Citations

Abstract Introduction: Large B-cell lymphoma (LBCL) harboring chromosomal rearrangements (double-hit, DH) or coexpression of MYC and BCL2 (double-expressor, DE) represents biologically aggressive subgroups with inferior clinical outcomes. Oncogene-driven DNA replication stress (ODRS) is a hallmark of MYC and BCL2 dysregulation; however, the DNA damage response (DDR) pathways that sustain replication and cell survival under ODRS remain incompletely defined in DLBCL. Identifying DDR dependencies induced by ODRS may reveal therapeutically actionable vulnerabilities in DH/DE-LBCL. Methods: We studied DH/DE-LBCL cell lines (OCILY1, SUDHL2, DOHH2), non-DH/DE-LBCL cell lines (HT, KARPAS422, HBL1), and benign retinal pigment epithelial (RPE) cells engineered to overexpress MYC and/or BCL2. ODRS and DNA damage were assessed by phosphorylation of RPA (S4/S8, S33) and H2AX histone (γH2AX), respectively, using immunofluorescence, immunohistochemistry, and western blot (WB). Activity of homologous recombination (HR), nonhomologous end-joining (NHEJ), and microhomology-mediated end joining (MMEJ) pathways were quantified with established reporter assays. POLΘ (gene: POLQ) expression, an MMEJ marker, was evaluated by qPCR and WB. DDR activity was assessed using genomic signatures from whole-exome sequencing data of 404 LBCL patients. In vitro sensitivity of LBCL cells to POLΘ and PARP inhibitors was assessed with publicly available small-molecule inhibitors using CellTiterGlo and clonogenic assays, and therapeutic efficacy was validated in a DH-LBCL xenograft model in NRG mice. Results: DH/DE-LBCL cells and primary tumors exhibited increased ODRS and persistent DNA damage compared with non-DH/DE-LBCL models, including accumulation of γH2AX in mitosis. Functional profiling revealed selective upregulation of MMEJ in MYC/BCL2-overexpressing cells, with minimal changes in HR or NHEJ activity. POLQ expression was significantly increased in DH/DE-LBCL cell lines and primary samples, and the ID6 signature (associated with MMEJ activity) was significantly enriched in DH-LBCL tumors compared to non-DH/DE-LBCL tumors. POLΘ inhibition demonstrated greater toxicity in DH/DE-LBCL and in MYC-overexpressing RPE cells compared to non-DH/DE-LBCL and wild-type RPEs. Combined inhibition of POLΘ and PARP resulted in marked synergy in vitro. The therapeutic efficacy of POLΘ inhibition, with the synthetic lethality of concurrent PARP inhibition, was validated in the DH-LBCL xenograft model. Concurrent POLΘ and PARP inhibition with small-molecule inhibitors induced an antitumor effect in vivo, with increased overall survival in mice compared with vehicle- or olaparib-single-agent-treated groups. Conclusions: ODRS in DH/DE-LBCL creates a selective dependence on MMEJ mediated by POLΘ. Therapeutic targeting of POLΘ, alone or in combination with PARP inhibition, exploits this vulnerability and represents a promising strategy for this high-risk LBCL subset. These findings provide a biological rationale for clinical translation of POLΘ-directed therapies in DH/DE-LBCL. Citation Format: Cecilia Ayala-Zambrano, Dongni Yi, Michelle Manske, Kimberly Gwin, Richar Kandasamy, Surendra Dasari, Anne Novak, Shuhei Asada, Xiaosheng Wu, Zhenkun Lou, Thomas Witzig, Jithma Abeykoon. c-MYC/BCL2 induced replication stress drives dependence on POLQ-mediated microhomology-mediated end joining in aggressive large B-cell lymphoma [abstract]. In: Proceedings of the Fifth AACR International Meeting on Advances in Malignant Lymphoma: From Discovery to Clinical Impact; 2026 Jun 24-27; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2026;7(3_Suppl):Abstract nr A029.

