Pathways In Diffuse Large B-cell Lymphoma Research Articles

BCL6 confers resistance to HDAC inhibitors in DLBCL

Diffuse large B-cell lymphoma (DLBCL) is an aggressive non-Hodgkin lymphoma with limited response to chemotherapy. Histone acetylation is reduced in DLBCL. Chidamide, a histone deacetylase inhibitor, shows promise in lymphomas but needs further investigation for DLBCL. Our study indicated that chidamide effectively suppresses DLBCL both in vitro and in vivo. High-throughput RNA sequencing analysis provided comprehensive evidence that chidamide markedly influences crucial signaling pathways in DLBCL, including the MAPK, MYC and p53 pathway. Additionally, we observed substantial variability in the sensitivity of DLBCL cells to chidamide, and identified that elevated expression of BCL6 might confer resistance to chidamide in DLBCL. Moreover, our investigations revealed that BCL6 inhibited chidamide-induced histone acetylation by recruiting histone deacetylase (HDACs), leading to drug resistance in DLBCL cells. Furthermore, we found that lenalidomide targeted BCL6 degradation through the ubiquitination pathway and restore the sensitivity of drug-resistant DLBCL to chidamide. Collectively, these findings provided valuable insights into the global impact of chidamide on DLBCL and highlight the potential of targeting HDACs as a therapeutic strategy for DLBCL. Identifying BCL6 as a biomarker for predicting the response to chidamide and the combination therapy with BCL6 inhibition has the potential to lead to more personalized and effective treatments for DLBCL patients.

HBx integration in diffuse large B-cell lymphoma inhibits Caspase-3-PARP related apoptosis

Diffuse large B-cell lymphoma (DLBCL) is the most common pathological type of non-Hodgkin lymphoma, and is closely associated with hepatitis B virus (HBV) infection status and hepatitis B X (HBx) gene integration. This project investigated the cellular biological effects and molecular mechanisms responsible for lymphomagenesis and the progression of HBx integration in DLBCL. The data showed that clinical DLBCL cells demonstrated HBx integration, and the sequencing analysis of integrated sites validated HBx integration in the constructed HBx-transfected cells. Compared with control cells, HBx-transfected cells had a significantly reduced proportion of mitochondrial membrane potential, signals of chromosomal DNA breaks, and proportion of apoptotic cells. Further studies found that this decreased apoptosis level was associated with a significant reduction of cleaved Caspase-3 and downstream poly ADP-ribose polymerase (PARP) proteins, revealing the molecular mechanisms of HBx-associated apoptosis in DLBCL. Animal experiments also demonstrated that the protein expression of cleaved Caspase-3 and PARP was prominently reduced in HBx-transfected cells from subcutaneous tumors in mice. Furthermore, the HBx-integrated cells in clinical tissues had significantly lower cleaved PARP levels than the HBx-negative samples. Therefore, HBx integration inhibits cell apoptosis through the Caspase-3-PARP pathway in DLBCL indicating a potential biomarker and therapeutic target in HBV related DLBCL.

Diffuse large B-cell lymphoma: the significance of CD8+ tumor-infiltrating lymphocytes exhaustion mediated by TIM3/Galectin-9 pathway

BackgroundOverexpression of T-cell immunoglobulin and mucin domain-containing protein 3 (TIM3) is related to the exhaustion of CD8+ tumor-infiltrating lymphocytes (TILs) in diffuse large B-cell lymphoma (DLBCL). However, the mechanism of TIM3-mediated CD8+TILs exhaustion in DLBCL remains poorly understood. Therefore, we aimed to clarify the potential pathway involved in TIM3-mediated CD8+TILs exhaustion and its significance in DLBCL.MethodsThe expression of TIM3 and its correlation with CD8+TILs exhaustion, the key ligand of TIM3, and the potential pathway of TIM3-mediated CD8+TILs exhaustion in DLBCL were analyzed using single-cell RNA sequencing and validated by RNA sequencing. The biological significance of TIM3-related pathway in DLBCL was investigated based on RNA sequencing, immunohistochemistry, and reverse transcription-quantitative polymerase chain reaction data. Finally, the possible regulatory mechanism of TIM3-related pathway in DLBCL was explored using single-cell RNA sequencing and RNA sequencing.ResultsOur results demonstrated that CD8+TILs, especially the terminally exhausted state, were the major clusters that expressed TIM3 in DLBCL. Galectin-9, mainly expressed in M2 macrophages, is the key ligand of TIM3 and can induce the exhaustion of CD8+TILs through TIM3/Galectin-9 pathway. Meanwhile, high TIM3/Galectin-9 enrichment is related to immunosuppressive tumor microenvironment, severe clinical manifestations, inferior prognosis, and poor response to CHOP-based chemotherapy, and can predict the clinical efficacy of immune checkpoint blockade therapy in DLBCL. Furthermore, the TIM3/Galectin-9 enrichment in DLBCL may be regulated by the IFN-γ signaling pathway.ConclusionsOur study highlights that TIM3/Galectin-9 pathway plays a crucial role in CD8+TILs exhaustion and the immune escape of DLBCL, which facilitates further functional studies and could provide a theoretical basis for the development of novel immunotherapy in DLBCL.

