DLBCL Cell Lines Research Articles

Identification of miR-451 target genes as prognostic markers in diffuse large B-cell lymphoma

ABSTRACT Background B-cell lymphoma, a diverse malignancy, is intricately regulated by multiple factors. MicroRNAs (miRNAs) have been demonstrated to be important regulators of the initiation and progression of human B-cell lymphoma, but their functions need to be further explored. Research design and methods Two B-cell lymphoma cell lines, Romas and HBL-1, were engineered to overexpress miR-451, with cell proliferation assessed via cell counting assays. Flow cytometry was used to study the effects of miR-451 on cell cycle and apoptosis. Bioinformatics analyses were performed using GEO datasets (GSE181063, GSE32918, GSE56315) to identify DEGs, and potential miR-451 target genes were predicted using tools like ENCORI, TargetScan, and R packages. A risk model for DLBCL prognosis was developed using Cox and LASSO regression. qRT-PCR validated the expression of these target genes. Results This study revealed that miR-451 inhibited cell proliferation, arrested the cell cycle, and induced apoptosis in human DLBCL cell lines. Bioinformatics analysis identified 9 target genes (MMP9, AQP9, RIN2, EOMES, LCP2, SELPLG, MAL, SOCS5, S1PR3) significantly associated with DLBCL prognosis, suggesting a potential mechanism by which miR-451 suppresses DLBCL development. Conclusions Our study indicates that a specific set of miR-451 target genes may significantly influence DLBCL patient outcomes.

Increased CCL2/CCR2 axis promotes tumor progression by increasing M2 macrophages in MYC/BCL2 double-expressor DLBCL

AbstractThe pathogenesis of myelocytomatosis oncogene (MYC) and B-cell lymphoma 2 (BCL2) double-expressor diffuse large B-cell lymphoma (DE-DLBCL) remains unclear. To investigate how MYC and BCL2 contribute to tumor aggressiveness, we analyzed tumors from 14 patients each with DE-DLBCL and non–DE-DLBCL using whole transcriptome sequencing. Validation was performed using publicly available data sets, tumor tissues from 126 patients, DLBCL cell lines, and a syngeneic mouse lymphoma model. Our transcriptome analysis revealed significantly elevated messenger RNA levels of C-C motif chemokine ligand 2 (CCL2) and C-C chemokine receptor type 2 (CCR2) in DE-DLBCLs when compared with non–DE-DLBCLs (adjusted P value < .05). Transcriptomic analysis of public data sets and immunohistochemistry corroborated these findings, indicating increased levels of M2 macrophages but a reduction in T-cell infiltration in DE-DLBCLs when compared with non–DE-DLBCLs (all P < .05). CCR2 expression was observed mainly in tumor-infiltrating macrophages and not in DLBCL cells. Increased expression of CCL2 and CCR2 was significantly associated with a poor prognosis in patients with DLBCL. In the in vitro analyses, MYChigh/BCL2high DLBCL cells showed higher CCL2 expression and secretion than MYClow/BCL2low cells. MYC and BCL2 increased CCL2 expression and secretion by upregulation of nuclear factor κB p65 in DLBCL cells, and CCL2 promoted M2 polarization of macrophages. In a mouse lymphoma model, CCL2 contributed to the immunosuppressive microenvironment and tumor growth of MYChigh/BCL2high tumors. We demonstrated that the increased CCL2/CCR2 axis confers aggressiveness to DE-DLBCL by increasing M2 polarization and can be a potential therapeutic target.

Abstract PR01: EZH2 inhibitors improve CAR-T therapy by enhancing lymphoma B cell immunogenicity and CAR-T cell functions

Abstract Although CAR-T19 therapy is widely used for relapsed/refractory B cell lymphomas, approximately half of patients experience post CAR-T relapse. The mechanisms underlying this resistance remain largely unknown and efforts to overcome this limitation are hindered by the lack of suitable pre-clinical models for study. To address these unmet needs, we generated a novel genetically engineered mouse model with conditional expression of Ezh2Y641F and BCL2 in germinal center (GC) B cells. Ezh2/BCL2 mice developed follicular lymphoma (FL) which histologically and transcriptionally recapitulates human FL. Moreover, we established a cell line derived from an Ezh2/BCL2 mouse that developed transformed FL (tFL). Injection of the cell line named “tFL-P6” in immunocompetent C57BL6 recipients resulted in rapid disease progression that histologically and transcriptionally mimics human tFL. Treatment of tFL-P6 with EZH2 inhibitors (EZH2i) didn’t affect proliferation and viability, however, the number of pretreated cells was significantly reduced when co-cultured with T cells. EZH2i pretreated tFL-P6 displayed extended interaction with T cells, as measured by live imaging. EZH2i reprogrammed tFL-P6 to restore T cell engagement genes such as ICOSL, ICAM1, OX40L, and integrins, as well as cytokines and chemokines involved in T cell recruitment, therefore enhancing immunogenicity. Notably, pretreated tFL-P6 cells were rejected by C57BL6 whereas they developed lethal disease in immunodeficient recipients. Pre-treatment of tFL-P6 as well as human DLBCL and PDX-derived cell lines significantly enhanced CAR-T19 cell killing effects in vitro. Administering CAR-T cells in mice engrafted with EZH2i-pretreated tFL-P6 significantly reduced the tumor burden and prolonged their survival (100% vs 30%, p&amp;lt;0.01). To assess the direct interactions between lymphoma cells and CAR-T cells in vivo, we performed intravital 2-photon imaging of popliteal lymph nodes using dTomato labeled CAR-T cells and GFP+ tFL-P6 cells. Pretreatment of tFL-P6 cells with EZH2i doubled the recruitment of CAR-T cells in the microenvironment and enhanced the duration of contact and surface engagement with CAR-T cells. To explore the impact of EZH2 inhibition on CAR-T cells, we manufactured CAR-T cells from splenic T cells of mice treated with EZH2i or vehicle for 14 days. Prior exposure to EZH2i didn’t affect proliferation and transduction during CAR-T production. Ex vivo assays with those CAR-T cells and tFL-P6 cells showed a superior killing effect and expansion of EZH2i-exposed CAR-T (p&amp;lt;0.001). Mice bearing tFL-P6 lymphomas displayed a longer survival when infused with EZH2i-pretreated CAR-T cells (p=0.06). Pretreatment with EZH2i resulted in an increased memory/effector ratio (p&amp;lt;0.01) and reduction of PD1+CD38+ exhausted CD8CAR-T cells (p&amp;lt;0.001). Overall, EZH2i improved CAR-T therapy by enhancing lymphoma cell immunogenicity and CAR-T cell functions. These results prompted the initiation of a clinical trial to evaluate the safety and efficacy of this combination in R/R B cell lymphomas (NCT05934838). Citation Format: Yusuke Isshiki, Xi Chen, Matt Teater, Ioannis Karagiannidis, Henna Nam, Amy Chadburn, Ari Melnick, Wendy Béguelin. EZH2 inhibitors improve CAR-T therapy by enhancing lymphoma B cell immunogenicity and CAR-T cell functions [abstract]. In: Proceedings of the Fourth AACR International Meeting on Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2024 Jun 19-22; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(3_Suppl):Abstract nr PR01.

