Relapse and treatment resistance remain critical obstacles in the clinical management of angioimmunoblastic T-cell lymphoma (AITL), limiting the success of current therapeutic strategies. Therefore, a comprehensive characterization of the tumor microenvironment (TME) in AITL is essential to enhance treatment efficacy. This study analyzed samples from 68 patients with AITL, stratified into three molecular subtypes based on immunoglobulin (IG) gene rearrangement and flow cytometry results. Utilizing single-cell RNA sequencing, the TME was profiled across subtypes A, B, and C, sampling multiple disease sites including the bone marrow, lymph nodes, and peripheral blood. Our findings revealed subtype-specific variations in cellular composition and transcriptional programs within the TME. Unlike other subtypes, subtype C was associated with a pronounced immunosuppressive environment at diagnosis and relapse. Additionally, it exhibited an enhanced response to Epstein–Barr virus infection, consistent with upregulated CD70 expression at relapse. Through the analysis of cellular communication networks, CD70, programmed cell death 1 (PDCD1), and inducible T cell costimulator (ICOS) were identified as promising immunotherapeutic targets in AITL. Finally, we delineated distinct cellular proportions and gene expression signatures characteristic of each subtype, providing a foundation for the development of tailored therapeutic interventions for patients with AITL.

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.

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.

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.

Introduction Most mantle cell lymphoma (MCL) patients still experience disease progression after treatment with BTK inhibitors (BTKis). Exploring targetable key genes and combination therapy strategies is crucial for improving the prognosis of such patients. Results Our results showed SMARCA2 deficiency was associated with MCL progression and BTKi resistance. In vitro and in vivo experiments confirmed that SMARCA2 downregulation reduced sensitivity to the BTKi, ibrutinib (IBN), through elevated ATP levels, increased oxygen consumption, and downregulated reactive oxygen species (ROS) levels. The ROS-inducing effect of IBN was attenuated in SMARCA2 deficient cell lines and patient samples. Joint analysis of RNA-seq and ATAC-seq data from cell lines with shSMARCA2 and RNA-seq from patient samples indicated the pathways that SMARCA2 regulates were enriched in ATP metabolism and in the mitochondrial inner membrane. SMARCA2 downregulation led to decreased accessibility to the introns of the TENT5C gene and increased H3K27me3 suppressive modification, leading to downregulated expression of TENT5C. TENT5C overexpression in shSMARCA2 cells restored ROS levels, reduced ATP levels, and resensitized cells to IBN. Mechanistic studies showed TENT5C/FAM46C colocalized with ATP5A1 in mitochondria, and FAM46C increased ATPase inhibitory factor (IF1) expression and its binding to ATP5A1 for inhibiting ATP synthesis. Our results also demonstrated the synergistic therapeutic effect of isoginkgetin and IBN by reducing ATP levels and inhibiting the BCR pathway, thereby reversing IBN resistance in vitro and in vivo. Conclusion Our study highlights that SMARCA2 regulates ATP metabolism by altering the accessibility and expression of TENT5C gene, thereby affecting BTKi resistance. Isoginkgetin combined with IBN provides a promising therapeutic opportunity for overcoming BTKi resistance by reducing ATP production in relapsed MCL.

Multiple myeloma (MM) remains an incurable hematologic malignancy. Despite tremendous advances in the treatment of this disease, about 10% of patients still have very poor outcomes with a median overall survival of less than 24 months. Our study aimed to underscore the critical mechanisms pertaining to rapid disease progression and provide novel therapeutic choices for these ultrahigh-risk patients. We utilized single-cell transcriptomic sequencing to dissect the characteristic bone marrow niche of patients who survived less than 2 years (EM24). Notably, enrichment of a LILRB4high pre-mature plasma-cell cluster was observed in EM24 patients compared to patients with durable remission. This cluster exhibited aggressive proliferation and a drug-resistance phenotype. High levels of LILRB4 promoted MM clonogenicity and progression. Clinically, high expression of LILRB4 was correlated with poor prognosis in both newly diagnosed MM patients and relapsed/ refractory MM patients. ATAC-sequencing analysis identified that pronounced chromosomal accessibility caused the elevation of LILRB4 on MM cells. CRISPR-Cas9 deletion of LILRB4 alleviated the growth of MM cells, inhibited the immunosuppressive function of myeloid-derived suppressive cells (MDSC), and further rescued T-cell dysfunction in the MM microenvironment. Greater infiltration of MDSC was observed in EM24 patients. We therefore generated an innovative T-cell receptor-based chimeric antigen receptor T cell, LILRB4-STAR-T. Cytotoxicity experiments demonstrated that LILRB4-STAR-T cells efficaciously eliminated tumor cells and impeded MDSC function. In conclusion, our study elucidates that LILRB4 is an ideal biomarker and promising immunotherapy target for high-risk MM. LILRB4-STAR-T-cell immunotherapy is promising against both tumor cells and the immunosuppressive tumor microenvironment in MM.

