DLBCL Cell Research Articles

Targeting Cellular Iron Homeostasis with Ironomycin in Diffuse Large B-cell Lymphoma.

Iron homeostasis represents a potential therapeutic target for high-risk patients with DLBCL that can be targeted with ironomycin to induce cell death and to sensitize tumor cells to conventional treatments.

A new role for the SRC family kinase HCK as a driver of SYK activation in MYD88 mutated lymphomas

AbstractThe SRC family kinase (SFK) HCK is transcriptionally upregulated and activated by mutated MYD88 (MYD88Mut), a key adaptor for Toll-receptor signaling. HCK activates BTK, AKT, and ERK in MYD88Mut lymphomas. SYK, a B-cell receptor (BCR) component, is activated in MYD88Mut lymphoma cells. Although the SFK LYN serves as a trigger for SYK activation in MYD88Mut ABC DLBCL cells, LYN activity is muted in MYD88Mut Waldenstrom macroglobulinemia (WM) cells. We therefore investigated a role for HCK in mediating SYK activation. Overexpression of wild-type (WT) (HCKWT) or gatekeeper mutated (HCKThr333Met) HCK in MYD88Mut lymphoma cells triggered SYK activation. Conversely, HCK knockdown reduced p-SYK in MYD88Mut lymphoma cells. Coimmunoprecipitation experiments showed that HCK was complexed with p-SYK in MYD88Mut BCWM.1 and TMD8 cells, but not in MYD88 WT Ramos cells. Rescue experiments in MYD88Mut lymphoma cells expressing HCKThr333Met led to persistent HCK and SYK activation and resistance to the HCK inhibitor A419259. Treatment of primary MYD88Mut WM cells with A419259 reduced p-HCK and p-SYK expression. Taken together, our findings show that SYK is activated by HCK in MYD88Mut B-cell lymphomas cells, broaden the prosurvival signaling generated by aberrant HCK expression in response to MYD88Mut, and help define HCK as an important therapeutic target in MYD88Mut B-cell lymphomas.

IRF8 is a transcriptional activator of CD37 expression in diffuse large B-cell lymphoma

AbstractDiffuse large B-cell lymphoma (DLBCL) represents the most common form of non-Hodgkin lymphoma (NHL) that is still incurable in a large fraction of patients. Tetraspanin CD37 is highly expressed on mature B lymphocytes, and multiple CD37-targeting therapies are under clinical development for NHL. However, CD37 expression is nondetectable in ∼50% of DLBCL patients, which correlates with inferior treatment outcome, but the underlying mechanisms for differential CD37 expression in DLBCL are still unknown. Here, we investigated the regulation of the CD37 gene in human DLBCL at the (epi-)genetic and transcriptional level. No differences were observed in DNA methylation within the CD37 promoter region between CD37-positive and CD37-negative primary DLBCL patient samples. On the contrary, CD37-negative DLBCL cells specifically lacked CD37 promoter activity, suggesting differential regulation of CD37 gene expression. Using an unbiased quantitative proteomic approach, we identified transcription factor IRF8 to be significantly higher expressed in nuclear extracts of CD37-positive as compared with CD37-negative DLBCL. Direct binding of IRF8 to the CD37 promoter region was confirmed by DNA pulldown assay combined with mass spectrometry and targeted chromatin immunoprecipitation (ChIP). Functional analysis indicated that IRF8 overexpression enhanced CD37 protein expression, while CRISPR/Cas9 knockout of IRF8 decreased CD37 levels in DLBCL cell lines. Immunohistochemical analysis in a large cohort of primary DLBCL (n = 206) revealed a significant correlation of IRF8 expression with detectable CD37 levels. Together, this study provides new insight into the molecular mechanisms underlying differential CD37 expression in human DLBCL and reveals IRF8 as a transcriptional regulator of CD37 in B-cell lymphoma.

Acquired Resistance to EZH2 Inhibitor GSK343 Promotes the Differentiation of Human DLBCL Cell Lines toward an ABC-Like Phenotype.

Diffuse large B-cell lymphoma (DLBCL) accounts for 40% of non-Hodgkin lymphoma, and 30% to 40% of patients will succumb to relapsed/refractory disease (rrDLBCL). Patients with rrDLBCL generally have low long-term survival rates due to a lack of efficient salvage therapies. Small-molecule inhibitors targeting the histone methyltransferase EZH2 represent an emerging group of novel therapeutics that show promising clinical efficacy in patients with rrDLBCL. The mechanisms that control acquired resistance to this class of targeted therapies, however, remain poorly understood. Here, we develop a model of resistance to the EZH2 inhibitor (EZH2i) GSK343 and use RNA-seq data and in vitro investigation to show that GCB (germinal center B-cell)-DLBCL cell lines with acquired drug resistance differentiate toward an ABC (activated B-cell)-DLBCL phenotype. We further observe that the development of resistance to GSK343 is sufficient to induce cross-resistance to other EZH2i. Notably, we identify the immune receptor SLAMF7 as upregulated in EZH2i-resistant cells, using chromatin immunoprecipitation profiling to uncover the changes in chromatin landscape remodeling that permit this altered gene expression. Collectively, our data reveal a previously unreported response to the development of EZH2i resistance in DLBCL, while providing strong rationale for pursuing investigation of dual-targeting of EZH2 and SLAMF7 in rrDLBCL.