Abstract PR02: Aging-associated molecular and cellular alterations determine the biology and clinical characteristics of DLBCL in elderly patients

Abstract About 30% of DLBCLs occur in patients over 75 years old. Due to comorbidities, this potentially curable disease is treated with a reduced dose chemoimmunotherapy regimen, associated with worse response rates and outcomes than the standard approach. Our data indicates that even those able to tolerate full-dose treatments have comparatively worse outcomes. We found that DLBCLs in older patients are more frequently of the ABC-DLBCL subgroup, and their microenvironment (TME) has infiltration of inflammatory and immunosuppressed cells, both characteristics independently associated with chemoimmunotherapy resistance. These features may imply we are facing different biologically diseases. To investigate this, we developed aged DLBCL murine models by implanting three murine lymphoma cells (harboring Setd2/BCL2, Ezh2/BCL2 and Tp53 mutations) in the spleen of young (3-4 months) and aged (21-23 months) mice and monitoring tumor growth by in vivo imaging. We also performed an integrative characterization of the TME from young and aged mice by single cell RNA-sequencing (coding and BCR/TCR), immunophenotyping (flow cytometry) and (MS)-based proteomics. For these two different murine DLBCL cell lines utilized, aged mice had higher tumor burden and showed significantly worse survival than younger mice. In addition, DLBCL cells isolated from aged mice displayed higher clonality and the activation of pro-inflammatory transcriptional programs than DLBCL cells isolated from younger mice. Regarding non-malignant TME B cells, CD11c+ T-bet+ Zeb2+ age-associated B cells (AABs) were significantly more abundant in aged v young mice. AABs displayed a secretory phenotype and transcriptional up-regulation of genes associated with pro-inflammatory programs such as IL-6, Stat3 and Nfatc1. TME T cells showed profound changes including increased infiltration of Foxp3+ Tregs and Pd1+ Cd8+ T cells, as well as the appearance of Cd8+ Gzk+ aged-associated T cells (ATT). Aged mice presented alterations in the myeloid compartment with expansion of Cd45+ Cd11b+ cells. Moreover, the TME of aged mice was enriched in tumor-associated macrophages polarized towards a pro-inflammatory phenotype as well as more abundance of Ly6C+ Ly6G+ myeloid-derived suppressor cells (MDSC). Aged TMEs were also characterized by increased stiffness and extracellular matrix changes affecting collagen and proteoglycans. These TME changes, were associated with intrinsic signaling and transcriptional changes in lymphoma cells like increased activation of NF-kB pathway. Overall, these alterations were compatible with the TME of elderly DLBCL patients showing a global increase in the proportion of inflammatory and immunosuppressive (IN-LME) category (41% in 75-85 y.o. vs. 26% in 50-65 y.o.) and specifically in Tregs, MDSC and CD8 PD1HIGH cells in 75-85 y.o. This data indicates that low-grade chronic inflammation that has been associated with age-related diseases determines molecular and cellular characteristics of the DLBCL in the elderly population. Citation Format: Nahuel Zamponi, Maria Victoria Revuelta, Rossella Marullo, Nicolas Di Siervi, Nikita Kotlov, Wendy Béguelin, Leandro Cerchietti. Aging-associated molecular and cellular alterations determine the biology and clinical characteristics of DLBCL in elderly patients [abstract]. In: Proceedings of the Fourth AACR International Meeting on Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2024 Jun 19-22; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(3_Suppl):Abstract nr PR02.

Effects of polyphyllin Ⅶ on proliferation, apoptosis and cell cycle of diffuse large B-cell lymphoma cells

The aim of this study was to investigate the effects of polyphyllin Ⅶ (PP Ⅶ) on proliferation, apoptosis, and cell cycle of diffuse large B-cell lymphoma (PLBCL) cell lines U2932 and SUDHL-4. The DLBCL cell lines were divided into a control group and a PPⅦ group, and experiments were conducted using MTT assay, flow cytometry, and Western blotting.Results showed that compared with the control group, PPⅦ significantly inhibited the proliferation of U2932 and SUDHL-4 cells (P<0.05). Apoptosis assays demonstrated that treatment with 0.50 and 1.00 µmol/L PP Ⅶ significantly increased the apoptosis rates of both cell lines (P<0.05), upregulated apoptosis-related proteins, and downregulated Bcl-2 protein level (P<0.05). Cell cycle analysis revealed that PPⅦ treatment led to an increase in G0/G1-phase cells (P<0.05) and a decrease in G2/M-phase cells (P<0.05), significantly downregulated cyclin D1, CDK4, CDK6, and survivin protein expression (P<0.05). In conclusion, PPⅦ exerted anti-lymphoma effects by inhibiting proliferation, promoting apoptosis, and inducing G0/G1 phase arrest in DLBCL cells.

Abstract 7598: The novel eIF4A inhibitor potently synergizes with BCL2 inhibitor in high grade B-cell lymphoma with MYC and BCL2 rearrangements through regulating UPR

Abstract Double hit lymphoma(DHL) is defined to be a chromosomal break with MYC gene combined with BCL2 or BCL6. MYC is an oncogene which proliferation the cells and BCL2 inhibit cell death. These combinations could be a cause of highly aggressive clinical features and poor survival of DLBCL. The WHO-HAEM5 renames the entity DLBCL/high-grade B-cell lymphoma with MYC and BCL2 rearrangements. Despite the dismal prognosis, no widely accepted standard treatment exists to the management of DHL. Therefore, new therapeutic approaches for these patients are urgently needed. Previously, we looked at the eIf4A inhibition was shown to downregulate MYC expression in lymphoma cell lines. Venetoclax, a highly selective inhibitor of BCL2, shows clinical activity for patients with DLBCL although there are concerns about increasing toxicity. Our hypothesis in the following: If eIF4A inhibitor preferentially inhibit the translation of oncogenes such as cMyc and venetoclax inhibit the BCL2 in DHL, then the combination of eIF4A inhibitor and BCL2 inhibitor will be highly effective for DHL and potentially safe for animals and humans. We tested the anti-lymphoma effect of CR-1-31-B, in combination with Venetoclax in lymphoma cell lines using flow cytometry. We performed cell apoptosis assays in increasing concentrations of Venetoclax, CR-1-31-B and a combination of the two drugs at 48 hours. To better understand molecular mechanisms underlying the observed synergistic effect in DLBCL-DHL, RNA-seq profiling was performed on cells treated with single drugs and their combinations, respectively. Venetoclax and CR1-31-B exhibited concentration and time dependent cytotoxicity in lymphoma cell lines at low nanomolar concentrations. Overall, the combination of eIF4A inhibitor and BCL2 inhibitor showed synergy in all kinds of DLBCL cell lines. Especially DHL cell lines were more sensitive to the drug combination and showed enhanced synergy compared to the non-DHL cell lines(p&amp;lt;0.0001). We analyzed RNA-seq profiling of cells treated with single drugs and their combinations and found that the transcriptional signature of the synergistic combination was unique relative to that of either constituent monotherapy. Gene set enrichment analysis was performed in the DHL and non-DHL group. The analysis demonstrates that known Unfolded protein response(UPR) part and metabolism, are enriched in DHL groups. This suggests that the UPR and metabolism pathway are closely related to the combination effect in DHL. These findings suggest that the combination of eIF4A inhibitor and BCL2 inhibitor have potent pharmacological activity in DHL lines, it was closely related with UPR. These combination is promising and novel treatment for high-grade B-cell lymphoma with MYC and BCL2 rearrangements. Further pre-clinical investigations of this combination for these patients are needed. Citation Format: So-Young Seol, Chung Hyun Park, Soo Jeong Kim, Haerim Chung, Hyunsoo Cho, Jin Seok Kim, Yu Ri Kim. The novel eIF4A inhibitor potently synergizes with BCL2 inhibitor in high grade B-cell lymphoma with MYC and BCL2 rearrangements through regulating UPR [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7598.

