TAS-117, a Novel Selective Akt Inhibitor Demonstrates Significant Growth Inhibition in Multiple Myeloma Cells in Vitro and in Vivo

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 942-942 ◽  
Author(s):  
Naoya Mimura ◽  
Hiroto Ohguchi ◽  
Diana Cirstea ◽  
Francesca Cottini ◽  
Gullu Topal Gorgun ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Drug Resistance ◽  
Cell Growth ◽  
Board Of Directors ◽  
Cell Lines ◽  
Akt Inhibitor ◽  
Advisory Committees ◽  
Significant Growth Inhibition

Abstract Abstract 942 The PI3K/Akt pathway mediates multiple myeloma (MM) cell growth and drug resistance, and targeting this molecule is a promising therapeutic option. In this study, we examined anti-MM activities of TAS-117 (TAIHO PHARMACEUTICAL CO., LTD., JAPAN), a selective potent Akt inhibitor in MM cell lines including MM.1S, MM.1R, OPM1 and H929 cells with high level of baseline Akt phosphorylation. TAS-117 induced significant growth inhibition in these cell lines, associated with downregulation of phosphorylation (Ser473 and Thr308) of Akt and downstream molecule FKHR/FKHRL1, without cytotoxicity in normal peripheral blood mononuclear cells. TAS-117 triggered G0/G1 arrest followed by apoptosis, evidenced by increased annexin V-positive cells, in both MM.1S and H929 cell lines. Apoptosis was further confirmed by cleavage of caspase-8, -3 and PARP. Interestingly, TAS-117 also induced: autophagy, evidenced by increased LC3-II; as well as endoplasmic reticulum (ER) stress, confirmed by induction of phospho-eIF2α, phospho-IRE1α and a molecular chaperone BiP/GRP78. Since the bone marrow (BM) microenvironment plays a crucial role in MM cell pathogenesis including drug resistance, we further examined the effect of TAS-117 in the presence of BM stromal cells (BMSCs). TAS-117 induced significant cytotoxicity in MM cells even in the presence of BMSCs, associated with downregulation of phospho-Akt. Importantly, TAS-117 inhibited secretion of IL-6 from BMSCs, and exogenous IL-6 and IGF-1 did not block cytotoxicity induced by this agent. We have previously shown the bortezomib activates Akt, and that Akt inhibition with bortezomib triggers synergistic MM cell cytotoxicity. TAS-117 enhanced bortezomib-induced cytotoxicity in MM.1S cells, associated with increased CHOP followed by PARP cleavage, suggesting that TAS-117 augments bortezomib-induced ER stress and apoptotic signaling. TAS-117 also enhanced cytotoxicity induced by other therapeutic agents (ie, rapamycin, dexamethasone, 17-AAG) in MM.1S cells. Finally, we examined anti-MM activities of TAS-117 in a xenograft murine model. Oral administration of TAS-117 for 14 days significantly inhibited growth of H929 plasmacytoma and was well tolerated. Taken together, the novel and selective Akt inhibitor TAS-117 blocks MM cell growth in vitro and in vivo, providing the preclinical framework for clinical evaluation of this agent to improve patient outcome in MM. Disclosures: Shimomura: TAIHO PHARMACEUTICAL CO., LTD.: Employment. Utsugi:TAIHO PHARMACEUTICAL CO., LTD.: Membership on an entity's Board of Directors or advisory committees. Anderson:Celgene, Millennium, BMS, Onyx: Membership on an entity's Board of Directors or advisory committees; Acetylon, Oncopep: Scientific Founder, Scientific Founder Other.

A Novel Acanthoic Acid Analog NPI-1342 Blocks IκB Kinase-α and Trigger In Vitro and In Vivo cytotoxicity in Multiple Myeloma Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1841-1841
Author(s):  
Dharminder Chauhan ◽  
Ajita V. Singh ◽  
Arghya Ray ◽  
Teru Hideshima ◽  
Paul G. Richardson ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Growth ◽  
Board Of Directors ◽  
Tumor Cells ◽  
Cell Lines ◽  
Inhibitory Effect ◽  
Advisory Committees ◽  
Acid Analog