Abstract Diffuse Large B Cell Lymphoma (DLBCL), the most common subtype of aggressive B-cell Non-Hodgkin Lymphoma (NHL), accounts for 30-40% of newly diagnosed NHL cases. Despite recent progress improving our understanding of molecular features of DLBCL, there is still a high relapse rate (~45%) among patients who undergo standard treatment (R-CHOP). Several groups have identified reproducible molecular clusters of DLBCL although, the biologic significance of many of the specific gene mutations within these clusters is poorly understood. DTX1 mutations are a feature of the BN2/C1 DLBCL classifiers and are among the mutations with unknown significance. BN2/C1 DLBCL are a mixture of ABC, GCB, and Unclassified cell of origin (COO) with a common signature being the presence of genetic alterations in genes known to regulate the NOTCH pathway; yet, these tumors lack transcriptional signatures of traditional NOTCH activation, suggesting a unique and undefined biology driven through non-canonical NOTCH pathway activation. DTX1 is an E3 ubiquitin ligase which directly binds the intracellular domain of the NOTCH receptor (NICD), targeting it for degradation, resulting in suppression of NOTCH pathway activity. We therefore hypothesize that DTX1 mutations lead to NOTCH pathway dysregulation and are a critical driver for a subset of BN2/C1 DLBCLs. Using whole exome sequencing (WES) data from 404 newly diagnosed DLBCL patients, we found 8% of patients with mutations in DTX1. Alterations in DTX1 were localized to the NICD binding domain, WWE1. In silico Alphafold modeling of DTX1 WWE1-interaction with NICD revealed that patient mutations clustered within predicted interacting surfaces with NICD. These findings lead to the hypothesis that mutations in the DTX1 WWE1 domain suppress its ability to bind to and degrade NICD, enhancing NOTCH pathway activation in DLBCL. To address this question, we first compared DTX1 protein expression by immunoblot in DTX1 WWE1 mutant (OCI-Ly3 and SUDHL2) and wildtype (WT) (OCI-Ly7, OCI-Ly19, SUDHL6, and U2932) DLBCL cell lines. We found higher expression of NICD1 (p=0.03) in OCI-Ly3 and NICD2 (p<0.0001) in SUDHL2 cell lines compared to WT. This increase was accompanied by an increase in NOTCH surface expression with higher NOTCH1 (p<0.0001) and NOTCH2 (p<0.0001) expression on OCI-Ly3 and SUDHL2 cells, respectively, compared to WT. To remove variability across cell lines we next used CRISPR to generate DTX1 knock out (KO) models using WT DTX1 DLBCL cell lines U2932 and OCI-Ly19. Analysis of NICD and NOTCH receptor protein expression confirmed our initial finding, showing an 8.5 and 4.8-fold increase of NICD1 and a 6 and 2.5-fold increase of NICD2 expression in the U2932 and OCI-Ly19 DTX1 KO cell lines, respectively, compared to empty vector (EV) controls. An increase in surface NOTCH1, and to a lesser extent NOTCH2, was seen in U2932 (p=0.01 and p=0.07, respectively) and OCI-Ly19 (p=0.001 and p=0.2, respectively) KO cells compared to EV controls. To identify pathways affected by DTX1 mutations, we next performed RNAseq on U2932 EV and KO cell lines. Of interest, we did not detect any significant changes in classical NOTCH driven genes (Hes1, Hey1, etc.), supporting prior studies on BN2/C1 tumors. However, we did identify potential non-canonical NOTCH-target genes that may contribute to lymphomagenesis. Gene expression in KO cells showed upregulation of genes involved in germinal center (GC) processes (AICDA, IRF4; p<0.001) and cellular growth and survival (IL6R, CCND2, STAT5B, BCL2; p<0.0001). Measurement of IL6R by flow cytometry and CyclinD2 by immunoblot further verified DTX1-mediated upregulation of IL6R (p<0.01) and CyclinD2 (p<0.01) for U2932 KO cells compared to EV control. We also observed downregulation of genes contributing to cell death (ex. BAX, CASP8, FAS; p<0.05) in KO cells compared to EV. Together, these novel findings support our hypothesis that DTX1 WWE1 mutations lead to dysregulation of the NOTCH pathway by driving overexpression of NICD and NOTCH, resulting in dysregulated expression of proteins involved in cell cycle, GC reaction, and apoptosis. We further provide new biologic insight into BN2/C1 DLBCL, ultimately highlighting a path toward novel therapeutic targets and the advancement of personalized medicine, as patients harboring these mutations may benefit from new therapies targeting inflammatory pathways, such as CELMoDs.

