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Despite significant progress in the treatment of patients with diffuse large B-cell lymphoma (DLBCL) and mantle cell lymphoma (MCL), the prognosis of patients with relapsed disease remains poor due to the emergence of drug resistance and subsequent disease progression. Identification of novel targets and therapeutic strategies for these diseases represents an urgent need. Here, we report that both MCL and DLBCL are exquisitely sensitive to transcription-targeting drugs, in particular THZ531, a covalent inhibitor of cyclin-dependent kinase 12 (CDK12). By implementing pharmacogenomics and a cell-based drug screen, we found that THZ531 leads to inhibition of oncogenic transcriptional programs, especially the DNA damage response pathway, MYC target genes and the mTOR-4EBP1-MCL-1 axis, contributing to dramatic lymphoma suppression in vitro. We also identified de novo and established acquired THZ531-resistant lymphoma cells conferred by over-activation of the MEK-ERK and PI3K-AKT-mTOR pathways and upregulation of multidrug resistance-1 (MDR1) protein. Of note, EZH2 inhibitors reversed resistance to THZ531 by competitive inhibition of MDR1 and, in combination with THZ531, synergistically inhibited MCL and DLBCL growth in vitro. Our study indicates that CDK12 inhibitors, alone or together with EZH2 inhibitors, offer promise as novel effective approaches for difficult-to-treat DLBCL and MCL.

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 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.

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.

Simple Summary Multiple myeloma (MM) is a cancer of antibody-producing plasma cells that remains incurable. These cells heavily depend on protein disulfide isomerase, PDIA1, for folding and structural integrity of antibodies and other secretory proteins to avoid unresolvable stress caused if they remain unfolded. High PDIA1 expression confers resistance to proteasome inhibitors and other stressors due to the gain in endoplasmic reticulum (ER) function, while maintaining or increasing vulnerability to PDIA1 inhibition. Here we report the identification and characterization of an orally bioavailable novel PDIA1 inhibitor CCF642-34 that is effective against multiple myeloma in pre-clinical models. PDIA1, the ER resident enzyme essential for the folding of disulfide bond-containing proteins, is upregulated in relapse and refractory myeloma. This increase in PDIA1 confers its sensitivity to CCF642-34, a structurally optimized PDIA1 inhibitor that induces apoptosis in myeloma cells but not in normal bone-marrow-derived CD34+ hematopoietic stem and progenitor cells. Abstract Multiple myeloma is a genetically complex hematologic neoplasia in which malignant plasma cells constantly operate at the maximum limit of their unfolded protein response (UPR) due to a high secretory burden of immunoglobulins and cytokines. The endoplasmic reticulum (ER) resident protein disulfide isomerase, PDIA1 is indispensable for maintaining structural integrity of cysteine-rich antibodies and cytokines that require accurate intramolecular disulfide bond arrangement. PDIA1 expression analysis from RNA-seq of multiple myeloma patients demonstrated an inverse relationship with survival in relapsed or refractory disease, supporting its critical role in myeloma persistence. Using a structure-guided medicinal chemistry approach, we developed a potent, orally bioavailable small molecule PDIA1 inhibitor CCF642-34. The inhibition of PDIA1 overwhelms the UPR in myeloma cells, resulting in their apoptotic cell death at doses that do not affect the normal CD34+ hematopoietic stem and progenitor cells. Bortezomib resistance leads to increased PDIA1 expression and thus CCF642-34 sensitivity, suggesting that proteasome inhibitor resistance leads to PDIA1 dependence for proteostasis and survival. CCF642-34 induces acute unresolvable UPR in myeloma cells, and oral treatment increased survival of mice in the syngeneic 5TGM1 model of myeloma. Results support development of CCF642-34 to selectively target the plasma cell program and overcome the treatment-refractory state in 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.

Inhibitors that target specific kinases or oncoproteins have become popular additions to or replacements for cytotoxic chemotherapies to treat many different types of cancer. However, many tumors lack a discernable target kinase and an amplified oncoprotein and/or rely on several cooperating mechanisms for progression. Thus, combinations of targeted therapies are essential for treating many cancers to avoid the rapid emergence of resistance. In this issue of the JCI, Ren et al. use an elegant kinase activity–profiling method and identify activity of the oncogene polo-like kinase-1 (PLK1) as an important driver of double-hit lymphoma (DHL), an aggressive subgroup of B cell lymphoma characterized by chromosomal translocations involving c-MYC and BCL2 or BCL6. Moreover, PLK1 activity was associated with MYC expression and poor prognosis in DHL patients. PLK1 inhibition with volasertib, alone and in combination with the BCL-2 inhibitor venetoclax, was efficacious in multiple DHL models, including mice harboring DHL patient–derived xenografts. Together, these data support PLK1 as a promising prognostic marker and therapeutic target for DHL.

Ibrutinib resistance, as a result of coordinated rewiring of signaling networks and enforced tumor microenvironment (TME)-lymphoma interactions, drives unrestrained proliferation and disease progression. To combat resistance mechanisms, we must identify the compensatory resistance pathways and the central modulators of reprogramming events. Targeting the transcriptome and kinome reprogramming of lymphoma cells represents a rational approach to mitigate ibrutinib resistance in B cell malignancies. However, with the apparent heterogeneity and plasticity of tumors shown in therapy response, a one size fits all approach may be unattainable. To this end, a reliable and real-time drug screening platform to tailor effective individualized therapies in patients with B cell malignancies is warranted. Here, we describe the complexity of ibrutinib resistance in B cell lymphomas and the current approaches, including a drug screening assay, which has the potential to further explore the mechanisms of ibrutinib resistance and to design effective individualized combination therapies to overcome resistance and disable aggressive lymphomas (see Outstanding Questions).

Background: Elotuzumab (ELO), an immunobiologic therapy targeting SLAMF7/CD319, is effective in the treatment of multiple myeloma (MM) with clinical indications in relapsed/refractory disease in combination with lenalidomide or pomalidomide. ELO binds SLAMF7 on the surface of MM cells to increase immune recognition while also binding SLAMF7 on NK cells resulting in the activation of an effector cell population capable of readily killing via antibody-dependent cellular cytotoxicity (ADCC) mechanisms. ELO efficacy has also been linked to enhanced antibody-dependent cellular phagocytosis (ADCP). Given the dependence of ELO activity on immune elements of the tumor microenvironment (TME), we hypothesized that characterization of the immunologic constituents of the immune TME (iTME) would provide cues capable of predicting clinical efficacy. Methods: Nineteen patients were enrolled on a single-center clinical trial run at Moffitt Cancer Center comparing clinical activity of ELO in combination with low-dose (10mg) vs high-dose (25mg) lenalidomide and dexamethasone in patients with biochemical relapse while on lenalidomide maintenance after first line therapy. Bone marrow aspirate (BMA) and peripheral blood (PB) samples we collected at baseline, after completion of 2 treatment cycles, and at time of progression. Cells isolated from the BMA were analyzed by multiparameter flow cytometry (MPFC) using 4 panels; 3 panels characterized lymphocytes according to maturation, activation and polarization (T H1, T H2, T H17 or T Reg) and a 4 th panel characterized myeloid elements. Data was acquired on a BD Symphony cytometer and analysis was performed using FlowJo software. Results: Patient samples were categorized according to initial therapeutic response. Responders (n=6) achieved >PR at best response, Progressors (n=5) were identified as patients whose disease markers increased >25% and Stable Disease (n=8) was identified as demonstrating Conclusion: Immune profiling of the myeloid and lymphoid components of the baseline BM iTME in MM prior to treatment with ELO reveals distinct patterns of cellular constitution associated with responsiveness or refractoriness to subsequent treatment. While NK cells are known to be important to the ELO mechanism of action, our data suggests that ELO activity is compromised in the absence of immature NK cells. This work suggests that measurable characteristics of the cellular components of the iTME may provide important cues to help predict ELO therapeutic efficacy. These observations will need to be confirmed in a larger data set. Disclosures Shain: Amgen Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sanofi Genzyme: Consultancy, Speakers Bureau; Adaptive Biotechnologies Corporation: Consultancy, Speakers Bureau; AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis Pharmaceuticals Corporation: Consultancy; BMS: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; GlaxoSmithLine, LLC: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen oncology: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Karyopharm Therapeutics Inc.: Honoraria, Research Funding. Baz: BMS, sanofi, Karyopharm, Janssen, AbbVie: Consultancy, Research Funding; Oncopeptides: Consultancy; GlaxoSmithKline: Consultancy, Honoraria; Merck: Research Funding. Nishihori: Novartis: Research Funding; Karyopharm: Research Funding.

