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

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.

Download Full-text

CYC065, a Potent Derivative of Seliciclib Is Active In Multiple Myeloma In Preclinical Studies

Blood ◽

10.1182/blood.v116.21.2999.2999 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2999-2999 ◽

Cited By ~ 1

Author(s):

Samantha Pozzi ◽

Diana Cirstea ◽

Loredana Santo ◽

Doris M Nabikejje ◽

Kishan Patel ◽

...

Keyword(s):

Cell Cycle ◽

Multiple Myeloma ◽

Cell Line ◽

Board Of Directors ◽

Cell Lines ◽

Preliminary Data ◽

Research Funding ◽

Preclinical Studies ◽

Dependent Manner ◽

Advisory Committees

Abstract Abstract 2999 Multiple myeloma (MM) is a treatable but incurable hematological malignancy and novel targeted therapies are under investigation. MM is characterized by dysregulation of the cell cycle, consequent to the overexpression of cyclins and their related kinases, the cyclins dependent kinases (CDK), a group of Ser/Thr proteine kinases. CDKs represent a promising therapeutic target, and inhibitors have been developed for anticancer treatment. We have previously studied seliciclib in the context of MM. CYC065, a second generation CDK inhibitor is the more potent derivative of seliciclib. It is mainly active on CDK 2, 5 and 9, involved in progression of the cell cycle and protein transcription. It has already shown promising results in preclinical studies in breast cancer and acute leukemia. We tested CYC065 in in vitro experiments in MM. Our preliminary data in 7 MM cell lines showed cytotoxicity of CYC065, both in MM cell lines sensitive as well as resistant to conventional chemotherapy, with an IC50 ranging between 0.06 and 2μ M, at 24 and 48h. Tritiated thymidine uptake assay confirmed the antiproliferative effects of CYC065 in MM, and its ability to overcome the growth advantage conferred by co-culture with bone marrow stromal cells derived from MM patients, and cytokines like interleukin 6 (10ng/ml) and insulin like growth factor-1 (50ng/ml). The anti-proliferative effect was evident both at 24 and 48h, starting at concentrations as low as 0.015μ M. The AnnexinV/PI assay in the MM1.s cell line confirmed CYC065's ability to induce apoptosis in a time dependent manner starting at 9 hours of treatment, at a concentration of 0.125 μ M, inducing 82% of apoptosis after 48h of exposure. Cell cycle analysis in the same MM1.s cell line showed an increase of subG1 phase, starting at 9 hours of treatment, at 0.125 μ M of CYC065. Preliminary results of western blot analysis confirmed the apoptotic effect of CYC065 in the MM1s cell line, highlighted by the cleavage of caspase 3, 8, 9 and PARP. The compound was tested in primary CD138+ cells isolated from three refractory MM patients, confirming its efficacy at 0.125 μ M, both at 24 and 48h. Comparative analysis in PBMCs from normal donors, for the evaluation of the drug toxicity is ongoing and will be presented. In conclusion our preliminary data confirm the efficacy of CYC065 in MM cell lines and primary MM cells, at nanomolar concentrations. Ongoing mechanistic and in vivo studies will delineate its role in the now increasing spectrum of CDK inhibitors in MM and better define its potential for clinical development in MM. Disclosures: Green: Cyclacel: Employment. Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Scadden:Fate Therapeutics: Consultancy, Equity Ownership, Patents & Royalties. Raje:Celgene: Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Research Funding; Acetylon: Research Funding.

Download Full-text

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

Blood ◽

10.1182/blood.v118.21.1841.1841 ◽

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.

Download Full-text

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

Blood ◽

10.1182/blood.v120.21.942.942 ◽

2012 ◽

Vol 120 (21) ◽

pp. 942-942 ◽

Cited By ~ 1

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.

