Patient Multiple Myeloma Cells Research Articles

Blockade of Deubiquitylating Enzyme USP1 Inhibits DNA Repair and Triggers Apoptosis in Multiple Myeloma Cells.

Purpose: The ubiquitin proteasome pathway is a validated therapeutic target in multiple myeloma. Deubiquitylating enzyme USP1 participates in DNA damage response and cellular differentiation pathways. To date, the role of USP1 in multiple myeloma biology is not defined. In the present study, we investigated the functional significance of USP1 in multiple myeloma using genetic and biochemical approaches.Experimental Design: To investigate the role of USP1 in myeloma, we utilized USP1 inhibitor SJB3-019A (SJB) for studies in myeloma cell lines and patient multiple myeloma cells.Results: USP1-siRNA knockdown decreases multiple myeloma cell viability. USP1 inhibitor SJB selectively blocks USP1 enzymatic activity without blocking other DUBs. SJB also decreases the viability of multiple myeloma cell lines and patient tumor cells, inhibits bone marrow plasmacytoid dendritic cell-induced multiple myeloma cell growth, and overcomes bortezomib resistance. SJB triggers apoptosis in multiple myeloma cells via activation of caspase-3, caspase-8, and caspase-9. Moreover, SJB degrades USP1 and downstream inhibitor of DNA-binding proteins as well as inhibits DNA repair via blockade of Fanconi anemia pathway and homologous recombination. SJB also downregulates multiple myeloma stem cell renewal/survival-associated proteins Notch-1, Notch-2, SOX-4, and SOX-2. Moreover, SJB induced generation of more mature and differentiated plasma cells. Combination of SJB and HDACi ACY-1215, bortezomib, lenalidomide, or pomalidomide triggers synergistic cytotoxicity.Conclusions: Our preclinical studies provide the framework for clinical evaluation of USP1 inhibitors, alone or in combination, as a potential novel multiple myeloma therapy. Clin Cancer Res; 23(15); 4280-9. ©2017 AACR.

Abstract 2525: Targeting myeloma cell-derived runx2 by miRNAs suppresses multiple myeloma growth and progression

Abstract In Multiple myeloma (MM), abnormal plasma cells accumulate in the bone marrow and spread to new bone sites. However, the mechanisms underlying this spread of MM cells remain unclear. Runx2 is a bone specific transcription factor upregulated in various human tumors, including MM. Our studies have previously demonstrated that Runx2 is a major driver of MM progression in bone. In the present study, our goal was to identify miRNAs targeting Runx2 to reduce tumor growth and dissemination of MM to new bone sites and asses their therapeutic potential. Expression analysis of a panel of miRNA's regulating Runx2 revealed an inverse relationship between Runx2 expression and two miRNAs: miR-342 and miR-363. miRNA expression analysis using the Gene Expression Omnibus database showed that miR-342 and miR-363 are highly expressed in plasma cells of normal donors (n= 3), while Runx2 is detected at very low levels. In contrast, MM cells of patients with newly diagnosed (n=23) and relapsed MM (n=17) expressed very little miR-342 and miR-363 but showed high levels of Runx2 expression. Reconstituting CAG human MM cells with synthetic miR-342, miR-363 or miR-342+miR-363 in combination reduced the expression of Runx2 and the metastasis-promoting Runx2 target genes RANKL, DKK1 and DMP1. Tumor cell viability and migration were also decreased compared to control in vitro. We then transfected miR-342, miR-363 or miR-342+miR-363 in mouse 5TGM1 MM cells expressing high levels of Runx2 and tested bone-homing and growth in syngenic C57Bl/KaLwRij mice by intravenous injection. Mouse sera were collected at week 2 and 4 after tumor cell injection and the levels of IgG2bκ, a marker of 5TGM1 cells, were measured by ELISA. The results showed significantly decreased IgG2bκ levels in mice bearing miR-342 or miR-363 transfected tumors compared to mice bearing control miR-scramble tumors. Additionally, miR-342+miR-363 co-transfection produced a synergistic effect on reducing tumor growth. Mechanistic studies demonstrated that miR-342 and miR-363 induced Runx2 suppression, results in inhibition of Runx2-downstream signaling pathways Akt/β-catenin/survivin, which are required for MM tumor progression. In conclusion, we have identified two novel miRNAs, miR-342 and miR-363, that negatively regulate Runx2 expression in MM cells. We further demonstrated that enhanced expression of miR-342 or miR-363 in MM cells inhibits MM growth in vivo. Thus, miR-342 and miR-363 are potential novel markers of MM prognosis and can be developed as new therapeutic drugs for MM treatment. Citation Format: Pramod S. Gowda, Mohammad Q. Hassan, Timothy N. Trotter, Yang Yang. Targeting myeloma cell-derived runx2 by miRNAs suppresses multiple myeloma growth and progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2525. doi:10.1158/1538-7445.AM2017-2525

Abstract 4049: The synergistic effect of melphalan and XPO1 inhibition in preclinical models of multiple myeloma

