Landgren, Ola

Abstract Studies have reported conflicting findings regarding the contribution of germline variants or somatic genomic drivers to racial disparities in multiple myeloma. To comprehensively investigate somatic drivers in relation to inherited genetics in multiple myeloma, we combined newly sequenced whole-genome sequencing data with publicly available datasets (total n = 1,286). Overall, we did not identify germline or somatic genomic differences that explain the different risk of developing multiple myeloma between patients with genetic similarity to African (AFR) or European (EUR) reference populations. A difference in the detectability and timing of APOBEC-associated and germinal center mutational activity was observed. Integrating epidemiologic data and mutational signature-based temporal estimates, we challenge the assumption that individuals in the AFR group develop multiple myeloma at a younger age. Finally, we demonstrate that, with equal access to efficacious therapies, patients in the AFR and EUR groups have equivalent clinical outcomes. Significance: Multiple myeloma is reported to occur at higher rates in individuals who self-identify as non-Hispanic Black. In this large dataset, genomic drivers occur at the same rate among ancestry groups, except for APOBEC mutagenesis. With equivalent therapy, clinical outcomes did not differ for patients grouped by genetic ancestry similarity.

Abstract Multiple myeloma (MM) with t(11;14)(CCND1;IGH) remains the only subset sensitive to the BCL2 inhibitor venetoclax. However, not all patients with t(11;14)(CCND1;IGH) respond to treatment, and some progress early after initial response. To investigate this, we examined 44 whole-genome and whole-exome sequencing data samples from 34 patients with t(11;14) MM treated with venetoclax. The presence of mutations in the RAS pathway was strongly associated with shortened progression-free survival (PFS) and was validated in an independent cohort of 21 patients with MM. The presence of 1q gain was also associated with shorter PFS in patients without RAS mutations. In 10 patients with paired prevenetoclax and postvenetoclax treatment samples, postvenetoclax progression was recurrently driven by the selection of genomic events in the BCL2/MCL1 and RAS pathways and of high-risk features (eg, loss of TP53 and CDKN2C). Overall, our study shows that comprehensive genomic profiling can identify most mechanisms underlying resistance to BCL2 inhibition in t(11;14)(CCND1;IGH) MM.

ABSTRACT Chimeric antigen receptor T-cell (CART) and T-cell engager (TCE) therapies targeting B-cell maturation antigen (BCMA) are transforming the treatment landscape for relapsed multiple myeloma (MM). However, despite impressive initial response rates, most patients eventually relapse. To investigate this unmet medical need, we applied whole-genome sequencing (WGS) to MM cells from cohorts of 102 relapsed patients treated with anti-BCMA CART and TCE therapies. Several genomic alterations were associated with clinical outcomes, particularly primary refractoriness, including high genomic complexity and mutations in genes regulating plasma cell identity, which predicted resistance to therapy. Single-cell RNA sequencing further revealed that MM cells from refractory patients exhibited high proliferation signatures and reduced expression of TNFRSF17 (encoding BCMA), while were less enriched for plasma cell–associated transcriptional programs, a phenomenon we term “plasma cell identity escape.” This profile was strongly associated with immune dysregulation of CD8 T cells including increased activation and exhaustion. This evolution of MM toward a more proliferative and lineage-divergent state, refractory to the anti-BCMA T-cell redirecting therapies, was functionally validated in preclinical MM mouse models. Collectively, our results comprehensively define the cellular and molecular mechanisms underlying primary resistance to anti-BCMA therapies.

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 Consumption of a Western diet and high body mass index (BMI) are risk factors for progression from premalignant phenotypes to multiple myeloma, a hematologic cancer. In the NUTRIVENTION trial (NCT04920084), we administered a high-fiber, plant-based diet (HFPBD; meals for 12 weeks, coaching for 24 weeks) to 23 participants with myeloma precursor states and elevated BMI. The intervention was feasible and improved quality of life and modifiable risk factors: metabolic (BMI, insulin resistance), microbiome (diversity, composition), and immune (inflammation, monocyte subsets). Disease progression trajectory improved (n = 2) or was stable. Findings were translated to Vk*MYC mice modeling the myeloma precursor state, in which a high-fiber diet (HFD) delayed disease progression through improved metabolism and microbiome composition, leading to increased short-chain fatty acid production that reinvigorated antitumor immunity and inhibited tumor growth. These effects from fiber consumption were independent of calorie restriction and weight loss. A HFD is a low-risk intervention that may delay progression to myeloma. Significance: A HFPBD in participants with precursor plasma cell disorders and an elevated BMI improved metabolic, microbiome, and immune biomarkers of disease. In a subset, it may delay progression to myeloma. In mouse models, a HFD delayed progression to myeloma independent of calorie restriction. See related commentary by Fairfield and Reagan, p. 623

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.

