Diffuse Large B-cell Lymphoma Proliferation Research Articles

Cuproptosis-related gene ATOX1 promotes MAPK signaling and diffuse large B-cell lymphoma proliferation via modulating copper transport.

Diffuse Large B-cell lymphoma (DLBCL) is a common subtype of non-Hodgkin lymphoma, highlighting the importance of studying susceptibility genes to develop personalized treatment strategies. While cuproptosis, caused by high levels of copper ions induced by ionophores, has been shown to affect cancer survival, its specific role in lymphoma is not yet clear. To investigate the involvement of upregulation-related genes in DLBCL, we employed bioinformatics techniques. Specifically, we analyzed the differentially expressed genes (DEGs) in the GSE25638 dataset using Weighted Gene Co-expression Network Analysis (WGCNA) and performed functional enrichment analysis. By building a Protein-Protein Interaction (PPI) network, candidate genes were identified. Gene Set Enrichment Analysis (GSEA) and Receiver Operating Characteristic (ROC) curve analysis were used to confirm the clinical diagnostic use of these genes. The effects of Antioxidant 1 (ATOX1) knockdown, CuCl2, and DCAC50 treatments on DLBCL cells and the activation of the Mitogen-Activated Protein Kinase (MAPK) pathway were investigated by conducting in vitro experiments. Bioinformatics and in vitro experiments confirmed elevated expression of ATOX1 in DLBCL cells and tumor samples. ATOX1 knockdown led to decreased cell proliferation and G2 cell cycle arrest in vitro. Additionally, Phosphorylated Extracellular Signal-Regulated Kinases 1 and 2 (P-ERK1/2) protein levels within the MAPK pathway were reduced as a result of ATOX1 knockdown, but these levels were recovered by CuCl2. Treatment with DCAC50 showed a dose-dependent antiproliferative effect in DLBCL cells, which was strengthened by ATOX1 knockdown. Our study demonstrated that ATOX1 may be important in DLBCL via controlling the MAPK pathway through copper transport, providing new insights into potential therapeutic strategies for DLBCL.

Abstract PO-034: Targeting metabolic plasticity and flexibility via SIRT3 inhibition to restrain nucleotides availability in DLBCLs

Abstract Cancer cells need metabolic adaptations to survive in hostile environment such as low nutrient and drug treatment conditions. DLBCLs (Diffuse large B cell lymphoma) originate from a metabolic competitive/challenging environment of germinal center (GC) when B cells undergo positive selections against antigens. However, it is largely unknown whether and how metabolic flexibility (the ability to use different nutrients) and plasticity (the ability to process the same nutrient differently) contribute to DLBCLs. Our previous study identified that SIRT3, the only mitochondrial protein deacetylase, is critical to survival and proliferation of DLBCLs by promoting glutaminolysis. Our mitochondrial localized SIRT3 inhibitor, YC8-02, has pan-toxicity to all DLBCL cells tested regardless of different subtypes with somatic mutations. Here, we observed glucose as an alternative nutrient source to compensate in the TCA cycle in SIRT3 deficient cells. DLBCLs exhibited resistance to SIRT3 inhibition in high glucose condition, while SIRT3 inhibited DLBCLs highly depend on glucose metabolism. The combination of YC8-02 with glucose metabolism inhibitors induces synergistic killing to DLBCLs. As glucose is the main carbon source of cells, it is unknown how DLBCLs manage the flexibility of using glucose for different purposes. Using in vivo metabolic imaging (MALDI), we observed that, in contrast to normal GC B cells, lymphomas specifically use the glucose for de novo nucleotide synthesis. SIRT3 inhibition decreased nucleotide levels in DLBCL cells in vitro and in vivo. The metabolic flux tracing showed that DLBCL cells requires SIRT3 activity to activate a glucose/serine/one-carbon metabolism pathway for de novo nucleotide biosynthesis, and Inhibition of SIRT3 decreased the glucose and serine being used for nucleotides in DLBCLs. Serine as another potent source for nucleotides can rescue DLBCLs from SIRT3 inhibition. Furthermore, we validated that overexpression of enzymes, like MTHFD2, in one carbon metabolism pathway can rescue the proliferation in SIRT3 deficient cells. High nucleotides levels in medium also help DLBCLs survival in YC8-02 treatments. To overcome the metabolic flexibility on environmental nucleotides, we used inhibitors of nucleoside transporters (ENT1/2), dipyridamole and draflazine, which can synergize with YC8-02 to kill DLBCLs in vitro and eradicate systemic lymphomas in vivo. In summary, our results illustrate that DLBCLs with active SIRT3 exhibit metabolic plasticity by repurposing glucose to nucleotide biosynthesis, while lymphoma cells can also reply on metabolic flexibility to manage nutrient usage and gain extracellular nucleotides for survival under SIRT3 inhibition. Our combination treatment SIRT3 and ENT inhibitors can be a promising therapy for DLBCLs by restraining their metabolic plasticity and flexibility. Citation Format: Meng Li, Noel R Park, Chris Chin, Coraline Mlynarczyk, Hening Lin, Shawn Davidson, Ari M Melnick. Targeting metabolic plasticity and flexibility via SIRT3 inhibition to restrain nucleotides availability in DLBCLs [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 PO-034.

Arsenic disulfide promoted the demethylation of PTPL1 in diffuse large B cell lymphoma cells.

