Therapeutic Options For Multiple Myeloma Research Articles

Identification of KDM4C as a gene conferring drug resistance in multiple myeloma.

Bortezomib (BTZ), a proteasome inhibitor, is a promising therapeutic option for multiple myeloma (MM) patients. However, drug resistance often occurs, leading to disease relapse and poor prognosis. In this study, we aimed to identify novel genes associated with drug resistance and investigate their roles in BTZ resistance. Through the screening of 26 genes frequently associated with chemosensitivity or drug resistance, we discovered that KDM4C, a histone demethylase, exhibited increased expression in BTZ-resistant MM cells compared to their sensitive counterparts. Overexpression of KDM4C enhanced the tolerance of a MM cell line to the drug, whereas the knockdown of KDM4C, using shRNA, increased the sensitivity of resistant cells to BTZ treatment. This suggests that KDM4C plays a pivotal role in conferring BTZ resistance. Our study offers fresh insights into BTZ resistance in MM and highlights KDM4C as a potential target for overcoming drug resistance.

Results of an Open-Label Phase 1 Study Repurposing Oral Metformin and Nelfinavir in Combination with Subcutaneous Bortezomib in Patients with Relapsed and/or Refractory Multiple Myeloma

Background: GLUT4 inhibition via the use of a protease inhibitor, such as nelfinavir (NEL), is an attractive therapeutic option in multiple myeloma (MM), given the dependence of MM cells on glucose. Furthermore, combining a GLUT4 inhibitor with an oxidative phosphorylation inhibitor, such as metformin (MET), may counter the possibility of therapeutic resistance to GLUT4 inhibition in MM cells, showing synergistic promise (Dalva-Aydemir et al., Clin Can Res 2015). In addition to GLUT4 inhibition, NEL also lowers the cytotoxicity resistance of MM cells against proteasome inhibitors such as bortezomib (BOR) (Driessen et al., Blood 2018). Thus, this study evaluated the safety and tolerability of repurposing MET and NEL, in combination with BOR for the treatment of relapsed/refractory MM (RRMM) that had progressed on all standard of care (SOC) therapies. Methods: This trial utilized a 3+3 dose escalation design with 3 dose levels planned for up to a maximum of six (21-day) cycles. MET was administered for 14/21 days and started at a dose of 500 mg BID with plans to be investigated at a maximum dose of 1000 mg BID. NEL was administered for 14/21 days and started at a dose of 1250 mg BID with plans to be investigated to a maximum dose of 2500 mg BID. SubQ BOR was administered on days 1, 8, and 15 of a 21-day cycle at a dose of 1.3 mg/m 2 to a portion of patients. Patients with RRMM were required to have had 2 prior lines of therapy and prior exposure to proteasome inhibitors (PIs), immunomodulators (IMiDs), and Anti-CD38 monoclonal antibodies. The primary objective was to determine the maximal tolerated dose (MTD) of this combination. The secondary objective was to evaluate the safety as well as the overall response rate (ORR) of this combination. This trial was funded by the Helen Diller Family Foundation. Results: From 4/2019 until 9/2022, nine patients were accrued with a median age of 65 (range: 42-81) that received a median of 7 prior lines of therapy (Range: 6-13). All but one patient had a prior autologous stem cell transplant. Only one patient was not triple-class refractory, and 2/3 rd (n = 6) also had prior anti-BCMA therapy. The first three patients received only MET (500 mg BID) and NEL (1250 mg BID) at the first dose level for up to one cycle before experiencing progressive disease and coming off the study. Given the limited activity of MET and NEL in the initial treatment of these three patients, two subsequent amendments to the protocol allowed for the upfront addition of BOR 1.3 mg/m2 to the original combination, as well as shortened the DLT evaluation period from two cycles to just one. Thus, the following six patients received MET (500 mg BID) and NEL (1250 mg BID) at the first dose level in addition to weekly BOR at 1.3 mg/m2. Four (44%) patients experienced grade 3+ hematological AEs and six (67%) experienced grade 3+ non-hematological AEs. The most common hematologic grade 3+ AE observed was anemia (44%). The nonhematologic grade 3+ AEs observed were dyspnea, fatigue, nausea, and vomiting (11% each). None of the AEs were possibly related to treatment, suggesting good overall safety of the combination. While eight of nine patients have since died of progressive disease, no deaths were attributable to the treatment. All six patients who received BOR upfront with MET and NEL were previously refractory to BOR in prior lines of therapy; however, none had received BOR as their last line of therapy prior to accrual to this study. Four of these six patients had progressive disease after one cycle of therapy and went off study. However, the remaining two were able to receive 2 or more cycles of therapy, with one experiencing an unconfirmed minor response (MR) in the first cycle and the other experiencing stable disease (SD) for all six cycles. The patient who experienced SD was safely able to transition to a subsequent clinical trial utilizing an anti-BCMA bispecific antibody therapy and remains alive at the time of this analysis. The study was closed before accrual to the next dose level was started. Conclusions: This is the first study to evaluate the safety and efficacy of this repurposed drug combination in this very difficult-to-treat population of RRMM. While no ORR was observed, identification of biomarkers used for selection of patients with no other SOC options who could experience at least SD responses may be beneficial in allowing this combination to slow or stabilize the disease progression until other novel therapies or clinical trials are available.

Bispecific Antibodies in Multiple Myeloma: Opportunities to Enhance Efficacy and Improve Safety.