Introduction: DARA is approved across lines of therapy for multiple myeloma, including in combination with standard-of-care regimens for NDMM. The CXCR4 receptor antagonist plerixafor is used in conjunction with granulocyte colony-stimulating factor (G-CSF) to increase stem cell mobilization for autologous stem cell transplant (ASCT) and can be given by upfront decision or as a rescue strategy. The phase 2 randomized GRIFFIN study (NCT02874742) evaluates frontline DARA in combination with lenalidomide, bortezomib, and dexamethasone (D-RVd) in transplant-eligible NDMM. In the primary analysis, more pts undergoing stem cell mobilization/collection in the D-RVd group received plerixafor compared with the RVd group (69.5% [66/95] vs 56.3% [45/80]) (Voorhees PM, et al. Blood. 2020). The phase 2 MASTER study (NCT03224507) evaluates DARA plus carfilzomib, lenalidomide, and dexamethasone (D-KRd) in transplant-eligible NDMM (Costa LJ, et al. EHA Library. 2020). Here, we present a summary of stem cell mobilization, collection yields, and ASCT data following frontline DARA-based induction therapy in GRIFFIN and MASTER. Methods: Eligible pts had NDMM and were candidates for ASCT. In GRIFFIN, pts were randomized 1:1 to receive D-RVd or RVd. Pts received 4 induction cycles (21 days) of lenalidomide (R; 25 mg PO on Days 1-14), bortezomib (1.3 mg/m 2 SC on Days 1, 4, 8, and 11), and dexamethasone (d; 40 mg PO QW) ± DARA (16 mg/kg IV QW in Cycles 1-4). After Cycle 4, pts underwent stem cell mobilization with G-CSF ± plerixafor, per institutional standards; if unsuccessful, chemo mobilization was permitted. Pts then received ASCT and subsequently 2 consolidation cycles (21 days) of D-RVd or RVd followed by maintenance therapy with R ± DARA. In the single-arm MASTER study, pts received 4 D-KRd induction cycles, ASCT, and 0, 4 or 8 D-KRd consolidation cycles followed by maintenance therapy with R, based upon achievement of minimal residual disease-negativity. In each 28-day cycle, all pts received carfilzomib (20/56 mg/m 2 IV QW), R (25 mg PO on Days 1-21), d (40 mg PO or IV QW), and DARA (16 mg/kg IV QW for Cycles 1-2, Q2W for Cycles 3-6, and Q4W for Cycles 7+). Mobilization was with G-CSF ± plerixafor as per institutional standards. Results: In GRIFFIN, among 207 (D-RVd, n=104; RVd, n=103) randomized pts, 91.3% (n=95) of D-RVd pts and 77.7% (n=80) of RVd pts underwent stem cell mobilization; of those mobilized, 98.9% (n=94) and 97.5% (n=78) underwent ASCT, respectively. In MASTER, 123 D-KRd pts enrolled and at last follow-up, 91.1% (n=112) underwent stem cell mobilization; of those mobilized, 98.2% (n=110) completed ASCT. In GRIFFIN, 46.3% (n=81) of mobilized pts received plerixafor upfront (D-RVd, 51.6%, n=49; RVd, 40.0%, n=32), and 18.3% (n=32 pts) received rescue plerixafor (D-RVd, 20.0%, n=19; RVd, 16.3%, n=13). In MASTER, 70.5% (n=79) D-KRd pts received upfront plerixafor and 25.9% (n=29) received rescue plerixafor. Median CD34 + cell yield was 8.3 × 10 6/kg for D-RVd and 9.4 ×10 6/kg for RVd in GRIFFIN, 6.0 ×10 6/kg for D-KRd in MASTER, and was numerically higher for pts who received upfront plerixafor. Median days for stem cell collection was 1 for pts receiving RVd and 2 for those receiving D-RVd or D-KRd. Median transplanted CD34 + cell count was 4.2 ×10 6/kg for D-RVd and 4.8 ×10 6/kg for RVd in GRIFFIN, and 3.2 ×10 6/kg for D-KRd in MASTER. In GRIFFIN, 93.7% of D-RVd pts and 98.8% of RVd pts reached the minimum institutional CD34 + threshold to perform a single ASCT, which was comparable to results in MASTER (95.5% of D-KRd pts) after first mobilization attempt; 85.3% of D-RVd pts, 92.5% of RVd pts, and 79.5% of D-KRd pts collected 2 times the minimum threshold of stem cells. Additional data by upfront and rescue plerixafor strategies are shown in the Table. Conclusion: The addition of DARA to proteasome inhibitor/immunomodulatory drug/dexamethasone-based induction therapy has a modest impact on stem cell mobilization, with a lower yield of stem cells and higher median number of days required for collection. Nonetheless, pts were able to undergo transplantation, and most pts collected sufficient stem cells for 2 transplants. Pts who received plerixafor by an upfront decision had numerically higher stem cell yields than pts who received plerixafor by a rescue strategy. An upfront plerixafor strategy for pts receiving DARA-based quadruplet induction therapy should be considered with allowance for additional days of apheresis as needed. Figure 1 Figure 1. Disclosures Chhabra: GSK: Honoraria. Costa: Janssen: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria, Speakers Bureau; Karyopharm: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; BMS: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding, Speakers Bureau. Kaufman: BMS: Consultancy, Research Funding; Fortis Therapeutics: Research Funding; Roche/Genetech, Tecnopharma: Consultancy, Honoraria; Sutro, Takeda: Research Funding; Genentech, AbbVie, Janssen: Consultancy, Research Funding; Novartis: Research Funding; Incyte, celgene: Consultancy; Tecnofarma SAS, AbbVie: Honoraria; Janssen: Honoraria; Incyte, TG Therapeutics: Membership on an entity's Board of Directors or advisory committees; Heidelberg Pharma: Research Funding; Amgen: Research Funding. Sborov: Sanofi: Consultancy; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; SkylineDx: Consultancy; GlaxoSmithKline: Consultancy. Reeves: Incyte Corporation: Honoraria; Takeda: Honoraria; Bristol-Myers Squibb: Speakers Bureau; Pharma Essentia: Consultancy, Honoraria. Rodriguez: Karyopharm: Consultancy, Speakers Bureau; Oncopeptides: Consultancy, Honoraria; Amgen: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau. Chari: Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Research Funding; Antengene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Shattuck Labs: Consultancy, Membership on an entity's Board of Directors or advisory committees; Secura Bio: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS/Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncopeptides: Consultancy, Membership on an entity's Board of Directors or advisory committees; Millenium/Takeda: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; Karyopharm: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen Oncology: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; GlaxoSmithKline: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sanofi Genzyme: Consultancy, Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Research Funding. Silbermann: Sanofi Genzyme: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Anderson: Celgene, BMS, Janssen, GSK, Karyopharm, Oncopeptides, Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Shah: Sutro Biopharma: Research Funding; Janssen: Research Funding; Indapta Therapeutics: Consultancy; CareDx: Consultancy; Sanofi: Consultancy; Kite: Consultancy; Poseida: Research Funding; Amgen: Consultancy; BMS/Celgene: Research Funding; Bluebird Bio: Research Funding; CSL Behring: Consultancy; GSK: Consultancy; Precision Biosciences: Research Funding; Teneobio: Research Funding; Oncopeptides: Consultancy; Nektar: Research Funding; Karyopharm: Consultancy. Bumma: Sanofi: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees. Holstein: Oncopeptides: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Genentech, GSK, Janssen, Secura Bio, Sorrento: Honoraria; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees. Jakubowiak: BMS: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Gracell: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; GSK: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees. Wildes: Carevive: Consultancy; Seattle Genetics: Consultancy; Sanofi: Consultancy; Janssen: Consultancy. Orlowski: CARsgen Therapeutics, Celgene, Exelixis, Janssen Biotech, Sanofi-Aventis, Takeda Pharmaceuticals North America, Inc.: Other: Clinical research funding; Asylia Therapeutics, Inc., BioTheryX, Inc., and Heidelberg Pharma, AG.: Other: Laboratory research funding; Asylia Therapeutics, Inc.: Current holder of individual stocks in a privately-held company, Patents & Royalties; Amgen, Inc., BioTheryX, Inc., Bristol-Myers Squibb, Celgene, Forma Therapeutics, Genzyme, GSK Biologicals, Janssen Biotech, Juno Therapeutics, Karyopharm Therapeutics, Inc., Kite Pharma, Neoleukin Corporation, Oncopeptides AB, Regeneron Pharmaceuticals, I: Membership on an entity's Board of Directors or advisory committees; Amgen, Inc., BioTheryX, Inc., Bristol-Myers Squibb, Celgene, EcoR1 Capital LLC, Genzyme, GSK Biologicals, Janssen Biotech, Karyopharm Therapeutics, Inc., Neoleukin Corporation, Oncopeptides AB, Regeneron Pharmaceuticals, Inc., Sanofi-Aventis, and Takeda P: Consultancy, Honoraria. Shain: Novartis Pharmaceuticals Corporation: Consultancy; Karyopharm Therapeutics Inc.: Honoraria, Research Funding; Janssen oncology: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sanofi Genzyme: Consultancy, Speakers Bureau; GlaxoSmithLine, LLC: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Adaptive Biotechnologies Corporation: Consultancy, Speakers Bureau; AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding. Cowan: Janssen: Consultancy, Research Funding; AbbVie: Consultancy, Research Funding; Sanofi: Consultancy, Research Funding; Cellectar: Consultancy; Harpoon: Research Funding; GSK: Consultancy; Secura Bio: Consultancy; BMS: Research Funding; Nektar: Research Funding. Dholaria: Takeda: Research Funding; Jazz: Speakers Bureau; MEI: Research Funding; Angiocrine: Research Funding; Poseida: Research Funding; Celgene: Speakers Bureau; Pfizer: Research Funding; Janssen: Research Funding. Pei: Janssen: Current Employment, Current equity holder in publicly-traded company. Cortoos: Janssen: Current Employment, Current equity holder in publicly-traded company. Patel: Janssen: Current Employment. Bartlett: Janssen: Current Employment. Vermeulen: Janssen: Current Employment, Current equity holder in publicly-traded company. Lin: Janssen: Current Employment. Richardson: AstraZeneca: Consultancy; Regeneron: Consultancy; Celgene/BMS: Consultancy, Research Funding; Oncopeptides: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; AbbVie: Consultancy; GlaxoSmithKline: Consultancy; Karyopharm: Consultancy, Research Funding; Protocol Intelligence: Consultancy; Janssen: Consultancy; Sanofi: Consultancy; Secura Bio: Consultancy; Jazz Pharmaceuticals: Consultancy, Research Funding. Voorhees: Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Secura Bio: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. OffLabel Disclosure: The specific regimen combination is not yet approved, but individual components are.