Promoter hypermethylation of the tumor suppressor gene is one of the well-studied causes of cancer development. The drugs that reverse the process by driving demethylation could be a candidate for anticancer therapy. This study was designed to investigate the effects of arsenic disulfide on PTPL1 methylation in diffuse large B cell lymphoma (DLBCL). We knocked down the expression of PTPL1 in two DLBCL cell lines (i.e., DB and SU-DHL-4 cells) using siRNA. Then the DLBCL proliferation was determined in the presence of PTPL1 knockdown. The methylation of PTPL1 in DLBCL cells was analyzed by methylation specific PCR (MSPCR). The effect of arsenic disulfide on the PTPL1 methylation was determined in DLBCL cell lines in the presence of different concentrations of arsenic disulfide (5 µM, 10 µM and 20 µM), respectively. To investigate the potential mechanism on the arsenic disulfide-mediated methylation, the mRNA expression of DNMT1, DNMT3B and MBD2 was determined. PTPL1 functioned as a tumor suppressor gene in DLBCL cells, which was featured by the fact that PTPL1 knockdown promoted the proliferation of DLBCL cells. PTPL1 was found hypermethylated in DLBCL cells. Arsenic disulfide promoted the PTPL1 demethylation in a dose-dependent manner, which was related to the inhibition of DNMTs and the increase of MBD2. Experimental evidence shows that PTPL1 functions as a tumor suppressor gene in DLBCL progression. PTPL1 hyper-methylation could be reversed by arsenic disulfide in a dose-dependent manner.

Abstract 5966: PRT2527, a novel highly selective cyclin-dependent kinase 9 (CDK9) inhibitor, has potent antitumor activity in combination with BTK and BCL2 inhibition in various lymphoid malignancies

Abstract CDK9 is a master regulator of transcription that modulates transcription elongation via phosphorylation of RNA polymerase II. Short-term inhibition of CDK9 depletes short-lived transcripts and labile proteins such as MCL1, BFL1 and MYC to promote cancer cell death. We previously described PRT2527, a novel, potent, highly selective CDK9 inhibitor with anti-leukemic activity in various preclinical models. PRT2527 is currently under evaluation in a Phase I clinical trial in patients with relapsed/refractory hematologic malignancies (NCT05159518). Here we demonstrate PRT2527 potently inhibits cancer cell growth and induces cell death in various models of Diffuse Large B Cell Lymphoma (DLBCL), Mantle Cell Lymphoma (MCL) and Chronic Lymphocytic Leukemia (CLL), through depletion of MCL1, BFL1 and MYC. Specifically, we show that brief treatment with PRT2527 (6h) potently induces apoptosis and inhibits proliferation in DLBCL, MCL and CLL cell lines. We also show that the addition of BTK inhibitors (BTKi) or BCL2 inhibitors (BCL2i) potentiates the apoptosis response induced by PRT2527 in vitro and leads to more robust and durable responses including complete tumor regressions in DLBCL CDX and PDX models in vivo. In all studies, the combination of PRT2527 with BTKi or BCL2i is well tolerated. Mechanistically, we show that BTK inhibition upregulates BMF, an endogenous inhibitor of BCL2 and BCL-xL, driving cells towards MCL1 and BFL1 dependency in TMD-8 cells. Concurrent depletion of MCL1 and BFL1 with PRT2527 leads to complete inhibition of Bcl-2- family mediated survival signaling, potently inducing cell death. In similar fashion, Venetoclax-driven BCL2 inhibition also potentiates the anti-tumor activity of PRT2527. Consistent with these findings in vitro, we observe reduced BFL1 and MCL1 as well as increased BMF in tumors of mice treated with BTKi and PRT2527 suggesting complete Bcl-2 family inhibition. We also demonstrate potent induction of apoptosis in peripheral blood mononuclear cells isolated from patients with both treatment-naïve and relapse/refractory CLL following brief treatment with PRT2527 (6h). Co-treatment with BTKi or BCL2i further enhances the apoptotic effect of PRT2527, similarly suggesting combinatorial benefit. Additionally, we characterize the activity of PRT2527 in models of BTKi relapsed/refractory MCL where increased CDK9 dependency has previously been described. In an Ibrutinib-resistant Mino CDX model, we observe increased tumor growth inhibition following treatment with PRT2527 compared to tumors derived from parental Mino cells, suggesting PRT2527 is efficacious in BTKi-resistant MCL. Altogether these data provide a rationale for evaluating PRT2527 in combination with BTK and BCL2 inhibitors for the treatment of patients with relapsed/refractory lymphoid malignancies. Citation Format: Norman Fultang, Ashley M. Schwab, Sophia McAneny-Droz, Diane Heiser, Peggy Scherle, Neha Bhagwat. PRT2527, a novel highly selective cyclin-dependent kinase 9 (CDK9) inhibitor, has potent antitumor activity in combination with BTK and BCL2 inhibition in various lymphoid malignancies [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 5966.

Tumor-derived Exosomal ENO2 Modulates Polarization of Tumor-associated Macrophages through Reprogramming Glycolysis to Promote Progression of Diffuse Large B-cell Lymphoma.

