Figure S2. The comparison of the expression of CDK4/6-cyclin D pathway genes based on age (A) and risk stratification (B). The Kaplan-Meier curve for CDK6 (C). The calibration curve of the nomogram for 1-year and 2-year overall survival (D).

Figure S3. Knockdown CDK4 and CDK6 via siRNA in HL-60 and molm13 cells (A). The effect of palbociclib and ATRA combination on cell cycle(B), cell proliferation (C), and cell apoptosis (D). * Represent when it compares with the control group, P<0.05. # Represent when it compares with the ATRA group, P<0.05.

BACKGROUND:Resistance to the proteasome inhibitor bortezomib is a major obstacle to the treatment of multiple myeloma (MM) and a major cause of relapse and death. Investigating the role of bortezomib resistance genes in MM is crucial. This study aimed to evaluate the potential of bortezomib resistance-related genes (BRGs) as prognostic biomarkers in MM. METHODS:The transcriptome data of bortezomib resistant myeloma cell lines, as well as the gene expression and clinical data of patients were downloaded from the Gene Expression Omnibus (GEO) database. Univariate Cox and least absolute shrinkage and selection operator (LASSO) Cox regression models were employed to screen variables and construct a multigene prognostic signature based on BRGs. Single-sample gene set enrichment analysis (ssGSEA) was performed to quantify the relative infiltration levels of immune cells. The pRRophetic algorithm was utilized to assess and infer the sensitivity of anti-MM chemotherapeutics. RESULTS:We identified 129 differentially expressed BRGs, with 25 associated with MM prognosis. Using the LASSO Cox regression model, we identified five key genes (IFI16, ARID5B, LTBP1, PNOC, CRIP1) and developed a bortezomib resistance model for risk stratification and prognosis prediction. Multivariate Cox regression analysis revealed that the risk score was an independent prognostic factor for overall survival (OS). Based on pRRophetic results, high-risk patients may be more sensitive to other chemotherapeutic agents, such as doxorubicin and etoposide. Additionally, we constructed a nomogram incorporating patient age, LDH, International Staging System (ISS), and BRGs, which demonstrated robust prognostic prediction capabilities. The receiver operating characteristic (ROC) values for 1-, 3-, and 5-years survival rates were 0.730, 0.734, and 0.775, respectively. We also validated the expression patterns of the five key genes in MM. IFI16 and CRIP1 expression levels were upregulated in relapsed patients, whereas ARID5B expression was decreased. PNOC and LTBP1 showed no significant differences. Notably, lower ARID5B expression was associated with poorer OS in patients. CONCLUSIONS:The BRGs signature is a reliable biomarker for predicting the prognosis of MM and helps optimize clinical decision-making for treatment, and identifies key gene ARID5B downregulation as an adverse prognostic factor in multiple myeloma.

BackgroundAlthough CD19 CAR-T cell therapy has improved outcomes in relapsed or refractory diffuse large B-cell lymphoma (DLBCL), responses are variable and predictive biomarkers are lacking. The triglyceride glucose-body mass index (TyG-BMI)—a surrogate of insulin resistance—has shown prognostic value in solid tumors, but its relevance to DLBCL and CAR-T efficacy remains unknown.MethodsWe retrospectively analyzed 204 newly diagnosed DLBCL patients treated with rituximab-containing regimens, stratified by median TyG-BMI. Prognostic associations were assessed by Kaplan-Meier and multivariate Cox regression, and a nomogram was constructed. In an exploratory sub-analysis, 20 patients who received CD19 CAR-T therapy were assessed. CAR-T cell persistence was measured by flow cytometry one week post-infusion and correlated with pre-infusion TyG-BMI by Pearson analysis. CRS and ICANS were graded per ASTCT criteria. Post-CAR-T survival was compared by Kaplan-Meier analysis stratified by median pre-infusion TyG-BMI.ResultsHigh TyG-BMI was independently associated with poorer PFS (HR, 2.146; 95% CI, 1.092–4.214; P = 0.027) and OS (HR, 2.531; 95% CI, 1.084–5.913; P = 0.032), and further stratified risk within IPI risk groups and across disease stages. A nomogram incorporating TyG-BMI, IPI, and bone marrow involvement predicted 1-, 3-, and 5-year OS (AUC: 0.886, 0.799, 0.772). In the CAR-T subcohort (n = 20), elevated pre-infusion TyG-BMI was significantly inversely correlated with CAR-T cell persistence at one week post-infusion (r = -0.570, P = 0.009). Patients with high pre-infusion TyG-BMI experienced inferior responses to CAR-T therapy, with shorter median PFS (5.0 vs. 19.5 months) and OS (39.0 vs. 81.0 months) compared with the low TyG-BMI group.ConclusionTyG-BMI is an independent prognostic factor in DLBCL that refines IPI-based stratification, and elevated pre-infusion TyG-BMI is associated with attenuated CAR-T cell persistence and inferior outcomes. This readily available metabolic index may aid risk assessment and treatment planning in DLBCL.

