Drug Resistance In Multiple Myeloma Research Articles

Preclinical Evaluation of the First in Class BMI1 Inhibitor PTC-028 Confirms Potent Efficacy to Target BMI1-Addiction in Multiple Myeloma

Background:Treatment modalities for multiple myeloma (MM) patients suffering from refractory or high-risk disease are still limited. Although translational research efforts previously identified key players and promising drug targets in MM biology (e.g. IRF4, MYC, FOXM1), direct modulation of these factors is impeded by the lack of clinically effective inhibitors. Initially linked to the pathogenesis of MM more than a decade ago, the polycomb group protein BMI1 represents a prominent intrinsic driver of MM without a suitable clinical grade inhibitor. We recently confirmed BMI1 overexpression as a hallmark of MM and proved its essential role in MM cell survival using a small molecule inhibitor that affects BMI1 transcript levels (PTC209). Based on these observations, here, we explored the anti-myeloma efficacy of the first in class BMI1 protein inhibitor PTC028 and confirmed its applicability to target a so far not drug-able key molecule in MM.Methods:The anti-MM efficacy of PTC028 (kindly provided by PTC Therapeutics Inc.) was studied in vitro using human MM cell lines (HMCLs) and primary MM cells. Candidate gene expression levels were analyzed by quantitative PCR (qPCR), Western Blot and/or flow cytometry (FC). Mechanistic studies were conducted via FC and immunoblot experiments. Confirmatory studies are employed using lentiviral transduction of BMI1 overexpression or knockout plasmids, respectively.Results:PTC028 induced a time-dependent reduction of BMI1 protein levels in HMCLs. Evaluation of the in vitro efficacy demonstrated potent anti-MM activity in all primary MM cells and HMCLs tested (median IC50: 39 nM, range: 11-102 nM). This activity persisted in the presence of bone marrow stromal cells and in proteasome inhibitor (PI) resistant HMCLs. Importantly, median IC50 values were significantly reduced compared to PTC209 (median IC50 680 nM, P <0.05) confirming superior activity of PTC028. We also observed a significant correlation between PTC028 IC50 values and the reduction of BMI1 protein levels in HMCLs (R=0.86, P=0.01), suggesting that a rapid drop of BMI1 levels could serve as predictive marker for treatment efficacy.Mechanistically, treatment with PTC028 led to an accumulation of cells in the G2/M phase of the cell cycle and induction of apoptosis (verified by the presence of cleaved PARP and depolarization of the mitochondrial membrane potential). To better segregate the sequence of these events we performed time course experiments with U266 and KMS-12-BM cells. This demonstrated rapid induction of G2/M cell cycle arrest (evident 6h post treatment) and subsequent induction of apoptosis. Western Blot analysis demonstrated accumulation of CDK1 and Cyclin B1 in line with the G2/M cell cycle stop. In addition, we evaluated MCL1 protein levels due to its known degradation during mitotic arrest and indeed observed a time dependent downregulation of this key MM survival factor. In contrast, neither BIM nor BAX were found to be deregulated. Further validation of these results was obtained by qPCR analysis. PTC028 induced rapid upregulation of mitosis associated genes (CCNB1, AURKA, BIRC5) 6h and 24h post treatment indicating mitotic arrest which was accompanied by MCL1 downregulation. No impact was found on the gene expression levels of BMI1 and other cell cycle (CCND1, CDKN2A, CDKN1A, CDKN1B) or apoptosis (BAX, BIM, NOXA) related genes.Due to the close interaction between BMI1, MYC and FOXM1 we were finally interested whether PTC028 also affects these key factors. Intriguingly, PTC028 treatment led to rapid downregulation of both proteins suggesting that targeting of BMI1 via PTC028 offers a novel strategy to attack this MM high-risk network.Conclusion:This study demonstrates potent preclinical activity of the first in class BMI1 inhibitor PTC028 in a panel of HMCLs, primary MM cells, PI-resistant HMCLs and in the presence of BMSCs. This suggests efficacy in the context of the MM microenvironment and drug resistance. Moreover, our data point to significant impact on a currently not targetable high-risk network consisting of BMI1, MYC and FOXM1. These results strongly support further evaluation of PTC028 in the clinical setting. Detailed results about the impact of PTC028 on BMI1 associated signaling networks as well as preliminary in vivo efficacy results will be provided at the meeting. DisclosuresWeetall:PTC Therapeutics: Employment. Branstrom:PTC Therapeutics: Employment. Zojer:Celgene, Janssen, Amgen, Takeda, BMS: Consultancy, Honoraria, Research Funding.

