Bortezomib-resistant Myeloma Cells Research Articles

The Oligosaccharyltransferase Complex: A Novel Vulnerability in Multiple Myeloma

Abstract Multiple myeloma (MM) is described as a clonal expansion of malignant plasma cells in the bone marrow characterized by several clinical symptoms: bone lesions, hypercalcemia, anemia, serum monoclonal gammopathy, immune suppression, and multiple organ failure. Despite the success of advanced treatments, disease relapse is common and incurable. Thus, there is an unmet clinical need to identify novel targets for relapsed/refractory MM treatment. The oligosaccharyltransferase (OST) complex catalyzes the transfer of N-glycan to a nascent translated protein which is a critical step in N-glycosylation, the most abundant post-translational modification of secretory and membrane-bound proteins. Aberrant expression of the OST complex or N-glycosylation is a diagnostic marker for solid tumors. However, the roles of the OST complex and N-glycosylation in MM development are virtually unknown. The mammal OST complex has two isoforms: OST-A and OST-B, with distinct catalytic subunits. Both isoforms share six subunits: OST4, RPN1, RPN2, DDOST, DAD1, and TMEM258. Analyzing the publicly available genomic data, we found that the shared subunits of the OST complex are abundantly expressed in MM cell lines and MM patient-derived cells. The expression levels of the OST subunits in relapsed MM patients are significantly higher than those in newly diagnosed MM patients. Intriguingly, patients expressing elevated levels of the OST complex are highly correlated with poor prognosis. These findings lead us to hypothesize that the OST complex might play a crucial role in MM. To address this hypothesis, we used the CRISPR/Cas9 system to knockout the OST's shared subunits and found that MM cells are dependent on the OST complex. Deletion of DDOST and DAD1 (two central structural subunits of the OST complex) impaired MM cell growth, arrested the cell cycle, and induced apoptosis. Using the subcutaneous xenograft model, we found that knockout of DDOST or DAD1 suppressed MM growth in vivo and extended the survival of tumor-bearing mice. Intriguingly, suppression of the OST complex's enzymatic activity by a specific inhibitor, NGI-1, recapitulated the phenotypes of the OST complex deletion. Of note, NGI-1 sensitizes the MM cells and bortezomib-resistant MM cells (both MM cell lines and primary patient cells) to bortezomib treatment. Mechanistically, we found that disruption of the OST complex in MM cells significantly suppressed transcriptomic signatures of MM pathology (NF-κB signaling, glycolysis, MYC targets, and cell cycle) and induced apoptosis pathway as well as inflammatory pathway (interferon alpha and gamma). Intriguingly, we found that all known genes responsible for the bortezomib-resistant phenotype were significantly downregulated upon the suppression of the OST complex. In conclusion, we identified the OST complex as a novel vulnerability in MM cells. Targeting the OST complex incorporation with Bortezomib might provide a novel and effective combined treatment for relapsed/refractory MM patients. We're currently investigating the underlying mechanisms of how the OST complex regulates MM pathology and developing patient-derived xenograft models to validate the therapeutic efficacy of combined treatment (NGI-1 and Bortezomib).

Deacetylation induced nuclear condensation of HP1γ promotes multiple myeloma drug resistance

Acquired chemoresistance to proteasome inhibitors is a major obstacle in managing multiple myeloma but key regulators and underlying mechanisms still remain to be explored. We find that high level of HP1γ is associated with low acetylation modification in the bortezomib-resistant myeloma cells using SILAC-based acetyl-proteomics assay, and higher HP1γ level is positively correlated with poorer outcomes in the clinic. Mechanistically, elevated HDAC1 in the bortezomib-resistant myeloma cells deacetylates HP1γ at lysine 5 and consequently alleviates the ubiquitin-mediated protein degradation, as well as the aberrant DNA repair capacity. HP1γ interacts with the MDC1 to induce DNA repair, and simultaneously the deacetylation modification and the interaction with MDC1 enhance the nuclear condensation of HP1γ protein and the chromatin accessibility of its target genes governing sensitivity to proteasome inhibitors, such as CD40, FOS and JUN. Thus, targeting HP1γ stability by using HDAC1 inhibitor re-sensitizes bortezomib-resistant myeloma cells to proteasome inhibitors treatment in vitro and in vivo. Our findings elucidate a previously unrecognized role of HP1γ in inducing drug resistance to proteasome inhibitors of myeloma cells and suggest that targeting HP1γ may be efficacious for overcoming drug resistance in refractory or relapsed multiple myeloma patients.

DUT enhances drug resistance to proteasome inhibitors via promoting mitochondrial function in multiple myeloma.

Acquired chemoresistance to proteasome inhibitors (PIs), such as bortezomib (BTZ), becomes an intractable obstacle in the management of multiple myeloma (MM) in the clinic, but the underlying mechanisms are still not well elucidated. In the current study, we established bortezomib-resistant (BR) myeloma cells and performed stable isotope labeling by amino acids in cell culture (SILAC) assay to screen profiled protein expression. The level of deoxyuridine triphosphatase (DUT), an important enzyme of nucleotide metabolism, increased in the BR MM cells. Retrospective analysis indicated patients with higher DUT expression had poorer responses to PI-based treatment and clinical outcomes. DUT knockdown by RNAi effectively minimized BTZ resistance in MM cells. Moreover, DUT knockdown was accompanied with the downregulation of proliferating cell nuclear antigen (PCNA), contributing to decelerating cell growth, as well as augmented apoptosis due to bortezomib treatment. In contrast, DUT overexpression in parental MM.1S and LP-1 cells enhanced BTZ resistance. Furthermore, acquired resistance to BTZ could trigger the modulation of mitochondrial metabolism and function, as evidenced by elevated expression of genes associated with mitochondrial metabolism, as well as altered oxygen consumption rate and adenosine triphosphate (ATP) production in BR MM cells. DUT inhibition partially attenuated mitochondrial modulation, and instead favored an early impairment of mitochondrial integrity upon BTZ exposure so as to restrict MM progression and overcome drug resistance to BTZ treatment both in vitro and in vivo. In conclusion, we unveiled previously unrecognized effects of DUT on acquired drug resistance of MM, thus manipulating DUT may be efficacious for sensitizing MM cells to PIs.

