Multidrug-resistant Multiple Myeloma Research Articles

Intracytoplasmatic BCMA Point Mutation and Teclistamab Resistance: Genomic Assessment of a Multidrug-Resistant Multiple Myeloma Patient

Introduction Proteasome inhibitors (PIs) are standard drugs for the treatment of Multiple Myeloma (MM), bispecific antibodies (BsAbs) and CART cells are rapidly and successfully being integrated into treatment regimens. Still, several current studies have shown that drug resistance remains one of the biggest clinical challenges. For instance, deletions in BCMA (alias TNFRSF17) were shown to confer a target loss and therefore resistance to BCMA CARTs (Da Via et al. Nat Med 2021, Samur et al. Nat Commun 2021) as well as to a CD3xBCMA bispecific T cell-engager (Truger et al. Blood Adv 2021). Besides the extracellular ligand-binding domain, BCMA further consist of a membrane spanning and a cytoplasmatic region that activates cell functions. To date not much is known in regard to alterations affecting the latter and their role in resistance. Here we present genetic profiling of a multi-refractory MM patient who relapsed to PIs, anti- BCMA (Teclistamab) and anti- GPRC5D (Talquetamab) BsAbs. Methods We performed FISH and a targeted capture-hybridization DNA sequencing panel (tchDNA-Seq) as previously described in Rosa-Rosa et al. Cancers 2022 in two sequential samples: first a bone marrow biopsy at diagnosis and second, 43 months later, a FFPE biopsy of the retroperitoneal mass of a progression plasmacytoma, after PI but prior Teclistamab treatment. The MM-specific tchDNA-Seq panel includes over 100 genes involved in MM progression, drug resistance, immunotherapy and canonical loci to cover IG rearrangements and frequent translocations. In order to predict the impact of the observed gene mutations on the protein structure, we generated 3D projections using the PyMOL molecular visualization system and references from the Protein Data Bank. The KinasePhos3 program was used to predict the most probable kinase to phosphorylate new, by the mutation generated, sites. Results Via FISH, the patient was stratified as a high-risk patient as 13% of the tumor cells showed a del17p at diagnosis, 97% a t(4;14) translocation, and 88% chromosomal gains for 1q21 and 11q13. The patient initially responded well to VRd+HCT. After 3.6 years he relapsed and developed an abdominal plasmacytoma. He was non-responsive to the following Isa-Kd treatment and relapsed again, two months after Teclistamab induction. To the subsequent PomCyDex and Talquetamab treatments he was primary refractory. In the plasmacytoma, four SNVs were detected: PSMC5 p.G152V with a VAF of 42%, BCMA p.C128Y (3%) and the two B2M mutations B2M p.S14F (47%) and B2M p.L107F (11%). At diagnosis, the VAF was 1% for PSMC5 p.G152V and B2M p.S14F, the other two SNVs were absent. In B2M, mutations in residues 13-16 have been associated with impaired protein expression and complex formation (Challa-Malladi et al. Cancer Cell 2011). The PSMC5 SNV was topologically identified within a glycine-rich loop in the ATP binding pocket of the proteasome in direct interaction with the ATP amine group, therefore likely impacting the proteolytic capacity increasing the PI tolerance. B CMA p.C128Y is located in the C-terminal cytoplasmatic tail within a highly conserved 25-aa protein segment (position 119 to 143) that has been shown to be necessary for TRAF association and NF-κB, Elk-1, and JNK activation (Hatzoglou et al. J Immunol 2000). A cysteine is mutated to a tyrosine. With high probability, this new site will be phosphorylated by kinases of the c-Src family (score 0.92), e.g. Fyn (0.71) or Lyn (0.66). Moreover, this new phosphotyrosine would become a binding site for the kinase SH2 domain (Diop et al. Int J Mol Sci 2022). Conclusions Here we report emerging mutations in PSMC5 and BCMA, that are likely causal for PI and Teclistamab resistance. PSMC5 p.G152 represents a mutational hotspot as four more PI-resistant patients with substitutions were recently reported in Haertle et al. Cancers 2023. The intracytoplasmatic BCMA SNV creates a new kinase phosphorylation and binding site resulting in aberrant downstream signaling. B2M forms part of the MHC1 molecules, responsible for antigen presentation to CD8+ T cells and the B2M serum level is a prognostic indicator in MM. However, the role of B2M alterations in disease progression and drug resistance needs to be elucidated. Predicting the biological impact based on 3D structural models remains speculative, thus, functional confirmation introducing the BCMA and B2M alterations in cell line models by genetic engineering is pending.