Abstract Introduction: Significant increases in response/survival have been seen over the past several years; however, multiple myeloma (MM) remains incurable. In this study we have demonstrated that the XPO1 inhibitors (XPO1i), selinexor (SEL) and KPT-8602, sensitize both wild-type and drug-resistant MM cells to melphalan (MEL) in preclinical models. Materials and Methods: We used the XPO1i SEL (300nM), KPT-8602 (300nM), and KOS-2464 (10nM) +/- MEL (5-20 μM) to treat human 8226, H929 and U266 MM cells, and MEL resistant 8226LR5 and U266LR6 cell lines and assayed for apoptosis. DNA damage was assayed by comet assay and γ-H2AX in H929 human MM cells. XPO1i/MEL-treated MM cells were assayed for P53, NFkB, IKKα, and Fanconi Anemia (FANC)/BRCA DNA repair proteins by Western blot and electrochemiluminescent immunoassay(ECL-I). We also treated cells from patients with newly diagnosed or relapsed/refractory MM with the XPO1i +/- MEL and assayed for apoptosis. NOD/SCID-γ (NSG) mice challenged with U226 or U266LR6 MM tumors were treated with XPO1i/MEL and assayed for tumor growth, survival, and toxicity. Results: The addition of the XPO1i’s SEL, KPT-8602 or KOS-2464 (P < 0.03) sensitized drug-resistant 8226LR5 and U266LR6 cells to MEL when compared to single-agent MEL. The XPO1i/MEL drug combination increased DNA damage (Comet assay/γ-H2AX) more than single agent MEL or XPO1i alone (P < 0.005). Western blot and ECL-I showed that XPO1i treatment can increase P53 and decrease NFkB, IKKα, FANC/BRAC pathway proteins including FANCD2 in MM cells. CD138+/light chain+ MM cells from newly diagnosed and relapsed/refractory MM patients were sensitized (20-fold and 5-10 fold respectively) by XPO1i to MEL. XPO1i/MEL combination treatment demonstrated a strong synergistic antitumor effect when compared to single-agent MEL (SEL/MEL, P = 0.0024 and KPT-8602/MEL, P = 0.0030) in NSG mice challenged with U266, with little toxicity as assessed by weight loss. Similar results were found with MEL resistant U266LR6 tumors in NSG mice. Conclusions: XPO1i's sensitized human MM cell lines, both parental and MEL resistant, and patient MM cells to MEL both in vitro and ex vivo, and in in vivo NSG mouse models. Our data show that the synergistic cell kill may be due to increased XPO1i/MEL-induced DNA damage. The mechanism of this synergy may be due to increased nuclear P53, in combination with decreased NFkB and IKKα, and decreased DNA repair activity of the FANC/BRCA pathway. Thus, combination therapies of XPO1i, especially the clinical compounds SEL and KPT-8602 +/- MEL, may have potential to improve the treatment outcomes of MM. The combination of XPO1i and melphalan are being investigated in the context of high-dose chemotherapy and autologous transplant (NCT 02780609). Citation Format: Joel G. Turner. The synergistic effect of melphalan and XPO1 inhibition in preclinical models of multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4049. doi:10.1158/1538-7445.AM2017-4049

Blockade of deubiquitylating enzyme Rpn11 triggers apoptosis in multiple myeloma cells and overcomes bortezomib resistance.

Proteasome inhibition is an effective therapy for multiple myeloma (MM) patients; however, the emergence of drug resistance is common. Novel therapeutic strategies to overcome proteasome inhibitor resistance are needed. In this study, we examined whether targeting deubiquitylating (DUB) enzymes upstream of 20S proteasome overcomes proteasome inhibitor resistance. Gene expression analysis, immunohistochemical studies of MM patient bone marrow, reverse transcription-PCR and protein analysis show that Rpn11/POH1, a DUB enzyme upstream of 20S proteasome, is more highly expressed in patient MM cells than in normal plasma cells. Importantly, Rpn11 expression directly correlates with poor patient survival. Loss-of-function studies show that Rpn11-siRNA knockdown decreases MM cell viability. Pharmacological inhibition of Rpn11 with O-phenanthroline (OPA) blocks cellular proteasome function, induces apoptosis in MM cells and overcomes resistance to proteasome inhibitor bortezomib. Mechanistically, Rpn11 inhibition in MM cells activates caspase cascade and endoplasmic stress response signaling. Human MM xenograft model studies demonstrate that OPA treatment reduces progression of tumor growth and prolongs survival in mice. Finally, blockade of Rpn11 increases the cytotoxic activity of anti-MM agents lenalidomide, pomalidomide or dexamethasone. Overall, our preclinical data provide the rationale for targeting DUB enzyme Rpn11 upstream of 20S proteasome to enhance cytotoxicity and overcome proteasome inhibitor resistance in MM.

DEK protein level is a biomarker of CD138positive normal and malignant plasma cells.

Overexpression of DEK oncogene is associated with increased proliferation of carcinoma cells and it is observed in several solid tumors due to the amplification of the 6p22.3 chromosomal region where DEK locates. Although the same chromosomal amplification occurs in multiple myeloma (MM), a plasma cell neoplasm, whether the expression and the copy number of the DEK gene are affected in MM remains elusive. We show that despite the increased copy number in CD138positive MM cells (4 out of 41 MM samples), DEK mRNA expression was down-regulated compared with that in CD138negative bone marrow (BM) cells of the same patients (P<0.0001). DEK protein was not detectable by immunohistochemistry (IHC) in CD138positive normal plasma cells or in malignant plasma cells of MM patients (n = 56) whereas it was widely expressed in normal and neoplastic B-cells. Stable knockdown or overexpression of DEK in CD138positive MM cell lines did not affect the proliferation and viability of the cells profoundly in the presence or absence of chemotherapeutic agent melphalan whereas knockdown of DEK moderately but significantly increased the expression level of CD138 (p<0.01). Decreased DEK expression in plasma cells suggests a potential role of this gene in plasma cell development and lack of detectable DEK protein by IHC could be used as a biomarker for normal and malignant plasma cells.

Expression of CD38 in myeloma bone niche: A rational basis for the use of anti-CD38 immunotherapy to inhibit osteoclast formation.