SMAD1 as a biomarker and potential therapeutic target in drug-resistant multiple myeloma

BackgroundSMAD1, a central mediator in TGF-β signaling, is involved in a broad range of biological activities including cell growth, apoptosis, development and immune response, and is implicated in diverse type of malignancies. Whether SMAD1 plays an important role in multiple myeloma (MM) pathogenesis and can serve as a therapeutic target are largely unknown.MethodsMyeloma cell lines and primary MM samples were used. Cell culture, cytotoxicity and apoptosis assay, siRNA transfection, Western blot, RT-PCR, Soft-agar colony formation, and migration assay, Chromatin immunoprecipitation (Chip), animal xenograft model studies and statistical analysis were applied in this study.ResultsWe demonstrate that SMAD1 is highly expressed in myeloma cells of MM patients with advanced stages or relapsed disease, and is associated with significantly shorter progression-free and overall survivals. Mechanistically, we show that SMAD1 is required for TGFβ-mediated proliferation in MM via an ID1/p21/p27 pathway. TGF-β also enhanced TNFα-Induced protein 8 (TNFAIP8) expression and inhibited apoptosis through SMAD1-mediated induction of NF-κB1. Accordingly, depletion of SMAD1 led to downregulation of NF-κB1 and TNFAIP8, resulting in caspase-8-induced apoptosis. In turn, inhibition of NF-κB1 suppressed SMAD1 and ID1 expression uncovering an autoregulatory loop. Dorsomorphin (DM), a SMAD1 inhibitor, exerted a dose-dependent cytotoxic effect on drug-resistant MM cells with minimal cytotoxicity to normal hematopoietic cells, and further synergized with the proteasomal-inhibitor bortezomib to effectively kill drug-resistant MM cells in vitro and in a myeloma xenograft model.ConclusionsThis study identifies SMAD1 regulation of NF-κB1/TNFAIP8 and ID1-p21/p27 as critical axes of MM drug resistance and provides a potentially new therapeutic strategy to treat drug resistance MM through targeted inhibition of SMAD1.

JunB is a key regulator of multiple myeloma bone marrow angiogenesis

Bone marrow (BM) angiogenesis significantly influences disease progression in multiple myeloma (MM) patients and correlates with adverse prognosis. The present study shows a statistically significant correlation of the AP-1 family member JunB with VEGF, VEGFB, and IGF1 expression levels in MM. In contrast to the angiogenic master regulator Hif-1α, JunB protein levels were independent of hypoxia. Results in tumor-cell models that allow the induction of JunB knockdown or JunB activation, respectively, corroborated the functional role of JunB in the production and secretion of these angiogenic factors (AFs). Consequently, conditioned media derived from MM cells after JunB knockdown or JunB activation either inhibited or stimulated in vitro angiogenesis. The impact of JunB on MM BM angiogenesis was finally confirmed in a dynamic 3D model of the BM microenvironment, a xenograft mouse model as well as in patient-derived BM sections. In summary, in continuation of our previous study (Fan et al., 2017), the present report reveals for the first time that JunB is not only a mediator of MM cell survival, proliferation, and drug resistance, but also a promoter of AF transcription and consequently of MM BM angiogenesis. Our results thereby underscore worldwide efforts to target AP-1 transcription factors such as JunB as a promising strategy in MM therapy.

AcidoCEST MRI Evaluates the Bone Microenvironment in Multiple Myeloma.

Multiple myeloma (MM) is an incurable disease of malignant plasma cells in the bone marrow (BM). Adaptive responses to hypoxia may be an essential element in MM progression and drug resistance. This metabolic adaptation involves a decrease in extracellular pH (pHe), and it depends on the upregulation of glucose transporters (GLUTs) that is common in hypoxia and in cancer cells. CEST MRI is an imaging technique that assesses pHe indirectly by the exchange rate of magnetic saturation transfer between labile protons on a solute and water. Thus, this study aimed to determine the feasibility of acidoCEST MRI for pHe measurement using an orthotopic mouse model of MM compared with GLUT1 immunofluorescence staining as a reference. Orthotopic BM engrafted MM xenografts were established in NSG/NOD mice using the human RPMI8226 myeloma cell line. AcidoCEST MRI was performed approximately 6 weeks after intravenous challenge, before and after intravenous administration of iopamidol. BM pHe values were generated via fitting the CEST spectrum with the Bloch-McConnell equations. Samples were decalcified, sectioned, and immunostained for GLUT1 expression. Pearson's correlation was used to assess the relationship between pHe and [H3O+] versus GLUT1 expression. Ten mice underwent acidoCEST MRI followed by immunofluorescent histologic analysis. A strong negative correlation was seen between pHe versus GLUT1 expression (r = - 0.75, p < 0.001). After transformation of pH to [H3O+], a strong positive correlation between [H3O+] and GLUT1 expression was observed (r = 0.8, p < 0.001). AcidoCEST MRI can measure the extracellular pH of bone marrow affected by multiple myeloma. In this MM orthotopic mouse model, pHe measured by acidoCEST MRI showed strong correlations with the metabolic phenotype of BM tumor assessed by immunofluorescent histological assessment of GLUT1 overexpression.

HMGB1 enhances chemotherapy resistance in multiple myeloma cells by activating the nuclear factor-κB pathway.

