Multiple Myeloma Cell Drug Resistance Research Articles

Interactions Between BAFF and BAFF Receptors Are Involved in Macrophage-Induced Bortezomib Resistance in Myeloma

Background:B-cell-activating factor (BAFF) is a member of the TNF family that critical for maintenance of B-cell development and homeostasis. BAFF receptor (BAFF-R), B-cell maturation antigen (BCMA) and transmembrane activator and CAML interactor (TACI) are three BAFF receptors. It has been reported that BAFF is expressed by neutrophils, monocytes, dentritic cells and macrophages and modulates the proliferation, survival and drug resistance of multiple myeloma (MM) cells. Our previous study showed that, macrophages protect MM cells from drug-induced apoptosis by direct interaction with MM cells. We hypothesized that BAFF/BAFF receptors play a role in macrophage-induced bortezomib resistance in myeloma.Methods: First, the expression levels of BAFF and its three receptors in primary MM cells, MM cell lines and peripheral blood monocyte(PBMC)-induced macrophages were detected by semiquantitative real time-polymerase chain reaction (qPCR),Western blot and flow-cytometry. Also the concentration of BAFF in the supernatants of MM patients' bone marrow, MM cell lines and macrophages were determined by ELISA. Second, Primary MM cells and MM cell lines were cocultured with macrophages for the indicated time (usually 4-6h and 24h), for some experiments, we added bortezomib to the coculture system. Cell viability and apoptosis of MM cells were verified by Cell Counting Kit-8(CCK8) after treated with recombinant human (rh) BAFF, BAFF neutralizing antibody and BAFF siRNA. The interactions between BAFF and its receptors are unveiled by flow-cytometry. Then, cell survival signaling activations that may confer MM drug resistance were examined by Western blot.Results: Two receptors of BAFF, TACI and BCMA were highly expressed in various MM cell lines. The expressions of BAFF in PBMC-induced macrophages were heterogeneous. Functional studies showed that rhBAFF promoted RPMI8226 and ARP1 myeloma cells growth (P<0.05) and protected them from bortezomib-induced apoptosis (P<0.05). Then we verified macrophage-mediated MM drug resistance by directly coculturing MM cells (ARP-1, RPMI8226) with PBMC-derived macrophages from healthy donors. The macrophage-induced bortezomib resistance was attenuated by neutralizing antibodies(P<0.05) and siRNA of BAFF(P<0.01) . Next we found that in MM cells cocultured with macrophages, bortezomib-induced PARP and caspase-3 cleavages were highly repressed and phosphorylated Src ,AKT and Erk1/2 were upregulated which indicated that BAFF-mediated MM drug resistance may be through ERK1/2 and Src pathway .In addition, BAFF induced activation of NF-κB2,a pathway critical for the growth and survival of these cells.Conclusions: Our data show that macrophage might induce drug resistance of MM cells by the interaction of BAFF and BAFF receptors, leading to a reduction in caspase proteins and subsequent activation of Src and Erk1/2 kinases and NF-κB2 pathways .These studies reveal a promising unknown role for BAFF/BAFF receptors, suggesting that targeting macrophage-MM interactions may represent a promising therapeutic modality. DisclosuresNo relevant conflicts of interest to declare.

The IKZF1-IRF4 Axis Regulates Macrophage Polarization and Macrophage-Mediated Anti-Tumor Immunity

In addition to antimicrobial activity, macrophages regulate tissue development, remodeling, and repair. In terms of neoplastic growth, tumor-associated macrophages (TAMs) are even considered pro-tumorigenic based on their angiogenic and T-cell suppressive properties. In multiple myeloma (MM) macrophages represent an abundant component of the stromal cell compartment and are believed to support proliferation, survival, and drug resistance of MM cells. Those pro-tumorigenic functions are partly instructed by T-helper (TH)-2 cytokines such as interleukin-4 (IL-4) and IL-10, which skew TAM differentiation towards an alternatively activated, or so-called M2-like, phenotype. In contrast, M1 macrophages generally considered as potent effectors in response to microbial products or interferon-γ (IFN-γ), are characterized by superior antigen-presentation, abundant production of pro-inflammatory cytokines such as interleukin-12 (IL-12), and consequently, promote a polarized type I immune response against infections as well as malignant cells. Transcriptional regulation is a key determinant for macrophage polarization. The Ikaros (IKZF1) transcription factor is critical for lymphoid development and is found in all hematopoietic progenitors as well as in T-, B-, NK-cells and macrophages. Interestingly, IKZF1 is overexpressed in MM and selectively degraded by lenalidomide, which is approved in MM therapy. The potential role of IKZF1 in modulating macrophage polarization has not been elucidated yet.Here, we show that IKZF1expression is found highly elevated in M2-like macrophages and in MM TAMs. IKZF1 deletion in human macrophages by small interfering RNA (siRNA) or by lenalidomide yields an upregulation of M1-specific cytokines (IL-12 and IL-1b), chemokines (CXCL10 and CCL5), and costimulatory molecules (CD86 and CD40) and leads to a potent TH1-TH17 response. In fact, lenalidomide-pretreated macrophages display strong tumoricidal effects when co-cultured with MM cell lines as opposed to their untreated counterparts promoting MM proliferation and viability. Utilizing immunoprecipitation-sequencing (ChIP-Seq) we reveal that IKZF1 governs IRF4 and IRF5 expression in human macrophages. Recent studies demonstrate that IRF4 controls M2 macrophage polarization, while IRF5 regulates the M1 phenotype respectively. We could show that a lenalidomid-mediated M1-phenotype induction is efficiently abrogated by IRF4 overexpression or IRF5 silencing.Overall, these findings unravel a novel role for IKZF1 in orchestrating macrophage polarization via the IRF4/IRF5 pathway. DisclosuresNo relevant conflicts of interest to declare.