Multiple myeloma (MM) is the second most common haematological neoplasm of adults in the Western world. Overall survival has doubled since the advent of proteosome inhibitors (PIs), immunomodulatory agents (IMiDs), and monoclonal antibodies. However, patients with adverse cytogenetics or high-risk disease as determined by the Revised International Staging System (R-ISS) continue to have poorer outcomes, and triple-refractory patients have a median survival of less than 1 year. Bispecific antibodies (BsAbs) commonly bind to a tumour epitope along with CD3 on T-cells, leading to T-cell activation and tumour cell killing. These treatments show great promise in MM patients, with the first agent, teclistamab, receiving regulatory approval in 2022. Their potential utility is hampered by the immunosuppressive tumour microenvironment (TME), a hallmark of MM, which may limit efficacy, and by undesirable adverse events, including cytokine release syndrome (CRS) and infections, some of which may be fatal. In this review, we first consider the means of enhancing the efficacy of BsAbs in MM. These include combining BsAbs with other drugs that ameliorate the effect of the immunosuppressive TME, improving target availability, the use of BsAbs directed against multiple target antigens, and the optimal time in the treatment pathway to employ BsAbs. We then discuss methods to improve safety, focusing on reducing infection rates associated with treatment-induced hypogammaglobulinaemia, and decreasing the frequency and severity of CRS. BsAbs offer a highly-active therapeutic option in MM. Improving the efficacy and safety profiles of these agents may enable more patients to benefit from these novel therapies and improve outcomes for patients with high-risk disease.

Chimeric antigen receptor T-cell therapy for multiple myeloma.

Multiple myeloma (MM) is a malignant plasma cell disorder that remains incurable for most patients, as persistent clonal evolution drives new mutations which confer MM high-risk signatures and resistance to standard care. The past two decades have significantly refashioned the therapeutic options for MM, especially adoptive T cell therapy contributing to impressive response rate and clinical efficacy. Despite great promises achieved from chimeric antigen receptor T-cell (CAR-T) therapy, the poor durability and severe toxicity (cytokine release syndrome and neurotoxicity) are still huge challenges. Therefore, relapsed/refractory multiple myeloma (RRMM), characterized by the nature of clinicopathologic and molecular heterogeneity, is frequently associated with poor prognosis. B Cell Maturation Antigen (BCMA) is the most successful target for CAR-T therapy, and other potential targets either for single-target or dual-target CAR-T are actively being studied in numerous clinical trials. Moreover, mechanisms driving resistance or relapse after CAR-T therapy remain uncharacterized, which might refer to T-cell clearance, antigen escape, and immunosuppressive tumor microenvironment. Engineering CAR T-cell to improve both efficacy and safety continues to be a promising area for investigation. In this review, we aim to describe novel tumor-associated neoantigens for MM, summarize the data from current MM CAR-T clinical trials, introduce the mechanism of disease resistance/relapse after CAR-T infusion, highlight innovations capable of enhanced efficacy and reduced toxicity, and provide potential directions to optimize manufacturing processes.

Discovery of the oncogenic MDM2, a direct binding target of berberine and a potential therapeutic, in multiple myeloma.

Recent studies have suggested the potency of berberine (BBR) for multiple cancer treatments, including multiple myeloma (MM). However, the direct target and underlying mechanism of BBR remain largely understood in MM. Here, we demonstrated that BBR inhibited cell proliferation and acted synergistically with bortezomib in MM.1S cells. BBR treatment induced MM cell cycle arrest by downregulating several cell cycle-related proteins. Murine double minute 2 (MDM2) as a BBR-binding protein was identified by surface plasmon resonance image (SPRi) analysis and molecular docking. Overexpression of MDM2 is associated with MM progression and a poor prognosis. Knockdown MDM2 by siRNA transfection can repress MM malignant progression and attenuate the BBR sensitivity to MM.1S cells. BBR treatment induced the degradation of MDM2 through the ubiquitin-proteasome system and reactivated P53/P21 in MM cells. Overall, our data has illustrated that MDM2, as a binding protein of BBR for the first time, may serve as a potential therapeutic option for MM.

Measurable Residual Disease Assessment in Multiple Myeloma: How Deep Is Enough?

The introduction of new and more effective therapeutic options for Multiple Myeloma (MM) has significantly deepened and prolonged patients’ remission. As currently used treatment protocols induce high rates of complete responses, Measurable Residual Disease (MRD) assessment has become essential to enhance the evaluation of treatment efficacy. Detection of MRD has improved with the development of highly sensitive and standardized techniques such as Next Generation Flow or Next Generation Sequencing, complemented by functional imaging techniques. These advances offer a valuable opportunity to further optimize criteria of response to treatment. Currently, extensive data demonstrate that MRD status is a valuable prognostic factor of survival. Since MRD represents a real measurement of disease burden, its incorporation in clinical trials to guide treatment decisions will certainly translate into clinical benefits. Sustained MRD negativity can be used to consider optimal candidates for treatment discontinuation, whereas MRD positive high-risk patients may have access to novel immunotherapeutic strategies such as bispecific drugs or CAR T cell therapy. In this review, we describe the available techniques to detect MRD, address the current data regarding MRD as a surrogate endpoint within clinical trials, examine how MRD can be introduced into the clinical management of MM patients, and discuss the future of MRD monitoring.

Survival and Outcomes of Newly Diagnosed Multiple Myeloma Patients Stratified by Transplant Status 2007-2018: Retrospective Analysis from the Canadian Myeloma Research Group Database

BackgroundConsiderable progress has been made in therapeutic options for multiple myeloma (MM). Understanding the current landscape of MM treatment options and associated outcomes in the real world is important in providing key insights into clinical and knowledge gaps which could be targeted for further optimization. MethodsThe Canadian Myeloma Research Group Database (CMRG-DB) is a prospectively maintained disease-specific database with >7000 patients. The objective of this study was to describe the trends in the treatment landscape and outcomes including early mortality, time to next treatment, and overall survival (OS) in each line of treatment stratified by autologous stem cell transplant (ASCT) receipt among newly-diagnosed MM patients in Canada between 2007 and 2018. ResultsA total of 5154 patients were identified among which 3030 patients (58.8%) received an upfront ASCT and 2124 (41.2%) did not. At diagnosis, the median age was 64 years and 58.6% were males. Bortezomib and lenalidomide were most frequently used (>50%) in first and second-line treatment respectively among both the ASCT and non-ASCT cohort. The median OS was 122.0 months (95% Cl 115.0-135.0 months) and 54.3 months (95% CI 50.8-58.8 months) for the ASCT and non-ASCT cohort respectively with an incremental decrease in OS in each subsequent line of treatment. ConclusionWe present the largest study to date in the Canadian landscape showing the characteristics, therapy usage, and outcomes among MM patients. This information will be critical in benchmarking current outcomes and provide key insight into areas of unmet needs and gaps for improvement of MM patients nationally.

