Immunomodulatory drugs (IMiDs) are a cornerstone of multiple myeloma treatment and newer cereblon E3 ligase modulating drugs (CELMoDs) are in clinical trials. However, a major barrier to improving patient outcomes is the inevitable development of resistance to these agents. To explore the mechanisms underlying IMiD/CELMoD resistance, and identify novel targets for treatment, human cell line models of acquired IMiD/CELMoD resistance were generated. Quantitative proteome analysis of these models identified common changes in lipid synthesis proteins and glucose labelling experiments confirmed altered lipid flux. Proteomic analysis of paired patient samples, from diagnosis and relapse on IMiD, suggested similar changes in lipid pathways. A genome wide CRISPR screen performed in a human multiple myeloma cell line with acquired CELMoD resistance identified dependencies in the lipid pathway genes Stearoyl-CoA Desaturase (SCD) and Membrane Bound Transcription Factor Peptidase Site 1 (MBTPS1). This work has led to novel insights into lipid pathway changes in the IMiD/CELMoD-resistant state which may represent targetable cancer cell vulnerabilities.

Abstract Multiple myeloma (MM) is characterized by its genomic complexity that includes recurrent driver single nucleotide variation (SNVs) alongside a high burden of structural variation (SV). Complete characterization of somatic variation in a MM genome is dependent on a technology that can accurately and sensitively detect both small and large variants. Here, we demonstrate performance of a novel linked-read approach, called LinkPrep™ technology, for high accuracy detection of both small and large somatic variants in four separate relapse MM genomes.Four MM patient-derived xenografts were profiled with Dovetail® LinkPrep™ kit and sequenced to ∼30X on an Illumina platform. Data was analyzed through the Dovetail Analysis Portal to detect SNVs/Indels (DeepSomatic), CNVs (Purple), and SVs (HiC-breakfinder and proprietary tools). Call sets were compared against other genomics technologies including WGS (80X tumor/ 30X normal), PacBio (15X), and OGM (400X).LinkPrep + Dovetail Analysis Portal returned high quality variant results for the four MM samples. LinkPrep data recalled all pathogenic SNV/Indels detected by 80X WGS (notable pathogenic variants were detected in NRAS, KRAS, TP53). LinkPrep data further detected SVs involving the IGH locus (inversions and translocations) in all four samples. Deeper analysis of one sample (M24) highlights that LinkPrep data detects genetic alterations common in MM including SNV mutations in NRAS and TP53 (both occurring at 100% VAF), IGH rearrangements, chr 1q gain, chr 1p loss of heterozygosity, trisomy of chr 5,7, and 15, and complex rearrangements involving chromosomes 2, 3, 4, 16, and 19. With the exception of a confident subclonal (1% SV-VAF) unbalanced translocation detected by LinkPrep, the remaining LinkPrep large SV calls were validated by other genomics methods. Underscoring the capability of LinkPrep for SV detection, LinkPrep calls at 30X contain >10-100 times more read support for every SV call over other technologies. Using the linked-read feature of LinkPrep data along with CNV segment calls and SV calls provided through the Dovetail Analysis Portal a candidate solution is derived for the complex rearrangement involving chromosomes 2, 3, 4, 16, and 19. This LinkPrep solution, refined down to base pair resolution for every breakpoint, is verified by 400X OGM data. Uniquely, LinkPrep data further enables visualization of the SV-reconstructed 3D genome and reveals neo-loops connecting enhancers with promoters to suggest enhancer hijacking mechanisms associated with the complex rearrangement.Together, these data demonstrate that LinkPrep linked-reads enable improved characterization of somatic variation in highly rearranged genomes, while simultaneously detecting epigenetic states -- all enabled with the accuracy and cost-effectiveness of short read sequencing. Citation Format: Lisa Munding, Nathan Becker, Enze Liu, Alexander Fortuna, Jonathan Torchia, Aneta Mikulasova, J. Zachary Sanborn, Brian Walker. Characterization of multiple myeloma genomes with LinkPrep assay enables detection of somatic variation and SV-driven interactions of the 3D genome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3248.

