Bone marrow stromal cells dictate lanosterol biosynthesis and ferroptosis of multiple myeloma.

Ferroptosis has been demonstrated a promising way to counteract chemoresistance of multiple myeloma (MM), however, roles and mechanism of bone marrow stromal cells (BMSCs) in regulating ferroptosis of MM cells remain elusive. Here, we uncovered that MM cells were more susceptible to ferroptotic induction under the interaction of BMSCs using in vitro and in vivo models. Mechanistically, BMSCs elevated the iron level in MM cells, thereby activating the steroid biosynthesis pathway, especially the production of lanosterol, a major source of reactive oxygen species (ROS) in MM cells. We discovered that direct coupling of CD40 ligand and CD40 receptor constituted the key signaling pathway governing lanosterol biosynthesis, and disruption of CD40/CD40L interaction using an anti-CD40 neutralizing antibody or conditional depletion of Cd40l in BMSCs successfully eliminated the iron level and lanosterol production of MM cells localized in the Vk*MYC Vk12653 or NSG mouse models. Our study deciphers the mechanism of BMSCs dictating ferroptosis of MM cells and highlights the therapeutic potential of non-apoptosis strategies for managing refractory or relapsed MM patients.

Round Table Discussion on Optimal Clinical Trial Design in Precursor Multiple Myeloma.

SUMMARY: While the current approach to precursor hematologic conditions is to "watch and wait," this may change with the development of therapies that are safe and extend survival or delay the onset of symptomatic disease. The goal of future therapies in precursor hematologic conditions is to improve survival and prevent or delay the development of symptomatic disease while maximizing safety. Clinical trial considerations in this field include identifying an appropriate at-risk population, safety assessments, dose selection, primary and secondary trial endpoints including surrogate endpoints, control arms, and quality-of-life metrics, all of which may enable more precise benefit-risk assessment.

ETV4-Dependent Transcriptional Plasticity Maintains MYC Expression and Results in IMiD Resistance in Multiple Myeloma.

Immunomodulatory drugs (IMiD) are a backbone therapy for multiple myeloma (MM). Despite their efficacy, most patients develop resistance, and the mechanisms are not fully defined. Here, we show that IMiD responses are directed by IMiD-dependent degradation of IKZF1 and IKZF3 that bind to enhancers necessary to sustain the expression of MYC and other myeloma oncogenes. IMiD treatment universally depleted chromatin-bound IKZF1, but eviction of P300 and BRD4 coactivators only occurred in IMiD-sensitive cells. IKZF1-bound enhancers overlapped other transcription factor binding motifs, including ETV4. Chromatin immunoprecipitation sequencing showed that ETV4 bound to the same enhancers as IKZF1, and ETV4 CRISPR/Cas9-mediated ablation resulted in sensitization of IMiD-resistant MM. ETV4 expression is associated with IMiD resistance in cell lines, poor prognosis in patients, and is upregulated at relapse. These data indicate that ETV4 alleviates IKZF1 and IKZF3 dependency in MM by maintaining oncogenic enhancer activity and identify transcriptional plasticity as a previously unrecognized mechanism of IMiD resistance. SIGNIFICANCE: We show that IKZF1-bound enhancers are critical for IMiD efficacy and that the factor ETV4 can bind the same enhancers and substitute for IKZF1 and mediate IMiD resistance by maintaining MYC and other oncogenes. These data implicate transcription factor redundancy as a previously unrecognized mode of IMiD resistance in MM. See related article by Welsh, Barwick, et al., p. 34. See related commentary by Yun and Cleveland, p. 5. This article is featured in Selected Articles from This Issue, p. 4.

Transcriptional Heterogeneity Overcomes Super-Enhancer Disrupting Drug Combinations in Multiple Myeloma.