OM301, a Synthetic Polypeptide Containing the p53TA (Transactivation) Domain, Impairs Mitochondrial Activity and Survival of Myeloma Cells

INTRODUCTIONAlthough the treatment of patients with multiple myeloma (MM) has dramatically improved, those with high-risk characteristics, including the deletion or mutation of the master tumor suppressor gene TP53 on chromosome 17, experience limited survival. OM301 is a synthetic polypeptide containing the p53TA (transactivation) domain, which prevents p53 degradation through inhibition of MDM2. Here, we demonstrate that OM301 has strong anti-MM activity in vitro and in vivo. RESULTSWe first assessed the cytotoxic effects of OM301 in MM cell lines with varying TP53 status (TP53 wild type: MM.1S, H929; TP53 mutated/null: L363, RPMI-8226, U266, JJN3, KMS11) and found that OM301 exerts significant cytotoxic effects at a concentration of ~5 µM in all cell lines we tested, while it was minimally toxic to human peripheral blood mononuclear cells. Next, using immunocompromised NSG mice models injected with MM.1S, we determined the in vivo efficacy of OM301 in three different studies. Many potent anticancer agents, particularly of peptide origin, show prominent anti-tumor effects but fail to sustain similar effects when given intraperitoneally because of poor absorption, distribution, metabolism and excretion properties. OM301 at an intraperitoneal dose of 20 mg/kg/body weight twice a day induced significant reduction in tumor size with respect to vehicle control, suggesting the stability of OM301 without any loss of its activity (n=7, p<0.0001). Accordingly, we investigated its effect in a disseminated NSG/MM.1S model and found that it significantly increased survival (p<0.0001) (see Figure).Because OM301 was designed to simulate the p53 interaction domain with MDM2, we first determined its effect on p53-MDM2 crosstalk using a p53-MDM2 co-Immunoprecipitation (co-IP) assay and compared it with effects from Nutlin-3a, a known inhibitor of p53-MDM2. The co-IP data showed that, unlike Nutlin-3a, OM301 does not inhibit the p53-MDM2 interaction. Thus, to confirm our findings, we first overexpressed MDM2 in HeLa cells, and, using MDM2-IP and p53-MDM2 co-IP, found similar observations. Additionally, OM301 also failed to induce endogenous upregulation of genes activated by p53, such as MDM2 and p21, as opposed to results from Nutlin-3a. RNA sequencing data also showed a distinctive OM301signature, as compared to Nutlin-3a in MM cells. While treatment of Nutlin-3a induced expression of p53-activated canonical genes, OM301-treated cells showed alterations in genes involved in inflammatory responses, c-Myc regulated genes, fatty acid metabolism, glucose metabolism, and oxidative phosphorylation, among others.Next, to dissect its underlying mechanism, we dual-tagged OM301 with fluorophores at the 3' and 5' ends to study its localization and its stability in MM cells. Indeed, OM301 was found to be stable and mainly localized in the cytosol. We then modified OM301 by biotinylation of its penetratin end and first verified its cytotoxic effect in different MM cell lines, which was similar to that of native OM301. The biotinylated OM301 was then immunoprecipitated using streptavidin beads. The streptavidin pull-down and subsequent proteomic analysis confirmed that OM301 does not interact with MDM2 but interacts with c-Myc and with proteins localized in mitochondria, including Bcl-2 and Bcl-2 family members such as Bclaf1, Bcl2L13, and Bcl2L1. Pull-down experiments and immunoblot analysis validated Bcl-2/OM301 interactions. To further evaluate the relative binding potentials of OM301, we performed molecular docking studies using the HPEPDOCK server (Yan et al., Nat Protoc. 2020;15:1829). Post-docking, the calculated docking scores for OM301 was -281, suggesting that OM301directly interacts with Bcl-2. Thus, we evaluated the effects of OM301 on mitochondrial function and physiology. Treatment with OM301 decreased mitochondrial membrane potential in different MM cell lines. OM301 also increased mitochondrial superoxide production and induced mitophagy and mitochondrial fission as seen by electron microscopy. CONCLUSIONHere, we report for the first time that OM301, although designed for p53-selective cells, may instead interact with Bcl-2, which in turn induces mitochondrial dysfunction, leading to cell death irrespective of their TP53 status. Our data suggest that OM301 may be a novel and effective therapeutic option for MM. [Display omitted] DisclosuresKrishnan: REGENERON: Consultancy; MAGENTA: Consultancy; BMS: Consultancy, Current equity holder in publicly-traded company, Speakers Bureau; JANSSEN: Consultancy, Research Funding; City of Hope Cancer Center: Current Employment; SANOFI: Consultancy; GSK: Consultancy; Amgen: Speakers Bureau. Marcucci: Novartis: Other: Speaker and advisory scientific board meetings; Agios: Other: Speaker and advisory scientific board meetings; Abbvie: Other: Speaker and advisory scientific board meetings.