INTRODUCTION: Despite proteasome inhibitors (PIs) improving multiple MM (MM) outcomes, patients often become resistant. Identifying mechanisms of resistance with translational potential are an urgent unmet clinical need. Preliminary studies from our group have identified that the therapeutically targetable acid ceramidase, ASAH1, is a key mediator of PI resistance and its presence in extracellular vesicles (EVs) derived from resistant MM cells, confers PI resistance on drug naïve MM cells. METHODS: Nanosight technology, transmission electron microscopy and immunoblot were used to define EVs. Viability and apoptosis assays were used to determine the effects of EVs and inhibitors on resistance acquisition/sensitization to PIs. LC-MS was used to interrogate EV cargo contents. Clinical relevance of ASAH1 was determined in multiple human data cohorts (M2GEN and MMRF CoMMpass). Genetic (shRNA) and pharmacological (ceranib-2) approaches were used to assess the role of ASAH1 mechanistically in vitro and in vivo using multiple isogenic naïve and PI resistant cell lines, patient derived CD138+ MM cells and NSG mouse models. RESULTS: Co-culture of sensitive MM cells with resistant MM-EVs alone significantly protected against PI cytotoxicity. Proteomic profiling revealed high levels of ASAH1 in EVs derived from PI resistant MM cells. Further, we observed ASAH1 is abundant in lysates of multiple PI resistant cell lines compared to their isogenic drug sensitive counterparts. In human datasets, high ASAH1 expression was noted in PI resistant MM patients compared to those newly diagnosed and correlated with significantly shorter survival times. Mechanistically, knockdown of ASAH1 led to reduced conversion of ceramide to sphingosine 1-phosphate (S1-P) and decreased expression/activity of the anti-apoptotic proteins MCL-1, BCL2 and BCL-xL and increases in pro-apoptotic BIM and NOXA. Notably, ASAH1 knockdown also significantly sensitized the cells to PI treatment and this effect was rescued by addition of exogenous S1-P. Pharmacological inhibition of ASAH1 with ceranib-2 also sensitized resistant cells to PI treatment and prevented EV mediated resistance transfer in vitro. This was recapitulated ex vivo with human clinical samples. Our orthotopic in vivo model using PI-resistant U266-PSR cells show that ceranib-2 is highly effective in limiting the growth of PI-resistant disease, protecting against MM induced bone disease, and increasing overall survival compared to both bortezomib and vehicle controls. CONCLUSION: We define the ceramidase ASAH1 as a novel, druggable target for the treatment of PI resistant MM. Disclosures Hampton: M2Gen: Current Employment. Siqueira Silva: AbbVie Inc.: Research Funding; Karyopharm Therapeutics Inc.: Research Funding. Shain: Janssen oncology: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sanofi Genzyme: Consultancy, Speakers Bureau; Karyopharm Therapeutics Inc.: Honoraria, Research Funding; Novartis Pharmaceuticals Corporation: Consultancy; GlaxoSmithLine, LLC: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Adaptive Biotechnologies Corporation: Consultancy, Speakers Bureau; AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Background: Multiple myeloma (MM) is twice as common in Blacks compared to Non-Hispanic (NH) Whites and Hispanics. While treatment and mortality differences have been reported for Black patients with MM compared to NH White patients, there is limited data on Hispanic populations. Furthermore, the factors driving observed differences in MM presentation and treatment responses by race and ethnicity are largely unknown. We investigated demographic, clinical, and molecular features, including tumor mutations and clonal hematopoiesis (CH), in a diverse population of patients with MM to elucidate mechanisms driving clinical disparities. Methods: Patients diagnosed with MM who consented to our institutional biorepository protocol were eligible for inclusion. Demographic and clinical data were obtained from cancer registry and abstracted from electronic medical records. MM tumor cells were purified from bone marrow aspirates by CD138 affinity chromatography. DNA was isolated from tumor cells and whole blood for each patient, and whole exome sequencing (WES) data was generated. Tumor somatic mutations were characterized using paired tumor-normal (blood) WES. CH was classified based on blood-derived somatic mutations, using paired tumors and reference populations as germline comparators. Outcomes included overall survival (OS; date of diagnosis to death/last contact) and progression-free survival (PFS; 1 st-line treatment start to 1 st disease progression/death). Results: A diverse group of MM patients (n=496) were included: NH White (80%), NH Black (10%) and Hispanic (9%). NH Black and Hispanic MM patients had a younger median age at diagnosis (57 and 53 yrs, respectively) compared to NH Whites (63 yrs, p = 0.0001; Fig A). There was no statistical difference in treatment categories received by race/ethnicity. NH Black patients had a longer time to hematopoietic cell transplant (HCT; 376 days) than NH White or Hispanic patients (248 and 270 days, respectively, p = 0.011). There was an improvement in OS for NH Black (HR 0.49, 95% CI 0.30-0.81) and Hispanic (HR 0.66, 95% CI 0.37-1.18) patients compared to NH White patients, but the association was not statistically significant in Hispanics. In univariable analysis, OS was also associated with age at diagnosis, International Staging System (ISS), treatment with HCT, and treatment regimen category. In multivariable analysis, after adjusting for age, ISS, HCT, and treatment category there was no longer a statistically significant association between OS and race/ethnicity. Although a worse PFS was present among Hispanic patients (adjusted HR 1.45, 95% CI 0.99-2.13), there was no statistically significant difference in PFS by race/ethnicity. The most mutated genes in MM tumors were KRAS (24%), NRAS (17%), TP53 (11%), DIS3 (9%), and BRAF (9%) (Fig B). Genes with significantly higher tumor mutation rates in Black compared to NH White patients were SP140 (12% v 4%, p = 0.026), AUTS2 (8% v 2%, p = 0.04), and SETD2 (6% v 1%, p = 0.037). IRF4 was most commonly mutated in Hispanics (11% v 3% in NH White and 0% in Black, p = 0.019). We identified CH using WES in 60 (12%) patients. The most CH mutations were in ASXL1, DNMT3A, and TET2. There was no difference in the prevalence of CH by race/ethnicity (p=0.8). There was a statistically significant difference in OS by race/ethnicity and CH status (Fig C). For NH Black patients, CH (HR 4.36, 95% CI 1.36-14.0) and age at diagnosis (HR 1.08, 95% CI 1.03-1.14) were associated with inferior OS (Fig C). After adjusting for age in multivariable analysis, the positive association with CH status among Black patients was no longer statistically significant (HR 2.72, 95% CI 0.48-15.4). A positive, but not statistically significant, association for PFS in NH White patients with CH was also noted (adjusted HR 1.38, 95% CI 0.95-2.0). Conclusions: This is the first study to examine differences in tumor mutation profiles, CH, and treatment among different racial and ethnic groups of patients diagnosed with MM. Our data suggest that age at diagnosis, tumor mutations, and CH may all contribute to clinical disparities observed in patients with MM. Efforts to expand our cohort and incorporate additional molecular biomarkers, epidemiologic characteristics, and clinical parameters are ongoing with the ultimate goal of elucidating targetable biological mechanisms to personalize management and optimize outcomes for diverse patients diagnosed with MM. Figure 1 Figure 1. Disclosures Hampton: M2Gen: Current Employment. Blue: WebMD: Consultancy; Janssen Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Siqueira Silva: AbbVie Inc.: Research Funding; Karyopharm Therapeutics Inc.: Research Funding. Baz: GlaxoSmithKline: Consultancy, Honoraria; BMS, sanofi, Karyopharm, Janssen, AbbVie: Consultancy, Research Funding; Oncopeptides: Consultancy; Merck: Research Funding. Nishihori: Novartis: Research Funding; Karyopharm: Research Funding. Shain: Janssen oncology: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Karyopharm Therapeutics Inc.: Honoraria, Research Funding; Sanofi Genzyme: Consultancy, Speakers Bureau; Novartis Pharmaceuticals Corporation: Consultancy; GlaxoSmithLine, LLC: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Adaptive Biotechnologies Corporation: Consultancy, Speakers Bureau; AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Introduction. Multiple myeloma (MM) is an all but incurable plasma cell malignancy without predictive biomarkers for approved therapies. Selinexor (SELI), a nuclear export inhibitor targeting exportin 1 (XPO1), is approved with dexamethasone (DEX) with promising SELI-combination studies ongoing. We investigated SELI combinations ex vivo to identify synergistic combinations and companion biomarkers. Methods. We established a platform to perform parallel RNA/exome sequencing and ex vivo drug sensitivity assessment on CD138+ cells from MM patient bone marrow aspirates. At the time of this analysis, 844 different samples with clinical, WES and RNA sequencing data were treated ex vivo with the following agents: SELI (n=75), DEX (192), pomalidomide (POM, 268), elotuzumab (ELO, 21), daratumumab (DARA, 117), SELI+DEX (22), SELI+POM (20), SELI+ELO (21), SELI+DARA (27). Cells were cultured with autologous macrophages, stroma, collagen matrix and patient-derived serum. Cell death (LD50 and AUC) was assessed through digital image analysis. Sequencing was performed through ORIEN/AVATAR. Links between non-synonymous mutations in coding genes and cell death were calculated using T-tests with multiple test correction. Results. Our analysis identified SELI+DEX (number of samples=60, p Conclusions. We observed ex vivo synergy between SELI and DEX, POM, ELO and DARA, and identified expression signatures and mutations associated with response to these agents. Ongoing analysis of additional samples is being performed to validate these results. Citation Format: Praneeth Reddy Sudalagunta, Mark B. Meads, Rafael Renatino Canevarolo, Maria Coelho Silva, Christopher Cubitt, Gabriel DeAvila, Raghunandan Reddy Alugubelli, Constantine N. Logothetis, Amit Kulkarni, Qi Zhang, Oliver Hampton, Christopher J. Walker, Yosef Landesman, Kenneth Shain, Ariosto Siqueira Silva. Characterization of synergistic selinexor combinations with dexamethasone, pomalidomide, elotuzumab, and daratumumab in primary MM cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1061.

Introduction Certain cancers have been shown to express a specific type of drug-resistance involving the export of drug target proteins from the nucleus of the cell to the cytoplasm. This mechanism of drug-resistance has been shown to be important in multiple myeloma (MM) and recently a nuclear export inhibitor, selinexor, has received approval by the FDA in MM. However, general nuclear export signal (NES) inhibitors like selinexor exhibit off-target toxicities when used in patients. We have begun to develop novel drugs that specifically prevent the export of the drug-target topoisomerase IIα (TOP2A). Methods In this study, we 1) generated an atomic homology model of human TOP2A to identify lead compounds targeting the NES of TOP2A; NES inhibitors (NESi), 2) examined binding specifically to TOP2A and inhibit nuclear export by immunofluorescence microscopy (IF) , nuclear-cytoplasmic fractionation, immunoprecipitation (IP), proximity ligation assay (PLA), and Biacore kinetic/affinity assay, and 3) tested the anticancer activity of these compounds in MM in vitro, ex vivo and in vivo MM. Results The atomic homology model of human TOP2A provided the basis for in silico molecular docking screenings of 139,735 small molecules (NCI) (MW < 500) to identify lead compounds targeting the NES of TOP2A. NESi compounds with the highest docking scores were assayed for their ability to induce apoptosis in human MM cells when used with the TOP2 inhibitor doxorubicin (DOX). We isolated four lead compounds that induced apoptosis when used with DOX and that inhibited nuclear export of TOP2A, as shown by IF and nuclear-cytoplasmic fractionation, IP, a PLA and Biacore kinetic/affinity assays. Inhibition by the lead compound, NCI-9138, was specific to TOP2A because p53 trafficking was unaffected and the inhibitor did not affect TOP2A protein expression or enzymatic function (decatenation). This NESi was found to sensitize human MM cells in vitro, in vivo mouse models, and patient MM cells exposed ex vivo to DOX but did not affect normal cell lines. These TOP2A-specific nuclear export inhibitors may potentially lead to a new approach in circumventing drug resistance. Conclusions Our previous studies have shown that TOP2A is exported from the nucleus of MM cells and that this is a significant cause of resistance to DOX therapy. In this study, we have shown that blocking export of TOP2A can sensitize human MM cells to DOX both in vitro, ex vivo in MM cells from patients, and in in vivo mouse models of MM. A drug that specifically inhibits the export of a single cancer-related protein has not been published. We intend to show that this unique approach to cancer-treatment may significantly advance the treatment of MM. This paper also demonstrates a proof of concept for the development of highly specific small molecule inhibitors that could be used as cancer therapeutics. Citation Format: Joel G. Turner, David Ostrov, Nicholas Lawrence, Jana Dawson, Juan Gomez, Alexis Bauer, Mark Meads, Alex Achille, Kenneth Shain. Novel small molecule inhibitors that target the exportin binding pocket of TOP2A for the treatment of multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1228.

