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Multiple myeloma promotes systemic skeletal bone disease that greatly contributes to patient morbidity. Resorption of type I collagen–rich bone matrix by activated osteoclasts results in the release of sequestered growth factors that can drive progression of the disease. Matrix metalloproteinase-13 (MMP13) is a collagenase expressed predominantly in the skeleton by mesenchymal stromal cells (MSC) and MSC-derived osteoblasts. Histochemical analysis of human multiple myeloma specimens also demonstrated that MMP13 largely localizes to the stromal compartment compared with CD138+ myeloma cells. In this study, we further identified that multiple myeloma induces MMP13 expression in bone stromal cells. Because of its ability to degrade type I collagen, we examined whether bone stromal–derived MMP13 contributed to myeloma progression. Multiple myeloma cells were inoculated into wild-type or MMP13–null mice. In independent in vivo studies, MMP13–null mice demonstrated significantly higher overall survival rates and lower levels of bone destruction compared with wild-type controls. Unexpectedly, no differences in type I collagen processing between the groups were observed. Ex vivo stromal coculture assays showed reduced formation and activity in MMP13–null osteoclasts. Analysis of soluble factors from wild-type and MMP13–null MSCs revealed decreased bioavailability of various osteoclastogenic factors including CXCL7. CXCL7 was identified as a novel MMP13 substrate and regulator of osteoclastogenesis. Underscoring the importance of host MMP13 catalytic activity in multiple myeloma progression, we demonstrate the in vivo efficacy of a novel and highly selective MMP13 inhibitor that provides a translational opportunity for the treatment of this incurable disease.Significance:Genetic and pharmacologic approaches show that bone stromal–derived MMP13 catalytic activity is critical for osteoclastogenesis, bone destruction, and disease progression.

All Supplementary Figures and Legends

Supplemental Figures and Tables to augment (1) the mathematical modeling (providing details on specific therapeutics); (2) patient characteristics (treatment history, clinical and predicted response), (3) synergistic activities identified by EMMA; and (4) data processing guides

Multiple myeloma remains treatable but incurable. Despite a growing armamentarium of effective agents, choice of therapy, especially in relapse, still relies almost exclusively on clinical acumen. We have developed a system, Ex vivo Mathematical Myeloma Advisor (EMMA), consisting of patient-specific mathematical models parameterized by an ex vivo assay that reverse engineers the intensity and heterogeneity of chemosensitivity of primary cells from multiple myeloma patients, allowing us to predict clinical response to up to 31 drugs within 5 days after bone marrow biopsy. From a cohort of 52 multiple myeloma patients, EMMA correctly classified 96% as responders/nonresponders and correctly classified 79% according to International Myeloma Working Group stratification of level of response. We also observed a significant correlation between predicted and actual tumor burden measurements (Pearson r = 0.5658, P < 0.0001). Preliminary estimates indicate that, among the patients enrolled in this study, 60% were treated with at least one ineffective agent from their therapy combination regimen, whereas 30% would have responded better if treated with another available drug or combination. Two in silico clinical trials with experimental agents ricolinostat and venetoclax, in a cohort of 19 multiple myeloma patient samples, yielded consistent results with recent phase I/II trials, suggesting that EMMA is a feasible platform for estimating clinical efficacy of drugs and inclusion criteria screening. This unique platform, specifically designed to predict therapeutic response in multiple myeloma patients within a clinically actionable time frame, has shown high predictive accuracy in patients treated with combinations of different classes of drugs. The accuracy, reproducibility, short turnaround time, and high-throughput potential of this platform demonstrate EMMA's promise as a decision support system for therapeutic management of multiple myeloma. Cancer Res; 77(12); 3336–51. ©2017 AACR.

Individual patient EMMA predicitons of actual responses to therapy (companion to Figure 3i).