Chemotherapy resistance is a main obstacle in the clinical chemotherapeutic treatment of multiple myeloma (MM). High-mobility group box 1 (HMGB1) has been revealed to be associated with the sensitivity of MM cells to chemotherapy, but how HMGB1 regulates chemotherapy resistance in MM has yet to be fully elucidated. In the present study, the exact molecular mechanism underlying HMGB1-mediated drug resistance in MM was explored using three chemotherapy-resistant MM cells (RPMI8226/ADR, RPMI8226/BOR and RPMI8226/DEX) that were successfully established. Reverse transcription-quantitative polymerase chain reaction revealed that the three chemotherapy-resistant MM cells exhibited a higher release of HMGB1 compared with the parental RPMI8226 cells. Interference with endogenous HMGB1 increased the sensitivity of drug-resistant MM cells to chemotherapy, which was supported by the low IC50 value and the enlargement of cell apoptosis. Furthermore, short hairpin (sh)RNA-transfected MM cells showed an obvious elevation in phosphorylated (p)-IKKα/β, p-IκBα and p-p65 in whole cell lysate and/or nucleus, and treatment of nuclear factor (NF)-κB activator reversed the effect of shHMGB1-mediated cell viability and apoptosis in MM cells. In conclusion, HMGB1 regulates drug resistance in MM cells by regulating NF-κB signaling pathway, suggesting that HMGB1 has the potential to serve as a target for MM treatment.

Emerging Therapeutic Strategies to Overcome Drug Resistance in Multiple Myeloma.

Simple SummaryMultiple myeloma is a deadly blood cancer, but fortunately drug development has substantially prolonged the lifespan of patients to average more than a decade after diagnosis with optimal therapy. As a result, the population of patients living with multiple myeloma has grown considerably. Through its course, patients suffer repeated relapses for which they require new lines of treatment. Currently, the key drug classes for treatment are immunomodulatory drugs, proteasome inhibitors, and monoclonal antibodies. The goal of this review is to summarize the understanding of the problem of resistance to these drugs, which is ultimately responsible for patient fatality. In addition, we will focus on how new agents that are promising in clinical trials overcome resistance.Multiple myeloma is a malignant plasma cell neoplasm that remains incurable and is ultimately fatal when patients acquire multi-drug resistance. Thus, advancing our understanding of the mechanisms behind drug resistance in multi-relapsed patients is critical for developing better strategies to extend their lifespan. Here, we review the understanding of resistance to the three key drug classes approved for multiple myeloma treatment: immunomodulatory drugs, proteasome inhibitors, and monoclonal antibodies. We consider how the complex, heterogenous biology of multiple myeloma may influence the acquisition of drug resistance and reflect on the gaps in knowledge where additional research is needed to improve our treatment approaches. Fortunately, many agents are currently being evaluated preclinically and in clinical trials that have the potential to overcome or delay drug resistance, including next-generation immunomodulatory drugs and proteasome inhibitors, novel small molecule drugs, chimeric antigen receptor T cells, antibody-drug conjugates, and bispecific antibodies. For each class, we discuss the potential of these strategies to overcome resistance through modifying agents within each class or new classes without cross-resistance to currently available drugs.

Targeting the GCK pathway: a novel and selective therapeutic strategy against RAS-mutated multiple myeloma

In multiple myeloma (MM), frequent mutations of NRAS, KRAS, or BRAF are found in up to 50% of newly diagnosed patients. The majority of the NRAS, KRAS, and BRAF mutations occur in hotspots causing constitutive activation of the corresponding proteins. Thus, targeting RAS mutation in MM will increase therapeutic efficiency and potentially overcome drug resistance. We identified germinal center kinase (GCK) as a novel therapeutic target in MM with RAS mutation. GCK knockdown (KD) in MM cells demonstrated in vitro and in vivo that silencing of GCK induces MM cell growth inhibition, associated with blocked MKK4/7-JNK phosphorylation and impaired degradation of IKZF1/3, BCL-6, and c-MYC. These effects were rescued by overexpression of a short hairpin RNA (shRNA)-resistant GCK, thereby excluding the potential off-target effects of GCK KD. In contrast, overexpression of shRNA-resistant GCK kinase-dead mutant (K45A) inhibited MM cell proliferation and failed to rescue the effects of GCK KD on MM growth inhibition, indicating that GCK kinase activity is critical for regulating MM cell proliferation and survival. Importantly, the higher sensitivity to GCK KD in RASMut cells suggests that targeting GCK is effective in MM, which harbors RAS mutations. In accordance with the effects of GCK KD, the GCK inhibitor TL4-12 dose-dependently downregulated IKZF1 and BCL-6 and led to MM cell proliferation inhibition accompanied by induction of apoptosis. Here, our data identify GCK as a novel target in RASMut MM cells, providing a rationale to treat RAS mutations in MM. Furthermore, GCK inhibitors might represent an alternative therapy to overcome immunomodulatory drug resistance in MM.