Patients with multiple myeloma (MM) have improved treatment options, including immunomodulatory drugs (IMiDs) and proteasome inhibitors (PIs). Despite their efficacy, increased rates of cardiovascular (CV) complications occur in patients exposed to some of these therapies. While previous research has focused on identifying the toxicities inherent to each specific agent, the CV side effects may be potentiated by the combination of PIs and IMiDs plus dexamethasone. We present a patient with MM with recurrent cardiotoxicity only when exposed to combination PI and IMiD-based therapy. We also review the literature in this context, and propose a potential algorithm for cardiotoxicity prevention in this population.

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.

Although recent advances in novel treatment approaches and therapeutics have shifted the treatment landscape of multiple myeloma, it remains an incurable plasma cell malignancy. Growing knowledge of the genome and expressed genomic information characterizing the biologic behavior of multiple myeloma continues to accumulate. However, translation and incorporation of vast molecular understanding of complex tumor biology to deliver personalized and precision treatment to cure multiple myeloma have not been successful to date. Our review focuses on current evidence and understanding of myeloma biology with characterization in the context of genomic and molecular alterations. We also discuss future clinical application of the genomic and molecular knowledge, and more translational research is needed to benefit our myeloma patients.

Summary Background Clonal haemopoiesis of indeterminate potential (CHIP) is an age-associated genetic event linked to increased risk of primary haematological malignancies and increased all-cause mortality, but the prevalence of CHIP in patients who develop therapy-related myeloid neoplasms is unknown. We did this study to investigate whether chemotherapy-treated patients with cancer who have CHIP are at increased risk of developing therapy-related myeloid neoplasms. Methods We did a nested, case-control, proof-of-concept study to compare the prevalence of CHIP between patients with cancer who later developed therapy-related myeloid neoplasms (cases) and patients who did not develop these neoplasms (controls). We identified cases from our internal biorepository of 123 357 patients who consented to participate in the Total Cancer Care biobanking protocol at Moffitt Cancer Center (Tampa, FL, USA) between Jan 1, 2006, and June 1, 2016. We included all individuals who were diagnosed with a primary malignancy, were treated with chemotherapy, subsequently developed a therapy-related myeloid neoplasm, and were 70 years or older at either diagnosis. For inclusion in this study, individuals must have had a peripheral blood or mononuclear cell sample collected before the diagnosis of therapy-related myeloid neoplasm. Controls were individuals who were diagnosed with a primary malignancy at age 70 years or older and were treated with chemotherapy but did not develop therapy-related myeloid neoplasms. Controls were matched to cases in at least a 4:1 ratio on the basis of sex, primary tumour type, age at diagnosis, smoking status, chemotherapy drug class, and duration of follow-up. We used sequential targeted and whole-exome sequencing and described clonal evolution in cases for whom paired CHIP and therapy-related myeloid neoplasm samples were available. The primary endpoint of this study was the development of therapy-related myeloid neoplasm and the primary exposure was CHIP. Findings We identified 13 cases and 56 case-matched controls. The prevalence of CHIP in all patients (23 [33%] of 69 patients) was higher than has previously been reported in elderly individuals without cancer (about 10%). Cases had a significantly higher prevalence of CHIP than did matched controls (eight [62%] of 13 cases vs 15 [27%] of 56 controls, p=0·024; odds ratio 5·75, 95% CI 1·52–25·09, p=0·013). The most commonly mutated genes in cases with CHIP were TET2 (three [38%] of eight patients) and TP53 (three [38%] of eight patients), whereas controls most often had TET2 mutations (six [40%] of 15 patients). In most (four [67%] of six patients) cases for whom paired CHIP and therapy-related myeloid neoplasm samples were available, the mean allele frequency of CHIP mutations had expanded by the time of the therapy-related myeloid neoplasm diagnosis. However, a subset of paired samples (two [33%] of six patients) had CHIP mutations that decreased in allele frequency, giving way to expansion of a distinct mutant clone. Interpretation Patients with cancer who have CHIP are at increased risk of developing therapy-related myeloid neoplasms. The distribution of CHIP-related gene mutations differs between individuals with therapy-related myeloid neoplasm and those without, suggesting that mutation-specific differences might exist in therapy-related myeloid neoplasm risk. Funding Moffitt Cancer Center.

Introduction: Despite some long-term remissions, eventual drug resistance in most patients remains a critical obstacle in the treatment of multiple myeloma (MM). The development of new drugs/drug combinations with novel mechanisms of action are needed for continued improvement in patient outcomes. Initiation of tumor cell death via activation of the intrinsic (mitochondrial) and/or extrinsic (death receptor) apoptotic signaling pathways has been shown to be an effective therapeutic strategy in MM. Venetoclax (Ven) is a selective, small-molecule inhibitor of BCL-2 that exhibits clinical activity in MM cells, particularly in patients harboring the t(11;14) translocation. Navitoclax (Nav) is a small-molecule that targets multiple antiapoptotic BCL-2 family proteins, including BCL-XL, BCL-2, and BCL-W to initiate the intrinsic apoptotic pathway. Eftozanermin alfa (Eftoza) is a novel, second generation TRAIL receptor agonist that induces cell death via death receptor pathways and is under investigation in multiple solid and heme malignancies. In addition, the pan-BET inhibitor mivebresib (Miv) and the BDII selective BET inhibitor ABBV-744 have shown synergistic activity with Ven in cell line models of multiple heme malignancies. Results reported here describe ex vivo drug sensitivities and functional genomic analyses of Ven, Nav, Eftoza, Miv, and ABBV-744 alone or in combination with standard-of-care agents, including bortezomib, carfilzomib, panobinostat, daratumumab, or pomalidomide. Methods: A high-throughput ex vivo drug screening assay using a coculture system of bone marrow (BM)-derived MM and stromal cells was used to assess the sensitivity of MM patient tumor cells (Figure 1A). Paired whole exome sequencing (WES) and RNA sequencing (RNA-seq) analyses were performed. Results: Primary MM patient specimens (n=52) were evaluated in the ex vivo platform, including treatment-naïve, early relapse (1-3 prior lines), and late relapse (4-8 prior lines) patients treated with proteasome inhibitors, immunomodulatory drugs, and monoclonal antibodies. As expected, t(11;14)-positive MM patient specimens were more sensitive than wildtype to Ven ex vivo (D AUC, -18.6, P=0.002), however MM cells harboring amp(1q) were more resistant than wildtype (D AUC, +5.07, P=0.032), suggesting MCL1 (1q21 gene locus) is a key resistance factor to Ven single-agent activity in MM. Gene set enrichment analysis identified B-cell receptor signaling (normalized enrichment score (NES), 1.96, adjusted P=0.010) and MYC pathway (NES, 1.95, adjusted P=0.010) overexpression as predictors of increased sensitivity to Ven ex vivo. A t(11;14) gene expression signature was also generated using a penalized regression model approach in an additional MMWG/ORIEN MM patient cohort (n=155). The t(11;14) predictive gene expression signature was confirmed by correlation with Ven AUC in the ex vivo model. Additional pathway analyses were performed to identify potential predictive markers of sensitivity/resistance for each single agent and drug combination. Although ex vivo activity of Nav was higher in t(11;14) specimens compared to non-t(11;14) (D AUC, -17.8, P=0.011), ex vivo activity in non-t(11;14) specimens was also observed, indicating additional anti-MM activity by cotargeting of BCL-XL and BCL-2. Both Miv and ABBV-744 showed single-agent activity ex vivo, however Miv demonstrated higher activity (median LD50=88.4nM), suggesting that pan-BET inhibition is more effective than BDII-specific BET inhibition in MM. Finally, a novel drug-combination effect analysis was used that identified novel synergistic ex vivo combinations including Ven and panobinostat (P=0.0013) and Eftoza with bortezomib (P=1.8E-7) or carfilzomib (P=7E-4). Additionally, single-agent induction of macrophage-mediated phagocytosis was observed in both Ven and daratumumab, which was synergistic when the 2 drugs were combined (Figure 1B). Conclusion: An ex vivo functional genomic screen of MM patient specimens demonstrated the usefulness of this approach to identify candidate drugs and potential predictive biomarkers for continued evaluation in clinical trials. This approach confirmed known mechanisms of drug sensitivity and identified new ones, including a novel characterized immune-mediated synergy between Ven and daratumumab, and potential combination strategy for Eftoza and proteasome inhibitors. Figure 1 Disclosures Siqueira Silva: Karyopharm: Research Funding; NIH/NCI: Research Funding; AbbVie: Research Funding. Kulkarni:M2GEN: Current Employment. Mitchell:AbbVie: Other: payment for bioinformatics analysis, Research Funding; M2GEN: Current Employment, Research Funding. Dai:Cygnal Therapeutics: Current Employment; M2GEN: Ended employment in the past 24 months. Hampton:M2GEN: Current Employment. Lu:AbbVie: Current Employment, Current equity holder in publicly-traded company. Modi:AbbVie: Current Employment, Other: may own stock or stock options. Motwani:AbbVie: Current Employment, Current equity holder in publicly-traded company. Harb:AbbVie: Current Employment, Other: may hold stock or stock options. Ross:AbbVie: Current Employment, Current equity holder in publicly-traded company. Shain:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; GlaxoSmithKline: Speakers Bureau; Sanofi/Genzyme: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Karyopharm: Research Funding, Speakers Bureau; AbbVie: Research Funding; Takeda: Honoraria, Speakers Bureau; Janssen: Honoraria, Speakers Bureau; Amgen: Speakers Bureau; Adaptive: Consultancy, Honoraria; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. OffLabel Disclosure: While this is a preclinical study, venetoclax for treatment of multiple myeloma is not an approved indication

