Abstract Mantle cell lymphoma (MCL) is a biologically heterogeneous B-cell malignancy. Although genomics and transcriptomics have delineated parts of the MCL disease spectrum, proteomics remains largely unexplored. Here, we conducted a comprehensive proteogenomic analysis integrating genomics, transcriptomics, and proteomics on peripheral blood samples from 27 patients with MCL and 4 healthy donors to investigate the translational and posttranslational dimensions of MCL. Our study identified 1296 downregulated and 468 upregulated proteins in MCL cells. The splicing pathways were significantly upregulated at both the mRNA and protein levels, suggesting a critical role for aberrant RNA splicing in MCL pathogenesis. Integration of proteomic data with genetic aberrations revealed immunoglobulin heavy chain variable mutational status and CCND1 mutation are associated with distinctive transcriptomic and proteomic profiles, which correspond to significant differences in clinical outcomes. A multiomics molecular stratification model incorporating proteomic data showed superior predictive power for patient survival compared with single-omics models (concordance index, 0.83 vs 0.74). This study provides, to our knowledge, the first comprehensive proteogenomic profile of MCL, offering novel insights into its molecular mechanisms and clinical behavior. The identification of molecular subtypes and prognostic protein signatures underscores the potential of proteomics to guide precision medicine strategies for MCL.

ABSTRACT:To elucidate the molecular basis underlying differential responses and resistance to ibrutinib in Waldenström macroglobulinemia (WM), we conducted a prospective phase 2 trial of ibrutinib monotherapy in treatment-naïve patients. A total of 74 sequential bone marrow (BM) aspirates from 17 patients, collected from baseline through 48 treatment cycles, were profiled using single-cell multiomics. BM cells were segregated primarily into B-cell/plasma cell and T-cell compartments. Longitudinal clonal tracking of malignant B cells/plasma cells identified 3 distinct evolutionary patterns: evolution (early clone contraction with late clone expansion and increasing genomic complexity), devolution (early clone expansion with late clone contraction and genomic simplification), and no evolution (stable clonal architecture). The evolution pattern was strongly associated with disease progression, whereas devolution correlated with durable clinical response. Transcriptomic profiling of resistant clones enabled development and validation of the Waldenström ibrutinib prediction (WIP) score, which predicted treatment response at baseline. Within the WIP signature, LYN emerged as a key regulator; LYN knockdown or inhibition significantly increased WM cell sensitivity to ibrutinib, suggesting a rational combination strategy. In parallel, GZMB+ CD8+ effector-memory T cells expanded after treatment in patients with progressive disease and coexisted with tumor evolution. These cells exhibited persistently impaired cytotoxic programs (eg, GNLY), a dedifferentiated memory-like state, elevated PDCD1 expression, and reduced T-cell receptor diversity. Together, this study provides, to our knowledge, the first single-cell framework of tumor clonal evolution and T-cell dysfunction under ibrutinib in WM, introduces the WIP score as a predictive biomarker for treatment response, and identifies actionable tumor-intrinsic and immune mechanisms driving resistance. This trial was registered at www.ClinicalTrials.gov as NCT02604511.

Waldenström macroglobulinemia (WM) is a rare indolent B-cell lymphoma with marked clinical and molecular heterogeneity. Clinical risk models, including IPSSWM, rIPSSWM, and MSSWM, were developed prior to the widespread use of Bruton tyrosine kinase inhibitors (BTKi), and their performance in the BTKi era remains uncertain. In addition, the prognostic impact of various genomic alterations is controversial. We retrospectively analyzed 453 symptomatic WM patients, including 203 who received non-BTKi therapy and 250 who received BTKi-based therapy. All three models significantly stratified prognosis in the non-BTKi cohort, with rIPSSWM showing the highest predictive accuracy, but none effectively predicted survival in BTKi-treated patients. Notably, among patients receiving first-line BTKi-based therapy, high-risk patients by any model achieved survival outcomes comparable to those of lower-risk patients, suggesting that upfront BTKi can overcome the adverse impact of high-risk clinical features. At the molecular level, MYD88 mutation was significantly associated with favorable outcomes exclusively in patients treated with first-line BTKi-based therapy, while CXCR4 and TP53 mutations predicted significantly inferior prognosis in both BTKi-based and non-BTKi cohorts. Our findings indicate that although clinical risk models remain relevant for patients receiving non-BTKi therapy, molecular features, especially MYD88, CXCR4, and TP53 mutations, provide superior prognostic insights for patients with BTKi-based regimens.