Abstract Description The efficacy of immunotherapy for splenic marginal zone lymphoma (SMZL) remains limited due to the strong immune suppression present in the tumor microenvironment (TME). Tregs play a key role in maintaining TME homeostasis by suppressing effector T cells and malignant B cells. Previously, we identified two Treg subsets CD26+ and CD161+ in SMZL. CD161+ Tregs were more effective at suppressing both CD8+T cells and malignant B cells, whereas CD26+Tregs primarily inhibited CD8+T cells. Patients with higher levels of CD26+ Tregs and fewer CD161+ Tregs tend to have worse outcomes. In this study, we found that cytokine-inducible SH2-containing protein (CISH), a known negative regulator of immune activation, is highly expressed in CD26+Tregs compared to CD161+ Tregs. Co-culturing B cells enhanced the expression of CISH and CD26 in CD4+CD25- Treg cells, knockdown of CISH in Tregs reduced CD26 expression and their immune suppression activity of CD8+T cells, while CRISPR knockout of CISH in Karpas 299 cells led to higher expression CD70, a molecule associated with less immunesuppression. Our findings suggest that increased CISH expression in Tregs could result in increased immune suppression and unfavorable outcome in SMZL, suggesting CISH is a potential therapeutic tagrget in SMZL by dempening immunosuppressive property of the Treg cells hence boosting the antitumor immunity. Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Oncogene expression can cause replication stress (RS), leading to DNA double-strand breaks (DSBs) that require repair through pathways such as homologous recombination, nonhomologous end-joining, and microhomology-mediated end-joining (MMEJ). Cyclin D1 (encoded by CCND1 ) is a well-known oncoprotein overexpressed in cancer; however, its role in RS is unknown. Using mantle cell lymphoma (MCL) as a naturally occurring model of cyclin D1 overexpression, we examined the impact of cyclin D1 on RS and DSB repair mechanisms. Cyclin D1 overexpression elevated RS, increased DNA damage, especially during mitosis, and caused specific upregulation of MMEJ. Furthermore, cyclin D1 activated polymerase theta ( POLQ ) transcription by binding its promoter loci, driving POLΘ-mediated MMEJ that is essential to withstand cyclin D1–induced RS. Moreover, concurrent ATM deficiency further intensified RS, enhanced POLQ expression, and heightened reliance on MMEJ-mediated DNA damage repair. Consequently, inhibition of POLΘ in cyclin D1–overexpressed settings further exacerbated RS, causing single-strand DNA gap accumulations and chromosomal instability, ultimately leading to apoptosis, an effect amplified in ATM-deficient cells. Targeting MMEJ via POLΘ inhibition is therefore an effective strategy in the context of cyclin D1 overexpression and ATM deficiency and may provide a unique therapeutic approach for treating MCL and other malignancies characterized by similar alterations.