Promising Anti-Mitochondrial Agents for Overcoming Acquired Drug Resistance in Multiple Myeloma

Multiple myeloma (MM) remains an incurable tumor due to the high rate of relapse that still occurs. Acquired drug resistance represents the most challenging obstacle to the extension of survival and several studies have been conducted to understand the mechanisms of this phenomenon. Mitochondrial pathways have been extensively investigated, demonstrating that cancer cells become resistant to drugs by reprogramming their metabolic assessment. MM cells acquire resistance to proteasome inhibitors (PIs), activating protection programs, such as a reduction in oxidative stress, down-regulating pro-apoptotic, and up-regulating anti-apoptotic signals. Knowledge of the mechanisms through which tumor cells escape control of the immune system and acquire resistance to drugs has led to the creation of new compounds that can restore the response by leading to cell death. In this scenario, based on all literature data available, our review represents the first collection of anti-mitochondrial compounds able to overcome drug resistance in MM. Caspase-independent mechanisms, mainly based on increased oxidative stress, result from 2-methoxyestradiol, Artesunate, ascorbic acid, Dihydroartemisinin, Evodiamine, b-AP15, VLX1570, Erw-ASNase, and TAK-242. Other agents restore PIs’ efficacy through caspase-dependent tools, such as CDDO-Im, NOXA-inhibitors, FTY720, GCS-100, LBH589, a derivative of ellipticine, AT-101, KD5170, SMAC-mimetics, glutaminase-1 (GLS1)-inhibitors, and thenoyltrifluoroacetone. Each of these substances improved the efficacy rates when employed in combination with the most frequently used antimyeloma drugs.

Targeting NSD2-mediated SRC-3 liquid\u2013liquid phase separation sensitizes bortezomib treatment in multiple myeloma

Development of chemoresistance is the main reason for failure of clinical management of multiple myeloma (MM), but the genetic and epigenetic aberrations that interact to confer such chemoresistance remains unknown. In the present study, we find that high steroid receptor coactivator-3 (SRC-3) expression is correlated with relapse/refractory and poor outcomes in MM patients treated with bortezomib (BTZ)-based regimens. Furthermore, in immortalized cell lines, high SRC-3 enhances resistance to proteasome inhibitor (PI)-induced apoptosis. Overexpressed histone methyltransferase NSD2 in patients bearing a t(4;14) translocation or in BTZ-resistant MM cells coordinates elevated SRC-3 by enhancing its liquid–liquid phase separation to supranormally modify histone H3 lysine 36 dimethylation (H3K36me2) modifications on promoters of anti-apoptotic genes. Targeting SRC-3 or interference of its interactions with NSD2 using a newly developed inhibitor, SI-2, sensitizes BTZ treatment and overcomes drug resistance both in vitro and in vivo. Taken together, our findings elucidate a previously unrecognized orchestration of SRC-3 and NSD2 in acquired drug resistance of MM and suggest that SI-2 may be efficacious for overcoming drug resistance in MM patients.

DNA Damage Response in Multiple Myeloma: The Role of the Tumor Microenvironment.

Simple SummaryMultiple myeloma (MM) is a plasma cell malignancy. Novel therapies have improved outcomes in MM patients; however, MM still remains incurable. MM cells present genomic instability, whose molecular basis is not fully understood. Recently, it has been reported that the DNA damage response (DDR) may influence genomic changes and drug resistance in MM. An abnormal DNA repair function may provide an alternative explanation for genomic instability. In this review, we show an overview of the DNA repair pathways and discuss the role of the tumor microenvironment in governing the DNA repair mechanisms. The tumor microenvironment factors, such as hypoxia and inflammation, and chemotherapeutic agents profoundly influence the DNA repair pathways in MM.Multiple myeloma (MM) is an incurable plasma cell malignancy characterized by genomic instability. MM cells present various forms of genetic instability, including chromosomal instability, microsatellite instability, and base-pair alterations, as well as changes in chromosome number. The tumor microenvironment and an abnormal DNA repair function affect genetic instability in this disease. In addition, states of the tumor microenvironment itself, such as inflammation and hypoxia, influence the DNA damage response, which includes DNA repair mechanisms, cell cycle checkpoints, and apoptotic pathways. Unrepaired DNA damage in tumor cells has been shown to exacerbate genomic instability and aberrant features that enable MM progression and drug resistance. This review provides an overview of the DNA repair pathways, with a special focus on their function in MM, and discusses the role of the tumor microenvironment in governing DNA repair mechanisms.