Background: Anti-CD38 monoclonal antibodies (mAbs) constitute a revolutionary class of targeted immunotherapeutic agents that have garnered significant attention due to their effectiveness in the treatment of multiple myeloma (MM). These antibodies exert their therapeutic effects through binding to CD38 on MM cells and inducing various cytotoxicity mechanisms. Clinically approved anti-CD38 mAbs, daratumumab and isatuximab, have become​ the ​foundation of therapy​ for MM. However, resistance ​develops in over 20% of patients undergoing this treatment. The concurrent administration of anti-CD38 mAbs with other anti-MM drugs has been shown to enhance therapeutic outcomes. We previously identified a novel I3MO derivative, I3MV-8b (compound 8b), which functions as a dual inhibitor of the proteasome and HDAC6, exhibiting strong anti-myeloma activity (Biomark Res. 2025)​. The current study reveals that treatment with compound 8b significantly upregulates CD38 expression on MM cells and restores immune cell function, thereby enhancing the efficacy of anti-CD38 mAbs in combating MM. Method & Results: ​First,​​ in the NK-humanized NSG mouse model, the combined treatment of compound 8b markedly improved the therapeutic efficacy of daratumumab (Dara). Using a co-culture system, we observed that​ compound 8b treatment augmented the efficacy of Dara-mediated NK cell cytotoxicity against MM in vitro. Subsequently, we evaluated the effect of 8b on CD38 expression in MM cell lines and primary patient-derived cells. RNA-seq analysis indicated that compound 8b significantly upregulated CD38 expression in MM cells, a finding confirmed​ by flow cytometry in both MM cell lines and CD138⁺ primary MM cells. Additionally, we investigated the combined administration of I3MO and HDAC6i, the principal pharmacophores of compound 8b, ​finding that this combination better enhanced CD38 expression on MM cells. Mechanistically, the inhibition of HDAC6 by compound 8b ​elevated histone acetylation levels, particularly H3K27 acetylation (H3K27ac). ATAC-seq analysis demonstrated that compound 8b treatment significantly enhanced chromatin accessibility, and H3K27ac ChIP-Seq ​analysis​ confirmed that compound 8b robustly facilitated CD38 transcription. ​Building on our previous investigations demonstrating I3MO's ability to inhibit USP7 and modulate protein stability,​​ we ​show​ that the I3MO group of compound 8b enhances the stabilization of the CD38 protein through USP7 inhibition. ​Furthermore, compound 8b exhibited notable immunomodulatory effects in the C57BL/KaLwRij myeloma mouse model. Multicolor spectral flow cytometry analysis indicated that treatment with compound 8b significantly increased the proportion of natural killer (NK) cells within the tumor immune microenvironment. Functionally, compound 8b augmented IFN-γ production in NK cells and downregulated the expression of the NK cell exhaustion marker TIGIT. These findings were corroborated by in vitro experiments, which showed that compound 8b treatment reduced TIGIT expression on NK cells, resulting in enhancing NK cell cytotoxicity and reducing NK cell fratricide. Conclusion: Our findings suggest that compound 8b enhances CD38 expression and modulates immune cell function, offering a promising strategy to potentiate anti-CD38 mAbs against MM.