Targeting EZH2 in Multiple Myeloma Could be Promising for a Subgroup of MM Patients in Combination with IMiDs

Multiple Myeloma is a B cell neoplasia characterized by the accumulation of clonal plasma cells within the bone marrow.Epigenetics is characterized by a wide range of changes that are reversible and orchestrate gene expression. Recent studies have shown that epigenetic modifications play a role in multiple myeloma (MM) by silencing various cancer-related genes.We investigated the epigenetic genes differentially expressed between normal bone marrow plasma cells (BMPC ; N=5) and MM plasma cells from patients (N=206). Using SAM (Significance Analysis of Microarrays) analysis, only 12 genes significantly differentially expressed between BMPC and MM cells (ratio > 2 and FDR (false discovery rate) < 5%) were identified, including the EZH2 histone methyltransferase. EZH2, the enzymatic subunit of Polycomb Repressive Complex 2, is a histone methyltransferases able to repress gene expression by catalyzing H3K27me3 histone mark. EZH2 overexpression has been associated with numerous hematological malignancies, including MM. We thus studied EZH2 role in MM physiopathology and drug resistance. EZH2 expression was analyzed in normal bone marrow plasma cells (BMPCs; N=5), primary myeloma cells from newly diagnosed patients (MMCs; N=206) and human myeloma cell lines (HMCLs; N=40) using Affymetrix microarrays. EZH2 gene is significantly overexpressed in MMCs of patients (median 574, range 105 - 4562) compared to normal BMPCs (median = 432; range: 314 - 563) (P < 0.01). The expression is even higher in HMCLs (median 4481, range 581 - 8455) compared to primary MMCs or BMPCs (P < 0.001). High EZH2 expression is associated with a poor prognosis in 3 independent cohorts of newly diagnosed patients (Heidelberg-Montpellier cohort - N=206, UAMS-TT2 cohort - N=345 and UAMS-TT3 cohort - N =158). Furthermore, GSEA analysis of patients with high EZH2 expression highlighted a significant enrichment of genes involved in cell cycle, downregulated in mature plasma cells vs plasmablasts, and EZH2 targets. Specific EZH2 inhibition by EPZ-6438 EZH2 inhibitor induced a significant decrease of global H3K27me3 in all the HMCLs tested (P < 0.01) and inhibited MM cell growth in 5 out of the 6 HMCLs tested. The inhibitory effect of EZH2 inhibitor on MM cell growth appeared at day 6 suggesting that it is mediated by epigenetic reprogramming. To confirm that EZH2 is also required for the survival of primary MMCs from patients, primary MM cells (n = 17 patients) co-cultured with their bone marrow microenvironment and recombinant IL-6 were treated with EPZ-6438. As identified in HMCLs, EZH2 inhibition significantly reduced the median number of viable myeloma cells by 35% (P = 0.004) from a subset of patients (n=9) while the other group (n=8) was resistant. Of interest, EPZ-6438 induced a significant global H3K27me3 decrease in both groups of patient. RNA sequencing of 6 HMCLs treated with EPZ-6438 combined with H3K27me3 ChIP analyses allowed us to create an EZ GEP-based score able to predict HMCLs and primary MM cells sensitivity to EZH2 inhibitors. We also observed a synergy between EPZ-6438 and Lenalidomide, a conventional drug used for MM treatment. More interestingly, pretreatment of myeloma cells with EPZ-6438 significantly re-sensitize drug-resistant MM cells to Lenalidomide. Investigating the effect of EPZ-6438/Lenalidomide combination in MMC, we identified that IKZF1, IRF4 and MYC protein levels were significantly more inhibited by the combination treatment (65.5%, 63.9% and 14.8% respectively) compared with Lenalidomide (51.5%, 43% and 2.2%) or EPZ-6438 (45.2%, 38.7% and 6.2%) alone.Clinical trials are ongoing with EZH2 inhibitors in lymphoma and could be promising for a subgroup of MM patients in combination with IMiDs. Furthermore, the EZ score enables identification of MM patients with an adverse prognosis and who could benefit from treatment with EZH2 inhibitors. DisclosuresGoldschmidt:Celgene: 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; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Millennium: Membership on an entity's Board of Directors or advisory committees, Research Funding; Chugai: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: 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; Amgen: Membership on an entity's Board of Directors or advisory committees. Hose:EngMab: Research Funding; Takeda: Other: Travel grant; Sanofi: Research Funding.

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.

Expansion of γδT Cells By New Generation IMiDs and Their Cytotoxicity Against Myeloma Progenitors

Although a therapeutic outcome with extended survival has been improved in patients with multiple myeloma (MM) owing to implementation new anti-MM agents, MM eventually relapses and still remains incurable. MM-initiating cells or MM progenitors are generally accepted to contribute to disease relapse through their drug-resistant nature. In order to overcome this drug resistance, various forms of immunotherapies have been studied. One such approach is the application of γδT cells. Vγ9Vδ2γδT (Vδ2 γδT) cells are important effectors in the first-line defense and tumor surveillance, and can be activated and expanded from peripheral blood mononuclear cells (PBMCs) upon treatment with zoledronic acid (Zol) and IL-2. New generation IMiDs, namely lenalidomide (LEN) and pomalidomide (POM), not only exert direct anti-MM actions, but also activate immune cells surrounding MM cells. In the present study, we aimed to clarify the effects of new generation IMiDs, LEN and POM, on activation and expansion of Vδ2 γδT cells in combination with Zol and the cytotoxic effects of ex-vivo expanded Vδ2 γδT cells against MM progenitors. After PBMCs isolated from normal donors were incubated for one week with Zol at 1 microM and LEN at 1 microM in combination, Vδ2 γδT cells were robustly expanded to be a majority of CD3-positive T cells as observed with Zol at 1 microM and IL-2 at 100 U/ml in combination. The expanded Vδ2 γδT cells expressed intracellular IFN-γ but not Foxp3 along with increased surface expression of NKG2D and DNAM-1, molecules known to be associated with NK or cytotoxic T cell-mediated cytotoxic activity, indicating induction of Th1-like Vδ2 γδT cells. In the same way as LEN, POM at 0.01, 0.1 and 1 microM was able to expand Vδ2 γδT cells from PBMCs isolated from all 10 normal donors. The expansion efficiency was slightly better with POM over LEN in some donors. The expansion efficiency with Zol plus these IMiDs appeared to be weaker in PBMCs from a majority of patients with MM than those from normal donors, as previously reported with Zol plus IL-2. Intriguingly, these IMiDs further expanded upon treatment with Zol plus IL-2 Vδ2 γδT cells from PBMCs from some donors. However, these IMiDs alone only marginally expanded Vδ2 γδT cells in the absence of Zol, suggesting costimulation of antigen-primed Vδ2 γδT cells by these IMiDs. Thus, LEN or POM appears to be able to expand Th1-like Vδ2 γδT cells in the presence of Zolwithout exogenous addition of IL-2. Such expanded Th1-like Vδ2 γδT cells exerted potent cytotoxic activity against MM cells. Interestingly, the expanded Vδ2 γδT cells also markedly minimized the sizes of side populations in RPMI8226 and KMS-11 cells, and suppressed their clonogenic capacity as determined by in vitro colony formation and tumorigenic capacity in SCID mice, suggesting targeting a drug-resistant clonogenic MM cells. Panobinostat, a pan-HDAC inhibitor, upregulated surface expression of NKG2D ligands, ULBPs and MICA/B, on MM cells, while impairing MM cells. Pretreatment with panobinostat may further potentiate the cytotoxic activity of Vδ2 γδT cells against MM cells. Combination with novel anti-MM agents warrants further study in terms of further enhancement of anti-MM effects with the ex-vivo expanded Th1-like Vδ2 γδT cells. The precise underlying mechanisms for ex-vivo expansion of Th1-like Vδ2 γδT cells should be clarified to make the best use of these new generation IMiDs with this unique and interesting phenomenon. DisclosuresNo relevant conflicts of interest to declare.