Developing Novel Targeted Therapies Using the High-Risk Vq Myeloma Model

Multiple myeloma (MM) is characterized by clonal expansion of malignant plasma cells (PCs) and aberrant production of monoclonal immunoglobulin detected as an M spike using serum protein electrophoresis. In the United States, MM represents ~15% of hematologic malignancies and is one of the few cancers increasing in incidence (e.g., 14,400 in 1996 to 32,270 in 2020, from SEER). Despite recent treatment advances, patients diagnosed with high risk/refractory MM (hrrMM) have an overall survival of only three years. Recently, our lab developed the first genetically engineered NrasQ61R; Myc-driven myeloma model (VQ mice). The VQ model recapitulates most biological and clinical features of hrrMM. VQ cells are serially transplantable into syngeneic recipients and can be propagated in vitro. As such, the VQ model could serve as a robust pre-clinical platform for drug screening and in vivo testing of new therapies to treat hrrMM. Whole exome sequencing (WES) of CD138+ cells from five primary VQ mice identified eight recurrently mutated genes. Of these eight genes, two have been previously identified as recurrently mutated in MM patients while the remainder appear to be unique to VQ myeloma. Meanwhile, RNA sequencing of CD138+ cells taken from both primary VQ mice and secondary VQ recipients show differential gene expression for five of these eight genes. Gene ontology analysis of recurrently mutated genes indicates that these genes play a role in interferon signaling, as well as receptor tyrosine kinase signaling. Here, we report the results of a high-throughput repurposing screen using the NCI's AOD IX library, which consists of 147 FDA-approved anti-cancer drugs. VQ cells were treated with 0.1 and 1µM of each compound in the presence or absence of 10nM of Trametinib (Tra), a potent MEK inhibitor. Screening results returned expected positive hits that were previously FDA approved for MM treatment (green dots, Panel 1B), such as proteasome inhibitors, histone deacetylase inhibitors, and chemotherapy drugs. Importantly, VQ cells were resistant to some compounds currently used to treat MM, including the BCL-2 inhibitor Venetoclax, the alkylating agent Melphalan, and the topoisomerase II inhibitor Etoposide (red dots, Panel 1B). De novo resistance to these therapies further indicates that the VQ cells model a multi-drug resistant MM. Two compounds were chosen for further testing: the protein translation inhibitor Omacetaxine (Oma) based on its single agent efficacy, and the multi-receptor tyrosine kinase inhibitor Ponatinib (Pot) based on its high synergy with Tra. Combination Index testing confirmed initial drug screening results but showed some antagonism between combination Oma and Pot treatment. We proceeded with in vivo testing of two novel combination therapies, in which 3-week treatment cycles were followed by a 1-week rest period between cycles: Three weeks of daily Tra and Pot treatment (TP), or two weeks of Tra and Pot treatment followed by one week of Oma (TPO) treatment. Vehicle (Veh) treated and Tra only treated mice were used as controls. Treatment efficacy was assessed against VQ-D1 MM, which grows primarily in the bone marrow (BM) and metastasizes to the spleen and liver of recipient mice. Four weeks post-treatment, myeloma growth in the BM was significantly reduced in both combo treatment groups, while MM burden in the spleen was also reduced. Overall, both TP and TPO treatment significantly extended survival compared to Veh or Tra treated mice, and significantly reduced liver extramedullary disease. To further test the range of Tra/Pot synergy in vivo, Tra dosage was reduced and treatment was administered daily. Interestingly, both low dose Tra monotherapy and TP combo therapy significantly extended survival compared to Veh control or Pot alone, and TP treated mice had increased survival compared to the Tra only treated mice. Taken together, our results show that low dose daily Trametinib treatment is superior to cycling dosage, and that Ponatinib treatment synergizes with both dosing schedules to extend survival in vivo. Our study demonstrates the utility of the VQ model for identifying new treatments for hrrMM and testing their efficacy in vivo. As part of our ongoing drug repurposing experiments, we have expanded our screening to a panel of 2,580 FDA-approved drugs, including but not limited to anti-cancer and anti-inflammatory drugs. Figure Disclosures Callander: Cellectar: Research Funding; University of Wisconsin: Current Employment.