Abstract Monoclonal antibodies targeting CD38 are a therapeutic mainstay in multiple myeloma (MM). Although they have contributed to improved outcomes, most patients still experience disease relapse, and little is known about tumor-intrinsic mechanisms of resistance to these drugs. Antigen escape has been implicated as a mechanism of tumor-cell evasion in immunotherapy. Yet, it is unknown whether MM cells can develop permanent resistance to anti-CD38 antibodies by acquiring genomic events leading to biallelic disruption of the CD38 gene locus. Here, we analyzed whole-genome and whole-exome sequencing data from patients 701 newly diagnosed MM, 67 patients at relapse with naivety to anti-CD38 antibodies, and 50 patients collected at relapse after anti-CD38 antibodies. We report a loss of CD38 in 10 of 50 patients (20%) after CD38 therapy, 3 of whom exhibited a loss of both copies. Two of these cases showed convergent evolution in which distinct subclones independently acquired similar advantageous variants. Functional studies on missense mutations involved in biallelic CD38 events revealed that 2 variants, L153H and C275Y, decreased binding affinity and antibody-dependent cellular cytotoxicity of the commercial antibodies daratumumab and isatuximab. However, a third mutation, R140G, conferred selective resistance to daratumumab, while retaining sensitivity to isatuximab. Clinically, patients with MM are often rechallenged with CD38 antibodies after disease progression and these data suggest that next-generation sequencing may play a role in subsequent treatment selection for a subset of patients.

Introduction Monoclonal antibodies (MoAbs) directed against CD38 are a mainstay in multiple myeloma (MM). Downregulation of CD38 expression is a resistance mechanism of antigenic escape, however, cell surface expression may return. While this tumor cell plasticity is likely driven by epigenetic mechanisms, it is unknown if MM cells can develop permanent resistance to anti-CD38 MoAb by acquiring genomic events leading to biallelic disruption of CD38 thus precluding re-treatment. Here, we report biallelic loss of CD38 as a recurrent mechanism of resistance, often via convergent evolution where distinct subclones are advantaged towards the same selective pressure. Methods We interrogated two sets of patients (Mayo Clinic; n = 31, Calgary University; n =19) with relapse following anti-CD38 MoAbs. Two additional cases of interest were selected from Heidelberg University. Whole genome sequencing (WGS, 60-100x) or whole exome sequencing (WXS), flow cytometry (flow), and bulk RNAseq were used to validate the effect of genomic events on CD38. Missense mutations predicted to affect CD38 MoAb binding were validated with site-directed mutagenesis of a CD38 plasmid, transduction into K562 MM cells, and CD38 MoAb binding assays. Results The prevalence of biallelic inactivation of CD38 at anti-CD38 MoAb relapse was 6% (3/50). An additional selected biallelic event was noted in the Heidelberg set. Each case is described in detail: MM-19 (Calgary) received daratumumab (Dara), lenalidomide, and dexamethasone (Dex), and then had a second exposure to Dara, following relapse. At this second relapse, WGS was performed on two independent extramedullary disease localizations in the liver. Both samples had unique, large deletions (>3 Mb) on chromosome 4 coupled with additional unique focal deletions involving CD38. Flow confirmed the presence of CD138+/CD38- plasma cell populations in both sites. A second case of convergent evolution was seen in a patient (Mayo) after seventh-line Dara/Len/Dex. WGS revealed a common deletion encompassing CD38. On the remaining allele, one subclone had a structural variation-mediated loss of CD38(cancer cell fraction; CCF 40%) while another harbored an L153H missense mutation (CCF 60%). Flow confirmed presence of two CD138+ clones; one with diminished CD38 expression. L153H was functionally validated to abrogate Dara binding, while maintaining CD38 expression. The third sample was collected after fifth-line Dara and sixth-line Dara/Bortezomib/Dex (Heidelberg) and represents another case of convergent evolution with a common loss of chromosome 4 and two subclones detected by WGS (P98Lfs*12, CCF 39% resulting in biallelic inactivation; R140G, CCF 61%). Bulk RNAseq revealed, in comparison to NDMM from CoMMpass, CD38 expression below the first decile. Notably, the R140G variant reduced binding affinity to Dara but not to Isatuximab (Isa). A fourth case (Mayo) relapsing after 6 months of Dara-based therapy similarly had a focal deletion of CD38 with an L18Sfs*16 frameshift deletion on the remaining allele associated with a 4.35-fold reduction in CD38 expression by RNAseq compared to pre-treatment. Interestingly, in 3/4 cases the CD38-negative clone emerged after a second exposure to anti-CD38 MoAb, suggesting emergence with the first exposure and then selection during the second. One other monoallelic event (C275Y; Heidelberg) was detected post-Isa. Copy number of the alternate allele is not known given targeted sequencing methodology, but the variant was seen to reduce binding affinity to both Dara and Isa. The prevalence of monoallelic loss of CD38 in newly diagnosed MM from MMRF CoMMpass was 7% (50/701) and two patients had mutations in CD38, but there were no cases of biallelic loss suggesting these events are driven by treatment pressure. Conclusion Distinction of the mode of CD38 loss is valuable for predicting outcome of re-challenge to anti-CD38 MoAbs. We see here an estimated 5-10% of anti-CD38 MoAb relapses are characterized by genomic antigen escape. Results here are in line with antigen escape to BCMA- and GPRC5D-directed immunotherapies (Lee et al, Nat Med 2023), suggesting that acquired antigen escape resulting from prolonged exposure to the selective pressures of immunotherapy is a recurrent mechanism of resistance in MM. Moreover, in select cases, non-overlapping resistance mutations provide rationale for switching agents at second exposure.