Macrophages can be polarized into functional classically activated (M1) or alternatively activated (M2) phenotype. Tumor-associated macrophages (TAMs) mainly exhibit M2 phenotype. Previous works determined that up-regulation of enolase 2 (ENO2) in diffuse large B-cell lymphoma (DLBCL) cells can promote macrophages to an M2-like phenotype, thereby consequently promoting the progression of DLBCL. Exosomes are a subset of extracellular vesicles, carrying various bioactive molecules, mediate signals transduction and regulate immune cells. In our study, we investigated the role and related mechanisms of DLBCL-derived exosomal ENO2 in regulating macrophage polarization during DLBCL progression via bioinformatics analysis and a series of experiments. The results of bioinformatics analysis indicated that high expression of ENO2 was positively correlated with DLBCL progression and macrophages M2/M1 ratio. ENO2 protein levels were increased in the exosomes of the sera of DLBCL patients and DLBCL cells. Moreover, the DLBCL-derived exosomes were assimilated by macrophages and then regulated macrophage polarization. The results of in vitro and in vivo experiments showed that DLBCL-derived exosomal ENO2 modulated macrophages polarization (increased M2 phenotype and decreased M1 phenotype), thereby promoting DLBCL proliferation, migration, and invasion. We then revealed that the modulation of macrophages polarization by DLBCL-derived exosomal ENO2 depended on glycolysis and was promoted through GSK3β/β-catenin/c-Myc signaling pathway. These findings suggested that DLBCL-derived exosomal ENO2 accelerated glycolysis via GSK3β/β-catenin/c-Myc signaling pathway to ultimately promote macrophages to an M2-like phenotype, which can promote the proliferation, migration and invasion of DLBCL, suggesting that exosomal ENO2 may be a promising therapeutic target and prognostic biomarker for DLBCL.

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

Introduction: N-acetyltransferase 10 (NAT10) is the only confirmed regulator to mediate the N4-acetylcytidine (ac4C) modification of mRNA and is crucial for mRNA stability and translation efficiency. However, its role in diffuse large B-cell lymphoma (DLBCL) development and prognosis has not yet been explored. Herein, we explored the functional significance and regulatory mechanism of NAT10 mediated mRNA ac4C modification in DLBCL, which is expected to propose novel therapeutic strategy. Methods: The expression levels of NAT10 were evaluated in DLBCL cell lines and lymph node biopsies tissues, and its prognostic value in patients was further analyzed. Stable NAT10 knockout DLBCL cell lines were constructed using CRISPR/Cas9 mediated genomic deletion. The biological functions of NAT10 in DLBCL cells were analyzed in vitro by cell proliferation and cell cycle assays. Xenograft models were used to analyze the effect of NAT10 on the tumorigenesis of DLBCL cells in vivo. RNA sequencing, acetylated RNA immunoprecipitation sequencing (acRIP-seq), LC-MS/MS, RIP and RNA stability analyses were performed to explore the mechanism by which NAT10 functions in DLBCL progression. Results: NAT10 was upregulated in DLBCL cell lines and tissues. NAT10 high expression was associated with poor patient prognosis. Knockout of NAT10 impaired cellular phase progression and proliferation of DLBCL. Further analysis revealed that NAT10-mediated ac4C acetylation of SLC30A9 transcript and subsequently stabilized SLC30A9 mRNA, leading to activation of AMPK signaling and thereby facilitating DLBCL progression (Figure 1). Keywords: aggressive B-cell non-Hodgkin lymphoma, diagnostic and prognostic biomarkers, genomics, epigenomics, and other -omics No conflicts of interests pertinent to the abstract.

Abstract LB023: Discovery, development and characterization of potent and selective USP11 inhibitors

Abstract Ubiquitin-mediated events are emerging as the gatekeeper in cell proliferation, and the recognition of deubiquitinating enzymes (DUBs) as the critical regulators of these processes is rapidly growing. Several studies in the last decade confirmed that the USP11 (Ubiquitin-Specific proteases 11) had promoted tumor growth and metastasis. Indeed, pan-cancer data analysis reveals enhanced expression of USP11 in numerous malignancies, including lymphoma. Our lab has established that USP11 deubiquitinates and stabilizes the translation initiation factor eIF4B (Eukaryotic Initiation Factor 4B) to promote eIF4B-dependent oncogenic translation and to facilitate the proliferation of diffuse large B-cell lymphoma (DLBCL). Brody and colleagues screened 2000 FDA- approved compounds and reported that the anti-neoplastic agent mitoxantrone is an effective non-specific inhibitor of USP11, which was later observed to target USP15. Thus, catalytically targeting USP11 provides substantial challenges as it encompasses high homology with the functional orthologs USP4 and USP15. Addressing this major limitation, Spiliotopoulos et al. identified a unique USP11-specific allosteric site without impeding USP15 and USP4 activities. Utilizing this platform, we performed a virtual screening of more than 10 million compounds from publicly available databases and identified 307 potential unique hits. The chemical library of these hits was assessed using an in-house optimized in-cell high throughput screening assay. The primary screening revealed that 16 of the hit compounds with EC50 less than 10μM. The two most potent, structurally different, USP11 inhibitors, RBF4 and RBF11, were further evaluated. In-vitro deubiquitinase activity assay of USP11 showed minimal inhibition on treatment with compounds (as we are targeting an allosteric site), which indicates that RBF4 and 11 do not bind to the catalytic domain. Thermal shift assays revealed that RBF4 preferentially stabilizes USP11 compared to other functional paralogues USP4 and USP15, which validates the selective USP11-RBF4 interaction. Further, treatment with RBF4 and 11 hampered DLBCL proliferation in a dose-dependent manner. Significantly, treatment with selected compounds depleted USP11-dependent oncogenic expression and, thus, the colony-forming capacity of DLBCL. These results show that these novel compounds can serve as an ideal tool to increase our understanding of USP11 biology and may emerge as an example of potent and selective USP11 inhibitors. Currently, we are evaluating their anti-cancer potency in pre-clinical models. Citation Format: Forum Kayastha, Bandish Kapadia, Noah B. Herrington, Anirban Roychowdhury, Glen E. Kellogg, Ronald B. Gartenhaus. Discovery, development and characterization of potent and selective USP11 inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB023.