Recent advancements in the treatment of multiple myeloma (MM), including immunomodulatory agents, monoclonal antibodies, and T cell-redirecting therapies, have significantly improved patient outcomes. However, MM remains incurable. The interaction between MM cells and non-malignant cells within the immunosuppressive tumor immune microenvironment (TiME) plays a critical role in disease progression, but it has not been fully elucidated. Our previous study reported that myeloid-derived suppressor cells (MDSCs), dysfunctional dendritic cells (DCs), and tumor-promoting osteoclasts (OCs) are enriched in the bone marrow of myeloma patients and play critical roles in T cell dysfunction. However, the underlying mechanisms remain incompletely understood (Front Immunol. 2022). A myeloid inhibitory receptor, LILRB4, was identified as highly expressed in a super high-risk subgroup of MM cells by single-cell RNA sequencing of patient samples. LILRB4 expression facilitated the generation of monocytic MDSCs and was associated with poor outcomes in MM patients (Haematologica 2024 & 2025). These findings suggest a potential mechanism by which LILRB4 drives an immunosuppressive microenvironment through the induction of immunosuppressive myeloid cells. Here, we further observed a significant reduction in erythroid-lineage commitment in MM patients with high LILRB4 expression, and we explored its molecular and cellular impact on the interaction between myeloma cells and the tumor microenvironment (TME). We report that LILRB4 is highly expressed in MM and activates the SHP2/ERK/AP-1 signaling cascade, thereby enhancing the expression of AP-1-associated chemokines and promoting the transdifferentiation of erythroid cells into immunosuppressive erythroid-derived myeloid cells (EDMCs). Through in vivo and in vitro analyses, we demonstrate that CD45⁺ erythroid progenitor cells (EPCs) in the MM microenvironment lose their erythroid differentiation potential and instead transdifferentiate into EDMCs. This reprogramming is driven by elevated levels of CCL3 secreted by LILRB4⁺ MM cells. Compared with the control group, deletion of CCL3 significantly inhibited myeloma cell growth, increased peripheral blood hemoglobin levels, reduced EDMC proportions in the bone marrow, enhanced IFN-γ secretion by CD8⁺ T cells, decreased the frequency of regulatory T cells (Tregs), and reduced the proportion of macrophages. Additionally, the ratio of monocytic MDSCs (M-MDSCs), which preferentially differentiate into tumor-associated macrophages (TAMs), to granulocytic MDSCs (G-MDSCs) was also decreased. EDMCs emerged as another major source of tumor-associated myeloid cells, further contributing to the suppressive TiME. These results provide a novel model of hematopoietic diversion in which myeloma “hijacks” CD45⁺ erythroid progenitor cells, redirecting them toward a myeloid fate to evade immune surveillance. EDMCs, as potent immunosuppressive players, strongly inhibit CD8⁺ T cell function and accelerate disease progression. Additionally, our findings establish a mechanistic link between LILRB4 and MM-associated anemia, a well-known poor prognostic factor.Our study therefore suggests that LILRB4 overexpression induces key molecular changes that enable macrophage inflammatory protein-1 alpha (MIP-1α/CCL3)-mediated formation of an immunosuppressive myeloid tumor microenvironment, thereby promoting MM cell growth and survival.