MSC Senescence-Related Genes Are Associated with Myeloma Prognosis and Lipid Metabolism-Mediated Resistance to Proteasome Inhibitors.

Complex carcinogenic mechanisms and the existence of tumour heterogeneity in multiple myeloma (MM) prevent the most commonly used staging system from effectively interpreting the prognosis of patients. Since the microenvironment plays an important role in driving tumour development and MM occurs most often in middle-aged and elderly patients, we hypothesize that ageing of bone marrow mesenchymal stem cells (BM-MSCs) may be associated with the progression of MM. In this study, we collected the transcriptome data on MM from The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO) databases. Differentially expressed genes in both senescent MSCs and MM tumour cells were considered relevant damaged genes. GO and KEGG analyses were applied for functional evaluation. A PPI network was constructed to identify hub genes. Subsequently, we studied the damaged genes that affected the prognosis of MM. Least absolute shrinkage and selection operator (lasso) regression was used to identify the most important features, and a risk model was created. The reliability of the risk model was evaluated with the other 3 GEO validation cohorts. In addition, ROC analysis was used to evaluate the novel risk model. An analysis of immune checkpoint-related genes, tumour immune dysfunction and exclusion (TIDE), and immunophenotypic scoring (IPS) were performed to assess the immune status of risk groups. pRRophetic was utilized to predict the sensitivity to administration of chemotherapeutic agents. We identified that MAPK, PI3K, and p53 signalling pathways were activated in both senescent MSCs and tumour cells, and we also located hub genes. In addition, we constructed a 14-gene prognostic risk model, which was analysed with the ROC and validated in different datasets. Further analysis revealed significant differences in predicted risk values across the International Staging System (ISS) stage, sex, and 1q21 copy number. A high-risk group with higher immunogenicity was predicted to have low proteasome inhibitor sensitivity and respond poorly to immunotherapy. Lipid metabolism pathways were found to be significantly different between high-risk and low-risk groups. A nomogram was created by combining clinical data, and the optimization model was further improved. Finally, real-time qPCR was used to validate two bortezomib-resistant myeloma cell lines, and the test confirmed that 10 genes were detected to be expressed in resistant cell lines with the same trend as in the high-risk cohort compared to nonresistant cells. Fourteen genes related to ageing in BM-MSCs were associated with the prognosis of MM, and by combining this genotypic information with clinical factors, a promising clinical prognostic model was established.

A clinically relevant pulse treatment generates a bortezomib-resistant myeloma cell line that lacks proteasome mutations and is sensitive to Bcl-2 inhibitor venetoclax

Proteasome inhibitors bortezomib and carfilzomib are the backbones of treatments of multiple myeloma, which remains incurable despite many recent advances. With many patients relapsing despite high initial response rates to proteasome inhibitor-containing regimens, it is critical to understand the process of acquired resistance. In vitro generated resistant cell lines are important tools in this process. The majority of previously developed bortezomib-resistant cell lines bear mutations in the proteasome PSMB5 sites, the prime target of bortezomib and carfilzomib, which are rarely observed in patients. Here we present a novel bortezomib-resistant derivative of the KMS-12-BM multiple myeloma cell line, KMS-12-BM-BPR. Unlike previously published bortezomib-resistant cell lines, it was created using clinically relevant twice-weekly pulse treatments with bortezomib instead of continuous incubation. It does not contain mutations in the PSMB5 site and retains its sensitivity to carfilzomib. Reduced load on proteasome due to decreased protein synthesis appears to be the main cause of resistance. In addition, KMS-12-BM-BPR cells are more sensitive to Bcl-2 inhibitor venetoclax. Overall, this study demonstrates the feasibility of creating a proteasome inhibitor resistant myeloma cell lines by using clinically relevant pulse treatments and provides a novel model of acquired resistance.

Novel Peptide-drug Conjugate Melflufen Efficiently Eradicates Bortezomib-resistant Multiple Myeloma Cells Including Tumor-initiating Myeloma Progenitor Cells.

Introduction of the proteasome inhibitor bortezomib has dramatically improved clinical outcomes in multiple myeloma. However, most patients become refractory to bortezomib-based therapies. On the molecular level, development of resistance to bortezomib in myeloma cells is accompanied by complex metabolic changes resulting in increased protein folding capacity, and less dependency on the proteasome. In this study, we show that aminopeptidase B, encoded by the RNPEP gene, is upregulated in bortezomib-resistant myeloma cell lines, and in a murine in vivo model. Moreover, increased RNPEP expression is associated with shorter survival in multiple myeloma patients previously treated with bortezomib-containing regimens. Additionally, expression is increased in plasma cell precursors, a B-lymphoid compartment previously associated with myeloma stem cells. We hypothesized that increased aminopeptidase B expression in aggressive myeloma clones may be used therapeutically toward elimination of the cells via the use of a novel peptide-drug conjugate, melphalan flufenamide (melflufen). Melflufen, a substrate of aminopeptidase B, efficiently eliminates bortezomib-resistant myeloma cells in vitro and in vivo, and completely suppresses clonogenic myeloma growth in vitro at subphysiological concentrations. Thus, melflufen represents a novel treatment option that is able to eradicate drug-resistant myeloma clones characterized by elevated aminopeptidase B expression.