Antitumor Effects of Poplar Propolis on DLBCL SU-DHL-2 Cells.

Propolis is resinous natural product produced by Western honeybees using beeswax and plant and bud exudates, which has a wide range of biological activities, including antioxidation, antibacterial, anti-inflammation, immune regulation, antitumor, and so on. Diffuse large B-cell lymphoma (DLBCL) is an aggressive cancer, and accounts for about 30% of all lymphomas. The effect of poplar propolis on DLBCL has not been reported. The IC50 of propolis on the proliferation of DLBCL SU-DHL-2 cell line and its proteins and gene expressions were detected by CCK-8 kit, label-free proteomic, and RT-PCR. The results showed that the IC50 of propolis at the 5 × l05/mL cell for 24 h was 5.729 μg/mL. Label-free-based proteomics analysis showed that there were 115 differentially expressed proteins (61 up-regulated and 54 down-regulated proteins) between IC50 dose-treated and solvent control groups. There were 32.47% differential proteins located in the nucleus, 20.78% in the cytoplasm, and 14.29% in mitochondria. The most significant different pathway (p = 0.0016) of protein enrichment was ferroptosis (including glutamate-cysteine ligase regulatory subunit, ferritin, and heme oxygenase). The relative expression trend of 17 of the total 22 genes selected according to proteomics results was in line with their encoded protein. The highest protein-protein interaction was serine/threonine-protein kinase PLK, which interacted with 16 differential proteins. In conclusion, poplar propolis inhibited SU-DHL-2 cells via ferroptosis pathway, accelerating cell death and down-regulated serine/threonine-protein kinase PLK1, affecting apoptosis of cell. This result provides a theoretical basis for the treatment of DLBCL using propolis.

Serine Protease 3 Promotes Progression of Diffuse Large B-Cell Lymphoma and Serves as a Novel Prognostic Predictor.

Diffuse large B-cell lymphoma (DLBCL) ranks among the most prevalent malignancies of the lymphohematopoietic system in adults. The PRSS (Serine Protease) protein family members had been reported to be involved in carcinogenesis as well as tumor progression. Here, we aimed to explore the expression profile of PRSS3 in DLBCL and investigate its clinical significance as well as detailed functions. We retrospectively enrolled 155 DLBCL patients from our hospital and tested protein expression level of PRSS3 through immunohistochemical staining. Accordingly, PRSS3 was highly expressed in certain DLBCL tissues. Chi-square test revealed that higher PRSS3 expression was correlated with advanced Ann Arbor stage, elevated serum LDH level, and higher International Prognostic Index. Moreover, univariate and multivariate analyses confirmed that higher PRSS3 can act as an independent unfavorable prognostic predictor for DLBCL. Two human DLBCL cell lines, SUDHL10 and OCI-LY3, were subjected for knockdown assays, followed by phonotype tests including proliferation and invasion. According to the cellular experiments, PRSS3-knockdown resulted in impaired DLBCL proliferation in the two cell lines above. Taken together, PRSS3 is a novel prognostic factor for DLBCL, which functions by multiple signaling pathways.

Effects of hsa-miR-28-5p on Adriamycin Sensitivity in Diffuse Large B-Cell Lymphoma.

Background Adriamycin (doxorubicin) is an important traditional drug that exhibits cytotoxicity in Diffuse Large B-cell Lymphoma (DLBCL). Doxorubicin affects the DLBCL cells at all stages of their cell cycle. Combined with our previous results, this study discovered that the overexpression of hsa-miR-28-5p inhibited the proliferation, promoted apoptosis, and triggered cell cycle arrest at the S-phase in DLBCL cells. However, the effect of (Homo sapiens, hsa)-microRNA (miR)-28-5p on doxorubicin sensitivity in DLBCL has not been investigated. This study aims to reveal the effects of hsa-miR-28-5p on doxorubicin sensitivity at the level of DLBCL cells. Methods To determine the optimal concentration of doxorubicin, different concentrations of doxorubicin were used to treat DLBCL cells. CCK-8 assay was used to detect the proliferation of DLBCL cells. The hsa-miR-28-5p-mimic NC and hsa-miR-28-5p mimic were transfected to doxorubicin-mediated DLBCL cells. Simultaneously, blank control groups were set up. The cells were cultured and transfected for 24 h. Next, each group was administered with different concentrations of doxorubicin and cultured again for 24 h to observe the effects of hsa-miR-28-5p on doxorubicin sensitivity at different times. The proliferation, early apoptosis, and late apoptosis in DLBCL cells were determined using soft agar colony-forming assay, mitochondrial membrane potential assay, and caspase-3 activity assay, respectively. The apoptosis and cell cycle were explored using Annexin V-PE/7-AAD and PI/RNase staining buffer, respectively. We speculated that PD-L1 might be involved in the effect of hsa-miR-28-5p on the sensitivity of adriamycin (doxorubicin) in the DLBCL cells. Hence, we performed immunohistochemistry (IHC) to determine PD-L1 expression within formalin-fixed paraffin-embedded (FFPE) samples from 52 DLBCL cases. Results The optimal concentration of doxorubicin targeting DLBCL cells was found to be 3.028 μmol/l. The effect of doxorubicin on DLBCL cells was time- and concentration-dependent. hsa-miR-28-5p mimic + doxorubicin remarkably decreased proliferation of DLBCL. DLBCL cell apoptosis rate was the highest in hsa-miR-28-5p mimic + doxorubicin group. Apart from that, hsa-miR-28-5p mimic plus doxorubicin had the best effect in promoting DLBCL cell apoptosis. After the intervention of hsa-miR-28-5p mimic + doxorubicin on DLBCL cells, the cell cycle was arrested in the S-phase and DNA synthesis was blocked. hsa-miR-28-5p mimic + doxorubicin could regulate the cycle of DLBCL cells. As a result, overexpression of hsa-miR-28-5p combined with doxorubicin is possibly involved in the development of DLBCL by affecting the proliferation, apoptosis, and cycle of DLBCL cells. PD-L1 showed an association with the prognosis of DLBCL patients. Combining with the literature, this suggested hsa-miR-28-5p may influence DLBCL occurrence and therapeutic effect by regulating the PD-L1 level. Conclusion The combination of hsa-miR-28-5p mimic and doxorubicin may be considered more effective in inhibiting growth, arresting the cell cycle, and promoting cell apoptosis of DLBCL cells compared to using doxorubicin alone. The effects of doxorubicin on DLBCL cells were found to be time- and concentration-dependent. The overexpression of hsa-miR-28-5p enhanced the effect of doxorubicin on DLBCL cells, which may be attributed to the regulation of PD-L1 levels.