Combination of Selinexor with BTK Inhibitor for Central Nervous System Diffuse Large B-Cell Lymphoma, Possible Mechanisms and Therapeutic Potential Exploration

Background: Primary central nervous system lymphoma (PCNSL) is a rare aggressive extra-nodal non-Hodgkin lymphoma (NHL) of which over 90% are diffuse large B-cell lymphoma (DLBCL). High-dose methotrexate (HD-MTX)-based regimens are the dominant therapy for newly diagnosed PCNSL, but the patients' complete remission rate (CRR) is merely about 50%. Bruton tyrosine kinase (BTK), a critical kinase in the B cell receptor (BCR) signaling pathway, contributes to the degradation of FOXO3a and promotes abnormal B cell proliferation. Recent studies revealed that BTK inhibitors (BTKi) play a role in various B cell lymphomas, including DLBCL, and have also shown promising efficacy in PCNSL. However, the clinical management of PCNSL is still challenging for its limited treatment options and poor prognosis, especially when patients developed acquired resistance to BTKi. Selinexor (KPT-330) is a first-ever oral small molecule Exportin-1 (nuclear output protein XPO1) inhibitor with a molecular weight of 443.31 Da, which can efficiently cross the blood-brain barrier. Currently Selinexor was found to promote the aggregation of FOXO3a and could synergistically inhibit PI3K/AKT and NF-κB pathways with BTKi to overcome acquired BTKi resistance. To investigate the therapeutic potential of Selinexor and BTKi in PCNSL, we examined the combination both in DLBCL cells and A20 transplantation mice, and further explored the clinical efficacy in PCNSL patients. Methods and Results: ABC-DLBCL cell lines (OCI-LY3 and OCI-LY10) were treated with Selinexor for 48 hours. Western blot analysis showed that Selinexor inhibited NF-κB, P-STAT3, and NFATc1 signaling pathways in DLBCL in vitro. Combining Selinexor(1μM) with ICP-022(2μM), a BTKi, resulted in more notable inhibition of the above tumor signaling pathways. A20 murine B-cell lymphoma transplantation models were divided into a control group, Selinexor monotherapy group, ICP-022 monotherapy group, and combination group. Tumor volume was measured after 30 days (Figures 1A), and the number of CD4+ T cells and CD8+ T cells and the corresponding cellular expression of PD-1 and TIM-3 in the spleen and tumor tissues of mice were detected. The results showed that the tumor volume in the combination group was minimized, and the number of CD4+ T cells and CD8+ T cells in spleens and tumors in this group was significantly increased, while PD-1 and TIM-3 expression was downregulated (Figures 1B-C). Based on these results, a prospective, open-label, single-arm, multicenter study was conducted with MSZ regimen (MTX, BTKi, Selinexor) for the treatment of PCNSL (Trial Registration Number: ChiCTR2200062154). Newly diagnosed primary and secondary central DLBCL patients (18-80 years old) received a 6-cycle MSZ regimen (HDMTX 3.5 g/m 2 3-h infusion d1, Zanubrutinib 160 mg Bid po, Selinexor 40 mg Biw po, 3 weeks per cycle) and evaluated by MRI/PET. 5 patients (2 GCB-DLBCL, 3 ABC-DLBCL) were enrolled, with a median age of 65 years (range: 54-73 years), 100% achieved metabolic remission (Figure 2A) and maintained long-term efficacy. 4 patients achieved early deep remissions of over 75% reduction with 1 cycle of treatment, and 1 patient had a complete remission after 2 cycles of treatment (Figures 2B). 3 patients chose radiation therapy as consolidation after the end of induction chemotherapy. 2 others were on maintenance therapy with Selinexor combined with Zanubrutinib, one of them has completed the two-year maintenance treatment and maintained sustained remission till now. Only 1 patient experienced several grade 3 adverse events (AEs) including anemia, decreased white blood cell and platelet counts, and elevated alanine aminotransferase.1 patient developed mild renal insufficiency, which recovered 6 months after the end of treatment. Patients were generally well-tolerated and there were no treatment-related deaths. Conclusion: This is the first report describing that Selinexor combined with BTKi can synergistically inhibit the DLBCL tumor signaling pathway and repairing the immune microenvironment. Correspondingly, in treatment of central DLBCL patients, the novel therapeutic strategy of MSZ regimen has achieved impressive results, with a CRR of 100%. Most patients attained early and rapid deep remissions with durable efficacy. This study provides theoretical support and preliminary clinical data for the combined application of Selinexor and BTK inhibitors.

17th International Conference on Malignant Lymphoma, Palazzo dei Congressi, Lugano, Switzerland, 13 - 17 June, 2023.

