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SIRT6 is one of seven human sirtuin genes and is known to act as an onco-suppressor gene in colorectal and ovarian cancers, although it is up-regulated in other cancers. Thus, SIRT6 is considered performing both tumor-suppressing and promoting roles. However, the association of SIRT6 with oral squamous cell carcinoma (OSCC) and its role in OSCC pathogenesis is currently unclear. This study aimed to investigate the expression of SIRT6 in patients with OSCC and its potential as a biomarker for early detection and prognosis prediction.Immunohistochemistry, quantitative real-time RT-PCR, and microarray analyses were performed to determine SIRT6 expression and its association with clinicopathological features in OSCC using clinical specimens.SIRT6 mRNA and protein expression levels were higher in OSCC tissues than in noncancerous tissues (p0.05). SIRT6 expression was predominant in patients aged ≥65 years and significantly correlated with shorter overall survival. In the microarray analysis, some SIRT6-associated genes, such as ANXA2, were up-regulated in OSCC.SIRT6 plays a role in tumor homeostasis, leading to a poor prognosis in OSCC. SIRT6 may represent a novel target not only for treatment, but also as a prognostic marker in OSCC.

Co-culture with MSC-derived BM-adipocytes inhibits spontaneous apoptosis of primary AMoL cells (S1); Co-culture with BM adipocytes activates p38MAPK cascade and protein folding in AMoL cells (S2); Network of proteins involved in the effects of BM-adipocyte co-culture in U937 cells (S3).

Leukemia cells in the bone marrow must meet the biochemical demands of increased cell proliferation and also survive by continually adapting to fluctuations in nutrient and oxygen availability. Thus, targeting metabolic abnormalities in leukemia cells located in the bone marrow is a novel therapeutic approach. In this study, we investigated the metabolic role of bone marrow adipocytes in supporting the growth of leukemic blasts. Prevention of nutrient starvation–induced apoptosis of leukemic cells by bone marrow adipocytes, as well as the metabolic and molecular mechanisms involved in this process, was investigated using various analytic techniques. In acute monocytic leukemia (AMoL) cells, the prevention of spontaneous apoptosis by bone marrow adipocytes was associated with an increase in fatty acid β-oxidation (FAO) along with the upregulation of PPARγ, FABP4, CD36, and BCL2 genes. In AMoL cells, bone marrow adipocyte coculture increased adiponectin receptor gene expression and its downstream target stress response kinase AMPK, p38 MAPK with autophagy activation, and upregulated antiapoptotic chaperone HSPs. Inhibition of FAO disrupted metabolic homeostasis, increased reactive oxygen species production, and induced the integrated stress response mediator ATF4 and apoptosis in AMoL cells cocultured with bone marrow adipocytes. Our results suggest that bone marrow adipocytes support AMoL cell survival by regulating their metabolic energy balance and that the disruption of FAO in bone marrow adipocytes may be an alternative, novel therapeutic strategy for AMoL therapy. Cancer Res; 77(6); 1453–64. ©2017 AACR.

Clinical characteristics of AMoL patients (S1); Quantitative expression of metabolites in U937 cells after BM-adipocyte co-culture and etomoxir treatment (S2); Genes whose expression was altered in U937 cells co-cultured with MSCs or BM-adipocytes and treated with etomoxir (S3); Genes whose expression was altered most often in U937 and THP1 cells by BM-adipocyte co-culture and treatment with etomoxir (S4); Proteins whose expression was altered in U937 cells by BM-adipocyte co-culture (S5).

Background: Anaplastic thyroid carcinoma (ATC) is a highly aggressive thyroid tumor with a poor prognosis. However, there are limited choices for ATC treatment. Recently, the effectiveness of antibody–drug conjugates has been demonstrated in various carcinomas. Whether the targets of antibody–drug conjugates are expressed in anaplastic thyroid carcinoma remains unclear. Methods: Fifty-four patients with ATC were enrolled in this study. Tissue microarrays were constructed using the archives of formalin-fixed paraffin-embedded tissue blocks. All sections were stained with the following antibody–drug conjugate targets: human epidermal growth factor receptor 2 (HER2), nectin-4, trophoblast cell surface antigen 2 (TROP-2), glycoprotein non-metastatic B (GPNMB), and B7-H3. Results: HER2 was negative in all tissues, whereas GPNMB and B7-H3 were expressed in most ATC tissues. TROP-2 and nectin-4 were expressed in 65% and 59% of ATC tissues, respectively. TROP-2 was expressed at significantly higher levels in ATC undifferentiated from papillary thyroid carcinoma than in ATC undifferentiated from follicular thyroid carcinoma and de novo ATC. In contrast, nectin-4 expression was markedly higher in patients with de novo ATC than in those with papillary and follicular thyroid carcinoma. Conclusions: TROP-2 and nectin-4 are potential therapeutic targets for ATC undifferentiated from papillary thyroid carcinoma and de novo ATC, respectively. GPNMB and B7-H3 potential for treating all types of ATC.