Introduction: Multiple myeloma (MM) with t(11;14)(q13;q32), resulting in CCND1;IGH rearrangement, remains the only subset amenable to targeted therapy with the BCL2 inhibitor, Venetoclax. Nevertheless, not all patients with this translocation respond to treatment and some progress early after initial response. The mechanisms behind this clinical heterogeneity are largely unknown. Methods: To investigate mechanisms of heterogeneity in response to Venetoclax, we interrogated 44 whole genome sequencing (WGS) and 24 whole exome sequencing data from a training cohort including 33 patients with t(11;14) MM treated with Venetoclax at the Mayo Clinic. In a validation cohort, we extracted single nucleotide variants (SNV) and indels from 17 MM patients with t(11;14) with available RNAseq data treated with Venetoclax at Emory University. Results: In the training cohort the median number of lines of therapy was 4 and Venetoclax drug regimens included combinations with dexamethasone, proteasome inhibitors, anti-CD38 agents, and immunomodulatory agents. After a median follow up of 30 months, the median progression free survival (PFS) was 9 months, and 13 patients (39%) achieved a complete remission (CR) to therapy, while 9 (27%) were refractory (i.e. no response or progressed within the first 60 days). For our driver discovery analysis, we integrated SNV, indels, copy number variants (CNV) and structural variants (SV). Deletion 17p (del17p) or TP53 mutation, MCL1 gain/amp, and hyper-APOBEC did not correlate with PFS in our cohort (p=0.59, p=0.71, and p=0.05, respectively). In contrast, mutations involving the MAPK pathway (NRAS, KRAS, BRAF, FGFR3) displayed the strongest association with short PFS, which was retained after adjusting for del17p/TP53 mutations, and 1q gain/amp (HR: 4.76, CI: 1.8-12.9, p=0.002). The association between suboptimal response and resistance to Venetoclax in MM patients with MAPK pathway mutations (n=7) was also observed in our validation cohort from Emory University (p=0.046). This is in line with what has been reported in functional studies in acute myeloid leukemia, where RAS pathway promotes MCL1 under the selective pressure of Venetoclax (Zhang et al. Signal Transduct Target Ther 2022). While MAPK mutations explained most cases of refractory patients (86%), the mechanisms through which initially responding patients subsequently progressed remain unclear. To investigate this, we interrogated 10 patients with pre- and post-treatment samples collected at disease progression (PD). We identified genomic events acquired/selected at PD of genes involved in the anti-apoptotic MCL1/BCL2 and MAPK pathways in 6/10 cases. One patient was refractory to Venetoclax and had double minutes involving BCL2, which caused multiple focal amplifications (>30 copies), likely reflecting extrachromosomal DNA. Another patient progressed with an acquired, focal deletion on BCL2L11. In two patients, the progressing clones harbored mutations in MAPK pathways (one BRAF and the other WNK2). Two patients who relapsed after sustained CR had major branching evolution where a previously undetectable focal deletion of PMAIP1 (NOXA) was seen at PD. Importantly, one of these deletions was a biallelic event. Venetoclax sensitivity assays conducted on PMAIP1 knockout in the MM t(11;14) cell line, KMS12PE, demonstrated significantly reduced cell death across all drug concentrations (p<0.05), validating the loss of NOXA as a mechanism of resistance to Venetoclax in MM. Finally, similarly with what was previously reported in chronic lymphocyte leukemia treated with Venetoclax (e.g., Herling et al Nat Comm 2018), CDKN2A/B monoallelic and biallelic focal deletions were selected post-Venetoclax, at PD in 4 patients while TP53 SNV/deletions were selected from subclonal to clonal in 2 patients at PD. Conclusion: In this study, we demonstrate that mutations involving the MAPK pathway identify a population of MM patients with t(11;14)(CCND1;IGH) who are unlikely to respond to Venetoclax. We also show that genomic events involving the MAPK and BCL2/MCL1 pathways are primarily responsible for disease progression after initial response. These findings highlight the critical role of comprehensive genomic profiling through WGS in identifying mechanisms of resistance to targeted therapies in MM, aiding patient selection for Venetoclax, and guiding the development of novel drug combinations.

