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Supplementary Figure 2 - PDF file 50K, Accessory cells protect MM cells from WT-1 specific CTLs. U266 cells were co-cultured with WT-1 specific T cells in presence and absence of different accessory cells. MM cell viability was determined 24h after addition of T cells by CS-BLI (A). Granzyme B production in the supernatant was determined by ELISA (B). Significance of inhibition of MM cell survival in the presence of accessory cells was tested by unpaired two tailed student's t test. *= p

Supplementary Figure 1 - PDF file 53K, Antigen specific, HLA-restricted and dose-dependent recognition of the MM cell lines U266 and UM9, but not of accessory cells by CD8+ and CD4+ mHag-specific CTLs. The CD4+ (A) and CD8+ (B) CTLs were co-cultured with the luc+ MM cell lines UM9 and U266 in different effector to target ratios. The HLA- and mHag typing of these MM cell lines are indicated. MM cell survival was determined 24 h after addition of T cells by CS-BLI. Results represent the mean values of triplicate cultures (+/- SEM). Results are representative of 3 independent assays. In (C and D) CD4+ (C) and CD8+ (D) CTLs were co-cultured with accessory cells. Accessory cell survival was determined 24h after addition of T cells by FACS annexine V/ propidium iodide staining of CD4 and CD8 negative cells in the co-cultures. Results are depicted as % surviving cells, measured as relative number annexin V/propidium iodide negative cells, compared to no T cells control

Supplementary Figure 3 - PDF file 55K, RGDw reduces adhesion of MM cells to accessory cells. Adherent HS-5 en HUVEC accessory cells were incubated with RGDw or an irrelevant peptide for 1hour. After washing away the unbound peptide, UM9 (A) or U266 (B) cells were added and incubated overnight. Plates were reversely centrifuged to determine adhesion to accessory cells. Results represent the mean values of triplicate cultures (+/- SEM). Differences in adhesion were tested by unpaired two tailed student's t test. *= p

Supplementary Figure 5 - PDF file 41K, Bortezomib does not improve the CTL mediated lysis of Fas negative L363 cellsL3L3 reactive CD8+CTLs were incubated at the indicated effector to target ratios with luc-transduced L3L3 cells alone or in the presence of various dilutions of bortezomib. The CTL mediated and bortezomib mediated lysis of L3L3 cells was determined after 48 hours by BLI. Results are depicted as % surviving cells using the triplicate cultures without T cells and bortezomib as the 100% survival control

Purpose: Cellular immunotherapy frequently fails to induce sustained remissions in patients with multiple myeloma, indicating the ability of multiple myeloma cells to evade cellular immunity. Toward a better understanding and effective therapeutic modulation of multiple myeloma immune evasion mechanisms, we here investigated the role of the tumor microenvironment in rendering multiple myeloma cells resistant to the cytotoxic machinery of T cells.Experimental Design: Using a compartment-specific, bioluminescence imaging-based assay system, we measured the lysis of luciferase-transduced multiple myeloma cells by CD4+ or CD8+ CTLs in the presence versus absence of adherent accessory cells of the bone marrow microenvironment. We simultaneously determined the level of CTL activation by measuring the granzyme B release in culture supernatants.Results: Bone marrow stromal cells from patients with multiple myeloma and healthy individuals, as well as vascular endothelial cells, significantly inhibited the lysis of multiple myeloma cells in a cell–cell contact-dependent manner and without substantial T-cell suppression, thus showing the induction of a cell adhesion-mediated immune resistance (CAM-IR) against CTL lysis. Further analyses revealed that adhesion to accessory cells downregulated Fas and upregulated the caspase-3 inhibitor survivin in multiple myeloma cells. Reconstitution of Fas expression with bortezomib enhanced the CTL-mediated lysis of multiple myeloma cells. Repressing survivin with the small-molecule YM155 synergized with CTLs and abrogated CAM-IR in vitro and in vivo.Conclusion: These results reveal the cell adhesion-mediated induction of apoptosis resistance as a novel immune escape mechanism and provide a rationale to improve the efficacy of cellular therapies by pharmacologic modulation of CAM-IR. Clin Cancer Res; 19(20); 5591–601. ©2013 AACR.

Supplementary Figure Legends - PDF file 73K, Legends for supplementary figures

Supplementary Figure 6 - PDF file 74K, Synergistic interaction of YM155 and CTLs. In A, the effect of YM155 on survivin and MCL-1 protein levels of MM cells is shown. The cells were cultured with YM155 during 24h and the western blot assays were carried out as indicated in material methods. Band intensities were quantified using image J software, calculated as relative values to the control protein tubuline and plotted as relative to the expression levels of cells cultured without YM155. In B, the type of interaction between YM155 and CTLs (see figure 5C-D) in the presence of accessory cells was analyzed using Compusyn software (version 1.0, 2004 �). In the plots (A) the CI values corresponding to the doses in figure 5 are shown. A CI value of 1 indicates synergy, additive effects or antagonism, respectively. In C the supernatants of assays as described in Fig 5C-D granzyme B release was measured using ELISA (B). Results are expressed as the mean values of the triplicate cultures. Error bars represent the SEM. Results are representative of 3 independent assays

Supplementary Figure 4 - PDF file 219K, FasL upregulation on CTLs upon MM cell recognition is not hampered by presence of accessory cells. CD4+ (A) and CD8+ (B) CTLs were cultured alone (unstimulated) or in the presence of tumor cells and in presence or absence of accessory cells as indicated. After 3 hours of co-incubation CTLs were evaluated for CD178 (Fas ligand) expression. Percentage of FasL expressing cells is indicated

Supplementary Figures 1-7 from Antimyeloma Activity of the Orally Bioavailable Dual Phosphatidylinositol 3-Kinase/Mammalian Target of Rapamycin Inhibitor NVP-BEZ235

PDF file - 160K

PDF file - 648K, Figure S1. TAS-117 is a novel potent and selective inhibitor of Akt kinases. Figure S2. Basal expression of phosphorylated Akt and Akt in MM cell lines. Figure S3. TAS-117 inhibits neither MEK/ERK nor JAK2/STAT pathway. Figure S4. TAS-117 modulates phenotype of side population (SP) cells and induces cytotoxicity in SP cells associated with Akt inhibition. Figure S5. TAS-117 overcomes cytoprotective effects conferred by cytokines by blocking Akt activation. Figure S6. TAS-117 treatment does not affect viability in BMSCs. Figure S7. TAS-117 triggers cleavage of caspase and PARP in MM cells. Figure S8. TAS-117 induces autophagosome formation in MM cells. Figure S9. TAS-117 in combination with bortezomib does not affect body weight of mice. Figure S10. TAS-117 in combination with carfilzomib induces synergistic cytotoxicity in RPMI8226 cells with low baseline of p-Akt, associated with inhibition of carfilzomib-induced p-Akt. Table S1. TAS-117 combination indices (CI) with bortezomib in MM.1S cells. Table S2. TAS-117 combination indices (CI) with bortezomib in RPMI8226 cells. Table S3. TAS-117 combination indices (CI) with carfilzomib in MM.1S cells. Table S4. TAS-117 combination indices (CI) with carfilzomib in RPMI8226 cells.