Leukemia cells in the bone marrow must meet the biochemical demands of increased cell proliferation and also survive by continually adapting to fluctuations in nutrient and oxygen availability. Thus, targeting metabolic abnormalities in leukemia cells located in the bone marrow is a novel therapeutic approach. In this study, we investigated the metabolic role of bone marrow adipocytes in supporting the growth of leukemic blasts. Prevention of nutrient starvation–induced apoptosis of leukemic cells by bone marrow adipocytes, as well as the metabolic and molecular mechanisms involved in this process, was investigated using various analytic techniques. In acute monocytic leukemia (AMoL) cells, the prevention of spontaneous apoptosis by bone marrow adipocytes was associated with an increase in fatty acid β-oxidation (FAO) along with the upregulation of PPARγ, FABP4, CD36, and BCL2 genes. In AMoL cells, bone marrow adipocyte coculture increased adiponectin receptor gene expression and its downstream target stress response kinase AMPK, p38 MAPK with autophagy activation, and upregulated antiapoptotic chaperone HSPs. Inhibition of FAO disrupted metabolic homeostasis, increased reactive oxygen species production, and induced the integrated stress response mediator ATF4 and apoptosis in AMoL cells cocultured with bone marrow adipocytes. Our results suggest that bone marrow adipocytes support AMoL cell survival by regulating their metabolic energy balance and that the disruption of FAO in bone marrow adipocytes may be an alternative, novel therapeutic strategy for AMoL therapy. Cancer Res; 77(6); 1453–64. ©2017 AACR.

// Kazumasa Sekihara 1, 2 , Kaori Saitoh 1 , Lina Han 3 , Stefan Ciurea 3 , Shinichi Yamamoto 1, 2 , Mika Kikkawa 4 , Saiko Kazuno 4 , Hikari Taka 4 , Naoko Kaga 4 , Hajime Arai 4 , Takashi Miida 1 , Michael Andreeff 3 , Marina Konopleva 3 , Yoko Tabe 1, 3, 5 1 Department of Laboratory Medicine, Juntendo University Graduate School of Medicine, Tokyo, Japan 2 Leading Center for the Development and Research of Cancer Medicine, Juntendo University Graduate School of Medicine, Tokyo, Japan 3 Section of Molecular Hematology and Therapy, Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA 4 Laboratory of Proteomics and Biomolecular Science, Research Support Center, Juntendo University Graduate School of Medicine, Tokyo, Japan 5 Department of Next Genertion Hematology Laboratory Medicine, Juntendo University Graduate School of Medicine, Tokyo, Japan Correspondence to: Yoko Tabe, email: tabe@juntendo.ac.jp Keywords: mantle cell lymphoma, mTORC1/2, selective inhibitor of nuclear export, XPO1, metabolism Received: May 31, 2016 Accepted: March 16, 2017 Published: March 27, 2017 ABSTRACT Mantle cell lymphoma (MCL) is an aggressive B-cell lymphoma with poor prognosis, characterized by aberrant expression of growth-regulating and oncogenic effectors and requiring novel anticancer strategies. The nuclear transporter exportin-1 (XPO1) is highly expressed in MCL and is associated with its pathogenesis. mTOR signaling, a central regulator of cell metabolism, is frequently activated in MCL and is also an important therapeutic target in this cancer. This study investigated the antitumor effects and molecular/metabolic changes induced by the combination of the small-molecule selective inhibitor XPO1 inhibitor KPT-185 and the dual mTORC1/2 kinase inhibitor AZD-2014 on MCL cells. AZD-2014 enhanced the KPT-185–induced inhibition of cell growth and repression of cell viability. The combination of KPT-185 and AZD-2014 downregulated c-Myc and heat shock factor 1 (HSF1) with its target heat shock protein 70 (HSP70). As a consequence, the combination caused repression of ribosomal biogenesis demonstrated by iTRAQ proteomic analyses. Metabolite assay by CETOF-MS showed that AZD-2014 enhanced the KPT-185–induced repression of MCL cellular energy metabolism through the TCA (Krebs) cycle, and further repressed KPT-185–caused upregulation of glycolysis. Thus the simultaneous inhibition of XPO1 and mTOR signaling is a novel and promising strategy targeting prosurvival metabolism in MCL.