Download Full-text

Selective HDAC6 Inhibition Via ACY-1215, Either Alone or in Combination with Bortezomib, Restores Osteoblast Function and Suppresses Osteoclast Differentiation in Multiple Myeloma

Blood ◽

10.1182/blood.v118.21.2908.2908 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2908-2908

Author(s):

Loredana Santo ◽

Teru Hideshima ◽

Andrew L. Kung ◽

Jen-Chieh Tseng ◽

David Tamang ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Growth ◽

Bone Formation ◽

Board Of Directors ◽

Bone Disease ◽

Research Funding ◽

Bone Homeostasis ◽

Advisory Committees ◽

Synergistic Cytotoxicity

Abstract Abstract 2908 Bone disease in multiple myeloma (MM) is due to the disruption of the delicate balance between osteoblast (OB)-mediated bone formation and osteoclast (OC)-mediated bone resorption. Agents that target both tumor cells and restore normal bone homeostasis can improve long-term disease control and prolong MM patient survival. It has been demonstrated that in vitro pan HDAC inhibitors accelerate OB maturation and suppress OC maturation, while bortezomib triggers OB activation and inhibits osteoclastogenesis. However it has recently been shown that vorinostat (SAHA), a non-selective HDAC inhibitor, causes bone loss in vivo by inhibiting immature OB. Here, we evaluated effects of a selective HDAC6 inhibitor ACY-1215 (Acetylon Pharmaceuticals, Inc), alone and in combination with bortezomib, on MM cell growth and related bone disease. ACY-1215 in combination with bortezomib has synergistic cytotoxicity due to simultaneous inhibition of the proteasome and aggresome pathways. We confirm the in vivo anti-MM activity of ACY-1215 in combination with bortezomib in two different xenograft mouse models: human MM injected subcutaneously; and luciferase-expressing human MM injected intravenously (disseminated MM model). Tumor growth was significantly delayed and overall host survival significantly prolonged in animals treated with combined therapy (34 vs 22 days, n=7, p<0.0011) in plasmacytoma model and (40 vs 17 days, n=12, p<0.0001) in disseminated model. Importantly, we show that ACY-1215 alone and in combination with bortezomib overcomes the proliferative effect of bone marrow stromal cells (BMSCs) and cytokines. MM cells stimulate OC formation and function, while inhibiting OB differentiation via both cell-to-cell contact and cytokine secretion. Therefore, osteoclastogenesis is an important therapeutic target in MM. In this context, we evaluated the effect of ACY-1215 (1μM) and bortezomib (2.5nM) on OCs generated from blood mononuclear cells stimulated with receptor activator of nuclear factor kappa B ligand (RANKL). ACY-1215 alone and in combination with bortezomib inhibited OC differentiation, evidenced by a decreased number of TRAP positive multinucleated cells and bone-resorbing activity. In addition, ACY-1215 (1μM) significantly decreased cell growth of mature OC in co-culture with MM cell lines. We next examined the effect of ACY-1215, alone and in combination with bortezomib, on downstream targets in RANKL/RANK signaling. ACY-1215 plus bortezomib inhibits transcription factors implicated in OC differentiation including p-ERK, p-AKT, c-FOS and NFATC1. Since there is decreased OB function and new bone formation in MM, we next assessed the effect of ACY-1215 on OB differentiation. ACY-1215, alone and in combination, enhanced OB differentiation, evidenced by increased alkaline phosphatase enzyme activity and alizarin red staining. In addition, we show increased mRNA expression of b-catenin, osteocalcin, Runx2 and Sp7 (OB differentiation markers) in immature OB triggered by ACY-1215. Finally, ACY-1215 was not toxic to PHA stimulated PBMCs, suggesting a favorable side effect profile and therapeutic index. Our studies therefore demonstrate that ACY-1215, alone and in combination with bortezomib, can inhibit osteoclastogenesis enhance osteoblastogenesis, and inhibit MM cell growth. Based upon these studies, ongoing clinical trials are examining the efficacy of ACY-1215 in relapsed MM and associated bone disease. Disclosures: Hideshima: Acetylon: Consultancy. Kung:Acetylon Pharmaceuticals, Inc.: Consultancy. Tamang:Acetylon Pharmaceuticals, Inc.: Employment. Yang:Acetylon Pharmaceuticals, Inc.: Employment. Jarpe:Acetylon Pharmaceuticals, Inc.: Employment. van Duzer:Acetylon Pharmaceuticals, Inc.: Employment. Mazitschek:Acetylon Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees. Bradner:Acetylon Pharmaceuticals, Inc.: Consultancy. Anderson:Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Acetylon Pharmaceuticals, Inc.: founder; Merck: Membership on an entity's Board of Directors or advisory committees. Jones:Acetylon Pharmaceuticals, Inc.: Employment. Raje:Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Acetylon: Research Funding.