Waldenström macroglobulinemia (WM) is a rare subtype of non-Hodgkin lymphoma characterized by malignant lymphoplasmacytic cells in the bone marrow (BM). To dissect the pathophysiology of WM, we evaluated clonal cells by mapping of B cell lymphomagenesis with adaptive and innate immune tumor microenvironment (TME) in the BM of WM patients using mass cytometry (CyTOF). In-depth immunophenotypic profiling of WM cells showed profound expansion of clonal cells in both un-switched and switched memory B cells and also plasma cells with aberrant expression variations. WM B lymphomagenesis was associated with reduction of most B cell precursors assessed with the same clonally restricted light chain and phenotypic changes. The immune TME was infiltrated by mature monocytes, neutrophils and adaptive T cells, preferentially subsets of effector T helper and effector and effector memory CTL cells that were associated with superior overall survival (OS), in contrast to progenitors of T cells and myeloid/monocytic lineage subsets that were suppressed in WM cohort. Moreover, decrease in immature B and NKT cells was related to worse OS in WM patients. Innate and adaptive immune subsets of WM TME were modulated by immune checkpoints, including PD-1/PD-L1PD-L2, TIGIT/PVR, CD137/CD137-L, CTLA-4, BTLA and KIR expression. The response of ibrutinib treatment to the reduction of clonal memory B cell was associated with high levels of immature B cells and effector memory CTL cells. This study demonstrates that CyTOF technology is a powerful approach for characterizing the pathophysiology of WM at various stages, predicting patient risk and monitoring the effectiveness of treatment strategies. This article is protected by copyright. All rights reserved.

The BCR/ABL preleukemic fusion gene (PFG) is one of the most frequent fusion genes in acute lymphoblastic leukemia (ALL) and was also detected in hematopoietic cells from umbilical cord blood (UCB) of healthy newborns. Since hematopoietic stem/progenitor cells (HSPC) are considered to be a critical cellular target for origination of leukemia, we have studied the presence of BCR/ABL PFG in expanded subpopulations of HSPC and differentiated cells from UCB of those healthy newborns, who have previously been tested positive for BCR/ABL by screening of their UCB mononuclear cells using RT-qPCR and FISH methods. We isolated cells from human UCB samples positive for BCR/ABL and negative controls. The isolated cells were sorted into 5 hematopoietic and progenitor cell subpopulations. We analyzed BCR/ABL in sorted and expanded subpopulations of UCB using FISH and RT-qPCR. We found that the number of BCR/ABL positive cells was similar in each studied subpopulation and the same as in differentiated lymphocytes. Our data showed that there is no specific subpopulation of hematopoietic and progenitor stem cells with an increased leukemogenic potential due to the presence of higher copies of BCR/ABL.

DNA double strand breaks (DSB) induced by ionizing radiation (IR) are usually measured using γH2AX/53BP1 DNA repair foci, that is considered to be the most sensitive assay for DSB analysis. While fluorescence microscopy (FM) is the gold standard for this analysis, imaging flow cytometry (IFC) may offer number of advantages such as lack of the fluorescence background, higher number of cells analyzed, and higher sensitivity in detection of DNA damage induced by IR at low doses. Along with appearance of γH2AX foci, the variable fraction of the cells exhibits homogeneously stained γH2AX signal resulting in so-called γH2AX pan-staining, which is believed to appear at early stages of apoptosis. Here, we investigated incidence of γH2AX pan-staining at different time points after irradiation with γ-rays using IFC and compared the obtained data with the data from FM. Appearance of γH2AX pan-staining during the apoptotic process was further analyzed by fluorescence-activated cell sorting (FACS) of cells at different stages of apoptosis and subsequent immunofluorescence analysis. Our results show that IFC was able to reveal dose dependence of pan-staining, while FM failed to detect all pan-staining cells. Moreover, we found that γH2AX pan-staining could be induced by therapeutic, but not low doses of γ-rays and correlate well with percentage of apoptotic cells was analyzed using flow cytometric Annexin-V/7-AAD assay. Further investigations showed that γH2AX pan-staining is formed in the early phases of apoptosis and remains until later stages of apoptotic process. Apoptotic DNA fragmentation as detected with comet assay using FM correlated with the percentage of live and late apoptotic/necrotic cells as analyzed by flow cytometry. Lastly, we successfully tested IFC for detection of γH2AX pan-staining and γH2AX/53BP1 DNA repair foci in lymphocyte of breast cancer patients after radiotherapy, which may be useful for assessing individual radiosensitivity in a clinically relevant cohort of patients.

Multiple myeloma (MM), a B cell malignancy characterized by clonal proliferation of plasma cells in the bone marrow, remains incurable despite the use of novel and conventional therapies. In this study, we demonstrated MM cell cytotoxicity triggered by realgar (REA; As4S4) nanoparticles (NREA) versus Arsenic trioxide (ATO) against MM cell lines and patient cells. Both NREA and ATO showed in vivo anti-MM activity, resulting in significantly decreased tumour burden. The anti-MM activity of NREA and ATO is associated with apoptosis, evidenced by DNA fragmentation, depletion of mitochondrial membrane potential, cleavage of caspases and anti-apoptotic proteins. NREA induced G2/M cell cycle arrest and modulation of cyclin B1, p53 (TP53), p21 (CDKN1A), Puma (BBC3) and Wee-1 (WEE1). Moreover, NREA induced modulation of key regulatory molecules in MM pathogenesis including JNK activation, c-Myc (MYC), BRD4, and histones. Importantly, NREA, but not ATO, significantly depleted the proportion and clonogenicity of the MM stem-like side population, even in the context of the bone marrow stromal cells. Finally, our study showed that both NREA and ATO triggered synergistic anti-MM activity when combined with lenalidomide or melphalan. Taken together, the anti-MM activity of NREA was more potent compared to ATO, providing the preclinical framework for clinical trials to improve patient outcome in MM.