7520 Background: Daratumumab (Dara) requires CD16-expressing natural killer (NK) cells for anti-myeloma activity. The use of bone marrow transplant (ASCT) results in prolonged immunosuppression, including lower levels of NK-cells. We evaluated if artificial intelligence (AI) predicted CD16 from routine hematoxylin and eosin (H&E) stained bone marrow biopsy slides could be used to guide Dara and transplant treatment strategies. Methods: We analyzed 212 newly diagnosed patients from the HealthTree registry treated with VRd (n=135; median follow-up 24.5 months (mo)) and D-VRd (n=77; 16.6 mo). H&E slides were processed using GigaTIME, a foundation model via zero-shot inference. Patients were dichotomized at median predicted CD16 and stratified by ASCT status. Subgroups: transplant-eligible (TE: age<65, ASCT; n=53), deferred (TD: age<70, no ASCT; n=34), and ineligible (TI: age≥70; n=17). Primary endpoint: time to next treatment (TTNT). Multivariate Cox regression adjusted for age, cytogenetic risk tested interaction between CD16, treatment and ASCT. Results: D-VRd achieved superior outcomes vs VRd (23.4% event rate vs 74.8%, p<0.001) despite lower ASCT use among D-VRd treated patients (50.6% vs 71.1%, p=0.005). In the VRd group, AI-predicted CD16 was highly prognostic in non-transplanted patients: High-CD16 achieved median TTNT 33.1 vs 5.1 mo for Low-CD16 (p=0.0053). ASCT rescued Low-CD16 patients (median 5.1 to 26.7 mo, p<0.001). Conversely, in D-VRd, CD16 did not stratify outcomes (p=0.48), indicating that Dara can rescue low-immune-fitness disease. Specifically, Dara rescued low CD16 non-transplanted patients from progression (86.8% 18-mo event-free survival for D-VRd versus 28.6% 18-mo event-free survival for VRd; p<0.001). High CD16 patients on D-VRd without ASCT achieved 86.3% event-free survival, non-inferior to ASCT (82.2%, p=0.21), supporting transplant deferral. The CD16×treatment×ASCT interaction was significant (HR 0.06, p=0.045). Among transplant-deferred patients, D-VRd improved 18-mo event-free rates vs VRd (70.5% vs 40%, p=0.029), with the largest benefit in Low CD16 (p=0.016). TI patients on D-VRd achieved 100% event-free at 18 mo vs 38.2% on VRd (p=0.011). Conclusions: We validated that AI-quantified CD16 from routine H&E guides Dara and transplant strategies in NDMM (n=212). The CD16×treatment×ASCT interaction (HR=0.06, p=0.045) confirms that CD16 predicts treatment benefit, not just prognosis. In VRd, low CD16 identifies a high-risk group (median TTNT 5.1 mo) needing intensification. D-VRd rescues low-immune-fitness patients (18-mo event-free survival: 86.8% vs 28.6% with VRd, p<0.001) and enables safe transplant deferral in high-fitness patients (82.2% with ASCT vs 86.3% without, p=0.21). This novel strategy enables precision medicine using standard H&E slides, with immediate clinical implications.