Discovery of selective small-molecule HDAC6 inhibitor for overcoming proteasome inhibitor resistance in multiple myeloma

Multiple myeloma (MM) has proven clinically susceptible to modulation of pathways of protein homeostasis. Blockade of proteasomal degradation of polyubiquitinated misfolded proteins by the proteasome inhibitor bortezomib (BTZ) achieves responses and prolongs survival in MM, but long-term treatment with BTZ leads to drug-resistant relapse in most patients. In a proof-of-concept study, we previously demonstrated that blocking aggresomal breakdown of polyubiquitinated misfolded proteins with the histone deacetylase 6 (HDAC6) inhibitor tubacin enhances BTZ-induced cytotoxicity in MM cells in vitro. However, these foundational studies were limited by the pharmacologic liabilities of tubacin as a chemical probe with only in vitro utility. Emerging from a focused library synthesis, a potent, selective, and bioavailable HDAC6 inhibitor, WT161, was created to study the mechanism of action of HDAC6 inhibition in MM alone and in combination with BTZ. WT161 in combination with BTZ triggers significant accumulation of polyubiquitinated proteins and cell stress, followed by caspase activation and apoptosis. More importantly, this combination treatment was effective in BTZ-resistant cells and in the presence of bone marrow stromal cells, which have been shown to mediate MM cell drug resistance. The activity of WT161 was confirmed in our human MM cell xenograft mouse model and established the framework for clinical trials of the combination treatment to improve patient outcomes in MM.

Advances Research on C-MYC Proto-oncogene in Multiple Myeloma -Review

Multiple myeloma(MM) as one of the most common tumors of hmatologic system, is characterized by malignant proliferation of plasma cells, and the chemotherapy is the main therapeutic method. MM is an incurable disease because of drug-resistance of MM cells. Although the pathogenesis of MM remains unknown, the chromosome abnormalities exit in half of the patients, particularly the highly expressed gene C-MYC. Furthermore, plenty of clinical researches indicated a high expression level of C-MYC implied worse progression and/or poor prognosis of MM. Recently, the work exploiting the compounds targeting MYC has made substantial progress, even in the MM therapy. In this article, briefly the recent advances of the research on C-MYC proto-oncogene in multiple myeloma are reviewed.

Study on the Association Between miRNA-202 Expression and Drug Sensitivity in Multiple Myeloma Cells.

An increasing amount of experimental evidence has shown that miRNAs play a causal role in hematologic tumorigenesis. In this study, we characterized the role of miR-202 in multiple myeloma (MM) drug sensitivity. The potential binding site of miR-202 and B cell-activating factor (BAFF) was confirmed by luciferase reporter assay. MM cells were transfected with miR-202 mimics and inhibitor. Cells growth was measured by WST-1 cell proliferation assay and Annexin V-FLUOS apoptosis assay. BAFF and miR-202 mRNA levels were measured by real-time PCR. Meanwhile, BAFF, Bcl-2 family survival proteins and MAPK pathway proteins were measured by Western blot. It was found that miR-202 was functioned as a modulator of BAFF expression. miR-202 over-expression sensitized MM cells to bortezomib (Bort) but less to Thalidomide (Thal) and dexamethasone (Dex). miR-202 mimics in combination with Bort inhibited MM cell survival more effectively as compared with Bort treatment alone. Our study also provided experimental evidence that JNK/SAPK signaling pathway was involved in the regulatory effect of miR-202 on drug resistance of MM cells. These results suggest that the regulatory mechanism of miR-202 expression may be a promising target for fine-tuning anti-myeloma therapy.

Drug Resistant Myeloma Cells Cause More Severe Bone Destruction in Myeloma

Background: Multiple myeloma (MM) is the second most common hematologic malignancy in the US and is characterized by osteolytic disease. More than 80% of MM patients have osteolysis, leading to in fractures, severe bone pain, spinal cord compression, and hypercalcemia, greatly compromising patient quality of life and resulting in mortality. While bone exists in a dynamic state between resorption and deposition, the molecular mechanisms underlying bone destruction in MM remain elusive. One contributing factor is heparanase (HPSE). In our work, we have previously shown that the expression of NEK2 and HPSE are closely correlated. We have also shown that high expression of chromosomal instability-related genes (e.g. NEK2) induces drug resistance and poor prognosis in MM. Though tumor burden is dramatically decreased in remission, bone destruction is poorly repaired, suggesting drug resistant MM cells may play a critical role in inducing bone destruction or in preventing the repair of bone disease in remission.Materials and Methods: Gene expression profiling (GEP) data and bone lytic lesions detected by MRI were collected from 333 newly diagnosed MM patients from the Total Therapy 2 (TT2) clinical trial. MM cell lines with stable overexpression or knockdown of NEK2 and/or HPSE were created using lentiviral delivery. Protein and mRNA expression was assessed by immunoblotting and real-time PCR, respectively. TRAP staining was used to assess osteoclast differentiation. In vivo studies were performed in the KaLwRij 5TGM1 MM mouse model in 3 different groups: empty vector (EV), NEK2 overexpression (NEK2 OE), or NEK2 OE with HPSE shRNA. Bone density and imaging assessment was performed via X-ray and microCT scanning. HPSE, NFκB, and USP7 contributions were examined using the specific small molecule inhibitors SST0001, BSM-345541, and P5091, respectively.Results: In correlating NEK2 expression with osteolytic lesion from 333 newly diagnosed MMs, we found the expression of NEK2 was significantly higher in MM patients with bone lytic lesion than those without lesions. NEK2 expression was also higher in patients with severe bone disease (7 or more lesions) than those fewer bone lytic lesions (< 7).To determine if NEK2 causes bone lytic lesions in MM, we performed in vivo studies injecting NEK2 OE 5TGM1 cells into KaLwRij mice. MicroCT showed clearly that bone volume and thickness of trabecular bone were decreased significantly in mice injected with NEK2 OE cells compared to EV cells. Strikingly, treatment with the HPSE inhibitor SST0001 dramatically rescued bone mass loss induced by NEK2 overexpression. Histological TRAP staining of decalcified bone sections revealed a significant increase in the number of osteoclasts in the bone surface area in mice injected with NEK2 OE cells compared to EV-treated mice, while addition of the SST0001 significantly reduced the number of osteoclast.Because osteoclast causes bone resorption, we examined the effect of applying conditioned media from MM cells with or without NEK2 overexpression to pre-osteoclast cells derived from MM patients, C57BL6 mice, and the pre-osteoclast RAW264.7 cell line. We found significantly increased mature osteoclasts (>3 nuclei) after culture with media collected from NEK2 MM cells compared to conditioned media collected from EV cells. Consistently, osteoclast differentiation-associated genes (i.e. CTSK, NFATc1, RANK, and TRAP) were upregulated in pre-osteoclasts cultured with NEK2 OE conditioned media. SST0001 suppressed osteoclast differentiation induced by NEK2, strongly suggesting that HPSE mediates NEK2 induced osteoclastogensis and bone destruction in MM. Mechanistic studies revealed that NEK2 directly binds to and activates USP7 resulting in deubiquitination of NFκB. Inhibition of NFκB decreased NEK2-induced HPSE expression induced by NEK2 at both the mRNA and protein level.Conclusions: Our findings suggest that high NEK2 expression may be a biomarker for bone disease in MM. We demonstrate that a subpopulation of drug-resistant, NEK2-overexpressing MM cells leads to increased production and activity of HPSE, and increased osteoclast differentiation contributing to severe bone disease. Mechanistically, we show that NEK2 interacts with USP7, stabilizing NFκB and upregulating HPSE. Our results demonstrate drug resistant MM cells may not only promote tumor cell survival but also contribute to bone destruction in MM. DisclosuresNo relevant conflicts of interest to declare.