Download Full-text

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

Blood ◽

10.1182/blood-2020-142580 ◽

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.

Download Full-text

CD38 Specific Chimeric Antigen Receptor KHYG-1 Natural Killer Cells: A Potential "Off the Shelf" Therapy for Multiple Myeloma

Blood ◽

10.1182/blood-2018-99-116403 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3261-3261 ◽

Cited By ~ 1

Author(s):

Arwen Stikvoort ◽

Subhashis Sarkar ◽

Renée Poels ◽

Niels WCJ van de Donk ◽

Sonja Zweegman ◽

...

Keyword(s):

Multiple Myeloma ◽

T Cells ◽

Board Of Directors ◽

Cell Lines ◽

Research Funding ◽

Nk Cell ◽

Advisory Committees ◽

Cd38 Expression ◽

Potential Safety

Abstract Chimeric Antigen Receptors (CARs) are engineered transmembrane proteins consisting of an antibody-derived antigen recognition domain linked to intracellular cell signaling domains. CAR engineered autologous T cells have been successful in the treatment of a variety of hematologic malignancies. However, several major caveats, including lack of universal donors, long manufacturing times, and absence of a donor in immunologically frail patients, have limited the successful translation of CAR-T cell based therapy to a larger pool of patients. A universal, easy to manufacture, "off the shelf" CAR-based product could potentially address these limitations and result in a lower cost of goods. Towards developing an "off the shelf" CAR-based therapy for Multiple Myeloma (MM), we explored the feasibility and preclinical efficacy of expressing CD38 CARs in KHYG-1 cells, a natural killer (NK) cell line, first established by Yagita et al from a patient with aggressive NK leukemia (Leukemia, 2000). To this end, we effectively transduced KHYG-1 cells with high-affinity CD38 CARs as well as our recently reported affinity-optimized CD38 CARs, which can readily target MM cells with high CD38 expression, while ignoring non-malignant cells with intermediate, low or no CD38 expression when brought to expression on T cells (Drent et al, Molecular Therapy 2017). Moreover, we assessed performance of first and second generation CARs, with co-stimulatory domains CD28 and 4-1BB, and found the combination of CD28/CD3ζ to lead to the best results. After expanding the CAR transduced KHYG-1 cells, we analyzed their phenotype and efficacy in MM by analyzing their cytotoxic activity against CD38+ and CD38- MM and AML cell lines (UM9/THP-1 and U266/HL60, respectively), and against primary MM cells. The CD38-CAR transduced KHYG-1 cells showed no phenotypic alterations, and at effector to target ratios as low as 1:1, induced a high cytotoxicity towards CD38+ cell lines as compared to mock or non-transduced KHYG-1, demonstrating the important contribution of the CD38 CAR on the KHYG-1 NK cell surface. CD38- cell lines were unaffected by both CD38-CAR transduced KHYG-1 cells and mock or non-transduced KHYG-1 cells, indicating the specificity towards CD38 of the CAR and thus the potential safety of the CD38-CAR KHYG-1 cell. Similarly, ex vivo assays using primary MM cells revealed superior cytotoxic activity of CD38-CAR KHYG-1 cells as compared to mock or non-transduced KHYG-1 cells (median 86,5% vs 14% at 1:1 E:T ratio, n=2, Figure 1A). Confirming our previous results we identified an affinity-optimized CD38-CAR which mediated strong primary MM cell cytotoxicity with little or no "off tumor" effect. Normal immune cells (B, T, monocytes), which were either CD38 negative or only intermediate positive, were unaffected (Figure 1B-D), suggesting the potential safety of the CAR-NK cell therapy for clinical applications. As clinical administration would require irradiation of CD38-CAR KHYG-1 cells, we tested the effect of irradiation on their proliferative and cytotoxicity potential. Irradiation with 10Gy, while drastically inhibiting proliferative activity and viability (50% survival after 3 days), did not affect cytotoxicity, suggesting that repeated administrations of irradiated, CD38-CAR transduced KYHG-1 cells may exert effective in vivo anti-tumor activity, which is currently being evaluated in appropriate in vivo models, specifically the humanized bone scaffold in vivo model published by Groen et al (Blood, 2012). In conclusion, we demonstrate that the incorporation of CAR technology into the immortal NK cell line KHYG-1 has enormous potential to become a safe and effective "off the shelf" therapy for MM. Disclosures Stikvoort: Onkimmune: Research Funding. Sarkar:Onkimmune: Research Funding. van de Donk:Amgen: Research Funding; Janssen Pharmceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Research Funding; Bristol-Myers Squibb: Research Funding; Celgene: Research Funding. Zweegman:Takeda: 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; Celgene Corp.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding. O'Dwyer: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; BMS: Research Funding; Celgene: Research Funding; Glycomimetics: Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees. Mutis:Gilead: Research Funding; Celgene: Research Funding; Novartis: Research Funding; OnkImmune: Research Funding; Genmab: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Download Full-text