Abstract In multiple myeloma (MM), minimal residual disease (MRD) is an established endpoint for accelerated drug approval but is limited by a need for serial invasive bone marrow (BM) biopsies, which may under‐sample spatial heterogeneity and result in false negativity. Furthermore, longitudinal (i.e., sustained) MRD negativity is emerging as a powerful tool for clinical decision‐making. To these ends, reliable systemic MRD assessment is a growing need. Next‐generation sequencing approaches for plasma cell‐free (cf) DNA generally fail to achieve adequate detection limits in low tumor fraction (TF) settings (i.e., MRD), but tumor‐informed approaches leveraging whole‐genome sequencing (WGS) have thus far achieved the lowest limits of detection (LODs). We therefore performed a longitudinal analysis of MRD assessed by serial WGS of plasma cfDNA as compared to clinical standard flow‐cytometric BM MRD in high‐risk smoldering MM. 25 baseline tumor WGS served to inform detection of disease in 87 sequential plasma samples. The median LOD across patients was 1.2 × 10−4 (range 9.0 × 10−5 – 2.1 × 10−4). TF was prognostic, and tumors with high‐risk genomics had higher baseline TF (P = 0.002) and eventual disease progression (n = 7, P < 0.001). Furthermore, dynamic changes in serially tracked TF portended outcome. In comparison to BM flow, cfDNA WGS was concordant in 33/45 (73.3%) MRD samples with consistent capture of BM‐positive cases, but added resolution to apparent false‐negative BM samples. Overall, despite the deeper LOD of localized BM flow, cfDNA WGS can detect MRD when BM flow did not, adding dynamic and systemic/spatial resolution to standard hyper‐local MRD assessment.

ABSTRACT:Here, we present the case for next-generation sequencing of samples from patients with multiple myeloma (MM), not only identify high-risk markers but also mutations and deletions relating to immunomodulatory drugs (IMiD), and more importantly to guide the sequencing of immunotherapy regimens in response to intrinsic antigen escape as a means of treatment resistance and relapse. This is a single-center study of CD138+ selected (n = 134) bone marrow aspirate samples from patients with smoldering MM (n = 11) and MM (newly diagnosed, n = 38; relapsed, n = 79). Samples were sequenced using a targeted panel in a clinical diagnostics laboratory. Data were analyzed for high-risk markers, treatment-related resistance mechanisms, and precision medicine targets to guide the future treatment of patients in the clinic. High-risk markers, including t(4;14), t(14;16), t(14;20), gain or amplification 1q, deletion of CDKN2C, and deletion or mutation of TP53, were identified in 15% samples from patients with newly diagnosed. At relapse, alterations in the cereblon degradation pathway were found in 24.3% of IMiD-treated patients. Deletions of 4p (CD38) were also enriched in patients who received anti-CD38 treatment (P = .03), which were mostly monoallelic. Deletions and mutations were detected in TNFRSF17-encoding B-cell maturation antigen (BCMA) in patients treated with anti-BCMA regimens, and the information was used to change the treatment of the patients. Targeted sequencing of diagnostic samples of patients with MM can be used for risk stratification and to monitor and adjust treatments as resistance mechanisms evolve.

Differential expression between normal chr1 and gain(1q) MM patients with t(4;14) translocation

Impact of additional chromosome 1 structural events on outcome A.Effect of amp(1q) with or without a total (TT) gain on PFS B. Effect of amp(1q) on OS

A key treatment for patients with multiple myeloma is high-dose melphalan followed by autologous stem cell transplant (ASCT). It can provide a deep response with long-term remission. However, some patients progress quickly, and it is not clear why. In this study, we performed single-cell RNA and T-cell receptor sequencing of the immune microenvironment of 40 patients before and after ASCT to determine if differences in the immune composition could define those who would progress. Clear differences in cell populations were identified in progressors, including increased T-cell infiltration, decreased T-cell receptor diversity, and decreased frequency of monocytes and CD56bright NK cells. We identified cell interactions that predicted progression, including increased frequency of CD8+ exhausted T cells and stromal cells and decreased frequency of CD56bright NK cells and plasmacytoid dendritic cells. We propose and validate a model of progression that can also be determined by flow cytometry. Together, these data highlight the importance of the immune microenvironment in understanding responses to ASCT.