Abstract Abstract 1841 Introduction: The dimeric Nuclear Factor-kappa B (NF-κB) transcription factor plays a key role during multiple myeloma (MM) cell adhesion-induced cytokine secretion in bone marrow stromal cells, which in turn triggers MM cell growth in a paracrine manner. NF-κB signaling pathway is mediated via canonical (IKK-α/IKK-β/NEMO-P50/65 or NF-κB1) and non-canonical (IKK-α/IKK-α/NIK-p52/RelB or NF-κB2) components. Prior studies have also linked constitutive activation of non-canonical NF-κB pathway to genetic abnormalities/mutation, allowing for an autocrine growth of MM cells. Other recent studies showed that constitutive NF-κB activity in tumor cells from MM patients renders these cells refractory to inhibition by bortezomib; and in fact, that bortezomib induces canonical NF-κB activity. These reports provided the impetus for the development of an agent with ability to modulate canonical and/or non-canonical NF-κB axis, allowing for a more robust and specific inhibition of NF-κB. Recent research and development efforts at Nereus Pharmaceuticals, Inc., have identified a novel small molecule acanthoic acid analog NPI-1342 as a potent NF-κB inhibitor. Here, we examined the effects of NPI-1342 on canonical versus non-canonical NF-κB signaling pathways, as well as its anti-tumor activity against MM cells using both in vitro and in vivo model systems. Methods: We utilized MM.1S, MM.1R, RPMI-8226, U266, KMS12PE, NCI-H929, OCI-MY5, LR5, Dox-40, OPM1, and OPM2 human MM cell lines, as well as purified tumor cells from patients with MM. Cell viability assays were performed using MTT and Trypan blue exclusion assays. Signal transduction pathways were evaluated using immunoblot analysis, ELISA, and enzymology assays. Animal model studies were performed using the SCID-hu model, which recapitulates the human BM milieu in vivo. Results: We first examined the effects of NPI-1342 on lipopolysaccharides (LPS)-induced NF-κB activity. Results showed that NPI-1342 inhibits LPS-stimulated NF-κB activity in vitro, as measured by phosphorylation of IkBa. To determine whether NPI-1342 triggers a differential inhibitory effect on IKKβ versus IKKα, MM.1S MM cells were treated with NPI-1342 for 48 hours, and protein lysates were subjected to kinase activity assays. NPI-1342 blocked IKKα, but not IKKβ or IKKγ phosphorylation. We next assessed whether the inhibitory effect of NPI-1342 on NF-κB activity is associated with cytotoxicity in MM cells. We utilized a panel of MM cell lines: at least five of these have mutations of TRAF3 (MM.1S, MM.1R, DOX40 and U266); one has no known NF-κB mutations (OPM2), and one has amplification of NF-κB1 (OCI-MY5). Treatment of MM cell lines and primary patient (CD138 positive) MM cells for 48 hours significantly decreased their viability (IC50 range 15–20 μM) (P < 0.001; n=3) without affecting the viability of normal peripheral blood mononuclear cells, suggesting selective anti-MM activity and a favorable therapeutic index for NPI-1342. NPI-1342-induced a marked increase in Annexin V+ and PI- apoptotic cell population (P < 0.001, n=3). Mechanistic studies showed that NPI-1342-triggered apoptosis in MM cells is associated with activation of caspase-8, caspase-9, caspase-3, and PARP cleavage. We next examined the in vivo effects of NPI-1342 in human MM xenograft models. For these studies, we utilized the SCID-hu MM model, which recapitulates the human BM milieu in vivo. In this model, MM cells are injected directly into human bone chips implanted subcutaneously in SCID mice, and MM cell growth is assessed by serial measurements of circulating levels of soluble human IL-6R in mouse serum. Treatment of tumor-bearing mice with NPI-1342 (20 mg/kg intraperitoneally, QD1-5 for 2 weeks), but not vehicle alone, significantly inhibits MM tumor growth in these mice (10 mice each group; P = 0.004). The doses of NPI-1342 were well tolerated by the mice, without significant weight loss. Finally, immunostaining of implanted human bone showed robust apoptosis and blockade of NF-κB in mice treated with NPI-1342 versus vehicle alone. Conclusions: We demonstrate the efficacy of a novel small molecule inhibitor of NF-κB NPI-1342 in MM using both in vitro and in vivo models. NPI-1342 blocks NF-κB activity with a preferential inhibitory activity against IKK-α component of NF-κB signaling. Our preclinical studies support evaluation of NPI-1342 as a potential MM therapy. Disclosures: Hideshima: Acetylon: Consultancy. Richardson:Millennium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Palladino:Nereus Pharmaceuticals, Inc: Employment, Equity Ownership. Anderson:Celgene: Consultancy; Millennium: Consultancy; Onyx: Consultancy; Merck: Consultancy; Bristol Myers Squibb: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Acetylon:; Nereus Pharmaceuticals, Inc: Consultancy.

Anti-Myeloma Activity of a Novel Free Radical Inducer Rrx-001

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4712-4712 ◽  
Author(s):  
Deepika Sharma Das ◽  
Ze Tian ◽  
Arghya Ray ◽  
Durgadevi Ravillah ◽  
Yan Song ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Clinical Trial ◽  
Drug Resistance ◽  
Board Of Directors ◽  
Tumor Cells ◽  
Cell Lines ◽  
Advisory Committees ◽  
Healthy Donors

Abstract Background and Rationale: Multiple Myeloma (MM) remains incurable despite the advent of novel drugs, highlighting the need for further identification of factors mediating disease progression and resistance. The bone marrow (BM) microenvironment confers growth, survival, and drug resistance in MM cells. Studies to date suggest an important role of BM hypoxia (low oxygenation) in MM cell survival, drug resistance, migration, and metastasis. Therapies targeting the MM cell in its BM milieu under hypoxic conditions may therefore achieve responses in patients resistant to various therapies. Recent studies led to the development of a novel aerospace-industry derived Phase 2 molecule RRx-001 with epigenetic and NO-donating properties. RRx-001 generates reactive oxygen and nitrogen species (RONS), which induces oxidative stress in tumor cells. Importantly, RRx-001 is also a potent vascular disrupting agent, which further provides rationale for utilizing RRx-001 as a therapeutic agent since tumor-associated angiogenesis is a characteristic of MM. A Phase I clinical trial has shown RRx-001 to have antitumor activity in heavily pretreated cancer patients and to be safe and well tolerated with no dose-limiting toxicities (Reid et al. J Clin Oncol 32:5s, 2014 suppl; abstr 2578). Here we examined the anti-MM activity of RRx-001 using in vitro and in vivo models of MM. Materials and methods: MM cell lines, patient MM cells, and peripheral blood mononuclear cells (PBMCs) from normal healthy donors were utilized to assess the anti-MM activity of RRx-001 alone or in combination with other agents. Drug sensitivity, cell viability, apoptosis, and migration assays were performed using WST, MTT, Annexin V staining, and transwell Inserts, respectively. Synergistic/additive anti-MM activity was assessed by isobologram analysisusing “CalcuSyn” software program. Signal transduction pathways were evaluated using immunoblotting. ROS release, nitric oxide generation, and mitochondrial membrane potential was measured as previously described (Chauhan et al., Blood, 2004, 104:2458). In vitro angiogenesis was assessed using matrigel capillary-like tube structure formation assays. DNMT1 activity was measured in protein lysates using EpiQuik DNMT1 assay kit. 5-methyl cytosine levels were analyzed in gDNA samples using methylflash methylated DNA quantification kit from Enzo life sciences; USA. For xenograft mouse model, CB-17 SCID-mice were subcutaneously inoculated with MM.1S cells as previously described (Chauhan et al., Blood, 2010, 115:834). Statistical significance of data was determined using a Student’st test. RRx-001 was obtained from RadioRx Inc., CA, USA; bortezomib, SAHA, and pomalidomide were purchased from Selleck chemicals, USA. Results: Treatment of MM cell lines (MM.1S, MM.1R, RPMI-8226, OPM2, H929, Dox-40 ARP-1, KMS-11, ANBL6.WT, ANBL6.BR, and LR5) and primary patient cells for 24h significantly decreased their viability (IC50 range 1.25nM to 2.5nM) (p < 0.001; n=3) without markedly affecting PBMCs from normal healthy donors, suggesting specific anti-MM activity and a favorable therapeutic index for RRx-001. Tumor cells from 3 of 5 patients were obtained from patients whose disease was progressing while on bortezomib, dexamethasone, and lenalidomide therapies. Moreover, RRx-001 inhibits proliferation of MM cells even in the presence of BM stromal cells. Mechanistic studies show that RRx-001-triggered apoptosis is associated with 1) induction of DNA damage response signaling via ATM/p53/gH2AX axis; 2) activation of caspases mediating both intrinsic and extrinsic apoptotic pathways; 3) increase in oxidative stress through release of ROS and generation of NO; and 4) decrease in DNA methyltransferase (DNMT1) enzymatic activity and global methylation levels. Furthermore, RRx-001 blocked migration of MM cells and angiogenesis. In vivo studies using subcutaneous human MM xenograft models show that RRx-001 is well tolerated and inhibits tumor growth. Finally, combining RRx-001 with bortezomib, SAHA, or pomalidomide induces synergistic anti-MM activity and overcomes drug resistance. Conclusion: Our preclinical studies showing efficacy of RRx-001 in MM disease models provide the framework for clinical trial of RRx-001, either alone or in combination, to improve outcome in relapsed and refractory MM patients. Disclosures Richardson: Oncopeptides AB: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees. Oronsky:RadioRx Inc, : Employment. Scicinski:RadioRx Inc,: Employment. Chauhan:Triphase Accelerator: Consultancy. Anderson:Celgene: Consultancy; Millenium: Consultancy; Onyx: Consultancy; Gilead: Consultancy; Sanofi Aventis: Consultancy; BMS: Consultancy; Oncopep/Acetylon: Equity Ownership.