Oncoprotein-driven human cancers exhibit replication stress (RS), leading to an increase in DNA double-strand breaks (DSB). These DSBs are repaired by one of the three main pathways - homologous recombination repair (HR), non-homologous end-joining (NHEJ), and microhomology-mediated end joining (MMEJ). Cyclin D1 (CCND1) is one of the most overexpressed oncoproteins and is universally overexpressed in mantle cell lymphoma (MCL). While CCND1 overexpression drives cells into the S-phase, its specific effects on RS and DSB repair pathways are poorly understood. Here, we investigated the impact of CCND1 overexpression on RS and DSB repair mechanisms in MCL. First, the expression level of CCND1 positively correlated with heightened RS, as indicated by elevated p-RPA-S33 and p-RPA S8 levels, and increased DNA damage, assessed by g-H2AX, especially in the mitotic phase, across diverse cell models, including MCL. Since CCND1-overexpressed cells proliferate rapidly, we hypothesized that some DNA repair pathways might be upregulated to meet the demand for repairing the excess RS-driven DSBs. Reporter assays demonstrated that the MMEJ pathway was specifically upregulated upon CCND1 overexpression, whereas no significant changes were observed in the HR and NHEJ pathways. Furthermore, CRISPR interference (CRISPRi)-mediated knockdown of CCND1 in MCL cells led to a significant reduction in MMEJ activity. This finding suggests the specificity of CCND1 in mediating the upregulation of the MMEJ pathway. Consistent with this, increased MMEJ activity, assessed via foci formation and immunoblotting of DNA polymerase theta expression (POLQ, encoded by POLQ gene), the primary polymerase for the MMEJ pathway, correlated with CCND1 overexpression, suggesting the crucial role of MMEJ in DSB repair processes under CCND1-overexpressed conditions. Sincethe MMEJ pathwayplays a pivotal role in repairing DSB during mitosis, we assessed the dependency of MCL cells on MMEJ-mediated DSB repair for cell survival. As expected, genetic deletion or pharmacological inhibition (ART558 and novobiocin) of POLQ in MCL cells led to increased RS and increased unrepaired DNA damage during mitosis, ultimately leading to chromosomal instability, mitotic catastrophe, and apoptosis. Additionally, the deficiency of POLQ in MCL cells resulted in increased levels of single-strand DNA, another critical biomarker for RS. Second, we found that ATM deficiency, the second most common genetic alteration after CCND1 overexpression in MCL, further increased the cellular dependence on MMEJ. Interestingly, the accumulation of unrepaired DNA damage during mitosis was notably increased in ATM-deficient CCND1-overexpressed cells. When POLQ was inhibited, a significant antitumor effect was evident in MCL cells, including ibrutinib-resistant MCL, both in vitro and in vivo, and this effect was enhanced by ATM deficiency. Further, simultaneous inhibition of both ATM (AZD0156) and POLQ was synthetic lethal in MCL cell lines and primary tumor cells from patients with MCL. Mechanistically, we observed the direct binding of CCND1 to the POLQ promoter and a corresponding upregulation of POLQ mRNA in CCND1-overexpressing cells. Consistently, a significant overexpression of POLQ mRNA was observed in primary MCL cells, compared to other non-Hodgkin lymphoma cells without CCND1 overexpression. Moreover, the positive correlation between POLQ and CCND1 expression was further validated using syngeneic B-lymphoma cells with and without CCND1 overexpression. Importantly, CRISPRi-mediated CCND1 knockdown reduced POLQ mRNA expression and the dependency of MCL cells on the MMEJ pathway for survival. In addition, ATM deficiency showed a further increase of endogenous POLQ expression and foci formation in cells with CCND1 overexpression, suggesting ATM negatively regulates the MMEJ pathway, especially in the CCND1 overexpressed background. In summary, CCND1 overexpression leads to a substantial increase in RS, intensifying the reliance on MMEJ-mediated mitotic DSB repair. Within MCL, POLQ is crucial in mitigating the adversities of CCND1-driven RS. Our data suggest that targeting the MMEJ pathway via POLQ inhibition, particularly in the presence of CCND1 overexpression and ATM deficiency, emerges as a promising therapeutic approach in oncoprotein-driven hematologic malignancies such as MCL.