Background: Anti-CD38 monoclonal antibodies (mAbs) have become a standard part of multiple myeloma (MM) therapy. Their activity is mostly mediated by NK cells through antibody-dependent cellular cytotoxicity (ADCC). Although the association of increased CD38+ NK cells with worse outcomes was demonstrated, the underlying mechanisms of resistance to anti-CD38 mAbs remain poorly understood. Aims: To identify immune effector cell subsets predictive of treatment efficacy and driving resistance to anti-CD38 mAbs. Methods: Bone marrow (BM) samples (N=111) of relapsed/refractory MM (RRMM) treated with anti-CD38-based regimens (CD38-IMID [N=64]; CD38-PI [N=33]; CD38-mono [N=14] were assessed at baseline (BASE; N=110) and progression (PD; N=23), using conventional EuroFlow 8-color MM panel, followed by high-dimensional spectral cytometryesigned for deep profiling of lymphoid subsets and their activation/exhaustion status. Subset proportions were expressed as percentage of lymphocytes, unless stated otherwise. Results: Patients were treated predominantly with daratumumab over isatuximab (92%, 82%, and 100% of CD38-IMID, CD38-PI, and CD38-mono regimens). Treatment groups differed by line of therapy (p <0.001), with CD38-IMID mainly used in line 2 (56%), CD38-PI in lines 2-3 (55%, 30%), and CD38-mono in line ≥4 (86%). Median progression-free survival (PFS) for the CD38-IMID group was 37 months, compared to CD38-PI (16 months; p = 0.012) and CD38-mono (3 months; p <0.001). First, RRMM BM (N=110) collected at BASE were assessed by the EuroFlow MM panel. Lymphocyte pool consisted of median 13.9% B (CD19+CD56-), 19.7% NK (CD19-CD56+), and 61.6% T cell (CD19-CD56-) lineage. 79.7% NK cells were CD38+, while only 29.2% T cells were CD38+. Elevated total NK cells were observed in the PD group (p=0.07). This difference was driven by increased CD38+ NK cell proportion (p <0.028). Lasso-penalized multivariable Cox regression adjusted for therapy group and all immune subsets identified higher percentages of CD38+ NK cells and mature B cells (CD19+CD38low/−CD81low) as significant predictors of PFS at BASE. Increased CD38+ NK cells were associated with worse PFS (HR 1.65, p=0.003), while higher levels of mature B cells correlated with improved PFS (HR 0.79, p=0.002). These findings remained significant also in uniform CD38-IMID group (p <0.007). To better characterize the immune landscape driving these associations, detailed subset distribution was explored using spectral cytometry at BASE (N=14). CD16-, CD16+CD57-, CD16+CD57+ cells represented median 1.68%, 4.26%, and 6.17% of lymphocytes. Comparing CD38+/- NK compartments, CD16+CD57− subset was significantly enriched in the CD38+ NK pool (p=0.006). Gating on 8 other key NK markers revealed that KLRB1+ (p=0.063) cells were also enriched in the CD38+ pool, whereas NKG2C+ cells were more frequent in the CD38- pool (p <0.001). To further investigate the impact of anti-CD38 mAbs on immune cells, paired BASE and PD samples were analyzed using conventional (N=23) and spectral (N=14) panels. Basic exploratory analysis using both methods showed that proportion of total T cells increased (p <0.017), while total B cells (p <0.012) and total NK cells (p=0.023) dropped at PD. Furthermore, both CD38+ NK and CD38+ T cells decreased (p <0.004). Finally, spectral cytometry was utilized in both BASE and PD to identify NK subsets most affected by anti-CD38 therapy. As expected, most reduced NK cells in lymphocytes were CD38+ (median drop, log2-fold change: 6.84%, 1.72; p=0.008), followed by CD16+CD57+ (5.85%; 1.31; p=0.023), KIR2DL+ (5.11%; 1.52; p=0.008), KLRB1+ (4.58%, 1.46; p=0.008), and CD16+CD57- cells (2.42%, 1.04; p=0.023). Conclusion: This study provides in-depth analysis of immune cells in anti-CD38 mAb-treated patients using conventional and spectral cytometry. Higher BASE proportion of CD38+ NK cells was associated with worse PFS, while higher levels of mature B cells predicted better outcomes. Post-therapy, CD38+ NK and CD38+ T cells, total NK cells, and total B cells decreased, while total T cells increased. Importantly, deeper profiling revealed CD16+CD57- and KLRB1+ cells among those enriched in the CD38+ NK pool at BASE. Post-treatment, CD16+CD57+, KIR2DL+, and KLRB1+ NK cells showed the greatest reduction. These results indicate a complex interplay of fratricide, resulting in preferential depletion of mature effector NK cell phenotypes by anti-CD38 therapy.