HOXD3 Up-regulating KDM5C Promotes Malignant Progression of Diffuse Large B-Cell Lymphoma by Decreasing p53 Expression

Background:Diffuse large B-cell lymphoma is a type of B-cell non-Hodgkin lymphoma with a high incidence. About one-third of patients are resistant or eventually relapse. The prognosis for patients with relapsed/resistant diffuse large B-cell lymphoma who need salvage therapy is not optimistic.Aims:To explore whether homebox D3 binding to lysine (K)-specific demethylase 5C promoted malignant progression of diffuse large B-cell lymphoma by decreasing p53 expression.Study Design:Cell culture study.Methods:The mRNA and protein expression of lysine (K)-specific demethylase 5C and homebox D3 in cells were respectively detected by real-time quantitative polymerase chain reaction analysis and Western blot. Real-time quantitative polymerase chain reaction analysis and Western blot were also applied to determine the transfection effects of shRNA-KDM5C or OeHOXD3 in OCI-Ly7 cells. After transfection, the cell viability, proliferation, and apoptosis were respectively analyzed by Cell Counting Kit-8 assay, EdU staining, and acridine orange—ethidium bromide staining. The interaction between homebox D3 and lysine (K)-specific demethylase 5C promoter was verified by the dual-luciferase reporter assay and chromatin immunoprecipitation (ChIP) assay.Results:Lysine (K)-specific demethylase 5C mRNA expression (HBL1 2.84 ± 0.29; SUDHL4 3.53 ± 0.21; OCI-Ly8 4.06 ± 0.24; OCI-Ly7 5.03 ± 0.28 vs. GM12878 1.00 ± 0.07; all P < .001) and protein expression (HBL1 1.52 ± 0.06; SUDHL4 1.77 ± 0.10; OCI-Ly8 2.34 ± 0.07; OCI-Ly7 2.78 ± 0.07 vs. GM12878 1.00 ± 0.07; all P < .001) in DLBCL cells were higher than that in GM12878 cells and showed the highest in OCI-Ly7 cells. Homebox D3 mRNA (OCI-Ly7 3.85 ± 0.17 vs. GM12878 1.00 ± 0.05; P < .001) and protein (OCI-Ly7 1.73 ± 0.10 vs. GM12878 1.00 ± 0.06; P < .001) expression were also highly expressed in OCI-Ly7 cells. Moreover, down-regulation of lysine (K)-specific demethylase 5C suppressed the viability and proliferation and enhanced the apoptosis of OCI-Ly7 cells. Knockdown of lysine (K)-specific demethylase 5C decreased the B-cell lymphoma 2 expression while increased the expression of Bax, cleaved caspase 3, cytochrome C, p53, and p21. The transcription factor homebox D3 was confirmed to interact with the lysine (K)-specific demethylase 5C promoter. Homebox D3 overexpression could reverse the regulating effect of down-regulation of lysine (K)-specific demethylase 5C on the OCI-Ly7 cells.Conclusion:Homebox D3 up-regulating lysine (K)-specific demethylase 5C promotes malignant progression of diffuse large B-cell lymphoma by decreasing p53 expression.

Inhibitors of Bcl-2 and Bruton\u2019s tyrosine kinase synergize to abrogate diffuse large B-cell lymphoma growth in vitro and in orthotopic xenotransplantation models

Numerous targeted therapies have been developed for diffuse large B-cell lymphoma, but the results of late-stage clinical trials were mostly disappointing and have led to very few new regulatory approvals. Here, we use single and combinatorial drug response profiling to show that the combined inhibition of the anti-apoptotic protein Bcl-2 and of the tyrosine kinase BTK with the small molecules venetoclax and ibrutinib efficiently kills DLBCL cells in vitro. High Bcl-2 expression due to either BCL2 amplifications or translocations, in conjunction with chronic active BCR signaling accurately predict responses to dual Bcl-2/BTK inhibition. Orthotopic xenotransplantation and patient-derived xenograft models confirm that the combinatorial is superior to single-agent treatment in reducing the lymphoma burden. Combinatorial treatment further efficiently overcomes both primary and acquired resistance to venetoclax, which we could link to reduced expression of the Bcl-2 family members Bcl-XL and Bcl-2A1 under ibrutinib. We found in a Swiss DLBCL cohort that ~15% of patients are projected to respond to the venetoclax/ibrutinib combination based on their high Bcl-2 expression and nuclear NF-κB localization. Our data show that drug sensitivities exposed by drug response profiling can be attributed to specific mutational signatures and immunohistochemical biomarkers, and point to combined Bcl-2/BTK inhibition as a promising therapeutic strategy in DLBCL.

ASB2 is a direct target of FLI1 that sustains NF-\u03baB pathway activation in germinal center-derived diffuse large B-cell lymphoma

BackgroundDiffuse large B-cell lymphoma (DLBCL) comprises at least two main biologically distinct entities: germinal center B-cell (GCB) and activated B-cell (ABC) subtype. Albeit sharing common lesions, GCB and ABC DLBCL present subtype-specific oncogenic pathway perturbations. ABC DLBCL is typically characterized by a constitutively active NF-kB. However, the latter is seen in also 30% of GCB DLBCL. Another recurrent lesion in DLBCL is an 11q24.3 gain, associated with the overexpression of two ETS transcription factors, ETS1 and FLI1. Here, we showed that FLI1 is more expressed in GCB than ABC DLBCL and we characterized its transcriptional network.MethodsGene expression data were obtained from public datasets GSE98588, phs001444.v2.p1, GSE95013 and GSE10846. ChIP-Seq for FLI1 paired with transcriptome analysis (RNA-Seq) after FLI1 silencing (siRNAs) was performed. Sequencing was carried out using the NextSeq 500 (Illumina). Detection of peaks was done using HOMER (v2.6); differential expressed genes were identified using moderated t-test (limma R-package) and functionally annotated with g:Profiler. ChIP-Seq and RNA-Seq data from GCB DLBCL cell lines after FLI1 downregulation were integrated to identify putative direct targets of FLI1.ResultsAnalysis of clinical DLBCL specimens showed that FLI1 gene was more frequently expressed at higher levels in GCB than in ABC DLBCL and its protein levels were higher in GCB than in ABC DLBCL cell lines. Genes negatively regulated by FLI1 included tumor suppressor genes involved in negative regulation of cell cycle and hypoxia. Among positively regulated targets of FLI1, we found genes annotated for immune response, MYC targets, NF-κB and BCR signaling and NOTCH pathway genes. Of note, direct targets of FLI1 overlapped with genes regulated by ETS1, the other transcription factor gained at the 11q24.3 locus in DLBCL, suggesting a functional convergence within the ETS family. Positive targets of FLI1 included the NF-κB-associated ASB2, a putative essential gene for DLBCL cell survival. ASB2 gene downregulation was toxic in GCB DLBCL cell lines and induced NF-κB inhibition via downregulation of RelB and increased IκBα. Additionally, downregulation of FLI1, but not ASB2, caused reduction of NF-κB1 and RelA protein levels.ConclusionsWe conclude that FLI1 directly regulates a network of biologically crucial genes and processes in GCB DLBCL. FLI1 regulates both the classical NF-κB pathway at the transcriptional level, and the alternative NF-κB pathway, via ASB2. FLI1 and ASB2 inhibition represents a potential novel therapeutic approach for GCB DLBCL.