MCL is an aggressive B-cell lymphoma with poor prognosis due to emergence of drug resistant populations and lymphoma progression. Ibrutinib is a bruton's tyrosine kinase (BTK) inhibitor that was shown to have high response rates in MCL patients. However, as the use of this drug continues to grow in MCL and other B-cell lymphomas, emergence of resistance and fatal progression are of increasing clinical concern. Currently the mechanisms driving IR are poorly understood and no recurrent driver mutations have been identified in MCL. We have modeled acquired resistance to ibrutinib and implemented chemical proteomics and a cell-based drug screen approaches in IR MCL lines and primary samples, we have shown that MCL cells become resistant to ibrutinib through a kinome-adaptive reprogramming mechanism that lead to constitutive activation of the PI3K/AKT/mTOR pathway with increased levels of Myc and sustained transcription activation. The acquired IR MCL cells have increased rates of growth and augmented adhesion to stromal cells. Collectively, our published and preliminary studies indicate that IR MCL relies on global transcriptome remodeling and subsequent kinome reprogramming, leading to molecularly and clinically aggressive phenotype and resistance to ibrutinib therapy. Rather than there being a single mechanism of acquired IR, kinase networks are rewired in a plethora of ways in MCL cells as they become resistant to ibrutinib treatment. Adaptive remodeling of the kinome creates therapeutic challenges, where even combination targeted kinase inhibitor treatments are unlikely to be successful. Here, using genomic, chemical proteomic and drug screen profiling, we report that enhancer remodeling-mediated transcriptional activation and adaptive signaling changes drive the aggressive phenotypes of IR. Accordingly, IR MCL cells are vulnerable to inhibitors of the transcriptional machinery and especially so to inhibitors of cyclin-dependent kinase 9 (CDK9), the catalytic subunit of the positive transcription elongation factor b (P-TEFb) of RNA polymerase II (RNAPII). Further, CDK9 inhibition disables reprogrammed signaling circuits and prevents the emergence of IR in MCL. Finally and importantly, we provided that a novel and facile ex vivo image-based functional drug screening platform could predict clinical therapeutic responses of IR MCL and identified vulnerabilities that can be targeted to disable the evolution of IR. Disclosures Shain: Amgen: Speakers Bureau; GlaxoSmithKline: Speakers Bureau; Adaptive: Consultancy, Honoraria; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Honoraria, Speakers Bureau; Sanofi/Genzyme: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda: Honoraria, Speakers Bureau; AbbVie: Research Funding; Karyopharm: Research Funding, Speakers Bureau. Shah:Kite/Gilead, Jazz, Incyte: Research Funding; Kite/Gilead, Celgene/Juno/BMS, Novartis, Pfizer, Amgen, Spectrum/Acrotech, Precision Biosciences, Beigene, AstraZeneca, Pharmacyclics/Jansen, Adaptive: Honoraria; NCCN: Vice-Chair, Acute Lymphoblastic Leukemia Working Group: Membership on an entity's Board of Directors or advisory committees; Kite/Gilead, Precision Biosciences, Novartis, AstraZeneca: Other: TRAVEL, ACCOMMODATIONS, EXPENSES; Moffitt Cancer Center: Current Employment.

Introduction. Multiple myeloma (MM) is an incurable plasma cell malignancy with a growing list of anti-MM therapeutics. However, the development of predictive biomarkers has yet to be achieved for nearly all MM therapeutics. Selinexor (SELI), a nuclear export inhibitor targeting exportin 1 (XPO1), has been approved with dexamethasone (DEX) in penta-refractory MM. Clinical studies investigating promising SELI- 3 drug combinations are ongoing. Here, we have investigated potential synergistic combinations of SELI and anti-MM agents in terms of ex vivo sensitivity, as well as paired RNAseq and WES to identify companion biomarkers. Methods. MM cells isolated from fresh bone marrow aspirates were tested for drug sensitivity in an organotypic ex vivo drug sensitivity assay, consisting of co-culture with stroma, collagen matrix and patient-derived serum. Single agents were tested at 5 concentrations, while two-drug combinations were tested at fixed ratio of concentrations. LD50 and area under the curve (AUC) were assessed during 96h-exposure as metrics for drug resistance. Drug synergy was calculated as a modified BLISS model. Matching aliquots of MM cells had RNAseq and WES performed through ORIEN/AVATAR project. Geneset enrichment analysis (GSEA) was conducted using both AUC and LD50 as phenotypes for single agents and combinations. Both curated pathways (KEGG and cancer hallmarks) and unsupervised gene clustering were used as genesets. Student t-tests with multiple test correction were used to identify non-synonymous mutations in protein coding genes associated with single agent or combination AUC. Results. For this analysis, a cohort of specimens from 103 patients (48% female, 4% Hispanic, 11% African American) was tested with SELI and/or DEX. with a median of 2 lines of therapy (0-12). A smaller cohort of 37 have been examined with SELI, pomalidomide (POM), elotuzumab (ELO) and daratumumab (DARA). Within this cohort we observed synergy between SELI and DEX, POM and ELO as shown in Figure 1. The volcano plot illustrates the number of samples, maximum drug concentration, as well as magnitude (x- axis) and significance (y- axis) of synergy. Although the SELI-DARA combination trended toward synergy, statistical significance was not achieved. To identify molecular mechanisms and biomarkers associated with sensitivity to SELI and SELI- combinations, we investigated paired RNAseq and WES with ex vivo sensitivity. Initially, we conducted GSEA on two cohorts of primary MM samples tested with SELI alone at 5µM (n=53) and 10µM (n=50). Cell adhesion (KEGG CAMS), inflammatory cytokines (KEGG ASTHMA), and epithelial mesenchymal transition (HALLMARK EMT) were associated with resistance in both cohorts, while the HALLMARK MYC TARGETS was associated with sensitivity (FWER p Conclusions. We observed ex vivo synergy between SELI and DEX, POM and ELO. Molecular analysis of matched ex vivo drug sensitivity, transcriptome and mutational profile identified environment-mediated drug resistance pathways positively correlated with SELI single agent resistance, as well as MYC regulated genes associated with ex vivo sensitivity. We also identified a list of mutations associated with SELI drug resistance and sensitivity, with special emphasis on two novel NES-containing genes, CEP290 and BCL7A. The next step of this project is to analyze transcriptional and mutational patterns associated with ex vivo synergy in the combinations here described, as putative biomarkers for future clinical investigation. Disclosures Shain: Amgen: Speakers Bureau; Adaptive: Consultancy, Honoraria; Karyopharm: Research Funding, Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; GlaxoSmithKline: Speakers Bureau; Janssen: Honoraria, Speakers Bureau; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sanofi/Genzyme: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda: Honoraria, Speakers Bureau; AbbVie: Research Funding. Kulkarni:M2GEN: Current Employment. Zhang:M2GEN: Current Employment. Hampton:M2GEN: Current Employment. Argueta:Karyopharm: Current Employment. Landesman:Karyopharm Therapeutics Inc: Current Employment, Current equity holder in publicly-traded company. Siqueira Silva:AbbVie: Research Funding; NIH/NCI: Research Funding; Karyopharm: Research Funding.

Introduction: DARA, a human IgGκ monoclonal antibody targeting CD38, is approved as monotherapy and in combination with standard-of-care regimens for relapsed/refractory multiple myeloma and NDMM. In the primary analysis of the phase 2 GRIFFIN study (NCT02874742) in patients with transplant-eligible NDMM, DARA plus RVd (D-RVd) significantly improved rates of stringent complete response (sCR) by the end of post-transplant consolidation therapy versus RVd (Voorhees P, Blood 2020). Here, we present updated efficacy and safety results following 12 months of maintenance therapy with lenalidomide (R) or DARA plus R (D-R). Methods: Patients with NDMM eligible for high-dose therapy (HDT) and autologous stem cell transplant (ASCT) were randomized 1:1 to RVd ± DARA, stratified by ISS stage and creatinine clearance rate. Patients received 4 induction cycles, HDT, ASCT, 2 consolidation cycles, and maintenance with R ± DARA for 24 months. During induction and consolidation, patients received R 25 mg PO on Days 1-14; V 1.3 mg/m2 SC on Days 1, 4, 8, and 11; and d 40 mg QW every 21 days. DARA 16 mg/kg IV was given on Days 1, 8, and 15 of Cycles 1-4 and Day 1 of Cycles 5-6. During maintenance (Cycles 7-32), patients received R 10 mg (15 mg in Cycles 10+ if tolerated) on Days 1-21 every 28 days ± DARA 16 mg/kg IV Q8W (or Q4W per patient decision after Amendment 2). The primary endpoint was rate of sCR at the end of post-ASCT consolidation per IMWG criteria, evaluated by a validated computer algorithm. Key secondary endpoints included progression-free survival (PFS) and rate of minimal residual disease (MRD) negativity (10-5 threshold per IMWG criteria) assessed by next-generation sequencing (clonoSEQ; Adaptive Biotechnologies). The primary hypothesis was tested at a 1-sided alpha of 0.10. All secondary analyses were evaluated using a 2-sided P value (alpha 0.05) and were not adjusted for multiplicity. Results: In total, 207 patients were randomized (D-RVd, n=104; RVd, n=103). Baseline demographics and disease characteristics were well balanced between arms. At the end of post-transplant consolidation (median follow-up, 13.5 months) in the response-evaluable population, the sCR rate favored D-RVd versus RVd (42.4% [42/99] vs 32.0% [31/97]; 1-sided P=0.0680). With additional D-R or R maintenance therapy, responses continued to deepen and remained higher for the D-RVd group versus the RVd group. At the 12-months-of-maintenance therapy data cut (median follow-up, 26.7 months), the sCR rate still favored D-RVd versus RVd (63.6% [63/99] vs 47.4% [46/97], 2-sided P=0.0253; Figure). MRD-negativity (10‒5) rates in the ITT population favored D-RVd versus RVd (62.5% [65/104] vs 27.2% [28/103], P Conclusions: After 26.7 months of median follow-up, the addition of DARA to RVd induction and consolidation, followed by D-R maintenance in patients with transplant-eligible NDMM continued to demonstrate deep and improved responses, including higher sCR and MRD negativity rates, compared with lenalidomide alone. Maintenance therapy increased sCR and MRD negativity rates, compared to post-consolidation rates. No new safety concerns were observed with longer follow-up. Support: Alliance Foundation Trials; https://acknowledgments.alliancefound.org; Janssen Oncology Disclosures Kaufman: Tecnopharma: Consultancy, Honoraria; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria; Sanofi/Genyzme: Consultancy, Honoraria; AbbVie: Consultancy; Amgen: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; TG Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Honoraria; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees. Sborov:University of Utah: Current Employment; Celgene, Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Personal fees. Reeves:Incyte: Honoraria; Takeda: Honoraria; Bristol Myers Squibb: Speakers Bureau. Rodriguez:BMS, Takeda, Amgen: Consultancy, Speakers Bureau. Chari:Janssen, Celgene, Novartis, Amgen, Bristol-Myers Squibb, Karyopharm, Sanofi, Genzyme, Seattle Genetics, Oncopeptides, Millennium/Takeda, Antengene, Glaxo Smith Kline, Secura Bio: Consultancy; Janssen, Celgene, Novartis, Amgen, Pharmacyclics, Seattle Genetics, Millennium/Takeda: Research Funding. Silbermann:Karyopharm: Consultancy; Janssen: Consultancy; Sanofi-Aventis: Consultancy, Research Funding. Costa:AbbVie: Consultancy; Sanofi: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Genentech: Consultancy; BMS: Consultancy, Honoraria. Anderson:Amgen: Consultancy, Honoraria, Research Funding; GSK: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Honoraria, Research Funding; Karyopharm: Consultancy, Honoraria, Research Funding. Shah:GSK, Amgen, Indapta Therapeutics, Sanofi, BMS, CareDx, Kite, Karyopharm: Consultancy; BMS, Janssen, Bluebird Bio, Sutro Biopharma, Teneobio, Poseida, Nektar: Research Funding. Efebera:Pharmacyclics: Research Funding; Ohio State University: Current Employment; Celgene: Research Funding; Takeda: Honoraria, Speakers Bureau. Holstein:Sorrento: Consultancy; Adaptive Biotechnologies: Consultancy; Takeda: Consultancy; GSK: Consultancy; Celgene: Consultancy; Genentech: Consultancy; Sanofi: Consultancy; Oncopeptides: Consultancy, Research Funding. Costello:Takeda, Celgene: Consultancy, Honoraria. Jakubowiak:Adaptive, Juno: Consultancy, Honoraria; AbbVie, Amgen, BMS/Celgene, GSK, Janssen, Karyopharm: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Wildes:Seattle Genetics: Consultancy; Carevive Systems: Consultancy; Janssen: Research Funding. Orlowski:Founder of Asylia Therapeutics, Inc., with associated patents and an equity interest, though this technology does not bear on the current submission.: Current equity holder in private company, Patents & Royalties; STATinMED Research: Consultancy; Sanofi-Aventis, Servier, Takeda Pharmaceuticals North America, Inc.: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen, Inc., AstraZeneca, BMS, Celgene, EcoR1 Capital LLC, Forma Therapeutics, Genzyme, GSK Biologicals, Ionis Pharmaceuticals, Inc., Janssen Biotech, Juno Therapeutics, Kite Pharma, Legend Biotech USA, Molecular Partners, Regeneron Pharmaceuticals, Inc.,: Honoraria, Membership on an entity's Board of Directors or advisory committees; Laboratory research funding from BioTheryX, and clinical research funding from CARsgen Therapeutics, Celgene, Exelixis, Janssen Biotech, Sanofi-Aventis, Takeda Pharmaceuticals North America, Inc.: Research Funding. Shain:BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Honoraria, Speakers Bureau; AbbVie: Research Funding; GlaxoSmithKline: Speakers Bureau; Adaptive: Consultancy, Honoraria; Sanofi/Genzyme: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda: Honoraria, Speakers Bureau; Karyopharm: Research Funding, Speakers Bureau; Amgen: Speakers Bureau. Cowan:Nektar: Research Funding; Janssen: Consultancy, Research Funding; Abbvie: Research Funding; Bristol-Myer Squibb: Research Funding; Celgene: Consultancy, Research Funding; Cellectar: Consultancy; Sanofi-Aventis: Consultancy. Lutska:Janssen: Current Employment. Bobba:Janssen: Current Employment. Pei:Janssen: Current Employment, Current equity holder in publicly-traded company. Ukropec:Janssen: Current Employment, Current equity holder in publicly-traded company. Vermeulen:Janssen: Current Employment, Current equity holder in publicly-traded company. Lin:Janssen Scientific Affairs: Current Employment, Current equity holder in publicly-traded company. Richardson:Celgene/BMS, Oncopeptides, Takeda, Karyopharm: Research Funding. Voorhees:TeneoBio: Other: Advisory Board; Oncopeptides: Consultancy, Honoraria; Novartis: Consultancy; Janssen: Other: Advisory Board; GSK: Honoraria; BMS: Other: Advisory Board; Adaptive Biotechnologies: Other: Advisory Board. OffLabel Disclosure: The specific regimen combination is not yet approved, but individual components are.