It is known that multiple myeloma (MM) cells express CD38 and that a recently developed human anti-CD38 monoclonal antibody Daratumumab mediates myeloma killing. However, the expression of CD38 and other functionally related ectoenzymes within the MM bone niche and the potential effects of Daratumumab on bone cells are still unknown. This study firstly defines by flow cytometry and immunohistochemistry the expression of CD38 by bone marrow cells in a cohort of patients with MM and indolent monoclonal gammopathies. Results indicate that only plasma cells expressed CD38 at high level within the bone niche. In addition, the flow cytometry analysis shows that CD38 was also expressed by monocytes and early osteoclast progenitors but not by osteoblasts and mature osteoclasts. Indeed, CD38 was lost during in vitro osteoclastogenesis. Consistently, we found that Daratumumab reacted with CD38 expressed on monocytes and its binding inhibited in vitro osteoclastogenesis and bone resorption activity from bone marrow total mononuclear cells of MM patients, targeting early osteoclast progenitors. The inhibitory effect was not observed from purified CD14+ cells, suggesting an indirect inhibitory effect of Daratumumab. Interestingly, all-trans retinoic acid treatment increased the inhibitory effect of Daratumumab on osteoclast formation.These observations provide a rationale for the use of an anti-CD38 antibody-based approach as treatment for multiple myeloma-induced osteoclastogenesis.

Leptin receptor antagonism of iNKT cell function: a novel strategy to combat multiple myeloma.

A hallmark of bone marrow changes with aging is the increase in adipocyte composition, but how this impacts development of multiple myeloma (MM) is unknown. Here, we report the role of the adipokine leptin as master regulator of anti-myeloma tumor immunity by modulating the invariant natural killer T (iNKT) cell function. A marked increase in serum leptin levels and leptin receptor (LR) expression on iNKT cells in MM patients and the 5T33 murine MM model was observed. MM cells and leptin synergistically counteracted anti-tumor functionality of both murine and human iNKT cells. In vivo blockade of LR signaling combined with iNKT stimulation resulted in superior anti-tumor protection. This was linked to persistent IFN-γ secretion upon repeated iNKT cell stimulation and a restoration of the dynamic antigen-induced motility arrest as observed by intravital microscopy, thereby showing alleviation of iNKT cell anergy. Overall our data reveal the LR axis as novel therapeutic target for checkpoint inhibition to treat MM.

HDAC3 regulates DNMT1 expression in multiple myeloma: therapeutic implications.

Epigenetic signaling pathways are implicated in tumorigenesis and therefore histone deacetylases (HDACs) represent novel therapeutic targets for cancers including multiple myeloma (MM). Although non-selective HDAC inhibitors show anti-MM activities, unfavorable side effects limit their clinical efficacy. Isoform- and/or class-selective HDAC inhibition offers the possibility to maintain clinical activity while avoiding adverse events attendant to broad non-selective HDAC inhibition. We have previously reported that HDAC3 inhibition, either by genetic knockdown or selective inhibitor BG45, abrogates MM cell proliferation. Here we show that knockdown of HDAC3, but not HDAC1 or HDAC2, as well as BG45, downregulate expression of DNA methyltranseferase 1 (DNMT1) mediating MM cell proliferation. DNMT1 expression was also regulated by c-Myc, and HDAC3 inhibition triggers degradation of c-Myc protein. Moreover, HDAC3 inhibition results in hyperacetylation of DNMT1, thereby reducing the stability of DNMT1 protein. Combined inhibition of HDAC3 and DNMT1 with BG45 and DNMT1 inhibitor 5-azacytidine (AZA), respectively, triggers synergistic downregulation of DNMT1, growth inhibition, and apoptosis in both MM cell lines and patient MM cells. Efficacy of this combination treatment was confirmed in a murine xenograft MM model. Our results therefore provide the rationale for combination treatment using HDAC3 inhibitor with DNMT1 inhibitor to improve patient outcome in MM.

Autocrine and Paracrine Interactions between Multiple Myeloma Cells and Bone Marrow Stromal Cells by Growth Arrest-specific Gene 6 Cross-talk with Interleukin-6

The pathogenesis of multiple myeloma (MM) has not yet been fully elucidated. Our microarray analysis and immunohistochemistry revealed significant up-regulation of growth arrest-specific gene 6 (Gas6), a vitamin K-dependent protein with a structural homology with protein S, in bone marrow (BM) cells of MM patients. ELISA showed that the serum levels of soluble Gas6 were significantly increased in the MM patients when compared with healthy controls. Gas6 was overexpressed in the human CD138-positive MM cell line RPMI-8226. Exogenous Gas6 suppressed apoptosis induced by serum deprivation and enhanced cell proliferation of the MM cells. The conditional medium from the human BM stromal cell line HS-5 induced cell proliferation and anti-apoptosis of the MM cells with extracellular signal-regulated kinase, Akt, and nuclear factor-κB phosphorylation, which were reversed by the neutralizing antibody to Gas6 or IL-6. The TAM family receptor Mer, which has been identified as a Gas6 receptor, was overexpressed in BM cells of MM patients. The knockdown of Mer by siRNA inhibited cell proliferation, anti-apoptosis, and up-regulation of intercellular cell adhesion molecule-1 (ICAM-1) in MM cells stimulated by an HS-5 cell-conditioned medium. Furthermore, the Gas6-neutralizing antibody reduced the up-regulation of IL-6 and ICAM-1 induced by a HS-5 cell-conditioned medium in MM cells. The present study provides new evidence that autocrine and paracrine stimulation of Gas6 in concert with IL-6 contributes to the pathogenesis of MM, suggesting that Gas6-Mer-related signaling pathways may be a promising novel target for treating MM.

Autologous bone marrow Th cells can support multiple myeloma cell proliferation in vitro and in xenografted mice.