Daratumumab with Pomalidomide and Dexamethasone at First Relapse in Relapsed and/or Refractory Multiple Myeloma (RRMM) Patients

Daratumumab with Pomalidomide and Dexamethasone at First Relapse in Relapsed and/or Refractory Multiple Myeloma (RRMM) Patients

Trial in Progress: Phase I Open-Label Study of Metformin and Nelfinavir in Combination with Bortezomib in Patients with Relapsed and/or Refractory Multiple Myeloma

Background:GLUT4 inhibition is an attractive therapeutic option in multiple myeloma (MM) given the dependence of MM cells on glucose transport. Furthermore, combining a GLUT4 inhibitor with an oxidative phosphorylation inhibitor in MM cells to counter the possibility of therapeutic resistance to GLUT4 inhibition has demonstrated synergistic promise (Dalva-Aydemir et al, Clin Can Res 2015). Using a GLUT4 inhibitor such as the HIV protease inhibitor nelfinavir is an attractive choice since it not only inhibits the transport function of GLUT4, but it also lowers the cytotoxicity resistance of MM cells against proteasome inhibitors such as bortezomib (Driessen et al, Blood 2018). Thus, in summary the FDA-approved oral HIV-PI nelfinavir, long used for the treatment of HIV disease, can bind to glucose transporters (GLUT4) on MM cells and reversibly inhibit glucose uptake, thus disrupting the Warburg effect. Nelfinavir can also enhance the activity of PIs such as bortezomib overcome its resistance in MM cells. Metformin, an oral FDA-approved drug for the treatment of Type II Diabetes has the ability to disrupt the electron transport chain of defective mitochondria within MM cells, thus blocking the ability of such mitochondria to generate energy and anabolic substrates as compensatory mechanisms required for proliferation in the setting of GLUT4 inhibition. Thus, this study will look at the safety and tolerability of repurposing the existing FDA-approved drugs for Type II diabetes and HIV in combination with bortezomib for the treatment of relapsed and/or refractory myeloma.Methods:This study is a phase 1 clinical trial of metformin and nelfinavir in combination with bortezomib for patients with relapsed and/or refractory myeloma. Part A of this trial is a 3+3 dose escalation design with 3 dose levels planned. Part B is a dose expansion cohort at the Maximal Tolerated Dose (MTD) determined in Part A. Up to a maximum of 36 patients will be enrolled and treated for a maximum of six 21-day cycles. Bortezomib is administered in its usual weekly subcutaneous dosing schedule of days 1, 8 and 15 of a 21-day cycle at a dose level of 1.3 mg/m2. Metformin will be administered for 14 days of a 21-day cycle and started at a dose level of 500 mg twice daily and will be investigated to a maximum dose of 1000 mg twice daily. Nelfinavir will also be administered for 14 days of a 21-day cycle and started at a dose level of 1250 mg twice daily and will be investigated to a maximum dose of 2500 mg twice daily. Key inclusion criteria include having relapsed/refractory myeloma with at least 2 prior lines of therapy and prior exposure to PIs, immunomodulators and Anti-CD38 monoclonal antibodies, adequate hematologic reserve, kidney function and liver function. This trial especially allows patients with non-secretory disease yet measurable by bone marrow assessments or cross-sectional imaging of plasmacytomas to be enrolled which constitutes an unmet need in the real-world MM population. Finally, this allows patients to be treated with a dexamethasone-free regimen. The primary objective of this trial is to determine the MTD of metformin and nelfinavir in combination with bortezomib. The secondary objective is to evaluate the safety and tolerability as well as the overall response rate (ORR) associated with this combination. Potential predictive biomarkers as well as resistance mechanisms using genomic RNA, immunohistochemistry and metabolomics-based assessment platforms will be evaluated in this study. Recruitment is ongoing with no safety concerns. This trial is registered on clinicaltrials.gov: NCT03829020. This investigator-initiated trial was funded by the generous gift of the Helen Diller Family Foundation. DisclosuresKumar: Astra-Zeneca: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bluebird Bio: Consultancy; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncopeptides: Consultancy; Antengene: Consultancy, Honoraria; Novartis: Research Funding; Carsgen: Research Funding; Roche-Genentech: Consultancy, Research Funding; Beigene: Consultancy; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Research Funding; Merck: Research Funding; KITE: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Tenebio: Research Funding; BMS: Consultancy, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Adaptive: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Research Funding. Ailawadhi: Karyopharm: Consultancy; AbbVie: Consultancy; Genentech: Consultancy; Takeda: Consultancy; GSK: Consultancy, Research Funding; Xencor: Research Funding; Cellectar: Research Funding; Medimmune: Research Funding; Ascentage: Research Funding; Pharmacyclics: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; BeiGene, Ltd.: Consultancy; Sanofi: Consultancy; Oncopeptides: Consultancy.

P-117: Multiple Myeloma treatment patterns and outcomes in the public and private healthcare systems in Brazil: one country, two worlds

<h3>Background</h3> Over the past two decades, the approval of several novel drugs has broadened therapeutic options for multiple myeloma (MM) patients, but has also raised concerns about increasing drug expenses and healthcare costs in general. Access to these treatments in public and private settings in Brazil differs significantly and it is unknown if shortcomings in treatment availability affect patient outcomes. The aim was to understand the patient characteristics, treatment patterns and MM patient outcomes in private and public settings in Brazil. <h3>Methods</h3> MMyBrave is an observational, retrospective study with no control group. Eligible patients were diagnosed with active MM between January 1, 2008 and December 31, 2016 (cut-off date), at 17 public and private medical centers in Brazil. The MM diagnosis was performed according to investigator assessment. Data collection started on June 2018, after ethical approval, and progressed until August 2019. Demographic and clinical characteristics were collected. Patients were stratified according to eligibility to transplant (intention-to-treat analysis) and treatment setting (public or private health systems). The overall survival (OS) was analyzed using the Kaplan-Meier (KM) curve and Cox's proportional-hazards model. <h3>Results</h3> A total of 943 patients were included. Private and public centers accounted for 44.9 and 55.1% of patients, respectively. The age (≥65 years) and female percentage at private and public centers were 64.2 and 63.4%; and 43.6 and 48.6, respectively. The International Staging System stages I, II, III and stage unknown were: 26.7, 22.6, 25.7 and 25.0 at private centers and 13.3, 20.2, 27.4, 39.1 at public centers. A total of 47.9% (50.2% private and 46.1% public) were eligible for autologous stem cell transplantation (ASCT). Proteasome inhibitor (PI) regimens (mainly bortezomib-based) were used by 52.8% at private centers (57.3% in ASCT eligible; 48.3% in ASCT ineligible). MM patients in the private health system had a higher overall survival (OS) than those in the public setting (75.9 x 63.3 months, p=0,007). <h3>Conclusion</h3> Real-world MM patient outcomes at private and public institutions in Brazil were quite different, with significantly higher OS in those followed in the Brazilian private healthcare system. Disease staging and treatment response were found as independent OS predictors, regardless reimbursement setting and ASCT eligibility. These preliminary results raise the hypothesis that the superior OS among patients in the private healthcare system may be due to a wider availability of therapies.