The Interaction of Bortezomib with P-Gp, MRP-1 and BCRP Drug Transporters: Implications for Therapeutic Applications of Bortezomib in Advanced Multiple Myeloma and Other Neoplasias.

Blood ◽

10.1182/blood.v114.22.1729.1729 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1729-1729

Author(s):

Melissa G Ooi ◽

Robert O'Connor ◽

Jana Jakubikova ◽

Justine Meiller ◽

Steffen Klippel ◽

...

Keyword(s):

Lung Cancer ◽

Multiple Myeloma ◽

Cell Line ◽

Board Of Directors ◽

Cell Lines ◽

Research Funding ◽

Significant Activity ◽

Cancer Resistance ◽

Advisory Committees ◽

Bortezomib Treatment

Abstract Abstract 1729 Poster Board I-755 Background Multidrug transporters are energy-dependent transmembrane proteins which can efflux a broad range of anticancer drugs and thereby play a role in resistance to the actions of substrate agents. Classically, three transporters, p-glycoprotein (Pgp; MDR-1; ABCB1), multidrug resistant protein-1 (MRP-1; ABCC1) and breast cancer resistance protein (BCRP; MXR; ABCG2), have been found to have the broadest substrate specificity and a strong correlation with drug resistance in vitro and in vivo in many models and forms of cancer. We have sought to characterize the interaction of bortezomib with these transporters and thereby explore the potential for these agents to play a role in resistance. Bortezomib is a novel proteosome inhibitor with significant activity in multiple myeloma, although subsets of patients remain refractory to the activity of the drug. Hence, better characterization of the interactions of this drug with classical resistance mechanisms may identify improved treatment applications. Methods and Results We investigated the role of these transporters by using isogenic cell line models which are resistant due to overexpression of a particular transporter: DLKP lung cancer cell line that overexpresses MRP-1; DLKP-A which overexpresses Pgp; and DLKP-SQ-Mitox which overexpresses BCRP. DLKP-A cells exhibited a 4.6-fold decrease in responsiveness to bortezomib compared to parental DLKP cells. In DLKP-SQ-Mitox, bortezomib-induced cytotoxicity was comparable to DLKP. When bortezomib was combined with elacridar, a Pgp and BCRP inhibitor, significant synergy was evident in DLKP-A (100% viable cells with single agent treatment versus 11% with the combination), but not DLKP-SQ-Mitox. Sulindac, an MRP-1 inhibitor, combined with bortezomib failed to produce any synergy in MRP-1 positive DLKP cells. Conversely, combination assays of Pgp substrate cytotoxics such as doxorubicin with Bortezomib were largely additive in nature. This indicates that bortezomib has little, if any, direct Pgp inhibitory activity, as combinations of a traditional Pgp inhibitor (such as