Figure S4. The effect of Palbociclib and ATRA combination on leukemia burden in vivo (A). Volcano plots of differentially expressed genes in HL-60 cells (B) and molm13 cells (C) from the control and palbociclib and ATRA combination groups.

Figure S1. The mutation frequency of CDK4/6-cyclin D pathway genes, CDKN1A and CDKN2A in 531 AML patients (A). The expression of CDKN1A and CDKN2A was compared between AML and healthy donors in the BeatAML database. * Represent P<0.05.

IntroductionBone marrow involvement (BMI) is a poor prognostic factor in diffuse large B cell lymphoma (DLBCL), and accurate evaluation of BMI is crucial for determining stages and prognosis. This study aimed to identify the most effective examinations for evaluating BMI in DLBCL, including positron emission tomography-computed tomography (PET/CT), immunoglobulin gene rearrangement (IGR), flow cytometry (FCM), bone marrow cytology (BMC) and bone marrow biopsy pathology (BMB), and to further explore its prognostic significance in DLBCL patients.MethodsThis retrospective study included 364 newly diagnosed DLBCL patients, all of whom underwent PET/CT, IGR, FCM, BMC, and BMB at diagnosis. Survival outcomes were analyzed via Kaplan-Meier and Cox regression models. Novel prognostic models incorporating combined IGR and BMB results were developed in a training cohort.ResultsCompared to other detection methods, Clonal IGR BMI-positive were found the highest rate of 114 patients (31.3%), and IGR BM involvement-positive patients of DLBCL had the worst survival outcomes, especially among patients in stages I to III (P<0.001). Notably, PET/CT existed some limitations in BMI diagnosis, particularly in stage IV patients (P>0.05). Additionally, the combination of IGR and BMB demonstrated superior prognostic predictive capability for the patients in stage IV (PP<0.001). Multivariate analysis further confirmed that double-positive BMI of IGR and BMB was an independent prognostic factors of PFS (P=0.026) and OS (P=0.042). In addition, the novel IPI and NCCN-IPI stratification models were established by incorporating the combination of IGR and BMB in training group. The C-index of novel models were increased when IGR and BMB were supplemented in our cohort.DiscussionOur results suggest that IGR is the most valuable methods for evaluating BMI compared to traditional detection methods. Adding the combination of IGR and BMB to the IPI and NCCN-IPI score may improve their predictive ability. In summary, IGR is essental for evaluation of BMI and provide an ideal method for disease staging and risk stratification in DLBCL patients in the rituximab era.