Mantle cell lymphoma (MCL) and diffuse large B-cell lymphoma (DLBCL) are aggressive hematologic malignancies characterized by the accumulation of lymphoid cells defective in cell apoptosis biology and function. The anti-apoptotic B-cell lymphoma 2 (BCL-2) family proteins are pivotal regulators of the mitochondrial apoptotic pathway and genetic aberrations in these genes are associated with lymphomagenesis and chemotherapeutic resistance. Notably, the anti-apoptotic myeloid cell leukemia 1 (MCL-1) protein is recurrently highly expressed in various kinds of non-Hodgkin's B-cell lymphomas and promotes the survival of lymphoma cells by counteracting pro-apoptotic protein activity. Collectively, these data support the hypothesis that MCL-1 plays a central role in B-cell lymphoma progression and drug resistance. Pharmacologically targeting MCL-1, therefore, represents an attractive strategy to combat these lymphomas. However, previous clinical and pre-clinical data suggest that treatment with single agent anti-BCL-2 family member therapy is associated with rapid acquisition of resistance. To this end, there is a great need to develop and apply selective small molecule MCL-1 inhibitors as part of a first-line therapy or upon emergence of tumor resistance characterized by upregulation of MCL-1 for lymphoma therapy. Here, we exploited the MCL-1 dependency in MCL and DLBCL by implementing pharmacogenomic and chemical proteomic approaches to investigate the molecular drug response and resistance mechanism to MCL-1 inhibitors. In anticipation of the evolution of MCL-1 inhibitor resistance, we modeled MCL-1 inhibitor resistance mechanisms by developing S63845 resistant lines with high doses of S63845 treatment for an extended period in MCL, DLBCL and MCL-derived lines. RNA sequencing and chemical proteomics on paired parental and resistant cells demonstrated that transcriptome and kinome reprograming linked to the MEK and ERK pathways contribute to MCL-1 inhibitor resistance via regulation of the BCL-2 family profile (BCL-2 and BIM), and as such, represent a novel targetable vulnerability in MCL-1 inhibitor resistant lymphoma. Additional analyses revealed synergistic activity of MCL-1 inhibitors (S63845, AZD5991) in combinations with inhibitors of MEK (Trametinib), ERK (SCH772984) and BCR (Ibrutinib) in MCL-1 inhibitor resistant MCL/DLBCL lines and primary samples. These results provide a strong rationale for further evaluation of MCL-1 inhibitor in combination with established therapy in the clinical setting and highlight a potential strategy for overcoming MCL-1 inhibitor resistance. Disclosures Shah: NCCN: Vice-Chair, Acute Lymphoblastic Leukemia Working Group: Membership on an entity's Board of Directors or advisory committees; Kite/Gilead, Precision Biosciences, Novartis, AstraZeneca: Other: TRAVEL, ACCOMMODATIONS, EXPENSES; Kite/Gilead, Jazz, Incyte: Research Funding; Moffitt Cancer Center: Current Employment; Kite/Gilead, Celgene/Juno/BMS, Novartis, Pfizer, Amgen, Spectrum/Acrotech, Precision Biosciences, Beigene, AstraZeneca, Pharmacyclics/Jansen, Adaptive: Honoraria. Shain:AbbVie: Research Funding; GlaxoSmithKline: Speakers Bureau; Takeda: Honoraria, Speakers Bureau; Amgen: Speakers Bureau; Sanofi/Genzyme: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Adaptive: Consultancy, Honoraria; Janssen: Honoraria, Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Karyopharm: Research Funding, Speakers Bureau.

Multiple myeloma (MM) is an incurable cancer of bone marrow-resident plasma cells, which evolves from a premalignant state, MGUS, to a form of active disease characterized by an initial response to therapy, followed by cycles of therapeutic successes and failures, culminating in a fatal multi-drug resistant cancer. The molecular mechanisms leading to disease progression and refractory disease in MM remain poorly understood. To address this question, we have generated a new database, consisting of 1,123 MM biopsies from patients treated at the H. Lee Moffitt Cancer Center. These samples ranged from MGUS to late relapsed/refractory (LR) disease, and were comprehensively characterized genetically (844 RNAseq, 870 WES, 7 scRNAseq), epigenetically (10 single-cell chromatin accessibility, scATAC-seq) and phenotypically (537 samples assessed for ex vivo drug resistance). Mutational analysis identified putative driver genes (e.g. NRAS, KRAS) among the highest frequent mutations, as well as a steady increase in mutational load across progression from MGUS to LR samples. However, with the exception of KRAS, these genes did not reach statistical significance according to FISHER's exact test between different disease stages, suggesting that no single mutation is necessary or sufficient to drive MM progression or refractory disease, but rather a common "driver" biology is critical. Pathway analysis of differentially expressed genes identified cell adhesion, inflammatory cytokines and hematopoietic cell identify as under-expressed in active MM vs. MGUS, while cell cycle, metabolism, DNA repair, protein/RNA synthesis and degradation were over-expressed in LR. Using an unsupervised systems biology approach, we reconstructed a gene expression map to identify transcriptomic reprogramming events associated with disease progression and evolution of drug resistance. At an epigenetic regulatory level, these genes were enriched for histone modifications (e.g. H3k27me3 and H3k27ac). Furthermore, scATAC-seq confirmed genome-wide alterations in chromatin accessibility across MM progression, involving shifts in chromatin accessibility of the binding motifs of epigenetic regulator complexes, known to mediate formation of 3D structures (CTCF/YY1) of super enhancers (SE) and cell identity reprograming (POU5F1/SOX2). Additionally, we have identified SE-regulated genes under- (EBF1, RB1, SPI1, KLF6) and over-expressed (PRDM1, IRF4) in MM progression, as well as over-expressed in LR (RFX5, YY1, NBN, CTCF, BCOR). We have found a correlation between cytogenetic abnormalities and mutations with differential gene expression observed in MM progression, suggesting groups of genetic events with equivalent transcriptomic effect: e.g. NRAS, KRAS, DIS3 and del13q are associated with transcriptomic changes observed during MGUS/SMOL=>active MM transition (Figure 1). Taken together, our preliminary data suggests that multiple independent combinations of genetic and epigenetic events (e.g. mutations, cytogenetics, SE dysregulation) alter the balance of master epigenetic regulatory circuitry, leading to genome-wide transcriptional reprogramming, facilitating disease progression and emergence of drug resistance. Figure 1: Topology of transcriptional regulation in MM depicts 16,738 genes whose expression is increased (red) or decreased (green) in presence of genetic abnormality. Differential expression associated with (A) hotspot mutations and (B) cytogenetic abnormalities confirms equivalence of expected pairs (e.g. NRAS and KRAS, BRAF and RAF1), but also proposes novel transcriptomic dysregulation effect of clinically relevant cytogenetic abnormalities, with yet uncharacterized molecular role in MM. Figure 1 Disclosures Kulkarni: M2GEN: Current Employment. Zhang:M2GEN: Current Employment. Hampton:M2GEN: Current Employment. Shain:GlaxoSmithKline: Speakers Bureau; Amgen: Speakers Bureau; Karyopharm: Research Funding, Speakers Bureau; AbbVie: Research Funding; Takeda: Honoraria, Speakers Bureau; Sanofi/Genzyme: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Honoraria, Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Adaptive: Consultancy, Honoraria; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Siqueira Silva:AbbVie: Research Funding; Karyopharm: Research Funding; NIH/NCI: Research Funding.