Multiple myeloma (MM) is a plasma cell malignancy where MM cell growth is supported by the bone marrow (BM) microenvironment with poorly defined cellular and molecular mechanisms. MM cells express CD40, a receptor known to activate autocrine secretion of cytokines and elicit proliferation. Activated T helper (Th) cells express CD40 ligand (CD40L) and BM Th cells are significantly increased in MM patients. We hypothesized that activated BM Th cells could support MM cell growth. We here found that activated autologous BM Th cells supported MM cell growth in a contact- and CD40L-dependent manner in vitro. MM cells had retained the ability to activate Th cells that reciprocated and stimulated MM cell proliferation. Autologous BM Th cells supported MM cell growth in xenografted mice and were found in close contact with MM cells. MM cells secreted chemokines that attracted Th cells, secretion was augmented by CD40-stimulation. Within 14 days of culture of whole BM aspirates in autologous serum, MM cells and Th cells mutually stimulated each other, and MM cells required Th cells for further expansion in vitro and in mice. The results suggest that Th cells may support the expansion of MM cells in patients.

Activation of NK cells and disruption of PD-L1/PD-1 axis: two different ways for lenalidomide to block myeloma progression.

Natural Killer (NK) cells play a critical role against tumor cells in hematological malignancies. Their activating receptors are essential in tumor cell killing. In Multiple Myeloma (MM) patients, NK cell differentiation, activation and cytotoxic potential are strongly impaired leading to MM escape from immune surveillance in tissues and bone marrow. Mechanisms used by MM to affect NK cell functions are mediated by the release of soluble factors, the expression of activating and inhibitory NK cell ligands, and the expression of immune check-point inhibitors. Lenalidomide represents an efficient clinical approach in MM treatment to improve patients’ survival. Lenalidomide does not only promotes tumor apoptosis, but also stimulates T and NK cells, thereby facilitating NK-mediated tumor recognition and killing. This occurs since Lenalidomide acts on several critical points: stimulates T cell proliferation and cytokine secretion; decreases the expression of the immune check-point inhibitor Programmed Death-1 (PD-1) on both T and NK cells in MM patients; decreases the expression of both PD-1 and PD-L1 on MM cells; promotes MM cell death and abrogates MM/stromal microenvironment cross-talk, a process known to promote the MM cell survival and proliferation. This leads to the inhibition of the negative signal induced by PD-1/PD-L1 axis on NK cells, restoring NK cell cytotoxic functions. Given the importance of an effective immune response to counteract the MM progression and the promising approaches using anti-PD-1/PD-L1 strategies, we will discuss in this review how Lenalidomide could represent an adequate approach to re-establish the recognition against MM by exhausted NK cell.

A phase I/IIa Study of the CD38 Antibody MOR202 in Combination With Pomalidomide or Lenalidomide in Patients With Relapsed or Refractory Multiple Myeloma

A phase I/IIa Study of the CD38 Antibody MOR202 in Combination With Pomalidomide or Lenalidomide in Patients With Relapsed or Refractory Multiple Myeloma

Abstract B08: Biomimetic Culture Platform for Ex Vivo Preservation of Patient-Derived Multiple Myeloma Cells

Abstract Multiple myeloma (MM) is an incurable B-cell malignancy with a typical survival of 5 to 7 years, and is characterized by: (1) the accumulation of IgG (κ or λ) producing monoclonal plasma cells in bone marrow (BM) and (2) the progression of metastatic osteolytic lesions in bones. We have developed a new approach for the extended culture (&amp;gt; 3 weeks) of difficult-to-preserve primary patient multiple myeloma cells (PMMCs) present in bone marrow aspirates, using an osteoblast (OSB)-derived 3D tissue scaffold constructed in a perfused microfluidic environment [1]. In contrast, the ex vivo viability of PMMCs cannot be preserved using conventional culture techniques beyond a few days. This biomimetic model was used to show, for the first time, that: (1) the adhesion of PMMCs to OSB, mediated by osteoblastic N-cadherin, and the long-term viability of OSBs were critical factors in maintaining the ex vivo viability and proliferative capacity of PMMCs; (2) perfusion flow enhances the survival of OSBs, and thus increases the long-term viability of PMMCs; and noteworthy, (3) PMMCs can acquire drug-resistance due to the adhesive interactions with OSBs, a phenomenon called cell adhesion-mediated drug-resistance (CAM-DR) [2]. These findings support that the in vitro model is capable of recapitulating critical MM tumor microenvironment associated with the survival and drug-resistance of PMMCs. Upon further development, our ex vivo model may provide a new means of selecting the best therapy for an individual patient based on the ex vivo response of his or her PMMCs to drug treatment options as well as novel immunotherapeutic treatments such as chimeric antigen receptor (CAR) T-cell therapy. This precision medicine approach is expected to provide a transformative means of: (1) reducing ineffective or unnecessary therapies; (2) minimizing excess toxicity and costs; and (3) inhibiting the development of tumor cells' cross-resistance to additional drugs, as a critical part of the long-term care of MM patients. Also, we anticipate that our approach can be used for: (1) preclinical evaluation of new therapeutics, (2) studying the development of drug-resistant PMMC subpopulations, (3) identifying novel therapeutic targets, and (4) developing ex vivo models of breast and prostate cancers that metastasize to BM via similar mechanisms. Currently, we are in the early stage of prospectively correlating the ex vivo chemosensitivity and resistance of PMMCs with short-term clinical response, as a means of validating the predictive capability of our ex vivo MM culture platform. We also plan to incorporate the 3D-reconstructed network of primary human osteocytes [3] to study interactions of osteocytes with PMMCs and other BMMCs including immune cells.