Selinexor Is an Effective Cancer Treatment in Hypoxic Conditions and Synergizes with Proteasome Inhibitors to Treat Drug Resistant Multiple Myeloma

Introduction: Multiple myeloma (MM) is a plasma cell malignancy, characterized by plasma cell accumulation in the bone marrow (BM) associated with lytic bone disease. Despite the implementation of novel therapies, more than 70% of MM patients relapse due to drug resistance. Low oxygenation (hypoxia) in the BM microenvironment, which was shown to decrease beyond physiologic BM conditions during the progression of MM, together with cellular and acellular components, plays a critical role in drug resistance. Increased levels of the nuclear exporter of tumor suppressors, exportin 1 (XPO1), was demonstrated in MM, and XPO1 inhibition showed a broad anti-tumor activity. In this study, we tested inhibition of XPO1 using the novel oral, covalent, slowly reversible inhibitor of nuclear export selinexor currently in Phase I/II clinical trials on sensitizing the hypoxia-inducible drug resistant MM cells both in vitro and in vivo.Methods: MM cell lines (MM1s, H929, OPM2 and U266) were treated with increasing concentrations of selinexor in normoxic (21% O2) and hypoxic (1% O2) conditions with or w/o bortezomib or carfilzomib. Assays included MTT proliferation Annexin V/PI staining for apoptosis, PI staining and immuniblots for cell cycle analysis.We tested the effects of selinexor as a single agent on tumor initiation in vivo, where MM1s-Luc-GFP cells were injected intravenously (IV) into SCID mice and mice were treated instantaneously with selinexor (15mg/kg) or vehicle (PVP + F-68) three times a week. We also tested the effect of selinexor (15mg/kg) as a single agent on tumor progression and survival in mice with established MM tumor. Furthermore, we studied the effects of selinexor on sensitization to bortezomib-resistant MM cells in vivo. MM1s-Luc-GFP cells were injected IV into SCID mice and tumors were allowed to grow for three weeks. Animals were then treated with a high-dose of bortezomib (1.5mg/kg twice a week) for two weeks to induce resistance to bortezomib. The mice were then randomly divided into 3 groups treated with (1) bortezomib (0.5mg/kg), (2) KPT-330 (10mg/kg), and (3) a combination of selinexor and bortezomib. Selinexor was administered by oral gavage three times a week (days 1, 3 and 5) and bortezomib was injected intraperitoneally twice a week (days 1 and 4). Tumor progression was imaged using bioluminescence imaging (BLI).Results: Selinexor, as a single agent, decreased proliferation, induced G1 cell cycle arrest and increased apoptosis of MM cells in both hypoxia and normoxia in a dose-dependent manner. Moreover, we found that the combination of selinexor with bortezomib or carfilzomib reversed the hypoxia-induced resistance in MM cells to proteasome inhibition and induced a synergistic effect on the inhibition of cell proliferation, G1 arrest and apoptosis, which was confirmed by response of respective cell signaling pathways as PI3K, MAPK and JNK.In vivo studies revealed that selinexor alone delayed tumor initiation and decreased by half existing tumor size as a single agent (p=0.0397), and significantly extended mice survival (53 days on vehicle versus 62 days on selinexor, p=000375). In addition, MM-bearing mice which developed resistance to bortezomib were resensitized by treatment of selinexor combined with bortezomib, decreasing tumor burden and showed considerably extended survival (half of the mice were alive at day 52) compared to mice treated with either selinexor (half of the mice were alive at day 49) or bortezomib alone (half of the mice were alive at day 40) (bortezomib versus selinexor p=0.0014, bortezomib versus combination treatment p=0.0004 as analyzed by Student t-test).Conclusions: The orally available inhibitor of nuclear export, selinexor, decreased survival of MM cells in vitro and delayed tumor initiation and tumor progression in vivo as a single agent. In addition, selinexor overcame hypoxia-induced resistance to proteasome inhibitors and resensitized MM cells to therapy both in vitro and in vivo, extending mice survival. These data provide a basis for future clinical trials to sensitize relapsed/refractory MM patients to therapy by inhibition of XPO1 with selinexor as well as combination studies of selinexor and proteasome inhibitors. A study of selinexor and carfilzomib is currently ongoing and early results that were published last year showed marked activity for this combination treatment in heavily pretreated MM patients. DisclosuresLandesman:Karyopharm: Employment. Azab:Karyopharm: Research Funding; Selexys: Research Funding; Verastem: Research Funding; Targeted Therapeutics LLC: Other: Founder and owner ; Cell Works: Research Funding.