Background: Multiple myeloma (MM) is a clonal plasma cell neoplasm typically associated with chronic therapy and resultant potential toxicities, including clonal cytopenias, myelodysplastic syndrome (MDS), or therapy-related myeloid neoplasms (tMN). Early identification of myelodysplasia is important for patient management and outcome. Next generation sequencing (NGS) is playing an ever increasing role in this field. Materials and Methods: The retrospective study was approved by Moffitt institutional review board (IRB). We searched our in-house NGS database with ~6000 patients and clinical databases to identify the patients with MM and sustained cytopenia with accompanying NGS data. The NGS results were analyzed for associations with myeloma and myelodysplasia. Results: Of the 196 identified patients identified (Table 1), there were 114 males (58%) and 82 females (42%) with a median age of 68 years. Eighty-four myeloma patients with cytopenia (43%) were found to have one or more somatic mutations and 112 patients (57%) showed no mutations. The most frequently mutated genes are as following: TP53 (12%), DNMT3A (8%), TET2 (6%), ASXL1 (5%), KRAS (5%), ETV6 (3%), RUNX1 (2%), CUX1 (2%), BCOR (2%), SF3B1 (2%), ZRSR2 (2%), EZH2 (2%), IDH2 (2%), SRSF2 (2%), and BRAF (1%). We divided the patients into four groups according their disease status at the time of NGS testing: 1) patients with myeloma but no myelodysplasia (MM_Only, 105 patients and 53.57%); 2) Patients with myelodysplasia but no overt residual myeloma (Myelodysplasia_Only, 14 patients, 7.14%); 3) Patients with both myeloma and myelodysplasia (MM+Myelodysplasia, 27 patients, 13.78%); 4) Patients with neither myeloma or myelodysplasia (Negative_for_Both, 50 patients, 25.51%). The "Myelodysplasia" in this study is defined as having either overt morphologic dysplasia (>10% of the lineage cells), or equivocal dysplasia but having myeloid-related (non-myeloma) cytogenetic abnormalities. NGS results were not included in the classification to assess the added diagnostic value of NGS. The Mutational profiles of the four disease groups are displayed in Figure 1 and compared in Table 1 and 2. The MM+Myelodysplasia group showed highest percentage of mutations (88.89% of patients tested), followed by Myelodysplasia_Only group (57.14%) and MM_Only group (35.24%), with Negative_for_Both group showing the lowest mutation rate (30.00%). The average number of somatic mutations/case also followed the same order: 1.63, 1.00, 0.48, and 0.36, respectively. Of the 196 patients, 58 patients (29.59%) had no morphologic dysplasia or myeloid-related cytogenetic abnormalities but showed one or more somatic mutations by NGS. These patients harbored clonal cytopenia of uncertain significance (CCUS) clones and would have been missed without NGS testing. Of these 58 patients, retrospective review actually identified 7 patients with morphologic dysplasia and were reclassified as MDS. Further mutational analysis revealed the following interesting findings. ASXL1, DNMT3A, KRAS, and SF3B1 mutations showed highest frequencies in MM+Myelodysplaisa group when compared with other 3 groups (Table 2), indicating a close association with myelodysplasia development in patients with persistent myeloma. In contract, among the 4 groups, RUNX1 mutations were most common in Myelodysplasia_only patients, suggesting a potential alternative pathway for myelodysplasia development in patient with myeloma in remission. It is possible that presence of myeloma clones create different evolution pressure on neoplastic myeloid clones. TP53 mutations were present in MM_Only group, but were much more frequent in patients with MM+Myelodysplasia and Myelodysplasia_only groups. The presence of TP53 mutations might therefore suggest increased risk for myelodysplasia. Finally, TET2 were similar between these groups and therefore not of significant diagnostic value. Conclusion: NGS testing is valuable in identifying CCUS, MDS, or tMN in myeloma patients, especially in those with no morphologic or cytogenetic abnormalities. Statistically significant differences are seen in the mutational profiles of the four groups of patients, suggestive of different roles in myelodysplasia development. Further studies are necessary to better distinguish the origin of these mutations as being derived from the myeloma versus the myeloid components of the disease. Disclosures Hussaini: Stemline: Consultancy; Amgen: Consultancy; Janssen: Consultancy; Adaptive: Consultancy; Boston Biomedical: Consultancy. Shain:Karyopharm: Research Funding, Speakers Bureau; AbbVie: Research Funding; Takeda: Honoraria, Speakers Bureau; Sanofi/Genzyme: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Speakers Bureau; GlaxoSmithKline: Speakers Bureau; Adaptive: Consultancy, Honoraria; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Honoraria, Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Nishihori:Novartis: Other: Research support to institution; Karyopharm: Other: Research support to institution.

Drug resistance remains a critical problem for the treatment of multiple myeloma (MM), which can serve as a specific example for a highly prevalent unmet medical need across almost all cancer types. In MM, the therapeutic arsenal has expanded and diversified, yet we still lack in-depth molecular understanding of drug mechanisms of action and cellular pathways to therapeutic escape. For those reasons, preclinical models of drug resistance are developed and characterized using different approaches to gain insights into tumor biology and elucidate mechanisms of drug resistance. For MM, numerous drugs are used for treatment, including conventional chemotherapies (e.g., melphalan or L-phenylalanine nitrogen mustard), proteasome inhibitors (e.g., Bortezomib), and immunomodulators (e.g., Lenalidomide). These agents have diverse effects on the myeloma cells, and several mechanisms of drug resistance have been previously described. The disparity of these mechanisms and the complexity of these biological processes lead to the formation of complicated hypotheses that require omics approaches for efficient and effective analysis of model systems that can then be interpreted for patient benefit. Here, we describe the combination of metabolomics and proteomics to assess melphalan resistance in MM by examining three specific areas: drug metabolism, modulation of endogenous metabolites to assist in therapeutic escape, and changes in protein activity gauged by ATP probe uptake.

Oprozomib is an oral proteasome inhibitor with activity in multiple myeloma (MM). Our phase 1b/2 study examined the safety and efficacy of oprozomib with dexamethasone in patients with relapsed and refractory MM. Oprozomib was administered with a 5/14 or 2/7 schedule with dexamethasone. Phase 1b primary objectives were to determine the maximum tolerated dose (MTD) and recommended phase 2 dose (RP2D) of oprozomib; phase 2 primary objectives were to determine overall response rate (ORR) and safety/tolerability of the RP2D. Between July 2, 2013, and August 29, 2016, data were available on 65 enrolled patients (5/14 schedule, n = 19; 2/7 schedule, n = 46). In phase 1b, MTD was 180 mg (5/14 schedule) and not reached (2/7 schedule); RP2D was 300 mg (2/7 schedule). In phases 1b and 2, ORR across dosing cohorts (210–330 mg) for the 2/7 schedule was 58.7% overall and 46.4% for bortezomib-refractory patients (n = 28). All patients reported ≥1 treatment-emergent adverse event (AE); the most common AEs were gastrointestinal. Grade ≥3 AEs occurred in 78.9% and 82.6% of patients on the 5/14 and 2/7 schedules, respectively. The oprozomib and dexamethasone combination has encouraging activity and could be an important MM therapy if gastrointestinal tolerability is improved.

e20043 Background: The diagnosis and upfront management of immunoglobulin light chain (AL) amyloidosis have greatly improved in recent years. However, little is known about the presentation, treatment, and outcome of these patients at first relapse/progression (R/P). Methods: All patients with AL amyloidosis who received salvage therapy for first R/P disease at Moffitt Cancer Center between 2008 and 2020 were included in this retrospective review. Definitions of hematologic and organ R/P were based on 2012 consensus. Overall survival was measured from the time of salvage to last follow up/death. Survival was assessed by Kaplan-Meier with log-rank comparison. Results: Sixty-nine patients were included. The median age at diagnosis was 62 years and 61% were male. Upfront therapy included high dose melphalan with autologous transplant in 36% and bortezomib in 52%. At salvage, 19% had disease refractory to upfront therapy and 40% had not achieved an organ response. The median time from upfront to salvage therapy was 22 months. Salvage regimens included proteasome inhibitors, daratumumab and immunomodulatory drugs in 55%, 13% and 22%, respectively. At least a very good partial response and organ response were achieved in 35% (22/62) and 39% (21/54) with measurable disease. The median overall survival was 60 months. Based on salvage indication, patients were classified into hematologic (n = 29) and organ R/P (n = 40), and the latter showed more frequent lambda-light chain disease (59% vs. 83%, p = 0.028) and low difference of involved-uninvolved free light chain at diagnosis (< 50 mg/L, 8% vs. 44%, p = 0.002). Negative prognostic markers for survival included bone marrow plasma cells ≥20% at diagnosis (median 17 months vs. not reached; p < 0.001) and organ, particularly cardiac R/P (median, 31 months vs. not reached; p = 0.003). Salvage ( p = 0.48) or prior regimens ( p = 0.11) did not impact post-salvage survival. Conclusions: Our study highlights the unmet need of salvage in R/P AL amyloidosis in a real-world setting, given the low rate of deep responses regardless of current salvage options. Patients with bone marrow plasma cells ≥20% at diagnosis and organ R/P at salvage had inferior survival, supporting use of intensive upfront regimens for the former and adjustment of therapy if deep response is not achieved.[Table: see text]

SUMMARY Ibrutinib, a bruton’s tyrosine kinase (BTK) inhibitor, provokes robust clinical responses in aggressive mantle cell lymphoma (MCL), yet many patients relapse with lethal Ibrutinib-resistant (IR) disease. Here, using genomic, chemical proteomic, and drug screen profiling, we report that enhancer remodeling-mediated transcriptional activation and adaptive signaling changes drive the aggressive phenotypes of IR. Accordingly, IR MCL cells are vulnerable to inhibitors of the transcriptional machinery and especially so to inhibitors of cyclin-dependent kinase 9 (CDK9), the catalytic subunit of the positive transcription elongation factor b (P-TEFb) of RNA polymerase II (RNAPII). Further, CDK9 inhibition disables reprogrammed signaling circuits and prevents the emergence of IR in MCL. Finally, and importantly, we find that a robust and facile ex vivo image-based functional drug screening platform can predict clinical therapeutic responses of IR MCL and identify vulnerabilities that can be targeted to disable the evolution of IR., In brief Zhao et al. conduct unbiased proteomic, enhancer, and transcriptional profiling in combination with high-throughput drug screening to identify super-enhancer and kinome remodeling as an Ibrutinib resistance mechanism. CDK9 and BRD4 are vulnerabilities for Ibrutinib resistance necessary to sustain super-enhancers, and inhibition of CDK9 or BRD4 prevents the emergence of resistance., Graphical Abstract