Translational Activation of ATF4 through Mitochondrial Anaplerotic Metabolic Pathways Is Required for DLBCL Growth and Survival.

Diffuse large B-cell lymphomas (DLBCL) are broadly dependent on anaplerotic metabolism regulated by mitochondrial SIRT3. Herein we find that translational upregulation of ATF4 is coupled with anaplerotic metabolism in DLBCLs due to nutrient deprivation caused by SIRT3 driving rapid flux of glutamine into the tricarboxylic acid (TCA) cycle. SIRT3 depletion led to ATF4 downregulation and cell death, which was rescued by ectopic ATF4 expression. Mechanistically, ATF4 translation is inhibited in SIRT3-deficient cells due to the increased pools of amino acids derived from compensatory autophagy and decreased glutamine consumption by the TCA cycle. Absence of ATF4 further aggravates this state through downregulation of its target genes, including genes for amino acid biosynthesis and import. Collectively, we identify a SIRT3-ATF4 axis required to maintain survival of DLBCL cells by enabling them to optimize amino acid uptake and utilization. Targeting ATF4 translation can potentiate the cytotoxic effect of SIRT3 inhibitor to DLBCL cells. SIGNIFICANCE: We discovered the link between SIRT3 and ATF4 in DLBCL cells, which connected lymphoma amino acid metabolism with ATF4 translation via metabolic stress signals. SIRT3-ATF4 axis is required in DLBCL cells regardless of subtype, which indicates a common metabolic vulnerability in DLBCLs and can serve as a therapeutic target.This article is highlighted in the In This Issue feature, p. 1.

SAMHD1 Performs Tumor-Promoting Effects in Diffuse Large B-Cell Lymphoma By Inhibiting DNA Damage and Sting-Mediated Cell Pyroptosis

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1), a mammalian triphosphohydrolase, plays a critical role in regulating DNA replication and damage repair. Dysregulation of SAMHD1 facilitates DNA damage-mediated cell proliferation, anti-tumor immune response and chemoresistance of cancer cells. Stimulator of interferon genes (STING) is a critical regulator of the innate immune response through the perception of DNA damage. Here, we investigated the functional significance of SAMHD1 and its regulatory effect on STING signaling.We first elucidated the expression level of SAMHD1 in DLBCL. Upregulation of SAMHD1 mRNA were identified in DLBCL cells (Figure 1A). High protein levels of SAMHD1 was validated in a cohort of newly diagnosed DLBCL patients (n=80). To further identify the biological functions of SAMHD1, SAMHD1-knockdown model was constructed in vitro. Loss-of SAMHD1 resulted not only in impaired proliferation but also in increased cell apoptosis and G0/G1 blockage (Figure 1B). To explore the function of SAMHD1 in vivo, xenograft DLBCL mice model was established. Mice bearing tumors with silenced SAMHD1 revealed delayed tumor growth and diminished tumor activity (Figure 1C).To further explore the functional mechanism of SAMHD1 in DLBCL, we performed RNA-sequencing (RNA-seq) in LY1 cells with and without SAMHD1-knockdown. Gene set enrichment analysis (GSEA) revealed significant enrichment of the cytosolic DNA-sensing pathway, which included an elevation of STING (Figure 2A). Reactome analysis identified the enrichment of STING-mediated introduction of host immune response, suggesting the potential regulation of SAMHD1 on STING (Figure 2B). Nervetheless, co-immunoprecipitation analysis couldn't found the direct interactions between SAMHD1 and STING (Figure 2C). As a DNA damage sensor, STING activation responses to the presence of double stranded DNA (dsDNA). Our results then showed that SAMHD1-knockdown significantly induced nuclear DNA damage and cytosolic dsDNA accumulation (Figure 2D and E), thereby triggering STING activation in DLBCL cells (Figure 2F).Next, we investigated the biological roles of STING in DLBCL. Genetic activation of STING impaired cell viability and enhanced cell death (Figure 3A-3B). To identify the form of cell death, morphological observation was performed and revealed that cell pyroptosis, represented by cell swelling with large bubbles, could be induced by elevated STING (Figure 3C). High levels of LDH were detected in STING overexpressed DLBCL cells, further verifying the emergence of cell pyroptosis (Figure 3D). To validate the involvement of STING in pyroptosis, we deleted STING by CRISPR/Cas9 genomic-editing system (Figure 3E). STING deletion not only alleviated proliferation inhibition (Figure 3F) but also restrained pyroptosis-related morphological changes and LDH release (Figure 3G and H). Expression of pyroptotic effectors were then detected to investigated the molecular mechanisms of pyroptosis. It was worth noting that the Bak/Bax-Caspase-3 mediated gasdermin E (GSDME), rather than ASC/NLRP3/Caspase-1 mediated GSDMD, was activated in STING overexpressed DLBCL cells (Figure 4A and B). Furthermore, silencing of GSDME significantly inhibited pyroptosis-induced morphological changes and LDH release (Figure 4C).The anti-tumor effects of STING agonists in DLBCL were further explored. Treatment of DMXAA, a STING agonist, significantly promoted pyroptosis in DLBCL cells (Figure 5A-5B), further resulting in suppressed proliferation in time- and dose-dependent manners in vitro (Figure 5C). Besides, DMXAA also contributes to the upregulation of immune checkpoints, especially programmed cell death protein 1 (PD-1). We further detected the synergistic effect of DMXAA and PD-1 inhibitor (BMS1166). Of note, combination of DMXAA and BMS1166 remarkably produced an enhanced anti-tumor effect on DLBCL cells in vitro (Figure 5D).In summary, our present study firstly demonstrates that SAMHD1 functions as an oncogene in DLBCL by inhibiting DNA damage induced STING activation. Intrinsic STING induces cell pyroptosis in DLBCL cells via activating the Bak/Bax-Caspase3-GSDME pathway. Moreover, investigation of drug combination highlights the synergistic effect of STING agonist and PD-1 inhibitor, providing a novel option for improving therapeutic effects of anti-PD-1 treatment in DLBCL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Differential Effects of Iberdomide Versus Revlimid on Leukocyte Trafficking, Immune Activation and DLBCL Tumor Cell Killing