Abstract 46: Mechanisms and impact of bystander killing by CAR T cells

Abstract While CD19-directed chimeric antigen receptor (CAR) T cell therapy has improved outcomes of patients with relapsed/refractory diffuse large B cell lymphoma (r/r DLBCL), a majority will suffer disease relapse following CAR T cell infusion. Therefore, understanding mechanisms of CAR T cell killing is critical to enhance therapeutic efficacy. Cytolysis of CAR T cell targets presumably involves recognition of CD19 antigen by the CAR followed by activation of the granzyme/perforin axis. However, bystander elimination of CD19-negative lymphoma cells by CAR T cells has been reported, indicating that an alternative pathway may be required for optimal killing. Through in-depth phenotyping of a large panel of human DLBCLs and in vitro killing assays, we observed that CD19 expression levels on DLBCL cells and vulnerability to CAR T cell killing were weakly correlated. Conversely, cell surface expression of the death receptor, FAS, on DLBCL cells was strongly associated with susceptibility to CAR T cell killing. Although FAS expression across DLBCL cell lines varied, it could be broadly enhanced following exposure to IFNγ. Potent CAR T cell-mediated cytolysis of CD19-negative, Fas-positive murine DLBCL cells required the presence of CD19-positive lymphoma cells in the culture. CRISPR/Cas9 targeting was employed to delete FAS and/or CD19 in isogenic human DLBCL cells to generate CD19+FAS+, CD19+FAS−, CD19−FAS+, and CD19−FAS− subsets, which enabled us to define the extent to which CD19 engagement by the CAR was required to affect killing via FAS. When CAR T cells were co-cultured with equal numbers of the four gene-modified DLBCL cells described above, CD19+FAS+ DLBCL cells were killed most effectively, followed by CD19+FAS− DLBCL cells, and CD19−FAS+ DLBCL cells. Double-negative DLBCL cells were poorly killed. This result suggested that CD19 engagement by the CAR was required for optimal DLBCL cell killing via FAS, and that CD19 and FAS need not necessarily be engaged on the same target cell. Future studies will determine if Fas/FasL interactions can be exploited by overexpression of FasL in CAR T cells and examination of DLBCL killing in vitro and in vivo. Citation Format: Joanna Chorazeczewski, Lishi Xie, Xiufen Chen, Sidney Wang, Justin Kline. Mechanisms and impact of bystander killing by CAR T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 46.

Abstract 6044: EPriL: A platform for the rapid identification of degraders of inhibitor-resistant kinases

Abstract Heterobifunctional kinase degraders (HKDs) are an important new class of therapeutic modalities, of which the first have recently entered clinical trials. HKDs are small molecules that comprise a kinase-of-interest binding moiety and an E3 ligase binding moiety connected via a spacer. HKDs are excellently suited to target kinases that have a scaffolding function, or that are resistant against conventional inhibitors. A HKD brings a kinase and an E3 ligase in proximity forming a ternary complex leading to ubiquitin-dependent catalytic degradation of the kinase of interest. This ternary complex formation is unique for each kinase which can make HKDs highly specific. To discover novel HKDs, we have developed a platform called Energetically Privileged Ligands (EPriLs), which is based on a macrocycle scaffold that binds non-covalently in the kinase ATP pocket with near-covalent affinity, and allows binding to kinases that have acquired known inhibitor resistance mutations. The macrocyclic structure allows attachment of E3 ligase ligands with multiple exit vectors. With the EPriL platform, we have developed novel HKDs for various clinically relevant kinase targets, including Bruton’s Tyrosine Kinase (BTK). BTK inhibitor drugs are used as first line treatment for chronic lymphocytic leukemia (CLL), Waldenström macroglobulinemia (WM) and as second line treatment for mantle cell lymphoma (MCL). Despite their great success in the clinic, many patients on BTK inhibitors relapse eventually, mostly because of the emergence of resistance mutations in the BTK protein. These include BTKC481S, which abrogates binding of covalent drugs, and BTKT474I, BTKL528W and BTKV416L which preclude binding of non-covalent inhibitors like pirtobrutinib. HKDs can combat resistance by degrading such BTK variants, and by removing the scaffold function of BTK. BTK degraders such as NX-5948 and NX-2127 are currently in clinical Phase 1. To show the applicability of the EPriL platform for the discovery of novel BTK degraders, we screened a library of more than 100 EPriL HKDs for biochemical binding to BTK, and tested their propensity to induce degradation of HiBiT-tagged BTKWT, BTKC481S and BTKT474I in HEK293 cell lines. The degrading activity of hits was verified through western blot on the BTK-dependent DLBCL cell line TMD8. The best degraders - based on potency and efficacy - were tested in cell proliferation assays on a panel of TMD8 BTK mutant cell lines. In these lines, endogenous BTK was mutated by CRISPR to BTKC481S, BTKT474I, BTKL528W or BTKV416L. Several BTK degraders had potent (&amp;lt; 20 nM IC50) antiproliferative activity on most TMD8 lines, including difficult-to-target variants such as BTKV416L and BTKL528W. The unique binding mode of the EPriL scaffold thus translates to broad activity on resistance variants, while its selectivity ensures minimal degradation of off-target tyrosine kinases as observed for other degraders. Citation Format: Michelle Muller, Jos de Man, Joost C. Uitdehaag, Freek van Cauter, Sander van Gemert, Milan Hoffmann, Yvonne G. van Mil, Winfried R. Mulder, Martine B. Prinsen, Jan Gerard Sterrenburg, Diep Vu, Joeri de Wit, Erik Ensing, Rogier C. Buijsman. EPriL: A platform for the rapid identification of degraders of inhibitor-resistant kinases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6044.

Mechanism about LMP1 of EB Virus Promoting Plasma Blast Differentiation of DLBCL Cell via mTORC1

To investigate possible mechanism on protien LMP1 expressed by EBV inducing plasmablast differentiation of DLBCL cell via the mTORC1 pathway. The expression levels of LMP1 protein, CD38 and the phosphorylation levels of p70S6K in EBV+ and EBV- DLBCL cell lines were detected by Western blot. Cell lines overexpressing LMP1 gene stablely were constructed and LMP1 gene was silenced by RNAi. The expression of LMP1 gene was verified by RT-qPCR. The expression levels of LMP1 and CD38 and the phosphorylation levels of p70S6K in each group were detected by Western blot. Compared with EBV-DLBCL cells, the expression of LMP1 was detected on EBV +DLBCL cells (P =0.0008), EBV +DLBCL cells had higher phosphorylation levels of p70S6K (P =0.0072) and expression levels of CD38(P =0.0091). Compared with vector group, the cells of LMP1OE group had higher expression levels of LMP1 and CD38 (P =0.0353; P <0.0001), meanwhile molecular p70S6K was phosphorylated much more(P =0.0065); expression of LMP1 mRNA was verified(P <0.0001). Compared with si-NC group, expression level of LMP1 protein(P =0.0129) was not detected and phosphorylated p70S6K disappeared of LMP1KO group (P =0.0228); meanwhile, expression of CD38 decreased,although there was no significant difference (P =0.2377). LMP1 promotes DLBCL cells plasmablast differentiation via activating mTORC1 signal pathway.