Adipocytes are the prevalent stromal cell type in adult bone marrow (BM), and leukemia cells continuously adapt to deficiency of nutrients acquiring chemoresistant profiles in the BM microenvironment. We have previously shown that fatty acid metabolism is a key energy pathway for survival of acute myeloid leukemia (AML) cells in the adipocyte-abundant BM microenvironment. The novel fatty acid β-oxidation (FAO) inhibitor avocatin B, an odd-numbered carbon lipid derived from the avocado fruit, induced apoptosis and growth inhibition in mono-cultured AML cells. In AML cells co-cultured with BM adipocytes, FAO inhibition with avocatin B caused adaptive stimulation of free fatty acid (FFA) uptake through upregulation of FABP4 mRNA, enhanced glucose uptake and switch to glycolysis. These changes reflect the compensatory response to a shortage of FFA supply to the mitochondria, and facilitate the protection of AML cells from avocatin B–induced apoptosis in the presence of BM adipocytes. However, the combination treatment of avocatin B and conventional anti-AML therapeutic agent cytarabine (AraC) increased reactive oxygen species and demonstrated highly synergistic effects on AML cells under BM adipocyte co-culture condition. These findings highlight the potential for combination regimens of AraC and FAO inhibitors that target bone marrow-resident chemoresistant AML cells.

We sought to gain a better understanding of the low-dose ionizing radiation (LDIR)-induced molecular changes in transformed pre-malignant cells in their microenvironment.The cellular response to LDIR was compared and contrasted using immortalized human Epstein-Barr virus-infected B-cells (EBV-B) in mono-culture, co-culture with human bone marrow derived stromal cells (MSC), or under the LDIR-induced bystander effect. The resulting alterations in protein and gene expression (including microRNA, miRNA) were evaluated by isobaric tags for relative and absolute quantification (iTRAQ) proteomics assay, western blot, cDNA array and quantitative reverse transcription polymerase chain reaction (RT-PCR), respectively.The miRNAs let7a, miR-15b, miR-16, and miR-21, and a lipid metabolic miRNA hub miR-23b, were upregulated after LDIR exposure in the mono-cultured EBV-B cells, but were downregulated in EBV-B cells co-irradiated with MSC. A lipid biosynthesis enzyme glycerol-3-phosphate acyltransferase, the common target of these miRNA, was downregulated at the level of protein and mRNA expression in the LDIR-exposed, mono-cultured EBV-B cells and upregulated MSC co-cultured EBV-B cells.These results suggest a putative miRNA regulatory mechanism controlling the LDIR-induced stress response, and illustrate that LDIR exposure, and the cell's microenvironment, can affect specific gene expression, both directly and indirectly, resulting in altered protein expression.

Acute myeloid leukemia (AML), the most common acute leukemia in adults, remains a therapeutic challenge. The phosphatidylinositol 3-kinase/AKT/mammalian target of rapamycin (PI3K/AKT/mTOR) signaling pathway is one of the key aberrant intracellular axes involved in AML. Areas covered: mTOR plays a critical role in sensing and responding to environmental determinants such as nutrient availability, stress, and growth factor concentrations; and in modulating key cellular functions such as proliferation, metabolism, and survival. Although abnormalities of mTOR signaling are strongly associated with neoplastic leukemic proliferation, the role of pharmacologic inhibitors of mTOR in the treatment of AML has not been established. Expert opinion: Inhibition of mTOR signaling has in general modest growth-inhibitory effects in preclinical AML models and clinical trials. Yet, combination of allosteric mTOR inhibitors with standard chemotherapy or targeted agents has a greater anti-leukemia efficacy. In turn, dual mTORC1/2 inhibitors, and dual PI3K/mTOR inhibitors show greater activity in pre-clinical AML models. Further, understanding the role of mTOR signaling in stemness of leukemias is important because AML stem cells may become chemoresistant by displaying aberrant signaling molecules, modifying epigenetic mechanisms, and altering the components of the bone marrow microenvironment.