BACKGROUND Multiple myeloma (MM) remains incurable due to inevitable therapeutic resistance, and Bortezomib (BTZ ) resistance is a major challenge. The interferon-inducible protein 16 (IFI16) is implicated in oncogenesis and drug resistance, but its role in MM BTZ resistance is unknown. Here,we identified IFI16 as a key mediator of drug resistance through bioinformatic analysis and clinical validation. METHODS Transcriptomic profiling of bortezomib-resistant MM cells (GSE160572, GSE136337, GSE2658, GSE9782) identified IFI16 as a top resistance-associated gene (HR>1, p<0.005). Validation cohorts included 78 MM patients (newly diagnosed/remission/relapsed) a nd iron-deficiency anemia controls. Functional studies utilized IFI16-silence down/overexpression models in H929/U266 cells and murine xenografts (subcutaneous/disseminated). mechanisms were performed by RNA-seq，WB and dual-luciferase reporter assays. KEY RESULTS Clinical Significance: IFI16 expression was significantly elevated in newly diagnosed (3.8-fold, p=0.002) and relapsed MM (2.9-fold, p=0.007) vs remission, correlating with bone lesions (r=0.5), renal impairment (r=0.47), and poor survival (HR=3.1, 95%CI 0.625-15.68). Functional Validation: IFI16 knockdown sensitized MM cells to bortezomib (IC50 decreased 2.2-fold, p<0.001), increased apoptosis (2.4-fold, p<0.001), and induced G0/G1 arrest. IFI16 overexpression conferred resistance (IC50 increased 1.6-fold, p<0.001) and reduced drug-induced apoptosis by14% (p=0.04). Mechanism: IFI16 activated Wnt/β- catenin signaling, increasing β-catenin (2.1-fold), p-GSK3β (2.5-fold), and cyclin D1(1.8-fold) while suppressing p21(3.1-fold); but IWP-2 reversed changes of the above protein and function resistance in IFI16-overexpressing cells. In Vivo Efficacy: IFI16 knockdown synergized with bortezomib, reducing tumor volume (decreased 70%, p<0.0001) and osteolytic lesions in murine models. Immunohistochemistry confirmed pathway suppression (β-catenin downregulated 70%; Ki67downregulated 50%). CONCLUSIONS & INNOVATION This study identifies that IFI16 is a prognostic biomarker and predictor of treatment failure and as a novel master regulator of MM pathogenesis and BTZ resistance. We also demonstrate that IFI16 exerts its oncogenic effects primarily through constitutive activation of the Wnt/β-catenin pathway. Targeting the IFI16/Wnt/β-catenin axis represents a promising strategy to overcome BTZ resistance and restore proteasome inhibitor sensitivity in high-risk MM.

Chemotherapy incorporating the proteasome inhibitor bortezomib (BTZ) has improved outcomes for patients with multiple myeloma (MM); however, resistance to chemotherapy and disease relapse remain significant challenges, closely associated with cellular senescence. This study investigated the key drivers of myeloma cell senescence and its role in MM progression. Flow cytometry assessed senescence-associated β-galactosidase (SA-β-gal) activity in myeloma cells from bone marrow samples of MM patients. BTZ was used to establish cell senescence model. RNA-seq identified key genes regulating myeloma cell senescence, which were verified by qPCR. After CBX7 knockdown and overexpression, SA-β-gal staining, CCK-8 assay, cell cycle assay, and colony formation assay were performed to investigate its effects on senescence. RNA-seq further identified downstream target genes and pathways, and small interfering RNA and ERK inhibitor were used to explore their effects. A xenograft mouse model was used to validate CBX7’s effect on myeloma cell senescence. Our results demonstrated that BTZ-based chemotherapy induces senescence in myeloma cells, with SA-β-gal activity linked to malignant proliferation. CBX7 was identified as a critical regulator of cellular senescence in myeloma cells. Elevated CBX7 levels were observed in newly diagnosed MM, decreased during remission, and increased again at relapse. CBX7 levels positively correlated with blast counts, creatinine, and β2-microglobulin levels, and negatively correlated with SA-β-gal activity. Functionally, CBX7 knockdown promoted BTZ-induced myeloma cell senescence and senescence-like growth arrest, whereas CBX7 overexpression had the opposite effect. Mechanistically, CBX7 regulates senescence-like growth arrest in myeloma cells via the ERK/STAT3/MIX1 axis. Silencing PIM1 or the ERK inhibitor U0126 mitigated CBX7-mediated myeloma cell senescence and enhanced the inhibitory effects of BTZ on cell viability and clone formation. In vivo, CBX7 knockdown enhanced BTZ-inhibited xenograft tumor growth. CBX7 is a pivotal target for regulating cellular senescence in myeloma cells, operating through a novel CBX7/ERK/PIM1 regulatory axis. Targeting CBX7 and its downstream pathways may augment the efficacy of standard chemotherapy.