Multiple myeloma (MM) is a cancer of the plasma cells characterized by excessive production of immunoglobulins and dependence on the protein degradation system, which makes proteasome inhibitors (PIs) an important treatment for MM patients. However, PI resistance remains an unsolved problem in MM. PIs directly target the 20S core particle (CP) of the proteasome, which is responsible for proteolysis. The 19S regulatory particle (RP) is responsible for recognizing and delivering ubiquitinated proteins to the 20S CP. We identified PSMD3/Rpn3, a scaffold subunit of the 19S RP essential for proteasome assembly, as a potential target in MM. Using preclinical in vitro and in vivo models, we demonstrated that targeting PSMD3 induces MM cell apoptosis and overcomes PI resistance. Mechanically, targeting PSMD3 disrupts protein homeostasis, impairs metabolic adaptation, and relieves immune suppression in MM. Bioinformatic analyses revealed that PSMD3 is highly expressed in MM patient samples and correlates with poor overall survival. IHC and immunoblotting confirmed elevated PSMD3 levels in MM patient plasma cells compared to healthy controls. siRNA-mediated PSMD3 knockdown significantly reduced the viability of multiple MM cell lines, including those resistant to bortezomib (ANBL6-BR), carfilzomib (AMO1-CFZR), and pomalidomide (H929-PomR), suggesting its role in overcoming drug resistance. Inducible PSMD3 knockout (PSMD3-iKO) in AMO1 and KMS11 cells suppressed proliferation, while re-expression of wild-type PSMD3 restored growth, confirming target specificity. PSMD3 silencing induced apoptosis and cell cycle arrest, validated by flow cytometry and immunoblotting. Given PSMD3's role in proteasome assembly, we evaluated proteasome function and found marked accumulation of K48-linked polyubiquitinated proteins and activation of ER stress pathways in PSMD3-depleted MM cells. A degron-linked reporter assay further confirmed impaired proteasome degradation. In-gel proteasome assays demonstrated that PSMD3 knockout disrupted 26S proteasome assembly without affecting 20S core activity, indicating a deficiency in 19S regulatory particle incorporation. Finally, in a xenograft mouse model, PSMD3 depletion significantly suppressed tumor growth and prolonged survival, validating its therapeutic relevance in vivo. To elucidate the mechanism underlying PSMD3 depletion–induced cell death, we performed quantitative proteomic analyses. Pathway enrichment analysis revealed significant alterations in mitochondrial-related processes including glycolysis and oxidative phosphorylation, indicating that PSMD3 knockdown disrupts mitochondrial homeostasis. Functional metabolic profiling using the Seahorse XF Analyzer demonstrated that PSMD3 silencing led to profound mitochondrial dysfunction, evidenced by elevated mitochondrial superoxide levels and a marked reduction in maximal respiratory capacity, spare respiratory capacity, and ATP-linked respiration. This mitochondrial impairment resulted in the release of mitochondrial double-stranded DNA (mtDNA) into the cytosol, which in turn activated the cGAS-STING pathway. Activation of this innate immune sensor triggered a robust type I interferon response, as evidenced by upregulation of interferon-stimulated genes (ISGs). Moreover, PSMD3 depletion induced hallmark features of immunogenic cell death (ICD), including surface exposure of calreticulin and release of high mobility group box 1 (HMGB1), both of which enhance the immunogenicity of MM cells and promote anti-tumor immune recognition. Our in vitro and in vivo data highlight the therapeutic potential of targeting PSMD3, a central regulator of protein homeostasis which links mitochondrial metabolism to immune surveillance in multiple myeloma. Targeting PSMD3 not only abrogates protein degradation but also disrupts energy metabolism and induces immunogenic cell death via cGAS-STING activation. These findings provide a compelling rationale for developing PSMD3-targeted strategies to overcome drug resistance and promote immune-mediated clearance of MM cells.

Immunomodulatory drugs (IMiDs) are a cornerstone of multiple myeloma (MM) therapy. However, drug resistance remains a major obstacle to cure the disease. Epigenetic dysregulation has been linked to disease progression and drug resistance, prompting the exploration of epigenetic-targeting drugs. Identifying new therapeutic targets is critical to overcoming treatment resistance and improving patient outcomes. We performed RNA sequencing (RNA-seq) on primary CD138+ bone marrow mononuclear cells (BMMCs) from MM patients with differential responses to IMiD-based combination immunotherapy. Analysis revealed that the E2F pathway, G2M pathway and MYC pathway were significantly activated in non-responders (n=8) compared to responders (n=8). By integrating our dataset with upregulated genes from D-KRd non-responders (Nat Med. 2021), we identified the epigenetic regulator PHF19 as the top correlated gene. PHF19 knockdown (KD) impaired MM cell proliferation, induced cell cycle arrest, and promoted apoptosis. RNA-seq of PHF19-KD cells showed significant downregulation of MYC targets, E2F signaling, G2M pathway, and IRF4-dependent pathways (as defined by the Shaffer signature), partially recapitulating the transcriptional profile of non-responders mentioned above. Consistent with this, RT-qPCR and Western blot confirmed reductions in IRF4 and MYC at both mRNA and protein levels upon PHF19 depletion. To elucidate PHF19's mechanistic role, we performed co-immunoprecipitation mass spectrometry (Co-IP/MS), which revealed PHF19 interactions with PRC2 complex components and unexpectedly, mRNA metabolic regulators. Subsequent ATAC-seq in PHF19-KD cells demonstrated widespread reductions in chromatin accessibility at multiple gene loci. Integrated RNA-seq/ATAC-seq analysis identified 391 overlapping genes enriched in cell cycle regulation, including IRF4 and MYC, suggesting that PHF19 sustains their expression by modulating transcriptional accessibility. Additionally, mRNA stability assays using actinomycin D revealed that IRF4 and MYC transcripts were destabilized in PHF19-KD cells, implicating PHF19 in post-transcriptional regulation. Our group previously linked PHF19 to resistance to proteasome inhibitors (PIs) and anti-CD38 monoclonal antibodies. Here, we assessed IMiD sensitivity via CCK-8 proliferation assays and flow cytometry-based apoptosis measurements. Strikingly, PHF19 depletion sensitized MM cells to IMiDs. Mechanistically, IMiDs treatment further suppressed IRF4 and MYC in PHF19-KD cells, whereas their levels remained stable in controls. PHF19 is overexpressed in immunotherapy non-responders and drives resistance by sustaining IRF4/MYC expression through dual epigenetic (chromatin remodeling) and post-transcriptional (mRNA stabilization) mechanisms. Targeting the PHF19-IRF4-MYC axis enhances IMiD sensitivity, positioning PHF19 as a promising epigenetic target to counteract myeloma progression and therapy resistance.