Supplementary Figures 1-14 from Targeting the Fanconi Anemia/BRCA Pathway Circumvents Drug Resistance in Multiple Myeloma

Supplementary Methods and Materials from Targeting the Fanconi Anemia/BRCA Pathway Circumvents Drug Resistance in Multiple Myeloma

The Fanconi anemia/BRCA (FA/BRCA) DNA damage repair pathway plays a pivotal role in the cellular response to replicative stress induced by DNA alkylating agents and greatly influences drug response in cancer treatment. We recently reported that FA/BRCA genes are overexpressed and causative for drug resistance in human melphalan-resistant multiple myeloma cell lines. However, the transcriptional regulation of the FA/BRCA pathway is not understood. In this report, we describe for the first time a novel function of the NF-κB subunits, RelB/p50, as transcriptional activators of the FA/BRCA pathway. Specifically, our findings point to constitutive phosphorylation of IκB kinase α and subsequent alterations in FANCD2 expression and function as underlying events leading to melphalan resistance in repeatedly exposed multiple myeloma cells. Inhibiting NF-κB by small interfering RNA, blocking the IκB kinase complex with BMS-345541, or using the proteasome inhibitor bortezomib drastically reduced FA/BRCA gene expression and FANCD2 protein expression in myeloma cells, resulting in diminished DNA damage repair and enhanced melphalan sensitivity. Importantly, we also found that bortezomib decreases FA/BRCA gene expression in multiple myeloma patients. These results show for the first time that NF-κB transcriptionally regulates the FA/BRCA pathway and provide evidence for targeting Fanconi anemia–mediated DNA repair to enhance chemotherapeutic response and circumvent drug resistance in myeloma patients. [Cancer Res 2009;69(24):9367–75]

Multiple myeloma (MM) is an osteolytic plasma cell malignancy that, despite being responsive to therapies such as proteasome inhibitors, frequently relapses. Understanding the mechanism and the niches where resistant disease evolves remains of major clinical importance. Cancer cell intrinsic mechanisms and bone ecosystem factors are known contributors to the evolution of resistant MM but the exact contribution of each is difficult to define with currentin vitroandin vivomodels. However, mathematical modeling can help address this gap in knowledge. Here, we describe a novel biology-driven hybrid agent-based model that incorporates key cellular species of the bone ecosystem that control normal bone remodeling and, in MM, yields a protective environment under therapy. Critically, the spatiotemporal nature of the model captures two key features: normal bone homeostasis and how MM interacts with the bone ecosystem to induce bone destruction. We next used the model to examine how the bone ecosystem contributes to the evolutionary dynamics of resistant MM under control and proteasome inhibitor treatment. Our data demonstrates that resistant disease cannot develop without MM intrinsic mechanisms. However, protection from the bone microenvironment dramatically increases the likelihood of developing intrinsic resistance and subsequent relapse. The spatial nature of the model also reveals how the bone ecosystem provides a protective niche for drug sensitive MM cells under treatment, consequently leading to the emergence of a heterogenous and drug resistant disease. In conclusion, our data demonstrates a significant role for the bone ecosystem in MM survival and resistance, and suggests that early intervention with bone ecosystem targeting therapies may prevent the emergence of heterogeneous drug resistant MM.

Syeda Saba Kareem,1 Neena Viswanathan,2 Solmaz Sahebjam,2 Nam D Tran,2 Tyra Gatewood,2 Katherine Tobon,1 Rachid Baz,1 Yolanda Piña,2 Kenneth H Shain,1,3 Sepideh Mokhtari2 1Malignant Hematology Department, Moffitt Cancer Center, Tampa, FL, USA; 2Neuro-Oncology Department, Moffitt Cancer Center, Tampa, FL, USA; 3Tumor Biology Department, Moffitt Cancer Center, Tampa, FL, USACorrespondence: Syeda Saba Kareem, Malignant Hematology Department, Moffitt Cancer Center, 12902 USF Magnolia Drive, Tampa, FL, 33612, Tel +850-321-4383, Fax +813-449-8246, Email syedasaba.kareem@moffitt.orgAbstract: Leukoencephalopathy in the setting of multiple myeloma (MM) is a rare demyelinating condition, with few reported cases in literature. Daratumumab is a CD38 targeted monoclonal antibody that has been widely used for the management of MM. In the absence of central nervous system (CNS) disease, many medication-induced leukoencephalopathy cases reported with MM, including daratumumab-induced, are associated with progressive multifocal leukoencephalopathy (PML) and John Cunningham (JC) virus. Currently, there are no reported cases of daratumumab-induced leukoencephalopathy among patients without CNS involvement or PML. We discuss 2 patients who developed leukoencephalopathy while receiving daratumumab-based therapy without evidence of PML or CNS disease. Both patients had baseline MRIs without significant white matter changes before daratumumab-based therapy. Patients began experiencing neurological deficits about 6 to 8 months after daratumumab-based therapy initiation. One patient passed away before being assessed for improvement of symptoms with daratumumab cessation. The second patient had some stabilization of symptoms after cessation; however, the leukoencephalopathy remained irreversible. As the class of anti-CD38 monoclonal antibodies expands in MM therapy, we highlight a potential treatment complication and the importance of detecting leukoencephalopathy early among patients receiving anti-CD38 therapy. We recommend vigilant monitoring of any new or worsening neurological symptoms to avoid serious complications of irreversible leukoencephalopathy.Keywords: anti-CD38, monoclonal antibody, neurotoxicity, plasma cell disorder, white matter changes, daratumumab, leukoencephalopathy, case report