Immunomodulatory Effects of HDACi in Combination with Checkpoint Blockade and Lenalidomide in the Immunosuppressive Multiple Myeloma Bone Marrow Microenvironment

Background: The interaction of myeloma (MM) cells with bone marrow (BM) accessory cells and/or the extracellular matrix induces genomic, epigenomic, and functional changes which promote tumor development, progression, cell adhesion mediated-drug resistance (CAM-DR), and immune suppression. To develop the most effective anti-MM treatment strategy and prevent tumor escape from immune recognition, both enhancing anti-MM effector immune response and overcoming MM-induced immune suppression is essential. Suppressive immune cells including myeloid derived suppressor cells (MDSC) and regulatory T cells (Treg) act as tumor promoters and suppressors of effector immune response, and therefore represent a significant barrier to current anti-tumor therapeutic strategies. We have recently shown that WT161, a novel small molecule inhibitor of HDAC6, alone or in combination with proteasome inhibitors induces MM cytotoxicity even in the bortezomib resistant MM cells. To target the MM BM immune suppressive microenvironment, we here have assessed the immunomodulatory effects of a novel triple-combination strategy using HDACi with checkpoint blockade and lenalidomide (Len) in MM BM ex vivo.Methods: Differential effects of Class I and Class II HDACi including pan-HDAC inhibitor Trichostatin A (TsA, 5-100nM), HDAC1 selective inhibitor Romidepsin (Rom, 1.5-15nM), HDAC3 inhibitor BG45 (10-40uM), HDAC6 inhibitor Tubastatin A HCl (TubA) and WT161 (0.5-10uM) alone or in combination with anti-PD1 (20ug/ml) and Len (0.5-1uM) were evaluated in MM-BM. HDACi direct effect on MM (MM1.S, H929, and RPMI8226) cell growth was measured by MTT. In the autologous coculture settings, BMMC from patients with newly diagnosed (ND)-MM and relapsed (R)-MM were cultured with HDACi alone or in combination with anti-PD1 and Len, with or without drug-wash-out for 1-5 days. Effector cell activation, proliferation, and cytotoxic activity was measured by flow cytometry analysis. Triple-combination effects on MDSC-mediated immune suppression was determined in cultures of autologous T cells with CD11b+CD14+HLA-DR- mMDSC, CD11b+CD14+HLA-DR-CD33+CD15+nMDSC, and CD11b+CD14+HLA-DR+APCs.Results: Healthy PBMC and MM cell line cultures demonstrated a dose-dependent toxicity and inhibition of proliferation in both CD4 and CD8 T cells induced by Rom; in contrast, TubA, WT161, and BG45 significantly induced T cell proliferation and cytotoxic activity even after wash out of HDACi. Additionally, HDAC6 inhibitors TubA and WT161 markedly increased expression of T cell exhaustion antigens TIM3 and LAG3, and these effects were partially reversed by drug washout. Moreover, triple-combination of HDACi with PD-1 blockade and Len significantly induced CD25+CD69+ activated T and NK cell mediated cytotoxicity of CD138+ MM cells. Interestingly, in the autologous co-cultures of MM-BMMC, TubA, BG45 and Rom induced CD25+CD69+ activated T cells, and the PD-1 blockade and Len further enhanced T cell activation. However, HDAC inhibitors, with the exception of HDAC3 inhibitor BG45, expanded immunosuppressive mMDSC populations within MM-BM myeloid compartment. Specifically, WT161 induced differentiation of myeloid cells by shifting CD14-HLA-DR-CD33+CD15+ nMDSCs to CD14+HLA-DR- mMDSCs and CD14+HLA-DR+APCs. Moreover, Rom significantly increased frequency of immunosuppressive IFNα producing plasmacytoid dendritic cells (pDCs), while decreasing Tregs, in MM-BM. Importantly, WT161 and TubA in combination with PD-1 blockade and Len reversed MDSC-mediated immunosuppressive activity, as measured by T cell proliferation. Finally, inhibition of HDAC6 induced MM cell cytotoxicity, and addition of PD-1 blockade and Len further enhanced TubA- and WT161 induced anti-MM immune responses.Conclusion: These data suggest that distinct classes of HDACi may induce differential immunomodulatory effects in the MM BM. Inhibition of HDAC6 induces changes in immune suppressor cells leading to enhanced anti-MM immune response, and addition of PD-1 blockade with Len further enhances HDAC6 inhibition-induced effector cell mediated anti-MM immune response. Ongoing analysis of triple combination of HDAC6 inhibition with checkpoint blockade and lenalidomide in the MM BM microenvironment will further define the role of HDAC6 in disease pathogenesis, and suggest novel immune-based epigenetic and targeted combination therapies for MM. DisclosuresHofmeister:Roche: Research Funding; Thrassos: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Research Funding; Takeda: Research Funding; Bristol-Myers Squibb: Research Funding; Celgene: Research Funding; Janssen: Research Funding. Bradner:Acetylon: Other: Scientific Founder; Novartis: Employment. Hideshima:Acetylon: Consultancy; C4 Therapeutics: Equity Ownership. Anderson:Oncopep: Other: scientific founder; Millenium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Other: scientific founder; Gilead Sciences: 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; MedImmune: Membership on an entity's Board of Directors or advisory committees. Gorgun:Bristol Mayer Squibb: Research Funding; Janssen: Research Funding; Merck: Research Funding.

Expression of histone deacetylase (HDAC) family members in bortezomib-refractory multiple myeloma and modulation by panobinostat.