A Novel SIRT1 Activator SIRT1720 Triggers In Vitro and In Vivo Cytotoxicity In Multiple Myeloma Via ATM-Dependent Mechanism

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3007-3007
Author(s):  
Dharminder Chauhan ◽  
Madhavi Bandi ◽  
Ajita V Singh ◽  
Teru Hideshima ◽  
Nikhil C. Munshi ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Board Of Directors ◽  
Cell Lines ◽  
Annexin V ◽  
Immunoblot Analysis ◽  
Advisory Committees ◽  
Rpmi 8226 ◽  
Sirt1 Activator

Abstract Abstract 3007 Background and Rationale: SIRT1 belongs to the silent information regulator 2 (Sir2) family of proteins and functions as NAD+-dependent deacetylase. Previous studies showed that resveratrol, a polyphenolic SIRT1 activator, inhibits tumorigenesis in various solid tumor and hematologic malignancies, including human multiple myeloma (MM) cells. This notion led to the discovery and development of more potent and selective pharmacological activators of SIRT1 as potential anti-cancer therapeutics. In this context, a recent medicinal chemistry research using high-throughput screening, and mass spectrometry identified SRT1720, a small molecule activator of SIRT1 that is structurally distinct from resveratrol. Here, we examined the anti-tumor activity of SRT1720 in MM cells using in vitro and in vivo model systems. Methods and Model: We utilized MM.1S, MM.1R, RPMI-8226, U266, KMS12BM, H929, and INA-6 (an IL-6 dependent) human MM cell lines, as well as purified tumor cells from patients with MM relapsing after prior therapies including lenalidomide or bortezomib. Cell viability, proliferation, and apoptosis assays were performed using trypan blue, MTT, thymidine incorporation, and Annexin V staining. Signal transduction pathways were evaluated using immunoblot analysis, ELISA, and enzymology assays. Results: We first confirmed the functional specificity of SRT1720 against SIRT1 using different experimental strategies. First, we utilized Fluor de Lys Deacetylase Assay to measure whether SRT1720 affects the SIRT1 deacetylase enzymatic activity. Treatment of MM.1R and RPMI-8226 MM cells with SRT1720 markedly increased the deacetylating activity; conversely, pre-treatment of cells with nicotinamide (NAM) - an inhibitor of SIRT1 – significantly blocked SRT1720-triggered deacetylating activity. Second, immunoblot analysis using antibodies specific against acetylated p53 (a known substrate of SIRT1) showed a marked decrease in acetylated state of p53 in SRT1720-treated MM cells. These findings in MM cells confirm SIRT1 as a selective target of SRT1720. We next examined the efficacyof SRT1720 in MM cells. Treatment of MM cell lines and primary patient cells for 24h significantly decreased their viability (IC50 range 3–7 uM) (P < 0.005; n=3) without markedly affecting the viability of normal peripheral blood mononuclear cells, suggesting specific anti-MM activity and a favorable therapeutic index for SRT1720. SRT1720-triggered apoptosis was confirmed in MM.1R and RPMI-8226 cells, evidenced by a marked increase in Annexin V+ and PI- cell population (P < 0.001, n=3). Importantly, SRT1720 induced apoptosis in MM cells even in the presence of bone marrow stromal cells. Mechanistic studies showed that SRT1720-triggered apoptosis in MM cells is associated with 1) activation of caspase-8, caspase-9, caspase-3, and PARP; 2) activation of pATM, CHK2, endoplasmic reticulum stress molecules pEIF2, and BIP; as well as an increase in reactive oxygen species (ROS); 3) inhibition of MM cell growth and survival pathway via NF-kB; and 4) inhibition of VEGF-induced migration of MM cells and associated angiogenesis. Importantly, blockade of pATM using a biochemical inhibitor KU-5593 significantly attenuated SRT1720-induced MM cell death (P value < 0.002; n=2). These data suggest that SRT1720-induced MM cell apoptosis is predominantly mediated by an ATM-dependent apoptotic pathway. We next examined the in vivo efficacy of SRT1720 using a human plasmacytoma xenograft mouse model. Treatment of tumor-bearing mice with SRT1720 (200 mg/kg, 5 days a week for three weeks), but not vehicle alone, significantly (P < 0.008) inhibits MM tumor growth in these mice. Finally, the combination of SRT1720 with bortezomib or dexamethasone triggered synergistic anti-MM activity. Conclusions: These preclinical studies provide the rationale for novel therapeutics targeting SIRT1 to improve patient outcome in MM. Disclosures: Munshi: Millennium Pharmaceuticals: Honoraria, Speakers Bureau. Richardson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees. Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

scholarly journals Dysfunctional HDAC8 Impacts Genomic Integrity and Is a Novel Therapeutic Target in Multiple Myeloma