Motivation Time-lapse microscopy is a powerful technique that relies on images of live cells cultured ex vivo that are captured at regular intervals of time to describe and quantify their behavior under certain experimental conditions. This imaging method has great potential in advancing the field of precision oncology by quantifying the response of cancer cells to various therapies and identifying the most efficacious treatment for a given patient. Digital image processing algorithms developed so far require high-resolution images involving very few cells originating from homogeneous cell line populations. We propose a novel framework that tracks cancer cells to capture their behavior and quantify cell viability to inform clinical decisions in a high-throughput manner. Results The brightfield microscopy images a large number of patient-derived cells in an ex vivo reconstruction of the tumor microenvironment treated with 31 drugs for up to 6 days. We developed a robust and user-friendly pipeline CancerCellTracker that detects cells in co-culture, tracks these cells across time and identifies cell death events using changes in cell attributes. We validated our computational pipeline by comparing the timing of cell death estimates by CancerCellTracker from brightfield images and a fluorescent channel featuring ethidium homodimer. We benchmarked our results using a state-of-the-art algorithm implemented in ImageJ and previously published in the literature. We highlighted CancerCellTracker’s efficiency in estimating the percentage of live cells in the presence of bone marrow stromal cells. Availability and implementation https://github.com/compbiolabucf/CancerCellTracker. Supplementary information Supplementary data are available at Bioinformatics online.

Multiple myeloma is an incurable hematological malignancy that impacts tens of thousands of people every year in the United States. Treatment for eligible patients involves induction, consolidation with stem cell rescue, and maintenance. High-dose therapy with a DNA alkylating agent, melphalan, remains the primary drug for consolidation therapy in conjunction with autologous stem-cell transplantation; as such, melphalan resistance remains a relevant clinical challenge. Here, we describe a proteometabolomic approach to examine mechanisms of acquired melphalan resistance in two cell line models. Drug metabolism, steady-state metabolomics, activity-based protein profiling (ABPP, data available at PRIDE: PXD019725), acute-treatment metabolomics, and western blot analyses have allowed us to further elucidate metabolic processes associated with melphalan resistance. Proteometabolomic data indicate that drug-resistant cells have higher levels of pentose phosphate pathway metabolites. Purine, pyrimidine, and glutathione metabolisms were commonly altered, and cell-line-specific changes in metabolite levels were observed, which could be linked to the differences in steady-state metabolism of naïve cells. Inhibition of selected enzymes in purine synthesis and pentose phosphate pathways was evaluated to determine their potential to improve melphalan's efficacy. The clinical relevance of these proteometabolomic leads was confirmed by comparison of tumor cell transcriptomes from newly diagnosed MM patients and patients with relapsed disease after treatment with high-dose melphalan and autologous stem-cell transplantation. The observation of common and cell-line-specific changes in metabolite levels suggests that omic approaches will be needed to fully examine melphalan resistance in patient specimens and define personalized strategies to optimize the use of high-dose melphalan.