Aim: Multiple myeloma (MM) is a hematological malignancy of antibody-producing mature B cells or plasma cells. The proteasome inhibitor, bortezomib, was the first-in-class compound to be FDA approved for MM and is frequently utilized in induction therapy. However, bortezomib refractory disease is a major clinical concern, and the efficacy of the pan-histone deacetylase inhibitor (HDACi), panobinostat, in bortezomib refractory disease indicates that HDAC targeting is a viable strategy. Here, we utilized isogenic bortezomib resistant models to profile HDAC expression and define baseline and HDACi-induced expression patterns of individual HDAC family members in sensitive vs. resistant cells to better understanding the potential for targeting these enzymes. Methods: Gene expression of HDAC family members in two sets of isogenic bortezomib sensitive or resistant myeloma cell lines was examined. These cell lines were subsequently treated with HDAC inhibitors: panobinostat or vorinostat, and HDAC expression was evaluated. CRISPR/Cas9 knockdown and pharmacological inhibition of specific HDAC family members were conducted. Results: Interestingly, HDAC6 and HDAC7 were significantly upregulated and downregulated, respectively, in bortezomib-resistant cells. Panobinostat was effective at inducing cell death in these lines and modulated HDAC expression in cell lines and patient samples. Knockdown of HDAC7 inhibited cell growth while pharmacologically inhibiting HDAC6 augmented cell death by panobinostat. Conclusion: Our data revealed heterogeneous expression of individual HDACs in bortezomib sensitive vs. resistant isogenic cell lines and patient samples treated with panobinostat. Cumulatively our findings highlight distinct roles for HDAC6 and HDAC7 in regulating cell death in the context of bortezomib resistance.

Intravenous Immunoglobulin G Suppresses Heat Shock Protein (HSP)-70 Expression and Enhances the Activity of HSP90 and Proteasome Inhibitors.

Intravenous immunoglobulin G (IVIgG) is approved for primary immunodeficiency syndromes but may induce anti-cancer effects, and while this has been attributed to its anti-inflammatory properties, IgG against specific tumor targets may play a role. We evaluated IVIgG alone, and with a Heat shock protein (HSP)-90 or proteasome inhibitor, using multiple myeloma and mantle cell lymphoma (MCL) cells in vitro, and with the proteasome inhibitor bortezomib in vivo. IVIgG inhibited the growth of all cell lines tested, induced G1 cell cycle arrest, and suppressed pro-tumor cytokines including Interleukin (IL)-6, IL-8, and IL-10. Genomic and proteomic studies showed that IVIgG reduced tumor cell HSP70-1 levels by suppressing the ability of extracellular HSP70-1 to stimulate endogenous HSP70-1 promoter activity, and reduced extracellular vesicle uptake. Preparations of IVIgG were found to contain high titers of anti-HSP70-1 IgG, and recombinant HSP70-1 reduced the efficacy of IVIgG to suppress HSP70-1 levels. Combining IVIgG with the HSP90 inhibitor AUY922 produced superior cell growth inhibition and correlated with HSP70-1 suppression. Also, IVIgG with bortezomib or carfilzomib was superior to each single agent, and enhanced bortezomib's activity in bortezomib-resistant myeloma cells. Moreover, IVIgG reduced transfer of extracellular vesicles (EVs) to cells, and blocked transfer of bortezomib resistance through EVs. Finally, IVIgG with bortezomib were superior to the single agents in an in vivo myeloma model. These studies support the possibility that anti-HSP70-1 IgG contained in IVIgG can inhibit myeloma and MCL growth by interfering with a novel mechanism involving uptake of exogenous HSP70-1 which then induces its own promoter.

Betulinic Acid Enhances the Sensitivity of Multiple Myeloma Cells to Bortezomib by the Inactivation of the AKT/mTOR Pathway

This study aimed to investigate the mechanism of betulinic acid on multiple myeloma cell resistance to bortezomib. To this end, the bortezomib-resistant RPMI-8226-R cells were generated by prolonged treatment of RPMI-8226 cells with increasing concentrations of bortezomib. Based on the measurements of cell viability and colony number, RPMI-8226-R cells exhibited enhanced resistance to bortezomib than RPMI-8226 cells. Treatment with betulinic acid resulted in increased sensitivity of RPMI-8226-R to bortezomib. When RPMI-8226-R cells were co-treated with bortezomib and betulinic acid, there was an increase in apoptosis rate, cleaved caspase-3, cleaved caspase-9 expression and the decrease in p-AKT/AKT and p-mTOR/mTOR levels. These results suggest that betulinic acid enhances the sensitivity of RPMI-8226-R cells to bortezomib by inhibiting the activation of the AKT/mTOR pathway in bortezomib-resistant multiple myeloma cells.

Abstract C052: FGF trapping impairs multiple myeloma growth through c-Myc degradation-induced mitochondrial oxidative stress