Accelerated hyperfractionated radiotherapy was performed as treatment for patients with T1 glottic cancer, and its utility was evaluated based on treatment outcomes and adverse effects. Fifty-eight men who had undergone radiotherapy were retrospectively reviewed. Tumor classification was Tis in 4 patients, T1a in 38, and T1b in 16. Histological examination revealed squamous cell carcinoma in 55 patients. Travel time from home to hospital was 0–1 hour for 24 patients, 1–2 hours for 9, and >2 hours for 25. Laser vaporization was performed prior to radiotherapy in 38 patients, and 19 patients received concurrent chemotherapy with an agent such as S-1. Patients were irradiated twice daily using an irradiation container. Most patients received a dose of 1.5 Gy/fraction up to a total of 60 Gy. The median overall treatment time was 30 days, with a median observation period of 59.6 months. A complete response was observed in all patients. The 5-year overall survival, disease-free survival, and local control rates were 97.2%, 93.2%, and 97.8%, respectively. Although grade 3 pharyngeal mucositis was observed in 2 patients, there were no other grade 3 or higher acute adverse events. As late toxicity, grade 2 laryngeal edema and grade 1 laryngeal hemorrhage were observed in 1 patient each, but no serious events such as laryngeal necrosis or laryngeal stenosis were observed. In conclusion, this treatment method brings excellent outcome and will substantially reduce the treatment duration among patients who need to stay at nearby hotels while undergoing treatment at hospitals in rural areas.

Mutations in the gene encoding Fms-like tyrosine kinase-3 (FLT3) are found in approximately 30% of acute myeloid leukemia (AML) patients, with two distinct groups of mutations; the internal tandem duplication mutations in juxtamembrane domain (FLT3-ITD) and the point mutations within the tyrosine kinase domains (FLT3-TKD). The second-generation tyrosine kinase inhibitor (TKI) quizartinib which primarily targets an inactive (type II) kinase conformation, has demonstrated responses in relapsed/refractory FLT3-ITD AML patients. However, duration of response to quizartinib is limited largely by secondary mutations in FLT3-TKD, most commonly at D835 activation loop, emerging in at least 20% of patients. To elucidate the alterations of transcript signatures of quizartinib-resistant FLT3-ITD/D835 mutants in AML, we performed cap analysis of gene expression (CAGE) sequencing utilizing 11 FLT3-ITD and 6 FLT3-ITD/D835 primary AML cells with wild type NPM1. CAGE identifies and quantifies the 5' ends of capped mRNA transcripts, which enables the identification of transcription start sites (TSS) and allows investigating promoter structures necessary for gene expression. The TSS of genes altered in AML cells with FLT3-ITD/D835 compared with FLT3-ITD were mapped, and CAGE detected upregulation of 124 genes and downregulation of 25 genes in FLT3-ITD/D835 cells (FDR < 0.05). Specifically, several genes closely linked with activation of MAPK signaling (MAPK6, TCF4), AKT/mTOR signaling (NBN) and both (TNFAIP6) were upregulated in the D835 acquired cells. Expression of inflammatory response cytokine genes IL1B and IL8 were also increased in FLT3-ITD/D835 cells. The FLT related functional pathway analysis by KEGG ontology indicated that Ras pathway with its downstream Raf/MAPK and PI3K/AKT/mTOR signaling including transcription factor NFKB are the most significantly activated pathways in FLT3-ITD/D835 cells compared to FLT3-ITD cells. No additional activation of STAT5 pathway, a direct downstream signaling of mutated FLT3, was found in FLT3-ITD/D835 cells. To assess the underlying molecular basis of transcription signature, we performed an unbiased search for enriched sequence motifs using HOMER software. HOMER revealed upregulation of 80 motifs and downregulation of 51 motifs in AML cells with FLT3-ITD/D835 compared with FLT3-ITD (q < 0.05). Motifs of AP-1, JUN, CEBP, ATF and NFKB were substantially enriched in FLT3-ITD/D835 compared to FLT3-ITD cells. ETS, SPI-1 and RUNX1 were the most common binding motifs altered (both increased and decreased) by D835 mutation acquisition. Of note, AP-1 modulates inflammatory response and is tightly regulated by MAPK. To validate the transcriptional changes and the highlighted gene ontology results of AML primary samples, we utilized paired isogenic FLT3-ITD or FLT3-ITD/D835Y transfected Ba/F3 cell lines. As predicted, type II kinase inhibitor quizartinib was active against FLT3-ITD Ba/F3 cells (IC50; 0.6nM) but not FLT3-ITD/D835Y Ba/F3 cells (IC50; > 5000nM). Type I kinase inhibitor crenolanib was highly active against both FLT3-ITD (IC50; 2.1nM) and FLT3-ITD/D835Y cells (IC50; 16.4nM). Focusing on FLT related Ras pathway, we first investigated MAPK activation in these cells. Immunoblot analysis demonstrated substantial reduction of phospho- (p-) ERK after quizartinib (5nM) or crenolanib (50nM) treatment both in FLT3-ITD and FLT3-ITD/D835Y cells, irrespective of their susceptibility. We next examined p-S6, the target of PI3K/AKT/mTOR and MAPK signaling, and observed that crenolanib but not quizartinib successfully diminished p-S6 expression in FLT3-ITD/D835Y cells. FLT3-ITD/D835 cells showed higher baseline expression of NFkB regulator p-IkB compared to FLT3-ITD cells, which was not repressed by crenolanib. In conclusion, our study showed that the acquired D835 mutation in FLT3-ITD AML cells facilitates transcriptional network activation to induce proinflammatory genes through Ras-MAPK-NF-κB/AP-1 pathway and Ras-PI3K-AKT-mTOR signaling. This transcriptional modulation promotes oncogenic pro-survival and proliferative signaling in AML cells and defines differential sensitivity against Type I and Type II kinase inhibitors. Disclosures Shah: ARIAD: Research Funding; BMS: Research Funding; Daiichi-Sankyo: Research Funding; Pfizer: Research Funding; Plexxikon: Research Funding. Konopleva:Reata Pharmaceuticals: Equity Ownership; Abbvie: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Stemline: Consultancy, Research Funding; Eli Lilly: Research Funding; Cellectis: Research Funding; Calithera: Research Funding.