Berberine is a bioactive isoquinoline alkaloid derived from many plants. Although berberine has been shown to inhibit growth and induce apoptosis of several tumor cell lines, its poor absorption and moderate activity hamper its full therapeutic potential. Here, we describe the synthesis of a series of 9-O-substituted berberine derivatives with improved antiproliferative and apoptosis-inducing activities. An analysis of novel berberine derivatives by EPR spectroscopy confirmed their similar photosensitivity and analogous behavior upon UVA irradiation as berberine, supporting their potential to generate ROS. Improved antitumor activity of novel berberine derivatives was revealed by MTT assay, by flow cytometry and by detection of apoptotic DNA fragmentation and caspase-3 activation, respectively. We showed that novel berberine derivatives are potent inhibitors of growth of HeLa and HL-60 tumor cell lines with IC50 values ranging from 0.7 to 16.7 &micro, M for HL-60 cells and 36 to &gt, 200 &micro, M for HeLa cells after 48 h treatment. Further cell cycle analysis showed that the observed inhibition of growth of HL-60 cells treated with berberine derivatives was due to arresting these cells in the G2/M and S phases. Most strikingly, we found that berberine derivative 3 (9-(3-bromopropoxy)-10-methoxy-5,6-dihydro-[1,3]dioxolo[4,5-g]isoquino[3,2-a] isoquinolin-7-ylium bromide) possesses 30-fold superior antiproliferative activity with an IC50 value of 0.7 &micro, M and 6-fold higher apoptosis-inducing activity in HL-60 leukemia cells compared to berberine. Therefore, further studies are merited of the antitumor activity in leukemia cells of this berberine derivative.

Berberine is a bioactive isoquinoline alkaloid derived from many plants. Although berberine has been shown to inhibit growth and induce apoptosis of several tumor cell lines, its poor absorption and moderate activity hamper its full therapeutic potential. Here, we describe the synthesis of a series of 9-O-substituted berberine derivatives with improved antiproliferative and apoptosis-inducing activities. An analysis of novel berberine derivatives by EPR spectroscopy confirmed their similar photosensitivity and analogous behavior upon UVA irradiation as berberine, supporting their potential to generate ROS. Improved antitumor activity of novel berberine derivatives was revealed by MTT assay, by flow cytometry and by detection of apoptotic DNA fragmentation and caspase-3 activation, respectively. We showed that novel berberine derivatives are potent inhibitors of growth of HeLa and HL-60 tumor cell lines with IC50 values ranging from 0.7 to 16.7 µM for HL-60 cells and 36 to >200 µM for HeLa cells after 48 h treatment. Further cell cycle analysis showed that the observed inhibition of growth of HL-60 cells treated with berberine derivatives was due to arresting these cells in the G2/M and S phases. Most strikingly, we found that berberine derivative 3 (9-(3-bromopropoxy)-10-methoxy-5,6-dihydro-[1,3]dioxolo[4,5-g]isoquino[3,2-a] isoquinolin-7-ylium bromide) possesses 30-fold superior antiproliferative activity with an IC50 value of 0.7 µM and 6-fold higher apoptosis-inducing activity in HL-60 leukemia cells compared to berberine. Therefore, further studies are merited of the antitumor activity in leukemia cells of this berberine derivative.

Specific niches within the tumor bone marrow (BM) microenvironment afford a sanctuary for multiple myeloma (MM) clones due to stromal cell-tumor cell interactions, which confer survival advantage and drug resistance. Defining the sequelae of tumor cell interactions within the MM niches on an individualized basis may provide the rationale for personalized therapies. To mimic the MM niche, we here describe a new 3D co-culture ex-vivo model in which primary MM patient BM cells are co-cultured with mesenchymal stem cells (MSC) in a hydrogel 3D system. In the 3D model, MSC with conserved phenotype (CD73+CD90+CD105+) formed compact clusters with active fibrous connections, and retained lineage differentiation capacity. Extracellular matrix molecules, integrins, and niche related molecules including N-cadherin and CXCL12 are expressed in 3D MSC model. Furthermore, activation of osteogenesis (MMP13, SPP1, ADAMTS4, and MGP genes) and osteoblastogenic differentiation was confirmed in 3D MSC model. Co-culture of patient-derived BM mononuclear cells with either autologous or allogeneic MSC in 3D model increased proliferation of MM cells, CXCR4 expression, and SP cells. We carried out immune profiling to show that distribution of immune cell subsets was similar in 3D and 2D MSC model systems. Importantly, resistance to novel agents (IMiDs, bortezomib, carfilzomib) and conventional agents (doxorubicin, dexamethasone, melphalan) was observed in 3D MSC system, reflective of clinical resistance. This 3D MSC model may therefore allow for studies of MM pathogenesis and drug resistance within the BM niche. Importantly, ongoing prospective trials are evaluating its utility to inform personalized targeted and immune therapy in MM.

// Gabriela Rozic 1 , Lena Paukov 1 , Jana Jakubikova 3 , Dikla Ben-Shushan 1 , Adrian Duek 1 , Adi Leiba 2, 4 , Abraham Avigdor 1, 2 , Arnon Nagler 1, 2 , Merav Leiba 1, 2 1 Division of Hematology and BMT, Sheba Medical Center, Tel Hashomer, Ramat Gan, Israel 2 Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel 3 Department of Medical Oncology, Jerome Lipper Multiple Myeloma Center, Harvard Medical School, Dana-Farber Cancer Institute, Boston, MA, USA 4 Department of Medical Education, Mount Auburn Hospital, Harvard Medical School, Cambridge, MA, USA Correspondence to: Merav Leiba, email: merav.leiba@sheba.health.gov.il Keywords: multiple myeloma, cell cycle, apoptosis, tubulin, AKT Received: March 01, 2016 Accepted: August 09, 2016 Published: August 23, 2016 ABSTRACT Despite advances in treatment, multiple myeloma (MM) remains incurable. Here we propose the use of STK405759, a novel microtubule targeting agent (MTA) and member of the furan metotica family for MM therapy. STK405759 inhibited tubulin polymerization in a cell-free system and in myeloma cells. This molecule had potent cytotoxic activity against several MM cell lines and patient-derived MM cells. Moreover, STK405759 demonstrated cytotoxicity against drug-resistant myeloma cells that overexpressed the P-glycoprotein drug-efflux pump. STK405759 was not cytotoxic to peripheral blood mononuclear cells, including activated B and T lymphocytes. This compound caused mitotic arrest and apoptosis of myeloma cells characterized by cleavage of poly (ADP-ribose) polymerase-1 and caspase-8, as well as decreased protein expression of mcl-1. The combination of STK405759 with bortezomib, lenalidomide or dexamethasone had synergistic cytotoxic activity. In in vivo studies, STK405759-treated mice had significantly decreased MM tumor burden and prolonged survival compared to vehicle treated- mice. These results provide a rationale for further evaluation of STK405759 as monotherapy or part of combination therapy for treating patients with MM.