Introduction: Revlimid (Rev), binds to CRL4 CRBN E3 ligase leading to recruitment and proteasomal degradation of transcription factors Aiolos and Ikaros. This inhibits proliferation of malignant B cells and stimulates activity of T, NK and macrophage cells, thereby providing clinical activity of Rev as a single agent and in combination with CD19/CD20 antibodies in DLBCL and FL. Iberdomide (Iber), a new CELMoD with enhanced substrate degradation compared to Rev, is currently being studied in clinical trials for B-NHL and MM. Presented here is extensive in vitro and in vivo characterization of immune enhancement and antitumor effects of Iber with direct comparison to Rev.Results: In a panel of DLBCL cell lines, comprising ABC and GCB-DLBCL models, Iber degraded Aiolos/Ikaros with faster kinetics and to a greater depth than Rev, which led to enhanced antiproliferative and cytotoxic effects. Iber acted in a cell of origin independent manner, whereas Rev is preferentially active in ABC-DLBCL. To examine the molecular effects of Iber and Rev in immune cells, we performed RNAseq and proteomic based analyses on Iber and Rev treated T, NK and monocyte cell populations. These experiments revealed a complex series of immunomodulatory activities including promotion of pro-inflammatory cytokine production, activation marker expressions and migratory machinery with a trend of Iber exhibiting greater enhancements. We confirmed these findings by demonstrating that secretion of chemoattractants for T cells, NK cells and monocytes including CXCL9, 10 and 11 (10-90% increase) and CCL8 (30% increase, p<0.01) were higher in PBMCs treated with Iber compared to Rev. Additionally, functional chemotaxis assays demonstrated that Iber and Rev increased the trafficking capacity of T-cells compared to DMSO alone, with Iber demonstrating a greater increase than Rev (46% vs 21%, p<0.01). Furthermore, Iber increased the proliferative capacity of CD8+ T and NK cells compared to Rev (10 and 3.6-fold vs 4 and 2.8-fold, respectively). Functional co-culture assays with DLBCL cells showed that Iber induced NK cell mediated killing of DLBCL cells to a greater extent than Rev and each molecule enhanced ADCC with Rituximab compared to vehicle controls. Translational data from clinical trials of a related CELMoD, Avadomide, revealed significant trafficking of immune cells such a T cells, NK cells and monocytes to the tumor microenvironment (TME). To examine the effects of Rev and Iber in an in vivo DLBCL GEMM model, we developed a humanized CRBN (hCRBN) mouse capable of facilitating proteasomal degradation of target substrates upon treatment with a CELMoD. The hCRBN mouse was then crossed with the Eμ-Myc DLBCL mouse model resulting in Eμ-Myc/hCRBN progeny that then developed disease. Splenocytes were collected and transplanted to recipient hCRBN mice. The tumor cells were allowed to engraft for 5 days upon which 3 daily doses of vehicle, Rev and Iber were given prior to the mice being sacrificed. Non-transplanted hCRBN mice served as controls. Similar to human disease, DLBCL cells remodeled the myofibroblast-immune network within lymph node and the splenic tissues including activated podoplanin (PDPN)-expressing fibroblastic reticular cells (FRCs) and diminished CD8+ T cells and CD11c+ DCs within the lymphoid TMEs. Treatment with Iber resulted in significantly enhanced infiltration of DCs and notably, cytolytic granzyme B positive T cells into the TME compared to Rev or vehicle treated mice (Figure 1). Additional characterization of the immune (T cell, NK and monocyte)-stroma TME is on-going and will be presented.Conclusion: Our data demonstrate that Iber is more potent in substrate degradation and functionality in anti-proliferative activity against DLBCL cell line models and at triggering immunostimulatory activities in multiple lymphoid and myeloid populations. Additionally, we generated a humanized CRBN mouse model that revealed the ability of CELMoDs in inducing immune-rich TMEs supporting rational combination strategies with immune focused agents being explored in lymphoma such as SIRPα blockade, CAR T and CD3xCD20 bispecifics. [Display omitted] DisclosuresNakayama: Bristol Myers Squibb: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Chiu: Bristol Myers Squibb: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Narla: Bristol Myers Squibb: Current Employment. Shakya: Bristol Myers Squibb: Current Employment. Gamez: Bristol Myers Squibb: Current Employment. Hagner: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Gandhi: Bristol Myers Squibb: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company.

Pirtobrutinib (LOXO-305) Is Active and Overcomes ERK Related Pro-Survival Signaling in Ibrutinib Resistant, BTK Cys481 Mutant Expressing WM and ABC DLBCL Lymphoma Cells Driven By Activating MYD88 Mutations