PD-1 Blockade Combined with R-CHOP in Patients with Newly Diagnosed EBV-Positive Diffuse Large B-Cell Lymphoma: A Retrospective Analysis

Background Epstein-Barr virus (EBV) -positive diffuse large B cell lymphoma (EBV+ DLBCL) is a relatively rare aggressive B-cell lymphoma. Most patients present with advanced disease, high incidence of extranodal involvement of 30% to 80% (Keane, Eur J Haematol, 2019), as well as poor prognosis when treated with immunochemotherapy. However, no consensus on standard treatment has been established by now. It was found that the expression of programmed death ligand-1 (PD-L1) was increased in EBV+ DLBCL (Kim, Cancer Res Treat, 2019). In addition, chronic EBV infection results in T-cell exhaustion that can be reversed by PD-1 blockade. Compared with PD-L1-positive EBV-negative DLBCL cell lines, the application of PD-1 blockade in PD-L1-positive EBV+ DLBCL cell lines can significantly restore T cell proliferation and activity(Quan, PLoS One, 2015), suggesting that targeting the PD-L1/PD-1 pathway represents a potential therapy for EBV+ DLBCL. Therefore, we retrospectively analyzed the data of nineteen patients with EBV+ DLBCL who were treated with PD-1 blockade combined with RCHOP in our center, hoping to provide reference for subsequent research. Methods Eligible pts had treatment-naive histologically confirmed diagnosis of EBV+ DLBCL. Additional eligibility criteria included at least one measurable lesion, acceptable haptic and hematological function. Pts with primary CNS lymphoma, primary mediastinal large B-cell lymphoma, secondary CNS involvement were excluded. Additional exclusion criteria included history of immune system disease, uncontrolled infection. Once deemed eligible, pts will receive six cycles of RCHOP combined with PD-1 blockade(21 days per cycle) as induction therapy and subsequently maintenance with PD-1 blockade for 1 year(21 days per cycle). End of treatment(EOT) responses were assessed by PET-CT according to the Lugano Classification 2014. The primary objective was to assess the overall response rate(ORR) at EOT. Results Between May 1, 2019, and Sep 1, 2022, 19 pts were enrolled. Pts' baseline demographics, clinical and pathological characteristics are shown in Table 1. The median age was 64 years old without significant gender difference. The majority of pts were intermediate- to high-risk by IPI. EBV DNA can be detected in blood serum at diagnosis in more than half of all patients. No patient was diagnosed with double expressor lymphoma among 18 specimens that were detected c-Myc and BCL-2 with immumohistochemical staining. EBV-encoded RNA (EBER) was positive(&amp;gt;80% of the tumor cells) in all specimens. PD-L1 positivity(&amp;gt;10% of the tumor cells) or CD30 positivity(&amp;gt;5% of the tumor cells) was also quite common. Besides, no double hit lymphoma was confirmed in patient with Fluorescence situ hybridization test(n=13). Treatment course and efficacy evaluation of all pts are summarized in Figure 1. All patients enrolled in this study had completed 6 cycles of induction therapy. Totally, the ORR at EOT was 94.7%, and all achieved CMR. The ORR of patients with PD-L1 positivity(n=10) was numerically higher than that of patients with negative PD-L1 expression (n=5) (100% vs 80%). EBV DNA detected in blood serum of thirteen patients have decreased to undetectable level after induction therapy. The immune-related adverse events (irAEs) were emphatically analyzed. One patient had recurrent fever after every dose of PD-1 blockade without obvious evidence of infection. Another patient developed grade 3 diarrhea since the second cycle of PD-1 blockade maintenance. Colonoscopy showed mucosal swelling and multiple superficial ulcers in the whole colon, which indicated immune colonitis. Maintenance therapy was therefore discontinued. No irAE was observed in the rest of patients. With a median follow-up time of 13.1m (7.2-38.5m), the median PFS and OS were not reached. Both of 1 year OS and PFS was 89.5%. Overall, 2 pts experienced disease progression (1 at EOT, 1 at 2 months after ASCT), 2 pts died (1 due to progressive disease, 1 due to COVID-19 infection ). Conclusions PD-1 blockade combined with R-CHOP as induction therapy in patients with newly diagnosed EBV+ DLBCL has demonstrated satisfactory response and limited irAEs. Longer follow-up is still warranted to confirm the long-term benefit, especially for patients with maintenance of PD-1 blockade. Still, we believe this therapeutic strategy can be regarded as a successful attempt at improving prognosis for pts with EBV+ DLBCL.

CD19 Intron Retention Is Mechanism of CAR-T Treatment Resistance in Non-Hodgkin Lymphoma

Background: Chimeric antigen receptor T-cell (CAR-T) therapy has become a viable treatment option for patients (pts) with relapsed or refractory diffuse large B-cell lymphoma (R/R DLBCL). Transcriptomic markers for CD19 directed CAR-T failure have been characterized in pre-treatment B-cell acute lymphoblastic leukemia (B-ALL) but not in DLBCL. Here, we report the identification of RNA alternative splicing (AS) events associated with CD19 directed CAR-T response in pre-treatment DLBCL pts and of RNA binding proteins (RNABP) regulating these AS events. Methods: To identify putative AS markers for CAR-T treatment outcome, we performed differential AS on bulk RNA-seq samples of pre-treatment tumor biopsies in pts that had a durable response (DR), defined via remission up to 9 months post-treatment, and pts that had a non-durable response (NDR) with rMATS-turbo (Shen et al. PNAS. 2014). Intron retention (IR) events within CD19 were quantified with a secondary, more robust algorithm to ensure reproducibility and accuracy. Survival analyses were done using the survival and survminer libraries in R. Validation assays for the downstream effects CD19 IR were conducted in EJ1, Jeko-1, OCI-Ly3, SU-DHL-6, and Toledo cell lines. The surface protein expression of CD19 was assayed via flow cytometry (FC) followed by Quantibrite PE (BD Biosciences, San Jose, CA) quantification. Total CD19 protein expression was measured via western blot (WB) in the aforementioned cell lines. Analyses of CD19 IR was done via RT-PCR followed by electrophoresis, and bands were quantified with iBright Analysis Software (Thermo Fisher Scientific, Carlsbad, CA). The effect of RNABP was assessed via the creation of CRISPR/Cas9 knockout (KO) cell lines followed by WB of the target gene and quantification of CD19 surface protein expression by FC. Results: Introns 2 and 6 of CD19 are more highly expressed in NDR pts. A combined metric estimating the proportion of normal CD19 isoforms that do not express introns 2 or 6 (normal CD19) was created and DR pts expressed normal CD19 isoforms significantly higher than NDR pts (Figure 1). Normal CD19 isoforms classification performance was assessed via leave one out cross validation and achieved an overall accuracy of 67.6%. Sensitivity (classifying NDR) was 61.1% and specificity (classifying DR) was 81.3%. The expression of normal CD19 relative to the median level within the cohort was significantly associated with progress free survival (Figure 2). The expression of CD19 intron 6 was negatively correlated with CD19 surface protein expression in DLBCL cell lines. Cell lines were sorted by cells that exhibited high CD19 surface expression (bright) and low expression (dim). Cell lines sorted into the dim category expressed higher levels of intron 6 than bright cells. The correlation of RNABP expression with CD19 intron 6 expression was done in this patient cohort and the NCICCR-DLBCL cohort. Six RNABP were significantly correlated in both cohorts (r 2 &amp;gt; 0.2 and p-value &amp;lt;0.01). NOP2, one of the RNABP that was positively correlated with intron 6 expression, was knocked out in Jeko-1 cell lines to validate the putative correlation with CD19 protein expression. The resulting knock out cells exhibited lower levels of intron 6 retention and higher CD19 protein expression by FC and WB than non-transformed cells. Conclusion: Pre-treatment tumors of R/R DLBCL patients who relapse before 9 months following CD19 directed CAR-T therapy have higher expression of CD19 isoforms that retain introns 2 and 6. The upregulated expression of the CD19 isoforms retaining these introns is correlated with CD19 surface protein expression and is a marker for sustained remission following CAR-T treatment. NOP2 is correlated with CD19 intron retention and surface protein expression and could be a target for modulating CD19 expression in conjunction with CAR-T treatment. Figure 1: The expression of normal CD19 isoforms in pre-treatment tumor samples of DR (red) and NDR patients (blue). ΔPSI denotes the difference in medians between the two groups. P-value yielded by a Wilcoxon test between the two groups is reported. Figure 2: Progress free survival (PFS) stratified by expression of normal CD19 isoforms. Red - above cohort median expression of normal CD19 isoforms. Blue - below cohort median expression of normal CD19 isoforms. P-value yielded via log-rank test.