Bruton tyrosine kinase inhibitor (BTKi) combined with rituximab-based chemotherapy benefits diffuse large B-cell lymphoma (DLBCL) patients. However, drug resistance is the major cause of relapse and death of DLBCL. In this study, we conducted a comprehensive analysis BTKi-resistance related genes (BRRGs) and established a 10-gene (CARD16, TRIP13, PSRC1, CASP1, PLBD1, CARD6, CAPG, CACNA1A, CDH15, and NDUFA4) signature for early identifying high-risk DLBCL patients. The resistance scores based on the BRRGs signature were associated with prognosis. Furthermore, we developed a nomogram incorporating the BRRGs signature, which demonstrated excellent performance in predicting the prognosis of DLBCL patients. Notably, tumor immune microenvironment, biological pathways, and chemotherapy sensitivity were different between high- and low-resistance score groups. Additionally, we identified TRIP13 as a key gene in our model. TRIP13 was found to be overexpressed in DLBCL and BTKi-resistant DLBCL cell lines, knocking down TRIP13 suppresses cell proliferation, promotes cell apoptosis, and enhances the apoptosis effect of BTKi on DLBCL cells by regulating the Wnt/β-catenin pathway. In conclusion, our study presents a novel BRRGs signature that could serve as a promising prognostic marker in DLBCL, and TRIP13 might be a potential therapeutic target for resistant DLBCL.

Multiple myeloma (MM) is an incurable hematological malignancy with poor survival. Accumulating evidence reveals that lactylation modification plays a vital role in tumorigenesis. However, research on lactylation-related genes (LRGs) in predicting the prognosis of MM remains limited. Differentially expressed LRGs (DELRGs) between MM and normal samples were investigated from the Gene Expression Omnibus database. Univariate Cox regression and LASSO Cox regression analysis were applied to construct gene signature associated with overall survival. The signature was validated in two external datasets. A nomogram was further constructed and evaluated. Additionally, Enrichment analysis, immune analysis, and drug chemosensitivity analysis between the two groups were investigated. qPCR and immunofluorescence staining were performed to validate the expression and localization of PFN1. CCK-8 and flow cytometry were performed to validate biological function. A total of 9 LRGs (TRIM28, PPIA, SOD1, RRP1B, IARS2, RB1, PFN1, PRCC, and FABP5) were selected to establish the prognostic signature. Kaplan-Meier survival curves showed that high-risk group patients had a remarkably worse prognosis in the training and validation cohorts. A nomogram was constructed based on LRGs signature and clinical characteristics, and showed excellent predictive power by calibration curve and C-index. Moreover, biological pathways, immunologic status, as well as sensitivity to chemotherapy drugs were different between high- and low-risk groups. Additionally, the hub gene PFN1 is highly expressed in MM, knocking down PFN1 induces cell cycle arrest, suppresses cell proliferation and promotes cell apoptosis. In conclusion, our study revealed that LRGs signature is a promising biomarker for MM that can effectively early distinguish high-risk patients and predict prognosis.

Philadelphia chromosome-positive B cell acute lymphoblastic leukemia [Ph(+) B-ALL] is a hematological malignancy with a poor prognosis. Epigenetic abnormalities, especially abnormal histone acetylation and microRNA (miRNA) dysregulation, are a group of epigenetic patterns that contribute to leukemia progression. However, their regulatory mechanisms in Ph(+) B-ALL have not been fully elucidated. In this study, we identified that miR-183-5p is significantly downregulated in Ph(+) B-ALL and associated with poor prognosis. Moreover, we found that the BCR-ABL fusion gene is a key target gene of miR-183-5p. MiR-183-5p directly targets the BCR-ABL gene and induces cell apoptosis via PTEN/AKT and c-MYC signaling pathways. In addition, a histone deacetylase inhibitor could mitigate the suppressive effects of HDAC2 on miR-183-5p by promoting promoter acetylation, thereby enhancing cell apoptosis. In conclusion, our results indicate that miR-183-5p is a potential biomarker and suggest that a novel HDAC2-miR-183-5p epigenetic circuitry regulation may be involved in the pathogenesis of Ph(+) B-ALL. Taken together, These findings provide new insights into the design of promising molecular-targeted drugs for Ph(+) B-ALL. MiR-183-5p directly targets the BCR-ABL fusion gene and promotes cell apoptosis through the PTEN/AKT and c-MYC signaling pathways. A histone deacetylase inhibitor could mitigate the suppressive effects of HDAC2 on miR-183-5p by promoting the acetylation, thereby enhancing cell apoptosis.