Multiple myeloma (MM) is a malignancy that is often driven by MYC and that is sustained by IRF4, which are upregulated by super-enhancers. IKZF1 and IKZF3 bind to super-enhancers and can be degraded using immunomodulatory imide drugs (IMiD). Successful IMiD responses downregulate MYC and IRF4; however, this fails in IMiD-resistant cells. MYC and IRF4 downregulation can also be achieved in IMiD-resistant tumors using inhibitors of BET and EP300 transcriptional coactivator proteins; however, in vivo these drugs have a narrow therapeutic window. By combining IMiDs with EP300 inhibition, we demonstrate greater downregulation of MYC and IRF4, synergistic killing of myeloma in vitro and in vivo, and an increased therapeutic window. Interestingly, this potent combination failed where MYC and IRF4 expression was maintained by high levels of the AP-1 factor BATF. Our results identify an effective drug combination and a previously unrecognized mechanism of IMiD resistance. SIGNIFICANCE: These results highlight the dependence of MM on IKZF1-bound super-enhancers, which can be effectively targeted by a potent therapeutic combination pairing IMiD-mediated degradation of IKZF1 and IKZF3 with EP300 inhibition. They also identify AP-1 factors as an unrecognized mechanism of IMiD resistance in MM. See related article by Neri, Barwick, et al., p. 56. See related commentary by Yun and Cleveland, p. 5. This article is featured in Selected Articles from This Issue, p. 4.

Smoldering Multiple Myeloma: Observation Versus Control Versus Cure.

Smoldering multiple myeloma (SMM) is an intermediate clinical stage in the spectrum of monoclonal plasma cell disorders. It represents a heterogeneous clinically defined condition in which some patients (approximately 50%) have monoclonal gammopathy of undetermined significance (premalignancy), and some (approximately 50%) have multiple myeloma (biologic malignancy). Using specific prognostic factors, patients with SMM, in whom malignant transformation has already likely occurred, can be identified. These patients are considered to have high-risk SMM. Patients with newly diagnosed high-risk SMM are candidates for early intervention with lenalidomide or lenalidomide plus dexamethasone for 2 years, or enrollment in clinical trials.

Mode of Progression in Smoldering Multiple Myeloma: A study of 406 patients.

The approach to patients with high-risk smoldering multiple myeloma (SMM) varies among clinicians; while some advocate early intervention, others reserve treatment at progression to multiple myeloma (MM). We aimed to describe the myeloma-defining events (MDEs) and clinical presentations leading to MM diagnosis among SMM patients seen at our institution. We included 406 patients diagnosed with SMM between 2013-2022, seen at Mayo Clinic, Rochester, MN. The 2018 Mayo 20/2/20 criteria were used for risk stratification. Median follow-up was 3.9 years. Among high-risk patients who did not receive treatment in the SMM phase (n=71), 51 progressed by last follow-up; the MDEs included: bone lesions(37%), anemia(35%), hypercalcemia(8%), and renal failure(6%); 24% met MM criteria based on marrow plasmacytosis (≥60%) and/or free light chain ratio (>100); 45% had clinically significant MDEs (hypercalcemia, renal insufficiency, and/or bone lesions). MM diagnosis was made based on surveillance labs/imaging(45%), testing obtained due to provider suspicion for progression(14%), bone pain(20%), and hospitalization/ED presentations due to MM complications/symptoms(4%). The presentation was undocumented in 14%. A high proportion (45%) of patients with high-risk SMM on active surveillance develop end-organ damage at progression. About a quarter of patients who progress to MM are not diagnosed based on routine interval surveillance testing.

High NEK2 expression in myeloid progenitors suppresses T cell immunity in multiple myeloma.

Multiple myeloma (MM) growth is supported by an immune-tolerant bone marrow microenvironment. Here, we find that loss of Never in mitosis gene A (NIMA)-related kinase 2 (NEK2) in tumor microenvironmental cells is associated with MM growth suppression. The absence of NEK2 leads to both fewer tumor-associated macrophages (TAMs) and inhibitory T cells. NEK2 expression in myeloid progenitor cells promotes the generation of functional TAMs when stimulated with MM conditional medium. Clinically, high NEK2 expression in MM cells is associated with increased CD8+ T effector memory cells, while low NEK2 is associated with an IFN-γ gene signature and activated T cell response. Inhibition of NEK2 upregulates PD-L1 expression in MM cells and myeloid cells. In a mouse model, the combination of NEK2 inhibitor INH154 with PD-L1 blockade effectively eliminates MM cells and prolongs survival. Our results provide strong evidence that NEK2 inhibition may overcome tumor immune escape and support its further clinical development.

Impact of cytogenetic abnormalities on the risk of disease progression in solitary bone plasmacytomas.