Abstract Multiple myeloma (MM) remains incurable because of chemotherapeutic resistance. Fibroblast growth factors (FGF) play a pivotal role in MM by acting as an autocrine/paracrine mitogen on plasma cells, bone marrow-derived endothelial cells and fibroblasts. Also, a recurrent chromosomal translocation t(4;14) is associated with FGFR3 upregulation in MM patients. Thus, agents able to hamper FGF signaling may represent a novel approach for MM treatment. Recently we have identified the PTX3-derived small molecule NSC12 as the first orally active antitumor pan-FGF trap. Here, the role of FGF/FGFR system in MM cell survival and the therapeutic potential of NSC12 were investigated. In vitro effects of NSC12 were tested on four human MM cell lines harboring (KMS-11 and OPM-2 cells) or not (U-266 and RPMI8226 cells) the t(4:14) translocation and on patient-derived MM cells (N = 26). In vivo effects of FGF blockade were investigated using subcutaneous (KMS-11 and RPMI8226 cells) and systemic (MM-1S cells) xenografts grown in NOD/SCID mice. In addition, gene expression profiling (GEP) and gene set enrichment analyses (GSEA) were performed in NSC12-treated MM cells. NSC12 blocked the proliferation of all human MM cell lines tested, causing inhibition of FGFR signaling, downregulation of the anti-apoptotic protein mcl-1 and caspase 3 activation. Cytofluorimetric analysis revealed a significant increase of apoptotic cells as early as 6 hrs after NSC12 treatment, with 100% of cell death after 24 hrs even when MM cells were co-cultured with patient-derived bone marrow stromal cells. In vivo, NSC12 blocked FGFR activation and reduced the growth of subcutaneous xenografts and MM cell dissemination in the BM. GEP and GSEA analyses of NSC12-treated cells revealed the upregulation of oxidative stress-induced genes and the downmodulation of c-Myc targets. Accordingly, NSC12 caused the rapid proteasomal degradation of c-Myc paralleled by GSH depletion, mitochondrial ROS production and depolarization, DNA damage and finally apoptosis in KMS-11 cells but not in KMS-11 cells expressing the undegradable mutated (T58A) form of c-Myc. Interestingly, these findings were confirmed on Bortezomib-resistant MM cells as well as on bone marrow-derived primary MM cells from newly diagnosed and relapsed/refractory patients, including plasma cells bearing the t(4;14) translocation obtained from high-risk MM patients. Our data reveal that FGF blockade triggers MM mitochondrial oxidative stress, DNA damage and apoptotic cell death via the proteasomal degradation of the c-Myc oncoprotein. Altogether, our findings dissect for the first time the mechanism by which the FGF/FGFR system plays a non-redundant role in MM cell survival and disease progression, and indicate that FGF targeting may represent a therapeutic approach for MM patients with poor prognosis and advanced disease stage. Citation Format: Roberto Ronca, Gaia Cristina Ghedini, Federica Maccarinelli, Antonio Sacco, Silvia Laura Locatelli, Eleonora Foglio, Sara Taranto, Elisabetta Grillo, Sara Matarazzo, Riccardo Castelli, Vanessa Desantis, Nadia Cattane, Annamaria Cattaneo, Marco Mor, Carmelo Carlo-Stella, Aldo Maria Roccaro, Marco Presta, Arianna Giacomini. FGF trapping impairs multiple myeloma growth through c-Myc degradation-induced mitochondrial oxidative stress [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C052. doi:10.1158/1535-7163.TARG-19-C052

Inhibition of Heme Oxygenase-1 Attenuates Bortezomib Resistance in Multiple Myeloma Via Down-Regulating Gas6 Production

Background Multiple myeloma (MM), one of the most prevalent hematological malignancies, is characterized by the proliferation of abnormal plasma cells. The proteasome inhibitor bortezomib is a frontline drug in the treatment of MM; however, most patients will develop acquired resistance to bortezomib, which has been a critical challenge in clinic. Our previous studies have indicated that inhibition of heme oxygenase-1 (HO-1) can attenuate drug resistance to MM. Moveover, emerging evidence suggests that growth arrest-specific gene 6 (Gas6) may regulate tumor microenvironment and serve as a novel target for cancer therapy. However, whether HO-1 mediated-Gas6 production involved in the development of bortezomib resistance in MM remains unclear. Aims The aims to perform the present study were to: 1) assess the association between intracellular HO-1 and Gas6 expression in the bone marrow of CD138+ cells of patients with MM; 2) test the role of HO-1 and Gas6 in the development of bortezomib resistance in MM; 3) explore whether HO-1 mediate Gas6 production and the potential mechanism. Methods Patients with different stages of MM (n=18) and healthy donors (n=18) at Affiliated Hospital of Guizhou Medical University were included in this study. Baseline clinical characteristic data and bone marrow samples were obtained from study participants. CD138+ plasma cells were purified from bone marrow samples. The recombinant lentiviral vector of HO-1 was constructed and subsequent transfected into U266 and RPMI8226 cells. The expressions of mRNA and protein levels were determined by real-time PCR and western blot, respectively. Secreted Gas6 was determined by ELISA. Flow cytometry was used to assess cell apoptosis. Results In human study, the HO-1 mRNA and protein level in CD138+ cells of patients with MM showed a significant increase compared with those in healthy group, respectively (P<0.01). With the development of MM, HO-1 mRNA and protein level were gradually increased from stage I to III. The similar findings were also observed in the expression of Gas6 mRNA and protein level (P<0.01). Interestingly, spearman correlation analysis showed that higher HO-1 mRNA and protein level were associated with increased Gas6 mRNA and protein level, respectively (P<0.05). To test whether HO-1 mediate Gas6 production, recombinant lentivirus-HO-1 vector and HO-1 siRNA were transfected into U266 and RPMI8226 cells to up/down-regulate HO-1 expression. Compared with control group, Gas6 mRNA and protein expression were significantly increased when U266 and RPMI8226 cells were up-regulated by recombinant lentivirus-HO-1 vector. Additionally, Gas6 mRNA and protein expression were also increased by the use of hemin (HO-1 inducer). In contrast, Gas6 expression presented a decrease in mRNA and protein level with the stimulation of HO-1 siRNA and HO-1 inhibitor ZnPPIX. Similar significantly trend was also observed in the culture medium. Moreover, the effect of HO-1 on bortezomib resistance in MM cells was markedly inhibited by the stimulation with Gas6 siRNA and Gas6 neutralizing antibody (the apoptosis rate in MM cells treated with PBS, Gas6 siRNA and Gas6 neutralizing antibody is 28%, 56% and 42%, respectively; P<0.01). These results indicated that HO-1 up-regulated Gas6 production in the development of bortezomib resistance in MM. Furthermore, to explore the potential mechanism how HO-1 regulates Gas6 production, MAPK, an important pathway in drug resistance, was investigated. U266 or RPMI8226 cells transfected with recombinant lentiviral vector of HO-1 were stimulated with p38MAPK inhibitor SB203580, the MEK inhibitor PD0325901, the JNK inhibitor SP600125, the PI3K inhibitor LY294002, and the STAT3 inhibitor S3I-201, and the HO-1 level of cells and Gas6 levels of cells and culture medium were determined. Findings showed that HO-1 level did not significantly change, while Gas6 expression in cells and Gas6 level in culture medium showed markedly decrease by the treatment with MAPK pathway inhibitor compared with control group. These results indicated that HO-1 mediated Gas6 production via MAPK pathway. Conclusion These results demonstrate that inhibition of HO-1 attenuates bortezomib resistance in MM via down-regulating Gas6 production. Targeting these pathological interactions holds promise for improve MM treatment. Disclosures No relevant conflicts of interest to declare.