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1610-1610
Author(s):  
Zuzana Chyra ◽  
Srikanth Talluri ◽  
Rao Prabhala ◽  
Mehmet K. Samur ◽  
Anil Aktas-Samur ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Growth ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Hdac Inhibitors ◽  
Dependent Manner ◽  
Dna Breaks ◽  
Advisory Committees

Abstract The histone modifications and associated changes in chromatin structure and function have emerged as important epigenetic mechanisms impacting gene expression and have significant translational relevance in cancers, including multiple myeloma (MM). Epigenetic intervention with histone deacetylases (HDACs) inhibitors is emerging as a promising therapeutic strategy in combination with current anti-myeloma agents. Although pan-HDAC inhibitors have been shown to be effective both in preclinical and clinical setting, they seem to be associated with toxicity. It is, therefore, extremely important to understand the biological and molecular roles of individual HDACs to then selectively target them to limit toxicities observed with pan-HDAC inhibitors. Based on our observation that elevated HDAC8 expression correlates with poor overall survival in MM patients in three different datasets including one publicly available dataset (GSE39754), we evaluated its functional role in MM. HDAC8, a member of class I HDAC isoenzymes, is responsible for the deacetylation of lysine residues on the N-terminal part of the core histones as well as non-histone proteins. We performed genetic modulation of HDAC8 by loss-of-function studies, using shRNA as well as siRNAs targeting HDAC8. Downregulation of HDAC8 in 3 different MM cell lines caused MM cell growth inhibition in a time-dependent manner which was associated with induction of cell apoptosis. Consistently, treatment with a selective and potent HDAC8 inhibitor (OJI-1) caused a significant inhibition of MM cell growth in a panel of 20 MM cell lines (IC50 = 80 nM) in a time- and dose-dependent manner, while having a minimal impact on six PBMC samples from healthy donors both in resting and activated state (IC50 = 150 nM). The mechanism of cell death was apoptosis as demonstrated by annexin-labeling. Importantly, both the HDAC8 knockdown and OJI-1 treatment inhibited DNA breaks as evidenced from γH2AX expression or a single cell gel electrophoresis method to visualize and quantitate DNA breaks. HDAC8 inhibition also caused inhibition of RAD51 foci and HR activity, as measured by strand-exchange assay. Interestingly, non-homologous end joining in MM cells was not impacted by these treatments. Consistent with these data, the overexpression of HDAC8 in MM as well as in normal cells increased DNA breaks and HR activity. Furthermore, the inhibition of HDAC8 (by knockdown and OJI-1) inhibited, whereas its overexpression increased genomic instability, as assessed by micronucleus assay, in surviving MM cells. We also demonstrate that HDAC8 interacts with RAD51 and impacts its acetylation. The treatment of MM cells with HDAC8 inhibitor (OJI-1) increased RAD51 acetylation. Next, we examined the in vivo efficacy of the HDAC8 conditional knockdown in a human xenograft mouse model, using H929 cells injected subcutaneously in SCID mice. HDAC8 knockdown not only caused a significant reduction in tumor growth but also increased survival (p=0.0016) compared to mice injected with control cells. Evaluation of tumors from these mice confirmed in vivo inhibition of DNA breaks and HR activity, and induction of apoptosis following HDAC8-knockdown. HDAC8 inhibitor OJI-1 also synergistically increased the cytotoxicity of existing MM drugs including dexamethasone, bortezomib and lenalidomide. In conclusion, our results demonstrate that elevated HDAC8 in MM cells is involved in inhibition of apoptosis but also contributes to increased DNA breaks and dysregulation of homologous recombination and genome stability. Therefore, HDAC8 is a novel target for therapeutic application in MM. Selective and potent HDAC8 inhibitor OJI-1 has shown a favorable therapeutic index with synergistic effect in combination with existing MM drugs. Disclosures Hajek: Pharma MAR: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Research Funding; BMS: Consultancy, Honoraria, Research Funding. Munshi: Janssen: Consultancy; Bristol-Myers Squibb: Consultancy; Amgen: Consultancy; Takeda: Consultancy; Celgene: Consultancy; Karyopharm: Consultancy; Abbvie: Consultancy; Adaptive Biotechnology: Consultancy; Oncopep: Consultancy, Current equity holder in publicly-traded company, Other: scientific founder, Patents & Royalties; Novartis: Consultancy; Pfizer: Consultancy; Legend: Consultancy.

scholarly journals Inhibition of Sialylation Impairs Adhesion on Madcam-1 and E-Selectin and Sensitize Multiple Myeloma Cells to Bortezomib in a Xenograft Mouse Model

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3204-3204
Author(s):  
Alessandro Natoni ◽  
Mariah Farrell ◽  
Heather Fairfield ◽  
Lucy Kirkham-McCarthy ◽  
Matt Macauley ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Drug Resistance ◽  
Board Of Directors ◽  
Research Funding ◽  
Immune Cell ◽  
Sialyl Lewis X ◽  
Advisory Committees ◽  
Selectin Ligands