Most patients with solitary bone plasmacytomas (SBP) progress to multiple myeloma (MM) after definitive radiation therapy as their primary treatment. Whether the presence of high-risk (HR) cytogenetic abnormalities by fluorescence in situ hybridization (FISH) in the clonal plasma cells, obtained either directly from the diagnostic SBP tissue or the corresponding bone marrow examination at the time of diagnosis, is associated with a shorter time to progression (TTP) to MM is unknown. This study evaluated all patients diagnosed with SBP at the Mayo Clinic from January 2012 to July 2022. The presence of del(17p), t(14;16), t(4;14), or +1q (gain or amplification) by FISH in clonal plasma cells was defined as HR. A total of 114 patients were included in this cohort, and baseline FISH was available for 55 patients (48%), of which 22 were classified as HR (40%). The median TTP to MM for patients with SBP and HR FISH was 8 months (95% confidence interval [CI], 6.3-26) compared with 42 months (95% CI, 25-not reached [NR]) in patients with SBP without HR FISH (P < .001). In a multivariate analysis, only HR FISH was a significant predictor for shorter TTP to MM, independent of minimal marrow involvement and an abnormal serum free light chain ratio at diagnosis. Deletion (17p) and gain 1q abnormalities were the most common FISH abnormalities responsible for the short TTP to MM. Thus, assessing for HR FISH abnormalities in clonal plasma cells derived from either the diagnostic SBP tissue or the staging bone marrow examination of patients with newly diagnosed SBP is feasible and prognostic for a shorter TTP to MM.

Prognostic value of early bone marrow MRD status in CAR-T therapy for myeloma.

Bone marrow (BM) assessment of minimal residual disease (MRD) is prognostic for survival in multiple myeloma (MM). BM is still hypocellular at month 1 post CAR-T, thus the value of MRD negative (MRDneg) status at this timepoint is unclear. We examined the impact of month 1 BM MRD status in MM patients who received CART at Mayo Clinic between 8/2016 and 6/2021. Among 60 patients, 78% were BM-MRDneg at month 1; and 85% (40/47) of these patients also had decreased to less than normal level of both involved and uninvolved free light chain (FLC < NL). Patients who achieved CR/sCR had higher rates of month 1 BM-MRDneg and FLC < NL. The rate of sustained BM-MRDneg was 40% (19/47). Rate of conversion from MRDpos to MRDneg was 5%(1/20). At month 1, 38%(18/47) of the BM-MRDneg were hypocellular. Recovery to normal cellularity was observed in 50%(7/14) with a median time to normalization at 12 months (range: 3-Not reached). Compared to Month 1 BM-MRDpos patients, patients who were BM-MRDneg had longer PFS irrespective of BM cellularity [PFS: 2.9 months (95% CI, 1.2-NR) vs. 17.5 months (95% CI, 10.4-NR), p < 0.0001]. Month 1 BM-MRDneg and FLC below normal were associated with prolonged survival. Our data support the continued evaluation of BM early post-CART infusion as a prognostic tool.

Response to COVID-19 Vaccination Post-CAR T Therapy in Patients With Non-Hodgkin Lymphoma and Multiple Myeloma.

COVID-19 adversely affects individuals with cancer. Several studies have found that seroconversion rates among patients with hematologic malignancies are suboptimal when compared to patients without cancer. Patients with non-Hodgkin lymphoma (NHL) and multiple myeloma (MM) are immunocompromised due to impaired humoral and cellular immunity in addition to prescribed immunosuppressive therapy. Chimeric antigen receptor T-cell (CAR T) therapy is now widely used for NHL and MM, but little is known about seroconversion rates after COVID-19 vaccination among these populations. We evaluated SARS-CoV-2 spike-binding IgG antibody levels following COVID-19 vaccination among NHL and MM CAR T therapy recipients. Out of 104 CAR T infusions, 19 patients developed known COVID-19 infection post-CAR T. We tested 17 patients that received CAR T for antibody spike titers post COVID-19 vaccination, only 29 % (n = 5) were able to mount a clinically relevant antibody response (>250 IU/mL).

Preclinical models for prediction of immunotherapy outcomes and immune evasion mechanisms in genetically heterogeneous multiple myeloma.