Combined Inhibition of HDAC and AKT As a Strategy to Overcome Multi-Drug Resistance in Patients with Multiple Myeloma

Nowadays, patients with multiple myeloma (MM) have multiple choices of therapy including monoclonal antibodies, proteasome inhibitors, and immunomodulatory drugs (IMiDs), whereas some patients still develop resistance to these drugs and require novel therapeutic modalities. Here, we focused on inhibition of HDAC and AKT to overcome drug resistance. Lenalidomide (Len) selectively binds to cereblon (CRBN), which mediates recruitment of specific substrates like IKZF1 to E3 ubiquitin ligase and subsequent degradation, resulting in downregulation of IRF-4 and c-Myc. Then, we developed Len-resistant myeloma cells by RNAi-mediated downregulation of CRBN. Treatment of these cells with HDAC inhibitors reduced IKZF1 mRNA, suggesting potential efficacy of HDAC inhibitors against CRBN-low expressing or mutated MM. According to the integrated database for expression profile and disease prognosis (GenomicScape, http://www.genomicscape.com), higher expression of MICA was significantly associated with better overall survival in MM. MICA is an NK cell-activating ligand and plays an important role in ADCC. We observed that ADCC activity of both daratumumab and elotuzumab against MM cells was enhanced in the presence of HDAC inhibitors, which was compatible with our previous data that HDAC inhibitors upregulated MICA mRNA expression via inhibition of IKZF1 (ASH2018 abstract #4435). We also observed that HDAC inhibitors upregulated MICA mRNA in CRBN-deficient cells, suggesting promise of the combination of HDAC inhibitors and monoclonal antibodies against Len-resistant MM. Len-resistance is also affected by phosphorylation status of GSK-3. PI3K/AKT pathway is frequently activated in MM cells, and AKT inactivates GSK-3 by direct phosphorylation, resulting in c-Myc stabilization. Enhanced phosphorylation of GSK-3 was observed in CRBN-deficient H929 cells after long-term culture with Len, and such a phosphorylation status of GSK-3 was correlated with less CRBN amount and higher Len concentration (Figure 1). Afuresertib, an AKT inhibitor, suppressed GSK-3 phosphorylation (p-GSK-3) with or without ACY-1215, an HDAC inhibitor, leading to a substantial decrease of c-Myc (Figure 2). On the other hand, CHIR 99021, a GSK-3 inhibitor, partially counteracted to cytotoxic effect of afuresertib on H929 cells (Figure 3). These results suggest that increased p-GSK-3 is involved in acquired Len-resistance, and that combined inhibition of HDAC and AKT can overcome Len-resistance through decreased p-GSK-3. Furthermore, we examined the efficacy of CUDC-907, a dual HDAC and PI3K inhibitor. CUDC-907 had a cytotoxic effect on the MM cell lines including those had low CRBN expression. Bortezomib, doxorubicin, and dexamethasone resistant MM cell lines were also sensitive to CUDC-907. CUDC-907 upregulated MICA mRNA expression, but downregulated IKZF1 mRNA expression. Treatment of RPMI-8226 cells with CUDC-907 enhanced the ADCC activity of daratumumab (Figure 4). Furthermore, CUDC-907 was effective on primary MM cells which were resistant to bortezomib and Len (Figure 5). Thus, dual inhibition of HDAC and AKT with or without monoclonal antibodies is a promising therapeutic approach to multi-drug resistant MM. Disclosures Imai: Celgene: Honoraria, Research Funding; Janssen Parmaceutical K.K.: Honoraria, Research Funding; Bristol-Myers Squibb: Research Funding. Futami:Torii Pharmaceutical: Research Funding. Ri:Janssen Pharmaceutical: Honoraria, Research Funding; Takeda Pharmaceutical: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Daiichi Sankyo: Research Funding; Ono Pharmaceutical: Honoraria, Research Funding; Kyowa Kirin: Research Funding; Chugai Pharmaceutical: Research Funding; Sanofi: Honoraria, Research Funding; Abbvie: Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; MSD: Research Funding; Novartis Pharma: Research Funding; Gilead Sciences: Research Funding; Astellas Pharma: Research Funding; Teijin Pharma: Research Funding. Yasui:TokioTHERA Holdings, Inc.: Equity Ownership. Iida:Teijin Pharma: Research Funding; Celgene: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Takeda: Honoraria, Research Funding; Astellas: Research Funding; Gilead: Research Funding; Sanofi: Research Funding; MSD: Research Funding; Abbvie: Research Funding; Kyowa Kirin: Research Funding; Chugai: Research Funding; Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Daichi Sankyo: Honoraria, Research Funding. Tojo:Torii Pharmaceutical: Research Funding; AMED: Research Funding.