ALKBH5-mediated N6-methyladenosine modification of TRERNA1 promotes DLBCL proliferation via p21 downregulation

Long noncoding RNAs (lncRNAs) have crucial functions in the tumorigenesis and metastasis of cancers. N6-methyladenosine (m6A) modification of RNA is an important epigenetic regulatory mechanism in various malignancies. Nevertheless, the mechanism of m6A-modified lncRNA in diffuse large B cell lymphoma (DLBCL) has remained poorly defined. In the present study, we showed that lncRNA TRERNA1 was associated with the poor prognosis of DLBCL patients. TRERNA1 with internal m6A modification was highly correlated with the demethylase ALKBH5 expression. We further demonstrated that TRERNA1 was a potential downstream target of ALKBH5-mediated m6A modification by m6A-RNA sequencing and m6A-RIP assays. Decreased m6A methylation of TRERNA1 regulated by ALKBH5 was shown to regulate cell proliferation in vitro and in vivo. The results of mechanism analyses revealed that TRERNA1 recruited EZH2 to epigenetically silence the expression of the cyclin-dependent kinases inhibitor p21 by H3K27me3 modification of its promoter region. In addition, ALKBH5 further inhibited p21 expression. Taken together, our results elucidate the functional roles and epigenetic alterations of TRERNA1 through m6A modification in DLBCL. TRERNA1, the expression of which is upregulated by ALKBH5, acts as a scaffold that decreases p21 expression. The results of the present study provide novel targets for the diagnosis and treatment of DLBCL.

PAK4 and NAMPT as Novel Therapeutic Targets in Diffuse Large B-Cell Lymphoma, Follicular Lymphoma, and Mantle Cell Lymphoma.

Simple SummaryNon-Hodgkin’s lymphomas (NHL) are cancers of the white blood cells. While some NHL subtypes, such as Diffuse large B-cell lymphoma (DLBCL) and mantle cell lymphoma (MCL), grow and spread aggressively, others, like follicular lymphoma (FL), are indolent in nature. Irrespective of how fast they grow, all NHL subtypes can spread to other organs in the body if not treated. In this study, we have demonstrated that the targeted inhibition of p21-activated kinase 4 (PAK4) and nicotinamide phosphoribosyl transferase (NAMPT) in different NHL subtypes by a novel, orally bioavailable, dual inhibitor KPT-9274 can lead to energy depletion, inhibition of cell proliferation, and ultimately apoptosis. KPT-9274 treatment shows potent anti-tumor effects in DLBCL and MCL subcutaneous xenograft models and enhances mice survival in a systemic FL model. Therefore, this study demonstrates the potential of targeting PAK4 and NAMPT by a small molecule inhibitor KPT-9274 for NHL therapy.Diffuse large B-cell lymphoma (DLBCL), grade 3b follicular lymphoma (FL), and mantle cell lymphoma (MCL) are aggressive non-Hodgkin’s lymphomas (NHL). Cure rates are suboptimal and novel treatment strategies are needed to improve outcomes. Here, we show that p21-activated kinase 4 (PAK4) and nicotinamide phosphoribosyl transferase (NAMPT) is critical for lymphoma subsistence. Dual targeting of PAK4-NAMPT by the Phase I small molecule KPT-9274 suppressed cell proliferation in DLBCL, FL, and MCL. Growth inhibition was concurrent with apoptosis induction alongside activation of pro-apoptotic proteins and reduced pro-survival markers. We observed NAD suppression, ATP reduction, and consequent cellular metabolic collapse in lymphoma cells due to KPT-9274 treatment. KPT-9274 in combination with standard-of-care chemotherapeutics led to superior inhibition of cell proliferation. In vivo, KPT-9274 could markedly suppress the growth of WSU-DLCL2 (DLBCL), Z-138, and JeKo-1 (MCL) sub-cutaneous xenografts, and a remarkable increase in host life span was shown, with a 50% cure of a systemic WSU-FSCCL (FL) model. Residual tumor analysis confirmed a reduction in total and phosphorylated PAK4 and activation of the pro-apoptotic cascade. This study, using various preclinical experimental models, demonstrates the therapeutic potential of targeting PAK4-NAMPT in DLBCL, FL, and MCL. The orally bioavailable, safe, and efficacious PAK4-NAMPT dual inhibitor KPT-9274 warrants further clinical investigation.