Background : Proteosome inhibitor (PI), immunomodulatory drug (IMiD), and anti- CD38-based quadruplet (quad) therapies have revolutionized the treatment of newly diagnosed (ND) multiple myeloma (MM) with unparalleled clinical benefit independent of patients' transplant eligibility. However, approximately 50% of patients do not attain minimal residual disease (MRD) negativity after initial combination therapy. Although high-dose melphalan with autologous stem cell transplant (HDM-ASCT) remains an option for some patients, others prefer a harvest and delayed approach. We were motivated to develop a study using an abbreviated fixed-duration (default 4 cycles) of the bispecific T cell redirecting monoclonal antibody linvoseltamab (Linvo) which targets CD3xBCMA as a strategy to deliver deep and durable responses after combination therapy (i.e. sustained MRD negativity 10-6). Methods :This is a single academic center investigator-sponsored Phase 2 trial in up to 25 evaluable patients with NDMM irrespective of HDM-ASCT eligibility who completed ≥4 cycles of standard triplet/quad-based combination therapy (IMiD and PI and/or anti-CD38). Patients must have attained a very good partial response (VGPR) or better but remain MRD positive by clonoSEQ with adequate organ function. The primary objective is to determine the MRD conversion rate after 4-6 cycles of Linvo. Key secondary objectives include sustained MRD negativity at 6, 12, and 24 -month milestones, progression-free survival, overall survival, and overall safety profile. Patients receive Linvo (28-day cycles) 200mg IV preceded by 2 step-up doses (5/25mg) weekly for cycles (C) 1-3 and every 2 weeks starting with C4 and beyond. After C4, bone marrow MRD is evaluated, and if positive, patients receive C5-6. After C4 or 6, patients return to standard management including maintenance therapy. Prophylaxis includes tocilizumab 8 mg/kg to prevent cytokine release syndrome (CRS) 1 hour prior to first dose, dexamethasone 40/10mg C1 only, and standard anti-microbial prophylaxis, IViG, and thromboembolism prophylaxis. The preplanned Simon 2-stage design stipulates >1 MRD negative response out of 15 patients before proceeding to stage 2. Results : As of July 23, 2025 (data cut-off), 19 patients (median follow up: 3.9 months) were enrolled with a median age of 60 years (range: 40-77) including 26% female; 11% Black, 47% Hispanic; 74% ECOG PS 0, 26% 1; 47% IgG, 21% IgA, 32% light chain only; 32% high-risk FISH (53% standard, 16% unknown). Prior to study entry, median combination cycles was 8 (range: 6-13) and 42% received KRd, 26% D-VRd, 21% D-KRd, 5% DRd, and 5% Isa-VRd. All patients were clonoSEQ MRD positive (84% VGPR; 16% sCR). As of data cut-off, 14 patients completed 4 cycles of Linvo and underwent MRD assessment with 100% (95%CI: 76.8-100%) achieving MRD negativity by both NGS clonoSEQ (10-6) and flow cytometry (10-5) (13 sCR; 1 VGPR). All patients remain alive, with no relapses and all after C4 have initiated lenalidomide-based maintenance therapy per treating physician. All 3 patients who reached the 6-month post Linvo MRD milestone remain MRD negative. Of the 19 patients who received Linvo to date, no patients developed any grade CRS or ICANS. Any grade adverse event (AE) occurred in 84% (74% Linvo related). Any grade related AEs occurring in >1 patient included: upper respiratory infection (3), rash (3), bone pain (2), cough (2), and neutropenia (2). Grade 3 AEs included neutropenia in 1 patient and infection (peritonsillar abscess) in another which was a serious AE (SAE). No other SAEs nor Grade 4 or 5 AEs occurred. Conclusion: Recent randomized studies of quad-therapies have shown unprecedented MRD negative responses (e.g. PERSEUS, ADVANCE, MIDAS) and in turn, HDM-ASCT may not confer further benefit in these patients. Given the recent clinical success of T cell redirecting bispecific antibodies, this study aims to use Linvo as immunoconsolidation for the fraction of patients who remain MRD positive. Thus far, the IMMUNOPLANT study shows that 100% of MRD assessed patients who completed abbreviated fixed-duration (default 4 cycles) of Linvo attained MRD negativity by both NGS clonoSEQ (10-6) and flow cytometry (10-5) and therefore has met its prespecified analysis threshold and continues to enroll in Part 2 (50 patients total). At the Meeting, we will present updated safety and efficacy data including MRD negative rates and durability (clinicaltrials.gov NCT06376526).