Pre-clinical evaluation of CD38 chimeric antigen receptor engineered T cells for the treatment of multiple myeloma.

Adoptive transfer of chimeric antigen receptor-transduced T cells is a promising strategy for cancer immunotherapy. The CD38 molecule, with its high expression on multiple myeloma cells, appears a suitable target for antibody therapy. Prompted by this, we used three different CD38 antibody sequences to generate second-generation retroviral CD38-chimeric antigen receptor constructs with which we transduced T cells from healthy donors and multiple myeloma patients. We then evaluated the preclinical efficacy and safety of the transduced T cells. Irrespective of the donor and antibody sequence, CD38-chimeric antigen receptor-transduced T cells proliferated, produced inflammatory cytokines and effectively lysed malignant cell lines and primary malignant cells from patients with acute myeloid leukemia and multi-drug resistant multiple myeloma in a cell-dose, and CD38-dependent manner, despite becoming CD38-negative during culture. CD38-chimeric antigen receptor-transduced T cells also displayed significant anti-tumor effects in a xenotransplant model, in which multiple myeloma tumors were grown in a human bone marrow-like microenvironment. CD38-chimeric antigen receptor-transduced T cells also appeared to lyse the CD38(+) fractions of CD34(+) hematopoietic progenitor cells, monocytes, natural killer cells, and to a lesser extent T and B cells but did not inhibit the outgrowth of progenitor cells into various myeloid lineages and, furthermore, were effectively controllable with a caspase-9-based suicide gene. These results signify the potential importance of CD38-chimeric antigen receptor-transduced T cells as therapeutic tools for CD38(+) malignancies and warrant further efforts to diminish the undesired effects of this immunotherapy using appropriate strategies.

PJ34, a poly(ADP-ribose) polymerase (PARP) inhibitor, reverses melphalan-resistance and inhibits repair of DNA double-strand breaks by targeting the FA/BRCA pathway in multidrug resistant multiple myeloma cell line RPMI8226/R.

There is still no ideal treatment for multidrug resistant multiple myeloma, looking for drugs which can reverse chemotherapy resistance and enhance curative effects of chemotherapy drugs becomes a problem that needs to be solved urgently. Poly(ADP-ribose) polymerase inhibitors appear to be an important tool for medical therapy of several malignancies. In the present study, we investigated the potential of the PARP-1 inhibitor PJ34, invitro, to further enhance the efficacy of the traditional chemotherapy drug melphalan in the multidrug-resistant multiple myeloma cell line RPMI8226/R. The effects of different concentrations of PJ34 and melphalan on cell proliferation were determined by the CCK-8 assay. The expressions of FA/BRCA pathway-related factors were detected by western blotting and RT-PCR. The percentage of cell apoptosis was measured with flow cytometry. DNA double-strand break (DSB) repair was quantified by γH2AX immunofluorescence. In addition, DNA damage repair at the level of the individual cell was determined by comet assay. Co-administration of PJ34 and melphalan had synergistic inhibitory effects on the proliferation of RPMI8226/R cells, suggesting more powerful antitumor activities. The apoptosis percentage also was increased more obviously by the treatment of melphalan plus PJ34. The activation of FA/BRCA pathway was inhibited by downregulation of related factors including FANCD2, BRCA2 and Rad51. PJ34 significantly increased the ratio of γH2AX-positive cells and the number of foci/cells. The comet tail rate of cells, tail length, tail moment and Olive tail moment all increased after PJ34 treatment in RPMI8226/R cells. These results indicate that PJ34 combined treatment with melphalan produces synergistic effects and reverses multidrug resistance of RPMI8226/R cells effectively. PJ34 cannot induce DNA damage directly, but it may increase the DNA damage induced by melphalan through inhibiting DNA damage repair. The suppression of FA/BRCA pathway may be the mechanism. Therefore, we suggest that PARP inhibitors may deserve future investigations as tools for medical treatment of multidrug resistant multiple myeloma.