Abstract Introduction Multiple myeloma (MM) is a cancer of clonal plasma cells that hijack the bone marrow (BM) niche to create a drug resistant, incurable malignancy. Aberrant sialylation has been linked to immune cell evasion, drug resistance, and metastasis in cancer; indeed sialyltransferases, including ST3GAL1, ST3GAL4 and ST3GAL6, are aberrantly expressed in many cancers (Glavey et al., 2014). We have previously shown that targeting ST3GAL6 in MM cells inhibits their ability to extravasate and colonize the BM in mouse models (Glavey et al., 2014). Moreover, we also showed that a subpopulation of MM cells expresses functional E-Selectin ligands which, upon expansion, gives rise to a more aggressive disease and resistance to bortezomib in mice (Natoni et al., 2017). Based off these findings, we herein investigated whether inhibiting sialylation in E-selectin-enriched MM cells with 3Fax-Neu5Ac, a small molecule sialyltransferase inhibitor, could alter the ability of these cells to home in the BM and restore bortezomib sensitivity in vivo. We hypothesized that inhibiting homing of MM cells to the BM will improve survival and that co-treatment with bortezomib and 3Fax-Neu5Ac will have a synergistic effect. Methods E-selectin ligands enriched MM1S cells (either positive or negative for GFP/Luciferase) were derived from parental cells by cell sorting using the HECA-452 antibody, which recognize sialofucosylated E-selectin ligands. We then determined the 3Fax-Neu5Ac dose and exposure times needed to decrease sialylation on these MM cells without causing toxicity. HECA-452-enriched MM1S cells were pretreated with 3Fax-Neu5Ac or vehicle for 7 days before being injected into SCID-beige mice and then treated with vehicle or bortezomib (0.3 mg/kg twice a week). Mice were analyzed via bioluminescence imaging (BLI) to monitor tumor progression and weighed twice a week. Mice were euthanized when they began to show paralysis under our IACUC protocol. 3Fax-Neu5Ac pretreated HECA-452 MM1S cells were also tested in vitro for their ability to adhere and roll on VCAM-1, MAdCAM-1 and E-Selectin under shear stress and to respond to bortezomib in co-culture with HS5 cells. Results Treatment of HECA-452 MM1S cells with 3Fax-Neu5Ac, at 300 μM for 7 days significantly reduced sialylation on these cells. Importantly, reducing sialylation with 3Fax-Neu5AC reduced tumor burden and increased survival, although this did not reach significance for survival (Figure 1A). Both vehicle- and 3Fax-Neu5Ac-treated cells significantly responded to bortezomib in the first 5 weeks of the in vivo study (Figure 1B). However, the HECA-452 MM1S cells did not show increased survival when treated with bortezomib suggesting an acquired mechanism of resistance in vivo. Importantly, pretreatment of the HECA-452 MM1S with 3Fax-Neu5Ac could improve survival of these mice preventing bortezomib resistance. In vitro, the HS5 stromal cells protected the HECA-452 MM1S cells from bortezomib and pretreatment with 3Fax-Neu5Ac partially reverted this protection. Moreover, the HECA-452 MM1S cells pretreated with 3Fax-Neu5Ac displayed reduced adhesion on MAdCAM-1 and E-selectin. Conclusions Sialylation plays an instrumental role in bone homing, BM colonization, and drug resistance of MM cells. Pretreatment of HECA-452 MM1S cells with 3Fax-Neu5Ac decreased their sialylation, restored sensitivity to bortezomib in vivo and prolonged survival in mice. This is likely because 3Fax-Neu5Ac pretreatment has multiple effects on MM cells including reducing cell adhesion mediated-drug resistance and adhesion to key molecules involved in BM homing such as MAdCAM-1 and E-selectin. The reduced adhesion on E-selectin is most likely due to the disruption of E-selectin ligands on the surface of MM cells as they require Sialyl Lewis X to function. Notably, we also found that de-sialylation impairs adhesion on MAdCAM-1 (3Fax-Neu5Ac vs DMSO P=0.038) which, together with E-selectin, is another critical BM homing receptor. This data suggests for the first time that sialylation may controls the affinity of integrin α4β7 and its counter-receptor MAdCAM-1. In turn, this would reduce BM homing and increase MM cells in the circulation were they are more prone to the cytotoxic effects of bortezomib. This study supports the importance of targeting sialylation in MM and provides a strong rationale for further clinical translation of this novel approach. Disclosures O'Dwyer: Glycomimetics: Research Funding; Celgene: Research Funding; BMS: Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Onkimmune: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding.

POU2AF1 As a Master Regulator of Oncogenic Transcription Factor Networks in Myeloma

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 18-19
Author(s):  
Ricardo De Matos Simoes ◽  
Ryosuke Shirasaki ◽  
Huihui Tang ◽  
Shizuka Yamano ◽  
Benjamin G Barwick ◽  
...  
Keyword(s):  
Cell Growth ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Rna Seq ◽  
Advisory Committees ◽  
Genome Scale ◽  
Crispr Activation

