in the wt-p53 JVM2 and Granta 519. However, GUT-70 induced apoptosis in mt-p53-bearing MINO and Jeko cells which was accompanied by only minimal cell cycle arrest. These findings suggest that apoptosis induction by GUT-70 in mt-p53 cells is in part independent from cell cycle arrest is known to protect cells from apoptosis. Moreover, the mt-p53 depletion by GUT-70 will further diminish its “gain of functions” for cell proliferation and anti-apoptosis. To determine if GUT-70 might potentiate the apoptotic effects of commonly used chemotherapeutic agents, we assessed the combination effects of GUT-70 with bortezomib (BTZ), a selective inhibitor of the 26S proteasome, and with doxoubicin (DOX), a conventional chemotherapeutic agent drug for MCL. Synergistic anti-proliferative effects of GUT-70 and BTZ or GUT-70 and DOX combinations were observed in both wt-p53 and mt-p53 MCL cells at 48 h post-exposure (combination index; GUT-70/Bortezomib; 0.59 for JVM2, 0.73 forMINO, GUT-70/Doxorubicin; 0.37 for JVM2, 0.35 for MINO). In conclusion, our results demonstrate that the novel anticancer agent tricyclic coumarin GUT-70, an Hsp90 inhibitor, has potential utility for mt-p53 bearing MCL cells, and in the combination therapy of MCL. Disclosures: No relevant conflicts of interest to declare.

Abstract 3390 In chronic myeloid leukemia (CML), the mechanisms of resistance to tyrosine kinase inhibitors (TKIs) beyond the Bcr-Abl mutations are not well understood. We have previously reported that TKI imatinib induces cell-surface expression of the chemokine receptor CXCR4, which results in enhanced migration towards CXCL12-producing BM stromal cells, promotes cell quiescence and development of the microenvironment-mediated, non-pharmacological drug resistance (Jin, Mol Cancer Ther 2008;7:48). Bcr-Abl tyrosine kinase directly activates Src-related kinase Lyn known to frequently localize in lipid raft plasma membrane microdomains and interact with CXCL12/CXCR4 signaling and is directly activated by p210Bcr-Abl. In this study, we investigated the effects of TKIs on the localization and interaction of CXCR4 and Lyn in the lipid rafts, and the role of lipid rafts as the signal transduction platform for CML cell migration. Confocal microscopy and discontinuous sucrose density gradient fractionation demonstrated that in CML cells CXCR4 primarily localized in the non-raft cell surface regions, while Lyn was present both in the lipid raft and non-raft fractions. In turn, the active, phosphorylated form (p-)LynTyr396 is present within the lipid rafts, while inactive p-LynTyr507 in non-raft fractions. Imatinib treatment under co-culture with mesenchymal stem cells (MSC) induced CXCR4 clustering in lipid raft fractions, which was directly co-immunoprecipitaed with Lyn. Under these culture conditions, imatinib repressed p-LynTyr507, but failed to deplete p-LynTyr396. Knock-down of Lyn by siRNA, Src inhibitor treatment, or lipid raft destruction by methyl-b cyclodextrin (MbCD) abrogated imatinib-induced KBM5 migration to MSCs and CXCL12 without affecting CXCR4 surface expression. Consistent with its effects on Src, dual Src/Abl kinase inhibitor dasatinib induced significantly less migration of CML cells to CXCL12 compared with imatinib or nilotinib (p =0.04). In summary, our data indicate that stromal cells interfere with inhibitory effects of TKI on active Lyn (p-Lyn)Tyr396 in CML cells and promote clustering of CXCR4 in lipid rafts where it co-localizes with p-LynTyr396 and facilitates migration of CML cells to the MSC monolayer. Lipid raft disruption by cholesterol depletion inhibit CML cells migration, suggesting that lipid rafts represent one of the key signaling modules responsible for interactions of CML cells with cells of BM niche. We propose that pharmacological disruption of lipid rafts may eliminate BM-resident CML cells through interference with microenvironment-mediated resistance. Disclosures: No relevant conflicts of interest to declare.