Activity of CDK1/2 Inhibitor LCQ195 Against Multiple Myeloma Cells.

Context: Conceptually, targeting proteins involved in cell cycle regulation is an appealing therapeutic strategy for multiple myeloma (MM), given the increased proliferative rate of biologically aggressive multi-drug resistant MM cells compared to not only their normal plasma cell counterparts, but also MM cells from earlier stages of the disease. Within the intricate network of signaling cascades and regulatory checkpoints involved in cell cycle progression and survival of neoplastic cells, cyclin-dependent kinases (CDKs) have emerged as intriguing therapeutic targets. We report the results of preclinical studies on the anti-MM activity of the selective CDK1/2 small molecule inhibitor NVP-LCQ195/AT9311 (Novartis Pharma/Astex Therapeutics).Methods/Results: In MTT colorimetric survival assays against a panel of 28 human MM cell lines, NVP-LCQ195 exhibited dose-dependent anti-MM activity, with IC50 values at or below ~0.5 μM (a pharmacologically relevant concentration) for 12 of 28 cell lines many of which are resistant to conventional or novel anti-MM therapies (e.g. dexamethasone, melphalan, thalidomide derivatives). NVP-LCQ195 was also active against primary MM cells isolated from heavily-pretreated/drug-resistant cases of MM. In marked contrast, the IC50 values of NVP-LCQ195 against non-malignant cells such as bone marrow stromal cells (BMSCs) and immortalized hepatocyte cells was >4 μM. In addition, both PHA-stimulated and unstimulated normal donor PBMCs were less sensitive than the majority of MM cell lines of our panel. Treatment with NVP-LCQ195 was able to overcome the protective effects conferred on MM cells by exogenous IL-6 (10ng/mL) or IGF-1 (50ng/mL), as well as by co-culture of MM cells with bone marrow stromal cells (BMSCs). NVP-LCQ195 was observed to trigger a distinct pattern of S-phase arrest, followed by eventual induction of apoptotic cell death. Mechanistic studies revealed that hsp90, B-raf, cyclin D1, and cyclin E2 levels decrease in response to NVP-LCQ195 treatment, while caspases- 3, -8 and PARP are cleaved within 16 and 24 hrs of drug treatment. We evaluated a series of combinations of this agent with conventional (e.g. dexamethasone, doxorubicin) and novel (e.g. bortezomib) anti-MM agents. No evidence of antagonism with any of these anti-MM agents was observed, indicating that combinations of NVP-LCQ195 with the aforementioned anti-MM agents can be feasible in clinical settings.Conclusion: Functional inhibition of proteins involved in cell cycle regulation remains an attractive approach for the treatment of MM. Treatment of MM cells with the CDK1/2 inhibitor NVP-LCQ195 induces, in a subset of MM cell lines, a distinct pattern of initial cell cycle arrest, anti-proliferative/pro-apoptotic molecular sequleae and eventual induction of cell death. Our results determine intriguing potential combination regimens for MM treatment. Further in vitro and in vivo studies are attempting to delineate molecular markers of MM cell responsiveness vs. resistance to NVP-LCQ195 and provide a framework for individualized treatment of select MM patients with this interesting class of compounds.

Dasatinib (BMS-354825): A Multi-Targeted Kinase Inhibitor with Activity Against Multiple Myeloma.