Splenic marginal zone lymphoma (SMZL) is a B-cell malignancy with a typically indolent course. However, some patients have more aggressive disease and initial treatment can range from observation to chemoimmunotherapy. The reason for this spectrum of disease behavior is incompletely understood but the tumor microenvironment (TME) likely plays a role as recent studies have shown that an optimized antitumor immune response in SMZL patients would allow the immune system to better target malignant B cells and irradicate the disease. Regulatory CD4+T cells (Tregs) are crucial in maintaining TME homeostasis by suppressing effector T cells and malignant B cells and in this study, we therefore explored the key factors influencing the function and development of intratumoral Tregs in SMZL patients. We identified two distinct Treg subsets in the TME of SMZL patients: CD26+Tregs and CD161+Tregs, each with unique phenotypic profiles, functions, and prognostic significance. CD161+Tregs suppress CD8+T cells and malignant B cells more effectively than CD26+Tregs, which only inhibit CD8+T cells. Clinically, patients with inferior outcomes have significantly higher numbers of CD26+Tregs and fewer CD161+Tregs. To delineate differences between these subsets, we performed Cellular Indexing of Transcriptomes and Epitopes Sequencing (CITE-seq) on biopsies from 7 SMZL patients. We found that cytokine-inducible SH2-containing protein (CISH) is the most highly expressed gene in CD26+Tregs compared to CD161+Tregs, aside from FOXP3. CISH has been reported to be a negative regulator of immune effector cell activation in tumor models. Although CISH deficiency leads to a loss of Foxp3 expression and reduced Treg suppressive function, the mechanism by which CISH modulates Treg function, especially immune suppression in hematologic malignancies, remains largely unknown. To further investigate the mechanism of CISH in Treg function in MZL patients, we conducted CITE-seq analysis on a larger cohort of MZL patients (n=12), divided into groups based on outcomes (EFS24 failed, n=4; EFS24 achieved, n=8). The CITE-seq analysis results showed significantly higher CISH expression in Tregs from patients with inferior outcomes (P=1.07E-06). We then explored factors influencing CISH expression and found that TCR engagement and treatment with cytokines (IL-15, IL-2, and IL-7) elevated CISH expression in Tregs. While we previously observed that lymphoma B cells could enhance Foxp3 expression in CD4+CD25-T cells, this study we found that B cells could also enhance CISH and CD26 expression in CD4+CD25-T cells in vitro, indicating that a high frequency of B cells in the environment may lead to increased CISH expression in Treg cells leading to an unfavorable outcome in patients. We further investigated the role of CISH in the function of Treg cells. Following CISH knockdown in Treg cells, we observed decreased CD26 expression and reduced suppressive function on CD8+ T cell proliferation. Using CRISPR-Cas9, we knocked out CISH in the Karpas 299 cell line, a human T cell lymphoma line that constitutively expresses CD25, Foxp3, and CISH. CISH-KO Karpas 299 cells showed significantly less apoptosis and Foxp3 expression compared to CISH-WT cells. Bulk RNA-seq analysis of CISH-KO and CISH-WT Karpas 299 cells revealed that CISH-KO cells showed enhanced DNA replication and cell cycle pathway activity. Taken together, our results suggest that high expression of CISH in Tregs may predict an unfavorable outcome in SMZL patients. Malignant B-cells in the TME of SMZL patients may promote expression of CD26 and CISH in Tregs, thereby modulating Treg function. Targeting CISH could decrease the immunosuppressive activity of Tregs in the TME, potentially optimizing antitumor immunity in SMZL patients.