The PI3K/Akt pathway plays a crucial role in the pathogenesis of multiple myeloma (MM) in the bone marrow (BM) milieu. However, efficacy of selective and potent Akt inhibition has not yet been fully elucidated. In this study, we, therefore, examined the biologic impact of selective and potent Akt inhibition by a novel allosteric inhibitor TAS-117. TAS-117 induced significant growth inhibition, associated with downregulation of phosphorylated Akt (p-Akt), selectively in MM cell lines with high baseline p-Akt. Cytotoxicity of TAS-117 was also observed in patient MM cells, but not in normal peripheral blood mononuclear cells. Importantly, TAS-117 induced significant cytotoxicity in MM cells even in the presence of BM stromal cells, associated with inhibition of IL6 secretion. Oral administration of TAS-117 significantly inhibited human MM cell growth in murine xenograft models. TAS-117 triggered apoptosis and autophagy, as well as induction of endoplasmic reticulum (ER) stress response with minimal expression of C/EBP homologous protein (CHOP), a fatal ER stress marker. Importantly, TAS-117 enhanced bortezomib-induced cytotoxicity, associated with increased CHOP and PARP cleavage and blockade of bortezomib-induced p-Akt, suggesting that TAS-117 augments bortezomib-induced ER stress and apoptotic signaling. Carfilzomib-induced cytotoxicity was similarly enhanced by TAS-117. Importantly, TAS-117 enhanced bortezomib-induced cytotoxicity in vivo, associated with prolonged host survival. Our results show that selective and potent Akt inhibition by TAS-117 triggers anti-MM activities in vitro and in vivo, as well as enhances cytotoxicity of proteasome inhibition, providing the preclinical framework for clinical evaluation of selective Akt inhibitors, alone and in combination with proteasome inhibitors in MM. Cancer Res; 74(16); 4458–69. ©2014 AACR.

Multiple myeloma (MM), the second most common hematologic malignancy worldwide, is a B cell malignancy characterized by high frequency of intra-clonal diversity within malignant plasma cells (PC) in the bone marrow (BM). To better understand the myeloma heterogeneity within its complex pathophysiology, we performed large-scale data-driven mass cytometry (CyTOF) analysis in cohort of 188 bone marrow (BM) samples from multiple myeloma (MM) patients compared to 10 age-matched healthy donors (HD). Our design focused on profiling of PC intra and inter-neoplastic heterogeneity based on molecular perturbations of transcriptional factors and signaling regulators and stemness-controlling markers ensuring development of B cell lymphopoiesis within myelomagenesis encompassing the different clinical spectra of pre-malignant/asymptomatic (16 MGUS and 25 SMM) and active symptomatic stages (43 NDMM and 104 relapsed or relapsed/refractory MM patients) of MM pathogenesis. Moreover, interaction of PC disease status with the immune ecosystem of myeloma microenvironment was evaluated as well. To distinguish tumor-driven specific immune changes from myeloma immune ecosystem, we observed that cell frequency of cytotoxic naïve and effector cells, g/dT, and early monocytes, myelocytes and erythroblasts immune subsets was significantly reduced in both premalignant and active MM stages. In contrast, mostly innate immune clusters including non-canonical monocytes, myeloblasts, and mature neutrophils, erythroblasts and platelets were present at a higher frequency across all MM stages versus HD. To evaluate cell distribution of B lymphopoiesis in MM disease stages, switched memory B cells and plasmablasts clusters were upregulated in premalignant stage MGUS compared to HD. Similar observations were detected in SMM and NDMM versus HD, with the highest abundance of PC clusters in NDMM. The downregulation of cell distribution in B cell progenies, immature and transitional B cells, and un-switched memory B cell clusters was observed in NDMM and relapsed/refractory MM patients. Furthermore, MM patients treated with Revlimid-Velcade-Dexamethasone therapy had decrease frequency of specific PC clusters and un-switched and transitional B cell clusters. In addition, our data revealed immunophenotyping aberrancies present not only in PC clusters but also across all myeloma B lymphomagenesis in BM samples from MM patients. In-depth characterization of malignant plasma cells, significant variations were detected in PC clusters of MM cohort based on different expression of IRF4, c-Myc, CD28, CD117, and FGFR-3, however with homogenous expression of sXBP1, and MMSET which differ in all 4 MM stages compared to HD. Significant upregulation of CD47 was showed in all PC clusters of MM cohort. Moreover, PC clusters differ in intra-clonal expression of self-renewing/stemness markers CD184, Notch-1, Oct3/4, KLF-4, Sox-2, and Nanog, supporting the idea of sub-clonal variations insight of MM tumor. This study might provide the rational for prediction of MM patient status and design of targeted therapy in MM on personalized bases. This work was supported by REA grant agreement No. 609427-SASPRO 0064/01/02, TRS-2015-00000170, APVV-16-0484 and VEGA 2/0076/17. Disclosures Hunter: Janssen: Consultancy. Jamroziak:Amgen: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding. Richardson:Amgen: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Research Funding. Kastritis:Prothena: Honoraria; Genesis: Honoraria; Takeda: Honoraria; Janssen: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Pfizer: Honoraria. Anderson:Sanofi-Aventis: Other: Advisory Board; Bristol-Myers Squibb: Other: Scientific Founder; Oncopep: Other: Scientific Founder; Amgen: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau.