Multiple myeloma (MM) is an incurable disease. Classical chemotherapeutics including bortezomib, melphalan, lenalidomide and thalidomide have greatly enhanced survival times. Unfortunately, patients typically relapse and become refractory with an average survival of 5 years post-diagnosis. Our emerging studies demonstrate a novel role for ULK3 in regulating autophagy in MM, a key program that sustains cell survival under times of stress and has been implicated as a major mechanism of proteasome inhibitor (PI) resistance. MM is known to be highly dependent on autophagy and, currently, specific ULK3 inhibitors are lacking. We posit that by targeting this marker in chemotherapy resistant MM patients, we can circumvent alternative metabolic routes and resensitize to standard of care proapoptotic therapy. We performed RNASeq analysis of CD138+ MM cells derived from patients across the disease stages spectrum (n=815) to confirm the role of ULK3 in disease progression and resistance to chemotherapy. We developed novel inhibitors SG3014/MA9060 that target multiple kinases including ULK3 (EC50 90nM) as well as BRD4. BRD4 is a known driver of MYC and its expression is increased in refractory MM. The BRD4 inhibitor, JQ1, effectively impairs the tumorigenic potential of MM but resistance has also been noted. We determined the efficacy of MA9060 for the treatment of CD138+ MM isolated from naive and refractory patients using a novel ex vivo high throughput platform developed at Moffitt.ULK3 is highly associated with MM stage of the disease. Refractory MM patients have increased autophagy activity with significantly higher expression of ULK3 in refractory patients and in drug resistant cell lines (immunoblotting U266 vs U266-PSR; RPMI-8226 vs RPMI-8226-B25; ABNL vs V10 resistant cells).Genetic ablation of ULK3 by siRNA in U266 and 8226 cell lines results in rapid cessation of the downstream autophagy proteins (ULK1, ATG13, pATG13) and MM cell death within 72h of transduction. Increased concentrations of autophagy inhibitors MA9060/SG3014 progressively decreased CMYC and ULK3 levels, as measured by immunoblotting in U266 cells. In vivo preclinical model of U266Luc tail vein injection proved our drugs are highly effective in reducing tumor dissemination and extending overall survival (CTRL untreated n=65 days vs MA9060 n=110). Importantly, we noted no overt toxicity and protected effect against myeloma-induced bone disease. This novel class of drug works synergistically with PI and can re-sensitize PI resistant disease to these effective therapies. We also show by EMMA ex vivo platform that MA9060 is highly effective for the treatment of CD138+ MM cells isolated from patients with refractory disease.ULK3 represents a novel target for treatment of MM refractory disease. Our dual inhibitors can increase overall survival in vivo and ex vivo, therefore we expect to quickly translate our novel molecules to the clinic. Citation Format: Marilena Tauro, Tao Li, Mark Meads, Praneeth R. Sudalagunta, Raghunandan R. Alugubelli, Nicholas J. Lawrence, Ernst Schonbrunn, Harshani Lawrence, Kenneth H. Shain, Conor C. Lynch. Novel autophagy inhibitory strategies to overcome chemotherapy resistance in multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 452.

Small-scale regulatory networks can model known biological processes; while large-scale genome-wide datasets can identify novel mechanisms. We developed a biophysical modeling framework that combines the accuracy of small-scale networks with the power of large-scale datasets. As a proof of principle, we implemented this framework on a cohort of 844 multiple myeloma (MM) patients’ (and 1092 TCGA breast cancer patients) z-normalized RNAseq data using t-Distributed Stochastic Neighbor Embedding to construct a disease-specific transcriptomic map, where genes closer to each other co-express within the cohort. Fuzzy c-means clustering is carried out to identify clusters of genes that are likely regulated by a common transcription factor (TF). We construct a gene regulatory network (GRN) for each cluster of co-expressing genes on a disease-specific transcriptomic map by identifying upstream TFs for each cluster using publicly available databases ENCODE and ChEA, and kinases that phosphorylate these TFs using PhosphoSitePlus and PhosphoPoint. An exhaustive list of TFs and kinases are reduced to a few key predictor variables using regression tree modeling for each gene in that cluster. This leads to a cascading network of kinases that phosphorylate TFs, which regulate expression of genes in a cluster. We derived a mechanistic model from first-principles to define functional relationships governing the GRN; where transcription, translation, and post-translational modifications are modeled using first-order reversible reaction kinetic equations. The patient-specific rate constants of the model are parametrized by single sample gene set enrichment analysis scores of key KEGG pathways like ribosome, protein synthesis, RNA degradation, etc. The system of differential equations, under steady-state, reduce to an algebraic equation that can predict the expression of every gene in a cluster from the expression of its upstream TFs and kinases alone, which is fitted to RNAseq data of 422 MM patients to estimate undetermined parameters. The remaining patients’ data is used to estimate the accuracy of the model using Pearson’s correlation (model predicted vs actual) coefficient, r. Out of 16,738 genes, 7,936 were predicted accurately (r>0.5), while the remaining genes were shown to have a significant overlap (hypergeometric test; p-value Citation Format: Praneeth Reddy Sudalagunta, Rafael Renatino Canevarolo, Mark Meads, Maria Coelho Silva, Xiaohong Zhao, Raghunandan Reddy Alugubelli, Joon-hyun Song, Erez Persi, Mehdi Damaghi, Kenneth H. Shain, Ariosto Silva. A novel gene regulatory network model identifies master regulators in cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4313.