Differential expression between normal chr1 and gain(1q) MM patients with t(11;14) translocation

ABSTRACT:We carried out a single-cell multiomic analysis on a series of MYD88-mutated Waldenström macroglobulinemia (WM) patients and identified 2 distinct subtypes of disease, memory B-cell (MBC)-like and plasma cell (PC)-like, based on their expression of key lineage defining genes. Biologically, the subtypes are characterized by their variable capacity to differentiate fully toward a PC and exhibit unique transcriptomic, chromatin accessibility, and genomic profiles. The MBC-like subtype is unable to differentiate beyond the MBC stage, upregulates key MBC genes, and is characterized by upregulated B-cell receptor and phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) signaling. In contrast, the PC-like subtype can partially differentiate toward a PC, upregulates key PC genes, has enhanced NF-κB signaling, and has an upregulated unfolded protein response. Pseudotime trajectory analysis of combined single-cell RNA sequencing and single-cell assay for transposase-accessible chromatin with sequencing supports the variable differentiation capacity of each subtype and implicate key transcription factors SPI1, SPIB, BCL11A, and XBP1 in these features. The existence and generalizability of the 2 disease subtypes were validated further using hierarchical clustering of bulk RNA-sequencing data from a secondary set of patients. The biological significance of the subtypes was further established using whole-genome sequencing, in which it was shown that CXCR4, NIK, and ARID1A mutations occur predominantly in the MBC-like subtype, and 6q deletions in the PC-like subtype. We conclude that the variable differentiation blockade seen in WM manifests itself clinically as 2 disease subtypes with distinct epigenetic, mutational, transcriptional, and clinical features, with potential implications for WM treatment strategies.

Multiple myeloma (MM) is an incurable malignancy characterized by mutated plasma cell clonal expansion in the bone marrow, leading to severe clinical symptoms. Thus, identifying new therapeutic targets for MM is crucial. The oligosaccharyltransferase (OST) complex transfers N-glycan to a nascent translated protein, a critical step in N-glycosylation. Aberrant expression of the OST complex or N-glycosylation is a diagnostic marker for solid tumors. However, the roles of the OST complex and N-glycosylation in MM development are unknown. We identified the OST complex as a novel vulnerability in MM cells. The OST complex is abundantly expressed in MM cell lines and patient-derived cells but exhibits minimal expression in most normal tissues. Elevated expression of this complex is associated with relapsed, high-risk MM and poor prognosis. Disrupting the OST complex suppressed MM cell growth and induced cell cycle arrest and apoptosis. Notably, we found that disruption of the OST complex by suppressing the genes known for the proteasome-resistant phenotype sensitizes the MM (both MM cell lines and primary patient cells) to bortezomib treatment. We tested this combination in the disseminated xenograft model, in which human MM cells migrate into the bone marrow of immunodeficient mice. We found that NGI-1 and bortezomib strikingly mitigate the MM progression compared with single drug treatment. Mechanistically, OST complex disruption downregulated MM pathological pathways (mTORC1 pathway, glycolysis, MYC targets, and cell cycle) and induced TRAIL-mediated apoptosis. Notably, MYC translation was robustly suppressed upon inhibiting the OST complex. Collectively, these results show the OST complex presents a novel target for MM treatment, and combining its inhibition with bortezomib offers a promising approach for relapsed MM patients.

