Yamashita, Yusuke

Abstract Background: Multiple myeloma (MM) is always preceded by monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM). While these precursor conditions are asymptomatic, they are not entirely benign and carry a lifelong risk of progression to MM. Unlike other cancers defined by pathology, malignant transformation from MGUS or SMM to MM has so far relied on demonstration of clinical end-organ damage or high tumor cell burden (i.e. SLiM criteria, SV Rajkumar Lancet Onc 2014), as morphology and cytogenetics cannot reliably distinguish them. Methods: To identify genomic features underlying clinical behavior in MGUS and SMM, a total of 374 patients with SMM (n= 290) or MGUS (n=84) with available whole exome (whole exome sequencing, WES, n=190) or whole genome sequencing (whole genome sequencing WGS, n=184) were included in the study. Overall, none of the patients had evidence of SLiM or CRAB criteria at the time of the biopsy. Based on the center of origin and availability of WES and WGS, 227 and 97 patients were used to form our study training and validation set. The study was designed in two parts: 1) defining a new classification for MM precursor conditions; 2) integrating genomics to existing prognostic score to improve accuracy in identifying patients at imminent risk of progression. Overall, 68 patients in the training set were either lost to follow-up or enrolled in a clinical trial and they were only included in the classification part of the study. Results: By leveraging the distribution of established MM-defining genomic events (Maura et al. JCO 2024) in the training set, we developed a workflow that differentiates MGUS and SMM into two genomically distinct entities: one with evidence of neoplastic transformation (i.e., genomic MM) and one without (i.e., genomic MGUS). Overall, 39% of MGUS and 91% of SMM cases had genomic evidence of neoplastic transformation in the training set (i.e., genomic MM). When we applied the same workflow and classification to the validation set, 46% and 83% of MGUS and SMM had evidence of neoplastic transformation, respectively. Importantly, none of the patients classified as genomic-MGUS progressed to MM in either the training or validation cohorts, with a median follow-up of 40 and 61 months, respectively. Moreover, all patients with IMWG 2/20/20 high risk SMM were classified as genomic MM in both the training and validation set. Interestingly, 5 out of 62 (8%) patients enrolled in early intervention trials for high risk SMM were genomically classified as genomic-MGUS. Overall, these data demonstrated that the MM genomic background and neoplastic transformation can be acquired very early in time, even before the SMM phase, in line with what observed in solid tumors. To avoid overestimating the prognostic impact of myeloma-defining genomic events—which are, by definition, enriched in the genomic-MM group compared to genomic-MGUS—we restricted our prognostic analysis to patients in whom transformation has already occurred (genomic-MM). In fact, distinct genomic drivers may predict malignant transformation but not necessarily the risk of imminent progression to MM once transformation has occurred. After running 5-fold cross validations corrected for IMWG 2/20/20, only presence of RAS mutations, genomic events involving MYC, presence of neoplastic copy number variant (CNV) signatures and APOBEC mutagenesis were predictive of early progression in the training set. Adding the presence of high-risk myeloma-defining genomic events to the IMWG 2/20/20 model (i.e., genomic-IMWG) significantly improves the accuracy of predicting MM progression in both the training (c-index 0.69 vs. 0.74) and validation sets (c-index 0.74 vs. 0.79), compared to the IMWG 2/20/20 alone. Conclusion: By leveraging comprehensive genomic profiling integrated with the clinical IMWG 2/20/20 risk score, we propose a novel classification and prognostication system for MM precursor conditions, which will better define patients who may benefit from early therapeutic intervention versus watch-and-wait monitoring.