Multiple myeloma promotes systemic skeletal bone disease that greatly contributes to patient morbidity. Resorption of type I collagen–rich bone matrix by activated osteoclasts results in the release of sequestered growth factors that can drive progression of the disease. Matrix metalloproteinase-13 (MMP13) is a collagenase expressed predominantly in the skeleton by mesenchymal stromal cells (MSC) and MSC-derived osteoblasts. Histochemical analysis of human multiple myeloma specimens also demonstrated that MMP13 largely localizes to the stromal compartment compared with CD138+ myeloma cells. In this study, we further identified that multiple myeloma induces MMP13 expression in bone stromal cells. Because of its ability to degrade type I collagen, we examined whether bone stromal–derived MMP13 contributed to myeloma progression. Multiple myeloma cells were inoculated into wild-type or MMP13–null mice. In independent in vivo studies, MMP13–null mice demonstrated significantly higher overall survival rates and lower levels of bone destruction compared with wild-type controls. Unexpectedly, no differences in type I collagen processing between the groups were observed. Ex vivo stromal coculture assays showed reduced formation and activity in MMP13–null osteoclasts. Analysis of soluble factors from wild-type and MMP13–null MSCs revealed decreased bioavailability of various osteoclastogenic factors including CXCL7. CXCL7 was identified as a novel MMP13 substrate and regulator of osteoclastogenesis. Underscoring the importance of host MMP13 catalytic activity in multiple myeloma progression, we demonstrate the in vivo efficacy of a novel and highly selective MMP13 inhibitor that provides a translational opportunity for the treatment of this incurable disease.Significance:Genetic and pharmacologic approaches show that bone stromal–derived MMP13 catalytic activity is critical for osteoclastogenesis, bone destruction, and disease progression.

While the past decade has seen meaningful improvements in clinical outcomes for multiple myeloma patients, a subset of patients do not benefit from current therapeutics for unclear reasons. Many gene expression-based models of risk have been developed, but each model uses a different combination of genes and often involve assaying many genes making them difficult to implement. We organized the Multiple Myeloma DREAM Challenge, a crowdsourced effort to develop models of rapid progression in newly diagnosed myeloma patients and to benchmark these against previously published models. This effort lead to more robust predictors and found that incorporating specific demographic and clinical features improved gene expression-based models of high risk. Furthermore, post challenge analysis identified a novel expression-based risk marker and histone modifier,PHF19, which featured prominently in several independent models. Lastly, we show that a simple four feature predictor composed of age, International Staging System stage (ISS), and expression ofPHF19andMMSETperforms similarly to more complex models with many more gene expression features included.Key pointsMost comprehensive and unbiased assessment of prognostic biomarkers in MM resulting in a robust and parsimonious model.Identification ofPHF19as the expression based biomarker most strongly associated with rapid progression in MM patients.