Adipocytes are the prevalent stromal cell type in adult bone marrow (BM), comprising approximately 60% of BM space in a 65-year old person. In BM environment, leukemia cells continuously adapt to deficient supply of nutrients and oxygen, acquiring quiescent and chemoresistant profiles. Fatty acid metabolism is one of the key energy pathways for AML survival (Samudio, J Clin Invest. 2010), and we previously demonstrated that AML cells activate oxidative phosphorylation and fatty acid oxidation (FAO) in the presence of BM-adipocytes (Tabe ASH 2015). These findings indicate the importance of FAO for AML cells survival under the adipocyte-abundant BM-microenvironment. A novel FAO inhibitor avocatinB, an odd-numbered carbon lipid derived from avocado fruit, has been recently shown to induce apoptosis and cell growth inhibition in AML cells (Lee, Cancer Res. 2015). In the present study, we investigated the molecular mechanisms of anti-leukemic effect of avocatinB in AML cells, utilizing THP1, OCI-AML3 and U937 AML cell lines co-cultured with human mesenchymal stem cells (MSC)-derived BM-adipocytes, mimicking the aging BM microenvironment. Treatment with avocatin B significantly induced ROS accumulation in U937 cells co-cultured with BM-adipocytes (MFI of ROS-sensitive dye; avocatinB (-) / (+); 164±50 / 581±85, p=0.04), whereas only minimum increase of ROS was observed in the absence of BM-adipocyte, indicating that avocatinB causes progressive oxidative damage in AML cells under the BM-adipocyte co-culture conditions. Of importance, avocatinB synergistically enhanced apoptotic effects of AraC in the presence of BM-adipocytes (combination index CI; adipocyte (-) / (+); THP1: 1.2 / 0.4, OCI-AML3: 0.7 / 0.3). Immunoblot analysis demonstrated that avocatinB activated the stress response kinase AMPK in THP1 and OCI-AML3 cells under BM-adipocyte co-culture conditions. AMPK is a crucial cellular energy sensor that regulates energy metabolism including FAO and gene transcription through mTOR inhibition. We therefore investigated the role of AMPK in avocatinB induced anti-leukemic effects on AML cells, utilizing AMPK knockdown (shAMPK) OCI-AML3 cells. shAMPK OCI-AML3 cells were significantly less sensitive to nutrient starvation-induced cell death in the absence of BM-adipocyte (p 1.0). In nsAMPK cells, but not in shAMPKcells BM-adipocyte co-culture upregulated p-4EBP1 and cMyc expression which was abrogated by avocatinB and AraC combination treatment accompanied by induction of cleaved caspase 3. In summary, FAO inhibitor avocatinB induces pro-apoptotic effects through AMPK-dependent inhibition of mTOR signaling that disrupts energy homeostasis and induces ROS accumulation in AML cells under BM-adipocyte co-culture conditions. The ability of avocatinB to selectively enhance anti-leukemic effects of AraC in the presence of BM-adipocytes suggests that the strategies targeting FAO warrant further exploration in elderly AML patients. Disclosures Konopleva: Reata Pharmaceuticals: Equity Ownership; Abbvie: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Stemline: Consultancy, Research Funding; Eli Lilly: Research Funding; Cellectis: Research Funding; Calithera: Research Funding.