The CXCL12gamma chemokine immobilized by heparan sulfate on stromal niche cells controls adhesion and mediates drug resistance in multiple myeloma

BackgroundThe survival and proliferation of multiple myeloma (MM) cells in the bone marrow (BM) critically depend on interaction with stromal cells expressing the chemokine CXCL12. CXCL12 regulates the homing to the BM niche by mediating the transendothelial migration and adhesion/retention of the MM cells. The gamma isoform of CXCL12 (CXCL12γ) has been reported to be highly expressed in mouse BM and to show enhanced biological activity compared to the ‘common’ CXCL12α isoform, mediated by its unique extended C-terminal domain, which binds heparan sulfate proteoglycans (HSPGs) with an extraordinary high affinity. Here, we investigated the expression of CXCL12γ in human BM and studied its functional role in the interaction of MM cells with BM stromal cells (BMSCs).MethodsWe assessed CXCL12γ mRNA and protein expression by human BMSCs using qPCR, flow cytometry, and immunohistochemistry. CRISPR-Cas9 was employed to delete CXCL12γ and the heparan sulfate (HS) co-polymerase EXT1 in BMSCs. To study the functional roles of BMSC-derived CXCL12γ and HSPGs in the interaction of MM cells with BMSCs cells, MM cell lines and primary MM cells were co-cultured with BMSCs.ResultsWe observed that CXCL12γ is expressed in situ by reticular stromal cells in both normal and MM BM, as well as by primary BMSC isolates and BMSC lines. Importantly, upon secretion, CXCL12γ, unlike the CXCL12α isoform, was retained on the surface of BMSCs. This membrane retention of CXCL12γ is HSPG mediated, since it was completely annulated by CRISPR-Cas9-mediated deletion of the HS co-polymerase EXT1. CXCL12γ expressed by BMSCs and membrane-retained by HSPGs supported robust adhesion of MM cells to the BMSCs. Specific genetic deletion of either CXCL12γ or EXT1 significantly attenuated the ability of BMSCs to support MM cell adhesion and, in addition, impaired their capacity to protect MM cells from bortezomib-induced cell death.ConclusionsWe show that CXCL12γ is expressed by human BMSCs and upon secretion is retained on their cell surface by HSPGs. The membrane-bound CXCL12γ controls adhesion of MM cells to the stromal niche and mediates drug resistance. These findings designate CXCL12γ and associated HSPGs as partners in mediating MM–niche interaction and as potential therapeutic targets in MM.

MiR-218 contributes to drug resistance in multiple myeloma via targeting LRRC28.

Multiple myeloma (MM) is a malignant neoplasm featured by obvious drug resistance and poor prognosis. MicroRNAs (miRNAs) are a class of small noncoding RNAs with crucial roles in many biological processes including cancer initiation and progression. The current study aims to investigate the pathogenic role and molecular mechanism of miRNAs in MM drug resistance. In the present study, The expression profile of miRNAs in MM samples was analyzed by microarray and real-time polymerase chain reaction. Protein expressions were detected by Western blot analysis. Cell apoptosis was detected by the Annexin V staining assay. The interaction between miRNA and the targeting mRNA was assessed using Dual luciferase reporter assay. Herein, we show that expression profile of miRNAs is deregulated in MM. miR-218, one of the most aberrational miRNAs in MM, is significantly decreased in MM cells compared to peripheral blood mononuclear cell (PBMC). Genetic manipulation reveals miR-218 control the response of MM cells to anticancer drug bortezomib (BTZ). Overexpression of miR-218 causes a significant aberrant genes expression including leucine rich repeat containing 28 (LRRC28). Mechanistic study shows that miR-218 control the drug response through mediating the expression of LRRC28 in MM cells. Overexpression of LRRC28 significantly reserves miR-218-mediated cell response to BTZ. Taken together, miR-218 is decreased in MM that contributes to BTZ resistance via targeting LRRC28, which might be used as a novel therapeutic target for multiple myeloma.

Signaling Pathway Mediating Myeloma Cell Growth and Survival.

Simple SummaryThe bone marrow (BM) microenvironment plays a crucial role in pathogenesis of multiple myeloma (MM), and delineation of the intracellular signaling pathways activated in the BM microenvironment in MM cells is essential to develop novel therapeutic strategies to improve patient outcome.The multiple myeloma (MM) bone marrow (BM) microenvironment consists of different types of accessory cells. Both soluble factors (i.e., cytokines) secreted from these cells and adhesion of MM cells to these cells play crucial roles in activation of intracellular signaling pathways mediating MM cell growth, survival, migration, and drug resistance. Importantly, there is crosstalk between the signaling pathways, increasing the complexity of signal transduction networks in MM cells in the BM microenvironment, highlighting the requirement for combination treatment strategies to blocking multiple signaling pathways.