BCMA Versus CD138 for Identifying Multiple Myeloma on Flow Cytometry

Introduction: Flow cytometry is an important tool in detecting multiple myeloma (MM), especially in cases of minimal residual disease (MRD). The reliability and stability of CD138, a marker used in many institutions on antibody panels to detect MM, has been questioned in recent studies, spurring the search for alternative markers. In this study, our aim was to compare BCMA (CD269) to CD138 in its ability to detect MM cells in flow cytometric analysis, in order to determine whether or not BCMA should be included in standard MM flow cytometry panels.Methods: In this retrospective study, 25 consecutive cases of multiple myeloma diagnosed on bone marrow biopsy (including cases of initial diagnosis, relapse, and MRD) were identified from our institution's CoPath database from November 2015 through September 2016. These cases included 21 patients. Four patients had two flow cytometry cases included; in these patients, the second bone marrow aspirate was obtained at least six weeks after the first.Cases were re-gated and analyzed (Kaluza Analysis program) with an 8-color flow cytometry panel containing the following markers: CD138, BCMA, CD45, CD19, CD3, CD14, CD64, GLY. All cases analyzed had also been run with a panel which included kappa/lambda to confirm the monoclonality of the plasma cell population identified. The same back-gating and Boolean gating strategy was used on all 25 cases. Boolean gates were used to exclude red cells and monocytes from the gated MM cells (i.e., cells expressing CD64, CD14, and GLY). BCMA and CD138 expression was compared on this gated population of MM cells. Data for coefficient of variation (CV) and geometric mean of both BCMA and CD138 expression were obtained from the Kaluza program for each case.Results: In our 25 cases, gating with BCMA expression was able to detect the same population of MM cells as CD138. In 7/25 (28%) cases, BCMA detected MM cells which were not detected by CD138. In 2/7 cases, the MM cells detected were CD138-negative, but BCMA-positive; both were cases of MRD. In 5/7 cases, the MM cells showed two populations, one positive for both BCMA and CD138, and the other only positive for BCMA (CD138-negative); four of these cases were MRD. The CV in 21/25 cases was larger for CD138 than BCMA. The average CV for BCMA was 62.12 (range 39.19-107.06), while the average CV for CD138 was 121.46 (range 35.86-264.34). In comparing the CVs for BCMA and CD138 across all cases, the difference was statistically significant (p = 0.00003). In comparing the geometric mean (mean fluorescence intensity) for BCMA and CD138, BCMA tended to stain more dimly than CD138, but was in a more consistent range, while CD138 showed a large variation in its staining intensity. The average geometric mean for BCMA was 3.71 (range 1.63-10.17), while the average for CD138 was 82.94 (range 0.44-639.39).Discussion: Our study shows that BCMA not only detects as many MM cells as CD138, but also has a lower coefficient of variation (p = 0.00003). The lower amount of variability in BCMA staining on MM cells makes this marker easier to use technically in terms of ease of gating around a more tightly clustered population of cells. The 7/25 cases which demonstrated CD138 negativity in all or part of the MM cell population detected also support previous studies which suggested that CD138 is a less reliable marker. 6/7 of these cases were MRD, showing that BCMA may be especially helpful in cases of MRD. Adding BCMA to MM antibody panels would help to ensure that these populations of CD138-negative MM cells would not be missed.In addition to its usefulness in diagnosis and monitoring of MM (especially MRD), new therapeutic options for MM are a reason to include BCMA in the more commonly used flow cytometry panels. For example, immunotherapies against CD38 necessitate the use of other markers when monitoring patients who have been treated with these agents. BCMA is also currently being targeted in chimeric antigen receptor (CAR) T-cell therapies for MM in clinical trials, another reason to routinely analyze MM cells for BCMA.Conclusion: Our study shows BCMA to be a more reliable marker for detecting MM cells than CD138, and supports a recommendation that BCMA be routinely used on flow cytometry panels to detect MM cells. DisclosuresGolightly:Stony Brook University, Alexion, Icon Laboratories, Enzo Labs: Consultancy, Employment, Speakers Bureau.

Immunotherapy in multiple myeloma

Despite available therapies, Multiple Myeloma (MM) remains an incurable hematologic malignancy. Over the past three decades, there have been tremendous developments in therapeutic options for MM. In regards to immunotherapy, Daratumumab was the first monoclonal antibody to receive FDA approval for multiple myeloma. Since then, other monoclonal antibodies such as elotuzumab and isatuximab have received FDA approval. Many clinical trials are underway investigating the efficacy of newer immunotherapies. This review summarizes recently presented and/or published data regarding this growing field, specifically regarding monoclonal antibodies, antibody-drug conjugates, bispecific antibodies, and trispecific antibodies.