The Hypoxia-Selective Epigenetic Agent, Rrx-001, Triggers Apoptosis and Overcomes Drug-Resistance in Multiple Myeloma Cells

Introduction The bone marrow (BM) microenvironment confers growth, survival, and drug resistance in multiple myeloma (MM) cells (Chauhan et al, Cancer Cell 2009, 16:309-323) BM hypoxia (low oxygenation) plays a role in promoting MM cell survival, drug resistance, migration, and metastasis. Novel therapies that selectively target the MM cell in its hypoxic BM milieu may therefore overcome conventional drug resistance. Recent studies led to the development of a novel aerospace industry-derived Phase 2 molecule RRx-001 with hypoxia-selective epigenetic and NO-donating properties. A Phase I clinical trial demonstrated promising evidence of anti-tumor activity in a heavily pretreated population with no dose-limiting toxicities (Reid et al. J Clin Oncol 32:5s, 2014 suppl; abstr 2578, Reid et al, Lancet Oncology, in press). RRx-001 is currently under investigation in multiple Phase II clinical trials. Here we examined both the mechanism of action and anti-MM activity of RRx-001 using in vitro and in vivo models of MM.Methods Cell viability, apoptosis, and migration assays were performed using MTT, Annexin V staining, and transwell Inserts, respectively. ROS and NO generation was measured as previously described (Chauhan et al., Blood, 2004, 104:2458). Synergistic anti-MM activity was assessed by isobologram analysisusing "CalcuSyn" software program. In vitro angiogenesis was assessed using matrigel capillary-like tube formation assays. DNMT1 activity was measured using DNMT1 assay kit. USP7 siRNA was purchased from Dharmacon. CB-17 SCID-mice were subcutaneously inoculated with MM.1S cells as previously described (Chauhan et al., Cancer Cell 2012, 11:345-358). Statistical significance of data was determined using a Student's t test. RRx-001 was obtained from EpicentRx, CA, USA; USP7 inhibitor P5091, bortezomib, SAHA, and pomalidomide were purchased from Selleck chemicals, USA.Results Treatment of MM cell lines (MM.1S, MM.1R, RPMI-8226, OPM2, H929, Dox-40 ARP-1, KMS-11, ANBL6.WT, ANBL6.BR, and LR5) and primary patient cells for 48h significantly decreased their viability (IC50 range 1.25nM to 2.5nM) (p < 0.05; n=3) without markedly affecting PBMCs from normal healthy donors, suggesting specific anti-MM activity and a favorable therapeutic index for RRx-001. Tumor cells from 3 of 5 patients were obtained from patients whose disease was progressing while on bortezomib, dexamethasone, and lenalidomide therapies. Moreover, RRx-001 inhibits proliferation of MM cells, even in the presence of BM stromal cells. Washout experiments showed that a short time (3h) exposure of MM cells to RRx-001 triggered irreversible cell death. RRx-001-triggered apoptosis is associated with: 1) induction of DNA damage response signaling via ATM/p53/gH2AX axis; 2) activation of caspases mediating both intrinsic and extrinsic apoptotic pathways; 3) increase in oxidative stress through release of ROS and generation of NO; and 4) decrease in DNMT1 activity and global methylation levels. Furthermore, RRx-001 blocked migration of MM cells and angiogenesis. Deubiqyitylating enzyme USP7 stimulates DNMT1 enzymatic activity. USP7-siRNA reduced DNMT1 activity and decreased MM cell viability. Importantly, the combination of USP7 inhibitor P5091 and RRx-001 triggered synergistic anti-MM activity associated with a robust decrease in DNMT1 activity, as well as increased degradation of USP7 substrate MDM2 and induction of downstream p21/p53 signaling axis. In vivo studies using a subcutaneous human MM xenograft model shows that RRx-001 is well tolerated, inhibits tumor growth, and enhances survival. Finally, combining RRx-001 with pomalidomide, bortezomib or SAHA induces synergistic anti-MM activity in p53-WT and p53-null MM cells, and overcomes drug resistance.Conclusion Our preclinical studies demonstrate that RRx-001, a ROS-mediated epigenetic inhibitor with anti-angiogenic properties selectively targets MM cells in vivo and synergizes with existing anti-MM agents to overcome therapeutic resistance. Our data also suggest a potential mechanism of action for RRx-001-induced epigenetic changes via USP7-DNMT1 complex and downstream p53/p21 signaling cascade. Collectively, these results provide a rationale for rapid translation of RRx-001, either alone or in combination, in a clinical trial of relapsed refractory MM. DisclosuresOronsky:epicentrx: Employment. Scicinski:epicentrx: Employment. Chauhan:Stemline Therapeutics: Consultancy.

Lenalidomide Enhances MOR202 Dependent Macrophage-Mediated Effector Functions Via the Vitamin D Pathway

Multiple myeloma (MM) is considered a chronic and incurable disease due to its highly complex and heterogeneous molecular abnormalities and the support from myeloma microenvironment factors. Macrophages are an abundant component of the stromal cell compartment and are believed to support proliferation, survival, and drug resistance of MM cells. Conversely, macrophages are key immune effector cells for the therapeutic effect of monoclonal antibodies and can directly eliminate tumor cells. However, myeloma-associated macrophages (MAMs) regularly fail to exert direct effector functions. Given their abundance in MM, an attractive therapeutic approach would be to stimulate their tumoricidal activity in order to promote antitumor immunity. Lenalidomide, an immunomodulatory agent that enhances antibody dependent cell mediated cytotoxicity (ADCC), has the potential to synergize with MOR202, an anti-CD38 monoclonal IgG1 antibody currently in phase I/IIa for the treatment of MM. Furthermore, vitamin D plays a key role in regulating effector functions of human macrophages. This is closely linked to the expression of the vitamin D-1-hydroxylase CYP27B1, which catalyzes the conversion of 25-hydroxy-vitamin D (25D) to the bioactive form 1,25-di-hydroxy-vitamin D (1,25D). We have previously shown, that vitamin D promotes tumoricidal activity of macrophages and improves the efficacy of rituximab-dependent cytotoxicity (Bruns et al., Sci. Transl. Med., 2015; Bittenbring et al., JCO, 2014). Therefore, we hypothesized that the combination of MOR202 with lenalidomide and MOR202 with 1,25D would enhance the MOR202- dependent macrophage-mediated effector functions against myeloma cells.Here we report that MAMs exhibit an altered vitamin D metabolism with a reduced expression of the vitamin D receptor (VDR) and CYP27B1, while the expression of CYP24A1, which catabolizes 1,25D, is increased. As a consequence MAMs cannot convert 25D into bioactive 1,25D. Given the importance of the vitamin D pathway for antibody mediated cytotoxicity, we screened several drugs for their ability to restore the vitamin D pathway in human macrophages. We found, by RNA-sequencing, that lenalidomide treatment modulates the phenotype of monocyte-derived macrophages and isolated MAMs, and that lenalidomide significantly increases the expression of the VDR, CYP27B1 and CAMP (cathelicidin antimicrobial peptide) in these macrophages. Furthermore, we demonstrate that isolated MAMs regularly fail to eliminate primary myeloma cells, and that the lack of effector functions can be overcome by treatment with lenalidomide and vitamin D. Moreover, we show that MOR202-dependent elimination of myeloma cells is enhanced by pre-treatment of isolated MAMs with lenalidomide and supplementation of vitamin D.In summary, these data show that the bioactive form of vitamin D (1,25D) is essential for the effector functions of MAMs and that the therapeutic activation of the vitamin D pathway by lenalidomide may restore their tumoricidal effector mechanisms. Furthermore, these findings highlight that stimulating the tumoricidal activity of MAMs, an abundant component of the stromal cell compartment, with lenalidomide and 1,25D is an attractive therapeutic approach in combination with the anti-CD38 monoclonal IgG1 antibody MOR202. DisclosuresBruns:Morphosys: Research Funding; Celgene: Research Funding.