Waldenström macroglobulinemia (WM), a malignant B-cell lymphoplasmacytic lymphoma, is a rare subtype of non-Hodgkin lymphoma representing about 1% of all cases. To better understand the WM pathogenesis, we performed large-scale data-driven proteomic profile of WM tumor cells associated with tumor-driven immune changes in the tumor microenvironment of 66 bone marrow (BM) samples from WM patients compared to 10 age-matched healthy donors (HD) by time-of-flight mass cytometry (CyTOF) technology. Our workflow has been designed based on extensive 3 CyTOF antibody panels to evaluate WM tumor within B cell lymphopoiesis concurrently with immune landscape of the tumor microenvironment in WM by state-of-art technology CyTOF. To map B cell lymphomagenesis in WM, we defined whole spectrum of maturation of B cell development, from hematopoietic stem cells and B cell precursors through immature B cells, transitional B cells, and naïve B cells together with memory un-switched and switched B cells, plasmablasts and plasma cells in BM samples of WM patients by positive and negative co-expression of 13 B cell-stage specific markers. Various immunophenotyping aberrancies within WM B lymphomagenesis were associated with WM clones characterized by significant increase of 11 B subset clusters from un-switched and switched memory B cells to plasma cells. Interestingly, WM clusters differ in intra-clonal expression of activation surface molecules (CD23, CD24, CD25, CD81, CD329, CD200, and CD319); transcriptional factors and regulators controlling B cell development (MYD88, Bcl-6, IRF-4, sXBP-1, and FGFR-3) and stemness-related markers (Oct3/4, Nanog, Sox-2, c-Myc, and Notch-1) in WM supporting the idea of sub-clonal heterogeneity insight of WM tumor. Moreover, decrease in cell frequency of B cell precursors (pro-B and pre-BI), naive B cells, and plasmablasts were observed in WM patients versus HD. To generate a comprehensive view of the tumor microenvironment, we observed significant upregulation of g/dT cells, CD4+ and CD8+ T effector cells, CD8+ T effector memory cells, monocytes, and neutrophils immune subsets and downregulation of immature T cells, CD8+ T naïve cells, plasmacytoid dendritic cells, myelo/mono progenitor clusters. Ibrutinib (IBRU) treatment has been effective in relapsed/refractory WM patients; therefore highest numbers of WM patients were receiving IBRU therapy in our cohort. IBRU treated WM patients had decreased frequency of naive B, CD4+ T naive cells and specific clusters of un-switched and switched memory B cells. Moreover, responder versus non-responders to IBRU therapy revealed increase of CD8+T effector memory cells. In sum, correspondence analysis reflecting data of each patient and immune subsets revealed stratification of WM patients with reflection on their clinical outcome, therefore providing the rational for prediction of WM patient status. This study was supported by APVV-16-0484 and VEGA 2/0076/17. Disclosures Hunter: Janssen: Consultancy. Richardson:Karyopharm: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Membership on an entity's Board of Directors or advisory committees. Kastritis:Amgen: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Takeda: Honoraria; Pfizer: Honoraria; Prothena: Honoraria; Genesis: Honoraria. Treon:BMS: Research Funding; Janssen: Consultancy; Pharmacyclics: Research Funding. Anderson:Celgene: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Sanofi-Aventis: Other: Advisory Board; Bristol-Myers Squibb: Other: Scientific Founder; Oncopep: Other: Scientific Founder; Amgen: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau.

Summary Jasmonates, plant stress hormones, have been demonstrated to be effective in killing various types of cancer cells. We therefore tested if methyljasmonate (MJ) has activity against multiple myeloma (MM) in vitro and in vivo. MM cell lines and primary MM tumour cells responded to MJ in vitro at concentrations that did not significantly affect normal haematopoietic cells, without stroma-mediated resistance. Brief MJ exposures of MM cells caused release of Hexokinase 2 (HK2) from mitochondria, rapid ATP depletion, perturbation of major intracellular signalling pathways, and ensuing mainly apoptotic cell death. Sensitivity to MJ correlated with lower cellular glucose consumption and lactate production, as well as lower intracellular protein levels of HK2, phosphorylated Voltage-dependent anion channel 2/3 (pVDAC2/3) and Aldo-keto reductase family 1 member C1 (AKR1C1), which represent potential biomarkers of responsiveness to MJ treatment, especially as AKR1C1 transcript levels also correlate with clinical outcome in bortezomib- or dexamethasone-treated MM patients. Interestingly, MJ synergized with bortezomib in vitro and prolonged survival of immunocompromised mice harbouring diffuse lesions of MM.1S cells compared to vehicle-treated mice (P = 0·0046). These studies indicate that jasmonates represent a new, promising strategy to treat MM.

Dendritic cells (DCs) and natural killer (NK) cells are central components of innate immunity for controlling tumor growth. The therapeutic effects of certain anti-myeloma drugs are partially mediated by targeting the innate immune response. In addition, novel types of natural compounds have been developed that efficiently modulate the activity of both the cellular and humoral compartments of immunity. MGN-3 is known as an activator of natural killer cells, inducer of apoptosis and cytokine production, and modulator of dendritic cell maturation and differentiation in vitro. We have performed a randomized, placebo-controlled study to examine the effects of MGN-3 on innate immune system parameters in 48 multiple myeloma patients. We performed immunophenotypic analysis of peripheral blood samples, determined NK cell activity, and assessed the cytokine profiles of plasma before and during 3 months of treatment. The results demonstrate a clear increase in NK activity in MGN-3-treated patients compared to the placebo group, an increased level of myeloid DCs in peripheral blood, and augmented concentrations of T helper cell type 1-related cytokines. The present study suggests that MGN-3 may represent an immunologically relevant product for activating innate immunity in multiple myeloma patients and warrants further testing to demonstrate clinical efficacy.

Lenalidomide is an amino-substituted derivative of thalidomide with direct antiproliferative and cytotoxic effects on the myeloma tumor cell, as well as antiangiogenic activity and immunomodulatory effects. Together with the introduction of bortezomib and thalidomide, lenalidomide has significantly improved the survival of patients with relapsed and refractory myeloma. The most common adverse events associated with lenalidomide include fatigue, skin rash, thrombocytopenia, and neutropenia. In addition, when lenalidomide is combined with dexamethasone or other conventional cytotoxic agents, there is an increase in the incidence of venous thromboembolic events. There is now evidence that continued treatment with lenalidomide has a significant impact on survival by improving the depth and duration of response. This highlights the value of adverse event management and appropriate dose adjustments to prevent toxicity, and of allowing continued treatment until disease progression. In this review, we will discuss the different lenalidomide-based treatment regimens for patients with relapsed/refractory myeloma. This is accompanied by recommendations of how to manage and prevent adverse events associated with lenalidomide-based therapy.