MYD88 mutations are common in B-cell malignancies including Waldenstrom Macroglobulinemia (WM) and ABC subtype of Diffuse Large B-cell Lymphoma (ABC DLBCL). Mutated MYD88 activates BTK, and triggers downstream pro-survival signaling that includes NF-kB and ERK (Yang et al, Blood 2013; Blood 2016). ERK related signaling triggers inflammatory cytokine production including IL-6 and IL-10 (Chen et al, Blood 2016). Ibrutinib covalently binds to BTK Cys481 and inactivates BTK and downstream NF-kB and ERK signaling. Ibrutinib is approved for the treatment of WM and is associated with high overall response rates (>90%) and long term progression free survival in WM though intolerance to therapy, as well as resistance related to acquired BTK Cys481 mutations frequently leads to treatment discontinuation. We therefore investigated a novel, non-covalent BTK-inhibitor, pirtobrutinib that binds to BTK at non-Cys481 amino acids (G473-K483). Pirtobrutinib showed highly selective anti-proliferative activity against MYD88 mutated WM (BCWM.1, MWCL-1) and ABC DLBCL (TMD-8 and HBL-1) versus MYD88 wild-type (OCI-Ly7, OCI-Ly19, Ramos, and RPMI-8226) cells, with marked apoptotic effect exhibited against primary MYD88 mutated WM cells at pharmacologically achievable levels (100-500 nM). Importantly, pirtobrutinib blocked BTK activity and overcame ibrutinib resistance in BCWM.1 WM and TMD-8 ABC DLBCL cells transduced to express both wild-type and mutated BTK (BTK Cys481Ser) with similar efficacy. The downstream signaling consequences of pirtobrutinib in vector only, wild-type and mutant BTK Cys481 expressing BCWM.1, MWCL-1, TMD-8 and HBL-1 cells was also examined. Treatment of vector only and wild-type BTK Cys481 expressing WM and ABC DLBCL cells with ibrutinib or pirtobrutinib abrogated both p-BTK and p-ERK signaling. In contrast, only pirtobrutinib blocked p-BTK and p-ERK signaling in mutant BTK Cys481 expressing WM and ABC DLBCL cells. In previous studies, we showed that inflammatory cytokine production that included IL-6 and IL-10 driven by ERK triggered ibrutinib resistance in wild-type BTK Cys481 MYD88 mutated lymphoma cells co-cultured with their mutated BTK expressing counterparts (Chen et al, BLOOD 2018). ERK-driven cytokine resistance to ibrutinib was postulated to explain how disease progression occurs in patients with modest variant expression of mutated BTK Cys481 (Woyach et al, JCO 2017; Xu et al, Blood 2017). Co-culture of BTK Cys481 mutated expressing TMD-8 cells with wild-type BTK expressing TMD-8 cells triggered resistance of the latter to ibrutinib. Treatment with pirtobrutinib blocked IL-6 and IL-10 production and overcame the protective effects conferred by BTK Cys481 mutated TMD-8 cells in these experiments. Lastly, oral administration of pirtobrutinib blocked p-BTK and p-ERK in BTK Cys481 mutated TMD-8 tumors xenografted in mice. Our findings therefore show that pirtobrutinib inhibits growth of MYD88 mutated lymphoma cells in a highly selective manner and can trigger apoptosis of primary WM patient BM lymphoplasmacytic cells at levels comparable to ibrutinib. Moreover, pirtobrutinib effectively blocked mutated BTK Cys481 driven BTK and ERK1/2 activation and produced similar cellular efficacy in both BTK wild-type and BTK Cys481 mutated cells. Pirtobrutinib also blocked the protective effect conferred to BTK wild-type cells through paracrine cytokines released by BTK Cys481Ser expressing cells. Lastly, pirtobrutinib blocked BTK and ERK1/2 activation in TMD8-BTK Cys481Ser xenografted mice. The findings support the development of pirtobrutinib in MYD88 driven lymphomas, including those resistant to ibrutinib on the bases of BTK Cys481 mutations. DisclosuresBranagan: Adaptive Biotechnologies: Consultancy; BeiGene: Consultancy; CSL Behring: Consultancy; Karyopharm: Consultancy; Pharmacyclics: Consultancy; Sanofi Genzyme: Consultancy. Castillo: Abbvie: Consultancy, Research Funding; BeiGene: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; Janssen: Consultancy; Roche: Consultancy; TG Therapeutics: Research Funding. Yang: Blueprint Medicines Corporations: Current Employment, Current holder of individual stocks in a privately-held company. Treon: BeiGene: Consultancy, Research Funding; Eli Lily: Research Funding; Abbvie/Pharmacyclics: Consultancy, Research Funding.

CC-99282 is a Novel Cereblon (CRBN) E3 Ligase Modulator (CELMoD) Agent with Enhanced Tumoricidal Activity in Preclinical Models of Lymphoma

CC-99282 is a novel, oral CELMoD ® agent currently under investigation in phase 1 clinical studies in patients with relapsed or refractory (R/R) non-Hodgkin lymphomas (NHL) and chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL). Mechanistically, CC-99282 interacts with the CRL4 CRBN E3 ubiquitin ligase substrate receptor CRBN to induce recruitment and ubiquitin-mediated proteasomal degradation of transcription factors Ikaros and Aiolos. The design intent for CC-99282 included efficient absorption, deep tissue distribution, and prolonged exposure to optimize activity in bulky lymphoma lesions. Recently, we reported that CC-99282 shows potent antitumor activity in different preclinical models of diffuse large B cell lymphoma (DLBCL; Lopez-Girona, et al. Hematol Oncol. 2021). Here, we provide an expanded analysis of CC-99282 activity as a monotherapy, as well as examine its synergistic activity with anti-CD20 antibody treatment, in preclinical models of NHL including DLBCL and follicular lymphoma (FL).Compared with existing agents targeting Ikaros/Aiolos that show activity in hematologic malignancies, such as lenalidomide, avadomide, and iberdomide (CC-220), CC-99282 induced a more rapid, deep, and sustained degradation of Ikaros/Aiolos, causing derepression of cyclin-dependent kinase (CDK) inhibitors and interferon-stimulated genes (IRF7, IFIT3, and DDX58), and the reduction of the highly critical oncogenic factors c-Myc and IRF4. These molecular changes were followed by potent, 10- to 100-fold enhanced, autonomous cell killing and induction of apoptosis (Figure). Our results show that these effects were independent of the cell of origin (activated B cell [ABC; TMD8 cell line], germinal center B cell [GCB; WSU-DLCL2 cell line], or primary mediastinal B cell lymphoma [PMBL] subtypes of DLBCL) or presence of high-risk chromosomal translocations (MYC, BCL2, and/or BCL6), as observed in a panel of 36 lymphoma cell lines that included DLBCL and FL cell lines. In vivo, CC-99282 demonstrated robust tissue distribution that favored target tissues and exhibited antitumor activity resulting in improved tumor regression and tumor-free animals in several lymphoma xenograft models, including an intracranial xenograft model. This strong antitumor activity was observed using various continuous and intermittent dosing paradigms.The potent, direct autonomous cell-killing activity of CC-99282 was augmented when CC-99282 was combined with the anti-CD20 antibody rituximab. In vitro combination studies of CC-99282 with rituximab in lymphoma cell lines demonstrated enhanced cell killing by human natural killer (NK) cells, macrophage-mediated phagocytosis, antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP). In FL and DLBCL cell lines, we showed that the combination of CC-99282 with rituximab resulted in increases in both NK-mediated ADCC and macrophage-mediated ADCP of up to 20% compared with rituximab treatment alone. In vivo, combination treatment with CC-99282 and rituximab induced dose-dependent tumor growth inhibition in WSU-DLCL2 and RL (FL) xenograft models. In the WSU-DLCL2 model, CC-99282 (1 mg/kg) or rituximab (10 mg/kg) monotherapy resulted in modest tumor growth inhibition, whereas the combination of CC-99282 (1 mg/kg) and rituximab (10 mg/kg) resulted in tumor regression in 100% of animals. Similar results were obtained in FL xenograft models using the RL cell line, where combinations of CC-99282 (1 mg/kg) with rituximab (25 mg/kg) induced complete tumor regression in 100% of animals.In conclusion, CC-99282 is a novel CELMoD agent with an improved substrate degradation profile compared with existing Ikaros/Aiolos-degrading agents. CC-99282 demonstrated enhanced antiproliferative and apoptotic activities across a broad range of lymphoma cells and a robust distribution profile that favors target tissues such as lymphoid organs. In addition, CC-99282 acts synergistically in combination with anti-CD20 monoclonal antibody treatment. Collectively, these data support the clinical investigation of CC-99282 as monotherapy and in combination with rituximab in patients with R/R NHL. [Display omitted] DisclosuresCarrancio: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Groocock: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Janardhanan: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Jankeel: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Galasso: Ryan Galasso: Current Employment, Current equity holder in publicly-traded company. Guarinos: Bristol Myers Squibb: Current Employment. Narla: Bristol Myers Squibb: Current Employment. Groza: Bristol Myers Squibb: Current Employment. Leisten: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Pierce: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Rolfe: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Lopez-Girona: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company.