Proteasome inhibitor induced SIRT1 deacetylates GLI2 to enhance hedgehog signaling activity and drug resistance in multiple myeloma.

Multiple myeloma (MM) is still incurable despite the successful application of proteasome inhibitors in clinic. Bortezomib represents the most common chemotherapy for MM, whereas acquired drug resistance and eventually developed relapse remain the major obstruction. In the current study, we established bortezomib-resistant myeloma cell lines and screened gene expression profiles using single cell RNA-sequencing. Resistant MM cells exhibited increased clonogenic potential, specific metabolic, and epigenetic signatures, along with the self-renewal signaling characteristic of MM stem-like cells. Aberrant activation of hedgehog (Hh) signaling was correlated with drug resistance and stem cell-like transcriptional program. The key transcriptional factor GLI2 of the Hh pathway was restricted in the high acetylation and low ubiquitination states in bortezomib-resistant myeloma cells. Further investigation revealed that SIRT1 deacetylates and stabilizes GLI2 protein at lysine 757 and consequentially activates the Hh signaling, and itself serves as a direct target of Hh signaling to format a positive regulating loop. Using combination screening with an epigenetic compound library, we identified the SIRT1 specific inhibitor S1541 and S2804 had very obvious synergetic antimyeloma effect. Sirt1 inhibition could partially impeded the Hh pathway and conferred bortezomib sensitivity in vitro and in vivo. Notably, elevated SIRT1 level was also a prominent hallmark for the resistant myeloma cells, and this expression pattern was confirmed in myeloma patients, but independent of RAS/RAF mutations. Clinically, SIRT1 expression in patients with complete response was suppressed but elevated in relapsed patients, and retrospective analysis showed patients with higher SIRT1 expression had poorer outcomes. In conclusion, the cooperation of SIRT1 and Hh is an important mechanism of drug resistance in myeloma, and therapeutics combining SIRT1 inhibitors will sensitize myeloma cells to proteasome inhibitors.

INHIBITION OF SPHINGOSINE KINASE 2 RESENSITISES BORTEZOMIB-RESISTANT MULTIPLE MYELOMA

Multiple myeloma, a haematological malignancy of the plasma cell, is still largely incurable. The introduction of proteasome inhibitors such as bortezomib has significantly improved patient survival, however bortezomib resistance is still a major hurdle which requires new therapeutic options. One such option may be inhibitors to sphingolipid metabolism. Sphingolipids are a family of lipids which can act as signalling molecules, influencing cellular pathways such as apoptosis. The enzyme sphingosine kinase is an especially attractive target in cancer, as it converts pro-apoptotic ceramide and sphingosine to pro-survival sphingosine-1-phosphate. We have previously shown that inhibition of sphingosine kinase 2 with K145 causes synergistic cell death when combined with bortezomib in bortezomib-naive myeloma cells, and thus we decided to test this combination in the bortezomib-resistant setting. To do so, bortezomib-resistant myeloma cell lines were generated and analysed. A 5TGM1 cell line generated to be resistant to bortezomib was also found to be resistant to carfilzomib, demonstrating cross-resistance that has also been seen in the clinic. Notably, these cells were found to possess a clinically-relevant mutation in the bortezomib-binding site of proteasomal subunit PSMB5. The SK2 inhibitor K145, when combined with either bortezomib or carfilzomib, caused synergistic cell death in the bortezomib-resistant cell lines tested. This was preceded by a significant increase in ER stress markers, as measured by Western blot, suggesting that this cell death may be induced by enhanced ER stress. When tested in vivo in a highly aggressive, bortezomib-resistant myeloma model, the combination of bortezomib and K145 gave a significant decrease in disease burden and increase in survival, suggesting this combination may be effective in bortezomib-resistant patients.

Curcumin Increases the Chemosensitivity of Multiple Myeloma to Bortezomib by Inhibiting the Notch1 Signaling Pathway

To evaluated the effect of curcumin on the bortezomib-resistant myeloma cells and the expression of Notch1 signaling pathway, in order to further explore its potential mechanism. Curcumin, bortezomib, and curcumin combined bortezomib were added into RPMI 8266, U266, 5 nmol/L bortezomib-resistant RPMI 8266 (RPMI 8226-V5R), 5 nmol/L bortezomib-resistant U 266 (U266-V5R) and CD138+ plasma cells respectively. The cell proliferation was measured by MTT assay. the apoptotic rate was determined by flow cytometry, and the Western blot was used to detect the expression of apoptosis-related proteins. Then, the expression of Notch1 in cells was inhibited by notch1 inhibitor DAPT and RNA interference, the above-motioned experiments should be repeated. Compared with single drug-treated groups, the treatment with 2 drugs could further inhibit cell proliferation, induce apoptosis and enhance the inhibition effect on notch1 signaling pathway (P<0.05), while the inhibiting Notch1 signaling pathway could reduce cell proliferation and increase the expression of cleaved caspase-3. Curcumin can increase chemosensitivity of myeloma cells to bortezomib, this effect may be related to the inhibition of Notch1.