Introduction: N6-methyladenosine (m6A) is the most abundant form of chemical modification in eukaryotes. m6A methylation is dynamically modulated by diverse types of regulators, including methyltransferases (‘writers’), RNA binding proteins (‘readers’), and demethylases (‘erasers’). Growing evidence has shown that m6A methylation plays an essential role in the development and progression of multiple cancers. However, the functions of m6A methyltransferase KIAA1429 in diffuse large B-cell lymphoma (DLBCL) remain undefined. Here, we aimed to unravel an epitranscriptomic mechanism mediated by the m6A methyltransferase KIAA1429 in DLBCL. Methods: Collecting lymph node biopsy samples from 60 de novo DLBCL patients and 20 reactive hyperplasia cases after informed consent. The biological function of KIAA1429 was evaluated using CRISPR/Cas9 mediated genomic deletion and CRISPR/dCas9-VP64 activation. RNA-seq, MeRIP-seq, RIP assays, luciferase activity assay, RNA stability experiments, and co-immunoprecipitation were performed to explore the regulatory mechanism of KIAA1429 in DLBCL. DLBCL cells were subcutaneously injected into SCID-Beige mice to establish xenograft models. The Institutional Animal Care guidelines were followed during the animal experiments. Results: m6A regulators were dysregulated in DLBCL, and a predictive model based on m6A score was developed. KIAA1429 expression was found to be elevated and associated with poor prognosis. KIAA1429 knockout inhibited DLBCL cell proliferation, induced cell cycle arrest in the G2/M phase, promoted apoptosis in vitro, and repressed tumor growth in vivo. Furthermore, KIAA1429 mediated m6A modification of CHST11 mRNA and recruited YTHDF2 to reduce the stability and expression of CHST11. By decreasing MOB1B expression, CHST11 inhibited Hippo-YAP signaling, reprogramming the expression of Hippo target genes (Figure 1). Conclusions: In summary, we identified for the first time that m6A methylation regulators were dysregulated, and its risk score could exert as an independent prognostic indicator in DLBCL. More importantly, our study identified a novel mechanism in which KIAA1429/YTHDF2-coupled epitranscriptional repression of CHST11 inactivates the Hippo-YAP pathway in DLBCL, highlighting the potential of KIAA1429 as a novel predictive biomarker and therapeutic target for DLBCL progression. Keywords: aggressive B-cell non-Hodgkin lymphoma, bioinformatics, computational and systems biology, genomics, epigenomics, and other -omics No conflicts of interests pertinent to the abstract.

Upregulation of USP25 promotes progression of human diffuse large B-cell lymphoma through blocking the ubiquitinated degradation of MDM2

Diffuse large B cell lymphoma (DLBCL) is a type of cancer that originates from abnormal B cells in the lymph nodes or other lymphoid tissues. Dysfunction of deubiquitinases is frequently implicated in malignant progression. This study planned to uncover the biological roles of deubiquitinase USP25 during DLBCL tumorigenesis. In this study we identified USP25 as a novel oncogene which is frequently upregulated in DLBCL and associated with dismal prognosis of patients. Moreover, USP25 silencing was found to inhibit DLBCL growth, migration, while induced an obvious increase in apoptosis in vitro. Meanwhile, USP25 could promote DLBCL tumour growth and lung metastasis in vivo. Mechanistically, the co-immunoprecipitation test provided a mechanistic explanation, showing that USP25 directly interacted with murine double minute 2 (MDM2) and MDM2 protein stability was maintained by USP25 mediated deubiquitination. In addition, overexpression of USP25 with C178A mutation failed to decrease its modification on MDM2 stability. Further mechanism-of-action studies demonstrated that USP25 promoted DLBCL progression via stabilizing MDM2 and consequently decreasing p53 expression. In addition, further analysis showed that the oncogenic effect of USP25 was relied on MDM2-p53 signaling pathway-mediated cell-cycle accelerating. Collective, USP25 was shown to be an important upstream regulator of the MDM2-p53 signaling pathway in DLBCL, and it has the potential to be employed as a novel target gene in the development of new therapeutic applications.

KIAA1429-mediated m6A modification of CHST11 promotes progression of diffuse large B-cell lymphoma by regulating Hippo–YAP pathway

BackgroundN6-methyladenosine (m6A) has been shown to participate in various essential biological processes by regulating the level of target genes. However, the function of m6A modification mediated by KIAA1429 [alias virus-like m6A methyltransferase-associated protein (VIRMA)] during the progression of diffuse large B-cell lymphoma (DLBCL) remains undefined.MethodsThe expression and clinical significance of KIAA1429 were verified by our clinical data. CRISPR/Cas9 mediated KIAA1429 deletion, and CRISPR/dCas9-VP64 for activating endogenous KIAA1429 was used to evaluate its biological function. RNA sequencing (RNA-seq), methylated RNA immunoprecipitation sequencing (MeRIP-seq), RNA immunoprecipitation (RIP) assays, luciferase activity assay, RNA stability experiments, and co-immunoprecipitation were performed to investigate the regulatory mechanism of KIAA1429 in DLBCL. Tumor xenograft models were established for in vivo experiments.ResultsDysregulated expression of m6A regulators was observed, and a novel predictive model based on m6A score was established in DLBCL. Additionally, elevated KIAA1429 expression was associated with poor prognosis of patients with DLBCL. Knockout of KIAA1429 repressed DLBCL cell proliferation, facilitated cell cycle arrest in the G2/M phase, induced apoptosis in vitro, and inhibited tumor growth in vivo. Furthermore, carbohydrate sulfotransferase 11 (CHST11) was identified as a downstream target of KIAA1429, which mediated m6A modification of CHST11 mRNA and then recruited YTHDF2 for reducing CHST11 stability and expression. Inhibition of CHST11 diminished MOB1B expression, resulting in inactivation of Hippo–YAP signaling, reprogramming the expression of Hippo target genes.ConclusionsOur results revealed a new mechanism by which the Hippo–YAP pathway in DLBCL is inactivated by KIAA1429/YTHDF2-coupled epitranscriptional repression of CHST11, highlighting the potential of KIAA1429 as a novel predictive biomarker and therapeutic target for DLBCL progression.