Background: Functional genomics studies based on CRISPR and shRNA have documented that multiple myeloma (MM) cells are preferentially dependent (compared to other neoplasias) on a series of TFs, including IKZF1 and IKZF3 (which are targeted by thalidomide derivatives) and others that are not amenable to degradation or small molecule inhibition. Transcriptional co-factors have been therapeutically targeted, for example, inhibitors of BRD4, a co-factor for pTEFB, can be used to down-regulate c-myc. Aim: To identify new transcriptional vulnerabilities in MM with an emphasis on transcriptional co-factors Methods: We integrated results from genome-scale studies using the AVANA library for loss-of-function by gene editing (in 19 MM lines) and the Calabrese library for CRISPR-mediated gene activation (in 5 MM lines) to identify critical transcriptional co-factors (co-TFs). RNA-Seq analysis was used to identify critical pathways affected by POU2AF1 activation and existing ChIP-Seq tracks in MM cells were reanalyzed. Results: POU2AF1 (OCA-B) was the most preferentially essential TF co-factor in MM cell lines vs. non-MM and one of top genes which, upon CRISPR activation in genome-scale studies, increased MM cell fitness in vitro. We further confirmed the role of this gene using focused libraries of sgRNAs against POU2AF1 in vitro and in an in vivo model of MM cell growth in bone marrow-like scaffolds "functionalized" with humanized mesenchymal bone marrow stromal cells to simulate the human BM. CRISPR activation of POU2AF1 is associated with increased MM cell growth. RNA-Seq of POU2AF1 activation in LP1 cells a transcriptional program characterized by upregulation of other genes that are preferentially essential for MM including PRDM1, SUPT7L, UBE2G2 and TSC1; broad-spectrum oncogenic dependencies (e.g KRAS) and genes known or proposed to be involved in the pathophysiology of MM or other neoplasias (e.g. RUNX2, FGFR3, SMO, CREB5, TNFRSF13B, MEF2D, PCGF2). POU2AF1 overexpression was also associated with down-regulation of CDKN1C; of MHC class II molecules and their transcriptional activator CIITA, suggesting that POU2AF1 activation could also contribute to increased MM growth in vivo by allowing escape from immune surveillance. ATAC-Seq data and genome-wide ChIPseq for H3K27Ac in MM cell lines indicate that chromatin surrounding the POU2AF1 locus was highly accessible, concordant with the consistent expression of this TF in MM cell lines and patient-derived cells. CoMMpass data showed that POU2AF1 expression was enhanced in a subset of MM patients at relapse compared to diagnosis. Motif analysis of ChIP-seq data for POU2AF1 identified significant overlap with motifs for TFs relevant to the POU family (e.g. Oct11, Oct2, Oct4); members of the ETS family (e.g. ELF1, Elf4, GABPA); and other TFs with roles in MM including c-myc; IRF4; NF-kappaB, PRDM1, RUNX2 and the POU2AF1 target CREB5. These data suggest a functional interaction between POU2AF1 and other MM-relevant TFs. The transcriptional signature of POU2AF1 activation is enriched for genes downregulated by suppression/inhibition of MM-preferential TFs or epigenetic regulators including IRF4, PRDM1, IKZF1/3 and DOT1L. POU2AF1 binding motifs are also enriched in the promoter regions of MM-preferential dependencies including several MM-preferential TFs. Conclusions: POU2AF1 is essential for MM cells in vitro and in vivo; has a significantly more pronounced and recurrent role as a dependency in MM compared to most other neoplasias; and can further drive MM cell growth, through its ability to interact with several TFs critical for MM, forming multi-protein functional complexes. These results establish POU2AF1 as a central component in the regulatory network of oncogenic TFs in MM and highlight the value of further exploring POU2AF1 as a therapeutic target in MM. Disclosures Downey-Kopyscinski: Rancho BioSciences, LLC: Current Employment. Tsherniak:Cedilla Therapeutics: Consultancy; Tango Therapeutics: Consultancy. Boise:AstraZeneca: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genetech: Membership on an entity's Board of Directors or advisory committees. Mitsiades:FIMECS: Consultancy, Honoraria; Ionis Pharmaceuticals, Inc.: Consultancy, Honoraria; Arch Oncology: Research Funding; Janssen/Johnson & Johnson: Research Funding; Karyopharm: Research Funding; TEVA: Research Funding; Takeda: Other: employment of a relative; Fate Therapeutics: Consultancy, Honoraria; Sanofi: Research Funding; Abbvie: Research Funding; EMD Serono: Research Funding.

Overcoming CD28-Mediated Multi-Drug Resistance With a Novel PIM2 Inhibitor

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1931-1931
Author(s):  
Carmen M. Baldino ◽  
Jayakumar R. Nair ◽  
Justin Caserta ◽  
Megan Murray ◽  
Stephane Dumas ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Drug Resistance ◽  
Board Of Directors ◽  
Cell Lines ◽  
Tumor Xenograft ◽  
Multi Drug Resistance ◽  
Employment Equity ◽  
Advisory Committees ◽  
Equity Ownership

Abstract Drug resistance in multiple myeloma (MM) is the major cause of treatment failure and is significantly mediated by pro-survival interactions with bone marrow microenvironment. A key myeloma receptor involved in this interaction is CD28, which has been largely characterized as the prototypic T cell costimulatory receptor. However, CD28 expression on myeloma cells is significantly correlated with disease progression, worse prognosis, and is significantly higher in the poor prognosis t(14;16) MAF subgroup. We now report that CD28 signaling mediates significant drug resistance and protects MM against death from multiple chemotherapeutics with different mechanisms of action – including dexamethasone, arsenic trioxide, melphalan or bortezomib. Inhibition of specific signaling (PI3K or Akt) or targets (Foxo3A or Bim) downstream of CD28 activation abrogates this protection. Unexpectedly, we found evidence that the PIM2 kinase (which is largely uncharacterized in MM but is also significantly overexpressed in the MAF subgroup) may be a previously unreported component of the CD28 pro-MM survival pathway. The novel small molecule PIM2 inhibitor JP_11646 (IC50 0.5 nM) abrogates CD28-mediated protection against apoptosis in MM cell lines in vitro, which we have not previously seen for any chemotherapeutic tested. In addition, blockade of CD28 activation sensitized MM cells significantly to JP_11646-induced death. Altogether, these data suggested that PIM2 inhibitors can overcome a major mechanism of multi drug resistance in MM. Jasco’s novel and selective pan-PIM inhibitor (JP_11646) has demonstrated biochemical IC50s of 24, 0.5 and 1 nM for PIM1, PIM2 and PIM3 respectively. The PIM mechanism of action has been confirmed through cell based transphosphorylation assays, where JP_11646 decreased PIM dependent phoshphorylation of the proapoptotic protein BAD at nM levels. JP_11646 increases apoptosis and decreases cell viability in multiple myeloma cell lines with the MAF translocations (<100 nM). JP_11646 is orally bioavailable and has demonstrated in vivo efficacy, inhibiting tumor growth by >80% in a MM1.S tumor xenograft study. These data provide solid rationale for further development of JP_11646 as a targeted therapy in MM, and specifically for patients exhibiting the MAF translocation. Disclosures: Baldino: Jasco pharmaceuticals: Employment, Equity Ownership, Founder and President Other, Membership on an entity’s Board of Directors or advisory committees. Caserta:Jasco Pharmaceuticals: Co-Founder Other, Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees. Dumas:Jasco Pharmaceuticals: Employment. Flanders:Jasco Pharmaceuticals: Employment. Lee:Jasco Pharmaceuticals: Research Funding.