Background Isothiocyanates, a family of phytochemicals found in cruciferous vegetables, have cytotoxic effects against several types of tumor cells. Multiple myeloma is a fatal disease characterized by clonal proliferation of plasma cells in the bone marrow. The growing body of preclinical information on the anti-cancer activity of isothiocyanates led us to investigate their anti-myeloma properties.Design and Methods We evaluated the anti-myeloma activity of the isothiocyanates, sulforaphane and phenethyl isothiocyanate, on a panel of human myeloma cell lines as well as primary myeloma tumor cells. Cell viability, apoptosis, cell cycle alterations and cell proliferation were then analyzed in vitro and in a xenograft mouse model in vivo. The molecular sequelae of isothiocyanate treatment in multiple myeloma cells were evaluated by multiplex analyses using bead arrays and western blotting.Results We observed that sulforaphane and phenylethyl isothiocyanate have activity against myeloma cell lines and patients‘ myeloma cells both in vitro and in vivo using a myeloma xenograft mouse model. Isothiocyanates induced apoptotic death of myeloma cells; depletion of mitochondrial membrane potential; cleavage of PARP and caspases-3 and -9; as well as down-regulation of anti-apoptotic proteins including Mcl-1, X-IAP, c-IAP and survivin. Isothiocyanates induced G2/M cell cycle arrest accompanied by mitotic phosphorylation of histone H3. Multiplex analysis of phosphorylation of diverse components of signaling cascades revealed changes in MAPK activation; increased phosphorylation of c-jun and HSP27; as well as changes in the phosphorylation of Akt, and GSK3α/β and p53. Isothiocyanates suppressed proliferation of myeloma cells alone and when co-cultured with HS-5 stromal cells. Sulforaphane and phenylethyl isothiocyanate enhanced the in vitro anti-myeloma activity of several conventional and novel therapies used in multiple myeloma.Conclusions Our study shows that isothiocyanates have potent anti-myeloma activities and may enhance the activity of other anti-multiple myeloma agents. These results indicate that isothiocyanates may have therapeutic potential in multiple myeloma and provide the preclinical framework for future clinical studies of isothiocyanates in multiple myeloma.

Recurrence of multiple myeloma (MM) after therapy suggests the presence of tumor-initiating subpopulations. In our study, we performed flow cytometry–based Hoechst 33342 staining to evaluate the existence of a MM population with stem-like features known as side population (SP) cells. SP cells exhibit substantial heterogeneity in MM cell lines and primary MM cells; express CD138 antigen in MM cell lines; display higher mRNA expression and functional activity of ABCG2 transporter; and have a higher proliferation index compared with non-SP cells. We observed evidence for clonogenic potential of SP cells, as well as the ability of SP cells to regenerate original population. Moreover, SP cells revealed higher tumorigenicity compared with non-SP cells. Importantly, lenalidomide decreased the percentage and clonogenicity of SP cells, and also induced phosphorylation changes in Akt, GSK-3α/β, MEK1, c-Jun, p53, and p70S6K in SP cells. Adherence to bone marrow stromal cells (BMSCs) increased the percentage, viability, and proliferation potential of SP cells. Lenalidomide and thalidomide abrogated this stimulatory effect of BMSCs and significantly decreased the percentage of SP cells. Our studies demonstrate a novel mechanism of action for lenalidomide, namely targeting SP fraction, providing the framework for new therapeutic strategies targeting subpopulations of MM cells including presumptive stem cells.

The transformation from monoclonal gammopathy of undetermined significance (MGUS) to multiple myeloma (MM) is thought to be associated with changes in immune processes. We have therefore used serologic analysis of recombinant cDNA expression library to screen the sera of MGUS patients to identify tumor-associated antigens. A total of 10 antigens were identified, with specific antibody responses in MGUS. Responses appeared to be directed against intracellular proteins involved in cellular functions, such as apoptosis (SON, IFT57/HIPPI), DNA and RNA binding (ZNF292, GPATCH4), signal transduction regulators (AKAP11), transcriptional corepressor (IRF2BP2), developmental proteins (OFD1), and proteins of the ubiquitin-proteasome pathway (PSMC1). Importantly, the gene responsible for the oral-facial-digital type I syndrome (OFD1) had response in 6 of 29 (20.6%) MGUS patients but 0 of 11 newly diagnosed MM patients. Interestingly, 3 of 11 (27.2%) MM patients after autologous stem cell transplantations showed responses to OFD1. We have confirmed T-cell responses against OFD1 in MGUS and observed down-regulation of GLI1/PTCH1 and p-β-catenin after OFD1 knock-down with specific siRNA, suggesting its functional role in the regulation of Hh and Wnt pathways. These findings demonstrate OFD1 as an important immune target and highlight its possible role in signal transduction and tumorigenesis in MGUS and MM.

The aim of this study was to compare the effect of a new synthetic isothiocyanate derivative, ethyl 4-isothiocyanatobutanoate (E-4IB) and cisplatin (CDDP) in CDDP-sensitive human ovarian carcinoma cell line (A2780) and its resistant subline (A2780/CP). In parental cells, in comparison to untreated cells, sequential administration of both compounds led to higher exosomal dye (LysoTracker Green DND-26) retention and to alterations of mitogen-activated protein kinases (MAPKs), JNK, ERK and p38, or Akt kinase accompanied by changes in several anti- and pro-apoptotic molecules and lysosomal protein LAMP-1, as detected by Western blotting. On the contrary, variant A2780/CP cells were resistant to CDDP- or to combined sensitizer (E-4IB)/inducer (CDDP)-related apoptosis induction and exerted minor changes in the levels of these molecules.

Background Previously, we validated capability of human adipose tissue-derived mesenchymal stem cells (AT-MSC) to serve as cellular vehicles for gene-directed enzyme prodrug molecular chemotherapy. Yeast fusion cytosine deaminase : uracil phosphoribosyltransferase expressing AT-MSC (CDy-AT-MSC) combined with systemic 5-fluorocytosine (5FC) significantly inhibited growth of human colon cancer xenografts. We aimed to determine the cytotoxic efficiency to other tumour cells both in vitro and in vivo. Methods CDy-AT-MSC/5FC-mediated proliferation inhibition against a panel of human tumour cells lines was evaluated in direct and indirect cocultures in vitro. Antitumour effect was tested on immunodeficient mouse model in vivo. Results Although culture expansion of CDy-AT-MSC sensitized these cells to 5FC mediated suicide effect, expanded CDy-AT-MSC/5FC still exhibited strong bystander cytotoxic effect towards human melanoma, glioblastoma, colon, breast and bladder carcinoma in vitro. Most efficient inhibition (91%) was observed in melanoma A375 cell line when directly cocultured with 2% of therapeutic cells CDy-AT-MSC/5FC. The therapeutic paradigm of the CDy-AT-MSC/5FC system was further evaluated on melanoma A375 xenografts on nude mice in vivo. Complete regression in 89% of tumours was achieved when 20% CDy-AT-MSC/5FC were co-injected along with tumour cells. More importantly, systemic CDy-AT-MSC administration resulted in therapeutic cell homing into subcutaneous melanoma and mediated tumour growth inhibition. Conclusions CDy-AT-MSC capability of targeting subcutaneous melanoma offers a possibility to selectively produce cytotoxic agent in situ. Our data further demonstrate beneficial biological properties of AT-MSC as a cellular vehicle for enzyme/prodrug therapy approach to molecular chemotherapy. Copyright © 2008 John Wiley & Sons, Ltd.