PD-1 Expression in Lymphoma Cells Mediates Cellular Proliferation By Engaging Phosphatase SHP-1/SHP-2

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&amp;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&amp;lt;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&amp;lt;0.05) and 24.5% (p&amp;lt;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&amp;lt;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&amp;lt;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.

Investigation of the Synergistic Combination of a Novel Celmod (CC-99282) and BET Inhibitors in Preclinical DLBCL

Background: Golcadomide (GOLCA; CC-99282) is a novel cereblon E3 ligase modulator (CELMoD), which binds to cereblon (CRBN), a substrate receptor of CRL4 CRBN E3 ubiquitin ligase, and induces proteasome-dependent degradation of Ikaros and Aiolos, transcription factors essential for B cell malignancies. GOLCA was shown to exhibit promising cell of origin independent activity in pre-clinical DLBCL models and is currently being investigated in early clinical trials (NCT03930953) for relapsed/refractory non-Hodgkin lymphomas (R/R NHL). Ikaros and Aiolos have previously been shown to have occupancy on the promoter of the proto-oncogene MYC, which is subsequently downregulated by CELMoDs in DLBCL models. Given MYC is a well-studied downstream target of BET inhibitors and a driver of lymphoma disease, we investigated the mechanistic synergy of anti-proliferative and pro-apoptotic effects of GOLCA in combination with BMS-986158, a potent BET inhibitor. Results: Dose titration and analysis of the anti-proliferative effects of DLBCL cell lines (SU-DHL-4, WSU-DLCL2 and CARNAVAL) treated with either GOLCA or BMS-986158 suggests significant single agent activity of either agent. Bliss synergy score was determined and strong synergism of the two drugs was observed on day 5, with maximal bliss score (14.3-29.8) centered at low doses (1.25 and 2.5 nM) of BMS-986158 and sub-lethal doses of GOLCA (5-50 nM). Similar synergistic effects were found when combining GOLCA with JQ1, another tool compound BET inhibitor, confirming BET inhibition potentiates the anti-DLBCL effects of the CELMoD agent. Incucyte-based live cell image confirmed the enhanced anti-proliferative capacity of the combination compared to single agents. EdU (5-ethynyl-2′-deoxyuridine)-based cell cycle analysis revealed an increased proportion of G1-phase cells in combination treatment (67%) compared to GOLCA (38%), BMS-986158 (43%) and DMSO (35.4%) in the GOLCA resistant SU-DHL-4 cells. To identify key pathways responsible for the observed synergistic combination effect, we performed transcriptomic analyses of SU-DHL-4 and WSU-DLCL2 cells treated with GOLCA/BMS-986158 for 72 hrs. Compared to either single agents, the combination treatment downregulated significantly more genes (~1000 and 500 genes in SU-DHL-4 and WSU-DLCL2, respectively), which were enriched with cell cycle pathways. Gene set enrichment analysis (GSEA) showed the top downregulated gene sets in combination treatment were E2F targets such as CHEK1 and G2M checkpoint molecules including AURKA and CDC45. Given the critical role of CDKN1A (gene encodes p21) in cell cycle arrest, we examined its expression by real-time PCR. We found treatment of GOLCA induced CDKN1A mRNA expression (16.4 and 25.1-fold in SU-DHL-4 and WSU-DLCL2, respectively); remarkably, this induction was further enhanced by the combination with BMS-986158 (40.1 and 128.5-fold in SU-DHL-4 and WSU-DLCL2, respectively). Western blot confirmed the synergistic induction of p21 in combination compared to single agents, with a concomitant reduction of phosphorylated Rb, E2F target genes (Chk1, SKP2 and E2F1) and MYC. Since the main targets of GOLCA and BMS-986158 are transcription factors (Ikaros and Aiolos) and transcriptional regulator (Brd4), respectively, we performed chromatin immunoprecipitation followed by sequencing (ChIP-seq) with antibodies targeting these three proteins along with various histone marks including H3K27ac and H3K4me3. Interestingly, the chromatin occupancies of Ikaros/Aiolos/Brd4 were highly overlapping in genes that are upregulated ( CDKN1A) and downregulated (e.g. CHEK1 and MYC) by the combination treatment. Further characterization of the interplay between Ikaros/Aiolos/Brd4 and their loci-specific interactomes may help elucidate the synergism at epigenetic levels. Conclusions: BET inhibition potentiates anti-proliferative effects of GOLCA in DLBCL cells through synergistic induction of p21 and inhibition of E2F signaling. The synergetic effects of BMS-986158 and GOLCA suggest potential clinical benefits of combining lower doses of single agents to achieve better efficacy with reduced risk of adverse effects.