Relative frequency of immature CD34+/CD90+ subset in peripheral blood following mobilization correlates closely and inversely with the absolute count of harvested stem cells in multiple myeloma patients

Background/Aim. Stem cells (SCs) guarantee complete/longterm bone marrow (BM) repopulation after SC-transplants. The aim of the study was to evaluate absolute count of total SCs (determined by ISHAGE-sequential-gating protocol ? SCish) and relative frequency of immature CD34+/CD90+ (CD90+SCish) subset in peripheral blood (PB) as predictive factors of mobilization and apheresis product (AP) quality. Methods. Mobilization included chemotherapy and granulocytegrowth- factor (G-CSF). Harvesting was performed by Spectra- Optia-IDL-system. The SCsish were determined as a constitutional part of CD34+ cells in the ?stem-cell-region? using FC- 500 flow-cytometer. In this study, the original ISHAGEsequential- gating protocol was modified by introduction of anti-CD90-PE monoclonal-antibody into the analysis of CD90 expression on SCish (CD90+SCish). The results were presented as a percentage of SCish per nucleated-cell count, absolute SCish count in ?L of the PB or the AP, percentage of the CD90+SCish expressed to SCish and absolute CD90+SCish count in ?L of the PB or the AP. Results. The absolute count of total SCish and CD90+SCish was significantly higher (p = 0.0007 and p = 0.0266, respectively) in the AP than in the PB samples. The CD90+SCish/total SCish indexes from PB were higher than indexes from the AP (p = 0.039). The relative frequency of CD90+SCish showed a highly significant inverse correlation with the absolute count of total SCish in both, the PB and AP (p = 0.0003 and p = 0.0013 respectively). The relative frequency of CD90+SCish from the PB also showed a significant (p = 0.0002) inverse relationship with total SCish count in the AP. Patients with less than 10% CD90+SCish in the PB had evidently higher (p = 0.0025) total SCish count in the AP. Conclusion. We speculate that lower CD90+SCish yield in the AP is not a consequence of an inferior collection efficacy, but most likely a result of several still not fully resolved immature SC cytomorphological/ biophysical features. Therefore, following the mobilization by chemotherapy G-CSF, some logical questions appear ? whether we should follow the absolute count of total SCish, or, whether we should test for relative frequency of CD90+SCish prior to harvesting. To reach the final conclusions, it is essential to conduct further controlled and larger investigations concerning the correlation of circulating and harvested SCs with patients' hematopoietic recovery.

Deubiquitylating Enzyme Rpn11/POH1/PSMD14 As Therapeutic Target in Multiple Myeloma

Introduction The ubiquitin proteasome pathway is a validated therapeutic target in multiple myeloma (MM), evidenced by the FDA approval of proteasome inhibitors bortezomib, carfilzomib, and ixazomib. However, these agents are associated with possible off-target toxicities and the eventual development of drug-resistance. Therapeutic strategies directed against deubiquitylating (DUB) enzymes upstream of the 20S proteasome may allow for more specific targeting of the UPS, with fewer off-target activities and toxicities. Rpn11 is a 19S-proteasome-associated DUB enzyme that facilitates protein degradation by the 20S proteasome core particle. Here we examined the role of Rpn11 in MM using both biochemical and RNA interference strategies.Materials and Methods Drug sensitivity, cell viability, and apoptosis assays were performed using WST, MTT, Annexin V staining, respectively. MM.1S MM cells were transiently transfected with control short interfering RNA (siRNA), RPN11 ON TARGET plus SMART pool siRNA using the cell line Nucleofector Kit V. In the xenograft mouse model, CB-17 SCID-mice were subcutaneously inoculated with MM.1S cells as previously described (Chauhan et al., Cancer Cell 2005, 8:407-419). Signal transduction pathways were evaluated using immunoblotting. Isobologram analysisand CalcuSyn software program were utilized to assesssynergistic/additive anti-MM activity. Statistical significance of observed differences were determined using a Student's t test. O-phenanthroline (OPA) was purchased from EMD Millipore, USA; and dex, lenalidomide, and pomalidomide were purchased from Selleck chemicals, USA.Results We found a statistically significant inverse correlation between Rpn11 levels and overall patient survival (p =0.022). Gene expression (GEP) analysisof Rpn11 showed a significantly higher level in patient MM cells versus normal plasma cells or PBMCs (p = 0.002 or p = 0.001 respectively). Immunohistochemical analysis of bone marrow biopsies from MM patients and normal healthy donors showed higher Rpn11 expression in MM cells than normal cells. Similarly, western blot analysis showed higher Rpn11 levels in MM cell lines and patient cells versus normal PBMCs.Rpn11 knockdown in MM cells significantly decreased cell viability (p < 0.001; n=3). To validate our siRNA data, we utilized Rpn11 inhibitor O-phenanthroline (OPA) (Verma et al., Science 2002, 298:611-5). Treatment of MM cell lines (MM.1S, MM.1R, RPMI-8226, ARP-1, Dox40, LR5, INA6, ANBL6.WT, and ANBL6.BR) and patient MM cells with OPA significantly decreased cell viability (IC50 range 8µM to 60µM; p < 0.001 for all cell lines; n=3) without markedly affecting PBMCs from normal healthy donors, suggesting selective anti-MM activity and a favorable therapeutic index for OPA. Importantly, the anti-MM activity of OPA was observed against tumor cells obtained from patients whose disease was progressing while on bortezomib, dexamethasone, and lenalidomide therapies. In concert with these data, the cytotoxicity of OPA was observed in MM cell lines sensitive and resistant to conventional and novel therapies. Furthermore, OPA inhibits proliferation of MM cells even in the presence of BM stromal cells or plasmacytoid dendritic cells (pDCs). OPA inhibits Rpn11 DUB activity without blocking 20S proteasome activities. Mechanistic studies show that OPA-triggered MM cell apoptosis is associated with 1) activation of caspases; 2) accumulation of polyubiquitinated proteins; 3); induction of ER stress; and 4) induction of autophagy. OPA-induced apoptosis occurs in a p53-independent manner, since OPA triggered apoptosis in both p53-null (ARP-1) and p53-mutant (RPMI-8226) MM cells. OPA inhibits MM cell growth in vivo and prolongs survival in a MM xenograft mouse model. Finally, combining OPA with lenalidomide, pomalidomide, or dex induces synergistic/additive anti-MM activity, and overcomes drug resistance.Conclusion Our preclinical data showing efficacy of OPA in MM models both validates targeting 19S proteasome-associated DUB Rpn11, and provides the framework for clinical evaluation of Rpn11 inhibitors to overcome proteasome inhibitor resistance and improve patient outcome in MM. DisclosuresMunshi:Celgene Corporation: Consultancy; Merck: Consultancy; Pfizer: Consultancy; Oncopep: Consultancy, Equity Ownership; Takeda: Consultancy. Chauhan:C4 Therapeutics: Equity Ownership; Epicent Rx: Consultancy; Oncopeptide AB: Consultancy; Stemline Therapeutics, Inc.: Consultancy. Anderson:Sonofi Aventis: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees; Oncopep: Other: Scientific Founder; Acetylon: Other: Scientific Founder; Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