The clinical utility of histone/protein deacetylase (HDAC) inhibitors in combinatorial regimens with proteasome inhibitors for patients with relapsed and refractory multiple myeloma (MM) is often limited by excessive toxicity due to HDAC inhibitor promiscuity with multiple HDACs. Therefore, more selective inhibition minimizing off-target toxicity may increase the clinical effectiveness of HDAC inhibitors. We demonstrated that plasma cell development and survival are dependent upon HDAC11, suggesting this enzyme is a promising therapeutic target in MM. Mice lacking HDAC11 exhibited markedly decreased plasma cell numbers. Accordingly, in vitro plasma cell differentiation was arrested in B cells lacking functional HDAC11. Mechanistically, we showed that HDAC11 is involved in the deacetylation of IRF4 at lysine103. Further, targeting HDAC11 led to IRF4 hyperacetylation, resulting in impaired IRF4 nuclear localization and target promoter binding. Importantly, transient HDAC11 knockdown or treatment with elevenostat, an HDAC11-selective inhibitor, induced cell death in MM cell lines. Elevenostat produced similar anti-MM activity in vivo, improving survival among mice inoculated with 5TGM1 MM cells. Elevenostat demonstrated nanomolar ex vivo activity in 34 MM patient specimens and synergistic activity when combined with bortezomib. Collectively, our data indicated that HDAC11 regulates an essential pathway in plasma cell biology establishing its potential as an emerging theraputic vulnerability in MM.

Multiple myeloma (MM) incidence, mortality, and survival vary by race and ethnicity, but the causes of differences remain unclear. We investigated demographic, clinical, and molecular features of diverse MM patients to elucidate mechanisms driving clinical disparities. This study included 495 MM patients (self-reported Hispanic, n = 45; non-Hispanic Black, n = 52; non-Hispanic White, n = 398). Hispanic and non-Hispanic Black individuals had an earlier age of onset than non-Hispanic White individuals (53 and 57 vs 63 years, respectively, P < .001). There were no differences in treatment by race and ethnicity groups, but non-Hispanic Black patients had a longer time to hematopoietic cell transplant than non-Hispanic White patients (376 days vs 248 days; P = .01). Overall survival (OS) was improved for non-Hispanic Black compared with non-Hispanic White patients (HR, 0.50; 95% CI, 0.31-0.81; P = .005), although this association was attenuated after adjusting for clinical features (HR, 0.62; 95% CI, 0.37-1.03; P = .06). Tumor mutations in IRF4 were most common in Hispanic patients, and mutations in SP140, AUTS2, and SETD2 were most common in non-Hispanic Black patients. Differences in tumor expression of BCL7A, SPEF2, and ANKRD26 by race and ethnicity were observed. Clonal hematopoiesis was detected in 12% of patients and associated with inferior OS in non-Hispanic Black patients compared with patients without clonal hematopoiesis (HR, 4.36; 95% CI, 1.36-14.00). This study provides insight into differences in molecular features that may drive clinical disparities in MM patients receiving comparable treatment, with the novel inclusion of Hispanic individuals.

We tested the hypothesis that using CXCR4 inhibition to target the interaction between the tumor cells and the microenvironment leads to sensitization of the tumor cells to apoptosis. Eligibility criteria included multiple myeloma (MM) patients with 1-5 prior lines of therapy. The purposes of the phase I study were to evaluate the safety and maximal-tolerated dose (MTD) of the combination. The treatment-related adverse events and response rate of the combination were assessed in the phase II study. A total of 58 patients were enrolled in the study. The median age of the patients was 63 years (range, 43-85), and 78% of them received prior bortezomib. In the phase I study, the MTD was plerixafor 0.32 mg/kg, and bortezomib 1.3 mg/m2 . The overall response rate for the phase II study was 48.5%, and the clinical benefit rate 60.6%. The median disease-free survival was 12.6 months. The CyTOF analysis demonstrated significant mobilization of plasma cells, CD34+ stem cells, and immune T cells in response to plerixafor. This is an unprecedented study that examines therapeutic targeting of the bone marrow microenvironment and its interaction with the tumor clone to overcome resistance to therapy. Our results indicate that this novel combination is safe and that the objective response rate is high even in patients with relapsed/refractory MM. ClinicalTrials.gov, NCT00903968.