The role of circulating miRNAs in multiple myeloma.

Multiple myeloma (MM) is a common malignant hematological disease. Dysregulation of microRNAs (miRNAs) in MM cells and bone marrow microenviroment has important impacts on the initiation and progression of MM and drug resistance in MM cells. Recently, it was reported that MM patient serum and plasma contained sufficiently stable miRNA signatures, and circulating miRNAs could be identified and measured accurately from body fluid. Compared to conventional diagnostic parameters, the circulating miRNA profile is appropriate for the diagnosis of MM and estimates patient progression and therapeutic outcome with higher specificity and sensitivity. In this review, we mainly focus on the potential of circulating miRNAs as diagnostic, prognostic, and predictive biomarkers for MM and summarize the general strategies and methodologies for identification and measurement of circulating miRNAs in various cancers. Furthermore, we discuss the correlation between circulating miRNAs and the cytogenetic abnormalities and biochemical parameters assessed in multiple myeloma.

Effective impairment of myeloma cells and their progenitors by blockade of monocarboxylate transportation.

Cancer cells robustly expel lactate produced through enhanced glycolysis via monocarboxylate transporters (MCTs) and maintain alkaline intracellular pH. To develop a novel therapeutic strategy against multiple myeloma (MM), which still remains incurable, we explored the impact of perturbing a metabolism via inhibiting MCTs. All MM cells tested constitutively expressed MCT1 and MCT4, and most expressed MCT2. Lactate export was substantially suppressed to induce death along with lowering intracellular pH in MM cells by blockade of all three MCT molecules with α-cyano-4-hydroxy cinnamate (CHC) or the MCT1 and MCT2 inhibitor AR-C155858 in combination with MCT4 knockdown, although only partially by knockdown of each MCT. CHC lowered intracellular pH and severely curtailed lactate secretion even when combined with metformin, which further lowered intracellular pH and enhanced cytotoxicity. Interestingly, an ambient acidic pH markedly enhanced CHC-mediated cytotoxicity, suggesting preferential targeting of MM cells in acidic MM bone lesions. Furthermore, treatment with CHC suppressed hexokinase II expression and ATP production to reduce side populations and colony formation. Finally, CHC caused downregulation of homing receptor CXCR4 and abrogated SDF-1-induced migration. Targeting tumor metabolism by MCT blockade therefore may become an effective therapeutic option for drug-resistant MM cells with elevated glycolysis.

Vaccine based immunotherapy as a strategy to bypass drug resistance in multiple myeloma

Clonal evolution under therapy leads to drug resistance and relapse in multiple myeloma (MM), requiring new treatment mo- dalities to control disease. Immunotherapy and specifically vacci- nation to induce anti-tumor CD8+ cytotoxic T-cells (CTLs) provides one such strategy, but susceptibility of drug resistant MM cells to CTL attack is as yet not defined. To examine this, a first requirement is identification of tumour-associated antigens in drug refractory MM. To this end, we had previously specifically evaluated cancer testis antigens (CTAs) in MM for retention at relapse post- therapy, and identified SPAG9 and MAGEC1 as frontline antigen targets for vaccines. Of these, SPAG9 is generic to relapse MM (expressed in w100% of cases) and MAGEC1 is essentially tumour-specific (expressed w60% of cases). An optimal strategy for vaccination as therapy is in disease remission to attack re-emerging MM cells that escape drug therapy. A second requirement is to model drug resistance in MM, and accordingly we have now generated derivatives of the XG2 MM cell line that are resistant to melphalan (XG2 M) or Bortezomib (XG2 600R3); the RPMI8226-derivative R5 line with multiple drug resistance was also available. A third requirement is to show effective lysis of drug resistant MM cells by antigen specific CTLs. To generate antigen-specific CTLs, we utilized pDOM-CTAepitope DNA vaccine delivery in HLA-A2 transgenic HHD mice. We examined two epitopes in respective vaccines, pDOM-SPAG9p1 and pDOM-MAGEC1p2. Both vac- cines elicited epitope specific T-cell responses in HHD measured by IFN release assays following prime and boost with electroporation. Significantly, using MM cell lines made chimeric for HHD MHC class I as targets, both vaccines induced CTLs in HHD mice that were able to effectively lyse primary (XG2 and others) and drug resistant MM cell lines (XG2M, XG2 600R3, R5) effectively. These data demonstrate that drug resistant MM cells are susceptible to CTL killing, substantiating a role for vaccination in MM to over- come drug resistance and hence prevent relapse.