elacridar) and doxorubicin would show marked synergy rather than just an additive effect in Pgp positive cells. To further characterize the extent of this interaction with Pgp, we conducted cytotoxicity assays in cell lines with varying levels of Pgp overexpression. NCI/Adr-res (ovarian cancer, high Pgp overexpression), RPMI-Dox40 (multiple myeloma, moderate Pgp overexpression) and A549-taxol (lung cancer, low Pgp overexpression). The combination of bortezomib and elacridar that produced the most synergy was in cell lines expressing moderate to high levels of Pgp expression. Cell lines with lower Pgp expression produced an additive cytotoxicity. We next examined whether bortezomib had any direct effect on Pgp expression. In RPMI-Dox40 cells, Pgp expression is reduced in a time-dependent manner with bortezomib treatment. Conclusions Our studies therefore show that bortezomib is a substrate for Pgp but not the other drug efflux pumps. In tumor cells expressing high levels of Pgp, the efficacy of bortezomib is synergistically enhanced by combinations with a Pgp inhibitor, while bortezomib treatment itself can reduce the expression of Pgp. This study suggests that in the subset of patients with advanced multiple myeloma or solid tumors which express high levels of Pgp, inhibition of its function could contribute to enhanced responsiveness to bortezomib. Disclosures Richardson: millenium: 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, speakers bureau up to 7/1/09; MLNM: speakers bureau up to 7/1/09. Mitsiades:Millennium Pharmaceuticals : Consultancy, Honoraria; Novartis Pharmaceuticals : Consultancy, Honoraria; Bristol-Myers Squibb : Consultancy, Honoraria; Merck &Co: Consultancy, Honoraria; Kosan Pharmaceuticals : Consultancy, Honoraria; Pharmion: Consultancy, Honoraria; PharmaMar: licensing royalties ; Amgen Pharmaceuticals: Research Funding; AVEO Pharma: Research Funding; EMD Serono : Research Funding; Sunesis Pharmaceuticals: Research Funding. Anderson:Celgene: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Millennium: Consultancy, Research Funding; Biotest AG: Consultancy, Research Funding.

Download Full-text

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

Blood ◽

10.1182/blood.v116.21.133.133 ◽

2010 ◽

Vol 116 (21) ◽

pp. 133-133 ◽

Cited By ~ 2

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.

Download Full-text

Lin28B/Let-7 Axis Regulates Multiple Myeloma Proliferation By Enhancing c-Myc and Ras Survival Pathways

Blood ◽

10.1182/blood.v122.21.273.273 ◽

2013 ◽

Vol 122 (21) ◽

pp. 273-273

Author(s):

Salomon Manier ◽

John T Powers ◽

Antonio Sacco ◽

Michaela R Reagan ◽

Michele Moschetta ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Proliferation ◽