Endocytic pathway inhibition attenuates extracellular vesicle-induced reduction of chemosensitivity to bortezomib in multiple myeloma cells

Extracellular vesicles (EVs), including exosomes and microvesicles, derived from bone marrow stromal cells (BMSCs) have been demonstrated as key factors in the progression and drug resistance of multiple myeloma (MM). EV uptake involves a variety of mechanisms which largely depend on the vesicle origin and recipient cell type. The aim of the present study was to identify the mechanisms involved in the uptake of BMSC-derived small EVs (sEVs) by MM cells, and to evaluate the anti-MM effect of targeting this process.Methods: Human BMSC-derived sEVs were identified by transmission electron microscopy, nanoparticle tracking analysis, and western blot. The effects of chemical inhibitors and shRNA-mediated knockdown of endocytosis-associated genes on sEV uptake and cell apoptosis were analyzed by flow cytometry. The anti-MM effect of blocking sEV uptake was evaluated in vitro and in a xenograft MM mouse model.Results: sEVs derived from BMSC were taken up by MM cells in a time- and dose-dependent manner, and subsequently promoted MM cell cycling and reduced their chemosensitivity to bortezomib. Chemical endocytosis inhibitors targeting heparin sulphate proteoglycans, actin, tyrosine kinase, dynamin-2, sodium/proton exchangers, or phosphoinositide 3-kinases significantly reduced MM cell internalization of BMSC-derived sEVs. Moreover, shRNA-mediated knockdown of endocytosis-associated proteins, including caveolin-1, flotillin-1, clathrin heavy chain, and dynamin-2 in MM cells suppressed sEV uptake. Furthermore, an endocytosis inhibitor targeting dynamin-2 preferentially suppressed the uptake of sEV by primary MM cells ex vivo and enhanced the anti-MM effects of bortezomib in vitro and in a mouse model.Conclusion: Clathrin- and caveolin-dependent endocytosis and macropinocytosis are the predominant routes of sEV-mediated communication between BMSCs and MM cells, and inhibiting endocytosis attenuates sEV-induced reduction of chemosensitivity to bortezomib, and thus enhances its anti-MM properties.

Chidamide, a subtype-selective histone deacetylase inhibitor, enhances Bortezomib effects in multiple myeloma therapy.

Drug resistance is the major cause for disease relapse and patient death in multiple myeloma (MM). It is an urgent need to develop new therapies to overcome drug resistance in MM. Chidamide (CHI), a novel oral HDAC inhibitor targeting HDAC1, 2, 3 and 10, has shown potential therapeutic effect in MM. In this study, we determined that CHI exhibited significant anti-tumor effect on MM cells both in vitro and in vivo, which was positively correlated with the expression of HDAC1. Meanwhile, CHI enhanced Bortezomib (BTZ) effects synergistically in MM cells and a combination of CHI with BTZ induced myeloma cell apoptosis and G0/G1 arrest in vitro and in vivo. Mechanistically, the synergistic anti-tumor effect of CHI and BTZ was related with the increased production of reactive oxygen species (ROS) dependent DNA damage and the changes of cell apoptosis and cycle pathways. Our data indicate that CHI may be a suitable drug to sensitize BTZ in MM cells, which provides novel insight into the therapy for MM patients.

Natural Killer Cells Activity Against Multiple Myeloma Cells Is Modulated by Osteoblast-Induced IL6 and IL-10 Production

Natural killer (NK) cells are part of the innate arm of the immune system; as such NK cells can be activated rapidly to target virus-infected and tumor cells without prior sensitization. The human NK-92 cell line is among the most widely used NK cells in preclinical research studies and have also been approved for clinical applications. In this study we evaluated how osteoblasts (OSB), which are bone forming cells and a key cellular component of the bone marrow microenvironment, modulate the cytotoxic activity of NK-92 cells against the MM.1S multiple myeloma (MM) cell line. Previous studies have shown that OSB confer drug resistance in MM and other cancers that metastasize to the bone marrow. We report here that in the presence of OSB, NK-92 cells secreted less perforin and granzyme A, resulting in a concomitant decrease in MM.1S cells killing. The physical presence of OSB and NK-92 cells in the cultures also induced IL-6 and IL-10 production; two cytokines which are known to impair the NK cell immunity against multiple myeloma and other cancers. OSB supernatant also conferred cytoprotection to MM.1S, suggesting a dual mechanism by which OSB may modulate both NK and MM cells. As NK cells and their chimeric antigen receptor-modified versions become more widely used in the clinic, our results suggest that understanding the role of OSB as potential immunoregulators of the NK cell mediated cytotoxic response in the bone marrow tumor microenvironment may provide new opportunities for enhancing the activity of this potent immunotherapeutic approach. Funding Information: This study was supported by the National Institutes of Health, grant#: 1R33CA212806-01A1. Declaration of Interests: The authors declare that they have no conflict of interest.

Tellurium compound provides pro-apoptotic signaling in drug resistant multiple myeloma

Multiple Myeloma, effectively treated by chemotherapeutic drugs, relapses due to drug resistance. We tested here the capacity of mesenchymal stromal cells, from the bone marrow of patients or from adipose tissue of healthy individuals, to induce drug resistance in Myeloma cell lines. We show that drug resistance can be achieved by factors secreted by the various MSC’s. Mass spectrometry analysis of MSC’s conditioned media revealed that fibronectin, was particularly instrumental in providing anti-apoptotic signals to MM cells. Moreover, we demonstrate that SAS ([octa-O-bis-(R,R)tartarate ditellurane]), an immunomodulator Tellurium compound, is not only able of blocking the physical interaction between MM cells and fibronectin but is also capable of re-sensitizing the cells to the chemotherapeutic drugs. Finally, we show that this re-sensitization is coupled with the blocking of pAKT induction, in MM cells, by the MSC’s. These results indicate that SAS may be useful in the treatment of drug resistant MM.

