ALBERT

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

Description: Adult T-cell acute lymphoblastic leukemia (T-ALL) has a poor prognosis associated with resistance and rapid relapses, necessitating novel therapeutic strategies. Malignant T cells require high concentrations of nutrients to sustain their increased rates of proliferation, and frequent activation of mTOR signaling, a central regulator of cell metabolism, has been shown in a high-risk subgroup of T-ALL with constitutive Notch activation (Chan, Blood, 2007;110,278). In this study, we analyzed the metabolic response mechanisms of Notch-activated aggressive T-ALL to the ATP-competitive mTOR inhibitor AZD2014 and to the combination of AZD2014 with a conventional chemotherapeutic agent, L-asparaginase (ASNase). To investigate the molecular modifications of key cellular metabolic processes induced by these agents, we undertook a comprehensive and quantitative analysis of charged metabolites by capillary electrophoresis mass spectrometry (CE-MS, Agilent Technologies) for metabolic profiling. We first assessed the anti-proliferative effects of the combination of AZD2014 and ASNase in CUTLL1, a T-ALL cell line with a Notch gain-of-function mutation. The medianinhibitory concentrations of AZD-2014 and ASNase were 250 nM and 0.005 U/mL, respectively (MTT assay, 48 hours). The combination of AZD2014 (100 nM) and ASNase (0.001 U/mL) caused apoptosis induction with increase in the % of sub-G1 fraction (Control: 5.5±1.8, AZD-2014: 9.5±2.1, ASNase: 10.9 ±1.5, AZD-2014/ASNase: 17.9 ±4.9; p

Description: Adipocytes are the prevalent stromal cell type in adult bone marrows (BM). With increasing age, BM stroma-resident mesenchymal stem cells (MSCs), increase their capacity to differentiate into adipocytes, which leads to the progressive accumulation of fat in the BM space. It is conceivable that the increased BM adipocyte content promotes leukemogenesis and negatively affects responsiveness to chemotherapy. We previously reported that free fatty acids (FFAs) promote the metabolic shift from pyruvate oxidation to fatty acid oxidation (FAO), which causes uncoupling of mitochondrial oxidative phosphorylation and promotes leukemia cell survival (Samudio, J Clin Invest. 2010). We further demonstrated the prominent antiapoptotic effects of BM-derived adipocytes co-cultured with cells from acute monocytic leukemia (AMoL), a poor-prognosis subtype of AML (Tabe ASH. 2013). Proteomic analysis with isobaric tags for relative and absolute quantification (iTRAQ) showed upregulation of protein folding pathways which increases the expression of antiapoptotic chaperone proteins HSP70 and HSP90, of integrin-mediated cell adhesion and migration pathways and downregulation of oxidative phosphorylation along with repression of cytochrome c.Metacore gene ontology (GO) analysis identified NF-kB, c-Jun, SP1, AP-1, and HMG as the potent relevant transcription factors that closely interact with and activate chaperone proteins, chromatin, and gene transcription. In this study, we characterized a gene signature linking transcription with metabolic changes that contribute to AMoL cell survival under conditions mimicking aging BM with prevalent adipocytes. We confirmed the antiapoptotic role of FFAs produced by the primary BM MSC-derived adipocytes via pharmacologic inhibition of FAO by etomoxir (EX), which inhibits fatty acid entry into the mitochondria. EX (50mM) treatment reversed the prosurvival effects of adipocytes on serum-starved U937 monoblast cells (% Annexin V, -/+ EX: mono-culture, 30.1±9.0 / 31.5±4.6; co-culture with adipocytes, 8.9±2.1 / 29.6±9.2; P=0.02). To assess the molecular links between metabolic pathways and gene expression triggered by BM adipocytes, we performed RNA-seq transcriptome analysis with a next generation sequencer system HiSeq1500 (Illumina) using TopHat software for alignment and Cufflinks software for identifying differential gene expression. RNA-Seq detected upregulation of 21 genes in U937 cells after co-culture with BM-derived adipocytes (false discovery rate,