The historical lack of preclinical models reflecting the genetic heterogeneity of multiple myeloma (MM) hampers the advance of therapeutic discoveries. To circumvent this limitation, we screened mice engineered to carry eight MM lesions (NF-κB, KRAS, MYC, TP53, BCL2, cyclin D1, MMSET/NSD2 and c-MAF) combinatorially activated in B lymphocytes following T cell-driven immunization. Fifteen genetically diverse models developed bone marrow (BM) tumors fulfilling MM pathogenesis. Integrative analyses of ∼500 mice and ∼1,000 patients revealed a common MAPK-MYC genetic pathway that accelerated time to progression from precursor states across genetically heterogeneous MM. MYC-dependent time to progression conditioned immune evasion mechanisms that remodeled the BM microenvironment differently. Rapid MYC-driven progressors exhibited a high number of activated/exhausted CD8+ T cells with reduced immunosuppressive regulatory T (Treg) cells, while late MYC acquisition in slow progressors was associated with lower CD8+ T cell infiltration and more abundant Treg cells. Single-cell transcriptomics and functional assays defined a high ratio of CD8+ T cells versus Treg cells as a predictor of response to immune checkpoint blockade (ICB). In clinical series, high CD8+ T/Treg cell ratios underlie early progression in untreated smoldering MM, and correlated with early relapse in newly diagnosed patients with MM under Len/Dex therapy. In ICB-refractory MM models, increasing CD8+ T cell cytotoxicity or depleting Treg cells reversed immunotherapy resistance and yielded prolonged MM control. Our experimental models enable the correlation of MM genetic and immunological traits with preclinical therapy responses, which may inform the next-generation immunotherapy trials.

Integrated analysis of next generation sequencing minimal residual disease (MRD) and PET scan in transplant eligible myeloma patients.

Minimal residual disease (MRD) assays allow response assessment in patients with multiple myeloma (MM), and negativity is associated with improved survival outcomes. The role of highly sensitive next generation sequencing (NGS) MRD in combination with functional imaging remains to be validated. We performed a retrospective analysis on MM patients who underwent frontline autologous stem cell transplant (ASCT). Patients were evaluated at day 100 post-ASCT with NGS-MRD and positron emission tomography (PET-CT). Patients with ≥ 2 MRD measurements were included in a secondary analysis for sequential measurements. 186 patients were included. At day 100, 45 (24.2%) patients achieved MRD negativity at a sensitivity threshold of 10-6. MRD negativity was the most predictive factor for longer time to next treatment (TTNT). Negativity rates did not differ according to MM subtype, R-ISS Stage nor cytogenetic risk. PET-CT and MRD had poor agreement, with high rates of PET-CT negativity in MRD-positive patients. Patients with sustained MRD negativity had longer TTNT, regardless of baseline risk characteristics. Our results show that the ability to measure deeper and sustainable responses distinguishes patients with better outcomes. Achieving MRD negativity was the strongest prognostic marker and could help guide therapy-related decisions and serve as a response marker for clinical trials.

Revisiting checkpoint inhibitors for myeloma: maintenance after stem cell transplant.

Multiple myeloma is a hematologic malignancy of plasma cells that manifests with bone marrow tumors causing lytic bone lesions. Autologous stem cell transplantation (ASCT) after high-dose chemotherapy and followed by prolonged maintenance therapy with lenalidomide (LEN) is an effective standard-of-care therapy for multiple myeloma. However, most patients ultimately relapse. Rational combination strategies that address immune dysfunction may prolong the durability of ASCT. In this issue of the JCI, Minnie and colleagues investigated the addition of a checkpoint inhibitor to LEN maintenance therapy after ASCT. They found that the immune checkpoint TIGIT was an optimal target in patient samples. In a syngeneic, immunocompetent multiple myeloma mouse model, blockade of TIGIT synergized with LEN maintenance by inducing immune protection, characterized in part by the expansion of polyfunctional T cells in the bone marrow. The treatment enhanced durable antimyeloma efficacy and has translatable implications.

Genomic profiling for clinical decision making in lymphoid neoplasms.