EZH1/2 Dual Inhibitor Valemetostat Tosylate (DS-3201b) Acts Differently from EZH2 Selective Inhibitor on Epigenetic Landscape to Exert Greater Anti-Tumor Effect Against Diffuse Large B-Cell Lymphoma

Enhancer of zeste homologous (EZH) 2 and its close homolog EZH1 are catalytic subunits of polycomb repressive complex (PRC) 2 protein complex, and play redundant and crucial role for the maintenance of transcriptional repression by tri-methylating histone H3 lysine 27 (H3K27). Hyper trimethylation of H3K27 has been associated with malignant lymphoma and myeloma progression, thus several small molecules suppressing PRC2 complex activity has been developed for hematological malignancy therapy. We have developed valemetostat tosylate (DS-3201b, also known as valematostat), a potent dual inhibitor of EZH1/2, and demonstrated its superior anti-proliferative effect against DLBCL cells to tazemetostat (EPZ-6438, E7438) a selective EZH2 inhibitor currently in clinic. In addition, valemetostat synergized with wide variety of 1st and 2nd line drugs used in DLBCL therapy both in vitro and in vivo proposing its potential combination opportunities. However, it is still elusive how valemetostat modulates epigenetic landscape and represses malignant B-cell proliferation more potently than selective EZH2 inhibitors. Therefore, impact on epigenetic landscape between valemetostat and tazemetostat was analyzed by RNA/ChIP-sequencing. Though these two inhibitors significantly reduced cellular global H3K27me3 level, we observed ectopic EZH1/2 accumulation in several tumor suppressor gene loci after tazemetostat treatment resulting in partial reduction in H3K27me3 and de-repression of silenced gene expression. Meanwhile valemetostat treatment evidently triggered gene expression by depleting H3K27me3 and enhancing H3K27Ac mark without inducing ectopic enrichment of EZH1/2, suggesting that valemetostat has a distinct effect on genome wide distribution of EZH1/2 from tazemetostat.In conclusion, these results suggest that valemetostat has a capacity of averting ectopic relocation of EZH1/2 on tumor suppressor genes mainly induced by EZH2 specific inhibition and thereby exerts greater anti-B cell tumor effect than EZH2 preferential inhibitor. A phase 2 clinical study of valemetostat is now ongoing for patients with Relapse/Refractory B-cell Lymphoma [ClinicalTrials.gov Identifier: NCT04842877] DisclosuresBanjo: Daiichi Sankyo Co., Ltd.: Current Employment. Hama: Daiichi Sankyo Co., Ltd.: Current Employment. Nosaka: Daiichi Sankyo Co., Ltd.: Current Employment. Takata: Daiichi Sankyo Co., Ltd.: Current Employment. Honma: Daiichi Sankyo Co., Ltd.: Current Employment. Kitagawa: Daiichi Sankyo Co., Ltd.: Current Employment. Yamamoto: Daiichi Sankyo RD Novare Co., Ltd.: Current Employment. Wada: Daiichi Sankyo RD Novare Co., Ltd.: Current Employment. Yoshida: Daiichi Sankyo RD Novare Co., Ltd.: Current Employment. Lim: Daiichi Sankyo RD Novare Co., Ltd.: Current Employment. Okamoto: Daiichi Sankyo RD Novare Co., Ltd.: Current Employment. Sato: Daiichi Sankyo RD Novare Co., Ltd.: Current Employment. Katayama: Daiichi Sankyo RD Novare Co., Ltd.: Current Employment. Sato: Daiichi Sankyo RD Novare Co., Ltd.: Current Employment. Goto: Daiichi Sankyo RD Novare Co., Ltd.: Current Employment. Abe: Daiichi Sankyo Co., Ltd.: Current Employment.