Tumor microenvironment-targeted nanoparticles loaded with bortezomib and ROCK inhibitor improve efficacy in multiple myeloma

Drug resistance and dose-limiting toxicities are significant barriers for treatment of multiple myeloma (MM). Bone marrow microenvironment (BMME) plays a major role in drug resistance in MM. Drug delivery with targeted nanoparticles have been shown to improve specificity and efficacy and reduce toxicity. We aim to improve treatments for MM by (1) using nanoparticle delivery to enhance efficacy and reduce toxicity; (2) targeting the tumor-associated endothelium for specific delivery of the cargo to the tumor area, and (3) synchronizing the delivery of chemotherapy (bortezomib; BTZ) and BMME-disrupting agents (ROCK inhibitor) to overcome BMME-induced drug resistance. We find that targeting the BMME with P-selectin glycoprotein ligand-1 (PSGL-1)-targeted BTZ and ROCK inhibitor-loaded liposomes is more effective than free drugs, non-targeted liposomes, and single-agent controls and reduces severe BTZ-associated side effects. These results support the use of PSGL-1-targeted multi-drug and even non-targeted liposomal BTZ formulations for the enhancement of patient outcome in MM.

A Novel Aryl-Guanidinium Based Compound Targets the STAT3 Signalling Pathway, Downregulates MCL-1 Expression and Induces Anti-Myeloma Activity

Multiple myeloma (MM) is a hematological malignancy characterized by the proliferation of malignant plasma cells in the bone marrow microenvironment. Increased understanding of the pathophysiology of MM and advances in therapeutics have led to an increased survival of patients in the past decade. However, despite the effectiveness of the first-line treatments, patients invariably relapse and become drug refractory due to genetic heterogeneity and induction of prosurvival signalling cascades in the bone marrow microenvironment. There is, therefore, a need for novel therapeutics which target key prosurvival signalling pathways in MM. The Janus kinase (JAK)/Signal transducer and activator of transcription 3 (STAT3) pathway in MM can be both constitutively active due to autonomous mutations or activated by growth and survival factors, such as interleukin-6 (IL-6), secreted by bone marrow stromal cells and other cells within the bone marrow microenvironment. STAT3 is upregulated in MM and increases the expression of genes involved proliferation, angiogenesis and evasion of apoptosis. STAT3 target genes include prosurvival BCL2 family members, such as MCL-1 which is overexpressed in MM, leading to apoptosis resistance and is associated with shorter patient survival. Several studies have shown promise in targeting prosurvival BCL2 family members with BCL2 homology domain 3 (BH3) mimetics in MM. We have previously described a member of a series of guanidinium based compounds which inhibits BRAF, a serine/threonine kinase component of the RAS/ERK signalling pathway, through a putative type-III allosteric mechanism [Diez-Cecilia et al, (2015) Bioorg. Med. Chem. Lett. 25, 4287-4292]. Here, we describe a new derivative of this series, VP79s, which exhibits cytotoxicity in MM cell lines with IC50 values in the low micromolar range; however, VP79s does not inhibit RAS/ERK signalling suggesting a novel mechanism of action. VP79s was found to induce apoptosis in NCI-H929 and U266B1 MM cell lines in a dose- and time-dependent manner. VP79s was more potent in inducing cell death in a panel of drug sensitive and drug resistant MM cell lines compared to standard and emerging therapeutic agents for MM. VP79s rapidly inhibited both constitutively active and IL-6-induced STAT3 signalling in MM cells. Examination of upstream mediators of STAT3 signalling suggested a mechanism for VP79s inhibition of STAT3 activation via inhibition of JAK2 phosphorylation and downregulation of the IL-6 receptors, CD126 and CD130. Analysis of downstream STAT3 targets demonstrated that treatment with VP79s resulted in a rapid time-dependent decrease in expression of MCL-1. VP79s was shown to overcome adhesion mediated drug resistance in MM cells in a co-culture model system with bone marrow stromal cells which induces MM cell resistance to treatment with the proteasome inhibitor bortezomib. Combining VP79s with bortezomib synergistically enhanced apoptosis in MM cells compared with single agent treatment. VP79s showed selective cytotoxicity in patient derived MM cells compared to normal donor lymphocytes and reduced the viability of MM patient samples in a dose-responsive manner suggesting its potential as an anti-MM therapeutic. In conclusion, the novel agent VP79s inhibits the JAK/STAT3 signalling pathway resulting in rapid downregulation of MCL-1 expression and inducing potent anti-MM activity. This mechanism may suggest a novel strategy for targeting MCL-1, an emerging target in MM. Disclosures No relevant conflicts of interest to declare.