Novel Protacs Demonstrate Dual Kinase Inhibition and Degradation of HCK and BTK, and Are Highly Active in MYD88 Mutated WM and ABC DLBCL Cells

Background: Activating mutations in MYD88 are prevalent in many B-cell malignancies, including Waldenström Macroglobulinemia (95-97%), primary CNS lymphoma (70-80%), ABC DLBCL (40%), marginal zone lymphoma (5-10%), and CLL (5-15%). Mutated MYD88 transcriptionally upregulates the SRC family member HCK, which in turn serves as a master signal for triggering multiple pro-survival cascades in mutated MYD88 lymphoma cells including BTK/NFKB, SYK, and ERK (Blood 127:3237-52; Blood Adv 4:141-153; Blood Adv 6:3332-38). In previous work, we developed and characterized a novel HCK kinase inhibitor, KIN-8194 (Blood 138:1966-79). KIN-8194 blocked both HCK and BTK and was active both in vitro and in vivo in MYD88 mutated xenograft lymphoma models, including BTK Cys481Ser ibrutinib resistant models. Proteolysis targeting chimeras (PROTACs) represent a novel approach for blocking kinase based signaling and may provide an advantage over kinase inhibitors with greater selectivity and sustained target inhibition. We therefore initiated a medicinal chemistry campaign to develop highly potent, selective, and bio-available HCK/BTK targeting PROTACS using KIN-8194 scaffold for the treatment of MYD88 driven lymphomas. Methods: We developed and characterized a large series of PROTACs using KIN-8194 as a scaffold. We rationally designed linkers to facilitate E3 ubiquitin ligase binding and improve physical properties leading to degraders with ideal oral pharmacokinetic properties and bioavailability. We characterized their kinome profiling by performing a KINOMEscan assay against a panel of 468 kinases at 1 uM ( Fig. 1). Subsequently, we conducted rigorous biological efficacy screenings using the ATP-based Cell Titer-Glo Luminescent Cell Viability Assay to identify the most potent compounds. Compounds with IC 50 scores lower than KIN-8194 were selected for further validation through flow cytometry based quantitative assays that verified apoptotic and cytotoxic effects on WM and MYD88-mutated lymphoma cell lines, as well as primary human peripheral blood mononuclear cells (PBMCs), utilizing Annexin V and Propidium iodide (PI) staining. Results: We identified and characterized several novel PROTACs from the KIN-8194 scaffold that exhibited potent dual inhibition of HCK and BTK kinase activity at 1 nM, as well as marked degradation of HCK and BTK by western blot analysis at dose levels as low as 100 nM in MYD88 mutated WM and ABC DLBCL cell lines. KINOMEscan profiling showed greater selectivity for novel PROTACs as exemplified in Fig. 1. Enhanced cell killing due to degradation of HCK and BTK versus inhibition alone was confirmed using respective PROTAC analogues with modified IMID warheads that do not bind cereblon. We observed suppression of downstream pro-survival NFKB and ERK signaling in response to the dual HCK/BTK PROTACs. Moreover, several HCK/BTK PROTACs (DFCI-004, -005, -006) demonstrated remarkable low IC 50 values (1-100 nM) in proliferation assays of MYD88 mutated BCWM.1 WM and TMD-8 ABC DLBCL cells. Importantly, the dual HCK/BTK PROTACs elicited higher levels of apoptosis in BCWM.1 WM and TMD-8 ABC DLBCL cells in comparison to the native KIN-8194 kinase inhibitor and showed sparing of healthy donor B- and T-cells akin to KIN-8194. Finally, murine PK studies showed that the HCK/BTK PROTACs DFCI-005 and -006 were highly bioavailable with F=40-42%, and C Max of 700-2700 nM at single oral dose ranges of 10-60 mg/kg. Conclusions: In these studies, we demonstrated the development and characterization of novel, dual HCK/BTK PROTACs that demonstrate potent and selective kinase inhibition and protein degradation of HCK and BTK. Importantly, the novel PROTACs showed enhanced apoptosis of MYD88 mutated WM and ABC DLBCL cells over the native kinase inhibitor KIN-8194 and sparing of healthy donor B- and T-cells. Lastly, novel HCK/BTK PROTACs exhibited high levels of bioavailability. Our studies therefore provide a framework for the advancement of dual HCK/BTK PROTACs for the treatment of MYD88 mutated lymphomas.

Inhibition of glutaminase-1 in DLBCL potentiates venetoclax-induced antitumor activity by promoting oxidative stress

AbstractDiffuse large B-cell lymphoma (DLBCL) is the most common lymphoma in adults, but first-line immunochemotherapy fails to produce a durable response in about one-third of the patients. Because tumor cells often reprogram their metabolism, we investigated the importance of glutaminolysis, a pathway converting glutamine to generate energy and various metabolites, for the growth of DLBCL cells. Glutaminase-1 (GLS1) expression was robustly detected in DLBCL biopsy samples and cell lines. Both pharmacological inhibition and genetic knockdown of GLS1 induced cell death in DLBCL cells regardless of their subtype classification, whereas primary B cells remained unaffected. Interestingly, GLS1 inhibition resulted not only in reduced levels of intermediates of the tricarboxylic acid cycle but also in a strong mitochondrial accumulation of reactive oxygen species. Supplementation of DLBCL cells with α-ketoglutarate or with the antioxidant α-tocopherol mitigated oxidative stress and abrogated cell death upon GLS1 inhibition, indicating an essential role of glutaminolysis in the protection from oxidative stress. Furthermore, the combination of the GLS1 inhibitor CB-839 with the therapeutic BCL2 inhibitor ABT-199 not only induced massive reactive oxygen species (ROS) production but also exhibited highly synergistic cytotoxicity, suggesting that simultaneous targeting of GLS1 and BCL2 could represent a novel therapeutic strategy for patients with DLBCL.

Lenalidomide in Combination with Etoposide Induced Ferroptotic Cell Death in DLBCL

Background ： Relapse/refractory(R/R) DLBCL after first-line immunochemotherapy have unfavorable outcomes, treatment of which remains challenging. The combination of multiple drugs in the second-line regimen improves efficacy but also increases toxicity, often leading to treatment-related adverse events (AEs) or dose reduction/interruption. This study aims to explore the effects of lenalidomide in combination with ibrutinib in diffuse large B-cell lymphoma and investigate its potential mechanisms. Methods: 1. From February 2019 to June 2023, patients aged 60 and above who received R 2-ICE regimen were analyzed for the efficacy and AE of the second-line therapy. 2. Human DLBCL cell lines SU-DHL-4 and U2932 were screened using the CCK-8 assay to demonstrate the synergistic cytotoxic effect of different drugs on DLBCL cells. Subsequently, various experiments were conducted to investigate whether oxidative damage occurred and explore the underlying mechanisms . These experiments included measurement of intracellular iron levels, MDA assay, detection of H 2O 2 and ·OH levels, ROS assay, assessment of mitochondrial membrane potential, and Hoechst staining. 3. SCID mouse model with subcutaneous xenografts of SU-DHL-4 cells in the right thigh was established. Different chemotherapy regimens, including PBS, R 2, R 2-ICE, and R 2-E, were administered to the mice, and the tumor-inhibitory effects and in vivo toxicity (evaluated based on body weight) of these treatments were observed. Results: 1. A total of 19 patients aged 60 years and above with relapsed/refractory diffuse large B-cell lymphoma (R/R DLBCL) received the R2-ICE regimen as second-line chemotherapy. They completed at least 2 treatment cycles, and the therapeutic efficacy was evaluated. Short-term efficacy: Overall Response Rate (ORR) was 81% (43.75% Complete Response [CR], 37.5% Partial Response [PR]). Long-term efficacy: Median follow-up of 30 months, with 1-year Progression-Free Survival (PFS) of 42.9% and 2-year PFS of 32.1%. One-year Overall Survival (OS) was 75%, and 2-year OS was 37.5%（Figure1A-B）. 2. Through CCK-8 cell proliferation assay, it was observed that lenalidomide，carboplatin and etoposide exerted time- and dose-dependent inhibitory effects on SU-DHL-4 and U2932 cells. Different IC50 concentrations of these drugs were chosen after 48 hours of treatment, and lenalidomide was administered in combination with etoposide or carboplatin. By calculating the CCK-8 cell survival rate and drug synergy formula, it was determined that lenalidomide in combination with etoposide had a synergistic inhibitory effect on DLBCL cell lines. Further CCK-8 experiments showed that ferroptosis inhibitor Fer-1-treated cells partially resisted the cytotoxic effect of lenalidomide in combination with etoposide on DLBCL cells (all differences were statistically significant, Figure 1. C-E). These findings indicate that lenalidomide in combination with etoposide suppresses DLBCL proliferation via the ferroptosis pathway. 3. Experiments of SU-DHL-4 cells were divided into four groups：control，len, eto，len+eto. By performing intracellular iron content detection, MDA assay, H2O2 and ·OH level detection, ROS experiment, mitochondrial membrane potential detection, and Hoechst staining, it was demonstrated that lenalidomide in combination with etoposide induces intracellular accumulation of ROS through an iron-dependent Fenton reaction, leading to cell membrane and DNA damage and DLBCL cell death(Figure 2). 4. SU-DHL-4 pretreated with Fer-1, and the experiments were divided into four groups: control, control + Fer-1, len+eto, len+eto+ Fer-1. By performing experiment mentioned above demonstrated that Fer-1 could reverse the oxidative damage induced by lenalidomide in combination with etoposide（Figure 3）. 5.In in vivo experiments, chemotherapy administered with PBS, R 2, R 2-E, and R 2-ICE regimens showed that R 2-E and R 2-ICE had similar tumor-suppressive effects and R 2-E were lower toxicity（Figure 4）. Conclusion: Lenalidomide combined with etoposide enhances intracellular reactive oxygenlevels through the ferroptosis pathway, resulting in synergistic killing of DLBCL cells through lipid and DNA oxidation damage. In vivo experiments confirmed that R 2-E showed similar antitumor effects with R 2-ICE and had lower toxicity.