Supplementary Table 8. The response rate by MYD88/CXCR4 genotype during first-line cytotoxic therapy.

Background: Mantle cell lymphoma (MCL) is an incurable lymphoma with high clinical heterogeneity. The application of BTK inhibitors (BTKi) has improved the prognosis of MCL patients, but most patients still experience disease progression within a few years, and those with relapsed MCL respond poorly to available salvage therapies. Elucidating the mechanisms of MCL resistance, early identification of resistant populations, and exploring targetable key genes and combination therapy strategies are crucial for improving the prognosis of such patients. Methods: In this study, we constructed stable cell lines with SMARCA2 knockdown and overexpression (Jeko-1 SMARCA2sh, Jeko-1 SMARCA2OE, Z138 SMARCA2sh, Mino SMARCA2sh) and induced ibrutinib-resistant cell lines (Z138-R, Z138-R SMARCA2OE). We verified the effects of SMARCA2 on cell proliferation, cell cycle, pathway abnormalities, and sensitivity to ibrutinib in vitro and in vivo. Transcriptome and ATAC-sequencing were performed with SMARCA2sh cell lines before and after treated with ibrutinib. The evaluation of mitochondrial function was achieved by detecting ATP release, oxygen consumption, mitochondrial membrane potential, and ROS production in SMARCA2sh and SMARCA2OE cells. The anti-MCL activity and mechanism of Isoginkgetin (IGK) monotherapy or combination with Ibrutinib were evaluated in SMARCA2sh or Ibrutinib resistant MCL cells in vitro and in vivo. Results: Preliminary studies [JCI 2022, doi: 10.1172/JCI153283] involving WES analysis of 134 MCL patient samples and RNA sequencing of 62 MCL patient samples found that SMARCA2 deletion or low expression was associated with MCL progression, BTKi resistance, and shorter patient survival. In vitro and in vivo experiments confirmed that SMARCA2 downregulation promoted MCL cell proliferation, extended the S phase of the cell cycle, and reduced sensitivity to ibrutinib. SMARCA2sh cells exhibited elevated ATP levels, increased oxygen consumption, and downregulated ROS levels. Ibrutinib inhibited ROS levels, but its inhibitory effect was diminished in SMARCA2sh cell lines, leading to Ibrutinib resistance. Patient-derived primary cell experiments confirmed that the inhibitory effect of ibrutinib on ROS was weakened in patients with SMARCA2 deletion. Joint analysis of RNA-seq and ATAC-seq data from cell lines with SMARCA2sh and RNA-seq from patient samples indicated that the pathway which SMARCA2 regulated were enriched in ROS production, ATP metabolism, and mitochondrial inner membrane-related pathways. Additionally, SMARCA2 downregulation led to decreased the accessibility of the intron regions of TENT5C genes, and higher H3K27me3 suppressive modification for downregulated expression of TENT5C. Overexpression of TENT5C in SMARCA2sh cell lines led to decreased cell proliferation, reduced ATP levels, upregulated ROS levels, and restored sensitivity to ibrutinib. These results were also validated by knocking down TENT5C in SMARCA2OE cell lines. Multicolor immunohistochemistry in tumor issue from mouse model and immunofluorescence assays of MCL cell line revealed co-localization of TENT5C/FAM46C with ATP5A and SLC25A5 in mitochondrial. Co-immunoprecipitation experiments further confirmed that FAM46C may regulate ATP synthesis and ADP/ATP transformation to affect drug resistance by interacting with ATP5A and SLC25A5 respectively. IGK has been reported to be effective in FAM46C knockout myeloma cell lines. Our in vitro experiments demonstrated the synergistic therapeutic effect of IGK and ibrutinib. Mechanistically, IGK inhibited ATP levels, restored ROS production, activated the BCR signaling pathway, thereby reversed ibrutinib resistance in SMARCA2sh and Z138-R cell lines. Finally, we used Cell Line-Derived Xenograft (CDX) and Patient-Derived Xenografts (PDX) models to further validate the synergistic effect of IGK and ibrutinib. Conclusion: Our study first highlights that SMARCA2 regulates the ATP metabolism by altering the accessibility and expression of the TENT5C gene, thereby affecting MCL proliferation and BTKi resistance. IGK enhances ibrutinib efficacy by reducing ATP production, and exhibits a synergistic effect with ibrutinib. These findings provide novel therapeutic targets and strategies to overcome BTK inhibitor resistance in MCL.