Common dependency pathway analysis

List of dysregulated oncogenes, tumor suppressor genes, and fusion proteins in the presence or absence of gain(1q) based on the Cancer Gene Census

Background: Chromothripsis, a catastrophic event of chromosomal shattering and reassembly, is a key driver of oncogenesis and is associated with poor prognosis in multiple myeloma (MM). However, the specific functional consequences and the molecular mechanisms by which it rewires the MM regulatory landscape remained largely unknown. Methods: Using previous definitions, we identified chromothripsis (≥10 breakpoints, ≥7 CN oscillations, ≥200 kb segment sizes), chromothripsis-like (5-9 breakpoints and 3-6 CN oscillation) and non-chromothripsis (WT) samples. We utilized multiomic data from the CoMMpass dataset (n=1,066) and an independent Indiana University (IU) dataset (n=134), as well as 14 patient-derived xenograft (PDX) models with short-/long-read whole genome sequencing (WGS, average depths 92x and 16x) to identify single nucleotide variations (SNVs), copy number (CN) abnormalities and structural variation (SV), as well as expression (>124 million reads), chromatin states (Cut&Tag-IT, >10M reads, H3K27ac, H3K4me1, H3K4me3, H3K9me3, H3K27me3, and H3K36me3), and Micro-C/LinkPrep (Dovetail Genomics; >900M reads) to identify 3D chromatin architecture/folding inside the cell and enhancer-target interactions. Results: We identified 417 chromothripsis events in 268 patients (25%) from the CoMMpass dataset. Events were identified more frequently in relapsed and refractory (RRMM) samples compared to newly diagnosed (NDMM) samples (30% vs. 22%), suggesting an association between chromothripsis and disease progression. The number of events found in chromosomes highly correlated with chromosome sizes (r=0.63, Pearson correlation). However, events were particularly enriched on chromosomes 1, and 17 (0.26 and 0.30 events per million bps). Breakpoints of these events were enriched on 17q and 1p (odds ratio=2.2 and 1.7, p=0.0003 and 0.001). Chromothriptic samples were associated with inferior progression-free (PFS; 970 vs. 1227 days, p=0.003) and overall survival (2176 vs. not reached, p=0.006) compared to WT. In chromothriptic samples, 34% exhibited breakpoints on >1 chromosome and an higher number of affected chromosomes correlated with worse PFS (≥3 vs. 2, p=0.03; ≥3 vs. 1, p=0.02). Meanwhile, CN oscillations plus breakpoints was an independent factor associated with inferior PFS(p=0.05,: 700 vs. 1358 days). Del(TENT5C), del(TP53)and t(4;14) were enriched in the chromothripsis group (32%, 18% and 20%, odds ratio=2.6, 3.4 and 2.4, p<0.001) and are associated with genomic instability. We applied the same approach and further identified 19 chromothriptic samples from the IU dataset, where t(4;14) and del(TP53) were also enriched (odds ratio=2.6 and inf.) We identified differentially expressed genes (DEGs) between the chromothripsis group and a subset of WT group with balanced frequencies of high-risk events (e.g. t(4;14) & del(TP53)) to generate a chromothripsis signature. Pathway analysis on 37 consistent DEGs between the two datasets identified ‘G2/M checkpoint’ and ‘interferon α/γ responses‘(p<0.05), suggesting selection for a growth advantage and activation of the cGAS/STING pathway. A neuron network model for predicting chromothripsis was trained with DEGs and reached performance of AUC=0.90 and AUPR=0.87. We utilized multi-omics data from established PDX models to further examine the functional consequences of chromothripsis events. Among 9 events detected in 7 samples, 5 events exhibited significantly enriched APOBEC mutational signature and kataegis within events. Copy number change alone did not account for changes in gene expression within the chromothripsis regions, with 71% of genes dysregulated through neo-TAD formation and rewiring of the epigenome, specifically through juxtaposition of super-enhancers and allele-specific expression associated with DNA methylation changes. Key dysregulated genes included MYCN (Log2FC=6.3), the tumor suppressor PMAIP1 (Log2FC=-4.9), and cell adhesion proteins (EPCAM/CD326, Log2FC=3.5) that were rarely expressed in other MM samples. Conclusion: We revealed novel molecular characteristics of chromothripsis and the mechanisms caused it. We determined that the number of events and CN abnormalities are associated with poor outcome and that epigenetic states are reprogrammed within the events. These findings advance the clinical risk stratification and reveal unique therapeutic vulnerabilities tied to chromothripsis-associated epigenetic dysregulation.