Abstract Differentiation therapy based on ATRA almost cured acute promyelocytic leukemia (APL). However, it is disappointing that ATRA is not effective against other acute myeloid leukemia (AML) subtypes. Developing new and effective anti-AML therapies that promote leukemia differentiation is necessary. The CDK4/6-cyclin D pathway is a key initiator of the G1–S phase transition, which determines cell fate. Herein, we investigated whether the CDK4/6 inhibitor palbociclib would synergize with ATRA to promote leukemia differentiation in vitro and in vivo. Our findings revealed that CDK4/6-cyclin D pathway genes were aberrantly expressed in AML, and we observed that palbociclib sensitized AML cells to ATRA-induced morphologic, biochemical, and functional changes indicative of myeloid differentiation. The combination of palbociclib and ATRA attenuated AML cell expansion in vivo. These enhanced differentiation effects may be associated with the regulation of transcription factors, including RARα, E2F1, and STAT1. Overall, our findings demonstrate that CDK4/6 inhibition sensitizes AML cells to ATRA and could guide the development of novel therapeutic strategies for patients with AML.

Abstract Background CD19-targeted chimeric antigen receptor T (CAR-T) cell therapy stands out as a revolutionary intervention, exhibiting remarkable remission rates in patients with refractory/relapsed (R/R) B-cell malignancies. However, the potential side effects of therapy, particularly cytokine release syndrome (CRS) and infections, pose significant challenges due to their overlapping clinical features. Promptly distinguishing between CRS and infection post CD19 target CAR-T cell infusion (CTI) remains a clinical dilemma. Our study aimed to analyze the incidence of infections and identify key indicators for early infection detection in febrile patients within 30 days post-CTI for B-cell malignancies. Methods In this retrospective cohort study, a cohort of 104 consecutive patients with R/R B-cell malignancies who underwent CAR-T therapy was reviewed. Clinical data including age, gender, CRS, ICANS, treatment history, infection incidence, and treatment responses were collected. Serum biomarkers procalcitonin (PCT), interleukin-6 (IL-6), and C-reactive protein (CRP) levels were analyzed using chemiluminescent assays. Statistical analyses employed Pearson’s Chi-square test, t-test, Mann–Whitney U-test, Kaplan–Meier survival analysis, Cox proportional hazards regression model, Spearman rank correlation, and receiver operating characteristic (ROC) curve analysis to evaluate diagnostic accuracy and develop predictive models through multivariate logistic regression. Results In this study, 38 patients (36.5%) experienced infections (30 bacterial, 5 fungal, and 3 viral) within the first 30 days of CAR T-cell infusion. In general, bacterial, fungal, and viral infections were detected at a median of 7, 8, and 9 days, respectively, after CAR T-cell infusion. Prior allogeneic hematopoietic cell transplantation (HCT) was an independent risk factor for infection (Hazard Ratio [HR]: 4.432 [1.262–15.565], P = 0.020). Furthermore, CRS was an independent risk factor for both infection ((HR: 2.903 [1.577–5.345], P < 0.001) and severe infection (9.040 [2.256–36.232], P < 0.001). Serum PCT, IL-6, and CRP were valuable in early infection prediction post-CAR-T therapy, particularly PCT with the highest area under the ROC curve (AUC) of 0.897. A diagnostic model incorporating PCT and CRP demonstrated an AUC of 0.903 with sensitivity and specificity above 83%. For severe infections, a model including CRS severity and PCT showed an exceptional AUC of 0.991 with perfect sensitivity and high specificity. Based on the aforementioned analysis, we proposed a workflow for the rapid identification of early infection during CAR-T cell therapy. Conclusions CRS and prior allogeneic HCT are independent infection risk factors post-CTI in febrile B-cell malignancy patients. Our identification of novel models using PCT and CRP for predicting infection, and PCT and CRS for predicting severe infection, offers potential to guide therapeutic decisions and enhance the efficacy of CAR-T cell therapy in the future.