Introduction: Non-Follicular Low-Grade B-Cell Lymphomas (LGBCL) are a subset of lymphomas that share overlapping clinical and histologic features. While most cases of LGBCL have an indolent course, some experience aggressive disease with no definitive biomarkers to identify aggressive cases at diagnosis. Significant advances have been made in the molecular classification of other lymphomas, yet molecular profiling and identification of mechanisms driving aggressive disease in LGBCL remain an unmet need. We previously used a multi-omic approach to characterize the molecular and immune landscapes of LGBCL tumors and identified a transcriptomic signature associated with cell proliferation and inferior overall survival (OS) in two independent cohorts (HR 7.82; 95% CI 2.4-25.4; p < 0.001 & HR 10.07; 95% CI 2.00-50.61; p = 0.005) and cases that transformed to DLBCL (Hopper et al., 2023). Here, we identify the oncoprotein DEK as a novel transcriptional regulator of this signature and use a functional genomic approach to further interrogate the role of DEK in B-cell lymphoma. Methods: Tumors from 64 newly-diagnosed/untreated LGBCL cases enrolled in the University of Iowa/Mayo Clinic Molecular Epidemiology Resource were included in this study (SMZL = 48, NMZL = 6, LPL = 5, B-NOS = 3, EMZL = 2). Tumor RNA-seq and paired tumor-normal WES were used to assess DEK expression and identify copy number alterations (CNAs), respectively. DEK deficient (KO) cell line models were generated using CRIPSR/Cas9. DEK expression was assessed by western blot (WB) and immunofluorescence (IF) using standard techniques. Proliferation was assessed by MTT assay. Results: To identify transcriptional regulators of our previously identified signature, we used the tool MAGIC to mine publicly available ChIP-seq data and identified DEK, an oncoprotein associated with tumorigenesis and poor prognosis in several cancers, as a key regulator of genes in our signature (p = 0.008). RNA-seq indicated that high levels of DEK expression associated with inferior OS in LGBCL (HR 2.99; 95% CI 1.21-7.36; p = 0.02). Additionally, we identified DEK as a target of copy number gains/amps in LGBCL (7% of cases), which corresponded to significantly higher DEK expression (p = 0.04). The association of DEK with aggressive lymphoma was confirmed when we assessed its expression in public cell line RNA-seq data and observed the highest expression in lines derived from aggressive lymphomas, which we further validated at the protein level in a panel of B-cell lymphoma cell lines by WB and IF confocal microscopy. Additionally, we detected nuclear localization of DEK, consistent with its role as a transcriptional regulator. As a model of DEK overexpression for functional characterization, we used the SUDHL6 lymphoma cell line, which has an amplification of DEK at 6p22.3 and expresses high levels of DEK protein. Using CRISPR technology, we generated DEK deficient SUDHL6 and tested the impact of loss of DEK expression. DEK KO cells proliferated significantly slower than WT SUDHL6 cells in vitro (p < 0.0001)(Panel A), indicating DEK plays a role in regulating proliferative capacity in lymphoma. To identify mechanisms responsible for reduced proliferation in DEK KOs, we assessed baseline protein expression levels of apoptosis proteins in WT versus KO cells. We found near complete abrogation of anti-apoptotic protein Bcl-2 and reduction of Bcl-xL expression in DEK KO cells (Panel B). These results indicate DEK may contribute to tumor proliferative capacity by regulating susceptibility to apoptosis. Conclusion: DEK has been implicated in regulation of gene expression, but its precise role has remained elusive, with little known about its role in lymphoma. Using an integrative molecular and functional genomics approach, we identified DEK as a novel oncoprotein in LGBCL that is upregulated in cases with inferior clinical outcome and is regulated by copy number gains/amps. DEK plays a role in regulating tumor proliferative capacity by altering susceptibility to apoptosis through regulation of Bcl-2 and Bcl-xL. Collectively, this data suggests high-risk LGBCL tumors may enrich for cells with DEK overexpression or CNAs as a mechanism driving oncogene addiction, highlighting DEK, and its downstream targets, as potential therapeutic targets in a subset of high-risk LGBCL.