The novel LSD1 inhibitor ZY0511 suppresses diffuse large B-cell lymphoma proliferation by inducing apoptosis and autophagy

Lysine-specific demethylase 1 (LSD1, also known as KDM1A) is an attractive agent for treatment of cancer. However, the anti-tumor effect of LSD1 inhibitors against diffuse large B-cell lymphoma (DLBCL) and the underlying mechanism are still unclear. Here, we report that KDM1A is overexpressed in human DLBCL tissues and negatively related to overall survival rate of DLBCL patients. ZY0511, a novel and potent LSD1 inhibitor developed by our group, inhibited the proliferation of human DLBCL cells. ZY0511 interacted with LSD1, induced methylation level of histone 3 lysine 4 and histone 3 lysine 9 in DLBCL cells. Mechanistically, transcriptome sequencing results indicated that ZY0511 induced the genes enrichment significantly related to cell cycle, autophagy, and apoptosis signaling pathways. Further study confirmed that ZY0511 blocked cell cycle at G0/G1 phase and expression of CDK4 and cyclin D1. ZY0511 decreased mitochondrial membrane potential and induced apoptosis, which can be reverted by a pan-caspase inhibitor, Z-VAD-FMK. Moreover, ZY0511 treatment significantly increased autophagy-associated marker proteins and autophagosomes formation in DLBCL cells. In vivo xenograft experiments confirmed that intraperitoneal administration of ZY0511 significantly suppressed SU-DHL-6 xenograft tumor growth in vivo. In conclusion, our findings identify that ZY0511 inhibits DLBCL growth both in vitro and in vivo via the induction of apoptosis and autophagy, and LSD1 inhibitor might be a promising strategy for treating DLBCL.

EGR1 Addiction in Diffuse Large B-cell Lymphoma.

Early growth response gene (EGR1) is a transcription factor known to be a downstream effector of B-cell receptor signaling and Janus kinase 1 (JAK1) signaling in diffuse large B-cell lymphoma (DLBCL). While EGR1 is characterized as a tumor suppressor in leukemia and multiple myeloma, the role of EGR1 in lymphoma is unknown. Here we demonstrate that EGR1 is a potential oncogene that promotes cell proliferation in DLBCL. IHC analysis revealed that EGR1 expression is elevated in DLBCL compared with normal lymphoid tissues and the level of EGR1 expression is higher in activated B cell-like subtype (ABC) than germinal center B cell-like subtype (GCB). EGR1 expression is required for the survival and proliferation of DLBCL cells. Genomic analyses demonstrated that EGR1 upregulates expression of MYC and E2F pathway genes through the CBP/p300/H3K27ac/BRD4 axis while repressing expression of the type I IFN pathway genes by interaction with the corepressor NAB2. Genetic and pharmacologic inhibition of EGR1 synergizes with the BRD4 inhibitor JQ1 or the type I IFN inducer lenalidomide in growth inhibition of ABC DLBCL both in cell cultures and xenograft mouse models. Therefore, targeting oncogenic EGR1 signaling represents a potential new targeted therapeutic strategy in DLBCL, especially for the more aggressive ABC DLBCL. IMPLICATIONS: The study characterizes EGR1 as a potential oncogene that promotes cell proliferation and defines EGR1 as a new molecular target in DLBCL, the most common non-Hodgkin lymphoma.

Clinical and Biological Subtypes of B-cell Lymphoma Revealed by Microenvironmental Signatures.

Diffuse large B-cell lymphoma (DLBCL) is a biologically and clinically heterogeneous disease. Transcriptomic and genetic characterization of DLBCL has increased the understanding of its intrinsic pathogenesis and provided potential therapeutic targets. However, the role of the microenvironment in DLBCL biology remains less understood. Here, we performed a transcriptomic analysis of the microenvironment of 4,655 DLBCLs from multiple independent cohorts and described four major lymphoma microenvironment categories that associate with distinct biological aberrations and clinical behavior. We also found evidence of genetic and epigenetic mechanisms deployed by cancer cells to evade microenvironmental constraints of lymphoma growth, supporting the rationale for implementing DNA hypomethylating agents in selected patients with DLBCL. In addition, our work uncovered new therapeutic vulnerabilities in the biochemical composition of the extracellular matrix that were exploited to decrease DLBCL proliferation in preclinical models. This novel classification provides a road map for the biological characterization and therapeutic exploitation of the DLBCL microenvironment. SIGNIFICANCE: In a translational relevant transcriptomic-based classification, we characterized the microenvironment as a critical component of the B-cell lymphoma biology and associated it with the DLBCL clinical behavior establishing a novel opportunity for targeting therapies.This article is highlighted in the In This Issue feature, p. 1307.