Low-dose ionizing radiation (LDIR, ≤ 0.1 Gy) which is typically associated with therapeutic and diagnostic radiological modalities, is known to induce remodeling of the stromal microenvironment. Whereas the biologic responses to LDIR are commonly described as a stress response, the carcinogenic potential of this environmental stressor remains unknown. Recently, ionizing radiation (IR)-induced alterations of miRNA expression that play a fundamental role in cell signaling events, have been demonstrated in various cell types (Marsit, Cancer Res. 2006). To assess a potential determinant influencing of LDIR induced miRNA alterations in pre-malignant cells in a microenvironment, we utilized immortalized pre-malignant Epstein-Barr virus infected-B (EBV-B) cells which are continuously proliferating in circulation or in lymph nodes. The LDIR system (0.1 Gy, 4MV X ray from a LINAC) was utilized in the in vitro co-culture of EBV-B and mesenchymal stromal cells (MSC) to mimic the lymph node stromal microenvironment. We confirmed that MSC protected co-cultured EBV-B cells from spontaneous apoptosis and caused accumulation of EBV-B cells in the G0/G1 phase (EBV-B; monoculture vs coculture with MSC; Sub G1% 42.0±2.4 vs 34.1±0.9 p< 0.01, G0/G1 % 42.6±2.1 vs 47.6±2.0, p To identify LDIR-induced modulation of specific miRNAs in EBV-B cells, a high-confidence list of 44 known LDIR-associated miRNAs from three separate screening studies of lymphoblastic cells was assembled (Cha et al., Oncol Rep.2009, Chaudhry et al., J Biomed Sci. 2010, Lhakhang et al., Comp Funct Genomics. 2012). From these miRNA, we focused on four miRNAs let-7a, miR-16, miR-19b and miR-21 that have been listed in more than two of the screens. Our studies showed that LDIR upregulated let-7a and miR-16 levels in irradiated mono-cultured EBV-B, but on the contrary downregulated all tested miRNAs in MSC co-cultured EBV-B cells (at 24-hour). We next investigated alterations of EBV-B gene expression by LDIR using the DNA microarray (Affymetrix). In mono-cultured EBV-B cells, cDNA microarray analysis detected upregulation of TGFB1 mRNA, and downregulation of the genes encoding a lipid biosynthesis enzyme glycerol-3-phosphate acyltransferase (GPAM), a growth factor amphiregulin (AREG), cell survival-/growth-related factors Rho-GTPase effector gene folmin-1 (FMN1), chemotaxis-inducing chemokine gene IL-8 and B-cell lymphoma 2 gene BCL2, all of which were confirmed by qRT-PCR. Downregulation of the GPAM protein was further shown on the protein level by western blot, and the ontology analysis demonstrated that LDIR caused the TGFbeta-dependent induction of the epithelial-mesenchymal transition (EMT) pathway in mono-cultured EBV-B cells. In contrast to the mono-culture condition, the GPAM mRNA and protein expression were upregulated in MSC co-cultured EBV-B cells. To determine the potential targets of LDIR-altered miRNAs, we next identified 79 genes that are commonly targeted by let-7a, miR-16, miR-19b, or miR-21 from a microrna.org database, and found that the expression changes of GPAM mRNA and protein were strikingly matched with miRNA profiling. Partial concordant changes of DNAJ(HSP40)A2 coding heat shock protein (HSP) 40 homolog, a cochaperone of HSP70s, CPEB3 coding RNA binding protein cytoplasmic polyadenylation element binding protein 3 (CEBP3) and its transcriptional target GLUR2 (glutamate receptor subunit), a receptor of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), were also observed in the microarray results. Since GPAM is a lipid-related metabolism gene which is known to be regulated by miR-27b and to negatively impact survival of breast cancer cells we next investigated the expression changes of miR-27b after LDIR in EBV-B cells. As expected, LDIR upregulated miR-27b in mono-cultured EBV-B cells, and downregulated in co-cultured EBV-B cells with MSC. Intriguingly, no significant changes of GPAM mRNA as well as tested miRNA expression was observed by 1Gy irradiation. In summary, we demonstrated that LDIR directly modulates EBV-B cells gene expression. Importantly, LDIR additionally impacts EBV-B cells indirectly through miRNA modulation by the neighboring stromal cells. Disclosures Konopleva: Novartis: Research Funding; AbbVie: Research Funding; Stemline: Research Funding; Calithera: Research Funding; Threshold: Research Funding.