Abstract 3110: Design, synthesis, and biological evaluation of transducin β-like protein 1 (TBL1) degraders

Abstract Transducin β-like protein 1 (TBL1) is an essential scaffold protein that participates in multiple critical signaling pathways, such as the Wnt/β-catenin pathway, where it protects β-catenin from ubiquitination and proteasomal degradation. Only one compound, BC-2059 (tegavivint, Iterion Therapeutics), has been reported to promote apoptosis by disrupting the TBL1/β-catenin interactions and show promising therapeutic effects in Wnt-driven cancers, such as colorectal cancer, breast cancer, and leukemia. However, recent studies showed that TBL1 modulates Wnt-regulated genes in a β-catenin-independent manner in diffuse large B-cell lymphoma (DLBCL). Although the involvement of β-catenin is still under debate, TBL1’s engagement in the antitumor effects of BC-2059 is certain and critical. In this study, we aimed to develop proof-of-concept BC-2059-based TBL1 selective degraders against DLBCL using the Proteolysis Targeting Chimeras (PROTACs) strategy and use them as chemical probes to investigate TBL1-related pathways in DLBCL. Two series (N- and O-linked) of BC-2059-based PROTACs were synthesized, and their cytotoxicity and protein degradation profiles were tested in cellular assays. Several compounds showed low micromolar to nanomolar cytotoxic activity. The TBL1 degradation of these PROTACs is currently underway. In the meantime, we modeled the ternary complex formation by developing a workflow where two binary complexes and a library of PROTACs having varied linker lengths/chemical compositions were modeled by PRosettaC. The protein-protein interactions of the generated ternary complexes were analyzed by Rosetta and the interactions between the linkers and the protein were calculated by Autodock 4. The results suggested that short linkers (8-14 heavy atoms) are more suitable for N-linked series degraders in terms of PROTAC-induced ternary complex formation, while long linkers (>18 heavy atoms) are preferable for O-linked series. Citation Format: Rui Yang, Lapo Alinari, Pui-kai (Tom) Li, Xiaolin Cheng. Design, synthesis, and biological evaluation of transducin β-like protein 1 (TBL1) degraders [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3110.

Immunotherapy for diffuse large B-cell lymphoma: current use of immune checkpoint inhibitors therapy

<abstract> <p>Patients diagnosed with diffuse large B-cell lymphoma (DLBCL) have high cure rates with current treatment options including immuno-polychemotherapy. However, around 30% of cases do not respond or develop relapse disease. For this, it is necessary to search for new therapeutic options. In recent years, therapy using chimeric antigen receptor (CAR) T-cells has been a strategy for those patients with LBDCG in progression or relapse, although only 30–40% of cases achieve durable remissions. The programmed death-1 (PD-1) receptor regulates the T-cell-mediated immune response through binding to its ligands (PD-L1). Some tumor cells present high expression of PD-L1, which down-regulates T-cell activation. The beneficial antitumor activity of PD-1 and PD-L1 has been widely demonstrated in certain solid organ malignancies. However, their utility in the treatment of lymphomas is complex. To date, different clinical trials have demonstrated its usefulness as an innovative therapeutic alternative in these tumors. In this review article, we evaluate the literature on the role of the PD-1/PD-L1 pathway in DLBCL and describe future strategies involving these new anticancer agents in this lymphoid neoplasm.</p> </abstract>

Phase 1 Study of Venetoclax, Ibrutinib, Prednisone, Obinutuzumab, and Lenalidomide in Combination with Polatuzumab (ViPOR-P) in Relapsed/Refractory B-Cell Lymphoma: Preliminary Analysis of Safety and Efficacy