Histone deacetylase (HDAC) inhibitors have been extensively investigated as therapeutic agents in cancer. However, the biological role of class IIa HDACs (HDAC4, 5, 7 and 9) in cancer cells, including multiple myeloma (MM), remains unclear. Recent studies show HDAC4 interacts with activating transcription factor 4 (ATF4) and inhibits activation of endoplasmic reticulum (ER) stress-associated proapoptotic transcription factor C/EBP homologous protein (CHOP). In this study, we hypothesized that HDAC4 knockdown and/or inhibition could enhance apoptosis in MM cells under ER stress condition by upregulating ATF4, followed by CHOP. HDAC4 knockdown showed modest cell growth inhibition; however, it markedly enhanced cytotoxicity induced by either tunicamycin or carfilzomib (CFZ), associated with upregulating ATF4 and CHOP. For pharmacological inhibition of HDAC4, we employed a novel and selective class IIa HDAC inhibitor TMP269, alone and in combination with CFZ. As with HDAC4 knockdown, TMP269 significantly enhanced cytotoxicity induced by CFZ in MM cell lines, upregulating ATF4 and CHOP and inducing apoptosis. Conversely, enhanced cytotoxicity was abrogated by ATF4 knockdown, confirming that ATF4 has a pivotal role mediating cytotoxicity in this setting. These results provide the rationale for novel treatment strategies combining class IIa HDAC inhibitors with ER stressors, including proteasome inhibitors, to improve patient outcome in MM.

Heat shock protein (HSP)90 inhibitors have shown significant anti-tumor activities in preclinical settings in both solid and hematological tumors. We previously reported that the novel, orally available HSP90α/β inhibitor TAS-116 shows significant anti-MM activities. In this study, we further examined the combination effect of TAS-116 with a RAS-RAF-MEK-ERK signaling pathway inhibitor in RAS- or BRAF-mutated MM cell lines. TAS-116 monotherapy significantly inhibited growth of RAS-mutated MM cell lines and was associated with decreased expression of downstream target proteins of the RAS-RAF-MEK-ERK signaling pathway. Moreover, TAS-116 showed synergistic growth inhibitory effects with the farnesyltransferase inhibitor tipifarnib, the BRAF inhibitor dabrafenib, and the MEK inhibitor selumetinib. Importantly, treatment with these inhibitors paradoxically enhanced p-C-Raf, p-MEK, and p-ERK activity, which was abrogated by TAS-116. TAS-116 also enhanced dabrafenib-induced MM cytotoxicity associated with mitochondrial damage-induced apoptosis, even in the BRAF-mutated U266 MM cell line. This enhanced apoptosis in RAS-mutated MM triggered by combination treatment was observed even in the presence of bone marrow stromal cells. Taken together, our results provide the rationale for novel combination treatment with HSP90α/β inhibitor and RAS-RAF-MEK-ERK signaling pathway inhibitors to improve outcomes in patients with in RAS- or BRAF-mutated MM.

A new synthetic isothiocyanate (ITC) derivative, ethyl 4-isothiocyanatobutanoate (E-4IB), appeared to be an effective modulator of cellular proliferation and potent inducer of apoptosis. In cooperation with cisplatin, this compound exerted synergistic effects in human ovarian carcinoma A2780 cells. In the present study we investigated in more detail E4IB-sensitisation for cisplatin-induced apoptosis. Sequential administration of both cytostatic agents led to increased intracellular platinum accumulation, glutathione level depletion and mitochondrial membrane potential dissipation. These events were accompanied with poly (ADP-ribosyl) polymerase cleavage, stimulation of caspase-3 activity, upregulation of p53, FasL and Gadd45alpha, cyclin B1 downregulation and an increase in mitogen-activated protein kinases JNK, ERK and p38 phosphorylation as well as PI3K level alterations. The presented results might have implications for developing new strategies aimed at therapeutic benefit of natural or synthetic ITCs in cooperation with various anticancer drugs.

N-nitrosomorpholine (NMOR) is a well-known hepatocarcinogen. Since this compound is representative of the group of indirect-acting N-nitrosamines, its metabolic activation should be essential. However, the mechanism of NMOR-induced carcinogenesis is still not completely clear. In this paper we tried to further our understanding of the genotoxic effects of NMOR. The central aim of this study was to elucidate to what extent NMOR requires metabolic activation. For evaluation of the mutagenicity of NMOR, V79 cells were used either in the presence or absence of the microsomal S9 fraction in the mutation assay and formation of reactive oxygen/nitrogen species (ROS/RNS) in Caco-2 cells treated with NMOR was measured by a fluorescent assay. A very weak rise of 6-thioguanine resistant mutations was observed in both NMOR-treated model cells, V79/-S9 and V79/+S9. A significant difference between the level of mutations in V79/-S9 and V79/+S9 cells was recorded on the 7th day of expression only. Data obtained by the fluorescent assay confirmed that NMOR caused generation of ROS/RNS. In summary, the presented results showed that NMOR might induce DNA damage not only indirectly by its activation by drug-metabolizing enzymes but also via direct formation of ROS/RNS.

Three diterpenoid quinones (royleanone – SAR 3, horminone – SAR 26, and acetyl horminone – SAR 43) isolated from the roots of Salvia officinalis L. were tested for their cytotoxic and DNA-damaging activity in human colon carcinoma cells Caco-2 and human hepatoma cells HepG2 cultured in vitro. Cytotoxicity was measured by the trypan blue exclusion technique and induction of apoptosis was evaluated by flow immunofluorocytometry after 30–300 min. exposure of HepG2 and Caco-2 cells to diterpenoid quinones and following 24 hr post-incubation in the culture medium. Induction of DNA breaks was measured after 60 min. exposure of cells to different concentrations of the compounds studied by the alkaline elution of DNA and by the Comet assay. Though all the quinones tested decreased the viability of the cells studied proportionally to the concentration and to the time of treatment (cytotoxicity=30–60%), the increased level of apoptotic nuclei comparable to the level of apoptotic nuclei induced by a topoisomerase I inhibitor was proved only in HepG2 cells treated with 1×10−4 mol/l SAR 26 or SAR 43. Either no or marginal increase of the level of apoptotic nuclei was observed in SAR 3-treated HepG2 cells and in SAR 3-, SAR 26- or SAR 43-treated Caco-2 cells. All compounds tested induced creation of DNA strand breaks in both cell types at concentrations >1×10−7–1×10−6 mol/l. The occurrence of DNA strand breaks at different pH values as well as the kinetics of DNA breaks rejoining were evaluated only in colonic cells Caco-2. The Comet assay processed in parallel at pH 13.0 and pH 12.1 showed that strand breaks detected in SARs-treated colonic Caco-2 cells originated from alkali-labile sites, as induced DNA lesions were converted to DNA strand breaks only under strong alkaline conditions. The kinetics of DNA rejoining revealed that SARs-induced DNA breaks were repaired very slowly.