Targeted DNA Damage Boost with Loncastuximab Tesirine in Combination with PARP Inhibitors in Diffuse Large B-Cell Lymphoma

MYC-over expressing lymphomas display inherent resistance to standard chemoimmunotherapy regimens and are characterised by genomic instability and constitutive activation of the DNA damage response (DDR) pathway. Loncastuximab tesirine (Lonca) is an antibody-drug conjugate directed against CD19, a surface antigen broadly expressed in B-cell malignancies. Upon CD19 binding, Lonca induces DNA damage by delivering a pyrrolobenzodiazepine dimer-based, DNA crosslinking warhead into target cells. Here we hypothesised that DDR inhibition could enhance DNA damage induced by Lonca in CD19-positive lymphoma cells. Since MYC-overexpressing cells mainly rely on PARP activity to attenuate endogenous MYC-induced replicative stress, we combined Lonca with Talazoparib (an FDA-approved PARP inhibitor), in a panel of 12 DLBCL cell lines (7 with MYC-rearrangement), and in vivo in a MYC/BCL-2-rearranged DLBCL PDX model. Lonca and Talazoparib showed significant antiproliferative activity in a dose and time dependent manner in most cell lines at clinically achievable concentrations. The highest sensitivity to both compounds was observed in a BRCA2 mutant cell line (DOHH2). These data are in line with the known synthetic lethality between BRCA mutations and platinum derivatives and PARP inhibitors. The addition of Talazoparib significantly enhanced the antiproliferative effects of Lonca in DLBCL cell lines irrespective of MYC/BCL2 rearrangements and TP53 genomic alterations. Of note, the highest efficacy of the combination was observed in MYC-rearranged and MYC-overexpressing cell lines. The combination of Lonca and Talazo sinergistically increased DNA damage as evaluated by comet assay, induced enhanced cell death and caused specific changes in cell cycle dynamics. Enhanced apoptosis was confirmed by Annexin/PI staining and caspase 3/7 cleavage, assessed by caspase Glo assay and Western Blot analyses. As previously shown (Fusani et al., ASH 2021), while Lonca and Talazoparib affected cell cycle inducing G2/M phase arrest, the combination specifically increased the percentage of cells arrested in the BrdU negative S phase of cell cycle. Upon combined treatment, we observed increased DDR activation with the evaluation of several DDR biomarkers such as phospho(p)-ATM, pCHK2 Thr68, pCHK1 Ser345, gH2AX. To better understand the molecular mechanism underlying this synergistic interaction, we performed RNA-Seq analysis in two representative cell lines (OCI-LY18 and SUDHL5). Interestingly while Lonca did not cause significant changes in gene expression profiling (GEP) after 24 hours of incubation, the combination resulted in specific GEP changes such as down-regulation of histone H2B and aurora kinase A (AURKA), genes mainly involved in S and G2/M transition of the cell cycle. Similar results were obtained by combining Talazoparib and different PARP inhibitors with the alkylating agent cisplatin, indicating a class effect. The synergistic interaction of Lonca and Talazoparib was further confirmed in vivo in a MYC/BCL-2 double hit PDX model. As previously shown (Fusani et al., ASH 2021), Lonca/Talazoparib combination did not result in enhanced DNA damage induction in PBMC-derived B cells from healthy donors. Importantly, PBMC-derived T cells from healthy donors did not show any sign of DNA damage accumulation upon exposure to Lonca, Talazoparib and the combination. These data provide the rationale for future therapeutic strategies based on selective induction of DNA damage in neoplastic B cells in combination with DDR inhibition in aggressive MYC-positive B cell lymphoma.

Combination of CDK4/6 Inhibitor Palbociclib and PI3K Inhibitor Idelalisib Synergistically Induces an Anti-Tumor Effect in B-Cell Lymphoma and Overcomes Ibrutinib Resistance