Purpose: The presence of hypoxia in the diseased bone marrow presents a new therapeutic target for multiple myeloma. Evofosfamide (formerly TH-302) is a 2-nitroimidazole prodrug of the DNA alkylator, bromo-isophosphoramide mustard, which is selectively activated under hypoxia. This trial was designed as a phase I/II study investigating evofosfamide in combination with dexamethasone, and in combination with bortezomib and dexamethasone in relapsed/refractory multiple myeloma. Patients and Methods: Fifty-nine patients initiated therapy, 31 received the combination of evofosfamide and dexamethasone, and 28 received the combination of evofosfamide, bortezomib, and dexamethasone. Patients were heavily pretreated with a median number of prior therapies of 7 (range: 2–15). All had previously received bortezomib and immunomodulators. The MTD, treatment toxicity, and efficacy were determined. Results: The MTD was established at 340 mg/m2 evofosfamide + dexamethasone with dose-limiting mucositis at higher doses. For the combination of evofosfamide, bortezomib, and dexamethasone, no patient had a dose-limiting toxicity (DLT) and the recommended phase II dose was established at 340 mg/m2. The most common ≥grade 3 adverse events (AE) were thrombocytopenia (25 patients), anemia (24 patients), neutropenia (15 patients), and leukopenia (9 patients). Skin toxicity was reported in 42 (71%) patients. Responses included 1 very good partial response (VGPR), 3 partial response (PR), 2 minor response (MR), 20 stable disease (SD), and 4 progressive disease (PD) for evofosfamide + dexamethasone and 1 complete response (CR), 2 PR, 1 MR, 18 SD, and 5 PD for evofosfamide + bortezomib + dexamethasone. Disease stabilization was observed in over 80% and this was reflective of the prolonged overall survival of 11.2 months. Conclusions: Evofosfamide can be administered at 340 mg/m2 twice a week with or without bortezomib. Clinical activity has been noted in patients with heavily pretreated relapsed refractory multiple myeloma.

Context.— Measurable (minimal) residual disease (MRD) is an independent prognostic factor for survival outcomes in patients with lymphoid and plasma cell malignancies and has been incorporated into consensus criteria regarding treatment response, strategy, and clinical trial endpoints. clonoSEQ (a next-generation sequencing [NGS]-MRD assay) uses multiplex polymerase chain reaction and NGS to identify clonotypic rearrangements at the immunoglobulin (Ig) H, IgK, IgL, T-cell receptor (TCR)-β, and TCR-γ loci, as well as translocated B-cell lymphoma 1/IgH and 2/IgH sequences for MRD assessment. Additionally, it can be used to confirm diagnoses of cutaneous T-cell lymphoma (CTCL). Objective.— To review the technical aspects of our experience using the clonoSEQ Assay in routine clinical practice. Design.— In this single-center experience, 390 patients with lymphoid and plasma cell malignancies were assessed with the NGS-MRD Assay at a central laboratory. Results.— Median time from arrival of the shipment to initiation of the assay (defined as captured in Adaptive's secure tracking system) was 2.1 hours. Overall, 317 patients had 1 or more samples submitted for sequence identification. Of these, 290 (91.5%) had trackable sequences identified. The median calibration rate of samples by malignancy (where n ≥ 10 samples, excluding CTCL samples) was 88.1%, across a variety of fresh and archived sample sources (177 of 201 samples). TCR-β and/or TCR-γ clonotypes were identified in 40 of 95 samples (42.1%) from 66 patients with suspected CTCL. Conclusions.— This NGS-MRD Assay is a valuable and sensitive tool for monitoring MRD in patients with plasma cell and lymphoid malignancies and assisting in the diagnosis of CTCL.

The plasma cell malignancy multiple myeloma (MM) evolves from a pre-malignant state and remains all but incurable due to emergence of therapy resistance. Despite intensive analyses, mechanisms driving MM progression and refractory disease are poorly understood. Integrating topologic, expression and epigenetic analyses of 1,016 patient specimens, we report super-enhancer core regulatory circuits (SECRCs) that drive and sustain MM epigenetic states. Reprogramming of cell identity and tumor microenvironment genes drive malignant conversion, while alterations in cell cycle and metabolic control genes cause refractory disease. Thus, select epigenetic states drive progression and therapy resistance, providing strategies to prevent and effectively treat MM.