The isothiocyanate, sulforaphane and the flavonoid, apigenin modulate gene expression including phase II detoxifying enzymes, such as glutathione S-transferases (GST) and UDP-glucuronosyltransferases (UGT). Using undifferentiated CaCo-2 cells, we have examined the interactions between sulforaphane and apigenin in the regulation of UGT and GST expression. We show that apigenin induces UGT1A1 transcription (4-fold) but not GSTA1, and that sulforaphane induces both UGT1A1 (3.7-fold) and GSTA1 (2.8-fold) transcription in both dose- and time-dependent manners. The combination of sulforaphane and apigenin resulted in a synergistic induction of UGT1A1 mRNA up to 12-fold, although this interaction was not seen for GSTA1. Nuclear factor kappa B (NF-kappaB) mRNA was induced by apigenin and sulforaphane (2.5- and 2-fold, respectively). NF-kappaB translocation inhibitor SN50 and phosphatidylinositol 3-kinase (PI3) inhibitor LY294002 decreased the induction of GSTA1 by sulforaphane almost to baseline level. However, the MEK inhibitor PD98059 enhanced significantly the induction of GSTA1 by sulforaphane. This suggests that NF-kappaB and PI3-kinase signaling pathways play a role in GSTA1 gene expression. Conversely, the induction of UGT1A1 transcription by sulforaphane was totally abolished by PD98059, although PD98059 slightly enhanced (20%) the induction of UGT1A1 by apigenin implying that the induction of UGT1A1 by sulforaphane is mediated by MAPK/extracellular signal-regulated kinase kinase, whereas UGT1A1 induction by apigenin may be associated with NF-kappaB translocation since the NF-kappaB translocation inhibitor, SN50 enhanced the induction of UGT by apigenin. The results show that UGT1A1 and GSTA1 are regulated by sulforaphane through different signal transduction pathways and the differences in the mechanisms of modulation of UGT1A1 transcription by sulforaphane and apigenin resulted in a synergistic effect between these two compounds in the induction of UGT1A1.

The antioxidant butylated hydroxyanisole (BHA) is a rodent carcinogen that also reduces the mutagenicity and carcinogenicity of other agents. In this study, we have evaluated possible mechanisms for the antimutagenicity of BHA by investigating its effects on N-methyl-N′-nitro-N-nitrosoguanidine (MNNG)-treated Chinese hamster V79 cells. Mutant frequency was determined using the hprt/V79 assay, while plating efficiency was used to measure cytotoxicity, and apoptosis was measured by flow immunofluorocytometry. In addition, DNA strand breaks and the kinetics of strand-break rejoining were investigated by the alkaline elution of DNA and by single-cell gel electrophoresis (SCGE). Although the higher concentration of BHA (0.5 mM) increased the cytotoxicity of MNNG and the lower concentration of BHA (0.25 mM) did not change it, both concentrations were antimutagenic in MNNG-treated cells, with the greater effect occurring at the lower BHA concentration. Neither BHA nor MNNG nor BHA + MNNG increased the level of apoptotic nuclei, and BHA did not change the level of MNNG-induced DNA strand breaks, though it did inhibit their rejoining. Determination of O6-methylguanine-DNA-methyltransferase (MGMT) activity confirmed that V79 cells do not synthesize active MGMT protein; MGMT activity was also undetectable after MNNG and BHA + MNNG treatment. The ability of BHA to reduce the level of MNNG-induced mutations did not correlate with cytotoxicity, induction of apoptosis, the level of DNA strand break induction, or MGMT activity. A modified SCGE assay showed that BHA significantly reduced the level of formamidopyrimidine-DNA-glycosylase + endonucleaseIII-sensitive sites, which at least partially are caused by oxidative DNA lesions. The results suggest that the protective effect of BHA on MNNG-induced mutagenicity is best explained by the antioxidative activity of BHA, which may scavenge free radicals that participate in MNNG-induced mutagenicity. Environ. Mol. Mutagen. 41:28–36, 2003. © 2003 Wiley-Liss, Inc.

Anti-tumor activities of selective HSP90α/β inhibitor, TAS-116, in combination with bortezomib in multiple myeloma

Purpose: Cellular immunotherapy frequently fails to induce sustained remissions in patients with multiple myeloma, indicating the ability of multiple myeloma cells to evade cellular immunity. Toward a better understanding and effective therapeutic modulation of multiple myeloma immune evasion mechanisms, we here investigated the role of the tumor microenvironment in rendering multiple myeloma cells resistant to the cytotoxic machinery of T cells. Experimental Design: Using a compartment-specific, bioluminescence imaging-based assay system, we measured the lysis of luciferase-transduced multiple myeloma cells by CD4+ or CD8+ CTLs in the presence versus absence of adherent accessory cells of the bone marrow microenvironment. We simultaneously determined the level of CTL activation by measuring the granzyme B release in culture supernatants. Results: Bone marrow stromal cells from patients with multiple myeloma and healthy individuals, as well as vascular endothelial cells, significantly inhibited the lysis of multiple myeloma cells in a cell–cell contact-dependent manner and without substantial T-cell suppression, thus showing the induction of a cell adhesion-mediated immune resistance (CAM-IR) against CTL lysis. Further analyses revealed that adhesion to accessory cells downregulated Fas and upregulated the caspase-3 inhibitor survivin in multiple myeloma cells. Reconstitution of Fas expression with bortezomib enhanced the CTL-mediated lysis of multiple myeloma cells. Repressing survivin with the small-molecule YM155 synergized with CTLs and abrogated CAM-IR in vitro and in vivo. Conclusion: These results reveal the cell adhesion-mediated induction of apoptosis resistance as a novel immune escape mechanism and provide a rationale to improve the efficacy of cellular therapies by pharmacologic modulation of CAM-IR. Clin Cancer Res; 19(20); 5591–601. ©2013 AACR.