Novel Flow Cytometry-Based Biomarkers Predict Recurrence in Myeloma Patients through Detection of MRD in the Bone Marrow and CTCs in Peripheral Blood

Introduction: The presence of a minimal residual disease (MRD) that remains after treatment is one of the main reasons for recurrence and relapse in multiple myeloma (MM) patients. Diagnosis of MRD is becoming vital to assess the effectiveness of the treatment as well as to predict survival among patients with complete remission. CD138 (syndecan-1) is the gold standard marker for detecting MM cells using multiparametric flow cytometry analysis or immunohistochemistry (IHC) staining of BM biopsies. However, the presence of highly clonogenic, drug resistant and stem-cell like CD138-negative MM cells have been previously demonstrated. Moreover, hypoxia which is known to drive MM progression, drug resistance and metastasis was shown to significantly decrease CD138 expression in MM cells. In this study, we utilized a novel set of biomarkers to detect MRD in the bone marrow as well as circulating tumor cells (CTCs) in the blood, in order to predict progression free survival (PFS) in MM patients defined as complete remission according to the detection of CD138+ cells in their bone marrow.Methods: To detect myeloma cells, we utilized a novel set of biomarkers, independent of CD138 expression and hypoxic state of MM cells (CD38+/CD3-/CD19-/CD14-/CD16-/CD123-) by two-color flow cytometry. We detected MM cells in the bone marrow of 25 patients with complete remission and very good partial response, whose bone marrow was defined as a CD138-negative (less than 0.5%). In order to retrospectively correlate the involvement of MM cells (%) with PFS, we used 24 months as a cut-off and compared the results acquired with the new method to traditional CD138-based flow or histology. In addition, based on the percentage of detected MM cells with the 2% cut-off, we analyzed the time to progression (months). Moreover, we detected MM cells in the peripheral blood from matching MM patients and performed analogous analysis. Furthermore, we also detected CTCs in 7 newly diagnosed patients and compared to 7 patients with progressive disease.Results: A study conducted on 25 patients with their bone marrow defined as a CD138-negative (less than 0.5%) showed that the novel strategy to detect MM cells was more precise than CD138-based flow or histology in predicting PFS. Patients who relapsed in less than 24 months had an average (±SEM) of 3.53±0.82%, while patients who relapsed later than 24 months had an average of 1.15±0.27% MM cells in the bone marrow when detected by the new method (p=0.004). In addition, the median PFS with <2% or ≥2% of MM cells detected by novel strategy was 31.6±2.8 and 18.5±2.2 months, respectively (p=0.004). Similarly, while CD138 detected very low amounts of CTCs and demonstrated no difference between fast and slow relapsing patients; we found that the new biomarkers detected significant differences in the number of circulating MM cells in patients who relapsed fast and patients who relapsed late or did not relapse. Patients who relapsed in less than 24 months had 1.74±0.42%, while patients who relapsed later than 24 months had 0.4±0.08% CTCs in the peripheral blood (p=0.01). In addition, we detected significantly more CTCs in patients with progressive disease (1.27±0.24%) compared to newly diagnosed MM patients (0.63±0.12%), respectively (p=0.03).Conclusions: The novel flow cytometry-based set of biomarkers provides an alternative strategy to detect MRD in the bone marrow and CTCs in the peripheral blood of MM patients, and both allowed prediction of PFS in MM patients. DisclosuresDe La Puente:Cellatrix LLC: Other: Co-founder. Vij:Amgen: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; Janssen: Consultancy; Jazz: Consultancy; Shire: Consultancy; Karyopharma: Consultancy; Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; Celgene: Consultancy. Azab:Targeted Therapeutics LLC: Other: Founder and owner; Cleave Bioscience: Research Funding; Verastem: Research Funding; Cell Works: Research Funding; Karyopharm: Research Funding; Vasculox: Research Funding; Glycomimetics: Research Funding; Cellatrix LLC: Other: Founder and owner; Selexys: Research Funding.