Description: Mantle cell lymphoma (MCL), which overexpresses cyclin-D1 through an alteration in the t(11;14)(q13;q32) chromosomal region, is associated with brief disease-free and overall survival durations characteristic of aggressive B-cell lymphomas. Bruton tyrosine kinase (BTK) has been identified as a key component of the B-cell antigen receptor (BCR) signaling pathway and is implicated in the pathogenesis of certain B-cell malignancies. Phase III clinical trials of BKT inhibitor ibrutinib in MCL patients have demonstrated clinical responses characterized by mobilization of tissue-resident MCL cells into the peripheral blood. However, since the time to maximum response with ibrutinib is relatively long and patients may become resistant to BTK inhibition, combination regimens that accelerate time to remission and increase depth of remission are of considerable interest. We hypothesized that combinations of ibrutinib with proapoptotic drugs that function independently of BCR signaling could yield synergistic anti-lymphoma interactions. Thus we investigated the antitumor effects and molecular mechanisms of simultaneous treatment with ibrutinib and selexinor, an oral selective inhibitor of nuclear export (SINE)(KPT-330, Karyopharm), or ABT-199, a selective Bcl-2 inhibitor. SINE agents exhibit antiproliferative and proapoptotic activities against MCL cells via inhibition of nuclear export of tumor suppresor proteins, transcription factors and oncogenic mRNAs and repression of ribosomal biogenesis (Tabe et al. ASH 2013). Selinexor showed promising anti tumor activity in agrresive lymphoma as part of ongoing Phase 1 study (ASCO 2014). ABT-199 has promising proapoptotic activity in relapsed/refractory CLL and NHL without inducing thrombocytopenia. In this study, we utilized four MCL cell lines: MINO, Z138, Jeko-1, and JVM2. Inhibition of BTK activity by ibrutinib resulted in reduction of cell proliferation in a dose-dependent manner with G0/G1 cell cycle arrest but no apoptosis induction (IC50 at 48 hrs by MTT: 5.4 mM for MINO, 3.5 mM for Z138, 0.5 mM for Jeko-1, 3.1 mM for JVM2). Western blot analysis demonstrated ibrutinib-induced downregulation of phospho-(p-)BTK, p-Akt, mTORC1 substrates p-S6K and p-4EBP1, and cyclin D1 expression. Single-agent selinexor induced cell growth inhibition with G0/G1 cell cycle arrest in a dose-dependent manner (IC50 ranging from 10 nM to 130 nM). The ibrutinib/selinexor combination resulted in further decrease of p-4EBP1 and cyclin D1 expression and downregulation of p-Rb, c-Myc, and Mcl-1, which translated into synergistic reduction of cell proliferation in three of the four tested cell lines (combination index [CI]: 0.4 for MINO, 0.2 for Jeko-1, 0.2 for JVM2). ABT-199 inhibited cell proliferation with apoptosis induction in MINO and Z138 cells (IC50: 1.5 nM for MINO, 17.5 nM for Z138), which synergistically enhanced the antiproliferative effects of ibrutinib (CI: 0.6 for MINO, 0.8 for Z138) with striking reductions of p-4EBP1, cyclin D1, p-Rb, and c-Myc expression along with induction of Bax and cleaved caspase-3. To investigate the molecular modifications of the cellular pathway network in response to BTK blockade by ibrutinib alone or in combination with selinexor or ABT-199, we employed the proteomic technology of isobaric tags for relative and absolute quantitation (iTRAQ). In MINO cells, iTRAQ identified 1,401 unique proteins. Ibrutinib induced downregulation of isoform BTK (p=0.02) and the cell cycle initiation of mitosis pathway (p=0.003) with decreases of ribosomal proteins and elongation factors. Combination with selinexor upregulated the apoptosis and oxidative stress–associated pathways with increases of cytochrome c, voltage-dependent anion channels, HSP10, and histone H1, all of which function as dynamic initiators of mitochondria-mediated apoptosis (p 〈 0.05). ABT-199 by itself induced upregulation of the apoptosis and oxidative stress–associated pathways (p 〈 0.001), and these effects were significantly enhanced by combination with ibrutinib. Taken together, our findings suggest that treatment with combinations of ibrutinib and selinexor or ABT-199 exerts synergistic antiproliferative effects through inhibition of mTOR signaling, downregulation of ribosomal biosynthesis, and induction of mitochondria-mediated apoptosis. These combinations warrant further evaluation in clinical trials in MCL patients. Disclosures Andreeff: Karyopharm: Research Funding.