Genes with increased dependency in cell lines with del(1p) [part A] Genes with increased dependency in cell lines with del(1p) [part B]

Bayes Factor output between genetic variables Correlation analysis between deletion region, gain regions, and other genetic events

Recurrent regions of chromothripsis and chromoplexy together with the candidate genes located within the regions. A. CHromothripsis B. Templated Instertion

Abstract Multiple myeloma (MM) is described as a clonal expansion of malignant plasma cells in the bone marrow characterized by several clinical symptoms: bone lesions, hypercalcemia, anemia, serum monoclonal gammopathy, immune suppression, and multiple organ failure. Despite the success of advanced treatments, disease relapse is common and incurable. Thus, there is an unmet clinical need to identify novel targets for relapsed/refractory MM treatment. The oligosaccharyltransferase (OST) complex catalyzes the transfer of N-glycan to a nascent translated protein which is a critical step in N-glycosylation, the most abundant post-translational modification of secretory and membrane-bound proteins. Aberrant expression of the OST complex or N-glycosylation is a diagnostic marker for solid tumors. However, the roles of the OST complex and N-glycosylation in MM development are virtually unknown. The mammal OST complex has two isoforms: OST-A and OST-B, with distinct catalytic subunits. Both isoforms share six subunits: OST4, RPN1, RPN2, DDOST, DAD1, and TMEM258. Analyzing the publicly available genomic data, we found that the shared subunits of the OST complex are abundantly expressed in MM cell lines and MM patient-derived cells. The expression levels of the OST subunits in relapsed MM patients are significantly higher than those in newly diagnosed MM patients. Intriguingly, patients expressing elevated levels of the OST complex are highly correlated with poor prognosis. These findings lead us to hypothesize that the OST complex might play a crucial role in MM. To address this hypothesis, we used the CRISPR/Cas9 system to knockout the OST's shared subunits and found that MM cells are dependent on the OST complex. Deletion of DDOST and DAD1 (two central structural subunits of the OST complex) impaired MM cell growth, arrested the cell cycle, and induced apoptosis. Using the subcutaneous xenograft model, we found that knockout of DDOST or DAD1 suppressed MM growth in vivo and extended the survival of tumor-bearing mice. Intriguingly, suppression of the OST complex's enzymatic activity by a specific inhibitor, NGI-1, recapitulated the phenotypes of the OST complex deletion. Of note, NGI-1 sensitizes the MM cells and bortezomib-resistant MM cells (both MM cell lines and primary patient cells) to bortezomib treatment. Mechanistically, we found that disruption of the OST complex in MM cells significantly suppressed transcriptomic signatures of MM pathology (NF-κB signaling, glycolysis, MYC targets, and cell cycle) and induced apoptosis pathway as well as inflammatory pathway (interferon alpha and gamma). Intriguingly, we found that all known genes responsible for the bortezomib-resistant phenotype were significantly downregulated upon the suppression of the OST complex. In conclusion, we identified the OST complex as a novel vulnerability in MM cells. Targeting the OST complex incorporation with Bortezomib might provide a novel and effective combined treatment for relapsed/refractory MM patients. We're currently investigating the underlying mechanisms of how the OST complex regulates MM pathology and developing patient-derived xenograft models to validate the therapeutic efficacy of combined treatment (NGI-1 and Bortezomib).