A New Role for the SRC Family Member HCK As a Driver of BCR/SYK Signaling in MYD88 Mutated Lymphomas

A New Role for the SRC Family Member HCK As a Driver of BCR/SYK Signaling in MYD88 Mutated Lymphomas

Preservation of T-Cell Stemness with a Novel Expansionless CAR-T Manufacturing Process, Which Reduces Manufacturing Time to Less Than Two Days, Drives Enhanced CAR-T Cell Efficacy

Preservation of T-Cell Stemness with a Novel Expansionless CAR-T Manufacturing Process, Which Reduces Manufacturing Time to Less Than Two Days, Drives Enhanced CAR-T Cell Efficacy

VIP152, a Selective CDK9 Inhibitor, Induces Complete Regression in a High-Grade B-Cell Lymphoma (HGBL) Model and Depletion of Short-Lived Oncogenic Driver Transcripts, MYC and MCL1, with a Once Weekly Schedule

VIP152, a Selective CDK9 Inhibitor, Induces Complete Regression in a High-Grade B-Cell Lymphoma (HGBL) Model and Depletion of Short-Lived Oncogenic Driver Transcripts, MYC and MCL1, with a Once Weekly Schedule

TP53 Mutations and RNA Binding Protein Musashi 2 Drive Resistance to PRMT5-Targeted Therapy in B-Cell Lymphoma

PRMT5 catalyzes the arginine symmetric demethylation of histone and non-histone proteins. PRMT5 is overexpressed in lymphoma and plays a key role in lymphomagenesis through the repression of several tumor suppressors, including P53, while promoting the expression of lymphoma-drivers, such as c-MYC.Currently, three PRMT5 inhibitors are being clinically evaluated in B-cell lymphoma patients, however, the mechanism of action of these molecules remain poorly understood. Thus, to identify the mechanism of action of the potent selective PRMT5 inhibitor, GSK3203591 (GSK-591), we performed a genome-wide CRISPR/Cas9 KO screen in the human mantle cell line, Z-138 and a validation CRISPR /Cas9 screen in the DLBCL cell line, OCI-LY19. Our genome-wide CRISPR screen identified 316 sensitizing genes and 89 genes involved in resistance. The top sensitizing gene was TP53, which validated our screens, as it is a well characterized target of PRMT5. Furthermore, we found that TP53 deletion and the hot spot TP53 R248Wmutation are biomarkers of resistance to GSK-591.RNA-binding protein MUSASHI 2 (MSI2) was identified as the top-ranked driver of resistance to GSK-591. MSI2 plays a key role in hematopoietic stem cells activation, myeloid leukemia and CLL, however, its function in lymphoma remains unknown. MSI2 is overexpressed in DLBCL patients (n=96). Relapsed MCL and DLBCL primary samples express high levels of both PRMT5 and MSI2. Furthermore, MSI2 depletion decreased cell viability and sensitized lymphoma cells to PRMT5 inhibitor in vitro and vivo. In contrast, overexpression of MSI2 reverted sensitivity to GSK-591 in lymphoma cells. Consistent with our genetic studies, inhibition of MSI2 using Ro 08-2750 (Ro) (Minuesa et al. Nat Commun. 2019), conferred sensitivity to GSK-591, and the combination therapy was synergistic inducing cytotoxicity.In order to identify specific downstream targets of MSI2 that may contribute to resistance to PRMT5 inhibition in lymphoma, we performed MSI2-HyperTRIBE, a recently discovered technology that allows mapping the MSI2 targeting network (Nguyen et al. Nat Commun. 2020). Using MSI2-HyperTRIBE, we were able to identify MSI2 targets in lymphoma cells. Moreover, we found that Ro treatment significantly blocked MSI2 binding activity, while GSK-591 had no effect on MSI2 targets.To uncover the mechanism involved in the synergy of PRMT5 and MSI2 inhibitors, we performed RNA-sequencing of Z-138 cells treated with GSK-591, Ro or the combination. Gene set enrichment analysis (GSEA) demonstrated a loss in the c-MYC pathway upon drug combination.MSI2 RNA-IP assays showed that MSI2 binds c-MYC and this interaction is disrupted by the combination of GSK-591 and Ro. We observed that the combination of MSI2 and PRMT5 inhibitors does not affect c-MYC mRNA levels but, rather, controls translation. Interestingly, in the human B cell line P493-6 that express a conditional, tetracycline-regulated c-MYC, the depletion of c-MYC significantly increased the anti-proliferative activity of GSK-591 and Ro alone or in combination.To further investigate whether there are other functionally relevant downstream targets of MSI2 promoting resistance to PRMT5 inhibition, we overlapped the differentially expressed genes upon treatment with GSK-591 and Ro (RNA-Seq), the Ro-direct targets in lymphoma (MSI2-HyperTRIBE) and the resistance genes to PRMT5 inhibition (whole-genome CRISPR screen) and we identified the anti-apoptotic protein BCL-2 as a common target of the PRMT5-MSI2 axis. We established first that the combination of GSK-591 and Ro blocked the binding of MSI2 to BCL-2 mRNA. We also found that the drug combination significantly decreased BCL-2 mRNA and protein abundance. Furthermore, Z-138 and OCI-LY19 BCL-2 KO cells are more sensitive to GSK-591 and Ro alone or in combination. Using venetoclax, we found that BCL-2 inhibition enhanced GSK-591 activity in vitro and in vivo. Thus, these data confirmed that BCL-2 is a key driver of resistance to PRMT5 inhibition.Overall, our study uncovered a novel oncogenic axis, PRMT5/MSI2/c-MYC/BCL-2 that drives resistance to PRMT5-targeted therapy in lymphoma. We demonstrated that TP53 LOF and MSI2 expression could be used as biomarkers for patient stratification. Moreover, we proposed two novel drug combination strategies, with venetoclax or a MSI2 inhibitor, to be considered in further clinical studies with PRMT5 inhibitors. DisclosuresBarbash: GlaxoSmithKline: Current Employment, Current equity holder in publicly-traded company. Batlevi: Viatris: Current holder of individual stocks in a privately-held company; Juno/Celgene: Consultancy; Pfizer: Current holder of individual stocks in a privately-held company; Bayer: Research Funding; ADC Therapeutics: Consultancy; TG Therapeutics: Consultancy; Medscape: Honoraria; BMS: Current holder of individual stocks in a privately-held company; Memorial Sloan Kettering Cancer Center: Current Employment; Kite Pharma: Consultancy; Life Sciences: Consultancy; Seattle Genetics: Consultancy; Karyopharm: Consultancy; Regeneron: Current holder of individual stocks in a privately-held company; Moderna: Current holder of individual stocks in a privately-held company; TouchIME: Honoraria; GLG Pharma: Consultancy; Dava Oncology: Honoraria; Xynomic: Research Funding; Roche/Genentech: Research Funding; Novartis: Research Funding; Epizyme: Research Funding; Janssen: Research Funding; Autolus: Research Funding. Younes: AZ: Current Employment, Other: Senior Vice President, Global Head of Haematology (Early and Late Stage) Oncology R&D at AstraZeneca.