Abstract 4434: Selinexor and melphalan combination therapy for the treatment of multiple myeloma

Abstract Introduction High-dose melphalan (MEL) followed by autologous stem cell transplant remains the standard of care for the treatment of multiple myeloma (MM). However, patients eventually develop drug resistance and die from progressive disease despite newer therapies using proteasome inhibitors and immunomodulatory drugs. The incurable nature of MM demonstrates the need for novel treatments. Our aim was to investigate whether MEL therapy could be improved by the addition of the XPO1 inhibitor selinexor (SEL) in drug-resistant and parental MM cells both in vitro and ex vivo. Materials and Methods SEL/MEL-treated human MM cells were assayed for cell viability (CT-Blue) and apoptosis by flow cytometry (activated caspase 3). Proximity ligation assays (PLD) were used to assess if XPO1-p53 binding was inhibited by SEL. Western blots of SEL-treated MM cells were performed for nuclear and total p53. MEL-resistant U266 (LR6) and 8226 (LR5) MM cell lines were developed by incremental exposure to MEL. MEL resistant and parental MM cells were treated in vitro with SEL +/- MEL and assayed for apoptosis and cell viability. Cells isolated from patients with newly diagnosed or relapsed MM, were treated with SEL +/- MEL and assayed for apoptosis. Results MM cell viability was decreased synergistically by SEL when used in combination with MEL, as shown by combination index (CI) values. Drug sequencing assays showed that concurrent treatment with MEL (10 μM) and SEL (300 nM) for 48 hours produced synergistic results in human H929 MM cells (CI value 0.079, n = 3). Sequential treatment, SEL for 24 hours followed by MEL for an additional 24 hours or the reverse sequence, also demonstrated synergy with CI = 0.208 (n = 3) and 0.142 (n = 3), respectively. Normal PBMCs (control) were unaffected by SEL/MEL treatment as shown by viability and apoptotic assays. PLD demonstrated that SEL blocks XPO1/p53 binding. Western blot data showed that the SEL treatment of MM cells increased nuclear and total p53. Drug-resistant LR5 and LR6 MM cells were found to be resistant to MEL when compared to parental cell lines. Both resistant MM cell lines were sensitized by SEL to MEL as shown by apoptosis assay (20-fold). CD138+/light chain+ MM cells derived from newly diagnosed and relapsed MM patients were also sensitized (5 to 10-fold) by SEL to MEL as demonstrated by apoptosis assay. Conclusions SEL synergistically improved the response of drug-resistant and parental MM cells to MEL in vitro and ex vivo. It is possible that this synergy may be due to an increase of nuclear p53 by SEL and the reported activation of p53 by MEL. Ongoing studies include in vitro experiments to investigate whether this drug combination reverses MEL resistance by the Fanconi Anemia/BRCA pathway, in vivo treatment of MM in NSG mice with SEL/MEL and a clinical trial using high-dose MEL in combination with SEL. Combination therapies using SEL and MEL may significantly improve the treatment outcomes of MM. Citation Format: Joel G. Turner, Jana L. Dawson, Christopher Cubitt, Taiga Nishihori, Claudio Anasetti, Melissa Alsina, Rachid C. Baz, Michael Kauffman, Sharon Shacham, William S. Dalton, Daniel M. Sullivan. Selinexor and melphalan combination therapy for the treatment of multiple myeloma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4434. doi:10.1158/1538-7445.AM2015-4434

Matrine and CYC116 synergistically inhibit growth and induce apoptosis in multiple myeloma cells.

To investigate whether CYC116 can potentiate matrine-dependent growth inhibition and apoptosis in multiple myeloma (MM) cells. The dose response relationship of matrine to dexamethasone-resistant and dexamethasone-sensitive MM cells was first established. Myeloma RPMI8226 cells were treated with matrine alone or combined with CYC116 for 24 h. Cell proliferation was measured using an MTT assay and apoptosis induction was evaluated by flow cytometry. Activation of the caspase pathway and expression of apoptosis regulator proteins were detected by Western blotting. Matrine significantly induced growth arrest and apoptosis in both drug-resistant and drug-sensitive MM cells. Treatment with the combination of matrine and CYC116 had a stronger cytotoxic effect on MM cells than did single drug treatments. Enhanced apoptosis observed following the combined treatment of matrine and CYC116 was associated with higher levels of activation of caspase-9, caspase-3, and poly adenosine diphosphate ribose polymerase (PARP) and down-regulation of the anti-apoptotic proteins Bcl-2 and Mcl-1 and the signaling proteins p-Akt and nuclear factor κB (NF-κB). CYC116 enhances the growth inhibitory and apoptotic effects of matrine on MM cells.

Interaction Between B7-H1 Molecules on Myeloma Cells and PD-1 Molecules on T Cells Induces Resistance to Antimyeloma Chemotherapy

Introduction: B7-H1 (also known as PD-L1 or CD274), a co-inhibitory molecule of the B7 family, is detected on various tumor cells and associated with tumor evasion from cytotoxic T lymphocyte (CTL)-mediated immune surveillance. Our previous study showed that B7-H1 expression levels on plasma cells from multiple myeloma (MM) patients were significantly upregulated compared with those cells from monoclonal gammopathy of undetermined significance patients and healthy volunteers. B7-H1-expressing MM cells had a proliferative advantage and were resistant to antimyeloma agents (Tamura et al. Leukemia 2013). However, it remains unknown whether cellular responses in B7-H1-expressing MM cells are affected by the interaction of B7-H1 molecules with their receptors, i.e., PD-1 and CD80. We thus investigated the reverse signal derived from B7-H1 binding to their receptors on MM cells and examined the clinical characteristics of B7-H1-highly positive MM patients in a multicenter study.Methods: 1) We established B7-H1-expressing MM cell lines called MOSTI expressing high levels of CD38 and CD138 from bone marrow mononuclear cells of myeloma patients and B7-H1-positive MM cells (B7-H1.KMS28PE) stably transfected with the B7-H1 gene. 2) B7-H1 knockdown MOSTI cell lines were obtained using B7-H1-specific short-hairpin RNA expressing a lentiviral vector. 3) The proliferative potential was examined by BrdU incorporation using flow cytometry (FCM) and the MTT assay. 4) Drug-induced apoptotic cells were stained with annexin V and propidium iodide (PI) and detected in FCM. 5) Magnetic Dynabeads were coupled with PD-1-Ig or CD80-Ig fusion proteins. B7-H1-expressing MM cells were co-cultured with the beads, and the binding capacity of the beads to B7-H1+ MM cells, drug sensitivity, and cell proliferation of B7-H1+ MM cells were analyzed. 6) We classified 105 cases with newly diagnosed MM into two groups according to B7-H1 expression levels on plasma cells and compared the clinical characteristics associated with prognosis between B7-H1-highly-expressing MM patients (n=43) and other patients (n=62).Results: 1) Knockdown of B7-H1 expression in MOSTI cells significantly suppressed cell proliferation and increased melphalan-induced apoptosis. These results demonstrated that B7-H1 expression is directly associated with aggressive myeloma behavior including cell growth and drug resistance. 2) B7-H1 molecules on MOSTI and B7-H1.KMS28PE cells bound more strongly to PD-1-Fc than to CD80-Fc. The binding of PD-1-Fc to MOSTI cells was inhibited by anti-B7-H1 antibody in a concentration-dependent manner. In MOSTI cells treated with PD-1-Fc beads, apoptosis induced by both melphalan and bortezomib was markedly inhibited in comparison with the cells treated with control Ig. PD-1-Fc bead-treated B7-H1.KMS28PE cells were also resistant to melphalan-induced apoptosis. However, CD80-Fc bead-treated cells did not show drug resistance. Resistance to antimyeloma agents via the reverse signal from PD-1 to MM cells was inhibited by the PI3K/AKT inhibitor LY294002. In Western blot analysis, phospho-AKT expression was significantly upregulated in PD-1-Fc-treated MM cells. However, the cell growth of PD-1-Fc-treated MOSTI cells was the same as that of control Ig-treated cells. These data indicate that the reverse signal delivered from B7-H1 expressed on MM cells bound to PD-1 induced the drug resistance of MM cells thorough the Akt signaling pathway. 3) Patients with B7-H1 highly-expressing MM cells tended to have the poor-risk cytogenetic abnormality t(4;14) (P=0.0703). Furthermore, fibroblast growth factor receptor 3 was significantly upregulated on MM cells in those B7-H1-highly positive patients (P=0.0141). Expression levels of CD56, CD45, and CD221, which were reported to be poor prognostic markers in MM, were siginificantly higher in B7-H1-highly positive patients compared with others.Conclusion: Our study revealed a new mechanism via which the interaction between B7-H1 on MM cells and PD-1 molecules not only inhibits tumor-specific CTLs but also induces the drug resistance of MM cells through the Akt signaling pathway. Furthermore, B7-H1 expression on MM cells may be associated with t(4;14) translocation and poor prognostic MM antigens. Thus, B7-H1 may be a reasonable target for immunotherapy. DisclosuresNo relevant conflicts of interest to declare.