scholarly journals TTI-281, an Orally Bioavailable Bromodomain and Extraterminal Domain (BET) Inhibitor, Is a Promising Candidate for the Treatment of Multiple Myeloma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2095-2095
Author(s):  
Zezhou Wang ◽  
Jaehyun Choi ◽  
Peter Dove ◽  
Chunlei Wang ◽  
Aaron D. Schimmer ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Board Of Directors ◽  
Therapeutic Agents ◽  
Vehicle Control ◽  
Advisory Committees ◽  
Bet Inhibitor ◽  
Low Toxicity ◽  
Orally Bioavailable

Abstract Although recent advances in the development of multiple myeloma (MM) therapies such as proteasome inhibitors and immunomodulatory agents have improved patient outcomes, MM remains incurable. Additional therapeutic agents with high efficacy, low toxicity and the convenience of oral administration are in high demand. BET inhibitors, such as JQ-1, have been considered as potential therapeutic agents for MM. In the present study, we report that TTI-281, an orally bioavailable BET inhibitor, displays anti-MM activity with a low toxicity profile in preclinical studies. First, TTI-281 was tested for binding and anti-tumor activity in vitro. BROMOscan and AlphaScreen assays demonstrated that TTI-281 bound to bromodomains of BRD2/BRD3/BRD4 with Kd values less than 10 nM. In MTS assays, TTI-281 inhibited the growth of MM cell lines (MM.1s, NCIH929, and RPMI-8826) with cell growth-inhibition (IC50) values less than 300 nM. Next, in vitro ADME screening and in vivo PK studies were conducted. Permeability assays using murine gastrointestinal epithelial cells indicated that TTI-281 had good permeability with little efflux liability (efflux ratio <1), suggesting favorable properties for oral absorption. Indeed, TTI-281 displayed excellent oral bioavailability in both mice and rats (93.1% and 91.8%, respectively). In addition, TTI-281 did not interfere with the metabolism of representative CYP isozyme substrates at concentrations up to 50 μM in pooled human liver microsomes. Data also suggested minimal potential for drug-drug interactions, allowing for the possible combination with first-line therapy to improve therapeutic and survival outcomes. Finally, TTI-281 was tested for anti-myeloma efficacy and tolerability in vivo. NOD-SCID mice (n=10/group) subcutaneously engrafted with the human myeloma cell line MM.1S were treated orally once daily for 21 days with different doses of TTI-281, vehicle control or the benchmark drug carfilzomib. TTI-281 reduced tumor growth in a dose-dependent manner in this MM xenograft model. At 30 mg/kg/day, TTI-281 led to a statistically significant decrease in tumor growth compared with the vehicle control and carfilzomib (reduced tumor volume: 67% after TTI-281 treatment vs 33% after carfilzomib treatment, p<0.0003). Furthermore, TTI-281 treatment was well tolerated, with no effect on body weight or other obvious toxicity. In summary, our preclinical data suggest that the orally available BET inhibitor TTI-281 has an excellent efficacy and safety profile, highlighting its potential as a promising drug candidate for myeloma therapy. Disclosures Wang: Trillium Therapeutics: Employment, Patents & Royalties. Choi:Trillium Therapeutics: Employment. Dove:Trillium Therapeutics: Employment, Patents & Royalties. Wang:Trillium Therapeutics: Employment. Schimmer:Novartis: Honoraria. Petrova:Trillium Therapeutics Inc: Employment, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Uger:Trillium Therapeutics: Employment, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Slassi:Trillium Therapeutics: Employment, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Dual Targeting of -TORC1 and -TORC2 as a New Strategy In the Treatment of Multiple Myeloma

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 133-133 ◽  
Author(s):  
Patricia Maiso ◽  
AbdelKareem Azab ◽  
Yang Liu ◽  
Yong Zhang ◽  
Feda Azab ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Bone Marrow ◽  
Board Of Directors ◽  
Cell Lines ◽  
Mechanism Of Action ◽  
Plasma Cells ◽  
Bone Marrow Microenvironment ◽  
Advisory Committees

Abstract Abstract 133 Introduction: Mammalian target of rapamycin (mTOR) is a downstream serine/threonine kinase of the PI3K/Akt pathway that integrates signals from the tumor microenvironment such as cytokines and growth factors, nutrients and stresses to regulate multiple cellular processes, including translation, autophagy, metabolism, growth, motility and survival. Mechanistically, mTOR operates in two distinct multi-protein complexes, TORC1 and TORC2. Activation of TORC1 leads to the phosphorylation of p70S6 kinase and 4E-BP1, while activation of TORC2 regulates phosphorylation of Akt and other AGC kinases. In multiple myeloma (MM), PI3K/Akt plays an essential role enhancing cell growth and survival and is activated by the loss of the tumor suppressor gene PTEN and by the bone marrow microenvironment. Rapamycin analogues such as RAD001 and CCI-779 have been tested in clinical trials in MM. Their efficacy as single agents is modest, but when used in combination, they show higher responses. However, total inhibition of Akt and 4E-BP1 signaling requires inactivation of both complexes TORC1 and TORC2. Consequently, there is a need for novel inhibitors that can target mTOR in both signaling complexes. In this study we have evaluated the role of TORC1 and TORC2 in MM and the activity and mechanism of action of INK128, a novel, potent, selective and orally active small molecule TORC1/2 kinase inhibitor. Methods: Nine different MM cell lines and BM samples from MM patients were used in the study. The mechanism of action was investigated by MTT, Annexin V, cell cycle analysis, Western-blotting and siRNA assays. For the in vivo analyses, Luc+/GFP+ MM.1S cells (2 × 106/mouse) were injected into the tail vein of 30 SCID mice and tumor progression was detected by bioluminescence imaging. Nanofluidic proteomic immunoassays were performed in selected tumors. Results: To examine activation of the mTOR pathway in MM, we performed kinase activity assays and protein analyses of mTOR complexes and its downstream targets in nine MM cell lines. We found mTOR, Akt, pS6R and 4E-BP1 are constitutively activated in all cell lines tested independently of the status of Deptor, PTEN, and PI3K. All cell lines expressed either Raptor, Rictor or both; excepting H929 and U266LR7 which were negative for both of them. Moreover, primary plasma cells from several MM patients highly expressed pS6R while normal cells were negative for this protein. We found that INK128 and rapamycin effectively suppressed phosphorylation of p6SR, but only INK128 was able to decrease phosphorylation of 4E-BP1. We observed that INK128 fully suppressed cell viability in a dose and time dependent manner, but rapamycin reached a plateau in efficacy at ± 60%. The IC50 of INK128 was in the range of 7.5–30 nM in the eight cell lines tested. Similar results were observed in freshly isolated plasma cells from MM patients. Besides the induction of apoptosis and cell cycle arrest, INK128 was more potent than rapamycin to induce autophagy, and only INK128 was able to induce PARP and Caspases 3, 8 and 9 cleavage. In the bone marrow microenvironment context, INK128 inhibited the proliferation of MM cells and decreased the p4E-BP1 induction. Importantly, treatment with rapamycin under such conditions did not affect cell proliferation. INK128 also showed a significantly greater effect inhibiting cell adhesion to fibronectin OPM2 MM1S, BMSCs and HUVECs compared to rapamycin. These results were confirmed in vivo. Oral daily treatment of NK128 (1.0 mg/kg) decreased tumor growth and improved survival of mice implanted with MM1S. Conclusion: Dual inhibition of TORC1 and TORC2 represent a new and promising approach in the treatment of MM and its microenvironment. The ability of INK128 to inhibit both TORC1 and TORC2 strongly supports the potential use of this compound in MM patients. Disclosures: Anderson: Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Ghobrial:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees.