The iron chelator deferoxamine decreases myeloma cell survival

ObjectiveThis study evaluated serum ferritin (SF) levels and investigated their relationships with various clinical markers in patients with multiple myeloma (MM). Furthermore, the effects and molecular mechanism of deferoxamine (DFO) in myeloma cells were studied.MethodsClinical data from 84 patients with MM were collected to evaluate SF content and its relationship with several important clinical parameters. MM1S and MM1R myeloma cells were chosen to investigate the effects of iron and DFO on cell survival and apoptosis.ResultsIncreased SF levels were detected in newly diagnosed patients, especially those with stage III disease or the κ isotype. SF content was positively correlated with β2-microglobulin, interleukin-6, and lactate dehydrogenase expression. Furthermore, patients with progressive or relapsed disease had higher SF levels. Importantly, iron chelation with DFO efficiently inhibited myeloma cell survival and accelerated apoptosis by regulating apoptosis-related genes.ConclusionsThe importance of SF for MM was highlighted. Additionally, it is suggested that DFO may be a good therapeutic option for MM.

Targeting NK Cell Inhibitory Receptors for Precision Multiple Myeloma Immunotherapy.

Innate immune surveillance of cancer involves multiple types of immune cells including the innate lymphoid cells (ILCs). Natural killer (NK) cells are considered the most active ILC subset for tumor elimination because of their ability to target infected and malignant cells without prior sensitization. NK cells are equipped with an array of activating and inhibitory receptors (IRs); hence NK cell activity is controlled by balanced signals between the activating and IRs. Multiple myeloma (MM) is a hematological malignancy that is known for its altered immune landscape. Despite improvements in therapeutic options for MM, this disease remains incurable. An emerging trend to improve clinical outcomes in MM involves harnessing the inherent ability of NK cells to kill malignant cells by recruiting NK cells and enhancing their cytotoxicity toward the malignant MM cells. Following the clinical success of blocking T cell IRs in multiple cancers, targeting NK cell IRs is drawing increasing attention. Relevant NK cell IRs that are attractive candidates for checkpoint blockades include KIRs, NKG2A, LAG-3, TIGIT, PD-1, and TIM-3 receptors. Investigating these NK cell IRs as pathogenic agents and therapeutic targets could lead to promising applications in MM therapy. This review describes the critical role of enhancing NK cell activity in MM and discusses the potential of blocking NK cell IRs as a future MM therapy.

Development of a Hydrashift 2/4 Isatuximab Assay to Mitigate Interference with Monoclonal Protein Detection on Immunofixation Electrophoresis in Vitro Diagnostic Tests in Multiple Myeloma

Introduction Evaluation of multiple myeloma (MM) response through quantification of M-protein, by serum protein electrophoresis (SPEP) and immuno-fixation electrophoresis (IFE) is challenging for clinical laboratories because therapeutic monoclonal antibodies (mAbs) can confound IFE when they converge with serum M-protein. This can be misleading when interpreting patients' response to therapy. Isatuximab (Isa), an IgG-kappa anti-CD38 mAb is approved based on the pivotal ICARIA-MM study in combination with pomalidomide (P) and dexamethasone (d), in the United States, the European Union, Canada, Australia, Switzerland and Japan for the treatment of adult patients with relapsed/refractory MM who have received at least two prior therapies including lenalidomide and a proteasome inhibitor. Recently, the Phase 3 IKEMA study evaluating Isa plus carfilzomib (K) and d met its primary endpoint at the first planned interim analysis, demonstrating significantly prolonged progression free survival (PFS) compared with Kd alone in patients with relapsed MM. Methods To overcome the interference mediated by Isa on the IFE test, we have developed an Isa-specific assay "HYDRASHIFT 2/4 Isa" using a highly specific rabbit, anti-Isa mAb. The HYDRASHIFT 2/4 Isa test is used in conjunction with the regular Hydragel IF procedure and the semi-automated Hydrasys 2 electrophoresis apparatus. The HYDRASHIFT 2/4 Isa test displaces Isa (IgG kappa) interference during the electrophoresis process by forming Isa/anti-Isa complex which is moved out of the gamma zone toward the alpha globulin fractions. Results: Sensitivity of the HYDRASHIFT 2/4 Isa assay was demonstrated on serum samples, including normal serum samples. Isa control and serum samples with different monoclonal components spiked with Isa (final concentrations in the range of 0.1 and 3.0 g/L) were analyzed with the HYDRASHIFT 2/4 Isa procedure used in conjunction with Hydragel 4 IF Acid Violet. The detection limit of Isa and/or the Isa / anti-Isa antibody complex visualized was 0.2 g/L. Importantly, efficient removal of Isa from the gamma globulin zone, with no trace signal evident, was demonstrated for all Isa concentrations tested, even for 3 g/L. Reproducibility of the HYDRASHIFT 2/4 Isa assay was also demonstrated between gels, between product lots, between instruments and on different test days. Ten different serum samples, including 1 normal serum sample and 9 serum samples with monoclonal components spiked with Isa at 1 g/L, were run using the HYDRASHIFT 2/4 Isa procedure in conjunction with Hydragel 4 IF Acid Violet. All samples gave 100% concordant results between gels on the different instruments and with different HYDRASHIFT 2/4 Isa lots. To evaluate specificity, 50 serum samples from MM patients were spiked with 1 g/L Isa and the anti-Isa mAb shifted Isa specifically with no impact on the patients' M-spike, demonstrating 100% specificity. Finally, the HYDRASHIFT 2/4 Isa test was evaluated on 15 samples from treated patients enrolled in Isa clinical trials at different therapy cycles. The evaluated samples demonstrated efficient removal of Isa from G and Kappa tracks on the gamma globulins zone and visualization of the Isa/anti-Isa complex on alpha zone and/or Isa on ELP track. For the pre-treatment samples, there was as expected no Isa detection, and no impact on M-protein was observed, further confirming the specificity of the assay toward Isa. Conclusions Therapeutic mAb inclusion in MM treatment regimens offer patients significant improvements in clinical outcomes. With rapid evolution of therapeutic options in MM, there is a clear need for a standardized and reliable method to ensure authentic IFE-based clinical assessment. Development of the Isa-specific HYDRASHIFT 2/4 Isa assay offers the advantage of high clinical utility due to the simplicity of the method as an add-on to the conventional IFE In vitro diagnostic test. Consequently, this assay will facilitate the correct assessment of clinical outcomes for patients receiving Isa as part of their MM treatment. Submission for global regulatory clearance for Isa-specific HYDRASHIFT 2/4 assay is planned in 2020. Disclosures Finn: Sanofi: Current Employment. Macé:Sanofi: Current Employment. Chu:Sanofi: Current Employment. van de Velde:Sanofi: Current Employment, Current equity holder in publicly-traded company. Melki:Sebia: Current Employment.