Autocrine and Paracrine Interactions Between Multiple Myeloma Cells and Bone Marrow Stromal Cells By Growth Arrest-Specific Gene 6 Crosstalk with Interleukin-6

The pathogenesis of multiple myeloma (MM) has not yet been fully elucidated. Our microarray analysis and immunohistochemistry revealed significant upregulation of growth arrest-specific gene 6 (Gas6), a vitamin K-dependent protein with a structural homology with protein S, in bone marrow (BM) cells of MM patients. ELISA showed that the serum levels of soluble Gas6 and interleukin (IL)-6were significantly increased in the MM patients when compared with healthy controls. Gas6 and TAM receptor tyrosine kinases Mer were overexpressed in human CD138-positive MM cell line RPMI-8226. Exogenous Gas6 and its signaling through Mer suppressed apoptosis induced by serum deprivation and enhanced cell proliferation of the MM cells. The conditioned medium from human BM stromal cell line HS-5 induced anti-apoptosis and cell proliferation of the MM cells with ERK, Akt and NF-kappaB phosphorylation, which were reversed by the neutralizing antibody to Gas6, IL-6 or Mer siRNA. Furthermore, the Gas6 neutralizing antibody reduced the upregulation of IL-6 and intercellular cell adhesion molecule-1 induced by a HS-5 cell-conditioned medium in MM cells. The present study provides new evidence that autocrine and paracrine stimulation of Gas6 in concert with IL-6 contributes to the pathology of MM, suggesting that Gas6/Mer signaling may be a promising novel target for treating MM. DisclosuresNo relevant conflicts of interest to declare.

Deubiquitylating Enzyme USP1 As Therapeutic Target in Multiple Myeloma

Introduction Deregulation of the ubiquitin-proteasome system (UPS) is linked to pathogenesis of various human diseases, including cancer. Targeting the proteasome is an effective therapy in multiple myeloma (MM) patients.Recent research efforts led to the discovery of newer agents that target enzymes modulating protein ubiquitin- conjugation/deconjugation rather than the proteasome itself, with the goal of generating more specific and less toxic anti-tumor therapies. Our prior studies have identified a role of deubiquitylating enzymes (DUBs) USP7, USP14, and UCHL5 in MM pathogenesis, and provided the rationale for targeting these DUBs in MM (Chauhan et al., Cancer Cell 2012, 11:345-358). Among DUBs, USP1 regulates DNA repair and the Fanconi anemia pathway by deubiquitylating two critical DNA repair proteins, FANCD2-Ub and PCNA-Ub. Additionally, USP1 stabilizes tumor-promoting inhibitor of DNA binding (ID) proteins. Here we examined the role of USP1 DUB in MM using both biochemical and RNA interference strategies.Methods We utilized MM cell lines, patient cells, and peripheral blood mononuclear cells (PBMCs) from normal healthy donors. Cell viability was assessed using WST and CellTiter-Glo assays. MM.1S cells were transiently transfected with control short interfering RNA (siRNA) USP1 ON TARGET plus SMART pool siRNA using the cell line Nucleofector Kit V. A biochemical inhibitor of USP1 SJB3-019A (SJB) was purchased from Medchem Express, USA. In vitro DUB enzymatic activity was assessed using Ubiquitin-AMC and Ubiquitin-Rhodamine assay kits, as well as Ub-CHOP-reporter and K48-linked Ubiquitin tetramers. Competitive Ub-VS probe labeling was performed, as previously described (Chauhan et al., Cancer Cell 2012, 11:345-358). Signal transduction pathways were evaluated using immunoblotting. Statistical significance of data was determined using a Student's t test.Results Gene expression profiling (GEP) analysis of USP1 showed significantly higher USP1 levels in patient MM cells versus normal plasma cells (p < 0.05).We found a statistically significant inverse correlation between USP1 levels and overall patient survival (p =0.036). Immunoblot blot analysis show higher USP1 levels in MM cell lines and patient cells compared to normal cells.USP1 knock-down reduced MM cell viability (p < 0.05).To validate our siRNA data, we utilized a novel USP1 inhibitor SJB3-019A (SJB). Analysis using Ub-Rhodamine, Ub-AMC and Ub-EKL reporter assays showed that SJB is a potent, specific, and selective inhibitor of cellular USP1 DUB activity (EC50=1μM), and does not inhibit other DUBs (USP2/USP5/USP7/USP14) or other families of cysteine proteases (UCH37) (EC50 >10 μM). SJB blocks labeling of USP1 with HA-Ub-VS probe in a concentration-dependent manner, but did not alter labeling of other DUBs with HA-Ub-VS. SJB inhibits USP1-mediated cleavage of K48-linked Tetra-ubiquitin chains, but not that mediated by USP2 or USP7. Treatment of MM cell lines (MM.1S, MM.1R, RPMI-8226, Dox-40, ARP1, KMS11, U266, ANBL6.WT, ANBL6.BR and LR5) and primary patient cells for 24h significantly decreases their viability (IC50 range 100nM to 500nM) (p< 0.05; n=3) without markedly affecting PBMCs from normal healthy donors, suggesting selective anti-MM activity and a favorable therapeutic index for SJB. Tumor cells from 4 patients whose disease was progressing while on bortezomib, dexamethasone, and lenalidomide therapies were sensitive to SJB. Furthermore, SJB3-019A inhibits proliferation of MM cells, even in the presence of BM stromal cells and plasmacytoid dendritic cells. Mechanistic studies show that SJB3-019A- triggered apoptosis is associated with 1) induction of cell cycle arrest via p21 upregulation; 2) activation of caspase 3, caspase-8 and caspase-9; 3) decreased homologous recombination activity and increased levels of Ub-FANCD2, Ub-FANCI, and Ub-PCNA; 4) defective DNA repair, evident by reduced RAD51; 5) degradation of USP1 and ID proteins; and 6) downregulation of Notch-1, Notch-2, SOX-4, and SOX-2 proteins. Finally, combination of SJB with lenalidomide, pomalidomide, HDACi ACY-241, or bortezomib induces synergistic anti-MM activity and overcomes drug resistance.Conclusion Our preclinical studies provide the framework for clinical evaluation of USP1 inhibitors, alone or in combination, as a potential novel MM therapy. DisclosuresMunshi:Celgene Corporation: Consultancy; Pfizer: Consultancy; Merck: Consultancy; Oncopep: Consultancy, Equity Ownership; Takeda: Consultancy. Chauhan:Stemline Therapeutics, Inc.: Consultancy; C4 Therapeutics: Equity Ownership; Oncopeptide AB: Consultancy; Epicent Rx: Consultancy. Anderson:Celgene Corporation: Consultancy; Millennium Pharmaceuticals: Consultancy; Novartis AG: Consultancy; Bristol-Myers Squibb:: Consultancy; Oncopep: Other: Scientific Founder; Acetylon: Other: Scientific Founder.