Preclinical Rationale for the Use of the Combined Treatment with the AKT Inhibitor Perifosine and the Multikinase Inhibitor Sorafenib in Hodgkin Lymphoma

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1653-1653
Author(s):  
Silvia Locatelli ◽  
Arianna Giacomini ◽  
Anna Guidetti ◽  
Loredana Cleris ◽  
Michele Magni ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Growth ◽  
Cell Line ◽  
Cell Lines ◽  
Drug Combination ◽  
Akt Inhibitor ◽  
Sorafenib Treatment ◽  
Responsive Cell

Abstract Abstract 1653 Introduction: A significant proportion of Hodgkin lymphoma (HL) patients refractory to first-line chemotherapy or relapsing after autologous transplantation are not cured with currently available treatments and require new treatments. The PI3K/AKT and RAF/MEK/ERK pathways are constitutively activated in the majority of HL. These pathways can be targeted using the AKT inhibitor perifosine (Æterna Zentaris GmBH, Germany, EU), and the RAF/MEK/ERK inhibitor sorafenib (Nexavar®, Bayer, Germany, EU). We hypothesized that perifosine in combination with sorafenib might have a therapeutic activity in HL by overcoming the cytoprotective and anti-apoptotic effects of PI3K/Akt and RAF/MEK/ERK pathways. Since preclinical evidence supporting the anti-lymphoma effects of the perifosine/sorafenib combination are still lacking, the present study aimed at investigating in vitro and in vivo the activity and mechanism(s) of action of this two-drug combination. METHODS: Three HL cell lines (HD-MyZ, L-540 and HDLM-2) were used to investigate the effects of perifosine and sorafenib using in vitro assays analyzing cell growth, cell cycle distribution, gene expression profiling (GEP), and apoptosis. Western blotting (WB) experiments were performed to determine whether the two-drug combination affected MAPK and PI3K/AKT pathways as well as apoptosis. Additionally, the antitumor efficacy and mechanism of action of perifosine/sorafenib combination were investigated in vivo in nonobese diabetic/severe combined immune-deficient (NOD/SCID) mice. RESULTS: While perifosine and sorafenib as single agents exerted a limited activity against HL cells, exposure of HD-MyZ and L-540 cell lines, but not HDLM-2 cells, to perifosine/sorafenib combination resulted in synergistic cell growth inhibition (40% to 80%) and cell cycle arrest. Upon perifosine/sorafenib exposure, L-540 cell line showed significant levels of apoptosis (up to 70%, P ≤.0001) associated with severe mitochondrial dysfunction (cytochrome c, apoptosis-inducing factor release and marked conformational change of Bax accompanied by membrane translocation). Apoptosis induced by perifosine/sorafenib combination did not result in processing of caspase-8, -9, -3, or cleavage of PARP, and was not reversed by the pan-caspase inhibitor Z-VADfmk, supporting a caspase-independent mechanism of apoptosis. In responsive cell lines, WB analysis showed that anti-proliferative events were associated with dephosphorylation of MAPK and PI3K/Akt pathways. GEP analysis of HD-MyZ and L-540 cell lines, but not HDLM-2 cells indicated that perifosine/sorafenib treatment induced upregulation of genes involved in amino acid metabolism and downregulation of genes regulating cell cycle, DNA replication and cell death. In addition, in responsive cell lines, perifosine/sorafenib combination strikingly induced the expression of tribbles homologues 3 (TRIB3) both in vitro and in vivo. Silencing of TRIB3 prevented cell growth reduction induced by perifosine/sorafenib treatment. In vivo, the combined perifosine/sorafenib treatment significantly increased the median survival of NOD/SCID mice xenografted with HD-MyZ cell line as compared to controls (81 vs 45 days, P ≤.0001) as well as mice receiving perifosine alone (49 days, P ≤.03) or sorafenib alone (54 days, P ≤.007). In mice bearing subcutaneous nodules generated by HD-MyZ and L-540 cell lines but not HDLM-2 cell line, perifosine/sorafenib treatment induced significantly increased levels of apoptosis (2- to 2.5-fold, P ≤.0001) and necrosis (2- to 8-fold, P ≤.0001), as compared to controls or treatment with single agents. CONCLUSIONS: Perifosine/sorafenib combination resulted in potent anti-HL activity both in vitro and in vivo. These results warrant clinical evaluation in HL patients. Disclosures: No relevant conflicts of interest to declare.

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