Clinical Controversies in the Management of Smoldering Multiple Myeloma.

Forty years ago this year, smoldering multiple myeloma was defined as a clinical entity that identifies a group of patients with a substantial burden of disease but with a relatively indolent natural history compared with symptomatic disease. Since then, there has been a revolution in the therapeutic options for multiple myeloma. The aim of this article is to describe recent advances in the identification of those patients who are at the highest risk of progression and whether they may benefit from therapy. Treatment of smoldering myeloma is an area of active debate and we present contrasting interpretations of the available trial data. We conclude by identifying the priorities for research that will help to clarify the management of this condition, which can be challenging for physicians and patients alike.

A pharmacodynamic model of clinical synergy in multiple myeloma.

BackgroundMultiagent therapies, due to their ability to delay or overcome resistance, are a hallmark of treatment in multiple myeloma (MM). The growing number of therapeutic options in MM requires high-throughput combination screening tools to better allocate treatment, and facilitate personalized therapy.MethodsA second-order drug response model was employed to fit patient-specific ex vivo responses of 203 MM patients to single-agent models. A novel pharmacodynamic model, developed to account for two-way combination effects, was tested with 130 two-drug combinations. We have demonstrated that this model is sufficiently parameterized by single-agent and fixed-ratio combination responses, by validating model estimates with ex vivo combination responses for different concentration ratios, using a checkerboard assay. This new model reconciles ex vivo observations from both Loewe and BLISS synergy models, by accounting for the dimension of time, as opposed to focusing on arbitrary time-points or drug effect. Clinical outcomes of patients were simulated by coupling patient-specific drug combination models with pharmacokinetic data.FindingsCombination screening showed 1 in 5 combinations (21.43% by LD50, 18.42% by AUC) were synergistic ex vivo with statistical significance (P < 0.05), but clinical synergy was predicted for only 1 in 10 combinations (8.69%), which was attributed to the role of pharmacokinetics and dosing schedules.InterpretationThe proposed framework can inform clinical decisions from ex vivo observations, thus providing a path toward personalized therapy using combination regimens.FundingThis research was funded by the H. Lee Moffitt Cancer Center Physical Sciences in Oncology (PSOC) Grant (1U54CA193489-01A1) and by H. Lee Moffitt Cancer Center's Team Science Grant. This work has been supported in part by the PSOC Pilot Project Award (5U54CA193489-04), the Translational Research Core Facility at the H. Lee Moffitt Cancer Center & Research Institute, an NCI-designated Comprehensive Cancer Center (P30-CA076292), the Pentecost Family Foundation, and Miles for Moffitt Foundation.

The Preclinical Activities of PTC596, a Novel Tubulin Binding Agent That Down-Regulates BMI1, Alone and in Combination with Bortezomib in Multiple Myeloma

Introduction: A novel tubulin binding agent PTC596, which is currently in clinical trials for solid tumors, was originally identified by its ability to kill cancer stem cells and to reduce BMI1 activity. PTC596 treatment results in hyperphosphorylation of the BMI1 protein and loss of BMI1 function as demonstrated by a reduction in H2A ubiquitination levels in a range of solid tumor lines. Subsequent studies have shown that the down-regulation of BMI1 protein is due to a G2/M arrest. In this study, we aimed to investigate the in-vitro and in-vivo anti-tumor activities of PTC596 and the combination with bortezomib in multiple myeloma (MM). Methods: For in-vitro evaluation, MTS and BrdU ELISA assays were performed using human MM cell lines. Approved by the Institutional Review Committee at Chiba University, primary myeloma cells and bone marrow stromal cells (BMSCs) were collected from the bone marrow of MM patients with informed consent. For in-vivo evaluation, the MM.1S subcutaneous xenograft model in NOG mice was used. To understand the mechanisms of action and target genes of the treatments, flow cytometry (FCM), western blotting, RNA-seq, and ChIP-seq were performed. Results: PTC596 induced significant cytotoxicity in all MM cell lines tested, including bortezomib-resistant OPM-2/BTZ and KMS-11/BTZ cells (CC50: 24-98 nM). PTC596 also suppressed cell proliferation when these cell lines were co-cultured with BMSCs. As expected, PTC596 reduced the levels of BMI1 protein and uH2A in a dose-dependent manner. Of note, PTC596 induced cell cycle arrest as detected by a BrdU FCM assay in MM cells and apoptosis as detected by annexin-V FCM in MM cell lines and primary myeloma cells. Moreover, oral administration of PTC596 twice a week for three weeks significantly inhibited the growth of MM.1S tumors implanted in immunodeficient mice and improved the survival of mice as compared with mice treated with vehicle only (p=0.0021). Of interest, bortezomib appeared to transcriptionally repress the expression of BMI1 and reduce the levels of uH2A. We then tested the efficacy of the combination of PTC596 with bortezomib in MM cells and found additive or synergistic effects when MM cell lines were co-cultured with BMSCs. Reductions in the levels of BMI1 protein and uH2A by PTC596 or bortezomib alone were significantly enhanced in the combination treatment. Furthermore, apoptosis induced by bortezomib was significantly enhanced by the combination with PTC596 as evidenced by increased annexin-positive cells detected in flow cytometric analysis and increased cleavage of caspases with reduction in MCL1 protein in western blotting. RNA-seq of MM.1S cells treated with PTC596 alone or in combination with bortezomib demonstrated repression of gene sets related to the cell cycle in either setting and enrichment of gene sets related to apoptosis in the combination. Ongoing analysis of our ChIP-seq data will reveal the direct targets of BMI1 in MM cells. Remarkably, oral administration of PTC596 combined with subcutaneous injection of bortezomib twice a week for five weeks significantly reduced MM.1S tumor growth in comparison to the control or either single treatment (p&lt;0.0001 vs. control, p=0.0003 vs. PTC596, p=0.0104 vs. bortezomib). Severe body weight loss was not observed in the combination-treated mice except one mouse which needed washout period until recovery. White blood cell count and hemoglobin level in peripheral blood of mice were not significantly changed during the treatment with PTC596 or even the combination. Survival of the host mice treated with the combination was significantly prolonged as compared with the control or either single treatment (p&lt;0.0001 vs. control, p=0.0002 vs. PTC596, p=0.0072 vs. bortezomib). Conclusions: Our results demonstrate that PTC596 alone and in combination with proteasome inhibition are potential novel therapeutic options in MM. The results of this study support the clinical evaluation of this promising therapeutic approach to improve the outcome of MM patients. Disclosures Sheedy: PTC Therapeutics: Employment. Weetall:PTC Therapeutics: Employment.