// Joel G. Turner 1 , Trinayan Kashyap 2 , Jana L. Dawson 1 , Juan Gomez 1 , Alexis A. Bauer 1 , Steven Grant 3 , Yun Dai 3 , Kenneth H. Shain 1, 4 , Mark Meads 1 , Yosef Landesman 2 , Daniel M. Sullivan 1, 5 1 Chemical Biology and Molecular Medicine Program, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL, USA 2 Karyopharm Therapeutics, Natick, MA, USA 3 Massey Cancer Center, Virginia Commonwealth University, Richmond, VA, USA 4 Department of Malignant Hematology, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL, USA 5 Department of Blood & Marrow Transplantation, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL, USA Correspondence to: Daniel M. Sullivan, email: dan.sullivan@moffitt.org Keywords: XPO1, bortezomib, carfilzomib, multiple myeloma, acquired drug resistance Received: July 26, 2016 Accepted: October 12, 2016 Published: October 28, 2016 ABSTRACT Acquired proteasome-inhibitor (PI) resistance is a major obstacle in the treatment of multiple myeloma (MM). We investigated whether the clinical XPO1-inhibitor selinexor, when combined with bortezomib or carfilzomib, could overcome acquired resistance in MM. PI-resistant myeloma cell lines both in vitro and in vivo and refractory myeloma patient biopsies were treated with selinexor/bortezomib or carfilzomib and assayed for apoptosis. Mechanistic studies included NFκB pathway protein expression assays, immunofluorescence microscopy, ImageStream flow-cytometry, and proximity-ligation assays. IκBα knockdown and NFκB activity were measured in selinexor/bortezomib-treated MM cells. We found that selinexor restored sensitivity of PI-resistant MM to bortezomib and carfilzomib. Selinexor/bortezomib treatment inhibited PI-resistant MM tumor growth and increased survival in mice. Myeloma cells from PI-refractory MM patients were sensitized by selinexor to bortezomib and carfilzomib without affecting non-myeloma cells. Immunofluorescence microscopy, Western blot, and ImageStream analyses of MM cells showed increases in total and nuclear IκBα by selinexor/bortezomib. Proximity ligation found increased IκBα-NFκB complexes in treated MM cells. IκBα knockdown abrogated selinexor/bortezomib-induced cytotoxicity in MM cells. Selinexor/bortezomib treatment decreased NFκB transcriptional activity. Selinexor, when used with bortezomib or carfilzomib, has the potential to overcome PI drug resistance in MM. Sensitization may be due to inactivation of the NFκB pathway by IκBα.

Pomalidomide and low-dose dexamethasone (PomDex) is standard treatment of lenalidomide refractory myeloma patients who have received >2 prior therapies. We aimed to assess the safety and efficacy of the addition of oral weekly cyclophosphamide to standard PomDex. We first performed a dose escalation phase 1 study to determine the recommended phase 2 dose of cyclophosphamide in combination with PomDex (arm A). A randomized, multicenter phase 2 study followed, enrolling patients with lenalidomide refractory myeloma. Patients were randomized (1:1) to receive pomalidomide 4 mg on days 1 to 21 of a 28-day cycle in combination with weekly dexamethasone (arm B) or pomalidomide, dexamethasone, and cyclophosphamide (PomCyDex) 400 mg orally on days 1, 8, and 15 (arm C). The primary end point was overall response rate (ORR). Eighty patients were enrolled (10 in phase 1 and 70 randomized in phase 2: 36 to arm B and 34 to arm C). The ORR was 38.9% (95% confidence interval [CI], 23-54.8%) and 64.7% (95% CI, 48.6-80.8%) for arms B and C, respectively (P = .035). As of June 2015, 62 of the 70 randomized patients had progressed. The median progression-free survival (PFS) was 4.4 (95% CI, 2.3-5.7) and 9.5 months (95% CI, 4.6-14) for arms B and C, respectively (P = .106). Toxicity was predominantly hematologic in nature but was not statistically higher in arm C. The combination of PomCyDex results in a superior ORR and PFS compared with PomDex in patients with lenalidomide refractory multiple myeloma. The trial was registered at www.clinicaltrials.gov as #NCT01432600.

The few studies that have examined rates of acute myeloid leukemia (AML) transformation in lenalidomide‐treated myelodysplastic syndrome (MDS) patients have been limited to deletion 5q MDS. The association between lenalidomide and subsequent primary malignancies (SPMs) in MDS patients has not been evaluated previously. We conducted a retrospective cohort study to evaluate the risk of both SPM and AML in association with lenalidomide. A cohort of MDS patients (n = 1248) treated between 2004 and 2012 at Moffitt Cancer Center were identified, and incident cases of SPM and AML transformation were ascertained. Using a nested case–control design, MDS controls were 1:1 matched to SPM (n = 41) and AML (n = 150) cases, on age and date of MDS diagnosis, gender, follow‐up time, IPSS, and del (5q). Associations between lenalidomide and (1) SPM incidence and (2) AML transformation were estimated with hazards ratios (HR) and 95% confidence intervals (CIs) in the cohort and odds ratios (OR) in the case–control analysis. SPM incidence did not differ significantly between cohort MDS patients treated with (0.7 per 100 person‐years) or without lenalidomide (1.4 per 100 person‐years) (HR = 1.04, 95% CI = 0.40–2.74), whereas a significantly reduced SPM risk was observed in the case–control sample (OR = 0.03, 95% CI =

With contemporary therapeutic strategies in multiple myeloma, heretofore unseen depth and rate of responses are being achieved. These strategies have paralleled improvements in outcome of multiple myeloma patients. The integration of the next generation of proteasome inhibitors and antibody therapeutics promise continued improvements in therapy with the expectation of consistent depth of response not quantifiable by current clinical methods. As such, there is a growing need to develop adequate tools to evaluate deeper disease response after therapy and to refine the response criteria including the minimal residual disease. Several emerging techniques are being evaluated for these purposes including multi-parameter flow cytometry, allele-specific oligonucleotide polymerase chain reaction, next-generation sequencing, and imaging modalities. In this review, we highlight the recent developments and evaluate advantages and limitations of the current technologies to assess minimal residual disease. We also discuss future applications of these methodologies in potentially guiding multiple myeloma treatment decisions.

Most patients with multiple myeloma (MM) respond well to initial therapy, but invariably relapse due to evolution of resistant phenotypes. Here we examine the evolutionary dynamics of proliferation of resistant MM phenotypes during therapy. By applying computational models to data from three clinical trials for newly diagnosed MM patients, we have quantified the size and level of chemoresistance of subpopulations within the tumor burden in 124 patients, prior to and during therapy. Subsequently, we used the computational models to explore an alternative strategy of “adaptive therapy” (AT), which includes defined treatment holidays, to improve the duration of “controlled disease” (CD). Simulations showed that AT could prolong CD in all three trials: 50.0% vs. 11.1% 50-month CD for a single agent approach in older adults (P = 0.0123), 80.4% vs. 58.8% 60-month CD for a multi-agent bortezomib based therapy (P = 0.0082), and 54.0% vs. 24.0% 60-month CD for a multi-agent lenalidomide based therapy (P < 0.0001). Increases in duration of CD resulted from the stabilization of tumor burden, which in turn would delay the growth of chemoresistant sub-populations in patients with partial (PR), or very good partial response (VGPR). These computational algorithms suggest that AT may provide an alternative and feasible therapeutic management strategy in MM.

Background: Multiagent 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. Methods: A 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. Findings: Combination 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. Interpretation: The proposed framework can inform clinical decisions from ex vivo observations, thus providing a path toward personalized therapy using combination regimens. Funding: This 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. Keywords: Clinical synergy, Multiple myeloma, Ex vivo, Pharmacodynamic model, High-throughput combination screening

The treatment of patients with Bing-Neel syndrome (BNS) is not standardized. We included patients with Waldenström macroglobulinemia (WM) and a radiologic and/or cytologic diagnosis of BNS treated with ibrutinib monotherapy. Response assessment was based on criteria for BNS from the 8th International Workshop for WM. Survival from BNS diagnosis (BNS survival), survival from ibrutinib initiation to last follow-up or death (ibrutinib survival), and time from ibrutinib initiation to ibrutinib discontinuation for toxicity, progression, or death (event-free survival [EFS]) were estimated. Twenty-eight patients were included in our study. The median age at BNS diagnosis was 65 years. Ibrutinib was the first line of treatment for BNS in 39% of patients. Ibrutinib was administered orally at a dose of 560 and 420 mg once daily in 46% and 54% of patients, respectively; symptomatic and radiologic improvements were seen in 85% and 60% of patients within 3 months of therapy. At best response, 85% of patients had improvement or resolution of BNS symptoms, 83% had improvement or resolution of radiologic abnormalities, and 47% had cleared the disease in the cerebrospinal fluid. The 2-year EFS rate with ibrutinib was 80% (95% confidence interval [CI], 58%-91%), the 2-year ibrutinib survival rate was 81% (95% CI, 49%-94%), and the 5-year BNS survival rate was 86% (95% CI, 63%-95%). Ibrutinib therapy is effective in patients with BNS and should be considered as a treatment option in these patients.

Combined lenalidomide and dexamethasone is a standard-of-care therapy for the treatment of older adults with multiple myeloma. Lenalidomide monotherapy has not been evaluated in newly diagnosed myeloma patients. We conducted a phase II study, evaluating a response-adapted therapy for older adults newly diagnosed with multiple myeloma without high-risk features who were ineligible for high-dose therapy and stem cell transplant. Patients were started on single-agent lenalidomide, and low-dose dexamethasone was added in the event of progressive disease, in a response-adapted approach. The primary endpoint was progression-free survival (PFS), and the International Myeloma Working Group's uniform response criteria were used to assess response and progression. Twenty-seven patients were enrolled, and 20 (74%) experienced a partial response or better to this response-adapted therapy. After a median follow-up of 69 months, the median PFS was 36 months [95% confidence interval (CI), 29·8 to not reached], and the median overall survival was 65 months (95% CI, 35·3 to not reached). Grade 3/4 adverse events were mainly haematological in nature. This response-adapted therapy in this patient population is feasible and results in durable responses that compare favourably with concurrent lenalidomide and dexamethasone. These results should be validated in prospective studies.