Background: Diffuse large B-cell lymphoma (DLBCL) is often curable with frontline chemotherapy; however, pts with relapsed/refractory (R/R) disease and those who fail CAR-T therapy have poor overall outcome. Oral small molecule targeted agents can disrupt key survival pathways in DLBCL, but fail to induce deep or durable responses as monotherapy. We have previously shown that ViPOR is safe and active in DLBCL, with preferential activity in non-GCB DLBCL and HGBCL-DH (Melani et al. Blood. 2020). Synergistic activity has also been demonstrated with MMAE, the chemotherapy payload of Polatuzumab (Pola), with ViPOR in pre-clinical DLBCL cell lines (unpublished data). We hypothesized that additional genotoxic stress with Pola will further leverage efficacy and time-limited, cyclic dosing will limit the hematologic toxicity of the regimen. Methods: R/R aggressive B-cell lymphoma pts with adequate organ function were eligible. In phase 1, a 3+3 design was used to determine the maximum tolerated dose (MTD) of 3 dose-levels (DLs) of Pola IV D2 and venetoclax (Ven) PO D2-14 (DL1: 1.4mg/kg + 600mg, DL2: 1.8mg/kg + 600mg, and DL3: 1.8mg/kg + 800mg) in combination with fixed-dose ibrutinib 560mg PO D1-14, prednisone 100mg PO D1-7, obinutuzumab 1000mg IV D1-2, and lenalidomide 15mg PO D1-14. An expansion cohort was included at the MTD for further analysis of safety and efficacy. ViPOR-P q21d x 6 cycles (C) was given without maintenance or planned consolidation. All pts received TLS, PCP, HSV, and G-CSF prophylaxis (ppx) with VTE ppx per investigator discretion. Baseline CT, PET, BM, and tumor biopsy were performed with CT scans after C2, 4, and 6 and PET after C6 or at time of suspected complete response (CR). Surveillance CT was performed q3m for 1y, q4m x 1y, q6m x 1y, then annually x 2y. Results: 15 pts were enrolled and treated in the dose-escalation cohort; 3 at DL1, 6 at DL2, and 6 at DL3. Lymphoma subtypes included DLBCL NOS (60%) and HGBCL-DH (40%). In 9 DLBCL NOS pts, 11% were GCB and 89% non-GCB subtype by IHC. Three (20%) pts had transformed disease. Median age was 51y (range 39-83), with stage III/IV disease in 80%, elevated LDH in 80%, and >2 extranodal sites of disease in 67%. Nine (60%) pts had high-intermediate/high-risk IPI. Median prior therapies were 2 (range 1-6), with prior BTKi in 27%, Pola in 7%, and CAR-T in 20%. Ten (67%) pts were primary refractory (i.e., <CR to frontline tx) and 8 (53%) of pts were refractory (i.e., <PR) to last tx. One dose-limiting toxicity (DLT) of prolonged G4 thrombocytopenia occurred at DL3, but no other DLTs were noted. Pola 1.8mg/kg and Ven 800mg were identified as the MTD and safe doses for use in the expansion cohort. Heme AEs were most common and included G3-4 (% cycles) thrombocytopenia (17%), anemia (7%), and neutropenia (5%). No cases of febrile neutropenia occurred across 58 cycles. The only non-heme G3-4 AE occurring in >10% pts was hypokalemia (29%), with all pts experiencing any grade hypokalemia requiring replacement. Other common non-heme AEs (% pts) included diarrhea (71%), increased LFTs (64%), nausea (50%), hypomagnesemia (43%), and rash (43%). VTE occurred in 7% with no a.fib or major bleeding observed. No events of TLS or treatment-related mortality occurred. Dose reductions and delays occurred in 3% and 5% of cycles, respectively. Of 12 pts off-tx, 7 completed all 6C, 3 experienced disease progression, and 1 each came off-tx per investigator discretion or unacceptable AE. Of 15 enrolled pts, 14 are evaluable for response with an overall response rate (ORR) and CR rate of 64% (9/14) and 36% (5/14), respectively (Fig 1A). With a median potential f/u of 7.4m, median TTR in responding pts was 1.35m, with 8 (89%) of 9 total responses ongoing (Fig 1B). One HGBCL-DH pt relapsed by imaging at 10.6m, 2 PR pts received consolidative XRT with allo-HSCT, and 2 PR pts remain on-tx. PFS and OS at 6m was 69.8% and 75%, respectively. Conclusion: ViPOR-P is safe without significant additional toxicity compared to ViPOR, and Pola 1.8mg/kg and Ven 800mg were taken forward in the ongoing expansion cohort. Most common G3-4 AEs were hematologic with no febrile neutropenia observed with the use of G-CSF ppx. Fixed-duration ViPOR-P x 6C without maintenance led to CR in 5 (42%) of 12 off-tx DLBCL pts with 80% CRs ongoing from 3.7-9.3m. A randomized phase 2 trial is planned to further elucidate the safety and efficacy of Pola in combination with ViPOR across R/R DLBCL and within pre-defined molecular and genetic subtypes. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Clinical significance of MYD88 non-L265P mutations in diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is a group of heterogeneous tumors with different molecular traits and clinical features. MYD88 is an oncogene that activates the nuclear factor κB pathway in DLBCL. MYD88 L265P mutation frequently occurs in DLBCL with poor prognosis, while the clinical significance of non-L265P mutations needs to be clarified. Next-generation sequencing was performed on a cohort of 356 patients with DLBCL to investigate the impact of MYD88 mutation. Ten MYD88 mutated variants were detected in 32% (114/356) of the cases. V217F, S219C, S222R, M232T, S243N, and T294P were identified as pathogenic variants. MYD88 non-L265P mutations occurred less than L265P mutation in DLBCL of the central nervous system and breast tissue. The coexistence of MYD88 non-L265P mutations with PIM1 mutation was also less than that of L265P mutation. The progression-free survival in patients with DLBCL with MYD88 non-L265P mutation was statistically better than in patients with MYD88 L265P mutation. The interpretation of variants of MYD88 mutation offers a precise guide for the management of DLBCL.

KIF23 is a potential biomarker of diffuse large B cell lymphoma: Analysis based on bioinformatics and immunohistochemistry.

Diffuse Large B Cell Lymphoma (DLBCL), the most common form of blood cancer. The genetic and clinical heterogeneity of DLBCL poses a major barrier to diagnosis and treatment. Hence, we aim to identify potential biomarkers for DLBCL.Differentially expressed genes were screened between DLBCL and the corresponding normal tissues. Kyoto Encyclopedia of Genes and Genomes and Gene oncology analyses were performed to obtain an insight into these differentially expressed genes. PPI network was constructed to identify hub genes. survival analysis was applied to evaluate the prognostic value of those hub genes. DNA methylation analysis was implemented to explore the epigenetic dysregulation of genes in DLBCL.In this study, Kinesin family member 23 (KIF23) showed higher expression in DLBCL and was identified as a risk factor in DLBCL. The immunohistochemistry experiment further confirmed this finding. Subsequently, the univariate and multivariate analysis indicated that KIF23 might be an independent adverse factor in DLBCL. Upregulation of KIF23 might be a risk factor for the overall survival of patients who received an R-CHOP regimen, in late-stage, whatever with or without extranodal sites. Higher expression of KIF23 also significantly reduced 3, 5, 10-year overall survival. Furthermore, functional enrichment analyses (Kyoto Encyclopedia of Genes and Genomes, Gene oncology, and Gene Set Enrichment Analysis) showed that KIF23 was mainly involved in cell cycle, nuclear division, PI3K/AKT/mTOR, TGF-beta, and Wnt/beta-catenin pathway in DLBCL. Finally, results of DNA methylation analysis indicated that hypomethylation in KIF23's promoter region might be the result of its higher expression in DLBCL.The findings of this study suggested that KIF23 is a potential biomarker for the diagnosis and prognosis of DLBCL. However, further studies were needed to validate these findings.