Introduction Poor diet quality, elevated body mass index (BMI), insulin resistance, microbiome dysbiosis, inflammation as well as immune dysfunction have all been implicated in progression from monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM) to multiple myeloma (MM). Plant-based diets have been associated with reduced risk of MGUS and MM in epidemiological studies. Whether a dietary intervention can impact these modifiable risk factors and delay disease progression has not been investigated. Methods We conducted a pilot, single-arm trial providing controlled high fiber plant based dietary (HFPBD) intervention for 12 weeks and health coaching for 24 weeks in 20 patients with MGUS/SMM with BMI≥25 (NCT04920084). The primary endpoint was feasibility (adherence and BMI reduction). Secondary and exploratory endpoints included quality of life, metabolic markers (insulin, adiponectin, leptin, LDL cholesterol), microbiome (diversity and composition by 16S and shotgun sequencing) and immune profiling in peripheral blood (flow cytometry and Olink inflammation panel) and bone marrow (scRNAseq and ATACseq). We also conducted an in vivo study in transgenic Vk*MYC mice fed a high fiber or control diet at the phase of mouse(m)SMM. The mice were monitored for the progression to active mMM. Differences in microbiome and immune response profiles between high fiber- and control-treated mice were assessed via microbiome 16S sequencing and flow cytometry. Results The HFPBD intervention was safe, feasible, improved quality of life and addressed modifiable risk factors - metabolic profile (improved BMI, insulin resistance, adiponectin leptin ratio), microbiome profile (increased alpha-diversity and butyrate producers) and immune (decreased inflammation and increased anti-inflammatory classical monocyte) subsets. A reduction in long-term progression trajectory was observed in 2 patients. Consistently, the high fiber diet delayed progression from mSMM to mMM in Vk*MYC mice increasing median progression-free survival from 12 weeks in the control arm to 30 weeks in the high fiber diet intervention arm. Additionally, in the intervention arm 40% of mice did not progress to mMM during the study period whereas all mice in the control arm progressed. Both human and mouse data showed that HFPBD modulated gut microbiota composition favoring the expansion of butyrate-producing bacteria. Additionally, short-chain fatty acids were increased in the feces of mice fed a high fiber diet. Integrated analysis from human bone marrow and peripheral blood analysis indicated that the dietary intervention reduced inflammatory biomarkers and skewed the immune response towards T helper and CD14+ monocytes. Consistently, the bone marrow of Vk*MYC mice fed high fiber diet was more infiltrated by IFNg-producing T lymphocytes while displaying less exhausted T cells and immunosuppressive myeloid cells. Conclusion This is the first interventional clinical trial and in vivo study to show that a HFPBD intervention may delay progression from MGUS/SMM to MM. To our knowledge there has been no similar dietary interventional study in hematological cancers or solid tumors. Together our in vivo and clinical data support the beneficial anti-inflammatory role of a HFPBD providing a link between diet, microbiota, and immune modulation to delay disease progression in MGUS/SMM.