Abstract 5215: Inhibition of PIM kinases targets synthetic vulnerabilities and enhances antigen presentation in B cell lymphoma

Abstract The PIM kinases are highly expressed in activated B-cell (ABC) diffuse large B-cell lymphoma (DLBCL). Oncogenic cooperation between PIMs and MYC has been demonstrated. Transgenic mice co-expressing Eμ-PIM and Eμ-MYC showed accelerated lymphomagenesis. Conversely, knockdown of PIMs dramatically decreased cMYC levels and lowered tumor incidence. Based on these preclinical data, a treatment strategy aiming at disrupting the oncogenic cooperation between PIMs and MYC may improve the outcome of DLBCL. Therefore, we treated a panel of DLBCL cell lines with increasing dose of the clinically relevant pan-PIM inhibitor (PIMi) AZD1208 (from 0.1 to 10μM) for 48 hours (Hrs), which resulted in a dose-dependent growth inhibition with a stronger efficacy in ABC DLBCL cell lines. The analysis of a CRISPR loss-of-function screening in three ABC (LY3, TMD8, HBL1) and three GCB (SUDHL-4, Pfeiffer, BJAB) DLBCL cell lines (Reddy et al, 2017) showed that PIM2 silencing led to significantly decreased viability irrespective of cell-of-origin, suggesting that this oncogene is essential for cell proliferation in DLBCLs. To identify the genes through which PIMs drive the lymphoma phenotype we performed gene expression profiling using 4 ABC DLBCL cell lines (RIVA, TMD8, SUDHL-2, U2932) treated with either DMSO or AZD1208 at 1μM for 4, 8 and 12 Hrs. We observed induction of 3,439 genes whereas 2,473 genes were downregulated. Gene pathway analysis showed that AZD1208 led to downregulation of genes regulated by MYC, including its known downstream p53 and NFKB target genes. On the other hand, AZD1208 treatment broadly induced MHC class II and antigen presentation genes as well as PI3K/AKT, cell cycle and glutaminase genes. Using a high-throughput screening approach, we found that the inhibitors of cell cycle (such as the BCL2 inhibitor venetoclax/ABT199 and the PLK4 inhibitor CFI-400945) and of glutaminase (CB839) enhanced the antiproliferative effect of AZD1208, whereas combinations with the PI3K/AKT/mTOR inhibitors had negligible synergistic effect. In conclusion, our study revealed previously unknown mechanisms of action of PIM inhibitors and provides a framework for future combination strategies. Citation Format: Patrizia Mondello, Chiara Tarantelli, Luciano Cascione, Alberto J. Arribas, Andrea Rinaldi, Anas Younes, Francesco Bertoni. Inhibition of PIM kinases targets synthetic vulnerabilities and enhances antigen presentation in B cell lymphoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5215.

The Role and Underlying Mechanism of Exosomal CA1 in Chemotherapy Resistance in Diffuse Large B Cell Lymphoma

Chemotherapy resistance plays a major role in treatment failure of diffuse large B cell lymphoma (DLBCL). Exosomes are closely related to tumor drug resistance. Herein, the expression of exosomal proteins in DLBCL and their roles in chemotherapy resistance of DLBCL are explored. Tandem mass tag labeling proteomics was used to perform proteomic profiling in exosomes from DLBCL patients’ serum. The expression of carbonic anhydrase 1 (CA1) in parental, chemo-resistant DLBCL cells and DLBCL patient exosomes was detected. Proliferation of DLBCL following CA1 knockdown was investigated both in vitro and in vivo, along with the effects on nuclear factor κB (NF-κB) and signal transducer and activator of transcription 3 (STAT3) pathways. We identified 54 differentially expressed proteins. We validated that the expression level of exosomal CA1 was higher in chemo-resistant DLBCL cells than in chemo-sensitive counterparts. Knockdown of CA1 inhibited the growth of DLBCL via inhibiting the activation of NF-κB and STAT3 signaling pathways both in vitro and in vivo. An increased expression level of exosomal CA1 was associated with poorer prognosis, and exosomal CA1 could be used as a biomarker to predict chemotherapeutic efficacy. Our study suggests that exosomal CA1 can promote chemotherapy resistance in DLBCL via the NF-κB and STAT3 pathways, and it can serve as a biomarker for DLBCL prognosis.

MYC-regulated lncRNA NEAT1 promotes B cell proliferation and lymphomagenesis via the miR-34b-5p-GLI1 pathway in diffuse large B-cell lymphoma

BackgroundLncRNA NEAT1 has been identified as a tumour driver in many human cancers. However, the underlying mechanism of lncRNA NEAT1 in diffuse large B-cell lymphoma (DLBCL) progression is unclear.MethodsThe expression levels of NEAT1, GLI1 and miR-34b-5p were detected by RT-qPCR and Western blotting in DLBCL tissues and cell lines. MTT and colony formation assays were performed to examine cell proliferation, while annexin-V staining and TUNEL assays were performed to measure cell apoptosis. The effect of NEAT1, GLI1 and miR-34b-5p on cell cycle-associated proteins was evaluated by Western blotting. Dual-luciferase reporter and RNA immunoprecipitation (RIP) assays were employed to investigate the interaction between NEAT1 and miR-34b-5p or GLI1 and miR-34b-5p. Moreover, chromatin immunoprecipitation (ChIP) was performed to demonstrate the interaction between MYC and NEAT1.ResultsNEAT1 and GLI1 were upregulated while miR-34b-5p was downregulated in DLBCL tissues and cell lines compared to normal controls. Knockdown of NEAT1 or overexpression of miR-34b-5p inhibited cell proliferation but promoted cell apoptosis. Overexpression of NEAT1 reversed GLI1-knockdown induced attenuation of cell proliferation. In other words, NEAT1 acted as a competing endogenous RNA (ceRNA), regulating the miR-34b-5p-GLI1 axis, further affecting the proliferation of DLBCL. Moreover, MYC modulated NEAT1 transcription by directly binding to the NEAT1 promoter.ConclusionWe revealed that MYC-regulated NEAT1 promoted DLBCL proliferation via the miR-34b-5p-GLI1 pathway, which could provide a novel therapeutic target for DLBCL.