Board Of Directors ◽

Cell Lines ◽

Research Funding ◽

Advisory Board ◽

Loss Of Function ◽

Advisory Committees ◽

Expression Levels ◽

Gain And Loss

Abstract Background MicroRNAs (miRNAs) play a pivotal role in tumorigenesis, due to their ability to target mRNAs involved in the regulation of cell proliferation, survival and differentiation. Lin28B is an RNA binding protein that regulates Let-7 miRNA maturation. Lin28B and Let-7 have been described to act as oncogenes or tumor suppressor genes, respectively, as demonstrated both in solid cancer and hematologic malignancies. However, the role of the Lin28B/Let-7 axis in Multiple Myeloma (MM) has not been studied. Method Lin28B level expression in MM patients was studied using previously published gene expression profiling (GEP) datasets. Let-7 expression levels were assessed in CD138+ primary MM cells and bone marrow stromal cells (BMSCs) by using PCR, as well as in circulating exosomes using miRNA array (Nanostring® Technology). Exosomes were collected from both normal and MM peripheral blood, using ultracentrifugation; and further studied by using electron microscopy and immunogold labeling for the detection of CD63 and CD81. The knockdown of Lin28B was performed on MM cell lines (U266, MM.1S, MOLP-8) by using a lentiviral Lin28B shRNA. Gain- and loss-of function studies for Let-7 were performed using Let-7 mimic and anti-Let-7 transfection in MM cell lines (MM1S, U266) and primary BMSCs. Cell proliferation has been evaluated by using thymidine assays. Effects of Let-7 and Lin28B on signaling cascades have been evaluated by western blot. Results Two independent GEP datasets (GSE16558; GSE2658) were analyzed for Lin28B expression, showing a significantly higher level in MM patients compared to healthy controls. In addition, high Lin28B levels correlated with a shorter overall survival (p = 0.0226). We next found that the Let-7 family members are significantly down-regulated in MM primary cells, particularly Let-7a and b (5 fold change, p < 0.05), as demonstrated by using qRT-PCR. Similarly, miRNA arrays showed a lower expression of Let-7-related miRNAs in circulating exosomes obtained from MM patients compared to healthy individuals. We further dissected the functional relevance of Lin28B in MM cells, by performing Lin28 knockdown (KD) in MM cell lines (U266, MOLP-8). This led to a significant decrease in MM cell proliferation associated with G1 phase cell cycle arrest. This was supported by up-regulation of Let-7 and down-regulation of c-Myc, Ras and Cyclin D1 in Lin28 KD MM cells. To further prove that Lin28B-dependent effects on MM cells are mediated by Let7, we next showed that let-7 gain- and loss-of-function studies regulate MM cell proliferation and Myc expression. Lin28B regulation in MM cells is dependent on Let-7, as demonstrated by an increase of both cell proliferation and c-Myc expression after anti-Let-7 transfection in the Lin28B KD cells. We therefore studied the regulation of Let-7 in MM cells through the interaction with BMSCs. Let-7 expression levels were significantly lower in BMSCs obtained from MM patients compared to healthy donors. Interestingly, the Let-7 expression level in MM cells was increased after co-culture with Let-7 over-expressing BMSCs, associated with a decrease of both cell proliferation and c-Myc expression. This suggests a potential transfer of Let-7 from BMSCs to MM cells. Conclusion This work describes a new signaling pathway involving Lin28B, Let-7, Myc and Ras in MM. Let-7 expression in MM cells is also regulated through the interaction of MM cells with BMSCs, leading to cell proliferation and Myc regulation in MM. Interference with this pathway might offer therapeutic perspectives. Disclosures: Leleu: CELGENE: Honoraria; JANSSEN: Honoraria. Daley:Johnson and Johnson: Consultancy, Membership on an entity’s Board of Directors or advisory committees; MPM Capital: Consultancy, Membership on an entity’s Board of Directors or advisory committees; Verastem: Consultancy, Membership on an entity’s Board of Directors or advisory committees; Epizyme: Consultancy, Membership on an entity’s Board of Directors or advisory committees; iPierian: Consultancy, Membership on an entity’s Board of Directors or advisory committees; Solasia, KK: Consultancy, Membership on an entity’s Board of Directors or advisory committees. Ghobrial:Onyx: Advisoryboard Other; BMS: Advisory board, Advisory board Other, Research Funding; Noxxon: Research Funding; Sanofi: Research Funding.