Supplementary Figure 9. The survival of patients according to IPSSWM risk model. (A) The overall survival of the whole cohort; (B) The overall survival in patients treated with BTKi-based therapy

Introduction: The maintenance of intracellular protein homeostasis is a critical biological process necessary for the survival of multiple myeloma (MM) cells. Tripartite motif (TRIM) proteins, a subfamily of the RING-type E3 ubiquitin ligase family, are integral to protein quality control. In this study, we examined the role of TRIM28, a member of the TRIM family, as a regulator of protein homeostasis in MM. Methods: Bioinformatic analysis was utilized to elucidate the association between TRIM28 and genes involved in the proteasome and autophagy mediated protein degradation pathway. ShRNA knockdown (KD) and overexpression (OE) experiments were conducted to investigate the role of TRIM28 in MM pathogenesis. ChIP-seq and IP-MS analyses were performed to identify potential downstream targets of TRIM28. Results: Firstly, our survival analysis revealed that MM patients exhibited poor outcomes with high levels of TRIM28 in both the MMRF-CoMMpass and in-house datasets, particularly in the context of bortezomib treatment. Overexpression of TRIM28 promoted cell proliferation and induced drug resistance in MM cell lines, while knock-down of TRIM28 had the opposite effect. Bioinformatic analysis demonstrated a strong correlation between TRIM28 expression and the expression of proteasome subunits and autophagy-related genes in MM datasets. RNA-seq data showed down-regulation of both the proteasome pathway and autophagy pathway after TRIM28 knockdown, leading to decreased proteasome activities and accumulation of ubiquitinated proteins. Immunofluorescence assay confirmed nuclear localization of TRIM28, while ChIP-seq identified its binding to proteasome gene promoters such as PSMB1, PSMD2, and PSMD4. Knock-down of TRIM28 reduced the expression of proteasome subunits, indicating its critical role in transcriptional activation of these genes in MM cells. Additionally, a decrease in autophagosome formation was observed in TRIM28 KD cells, indicating the involvement of TRIM28 in autophagy activity. Further IP-MS and Co-IP assays demonstrated the interaction between TRIM28 and 14-3-3ζ. A significant reduction in the polyubiquitination level of 14-3-3ζ was noted in TRIM28 KD cells. The 14-3-3ζprotein serves as an adaptor that modulates cellular signaling by binding to a wide array of proteins. Therefore, acting as an E3 ligase, TRIM28 promotes ubiquitin-dependent degradation of 14-3-3ζ, thereby enhancing autophagy activity in MM. These findings underscore the crucial role of TRIM28 in maintaining protein homeostasis by regulating both proteasome and autophagy activities involved in myeloma pathogenesis. Conclusion: Our research has provided new and important insights into the crucial role of TRIM28 in maintaining protein homeostasis and its involvement in myeloma pathogenesis. TRIM28 activates the proteasome and autophagy pathways, making it a potential promising therapeutic target in MM.