Multiple myeloma is a malignant neoplasm of plasma cells that accumulate in bone marrow, leading to bone destruction and marrow failure. This manuscript discusses the management of patients with solitary plasmacytoma, smoldering multiple myeloma, and newly diagnosed multiple myeloma.

Interactions between the inhibitor of apoptosis protein antagonist LCL161 and the histone deacetylase inhibitor panobinostat (LBH589) were examined in human multiple myeloma (MM) cells. LCL161 and panobinostat interacted synergistically to induce apoptosis in diverse MM cell lines, including those resistant to bortezomib (PS-R). Similar interactions were observed with other histone deacetylase inhibitors (MS-275) or inhibitors of apoptosis protein antagonists (birinapant). These events were associated with downregulation of the noncanonical (but not the canonical) NF-κB pathway and activation of the extrinsic, caspase-8–related apoptotic cascade. Coexposure of MM cells to LCL161/LBH589 induced TRAF3 upregulation and led to TRAF2 and NIK downregulation, diminished expression of BCL-XL, and induction of γH2A.X. Ectopic expression of TRAF2, NIK, or BCL-XL, or short hairpin RNA TRAF3 knock-down, significantly reduced LCL161/LBH589 lethality, as did ectopic expression of dominant-negative FADD. Stromal/microenvironmental factors failed to diminish LCL161/LBH589–induced cell death. The LCL161/LBH589 regimen significantly increased cell killing in primary CD138+ cells (N = 31) and was particularly effective in diminishing the primitive progenitor cell–enriched CD138–/19+/20+/27+ population (N = 23) but was nontoxic to normal CD34+ cells. Finally, combined LCL161/LBH589 treatment significantly increased survival compared with single-agent treatment in an immunocompetent 5TGM1 murine MM model. Together, these findings argue that LCL161 interacts synergistically with LBH589 in MM cells through a process involving inactivation of the noncanonical NF-κB pathway and activation of the extrinsic apoptotic pathway, upregulation of TRAF3, and downregulation of TRAF2/BCL-XL. Notably, this regimen overcomes various forms of resistance, is active against primary MM cells, and displays significant in vivo activity. This strategy warrants further consideration in MM.

Lenalidomide, bortezomib, and dexamethasone (RVd) followed by autologous stem cell transplantation (ASCT) is standard frontline therapy for transplant-eligible patients with newly diagnosed multiple myeloma (NDMM). The addition of daratumumab (D) to RVd (D-RVd) in transplant-eligible NDMM patients was evaluated. Patients (N = 207) were randomized 1:1 to D-RVd or RVd induction (4 cycles), ASCT, D-RVd or RVd consolidation (2 cycles), and lenalidomide or lenalidomide plus D maintenance (26 cycles). The primary end point, stringent complete response (sCR) rate by the end of post-ASCT consolidation, favored D-RVd vs RVd (42.4% vs 32.0%; odds ratio, 1.57; 95% confidence interval, 0.87-2.82; 1-sided P = .068) and met the prespecified 1-sided α of 0.10. With longer follow-up (median, 22.1 months), responses deepened; sCR rates improved for D-RVd vs RVd (62.6% vs 45.4%; P = .0177), as did minimal residual disease (MRD) negativity (10−5 threshold) rates in the intent-to-treat population (51.0% vs 20.4%; P < .0001). Four patients (3.8%) in the D-RVd group and 7 patients (6.8%) in the RVd group progressed; respective 24-month progression-free survival rates were 95.8% and 89.8%. Grade 3/4 hematologic adverse events were more common with D-RVd. More infections occurred with D-RVd, but grade 3/4 infection rates were similar. Median CD34+ cell yield was 8.2 × 106/kg for D-RVd and 9.4 × 106/kg for RVd, although plerixafor use was more common with D-RVd. Median times to neutrophil and platelet engraftment were comparable. Daratumumab with RVd induction and consolidation improved depth of response in patients with transplant-eligible NDMM, with no new safety concerns. This trial was registered at www.clinicaltrials.gov as #NCT02874742.