Inhibition of PIM Kinases Targets Synthetic Vulnerabilities and Enhances Antigen Presentation in B-Cell Lymphoma

The PIM kinases are highly expressed in activated B-cell (ABC) diffuse large B-cell lymphoma (DLBCL). Oncogenic cooperation between PIMs and MYC has been demonstrated. Transgenic mice co-expressing Em-PIM and Em-MYC showed accelerated lymphomagenesis. Conversely, knockdown of PIMs dramatically decreased cMYC levels and lowered tumor incidence. Based on these preclinical data, a treatment strategy aiming at disrupting the oncogenic cooperation between PIMs and MYC may improve the outcome of DLBCL. Therefore, we treated a panel of DLBCL cell lines with increasing dose of the clinically relevant pan-PIM inhibitor (PIMi) AZD1208 (from 0.1 to 10μM) for 48 hours (Hrs), which resulted in a dose-dependent growth inhibition with a stronger efficacy in ABC DLBCL cell lines. (Figure 1A)The analysis of a CRISPR loss-of-function screening in three ABC (LY3, TMD8, HBL1) and three GCB (SUDHL-4, Pfeiffer, BJAB) DLBCL cell lines (Reddy et al, 2017) showed that PIM2 silencing led to significantly decreased viability irrespective of cell-of-origin (Figure 1B), suggesting that this oncogene is essential for cell proliferation in DLBCLs. To identify the genes through which PIMs drive the lymphoma phenotype we performed gene expression profiling using 4 ABC DLBCL cell lines (RIVA, TMD8, SUDHL-2, U2932) treated with either DMSO or AZD1208 at 1μM for 4, 8 and 12 Hrs. We observed induction of 3,439 genes whereas 2,473 genes were downregulated. (Figure 1C) Gene pathway analysis showed that AZD1208 led to downregulation of genes regulated by MYC, including its known downstream p53 and NFKB target genes. On the other hand, AZD1208 treatment broadly induced MHC class II and antigen presentation genes as well as PI3K/AKT, cell cycle and glutaminase genes. (Figure 1D) Using a high-throughput screening approach, we found that the inhibitors of cell cycle (such as the BCL2 inhibitor venetoclax/ABT199 and the PLK4 inhibitor CFI-400945) and of glutaminase (CB839) enhanced the antiproliferative effect of AZD1208, whereas combinations with the PI3K/AKT/mTOR inhibitors had negligible synergistic effect. (Figure 1E) In conclusion, our study revealed previously unknown mechanisms of action of PIM inhibitors and provides a framework for future combination strategies. Disclosures Younes: Xynomics: Consultancy; Biopath: Consultancy; Genentech: Research Funding; AstraZeneca: Research Funding; Syndax: Research Funding; BMS: Research Funding; HCM: Consultancy; Celgene: Consultancy, Honoraria; Epizyme: Consultancy, Honoraria; Takeda: Honoraria; Roche: Consultancy, Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Curis: Honoraria, Research Funding; Merck: Honoraria, Research Funding; Abbvie: Honoraria; Pharmacyclics: Research Funding. Bertoni:Nordic Nanovector ASA: Research Funding; Acerta: Research Funding; ADC Therapeutics: Research Funding; Bayer AG: Research Funding; Cellestia: Research Funding; CTI Life Sciences: Research Funding; EMD Serono: Research Funding; Helsinn: Consultancy, Research Funding; ImmunoGen: Research Funding; Menarini Ricerche: Consultancy, Research Funding; NEOMED Therapeutics 1: Research Funding; Oncology Therapeutic Development: Research Funding; PIQUR Therapeutics AG: Other: travel grant, Research Funding; HTG: Other: Expert Statements ; Amgen: Other: travel grants; Astra Zeneca: Other: travel grants; Jazz Pharmaceuticals: Other: travel grants.

The contribution of MYC and PLK1 expression to proliferative capacity in diffuse large B-cell lymphoma

Polo-like kinase-1 (PLK1) regulates the MYC-dependent kinome in aggressive B-cell lymphoma. However, the role of PLK1 and MYC toward proliferation in diffuse large B-cell lymphoma (DLBCL) is unknown. We use multiplexed fluorescent immunohistochemistry (fIHC) to evaluate the co-localization of MYC, PLK1 and Ki67 to study their association with proliferation in DLBCL. The majority (98%, 95% CI 95–100%) of MYC/PLK1-double positive tumor cells expressed Ki67, underscoring the key role of the MYC/PLK1 circuit in proliferation. However, only 38% (95% CI 23–40%) and 51% (95% CI 46–51%) of Ki67-positive cells expressed MYC and PLK1, respectively. Notably, 40% (95% CI 26–43%) of Ki67-positive cells are MYC- and PLK-negative. A stronger correlation exists between PLK1 and Ki67 expression (R = 0.74, p < .001) than with MYC and Ki67 expression (R = 0.52, p < .001). Overall, the results indicate that PLK1 has a higher association than MYC in DLBCL proliferation and there are mechanisms besides MYC and PLK1 influencing DLBCL proliferation.