Biological and Clinical Significance of PIM1 Genetic Alterations in Diffuse Large B-Cell Lymphoma

Background: PIM1 is a somatic hypermutation gene in diffuse large B-cell lymphoma (DLBCL) and its inhibitors exhibit a great value in application in multiple types of lymphoma. Nevertheless, investigation on its genetic alterations, biological characteristics, clinical significance and response to drugs is still lacking.Methods: We integrated the genome sequencing (discovery cohort, n=162; validation cohort, n=1001) and transcriptome sequencing (discovery cohort, n=140; validation cohort, n=928) to capture more detailed insights into the potentially biological functions of PIM1 genetic alterations, and analyzed their relationship with biological characteristics and clinical value which provide a possibility for risk stratification and therapeutic exploitation for patients with DLBCL.Results: PIM1 mutations were identified in 28.4% of DLBCL patients and significantly correlated with higher IPI scores (P=0.013), disease relapse (P=0.031) and CNS and/or testis involvement (P=0.001), as well as inferior PFS (P=0.022) and OS (P=0.0022). Multivariate analysis revealed that PIM1 mutation status was an independent poor prognostic factor (HR=2.86; 95% CI, 1.40-5.84; P=0.004). The most frequent PIM1 mutation type was missense mutations (84.1%), followed by frameshift deletions and nonsense mutations. During the distribution of base substitutions, C > T base substitution was predominant mutation type (54.4%), followed by C >G transversion (29.3%). During different exons, exon 4 of PIM1 was most often mutated. PIM1 mutations significantly co-occurred with the mutations of SETD1B (P<0.001) and CD79B (P=0.001), and was mutually exclusive to the SPEN mutation (P=0.024). We also explored the relationship between PIM1 mutations and genes distributed on previously reported signaling pathways in DLBCL and uncovered that mutations in MYD88 (P<0.001) and PRDM1 (P<0.001) involved in the NF-κB pathway were significantly enriched in the patients with PIM1 mutations. Unsurprisingly, patients with PIM1 mutations exhibited higher mutation frequencies in CD79B (P=0.001) involved in BCR pathway. PIM1 mutations were involved in immunoglobulin-related immune response, complement activation, B cell mediated immunity, B cell activation, antigen binding, and cytokine activity, which contributed to the signaling pathways of tumor microenvironment (e.g. cytokine-cytokine receptor interaction, chemokine signaling pathway, IL-17 signaling pathway, TNF signaling pathway), JAK-STAT and NF-κB. By MCODE analysis, five significant modules were obtained from the protein-protein interaction (PPI) network. Finally, a PIM1 mutation-related gene signature consisting of some independent prognostic factors was developed. According to the risk score, patients with high-risk score exhibited significantly shorter OS (P=0.0016) and PFS (P<0.001). The areas under the curve (AUCs) for the predictions of 1-, 3-, and 5-year OS were respectively 0.69, 0.70, and 0.72, suggesting that the risk score based on the PIM1 mutation-related gene signature had satisfactory sensitivity and specificity. We further found that compared to patients with low-risk score, patients with high-risk score had higher sensitivity to some drugs of targeting the immune microenvironment, including TGFβ receptor inhibitors SB525334 (P<0.0001) and SB505124 (P<0.0001) and immunomodulator Lenalidomide (P=0.041), as well as NF-κB inhibitors Parthenolide (P<0.0001) and TPCA-1 (P<0.0001) and JAK inhibitors Ruxolitinib (P=0.014) and TG101348 (P=0.0053), accompanying with significantly lower IC50 values. In addition, another common chemotherapeutic drug Gemcitabine was also predicted to be more sensitive for patients with high-risk score (P=0.047). Other targeted drugs such as Aurora kinase inhibitors VX-680 (P<0.0001) and ZM-447439 (P=0.014), Bcl-2 inhibitors Obatoclax Mesylate (P=0.00036) and Navitoclax (P<0.0001), and CDK inhibitors Roscovitine (P=0.0012), AT-7519 (P=0.0033), PHA-793887 (P<0.0001) and THZ2-49 (P=0.0053) also exhibited higher drug sensitivity for patients with high-risk score.Conclusions: PIM1 mutations play a vital role in patient risk stratification and provide novel insights into therapeutic decision making for DLBCL patients with high-risk score. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Biomarkers and novel therapeutic approaches for diffuse large B-cell lymphoma in the era of precision medicine.

Despite the great efforts for better treatment options for diffuse large B-cell lymphoma (DLBCL) (most common form of non-Hodgkin lymphoma, NHL) to treat and prevent relapse, it continues to be a challenge. Here, we present an overview of DLBCL and address the diagnostic assays and molecular techniques used in its diagnosis, role of biomarkers in detection, treatment of early and advanced stage DLBCL, and novel drug regimens. We discuss the significant biomarkers that have emerged as essential tools for stratifying patients according to risk factors and for providing insights into the use of more targeted and individualized therapeutics. We discuss techniques such as gene expression studies, including next-generation sequencing, which have enabled a more understanding of the complex pathogenesis of DLBCL and have helped determine molecular targets for novel therapeutic agents. We examine current treatment approaches, outline the findings of completed clinical trials, and provide updates for ongoing clinical trials. We highlight clinical trials relevant to the significant fraction of DLBCL patients who present with complex cases marked by high relapse rates. Supported by an increased understanding of targetable pathways in DLBCL, clinical trials involving specialized combination therapies are bringing us within reach the promise of an effective cure to DLBCL using precision medicine. Optimization of therapy remains a crucial objective, with the end goal being a balance between high survival rates through targeted and personalized treatment while reducing adverse effects in DLBCL patients of all subsets.