Comprehensive Characterization of the Epigenetic Landscape in Multiple Myeloma

Multiple myeloma (MM) is a B cell neoplasia characterized by the accumulation of clonal plasma cells within the bone marrow. Epigenetic modifications, including DNA methylation and histone post-translational changes, are involved in MM development and progression, but also in drug resistance. We have derived a large cohort of patient-derived HMCLs that remain dependent on the addition of exogenous MM bone marrow growth factors, reflecting primary tumor conditions. We have described their molecular diversity by analyzing the gene expression profile and mutational landscape and have showed that HMCL molecular diversity reflects part of the molecular heterogeneity of primary MM cells. However, the epigenetic landscape of HMCLs has never been described. A comprehensive characterization of the epigenetic landscape of HMCLs would advance our understanding of MM pathophysiology and may attempt to identify new therapeutic targets. In our study, we presented the epigenetic landscape of HMCLs. We performed chromatin immunoprecipitation sequencing (ChIP-seq) to analyze changes of the histone marks (H3K9me3 to follow heterochromatin, H3K4me1 and H3K27ac to follow enhancer activity, H3K4me3 and H3K36me3 to follow active transcription and H3K27me3 to follow Polycomb-silenced chromatin) on 16 HMCLs, representative of the molecular heterogeneity of MM. The differential analysis of histone modification profiles of HMCLs highlighted links between histone modifications and cytogenetic abnormalities or recurrent mutations. H3K4me3 and H3K27me3 profile analysis revealed specific clusters of HMCL related to 1q gain and t(4;14) translocation, respectively. These two cytogenetic abnormalities lead to deregulation of epigenetic player expression (e.g. SETDB1 and MMSET) and thus, could alter histone modification profile. Using histone modifications associated to enhancer regions (H3K4me1 and H3K27ac), we identified super-enhancers (SE) associated with genes involved in the biology of MM. 607 to 2510 predicted super-enhancers per HMCL were identified, including MAF, MYC, CCND1, CCND2, TRAF3 or NSD2. These super-enhancers differ from typical enhancers in both size and H3K4me1 and H3K27ac levels. The SE-associated genes identified in HMCLs with a prognostic value in two independent cohorts of newly diagnosed patients (CoMMpass cohort; N = 674 and Montpellier cohort; N = 69 with RNA-seq data) were used to build a score predicting MM patient outcome (Figure 1). Moreover, among the 28 genes that compose the risk-score (BSG, HK2, HNRNPC, HSPA9, IL10, ILF3, LDHB, MDH1, MYBPC2, NCL, NUDC, PARP1, PDIA6, PRPS1, RPL8, RPL13A, RPL27A, RPL35, SF3B2, SLC7A5, SLC25A39, SMARCA4, SPN, STC2, THY1, TNPO2, TPR), public datasets of RNAi and CRISPR/Cas9 screening revealed four genes (YWHAQ, IL10, HK2 and THY1) identified as significant essential myeloma genes, suggesting that they could represent potential therapeutic targets. We also identified promoters of genes characterized by a co-localization of H3K9me3 and H3K27me3 repressive marks in HMCLs. We evaluated the prognostic value of these genes in the CoMMpass and Montpellier cohorts, and selected genes associated with poor outcome when their expression is low in MM cells of patients (ARHGEF5, BIVM, DEF8, GRID2IP, HDAC9, HSPA1L, KDM4C, NLRP2, P4HA3, PAG1, PM20D1, RMND5A, SEMA6A, SFMBT2, THEMIS2, TPRKB, ZFP2 and ZNF5188B) underlining potential new tumor suppressor genes. These potential tumor suppressor genes associated with repressive histone marks were used to build a second risk score splitting MM patients in low- and high-risk groups in CoMMpass and Montpellier cohorts. Finally, we explored H3K4me3 marks comparing drug-resistant and -sensitive HMCLs (N = 16) to identify regions involved in drug resistance. From these data, we developed epigenetic biomarkers based on this H3K4me3 modification predicting lenalidomide and romidepsin HDCAi response. This study provides a comprehensive characterization of the MM epigenetic landscape representing unique resources for future biological studies and could help to identify novel critical epigenetic modifications involved in MM progression and drug resistance. Furthermore, risk-scores based on super enhancers and repressive regions together with epigenetic biomarkers of drug response could represent new tools for precision oncology in MM. Disclosures De Boussac: Diag2Tec: Current Employment. Bruyer:Diag2Tec: Current Employment. Vincent:janssen: Membership on an entity's Board of Directors or advisory committees, Other: Congress support; Celgene: Membership on an entity's Board of Directors or advisory committees, Other: Congress support; takeda: Membership on an entity's Board of Directors or advisory committees, Other: Congress support. Moreaux:Diag2Tec: Consultancy.