Background: Acute myeloid leukemia (AML) is an incurable hematological malignancy. The triglyceride-glucose (TYG) index is a surrogate marker for insulin resistance, but its relationship with AML prognosis remains unclear. This study aimed to investigate the prognostic value of the TYG index in AML. Methods: We retrospectively analyzed laboratory test data of 158 initially diagnosed AML patients and calculated the TYG index. The association between TYG index and patient prognosis was evaluated by survival curve. We constructed a prognostic nomogram through multivariate Cox regression analyses, and its performance was evaluated using calibration curves, the C-index, and the area under the curve (AUC). Results: A total of 158 AML patients, aged between 18 and 83 years (mean age 50.48 years), were included in this study from January 2017 to June 2023. Of these, 131 patients (82.9%) achieved CR after one or two courses of induction chemotherapy. 81 patients (51.27%) were male, 71 patients (44.94%) relapsed and 89 patients (56.32%) died at the end of follow-up. The median follow-up time was 27 months. According to the 2017 European Leukemia Network (ELN2017) genetic risk stratification, 44 (27.85%), 59 (37.34%), and 55 (34.81%) of patients were classified into favorable, intermediate, and adverse risk groups, respectively. Fasting glucose and fasting triglycerides were measured at diagnosis, with a median TYG index level of 1.28. Patients were divided into high and low TYG index groups based on median value. We investigated the impact of TYG index on RFS and OS. Patients with high TYG index had significantly inferior RFS and OS compared to those with low TYG index (5-year RFS: 52.9% vs. 16.2%, log-rank P<0.001; 5-year OS: 42.6% vs. 20.8%, log-rank P<0.01). We further investigated whether the TYG index could stratify patients within each ELN risk group. In the intermediate risk group, patients with high TYG index also had significantly inferior RFS and OS compared to those with low TYG index patients (5-year RFS: 53.6% vs. 14.4%, log-rank P<0.01; 5-year OS: 43.9% vs. 23.2%, log-rank P<0.05). Similar results were observed in the adverse risk groups for RFS (2-year RFS: 51.5% vs. 9.3%, log-rank P<0.05) but not for OS. Although there was no statistically significant difference in the favorable risk group, there was a trend toward worse RFS and OS for patients with high TYG index. In particular, among the 22 patients with AML-ETO positivity in the favorable risk group, those with high TYG index had significantly inferior RFS and OS compared to those with low TYG index (3-year RFS: 91.7% vs. 14.6%, log-rank P<0.01; 5-year OS: 85.1% vs. 28.6%, log-rank P<0.05). Multivariate analysis showed that TYG index was an independent prognostic factor for both OS (HR 1.502, 95%CI 1.139-1.981) and RFS (HR 1.526, 95%CI 1.110-2.099) in AML patients. A new prognostic nomogram was developed, incorporating the TYG index, age, and ELN2017. This nomogram demonstrated high predictive accuracy for 1-year, 3-year, and 5-year OS in AML patients, with AUCs of 0.789, 0.764, and 0.798, respectively. Conclusions: The TYG index is an independent prognostic factor for AML. The new nomogram, which combines TYG index, age, and ELN2017 genetic risk stratification, provides a quick and simple method with high clinical value for predicting the prognosis of AML patients.