Development of Humanized Diffuse Large B-Cell Lymphoma Mouse Models

IntroductionDLBCL is a commonly diagnosed, aggressive non-Hodgkin's lymphoma with ~40% of patients experiencing refractory or relapsed disease. Development of alternative therapies that target molecular features defining these unresponsive tumors is an active area of research to significantly advance the field and improve these patient's clinical management. However, few DLBCL animal models exist to test the efficacy of newly developed treatments, and are restricted to transgenic or xenograft mice that often fail to recapitulate the heterogeneous sub-classifications of this complex disease. While transgenic mice allow for spontaneous tumor formation, these models rely on inducing expression of specific oncogenes that drive a select group of DLBCL. The xenograft model offers several advantages, such as reproducing late-stage disease and shortening the model development time, but consist of implanting the tumor cells in a localized region or subcutaneously into immune-deficient mice. Despite some benefits of the transplant approach, these models are limited by their engraftment reproducibility and interactions with host micro-environments. Here, we explored the utility of humanizing Nod-Scid-IL2Rg null (NSG) mouse strains with factors associated with enhancing myeloid and lymphoma cell growth to establish a pipeline for rapid, reliable generation of in vivo DLBCL models.MethodsWe transduced the well-established human DLBCL cells, U2932, with the luciferase (Luc)-EGFP gene. The Luc-expressing (U2932-Luc) tumor cells were sorted for GFP positivity (GFP +) and expanded. The U2932-Luc cells (1 x 10 6/100µl PBS) were injected IV via tail vein into 8~12-week-old mice of various humanized NSG strains (representing equal numbers of each sex). NSG mice were humanized by transgenic expression of human cytokines (either human IL6 alone or IL6 plus SCF, GM-CSF, and IL-3) with the CMV promoter. Each experiment included of U2932-Luc cell transplanted group and control groups. We assessed U2932-Luc cell engraftment and growth by weekly in vivo imaging (IVIS 200 Imager, Perkin Elmer). To evaluate the organ specific engraftment/progression, we confirmed engraftment by bioluminescence imaging at the 2 nd week, then euthanized one mouse per week. At 15 minutes before euthansia, luciferin was injected via intraperitoneal injection. Following euthanasia, the organs were excised and underwent ex vivo IVIS imaging. The spleen, lungs, and liver were then fixed with 10% formalin and embedded in paraffin. Sections were stained with hematoxylin and eosin, and an anti-CD20 antibody to evaluate the tumor morphology using a Zeiss AXIO Imager M2 microscope (Zeiss, Nashville, TN). All other mice were monitored for survival and the median survival between the IL6 and IL6/SGM3 mice were compared using the Log-rank test.ResultsSimilar to previously reported DLBCL humanized strain (MISTRG) (Hashwah, 2019), we used the IL6/SGM3 expressing strain. However, our studies also included the IL6 only humanized strain. We found that both the IL6 and IL6/SGM3 strains were highly permissive to DLBCL growth. The IL-6 strain exhibited a heightened growth of U2932 cells relative to the IL-6/SGM3 mice. As shown in Figure 1, the IL6 mice survived longer than IL6/SGM3 mice. Significant difference between the median survival of IL6 and IL6/SGM3 mice i.e. 48 days vs 42 days was observed (p < 0.0482). The organ specific evaluation demonstrated that U2932-Luc cells were initially engrafted and grew in the lung, liver, and spleen. Subsequently, U2932 cells were found in the skeleton, ovary, and brain. Of note, we detected significantly enlargements of the kidney, spleen, and ovary at the terminal stage.ConclusionsOur humanized mouse model approach of using U2932 human DLBCL cells transduced with the Luc gene in the NSG-IL6 and NSG-IL6/SGM3 mice reproduced the clinical features of an aggressive DLBCL that paralleled the original patient. This model will provide a new tool to enable expansion of patient samples while overcoming the current limitations of DLBCL xenografts and transgenic mice. The ability to maintain growth of patient-derived samples within clinically relevant locations has great potential to more accurately test patient-specific, personalized treatment strategies. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

BTG1 Mutation Promotes Aggressive Lymphoma Development By Lowering the Threshold to MYC Activation and Generating “Super-Competitor” B Cells

BTG1 Mutation Promotes Aggressive Lymphoma Development By Lowering the Threshold to MYC Activation and Generating “Super-Competitor” B Cells

Targeting Asparagine and Serine Metabolism in Germinal Centre-Derived B Cells Non-Hodgkin Lymphomas (B-NHL).

BL and DLBCL are subtypes of B-cell lymphomas that arise from germinal centre B lymphocytes. Differentiation between BL and DLBCL is critical and can be challenging, as these two types of cancer share the same morphological, immunophenotypic, and genetic characteristics. In this study, we have examined metabolism in BL and DLBCL lymphomas and found distinctive differences in serine metabolism. We show that BL cells consume significantly more extracellular asparagine than DLBCL cells. Using a tracer-based approach, we find that asparagine regulates the serine uptake and serine synthesis in BL and DLBCL cells. Calculation of Differentially Expressed Genes (DEGs) from RNAseq datasets of BL and DLBCL patients show that BL cancers express the genes involved in serine synthesis at a higher level than DLBCL. Remarkably, combined use of an inhibitor of serine biosynthesis pathway and an anticancer drug asparaginase increases the sensitivity of BL cells to extracellular asparagine deprivation without inducing a change in the sensitivity of DLBCL cells to asparaginase. In summary, our study unravels metabolic differences between BL and DLBCL with diagnostic potential which may also open new avenues for treatment.