MDM2-P53 Signaling Pathway-Mediated Upregulation of CDC20 Promotes Progression of Human Diffuse Large B-Cell Lymphoma.

BackgroundCell-division cycle 20 (CDC20) is overexpressed in a variety of tumor cells and is negatively regulated by wild-type p53 (wtp53). Our previous study uncovered that CDC20 was upregulated and associated with poor outcome in diffuse large B-cell lymphoma (DLBCL) based on bioinformatics analysis. Dysregulation of the MDM2-p53 is a major mechanism to promote DLBCL. Thus, we hypothesized that CDC20 could be a downstream gene of the MDM2-p53 signaling pathway. However, the clinical significance and mechanistic role of a novel MDM2-p53-CDC20 signaling pathway in DLBCL have still remained unclear.Materials and MethodsRT-qPCR was performed in MDM2 knocked down (KD) and control (Ctrl) OCI-Ly3/OCI-Ly10 cells to investigate whether CDC20 was a downstream gene of the MDM2-p53 pathway. The effects of CDC20 on cell proliferation, cell cycle and apoptosis were assessed, as well as the role of CDC20 in suppressing tumorigenicity in vivo. Furthermore, we also investigated the roles of CDC20 and MDM2 in progression of DLBCL and the underlying mechanisms.ResultsThe results of RT-qPCR revealed that CDC20 was downregulated while TP53 was upregulated in MDM2 KD OCI-Ly3 and OCI-Ly10 cells. It was unveiled that the expression levels of CDC20 and MDM2 were upregulated in DLBCL tissues and cells, and high CDC20 expression was correlated with adverse clinical features and poor outcome. Functional assays showed that downregulation of CDC20 could inhibit proliferation, induce apoptosis and cell cycle arrest in vitro. In addition, inactivation of the MDM2-p53 pathway by downregulation of MDM2 restored wtp53 expression level and reduced CDC20 protein level in OCI-Ly3 and OCI-Ly10 cells. Besides, targeting CDC20 was found to suppress tumorigenesis of DLBCL in vivo.ConclusionCDC20 was identified as a key downstream gene of the MDM2-p53 signaling pathway in DLBCL and may be used as a novel target gene to guide therapeutic applications.

Comprehensive characterization of driver genes in diffuse large B cell lymphoma.

Diffuse large B cell lymphoma (DLBCL) is the most common hematological malignancy and is one of the most frequent non-Hodgkin lymphomas. Large-scale genomic studies have defined genetic drivers of DLBCL and their association with functional and clinical outcomes. However, the lymphomagenesis of DLBCL is yet to be fully understood. In the present study, four computational tools OncodriveFM, OncodriveCLUST, integrated Cancer Genome Score and Driver Genes and Pathways were used to detect driver genes and driver pathways involved in DLBCL. The aforementioned tools were also used to perform an integrative investigation of driver genes, including co-expression network, protein-protein interaction, copy number variation and survival analyses. The present study identified 208 driver genes and 31 driver pathways in DLBCL. IGLL5, MLL2, BTG2, B2M, PIM1, CARD11 were the top five frequently mutated genes in DLBCL. NOTCH3, LAMC1, COL4A1, PDGFRB and KDR were the 5 hub genes in the blue module that were associated with patient age. TP53, MYC, EGFR, PTEN, IL6, STAT3, MAPK8, TNF and CDH1 were at the core of the protein-protein interaction network. PRDM1, CDKN2A, CDKN2B, TNFAIP3, RSPO3 were the top five frequently deleted driver genes in DLBCL, while ACTB, BTG2, PLET1, CARD11, DIXDC1 were the top five frequently amplified driver genes in DLBCL. High EIF3B, MLH1, PPP1CA and RECQL4 expression was associated with decreased overall survival rate of patients with DLBCL. High XPO1 and LYN expression were associated with increased overall survival rate of patients with DLBCL. The present study improves the understanding of the biological processes and pathways involved in lymphomagenesis. The driver genes, EIF3B, MLH1, PPP1CA, RECQL4, XPO1 and LYN, pave the way for developing prognostic biomarkers and new therapeutic strategies for DLBCL.

Pevonedistat, a NEDD8‐activating enzyme inhibitor, induces apoptosis and augments efficacy of chemotherapy and small molecule inhibitors in pre‐clinical models of diffuse large B‐cell lymphoma

We studied the biological activity of pevonedistat, a first‐in‐class NEDD8‐activating enzyme (NAE) inhibitor, in combination with various cytotoxic chemotherapy agents and small molecule inhibitors in lymphoma preclinical models. Pevonedistat induced cell death in activated B‐cell (ABC) diffuse large B‐cell lymphoma (DLBCL) cell lines and to a lesser degree in germinal center B‐cell (GCB) DLBCL cell lines. In pevonedistat sensitive cells, we observed inhibition of NF‐κB activity by p65 co‐localization studies, decreased expression of BCL‐2/Bcl‐XL, and upregulation of BAK levels. Pevonedistat enhanced the activity of cytarabine, cisplatin, doxorubicin, and etoposide in ABC‐, but not in the GCB‐DLBCL cell lines. It also exhibited synergy with ibrutinib, selinexor, venetoclax, and A‐1331852 (a novel BCL‐XL inhibitor). In vivo, the combination of pevonedistat and ibrutinib or pevonedistat and cytarabine prolonged survival in SCID mice xenograft models when compared with monotherapy controls. Our data suggest that targeting the neddylation pathway in DLBCL is a viable therapeutic strategy and support further clinical studies of pevonedistat as a single agent or in combination with chemotherapy or novel targeted agents.