With the introduction of large-scale molecular profiling methods and high-throughput sequencing technologies, the genomic features of most lymphoid neoplasms have been characterized at an unprecedented scale. Although the principles for the classification and diagnosis of these disorders, founded on a multidimensional definition of disease entities, have been consolidated over the past 25 years, novel genomic data have markedly enhanced our understanding of lymphomagenesis and enriched the description of disease entities at the molecular level. Yet, the current diagnosis of lymphoid tumors is largely based on morphological assessment and immunophenotyping, with only few entities being defined by genomic criteria. This paper, which accompanies the International Consensus Classification of mature lymphoid neoplasms, will address how established assays and newly developed technologies for molecular testing already complement clinical diagnoses and provide a novel lens on disease classification. More specifically, their contributions to diagnosis refinement, risk stratification, and therapy prediction will be considered for the main categories of lymphoid neoplasms. The potential of whole-genome sequencing, circulating tumor DNA analyses, single-cell analyses, and epigenetic profiling will be discussed because these will likely become important future tools for implementing precision medicine approaches in clinical decision making for patients with lymphoid malignancies.

The genetic heterogeneity and drug resistance mechanisms of relapsed refractory multiple myeloma.

Multiple myeloma is the second most common hematological malignancy. Despite significant advances in treatment, relapse is common and carries a poor prognosis. Thus, it is critical to elucidate the genetic factors contributing to disease progression and drug resistance. Here, we carry out integrative clinical sequencing of 511 relapsed, refractory multiple myeloma (RRMM) patients to define the disease's molecular alterations landscape. The NF-κB and RAS/MAPK pathways are more commonly altered than previously reported, with a prevalence of 45-65% each. In the RAS/MAPK pathway, there is a long tail of variants associated with the RASopathies. By comparing our RRMM cases with untreated patients, we identify a diverse set of alterations conferring resistance to three main classes of targeted therapy in 22% of our cohort. Activating mutations in IL6ST are also enriched in RRMM. Taken together, our study serves as a resource for future investigations of RRMM biology and potentially informs clinical management.

Phase 2 trial of ixazomib, cyclophosphamide, and dexamethasone for previously untreated light chain amyloidosis.

Bortezomib, a proteasome inhibitor (PI), has shown efficacy in the treatment of newly diagnosed and relapsed light chain (AL) amyloidosis, and is often used in combination with cyclophosphamide and dexamethasone. Ixazomib is the first oral PI to be approved in routine practice but has not yet been evaluated in the upfront treatment setting. Newly diagnosed AL amyloidosis patients with measurable disease and adequate organ function were enrolled. The primary objective was to determine the hematologic response rate of ixazomib in combination with cyclophosphamide and dexamethasone. Treatment was given for 12 cycles, followed by ixazomib maintenance until progression. Thirty-five patients were included; their median age was 67 years, and 69% were male. Major organ involvement included heart (66%) and kidneys (54%). A median of 4 induction cycles (range, 1-12) were administered. The overall hematologic response to induction was 63% and included complete response in 11.4% and very good partial response in 37.1% of patients. One patient was upstaged to complete response during maintenance. The most common reason for going off study was the institution of alternate therapy (61%). With a median follow-up of 29.7 months for the living patients, the 2-year progression-free survival and overall survival were 74% and 78%, respectively. The median time to alternate therapy was 7.5 months. Grade ≥3 hematologic and nonhematologic adverse events occurred in 23% and 49% of patients. Given ixazomib's favorable toxicity profile, which is an important advantage for the typically frail AL population, further evaluation of ixazomib in other combinations in the upfront setting is warranted. This trial was registered at www.clinicaltrials.gov as #NCT01864018.

Genetic subtypes of smoldering multiple myeloma are associated with distinct pathogenic phenotypes and clinical outcomes.

Smoldering multiple myeloma (SMM) is a precursor condition of multiple myeloma (MM) with significant heterogeneity in disease progression. Existing clinical models of progression risk do not fully capture this heterogeneity. Here we integrate 42 genetic alterations from 214 SMM patients using unsupervised binary matrix factorization (BMF) clustering and identify six distinct genetic subtypes. These subtypes are differentially associated with established MM-related RNA signatures, oncogenic and immune transcriptional profiles, and evolving clinical biomarkers. Three genetic subtypes are associated with increased risk of progression to active MM in both the primary and validation cohorts, indicating they can be used to better predict high and low-risk patients within the currently used clinical risk stratification models.