Panobinostat Potentiates Melphalan-Induced Apoptosis of Myeloma Cells and Minimizes Untoward Side Effects in Multiple Myeloma

Introduction: Studies demonstrating the dynamic interplay between protein acetylation status, cell cycle progression and apoptosis in multiple myeloma (MM) cells have provided a strong rationale for the use of the nonselective histone deacetylase inhibitor (HDACi) panobinostat, as a therapeutic option for MM. However, despite the promising preclinical data, the clinical responses achieved after treatment of MM patients with single-agent panobinostat have been disappointing. On the other hand, this HDACi has been shown to function synergistically with a range of structurally and functionally diverse chemical compounds, including genotoxic drugs, biologically active polypeptides, and novel immune therapies. Since a better understanding of the molecular pathways targeted by panobinostat in MM cells may provide a better rationale for the selection of new drug combinations with synergistic potential, herein we investigate the mechanistic basis for the action of the combination treatment of panobinostat and melphalan in MM. Methods: Malignant bone marrow plasma cells (BMPCs) and peripheral blood mononuclear cells (PBMCs) of 26 unselected newly diagnosed MM patients (12F/14M; median age 60 years, range 42-66) were analyzed. PBMCs from 25 apparently healthy controls were examined in parallel. Cells were pre-treated or not with 5nM panobinostat for 24h, then treated with 100μg/ml melphalan for 5min and critical DNA damage response and repair (DDR/R) signals, including fundamental DNA repair mechanisms [nucleotide excision repair (NER) using Southern blot analysis; interstrand cross-links repair (ICL/R) using quantitative-PCR; double-strand breaks repair (DSB/R) using immunofluorescence quantification of γH2AX foci by confocal microscopy], cells' viability, cytotoxicity and apoptosis rates were evaluated. Moreover, in untreated cells, chromatin organization (using micrococcal nuclease digestion-based analysis) and the levels of certain DNA repair proteins associated with NER and DSB/R mechanisms (using western blot) were also analyzed. The study was approved by the Institutional review board of Alexandra Hospital and all subjects provided informed consent. The study was conducted according to the Declaration of Helsinki. Results: Following ex vivo panobinostat-only treatment, both BMPCs and PBMCs showed acetylation of histone H4 and significant relaxation of chromatin structure. On the other hand, only BMPCs showed a dramatic panobinostat-induced decrease in the levels of critical DNA repair proteins associated with NER (XPC, ERCC3, DDB2) and DSB/R (RAD50, MRE11, KU70, KU80, DNA-PK) mechanisms. No significant effect of panobinostat treatment on the DNA repair proteins was observed in PBMCs from the same ΜΜ patients and in PBMCs from healthy controls. In BMPCs from MM patients, panobinostat potentiates melphalan cytotoxicity, through the inhibition of critical DNA repair mechanisms and the resulting intensification of melphalan-induced DNA damage. Indeed, combined treatment of BMPCs with panobinostat and melphalan significantly reduced the NER, ICL/R and DSB/R capacity of the cells, resulting in significantly increased DNA damage burden compared with melphalan-only treatment (all P&lt;0.01). Moreover, co-treatment of BMPCs with panobinostat and melphalan resulted in significant enhancement of the cytotoxicity and apoptosis rates of the cells, while it significantly reduced BMPCs' viability compared with melphalan-only treatment (P&lt;0.001). In contrast to the BMPCs results, panobinostat pre-treatment of PBMCs from the same patients reduced melphalan-induced toxicity through amelioration of the DNA repair capacity of these cells and the resulting lower melphalan-induced DNA damage burden. Indeed, co-treatment of PBMCs with panobinostat and melphalan significantly increased the NER, ICL/R and DSBs/R capacity of the cells, decreased DNA damage burden, reduced cytotoxicity and apoptosis rates, while it significantly increased PBMCs' viability compared with melphalan-only treatment (all P&lt;0.05). Conclusion: Our data demonstrate that panobinostat potentiates melphalan-induced apoptosis of myeloma cells, and in parallel it helps minimize untoward side effects, thus paving the way to combination therapies with lower side effects and improved anti-myeloma efficacy. Disclosures Dimopoulos: Sanofi Oncology: Research Funding. Terpos:Medison: Honoraria; Takeda: Honoraria, Other: Travel expenses, Research Funding; Janssen: Honoraria, Other: Travel expenses, Research Funding; Genesis: Honoraria, Research Funding; Celgene: Honoraria; Amgen: Honoraria, Other: Travel expenses, Research Funding.