β-Elemene suppresses tumor growth of diffuse large B-cell lymphoma through regulating lncRNA HULC-mediated apoptotic pathway.

Background: Diffuse large B-cell lymphoma (DLBCL) is considered the most common aggressive subtype of lymphoma. A number of DLBCL patients fail to achieve a response to currently available therapies or develop resistance. β-Elemene is derived from herb Curcuma wenyujin, and exhibits anti-tumor activity in both solid and non-solid tumors through modulating several molecular signaling pathways. We aimed to explore the role of β-elemene in DLBCL treatment and elucidate the involved mechanism.Materials and methods: Cell viability, apoptosis and expressions of related proteins were assessed and in vivo study were performed to determine the tumor suppressive effect of β-elemene and explore the molecular mechanisms.Results: β-Elemene significantly suppressed the viability of DLBCL cells, and β-elemene down-regulated the lncRNA HULC expression and regulated key pro-apoptotic and anti-apoptotic proteins to induce significant apoptosis of DLBCL cells. HULC overexpression could decrease the β-elemene induced apoptosis, while HULC knockdown increased the apoptosis in DLBCL cells. In vivo study further confirmed that β-elemene could suppress the growth of DLBCL xenograft and regulate the HULC expression and the critical proteins of the apoptotic pathway.Conclusion: β-Elemene performs as a tumor suppressor and modulator of HULC-mediated apoptotic pathway in DLBCL and will be an alternative candidate for clinical application.

TCL1 as a hub protein associated with the PI3K/AKT signaling pathway in diffuse large B-cell lymphoma based on proteomics methods

This study aimed to investigate the hub protein related to the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT) signaling pathway in diffuse large B-cell lymphoma (DLBCL). We used proteomics methods (iTRAQ) to explore the differentially expressed proteins in the non-germinal center B-cell -like (non-GCB) DLBCL in our previous study. In this study, a total of 137 formalin-fixed paraffin-embedded DLBCL tissue samples were analyzed via immunohistochemistry to verify the expression of TCL1, AKT1 + 2+3, IKKβ and to determine the differentially expressed proteins associated with the PI3K/AKT signaling pathway. Spearman correlation was used to analyze the relationship between these proteins, and survival analysis was used to investigate their effects on prognosis. Immunohistochemistry analysis indicated that TCL1, AKT1 + 2+3, and IKKβ were highly positively expressed in DLBCL. Results showed that the expression of TCL1 was related to ethnicity (p = 0.022), primary site (p = 0.045), Ann Arbor stage (p = 0.037), the International Prognostic Index (p = 0.005), β2-microglobulin (p = 0.030), BCL2 expression (p < 0.001), and Ki-67 expression (p = 0.008). A positive correlation was found between TCL1 and AKT1 + 2+3 (p < 0.001; r = 0.475). A positive correlation was also found between AKT1 + 2+3 and IKKβ (p < 0.001; r = 0.342). In survival analysis, anemia, non-treatment with R‑CHOP, positive TCL1 expression, and Ki-67 expression≥50% independently predicted short progression-free survival and overall survival in the total cohort (p < 0.05). Thus, TCL1 as a hub protein is associated with the PI3K/AKT signaling pathway in DLBCL. TCL1 expression indicated a poor prognosis in patients with DLBCL. With further studies, TCL1 may be established as a reliable prognostic biomarker and potential immunotherapeutic target for improving therapeutic efficacy for DLBCL in the future.

Pharmacological DNA demethylation restores SMAD1 expression and tumor suppressive signaling in diffuse large B-cell lymphoma

AbstractThe sphingosine-1-phosphate (S1P) receptor S1PR2 and its downstream adaptor Gα13 are recurrently mutationally inactivated in the germinal center B-cell subtype of diffuse large B-cell lymphoma (DLBCL) and are silenced by the S1PR2 repressor FOXP1 in the activated B-cell like subtype of the disease. Loss of S1PR2 signaling relieves the germinal center confinement that is maintained by an S1P gradient and allows cells to resist S1P-induced apoptosis. We have shown previously that S1PR2 expression is induced in normal B cells through a newly described transforming growth factor-β (TGF-β)/TGF-βRII/SMAD1 signaling axis that is inactivated in >85% of DLBCL patients. DLBCL cell lines lacking S1PR2, TGFBRII, or SMAD1 as the result of genomic editing all have a strong growth advantage in vitro, as well as in subcutaneous and orthotopic xenotransplantation models. Here, we show that the TGF-β signaling pathway in DLBCL is blocked at the level of SMAD1 in DLBCL cell lines and patient samples by hypermethylation of CpG-rich regions surrounding the SMAD1 transcription start site. The pharmacologic restoration of SMAD1 expression by the demethylating agent decitabine (DAC) sensitizes cells to TGF-β–induced apoptosis and reverses the growth of initially SMAD1− cell lines in ectopic and orthotopic models. This effect of DAC is reduced in a SMAD1-knockout cell line. We further show that DAC restores SMAD1 expression and reduces the tumor burden in a novel patient-derived orthotopic xenograft model. The combined data lend further support to the concept of an altered epigenome as a major driver of DLBCL pathogenesis.