Inhibition of thioredoxin activates mitophagy and overcomes adaptive bortezomib resistance in multiple myeloma

BackgroundAlthough current chemotherapy using bortezomib (Velcade) against multiple myeloma in adults has achieved significant responses and even remission, a majority of patients will develop acquired resistance to bortezomib. Increased thioredoxin level has been reported to be associated with carcinogenesis; however, the role of thioredoxin in bortezomib drug resistance of myeloma remains unclear.MethodsWe generated several bortezomib-resistant myeloma cell lines by serially passaging with increased concentrations of bortezomib over a period of 1.5 years. Thioredoxin expression was measured by real-time PCR and western blot.ResultsThe role of thioredoxin in the survival of bortezomib-resistant myeloma cells was determined by specific shRNA knockdown in vitro and in vivo. Thioredoxin inhibitor (PX12) was used to determine the effectiveness of thioredoxin inhibition in the treatment of bortezomib-resistant myeloma cells. The effect of thioredoxin inhibition on mitophagy induction was examined. The correlation of thioredoxin expression with patient overall survival was interrogated. Thioredoxin expression was significantly upregulated in bortezomib-resistant myeloma cells and the change correlated with the increase of bortezomib concentration. Thioredoxin gene knockdown using specific shRNA sensitized bortezomib-resistant myeloma cells to bortezomib efficiency in vitro and in vivo. Similarly, pharmacological inhibition with PX12 inhibited the growth of bortezomib-resistant myeloma cells and overcame bortezomib resistance in vitro and in vivo. Furthermore, inhibition of thioredoxin resulted in the activation of mitophagy and blockage of mitophagy prevented the effects of PX12 on bortezomib-resistant myeloma cells, indicating that mitophagy is the important molecular mechanism for the induction of cell death in bortezomib-resistant myeloma cells by PX12. Moreover, inhibition of thioredoxin resulted in downregulation of phosphorylated mTOR and ERK1/2. Finally, thioredoxin was overexpressed in primary myeloma cells isolated from bortezomib-resistant myeloma patients and overexpression of thioredoxin correlated with poor overall survival in patients with multiple myeloma.ConclusionsOur findings demonstrated that increased thioredoxin plays a critical role in bortezomib resistance in multiple myeloma through mitophagy inactivation and increased mTOR and ERK1/2 phosphorylation. Thioredoxin provides a potential target for clinical therapeutics against multiple myeloma, particularly for bortezomib-resistant/refractory myeloma patients.

Abstract A136: KDM5A activity inhibition by 2-PCPA: overcoming tolerance to bortezomib

Abstract Purpose: The proteasome inhibitor bortezomib has shown impressive clinical activity alone and in combination with conventional and other novel agents for myeloma treatment. Although bortezomib is known to be a selective proteasome inhibitor, the downstream mechanisms of cytotoxicity and drug resistance remain to be understood. However, resistance to bortezomib as a single agent develops in the majority of patients, and activity in other malignancies has been less impressive. To overcome bortezomib resistance, we compared differential gene expression profiles of bortezomib-resistant and bortezomib-sensitive IM-9 cell lines in response to bortezomib, and expressions of KDM5A genes were significantly increased. Methods: The differential gene expression profiles of bortezomib-resistant IM-9 and bortezomib-sensitive IM-9 cell lines in response to bortezomib were performed using Affymetrix GeneChip. To confirm the results, real-time PCR and Western blot analysis were performed. Bortezomib-resistant IM-9 cells were treated with or without bortezomib and&amp;lt;/&amp;gt;or histone demethylase inhibitor, trans-2-phenylcyclopropylamine hydrochloride (2-PCPA). Moreover, level of histone methylation was examined by Western blot analysis. Results: At concentrations that effectively inhibited proteasome activity (maximum dose with 100nM), bortezomib induced cell death in bortezomib-sensitive IM-9 cells, but not in bortezomib-resistant IM-9. In comparison of differential gene expression profiles between bortezomib-resistant IM-9 and bortezomib-sensitive IM-9 cells, we showed overexpression of KDM5A, which is associated with chromatin-mediated reversible drug-tolerant state, Moreover, 2-PCPA overcomes bortezomib resistance in a dose-dependent manner. The histone demethylation at H3K4, H3K9, and H3K27 was suppressed with 2-PCPA. Conclusion: Histone lysine-specific demethylase inhibitor, 2-PCPA, with bortezomib may be useful for overcoming bortezomib resistance in myeloma cells by suppression of KDM3A and KDM5A activities. Citation Format: Yasuhito Terui, Yuji Mishima, Kiyohiko Hatake. KDM5A activity inhibition by 2-PCPA: overcoming tolerance to bortezomib [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A136.