A Phase I/IIa Study of the CD38 Antibody MOR202 Alone and in Combination with Pomalidomide or Lenalidomide in Patients with Relapsed or Refractory Multiple Myeloma

A Phase I/IIa Study of the CD38 Antibody MOR202 Alone and in Combination with Pomalidomide or Lenalidomide in Patients with Relapsed or Refractory Multiple Myeloma

Arginine Methylation Mediated By PRMT5 Has Significant Biological and Clinical Impact in Multiple Myeloma

Arginine-specific methyltransferases critically regulate cellular homeostasis by dictating the biological outcome of target proteins. Among them, Protein Arginine Methyltransferase 5 (PRMT5) has attracted growing interest due to its role as an enzyme mediating epigenetic regulation of anti-cancer target genes, as well as in methylation of non-histone proteins involved in growth-regulating and survival pathways including p53. However, little is known about its biologic function in multiple myeloma (MM).To first evaluate the clinical significance of PRMT5 in MM pathogenesis, we analyzed RNA-seq data from newly-diagnosed MM patients and identified highly upregulated PRMT5 in 320 patients' CD138+ cells compared to 16 samples of normal bone marrow (BM) plasma cells. Additional analysis of PRMT5 expression in two independent datasets also showed further PRMT5 mRNA upregulation during progression of MM. Immunohistochemical staining also confirmed elevated expression of PRMT5 in BM biopsies from MM patients as compared to healthy individuals and monoclonal gammopathy of undetermined significance (MGUS). Moreover, analysis of the prognostic significance of PRMT5 expression in MM patients enrolled on IFM/DFCI 2009 clinical study showed that high PRMT5 expression was associated with poor prognosis in terms of both event free (p= 0.016) and overall survival (p=0.018). Consistently, we also found upregulated PRMT5 expression at both mRNA and protein levels in MM cell lines (N=11) and patients CD138+ MM cells (N=3) as compared to PBMCs from healthy volunteers, associated with a parallel increase of cellular symmetric arginine di-methylation (SDMA) substrates. Interestingly, genetic depletion of PRMT5 in H929 (p53wt) and KMS11 (p53null) MM cell lines by shRNA decreased SDMA levels, associated with cell growth inhibition in a p53-independent manner. Likewise, pharmacological inhibition of PRMT5 with the small molecule inhibitor EPZ015666 triggered decreased SDMA levels, cell growth, survival, and clonogenicity, as well as induction of caspase-dependent apoptosis in MM cell lines. Moreover, although PRMT5 and SDMA levels were increased in MM cells cultured in the presence of BM stromal cell supernatant, cytotoxic activity of EPZ015666 was maintained. Notably, drug treatment significantly impaired cell proliferation of patient MM cells (n=2) even in the presence of BM mononuclear or stromal cells, without toxicity on normal PBMCs. At the level of gene expression modulation, PRMT5 inhibition was associated with downregulation of NF-kB-dependent transcription, evidenced by both gene set enrichment analysis (GSEA) and Ingenuity Upstream Regulator Analysis. Moreover, analysis of protein levels confirmed reduction of both canonical and non-canonical NF-kB pathways, evidenced by significantly decreased NF-kB DNA binding activity by ELISA. Importantly, Mass Spectrometry analysis identified TRIM21 as a new PRMT5 interactor; and EPZ015666-treated cells showed that PRMT5 methylates TRIM21 evidenced by WB analysis. Since TRIM21 mediates monoubiquitination of IKKbeta, thereby triggering its selective autophagy-mediated degradation, we next analyzed EPZ015666 effects on IKKbeta. Treatment increased both monoubiquitination of IKKbeta and the formation of IKKbeta-TRIM21-pBECLIN1-pULK1 autophagic complexes. Conversely, inhibition of autophagosome formation by 3-methyladenine abrogated the anti-MM activity of EPZ015666 and IKKbeta degradation, indicating that selective autophagic degradation of IKKbeta and inhibition of NF-kB signaling mediates EPZ015666-triggered anti-MM activity. Consistent with this view, confocal microscopy analysis also confirmed co-localization of IKKbeta in the autophagosome after EPZ015666 treatment. Finally, stable silencing of TRIM21 in MM cell lines significantly abrogated the anti-proliferative effect of EPZ015666. Collectively, these data delineate arginine methylation as a new control mechanism of MM cell growth, and demonstrate that inhibiting PRMT5 decreases tumor cell survival via blockade of NF-kB signaling, even in the context of the BM milieu. These data demonstrate the biologic and prognostic significance of PRMT5 in MM pathogenesis, and provide the rationale for novel therapies targeting PRMT5 to improve patient outcome in MM. DisclosuresHideshima:Acetylon: Consultancy; C4 Therapeutics: Equity Ownership. Munshi:Takeda: Consultancy; Celgene Corporation: Consultancy; Merck: Consultancy; Pfizer: Consultancy; Oncopep: Consultancy, Equity Ownership. Anderson:Gilead: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Acetylon: Equity Ownership; Oncoprep: Equity Ownership; Oncoprep: Equity Ownership; Acetylon: Equity Ownership; Millennuim: Membership on an entity's Board of Directors or advisory committees; Millennuim: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Equity Ownership; C4 Therapeutics: Equity Ownership; Bristol Myers Squibb: 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.