Abstract 777 Hypoxia and the interactions with bone marrow (BM) stromal cells have emerged as important mechanisms of leukemia cell survival and chemoresistance. The PI3K-Akt-mTOR-HIF1a signaling pathway has been shown to be activated in response to hypoxia in several cancer models including AML. Therefore, effective targeting of PI3K/Akt signaling pathway could suppress AML cell survival in the hypoxic BM microenvironment. Furthermore, concomitant intra-pathway blockade, or inhibition of the key pro-survival pathways may be more effective. In this study, we investigated the anti-leukemia effects and molecular mechanisms of apoptosis induction in the context of hypoxic bone marrow microenvironment by simultaneous use of a selective Class I PI3K inhibitor GDC-0941 (Genentech) with BH3 mimetic ABT-737 (Abbott) or FLT3 inhibitor sorafenib in AML cells. For hypoxia experiments, AML cells with FLT3-ITD mutation and wild-type FLT3 (FLT3-ITD: MOLM13, MV4;11, wt-FLT3: HL60) were cultured under 1.0% O2 for at least 14 days to assure their continuous proliferation and survival. Under hypoxia, more cells accumulated in G0/G1 phase, indicating that hypoxic conditions promote cell cycle quiescence in leukemic cells. GDC-0941 treatment in normoxia resulted in a reduction of cell proliferation with G0/G1 cell cycle arrest and minimal apoptosis induction, in a time and concentration-dependent manner (IC50 at 48 hrs; 0.7 μM for HL-60, 0.9 μM for MOLM13, 0.7 μM for MV4;11). In hypoxia, GDC-0941 further enhanced cell growth inhibition and G0/G1 cell cycle arrest. Importantly, co-culture with BM mesenchymal stroma cells (MSC), which protected AML cells from cytarabine induced apoptosis, did not affect cell growth inhibition by GDC-0941 both in normoxia and hypoxia. Combined treatment of GDC-0941 synergistically enhanced the ABT-737- or sorafenib induced apoptosis under both normoxic- and hypoxic conditions (p We next examined the molecular mechanisms of apoptosis induction by simultaneous blockade of PI3K and mutant FLT3 in MOLM13 cells. GDC-0941 inhibited phospho-AktSer473, irrespective of oxygen tension. Hypoxia increased/induced phosphorylation of AktSer473, and of the pro-survival serine/threonine-protein kinase Pim-1, known to promote hypoxia-induced chemoresistance. Pim-1 is a downstream target of Akt and FLT-3 signaling that promotes cell survival and inhibits apoptosis through Bcl-2-dependent mechanisms. While GDC-0941 or ABT-737 alone did not affect hypoxia-induced Pim-1 levels, the GDC-0941/ABT-737 combination down-regulated Pim-1 by 86% (24 hrs). Treatment with sorafenib induced Pim-1 down-regulation by itself by 70%. Under hypoxia, expression levels of Bcl-2 family proteins Bcl-2 and BIM were not significantly changed by any treatment and cell culture conditions. In turn, Mcl-1 expression was upregulated by MSC co-cultures. Further, sorafenib alone but not GDC-0941 or ABT-737 decreased Mcl-1 protein levels, which was reversed by MSC co-culture in hypoxia; in turn, the GDC-0941/sorafenib or GDC-0941/ABT-737 combination potently suppressed Mcl-1 expression under MSC/hypoxia conditions. These changes were associated with the observed apoptotic responses. In summary, our findings indicate that hypoxic conditions of BM microenvironment result in stimulation of the pro-survival Akt and PIM kinase pathways and increase anti-apoptotic Mcl-1 protein in AML cells with mutated FLT3. In turn, simultaneous blockade of PI3K and FLT3 pathways, or of PI3K and Bcl-2 results in inhibition of these signaling modules and synergistic induction of apoptosis in AML. This data suggest that combined inhibition of Bcl-2 with PI3K or FLT3-ITD may constitute a targeted approach to eradicate chemoresistant AML cells sequestered in the hypoxic BM niches. Disclosures: No relevant conflicts of interest to declare.