Background: DNA mismatch repair (MMR) deficiency (dMMR), which leads to genomic and microsatellite instability, is a biomarker that predicts response to immunotherapies and has variable prognostic effects for chemotherapies in solid tumors. The dMMR can be detected using gene sequencing or immunohistochemistry for four essential MMR proteins: MSH6, MSH2, MLH1, and PMS2. Diffuse large B-cell lymphoma (DLBCL) is the most common aggressive lymphoma. Previous studies have suggested the role of MMR gene variants in DLBCL lymphomagenesis, yet the prognostic role of dMMR in DLBCL has not been well studied. Methods: We performed targeted next-generation sequencing and immunohistochemistry for MSH6, MSH2, MLH1, and PMS2 in a large cohort of DLBCL patients treated with standard chemoimmunotherapy. Gene expression profiling (GEP) was performed using the Affymetrix GeneChip Human Genome HG-U133 Plus 2.0 microarray (data in GSE31312). Fluorescent multiplex immunohistochemistry (mIHC) was performed using MultiOmyx multiplexing immunofluorescence staining protocols and antibodies against 13 immune markers. We investigated the frequencies of dMRR and MMR protein expression and correlated the mutation status and expression levels with patient survival and DLBCL biology, including the number of mutated genes, GEP data, previously analyzed DLBCL biomarkers, and immune cell immunophenotypes. Results: MMR gene mutations and loss of MMR protein expression were infrequent in DLBCL (Figure A) and did not show a significant prognostic impact. MMR proteins were commonly expressed in DLBCL samples and the germinal centers of reactive tonsil controls (Figure B), with higher mean and median percentages of tumor cells expressing MSH6 and MLH1 proteins than in the solid tumor samples used for comparison. High expression of MMR proteins was associated with Ki-67, MYC, and p53 overexpression as determined by immunohistochemistry. GEP analysis identified significantly upregulated genes involved in the mitotic cell cycle and DNA metabolism and prominent downregulated immune gene signatures in DLBCL with high MMR protein expression. Fluorescent mIHC confirmed the associations with decreased T cell abundance in MSH6/MSH2/MLH1/PMS2 highly expressing DLBCL (Figure C) independent of p53 and MYC expression status, whereas MSH6/ MLH1 mutations were associated with increased T cell frequencies. High versus low expression of MSH6, MLH1, and PMS2 showed significant unfavorable prognostic effects in DLBCL when the optimal cutoffs were used for high expression. However, when the median percentages were used as cutoffs, the prognostic effects were not significant; nonetheless, MSH6 and PMS2 high expression showed significant adverse prognostic effects in the MYC¯ DLBCL subset. Interestingly, when we used Ki-67, MYC + or BCL2 + percentages in DLBCL assessed by immunohistochemistry to subtract MMR protein percentages and then correlated the differences to survival, we found significant favorable prognostic impact by higher percentage expression differences between MMR proteins and BCL2, or by intermediately higher differences between MMR proteins and MYC. For MSH2/MLH1/PMS2, the higher percentages compared with MYC/BCL2 showed no significant correlations with T cell infiltration levels, whereas the higher differences between MSH6 and MYC/BCL2 were associated with significantly lower T cell frequencies (Figure D). Conclusions: This study revealed that high expression of MMR proteins is a common feature of DLBCL associated with lower tumor-infiltrating T cells, MYC and p53 overexpression, and context-dependent prognostic effects. In contrast, dMMR and loss of MMR proteins are infrequent and have no significant prognostic effects in DLBCL treated with standard chemoimmunotherapy. These results may have implications for understanding DLBCL biology and the low efficacy of PD-1 blockade immunotherapy in DLBCL.