Mantle cell lymphoma (MCL) is an aggressive B-cell lymphoma characterized by the aberrant expression of several growth-regulating, oncogenic effectors. Exportin 1 (XPO1) mediates the nucleocytoplasmic transport of numerous molecules including oncogenic growth-regulating factors, RNAs, and ribosomal subunits. In MCL cells, the small molecule KPT-185 blocks XPO1 function and exerts anti-proliferative effects. In this study, we investigated the molecular mechanisms of this putative anti-tumor effect on MCL cells using cell growth/viability assays, immunoblotting, gene expression analysis, and absolute quantification proteomics. KPT-185 exhibited a p53-independent anti-lymphoma effect on MCL cells, by suppression of oncogenic mediators (e.g., XPO1, cyclin D1, c-Myc, PIM1, and Bcl-2 family members), repression of ribosomal biogenesis, and downregulation of translation/chaperone proteins (e.g., PIM2, EEF1A1, EEF2, and HSP70) that are part of the translational/transcriptional network regulated by heat shock factor 1. These results elucidate a novel mechanism in which ribosomal biogenesis appears to be a key component through which XPO1 contributes to tumor cell survival. Thus, we propose that the blockade of XPO1 could be a promising, novel strategy for the treatment of MCL and other malignancies overexpressing XPO1.

Hyperthermia (HT) improves the efficacy of anti-cancer radiotherapy and chemotherapy. However, HT also inevitably evokes stress responses and increases the expression of heat-shock proteins (HSPs) in cancer cells. Among the HSPs, HSP70 is known as a pro-survival protein. In this study, we investigated the sensitizing effect of pifithrin (PFT)-μ, a small molecule inhibitor of HSP70, when three human prostate cancer cell lines (LNCaP, PC-3, and DU-145) were treated with HT (43°C for 2 h). All cell lines constitutively expressed HSP70, and HT further increased its expression in LNCaP and DU-145. Knockdown of HSP70 with RNA interference decreased the viability and colony-forming ability of cancer cells. PFT-μ decreased the viabilities of all cell lines at one-tenth the dose of Quercetin, a well-known HSP inhibitor. The combination therapy with suboptimal doses of PFT-μ and HT decreased the viability of cancer cells most effectively when PFT-μ was added immediately before HT, and this combination effect was abolished by pre-knockdown of HSP70, suggesting that the effect was mediated via HSP70 inhibition. The combination therapy induced cell death, partially caspase-dependent, and decreased proliferating cancer cells, with decreased expression of c-Myc and cyclin D1 and increased expression of p21^, indicating arrest of cell growth. Additionally, the combination therapy significantly decreased the colony-forming ability of cancer cells compared to therapy with either alone. Furthermore, in a xenograft mouse model, the combination therapy significantly inhibited PC-3 tumor growth. These findings suggest that PFT-μ can effectively enhance HT-induced antitumor effects via HSP70 inhibition by inducing cell death and arrest of cell growth, and that PFT-μ is a promising agent for use in combination with HT to treat prostate cancer.