Multiple myeloma (MM) shows inherent clinical and biological heterogeneity, leading to variable treatment responses and outcomes. The complex molecular landscape of MM makes precise risk stratification through clinical genetic testing difficult. Thus, identifying better biomarkers is essential to enhance existing stratification methods and guide personalized therapy decisions. Here, we systematically analyzed the intratumor heterogeneity of tumor cells from 12 newly diagnosed MM patients with different outcomes at single-cell resolution, especially those with an overall survival of less than 2 years, considered extremely high-risk in the real world. Among the eight heterogeneous tumor cell subclusters in these patients' myeloma cells, a particularly aggressive subset was discovered, characterized by severe chromosomal instability, high-level drug resistance, and high-risk genes. Survival analysis indicated that a high rate of this aggressive cell subset was associated with poor outcomes of the patients. We identified seven genes (LILRB4, CD74, TUBA1B, CCND2, HIST1H4C, ITGB7, and CRIP1) with extremely high expression within this subset of aggressive myeloma cells. Multivariate Cox analysis showed that the seven-gene signature score was the worst factor for patients' outcome independently of aberrant cytogenetics and International Staging System stage. We then established an integrated risk stratification model combined with the seven- gene signature score. This model significantly improved the risk discrimination capabilities, especially in distinguishing the ultra-high-risk myeloma patients with the worst outcome in our cohort, and was validated in five independent datasets of MM patients. We further devised a simple digital polymerase chain reaction method for feasible quantification of the seven-gene signature, which still significantly differentiated the survival of MM patients and has considerable value for clinical application. Overall, this integrated risk-scoring model derived from single-cell RNA-sequencing data was significantly associated with a more advanced stage of myeloma, facilitating guided risk-adapted treatment strategies for such ultra-high-risk patients.

Maintaining protein homeostasis is vital for multiple myeloma (MM) cell survival. Indirubin- 3-monoxime (I3MO), a potential MM therapeutic, inhibits proteasome activity, while histone deacetylase 6 (HDAC6) regulates autophagy. We developed I3MV- 8b, an I3MO derivative, integrating an HDAC6 inhibitor moiety to enhance dual inhibition of proteasome and autophagy pathways. The anti-MM effects of I3MV- 8b were tested in vitro and in vivo. To identify downstream targets, RNA-seq and dual-luciferase reporter assays were performed. Additionally, ChIP-seq and IP-MS techniques were employed to elucidate the underlying molecular mechanism. I3MV- 8b significantly suppressed MM cell proliferation and induced apoptosis. Combined with proteasome inhibitors, I3MV- 8b enhanced cytotoxicity by concurrently inhibiting proteasome and autophagy pathways. It reduced TRIM28 transcription, correlating with lower expression of proteasome subunits and autophagy-related genes. ChIP-seq revealed that TRIM28 binds to proteasome gene promoters, and its knockdown decreased proteasome subunit expression and activity. TRIM28 knockdown also impaired autophagosome formation. IP-MS and Co-IP assays showed TRIM28 interacted with 14–3 - 3ζ, a negative regulator of autophagy, promoting its ubiquitination and degradation. This interaction reduced autophagy regulation, further sensitizing cells to treatment. I3MV- 8b offers a novel dual inhibition strategy targeting proteasome and autophagy, presenting a promising therapeutic option for MM.