Abstract A176: Quantitative parabolic optimization platform for the design of epigenetic-based drug combinations against bortezomib-resistant multiple myeloma

Abstract Proteasome inhibitor-based drug combinations have improved multiple myeloma median survival beyond 5 years. Multiple myeloma, however, remains incurable as patients inevitably relapse and become resistant to proteasome inhibitors, such as bortezomib. Thus, there is a need to identify improved drug combinations that may serve as later lines of treatment against bortezomib-resistant multiple myeloma. Epigenetic inhibitors have been explored in treating advanced multiple myeloma; however, clinical trials have shown mixed results. There is increasing evidence from these clinical trials that epigenetic-based therapies may see greater benefit as part of drug combinations rather than as monotherapies. Rationally identifying optimal epigenetic-based drug combinations against aggressive cancers, such as bortezomib-resistant multiple myeloma, however, remains a challenge. Utilizing a quantitative parabolic optimization platform (QPOP), optimal drug combinations against bortezomib-resistant multiple myeloma were identified. QPOP analysis uses quantifiable phenotypic outputs to identify significant effects amongst a set of drugs using a minimal number of experiments through parabolic response surface mapping. By comparing therapeutic response of bortezomib-resistant multiple myeloma cells to bortezomib-sensitive multiple myeloma cells as well as normal control cells, a series of drug combinations were identified from an initial pool of 129 FDA approved oncology drugs. QPOP does not rely on molecular mechanism modeling but rather uses experimental data to rationally identify optimal drug combinations. As a result, targetable molecular mechanisms of cancer progression can be identified without any previous assumptions or bias. Thus, QPOP revealed that drug resistance in multiple myeloma may involve specific epigenetic changes as identified by top-ranked epigenetic inhibitor containing drug combinations. While it is known that DNA hypermethylation accumulates during multiple myeloma progression, reversal of DNA hypermethylation by DNA methyltransferase inhibitors as monotherapies had no significant therapeutic effect in vitro or in vivo. DNA hypermethylation can be targeted, however, with the appropriate drug combinations to effectively treat in vitro and in vivo models of bortezomib-resistant multiple myeloma, as well as ex vivo primary multiple myeloma cells from a wide range of patients. QPOP analysis also revealed that optimal drug combinations for other classes of epigenetic inhibitors, such as HDAC inhibitors, change from naïve multiple myeloma to drug-resistant multiple myeloma. Thus, QPOP-based drug combination optimization not only revealed effective epigenetic-based drug combinations but also the molecular mechanisms by which epigenetic modifiers affect multiple myeloma disease progression. This work highlights the need for rational identification of different epigenetic-based drug combinations throughout the progression of multiple myeloma. Beyond multiple myeloma, this platform can be used to identify optimal drug combinations for other cancers as well as provide insight into therapeutically targetable molecular mechanisms that drive cancer progression in various cancer subtypes. Citation Format: Edward Kai-Hua Chow. Quantitative parabolic optimization platform for the design of epigenetic-based drug combinations against bortezomib-resistant multiple myeloma [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A176.

Truncated protein tyrosine phosphatase receptor type O suppresses AKT signaling through IQ motif containing GTPase activating protein 1 and confers sensitivity to bortezomib in multiple myeloma.

Proteasome inhibitors are an important part of our chemotherapeutic armamentarium against multiple myeloma, but the vast majority of patients eventually develop drug-resistant disease through incompletely understood mechanisms. Comparison of gene expression profiles (GEPs) of bortezomib-resistant (BR) myeloma cell lines with their drug-naïve counterparts revealed decreased expression of truncated Protein tyrosine phosphatase receptor-type O (PTPROt) in BR cells. Over-expression of wild-type PTPROt in drug-naïve and BR cells reduced myeloma cell proliferation, induced apoptosis, and sensitized cells to bortezomib and to alkylating agents. PTPROt expression reduced AKT phosphorylation and activity, and sensitized to pharmacologic AKT pathway inhibitors, but this was not the case for a substrate-trapping catalytic domain-inactivating mutant. Co-immunoprecipitation and mass spectrometry studies identified IQ motif containing GTPase activating protein 1 (IQGAP1) as a PTPROt binding partner, and PTPROt reduced tyrosine phosphorylation of IQGAP1, providing a link to AKT activity. Analysis of clinically annotated GEP databases identified high PTPROt expression as being related to an increased likelihood of achieving complete remission with bortezomib therapy, while low expression was linked to a greater likelihood of disease progression. Finally, high PTPROt expression associated with prolonged median overall survival in patients receiving bortezomib-based therapy in the front-line or relapsed and/or refractory settings. Taken together, these data identify PTPROt suppression as a novel mechanism of myeloma resistance to bortezomib in myeloma cell lines, and also support the possibility that PTPROt expression could be used as a biomarker to predict outcomes with bortezomib, and by which to select patients for therapy with AKT inhibitors.

Hsa-miR-631 resensitizes bortezomib-resistant multiple myeloma cell lines by inhibiting UbcH10.

Although bortezomib (BTZ) remains a first-line agent for multiple myeloma (MM) therapy, the development of BTZ resistance has become an indicator of poor prognosis in MM patients. It is thus urgent to develop strategies to restore the vulnerability of MM to BTZ. This study demonstrated, for the first time, that UbcH10 is highly expressed in BTZ-resistant myeloma cell lines U-266/BTZ, NCI-H929/BTZ and RPMI-8226/BTZ, which is attributed to the inactivation of post-transcriptional control. The in-depth study revealed that during the development of BTZ resistance in these cells, the hsa-miR-631 levels were decreased, which resulted in the increased expression of the target gene UbcH10. We also found that the multiple drug-resistant protein MDR1 exhibited a positive correlation with UbcH10 due to the reduced ubiquitination of MDR1, which was caused by high UbcH10 expression. Following overexpression of miR-631, both BTZ sensitivity and BTZ-induced apoptosis were enhanced in the resistant cells. Meanwhile, resensitization by miR-631 overexpression was blocked by exogenous expression of UbcH10, which was not regulated by intracellular miR-631. In conclusion, the miR-631/UbcH10/MDR1 pathway is closely associated with the development of BTZ resistance in myeloma cells, and the overexpression of miR-631 can significantly improve BTZ sensitivity in resistant myeloma cells.