Myeloid-derived suppressor cells (MDSCs) are implicated in the regulation of immune responses closely associated with poor clinical outcomes in cancer. However, the MDSC subtypes in non-Hodgkin's lymphoma (NHL) have not been systematically investigated. So, we investigated the percentage of MDSC subsets in 78 newly diagnosed NHL patients by flow cytometry. The results showed that all MDSC subsets increased in NHL patients compared with healthy donors. Notably, MDSCs, monocytic MDSCs, and CD14 + CD66b + MDSCs significantly increased in NHL patients compared with those with lymphadenitis donors. polymorphonuclear MDSCs (PMN-MDSCs), early-stage MDSCs (e-MDSCs), and the International Prognostic Index were independent risk factors for poor clinical efficacy and were involved in constructing the nomogram for predicting clinical efficacy. Progression-free survival (PFS) was significantly shorter in patients with high level of MDSC subsets, and PMN-MDSCs emerged as an independent prognostic factor for PFS. PMN-MDSCs, e-MDSCs, and the International Prognostic Index were involved in constructing the nomogram for predicting PFS. Patients with a higher percentage of MDSCs, PMN-MDSCs, e-MDSCs, and CD14 + CD66b + MDSCs experienced a shorter overall survival compared with those with lower percentages. In addition, research on mechanisms found that T cell function was suppressed and mediated by the expansion of MDSCs via involving arginase-1 and interleukin-10 in vitro and in vivo. In conclusion, our study demonstrates that the increased circulating MDSC subsets predict poor clinical efficacy and prognosis in NHL, potentially involving T cell suppression through MDSC subset expansion. These findings indicate the potential of MDSC subsets as comprehensive diagnostic, prognostic biomarkers, and therapeutic targets for NHL.

Background: Bruton tyrosine kinase inhibitor (BTKi) combined with rituximab-based chemotherapy benefits diffuse large B-cell lymphoma (DLBCL) patients. However, drug resistance is a major cause of relapse and death in DLBCL. This study aims to explore the relationship between BTKi-resistance related genes (BRRGs) and DLBCL prognosis, and to clarify the function and mechanism of key resistance genes. Results: In this study, we analyzed transcriptome data from parental and ibrutinib-resistant clone cell lines (GSE138126) and identified 1186 differentially expressed genes (DEGs), with 552 upregulated and 634 downregulated in the ibrutinib-resistant group. The GSE31312 dataset was used as the training cohort, while GSE87371 and GSE10846 served as external validation cohorts. Univariate Cox regression analysis identified 247 BRRGs associated with overall survival (OS). LASSO regression and tenfold cross-validation further identified 10 key BRRGs (CARD16, TRIP13, PSRC1, CASP1, PLBD1, CARD6, CAPG, CACNA1A, CDH15, and NDUFA4) to construct a prognostic signature. The resistance score formula based on 10-BRRGs is: Resistance Score=(0.2242×CARD16)+(0.0064×TRIP13)+(0.0174×PSRC1)+(0.0276×CASP1)+(0.0118×PLBD1)+(0.0627×CARD6)+(−0.0394×CAPG)+(−0.0661×CACNA1A)+(−0.3578×CDH15)+(0.6447×NDUFA4). Patients were divided into high- and low-resistance score groups based on the median resistance score. Kaplan-Meier survival analysis showed that patients with high-resistance scores had worse OS compared to those with low-resistance scores in both the training and validation cohorts. Additionally, the BRRGs signature exhibited good predictive efficacy for 1-year, 3-year, and 5-year OS, with AUC values of 0.731, 0.761, and 0.777, respectively. Notably, tumor immune microenvironment, biological pathways, and chemotherapy sensitivity were different between high- and low-resistance score groups. Furthermore, we identified TRIP13 as a key resistance gene in this signature and found that TRIP13 was overexpressed in DLBCL and BTKi-resistant DLBCL cell lines. Knockdown of TRIP13 inhibited cell proliferation, promoted apoptosis, and enhanced the apoptotic effect of BTKi in vivo and in vitro. Mechanistically, these effects were mediated through the Wnt/β-catenin signaling pathway. Conclusion: Our study presents a novel BRRGs signature that could serve as a promising prognostic marker in DLBCL. Additionally, TRIP13 might be a potential therapeutic target for resistant DLBCL.

Supplementary Figure S4. Expressions of ADAM9 in ESCC specimens, and interference efficiencies of siRNAs against ADAM9.