The genetic heterogeneity of multiple myeloma (MM) and the evolution of the disease as it progresses result in a multiplicity of proliferative/anti-apoptotic pathways that can operate in MM cells, particularly within the context of their interaction with the bone marrow (BM) microenvironment. Collectively, these factors can contribute to de novo or acquired refractoriness of MM cells to diverse conventional and/or novel anti-MM therapeutics. To counteract the multiplicity of pathways potentially implicated in the control of MM cell resistance to drug-induced apoptosis, we have explored the use of multi-targeted small-molecule inhibitors (e.g. kinase inhibitors), clinical levels of which can simultaneously suppress the expression of multiple targets. Here we describe studies on the novel, oral, multi-targeted kinase inhibitor dasatinib (BMS-354825, Bristol Myers Squibb), which inhibits BCR-ABL, SRC, c-KIT, PDGF-R, and ephrin (EPH) receptor kinases. Although BCR-ABL and c-KIT are not primary oncogenes driving MM proliferation and survival, we studied dasatinib because of: a) emerging data from our laboratory (CS Mitsiades, unpublished observations) on patterns of expression/function patterns of EPH receptors in MM cell lines and primary tumor specimens; and b) the roles of PDGF-R and SRC in tumor-microenvironment interactions, e.g. pericytes/endothelial cells in angiogenesis and osteoclast-mediated bone resorption, respectively. In vitro, we found that dasatinib significantly suppresses, at clinically achievable sub-μM concentrations, the viability of MM cell lines (including lines resistant to conventional or other novel anti-MM agents); primary tumor specimens from multi-drug resistant MM patients; as well as MM cells co-cultured with BM stromal cells. Mechanistic studies showed that dasatinib can induce caspase-8 and -12 activation and sensitize primary MM cells to agents activating caspase-9 (e.g. Dex and bortezomib). Even though IC50 values were higher in MM cells than BCR-ABL+ CML cells, the IC50 of dasatinib was <100 nM in 8/15 MM cell lines tested, suggesting substantial sensitivity to dasatinib in at least a subset of MM. Interim analyses correlating the baseline transcriptional profiles of MM cells with their degree of responsiveness to low nM levels of dasatinib showed that increased responsiveness to this inhibitor correlated with increased baseline expression of diverse proliferative/anti-apoptotic genes, including transcriptional regulators (e.g. MAF, MAFF, NFYC, PML, YY1, DAXX), cell surface receptors (e.g. EPH receptor B4, CXCR4), proteasome subunits (PSMC3, PSMD12, PSME2) and regulators of apoptosis (e.g. CIAP1, IKKe). Ongoing expansion of the panel of tested MM cells is expected to further refine this molecular signature of dasatinib responsiveness. Ongoing in vivo studies are addressing the optimal dose, schedules and sequence of administration of dasatinib in combination with other anti-MM agents. These studies, coupled with our molecular profiling efforts to identify a molecular signature of dasatinib responsiveness, will inform the design of future clinical trials of dasatinib or its analogs for the treatment of MM patients.

Modulation of multidrug-resistant multiple myeloma by cyclosporin

Resistance to chemotherapy in refractory multiple myeloma is frequently associated with expression of multidrug resistance (MDR). In resistant cells, intracellular accumulation of doxorubicin and vincristine does not occur because the MDR- 1 gene product, a membrane glycoprotein (PgP), is an energy-dependent efflux pump. Cyclosporin is one of several non-cytotoxic drugs that can block the function of PgP. In a prospective study, we assessed the possibility that cyclosporin could be used clinically to modulate MDR. We studied 21 patients with multiple myeloma; disease had progressed during primary chemotherapy in 6 and was resistant to VAD (vincristine, doxorubicin, dexamethasone) in 15. The patients received cyclosporin by continuous infusion during VAD treatment; there were three cyclosporin dosage groups (5, 7·5, 10 mg/kg daily). Serum cyclosporin concentrations adequate for MDR modulation were reached in all patients receiving 7·5 or 10 mg/kg daily. 47% (7) of the VAD-refractory patients and 48% (10) of the whole group responded to VAD. Before treatment, MDR-1expression was present in 12 patients. After VAD plus cyclosporin, no MDR- 1-positive plasma cells were present in 6 of 8 patients tested. The response rate in MDR- 1-positive patients was 58% compared with 33% in all our patients. Toxic effects were mild and reversible and did not include nephrotoxic or serious cardiovascular side-effects. 12 months after the start of treatment, survival was 85%, and disease-free survival at a median of 9 months after the response was 65%. Thus, in multiple myeloma clinical resistance to VAD can be circumvented by cyclosporin, which enables the cytotoxic drugs to eliminate resistant myeloma cells.