Download Full-text

Expression Of Truncated Protein Tyrosine Phosphatase, Receptor Type, O (PTPROt) Influences Multiple Myeloma Sensitivity To Bortezomib Through Akt Signaling, and Is a Favorable Clinical Prognostic Factor

Blood ◽

10.1182/blood.v122.21.2520.2520 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2520-2520

Author(s):

Hua Wang ◽

Veerabhadran Baladandayuthapani ◽

Zhiqiang Wang ◽

Jiexin Zhang ◽

Heather Yan Lin ◽

...

Keyword(s):

Multiple Myeloma ◽

Board Of Directors ◽

Cell Lines ◽

Research Funding ◽

Myeloma Cell ◽

Advisory Committees ◽

Over Expression ◽

Resistant Disease ◽

Drug Naïve ◽

Rpmi 8226

Abstract Background Proteasome inhibitors such as bortezomib and carfilzomib are an important part of our current chemotherapeutic armamentarium against multiple myeloma, and have improved outcomes in the up-front, relapsed, and relapsed/refractory settings. Their efficacy has been demonstrated both as single agents, and as part of rationally designed combination regimens, but they are at this time used empirically, since biomarkers to identify patients who would most or least benefit from their application have not been clinically validated. Moreover, the vast majority of patients eventually develop drug-resistant disease which precludes further proteasome inhibitor use through mechanisms that have not been fully elucidated. Methods We compared gene expression profiles (GEPs) of a panel of bortezomib-resistant myeloma cell lines and their vehicle-treated, drug-naïve counterparts to identify significant changes associated with drug resistance. The list of genes whose expression was changed by at least 2-fold was compared with independent RNA interference studies whose goal was to identify genes whose suppression conferred drug resistance. Further validation of genes of interest was pursued in a panel of myeloma cell lines, and in clinically annotated GEP databases. Results Suppression of PTPROt expression was noted in bortezomib-resistant RPMI 8226 and ANBL-6 myeloma cells compared to isogenic, drug-naïve controls, and this was confirmed by quantitative PCR. Overexpression of PTRPOt in RPMI 8226, ANBL-6 and other myeloma cell lines was by itself sufficient to increase the level of apoptotic, sub-G0/G1 cells compared to vector controls, or cells expressing a phosphatase-dead PTPROt mutant. Moreover, PTPROt enhanced the ability of bortezomib to reduce myeloma cell viability, in association with increased activation of caspases 8 and 9. Exogenous over-expression of PTPROt was found to reduce the activation status of Akt, a known anti-apoptotic pathway that reduces bortezomib activity, based on Western blotting with antibodies to phospho-Akt (Ser473), and Akt kinase activity assays. Notably, we also found that exogenous over-expression of PTPROt resulted in increased expression levels of p27Kip1. Interestingly, array CGH data from studies of myeloma cell lines and primary cells showed that the PTPROt gene was located in a genomic region with a high propensity for loss. Analysis of the Total Therapy databases of GEP and patient outcomes available on the Multiple Myeloma Genomics Portal showed that higher than median expression of PTPROt was associated with better long-term survival (P=0.0175). Finally, analysis of the Millennium Pharmaceuticals database of studies of bortezomib in the relapsed and relapsed/refractory setting showed high PTRPOt expression was more frequently seen in patients who achieved complete remission (P<0.01), and was associated with a better median overall survival (P=0.0003). Conclusions Taken together, the data support the possibility that high expression of PTPROt is a good prognostic factor for response to bortezomib-containing therapies, and that this may occur through modulation by PTPROt of the Akt pathway. Moreover, they suggest that strategies to enhance the expression of PTPROt should be investigated to restore bortezomib sensitivity in patients with proteasome inhibitor-resistant disease. Disclosures: Orlowski: Bristol-Myers Squibb: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Millennium: The Takeda Oncology Company: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Onyx: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Resverlogix: Research Funding; Array Biopharma: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Genentech: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Merck: Membership on an entity’s Board of Directors or advisory committees.

Download Full-text