ABSTRACT:Multiple myeloma (MM), a clonal plasma cell malignancy characterized by high chromosomal instability and inevitable relapse. Increased understanding of immune dysregulation and suppression during MM progression has led to the development of various immunotherapies over the past two decades. Immunotherapeutic strategies, including immunomodulatory imide drugs, monoclonal antibodies, immune checkpoint inhibitors, antibody-drug conjugates, chimeric antigen receptor T cells, and bispecific T cell engagers, have been evaluated in numerous clinical trials and demonstrated significant clinical efficacy, particularly in patients with relapsed and refractory MM. However, despite these substantial advances in immunotherapy, heavily pretreated patients continue to face challenges due to limited therapeutic options and the emergence of multiple drug resistance. Therefore, it is imperative to identify new targets and develop additional treatments aimed at preventing immune escape while enhancing the efficacy of existing immunotherapies.

Monocyte‐derived cells, including osteoclasts, dendritic cells, and macrophages, are key components of the immunosuppressive tumor microenvironment in multiple myeloma (MM). However, the mechanisms linking monocyte dysfunction to immune evasion remain incompletely understood. In this study, single‐cell RNA sequencing (scRNA‐seq) of peripheral blood (PB) and bone marrow (BM) monocytes was performed from healthy donors (HDs) and MM patients to generate a comprehensive single‐cell transcriptional map. Although PB and BM monocytes displayed comparable cellular compositions, MM monocytes exhibited marked transcriptional alterations, most prominently within the type I interferon (IFN) signaling pathway. Trajectory analyses revealed IFN‐driven disruptions in monocyte differentiation and developmental trajectories in both PB and BM compartments. Functional co‐culture assays demonstrated that activation of the type I IFN pathway enhanced MM cell proliferation, suggesting that IFN‐mediated monocyte reprogramming facilitates tumor progression. In an independent validation cohort, longitudinal sampling before and after induction therapy confirmed that anti‐myeloma treatment alleviated the excessive IFN response of BM monocytes. Collectively, these findings uncover a mechanistic link between aberrant IFN activation and monocyte dysregulation in MM, providing new insights into immune dysfunction and highlighting the IFN pathway as a potential therapeutic target to restore anti‐tumor immunity.

The activated B-cell-like subtype of diffuse large B-cell lymphoma (ABC-DLBCL) displays a worse outcome than the germinal center B-cell-like subtype (GCB-DLBCL). Currently, targeting the tumor microenvironment (TME) is the most promising approach to cure DLBCL with profound molecular heterogeneity; however, the factors affecting the tumor-promoting TME of ABC-DLBCL remain elusive. Here, cytokine interleukin-16 (IL-16) is expressed in tumor cells of ABC-DLBCL and secreted by the cleavage of active caspase-3. The serum IL-16 levels are not only a sensitive marker of treatment response, but also positively correlated with unfavorable prognosis in DLBCL patients. While IL-16 shows few direct promotional effects on tumor cell growth in vitro, its bioactive form significantly promotes tumor progression in vivo. Mechanically, IL-16 increases the infiltration of macrophages by the chemotaxis of CD4+ monocytes in the TME, enhancing angiogenesis and the expression of cytokine IL-6 and IL-10, as well as decreasing T-cell infiltration to accelerate tumor progression. This study demonstrates that IL-16 exerts a novel role in co-ordinating the bidirectional interactions between tumor progression and the TME. IMM0306, a fusion protein of CD20 mAb with the CD47 binding domain of SIRPα, reverses the tumor-promoting effects of IL-16, providing new insights into treatment strategy in ABC-DLBCL.