PYK2 Inhibitors Sensitize Hypoxia-Induced Drug Resistant Multiple Myeloma Cell

Introduction: Multiple myeloma (MM) is a plasma cell malignancy, characterized by plasma cell accumulation in the bone marrow (BM) and hyperproduction of immunoglobulin IgG. Despite the implementation of novel therapies, more than 70% of MM patients relapse due to drug resistance and micro-residual disease attributed to cancer stem cells. The hypoxic nature of the BM plays a critical role in MM cells acquiring a stem cell-like phenotype, and together with cellular and acellular components of the BM microenvironment contributes to drug resistance leading to minimal-residual disease. In this study, we tested inhibition of proline-rich tyrosine kinase 2 (PYK2), using VS-4718 and VS-6063, on reversing the hypoxia-inducible stem cell-like phenotype of MM cells and sensitizing them to therapy both in vitro and in vivo.Methods: VS-4718 and VS-6063 FAK/PYK2 inhibitors were obtained from Verastem, Inc. MM cell lines (MM1s, H929 and RPMI8226) were treated with bortezomib or carfilzomib under normoxic (21% O2) or hypoxic (1% O2) conditions, in the presence or absence of the FAK/PYK2 inhibitors VS-4718 or VS-6063. MM cells were analyzed for cell proliferation by MTT assay; apoptosis was analyzed by Annexin V/PI staining and analyzed by flow cytometry; and cell signaling associated with cell proliferation and apoptosis was analyzed by western blotting.Furthermore, we have tested the effect of the PYK2 inhibitors on sensitization of bortezomib-resistant MM cells in two models in vivo. In the first model, MM1s-Luc-GFP cells were injected intravenously into SCID mice and tumors were allowed to grow for four weeks; animals were then treated with a low-dose of bortezomib (0.5mg/kg twice a week) for two weeks to induce resistance to bortezomib. The mice were divided into 6 groups treated with (1) vehicle, (2) VS-4718 (50mg/kg) alone, (3) VS-6063 (50mg/kg) alone (4) bortezomib alone, (5) combination of VS-4718 and bortezomib, and (6) combination of VS-6063 and bortezomib, where VS-4718 and VS-6063 were administered by oral gavage twice a day every day. Mice survival was then followed for four more weeks.In the second model, H929 cells were injected subcutaneously into SCID mice. When tumors reached a mean of ~128 mm3 mice were randomized into five groups and treated with (1) vehicle, (2) VS-4718 (50mg/kg) alone, (3) bortezomib (1mg/kg) alone, (4) combination of VS-4718 and bortezomib (concurrently), and combination of VS-4718 and bortezomib (sequentially to simulate minimum residual disease). VS-4718 was administered by oral gavage twice a day and tumor growth was measured twice weekly using calipers.Results: We found that FAK/PYK2 inhibitors, VS-4718 and VS-6063, decreased proliferation and increased apoptosis of MM cells in both hypoxia and normoxia as single agents. Moreover, we found that hypoxia induced drug resistance to bortezomib and carfilzomib in MM cells. The combination of the two PYK2 inhibitors with either of the proteasome inhibitors reversed the hypoxia-induced resistance in MM cells and induced a synergistic effect with proteasome inhibitors on inhibition of cell proliferation, apoptosis, and cell signaling related to proliferation and apoptosis. In vivo study revealed that mice that developed MM disease with resistance to bortezomib were resensitized by treatment with the PYK2 inhibitors and showed significantly extended survival compared to mice treated with bortezomib alone. Furthermore, in mice treated with bortezomib only, tumors progressively re-grew after bortezomib treatment ended. While, in the bortezomib and VS-4718 combination treated mice and mice treated with VS-4718 in minimal residual setting (MRD) tumor grow-back (relapse) was prevented after cessation of bortezomib treatment.Conclusions: We report that FAK/PYK2 inhibitors, VS-4718 and VS-6063, decreased proliferation and increased apoptosis of MM cells as single agents. Hypoxia induced resistance to proteasome inhibitors and the PYK2 inhibitors resensitized MM cell to therapy in vitro and in vivo. Moreover, FAK/PYK2 inhibitor VS-4718 was able to prevent relapse in in vivo MM model simulating minimal residual disease. These data provide a basis for future clinical trials to sensitize relapsed/refractory MM patients to therapy by FAK/PYK2 inhibitors and their use to reduce relapse post front line treatment in an MRD setting. DisclosuresTrombino:Verastem: Employment. Padval:Verastem: Employment. Azab:Verastem: Research Funding.

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

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