Gupta, Vikas A.

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.

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.

Background: Multiple myeloma (MM) is an incurable plasma cell malignancy with several recurrent primary cytogenetic abnormalities including t(11,14)(q13;q32), resulting in increased expression of CCND1. Although 40% of patients with t(11;14) MM respond favorably to the BCL-2 inhibitor venetoclax (ven), 60% fail to achieve an objective response. Previous studies have suggested upregulation of PMAIP1, encoding the pro-apoptotic NOXA protein, in association with ven sensitivity (Leblay, Blood, 2024). Here we sought to evaluate the role for NOXA in ven response in t(11;14) MM. Methods: We analyzed whole exome, whole genome and RNA sequencing data of CD138+ tumor cells from 1,359 patients with MM in the CoMMpass and Mayo Clinic cohorts. The histone mark H3K27ac was assessed in t(11;14)+ KMS12PE and U266B1 human myeloma cell lines using ChIP-seq. Gene expression and protein levels of PMAIP1 (NOXA) were also detected by qRT-PCR and Western blot, respectively. Live cell detection was evaluated by Annexin V and PI using flow cytometry and by YoYo3 dye using IncuCyte following treatment with ven. Results: The t(11;14) MM subtype was associated with a 1.6-5.5-fold increased expression of PMAIP1 compared to non-t(11,14) MM in the CoMMpass and Mayo Clinic cohorts (p<0.0001). These findings are consistent with a significant correlation between CCND1 and PMAIP1 expression in both cohorts (p<0.001). To evaluate whether cyclin D1 is directly associated with increasing expression of PMAIP1 in t(11;14) MM, we generated a cyclin D1 knock out (KO) version of the U266B1 line using CRISPR/cas9. Two independent U266B1 KO cell lines showed reduced expression of PMAIP1 and NOXA protein compared to the WT U266B1 consistent with a role for cyclin D1 in regulating PMAIP1 expression. In addition, the KO lines had a 4.8-fold reduced H3K27ac signal at the PMAIP1 locus in comparison to the WT control suggesting a role for cyclin D1 in promoting H3K27ac. We previously observed increased expression of CCND1 in association with the G allele compared to the A allele of rs9344 in both the CoMMpass and Mayo Clinic cohorts (Leukemia, in press). We next asked whether the G risk allele of rs9344 within CCND1 impacted gene expression and H3k27ac levels at the PMAIP1 locus. To study the allele specific effect of CCND1, we used the CRISPR/cas9 edited KMS12PE WT (rs9344 AA) and Knock In (KI) (rs9344 GG) cell lines. In the KI cells, the G allele was accompanied by an ~2-fold increased expression of PMAIP1 and NOXA protein detected by western blot (<0.05). Increased PMAIP1 expression in KI lines was also accompanied by a 2.2-fold increased H3K27ac further suggesting a role for the G allele of rs9344 in promoting H3K27ac and gene expression at the PMAIP1 locus. Finally, we evaluated the consequence of PMAIP1 expression levels in relation to ven response. Increased expression of PMAIP1 and elevated levels of NOXA protein in the KMS12PE KI lines was associated with increased sensitivity to ven resulting in ~50% increase in cell death of KI vs. WT lines (p<0.02). Conclusion: We demonstrate a role for cyclin D1 in regulating PMAIP1 expression through H3K27ac resulting in increased NOXA protein. Levels of NOXA are associated with the G allele of rs9344 and increased venetoclax response.

Abstract Waldenstrom macroglobulinemia (WM) and its precursor IgM gammopathy are distinct disorders characterized by clonal mature IgM-expressing B-cell outgrowth in the bone marrow. Here, we show by high-dimensional single-cell immunogenomic profiling of patient samples that these disorders originate in the setting of global B-cell compartment alterations, characterized by expansion of genomically aberrant extrafollicular B cells of the nonmalignant clonotype. Alterations in the immune microenvironment preceding malignant clonal expansion include myeloid inflammation and naïve B- and T-cell depletion. Host response to these early lesions involves clone-specific T-cell immunity that may include MYD88 mutation–specific responses. Hematopoietic progenitors carry the oncogenic MYD88 mutations characteristic of the malignant WM clone. These data support a model for WM pathogenesis wherein oncogenic alterations and signaling in progenitors, myeloid inflammation, and global alterations in extrafollicular B cells create the milieu promoting extranodal pattern of growth in differentiated malignant cells. Significance: These data provide evidence that growth of the malignant clone in WM is preceded by expansion of extrafollicular B cells, myeloid inflammation, and immune dysfunction in the preneoplastic phase. These changes may be related in part to MYD88 oncogenic signaling in pre–B progenitor cells and suggest a novel model for WM pathogenesis. This article is highlighted in the In This Issue feature, p. 549