Introduction: Aberrant activity of cyclin-dependent kinases 4/6 (CDK4/6) is frequently detected in B-cell non-Hodgkin's lymphoma (B-NHL), indicating the therapeutic implications of CDK4/6 inhibitors for B-cell malignancies. What's more, the introduction of agents targeting B cell receptor signaling, especially phosphatidylinositol 3-kinase (PI3K) isoform-specific inhibitors, is rapidly changing how B cell malignancies are treated. In this study, we aim to investigate whether the combination of CDK4/6 inhibitor palbociclib (PAL) and PI3Kδ inhibitor idelalisib (IDE) has synergistic anti-tumor effects in B cell lymphoma. Besides, we also evaluated the effect of co-administration PAL and IDE in Bruton's tyrosine kinase inhibitor (BTKi) resistant model. Methods: In vitro, cell viability was analyzed using Cell Titer-Glo ®️ Luminescent Cell Viability Assay. Flow cytometry was used to detect apoptosis and cell cycle arrest induction. Western Blotting analysis was used to detect the changes in essential proteins in related signaling pathways. We also conducted RNA-seq to evaluate the whole transcriptome changes brought by co-treatment with PAL and IDE. The synergistic anti-tumor effects of PAL and IDE were also evaluated in vivo. BTKi secondary resistant cell lines were generated by long-time exposure to ibrutinib and BTK C481S mutation cell line was conducted by lentivirus infection and puromycin selection. Results: Firstly, we analyzed diffuse large cell lymphoma (DLBCL) gene expression profile results from GEO database, and there were more than 50% patients carried cell cycle-related genes alterations, especially for CDKN2A-CDK4/6/CCND1-Rb machinery. Then, it was noteworthy that the high level of CDK4/6 mRNA and protein expression correlated with worse survival of DLBCL patients. Next, we illustrated that pharmacological inhibition of CDK4/6 kinase activity induced inhibition of cell proliferation, tumor cell apoptosis, and cell senescence. Meanwhile, PAL treatment upregulated the expression of PI3Kδ, p-AKT (S473), and p-mTOR (S2448), suggesting compensatory activation of the PI3K-AKT-mTOR signaling pathway. Thus, combination treatment of PAL and PI3K inhibitor IDE was assessed, and this combination synergistically induced a more potent anti-proliferative effect in several B-NHL cell lines, including mantle cell lymphoma cell lines (Z-138 and Jeko-1), DLBCL cell lines (U2932 and OCI-Ly8) and Burkitt lymphoma cell line (Raji). Meanwhile, co-administration treatment could also trigger synergistic anti-tumor activity in BTK inhibitor ibrutinib-resistant HBL-1 BTK C481S and HBL-1 IR cells (long time exposure to ibrutinib). Next, the flow cytometry results indicated that the B-NHL cells became apoptotic after the PAL and IDE treatment. The activation of apoptosis-related proteins (cleaved-caspase3 and cleaved-PARP) and decrease of anti-apoptosis proteins expression (BCL-2, BCL-xL, XIAP, and MCL-1) were induced by combination of CDK4/6 and PI3K inhibitor treatment. The combination of PAL and IDE also substantially increased the cell population of G0/G1 phase and elevated expression of CDK4, CDK6, and CyclinD1. Furthermore, the gene expression profile analysis demonstrated that the mRNA expression level of PLK1 was significantly decreased by co-administration treatment, rather than in PAL or IDE treatment alone. Finally, the combination treatment also showed synergistic anti-tumor activity in multiple tumor-bearing mice models. Conclusion: The combination of CDK4/6 inhibitor palbociclib and PI3K inhibitor idelalisib synergistically induced anti-tumor activity in B-cell lymphoma through downregulation of PLK1 expression, suggested a new combination direction for the treatment of B-NHL and even BTK inhibitor-resistant patients.

Preclinical Study of DZD8586, a Non-Covalent LYN/BTK Dual Inhibitor with Excellent BBB Penetration, for the Treatment of B-Cell Non-Hodgkin Lymphoma (B-NHL)

Introduction Bruton's Tyrosine Kinase (BTK) inhibitors have been approved for the treatment of B-NHL, such as CLL and MCL. However, resistance to BTK inhibitors still inevitably occurred, and different types of mutations were identified post covalent and non-covalent BTK inhibitor treatment. Although BTK inhibitors showed some activities in the treatment of relapsed or refractory non-germinal center B-cell (non-GCB) DLBCL, no drugs have been approved due to unsustainable tumor response. It is hypothesized that blockage of BTK pathway alone is not sufficient to achieve optimal anti-tumor efficacy in DLBCL. Due to limited blood-brain barrier (BBB) penetration of the current therapies, treatment of central nervous system lymphoma (CNSL) remains a clinical challenge. DZD8586 is a rationally designed, oral, non-covalent, LYN and BTK dual inhibitor with excellent BBB penetration as a potential treatment option for B-NHL. Here we report the preclinical activity of DZD8586 in lymphoma cell lines and animal models which supports its clinical development to address the above unmet medical needs. Methods The enzymatic activity of DZD8586 against multiple kinases was assessed at Eurofins panel with purified enzymes. TMD-8-IbruR cells carrying the C481S mutation were generated by stepwise induction of increasing concentrations of Ibrutinib. Cells carrying pirtobrutinib resistance mutations were obtained by transfection of different BTK mutations into RI-1 cells using lentivirus. Cell growth inhibition was determined by CellTiter-Glo. A pBTK enzyme-linked immunosorbent assay (ELISA) was used to evaluate the pharmacodynamic profile of DZD8586. Western blot was used to test the modulations of signaling pathways with DZD8586 treatment. Animal models were established by subcutaneous and intracranial inoculation of tumor cells, respectively, and animals were treated with DZD8586 or other BTK inhibitors. The anti-tumor efficacy was assessed by comparing the change in mean tumor volume between the different treatment groups and vehicle controls. Results Kinase panel profiling showed that DZD8586 potently inhibited LYN and BTK, with good selectivity against other kinases. In cell lines expressing C481X and pirtobrutinib resistance mutations, DZD8586 demonstrated concentration dependent anti-proliferative effects, with similar GI 50s among cells carrying different BTK mutations. In a panel of DLBCL cell lines, DZD8586 exhibited potent cell growth inhibition and induced cell death in a variety of cell lines, and its effect correlated with modulations of both LYN and BTK pathways. In both subcutaneous and CNS tumor models, DZD8586 exhibited profound tumor growth inhibition in a dose-dependent manner, and induced tumor regression at the doses of 50 mg/kg and above. There was a good correlation between plasma concentrations of DZD8586 and modulations of pBTK in whole blood and tumor tissue. DZD8586 has good cell permeability and is not significantly transported by P-gp and BCRP expressed at the BBB. The K puu,CSF of DZD8586 in rat and monkey were 1.2 and 1.3, respectively, suggesting its excellent BBB penetration in humans. Conclusion DZD8586 is a novel non-covalent LYN/BTK dual inhibitor, which could overcome resistance mutations to the approved covalent and non-covalent BTK inhibitors, and derive clinical benefit to patients with DLBCL and CNSL by blocking both BTK-dependent and BTK-independent signaling pathways with excellent BBB penetration.

MYD88L265P augments proximal B-cell receptor signaling in large B-cell lymphomas via an interaction with DOCK8

AbstractDiffuse large B-cell lymphoma (DLBCL) is a clinically and genetically heterogeneous disease with at least 5 recognized molecular subtypes. Cluster 5 (C5)/MCD tumors frequently exhibit concurrent alterations in the toll-like receptor (TLR) and B-cell receptor (BCR) pathway members, MYD88L265P and CD79B, and have a less favorable prognosis. In healthy B cells, the synergy between TLR and BCR signaling pathways integrates innate and adaptive immune responses and augments downstream NF-κB activation. In addition, physiologic TLR9 pathway engagement via MYD88, protein tyrosine kinase 2 (PYK2), and dedicator of cytokinesis 8 (DOCK8) increases proximal BCR signaling in healthy murine B cells. Although C5/MCD DLBCLs are selectively sensitive to Bruton tyrosine kinase (BTK) inhibition in in vitro studies and certain clinical trials, the role of mutated MYD88 in proximal BCR signaling remains undefined. Using engineered DLBCL cell line models, we found that concurrent MYD88L265P and CD79B alterations significantly increased the magnitude and duration of proximal BCR signaling, at the level of spleen tyrosine kinase and BTK, and augmented PYK2-dependent DOCK8 phosphorylation. MYD88L265P DLBCLs have significantly increased colocalization of DOCK8 with both MYD88 and the proximal BCR-associated Src kinase, LYN, in comparison with MYD88WT DLBCLs, implicating DOCK8 in MYD88L265P/proximal BCR cross talk. Additionally, DOCK8 depletion selectively decreased proximal BCR signaling, cellular proliferation, and viability of DLBCLs with endogenous MYD88L265P/CD79BY196F alterations and increased the efficacy of BTK blockade in these lymphomas. Therefore, MYD88L265P/DOCK8-enhanced proximal BCR signaling is a likely mechanism for the increased sensitivity of C5/MCD DLBCLs to BTK blockade.