Introduction: Despite the improved clinical outcomes with novel therapies in patients with chronic lymphocytic leukemia (CLL), Richter transformation (RT) of CLL remains a major clinical challenge. Our group showed a selective response (~40%) with the PD-1 blocking antibody pembrolizumab in patients with RT, particularly after prior exposure to ibrutinib (Ding et al, Blood, 2017). It has been established that PD-1 inhibits cytotoxic T-cell functions via the interaction with its ligands, PD-L1 and PD-L2. However, the role of PD-1 in tumor B-cells is understudied. Here we show the expression and functional implications of the PD-1 signaling axis in B-cell pathobiology. Methods: CLL-involved lymph node (LN) (n=26) and RT (with diffuse large B-cell lymphoma [DLBCL] histology) -involved LN (n=20) samples were tested for PD-1 expression by immunohistochemistry (IHC) (mouse clone NAT105, Abcam). Then, PD-1 expression was examined in 10 lymphoma cell lines and 1 CLL-like cell line (MEC-1) by both flow cytometry (FC) and Western blot (WB) analysis. PD-1 overexpression using pLEX-lentiviral packaging system (Thermo Scientific) was evaluated for its impact on in vitro cell cycle regulation and in vivo tumor growth. CRISPR/CAS9 mediated PD-1 knockdown evaluated on cell growth in MEC-1 and Mino cells. Role of PD-1 was further examined on the regulation of cell signaling pathways using human phospho kinase array kit (R&D) and WB analysis. Additionally, gene expression signatures were evaluated by RNA-seq using pLEX-lentiviral-PD-1 transfected DLBCL cell lines OCI-LY7 and OCI-LY19. Results: PD-1 expression was significantly increased in RT-LN compared to CLL-LN (mean ± SEM in RT vs. CLL, 30.6% ± 4.7% vs. 11.5% ± 2.8%, p<0.001) as determined by IHC. PD-1 expression was highest in patients with RT where the last prior CLL therapy was ibrutinib. To test the role of PD-1 in tumor B-cells, its expression was assessed in lymphoma and CLL cell lines. The expression of PD-1 was found to be variable by both FC and WB analysis, but at very low-levels in DLBCL cell lines OCI-LY7 and OCI-LY19. Constitutive lentiviral (pLEX-PD-1)-mediated overexpression of PD-1 in OCI-LY7 and OCI-LY19 cells led to increased cell growth (1.4 and 1.9 fold, respectively, compared to original lines after 4 days of culturing of equal number of cells), which was further confirmed by cell cycle analysis which showed an increase in S phase by 20.4% (p<0.05) and 24.5% (p<0.05) in OCI-LY7 and OCI-LY19 cells, respectively. When the luciferase+ PD-1 overexpressing OCI-LY7 cells (1X10 6) were injected intravenously into NSG mice, increased tumor growth (p<0.01) was observed by bioluminescence imaging after 22 days of follow-up. PD-1 knockdown in MEC-1 and Mino cells (high basal PD-1 expression) led to significant inhibition of cell growth (85% and 79.5%, respectively, p<0.05). Using phospho-kinase array, an overall decrease of phosphorylation was detected on multiple sites of p53 (S392, S15, S46) and Chk-2 (T68) in PD-1 overexpressing OCI-LY7 and OCI-LY19 cell lines. This finding was further confirmed by WB analyses. In addition, decreased total p53 and increased phosphorylation on both SHP-1 and SHP-2 were found in PD-1 overexpressing OCI-LY7 and OCI-LY19 cell lines. SHP-1 (TPI-1) and SHP-1+SHP-2 (TPI-1+TNO155) inhibitor treatment promoted OCI-LY19 cell death and led to recovery of phosphorylation on ATM, Chk-2 and p53 in these cells. RNA sequencing data in PD-1 overexpressing OCI-LY7 and OCI-LY19 cells revealed increased expression of genes that promote cell cycle progression and DNA replication. Conclusion: Our data showed robust PD-1 expression in patients with Richter transformation from CLL to DLBCL. PD-1 overexpression in DLBCL lymphoma cell lines enhanced cell proliferation in vitro and in vivo. Further investigation identified that PD-1 modified the phosphorylation/function of SHP phosphatases and thereby regulated p53 pathways ( Figure 1). These findings indicate that RT with DLBCL cells have the ability to “hijack” the PD-1 pathway which results in downregulation of p53 mediated DNA repair. This novel finding provides for new strategies to further evaluate the interaction of PD-1 signals with DNA repair pathways and suggest potential novel targets for RT.