Abstract Introduction: Despite remarkable advances in treatment, patients with multiple myeloma (MM) continue to suffer relapse. Therefore, the development of novel therapeutic strategies is required. The SWI/SNF chromatin remodeling complex, especially its core ATPase subunits SMARCA2/4, is essential for super-enhancer function and MM cell survival. Furthermore, NSD2, which is overexpressed in t(4;14)-positive MM, has been reported to interact with SMARCA2/4, suggesting differential sensitivity to SMARCA2/4 inhibition. While ATPase inhibitors and PROTAC degraders targeting SMARCA2/4 have demonstrated promising preclinical efficacy in MM and other malignancies, their broader therapeutic relevance remains to be clarified. Methods: To support the clinical development of mSWI/SNF antagonists for MM, we first performed PRISM-based profiling of the anti-proliferative effects of both an mSWI/SNF ATPase PROTAC degrader and a catalytic inhibitor across a panel of 888 cancer cell lines from diverse lineages. Lineage enrichment analysis identified plasma cell myeloma as one of the most sensitive malignancies, with the PROTAC degrader demonstrating particularly potent effects. These were followed by in-depth studies in 35 human myeloma cell lines (HMCLs) using FHD-286 (SMARCA2/4 ATPase inhibitor), AU-24118 (CRBN-based degrader), and AU-15330 (VHL-based degrader). Cell viability was assessed after 5-day treatment by flow cytometry with Annexin V and Live/Dead staining. Additional assays were performed in hCRBN-engineered murine plasmacytoma cell lines and Vk*MYChCRBN MM mouse models. Chromatin profiling employed CUT&Tag with SMARCA2/4 and IKZF1/3 antibodies. Results: FHD-286 and AU-24118 showed potent anti-myeloma activity across most cell lines (median IC₅₀: 4.5 nM and 5.9 nM, respectively). HMCLs with t(4;14), as compared to those without, were more sensitive to FHD-286 (p= 0.0064), with a similar trend for AU-24118 (p=0.09). AU-24118 log2 IC₅₀ values correlated with those of FHD-286 (r=0.62, p=0.0002) and with pomalidomide (r=0.61, p=0.0002), suggesting a shared biology. Indeed, CUT&Tag analysis for SMARCA2/4 and the pomalidomide targets IKZF1/3 showed these factors co-localized at most sites throughout the genome in MM.1S cells, including at DUSP22/IRF4, POU2AF1, and IgH super-enhancers. Treatment with AU-15330, depleted SMARCA2/4 off chromatin at >80% of sites within 8 hours (FDR <0.01), and somewhat surprisingly also depleted IKZF1/3 off chromatin at 70% of bound regions, indicating IKZF1/3 function is dependent upon SWI/SNF. Resultantly, combination studies of SWI/SNF targeting agents and pomalidomide showed a lack of synergy and in some cases antagonism, highlighting the importance of careful evaluation when considering combination with pomalidomide. We and others have demonstrated that murine cells are IMiDs resistant due to species-specific differences in CRBN. Using three distinct mouse plasmacytoma cell lines (+/-hCRBN), hCRBN-expressing cells showed a 100-fold greater sensitivity to AU-24118 compared hCRBN- cell lines. In vivo safety testing of AU-24118 using hCRBN transgenic mice exhibited early mortality within 7 days at doses ≥1.875 mpk and within 3 days at doses ≥7.5 mpk, accompanied by rapid body weight loss. In contrast, a preliminary efficacy study using de novo Vk*MYChCRBN mice, AU-24118 administered at 1 mpk three times per week was well tolerated and led to approximately 40% reduction in serum M-protein within 2 weeks (p<0.05), with no notable weight loss, demonstrating a therapeutic window. Conclusions: Broad PRISM profiling confirms lineage-specific vulnerability of MM to SWI/SNF inhibition, with in vitro and in vivo validation supporting selective efficacy in high-risk t(4;14)-positive subtypes. FHD-286, AU-15330, and AU-24118 demonstrated strong in vitro activity, particularly in t(4;14)-positive HMCLs. This activity was due, in part, to chromatin compaction and decommissioning of lineage and oncogenic enhancers as well as eviction of IKZF1/3 off chromatin. AU-24118 showed high selectivity, with picomolar range IC₅₀ in sensitive HMCLs. Due to species-specific CRBN differences, accurate toxicity evaluation of AU-24118 requires hCRBN-expressing models, and preliminary data indicate in vivo efficacy in the highly predictive Vk*MYChCRBN model. These findings support the potential of SMARCA2/4 targeting agents, especially AU-24118, as promising therapies for MM, particularly in high-risk t(4;14)-positive cases.

Multiple myeloma (MM) is consistently preceded by monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM). While these precursor conditions are asymptomatic, they are not entirely benign and carry a lifelong risk of progression to MM. Unlike other cancers defined by pathology, malignant transformation from MGUS or SMM to MM has so far relied on demonstration of clinical end-organ damage as morphology and cytogenetics cannot reliably distinguish them. In this study, using genomic data from 374 patients with MGUS or SMM (277 training, 97 validation), to our knowledge, we demonstrate for the first time the ability to identify malignant transformation in MGUS and SMM. We introduce the concept of genomic MM and genomic MGUS to differentiate the subsets of MGUS and SMM that are biologically malignant with genomic features indistinguishable from MM from the subset that is premalignant and unlikely to progress to malignancy. Importantly, we find that most SMM has biological features of malignant transformation indistinguishable from MM. As expected, this subset that we consider having genomic MM is associated with a high risk of progression to MM although some patients remained progression-free beyond 5 years. Conversely, 60% of MGUS and 10% of SMM have no evidence of malignant transformation (genomic MGUS), with no progression during follow-up. Integration of genomic features with the 2/20/20 International Myeloma Working Group model significantly improved the prediction of progression among genomic MM. These findings support the use of genomic criteria to refine the classification and the risk stratification in myeloma precursor conditions.