Z138 Cells Research Articles

A genetically engineered mouse/human chimeric antibody targeting CD99 enhances antibody-dependent cellular phagocytosis against human mantle cell lymphoma Z138 cells

Background: Mantle cell lymphoma (MCL) is an aggressive form of B-cell nonHodgkin lymphoma. The elimination of MCL cells via phagocytosis is essential for cancer eradication. Therefore, discovering novel targeted antibodies that can induce phagocytosis is needed. We have demonstrated that our in-houseproduced mouse anti-CD99 mAb clone MT99/3 could induce potent anticancer activities against MCL cell lines in both in vitro and in vivo mouse xenograft models. Nevertheless, for use in humans, the mouse mAb needs to be transformed into a mouse/human chimeric mAb that contains a human Fc region to activate human immune effector functions, especially macrophage-mediated phagocytosis. Antibody-dependent cellular phagocytosis (ADCP) mediated by mouse/human chimeric mAb MT99/3 against MCL has not been previously reported. Objective: This study aimed to genetically engineer a mouse/human chimeric antibody against human CD99 derived from mouse mAb MT99/3 and to evaluate its effect in mediating the ADCP mechanism for eradicating MCL cells in vitro using monocyte-derived macrophages. Materials and methods: The expression plasmid to produce chimeric anti-CD99 antibody, ChAbMT99/3, was constructed by fusing the variable domains of mouse mAb MT99/3 with the constant domains of human IgG1 and the constant domains of kappa light chain. ChAbMT99/3 was expressed in the stable human expression system based on HEK293T cells. ChAbMT99/3 was purified from the culture supernatant of ChAbMT99/3-expressing HEK293T cells using Protein G chromatography. The purity and structure of ChAbMT99/3 were verified by SDS-PAGE and western blotting. The binding specificity and activity were determined by staining with cells expressing recombinant and native human CD99. The anticancer activity of ChAbMT99/3 in mediating the ADCP mechanism against MCL cell line Z138 using human monocytederived macrophages was evaluated. Results: We successfully constructed the plasmid to produce ChAbMT99/3. Human HEK293T cells stably expressing ChAbMT99/3 were established. The ChAbMT99/3- expressing HEK293T cells could secrete ChAbMT99/3 into the culture supernatant. The high purity and complete IgG structure of ChAbMT99/3 were obtained from the purification process. Crucially, this chimeric antibody retained its binding reactivity to recombinant and native human CD99. In addition, the produced ChAbMT99/3, upon binding to MCL cells, significantly enhanced ADCP against MCL cell line Z138 in a dose-dependent manner. Conclusion: The production of a mouse/human chimeric antibody against human CD99 derived from mouse mAb MT99/3 was successful. The engineered antibody could mediate ADCP activity against MCL cells. The produced ChAbMT99/3 might be a promising therapeutic candidate for MCL treatment.

Novel 1,3,4-trisubstituted Pyrazolopyrimidine Derivatives Show Potent Antiproliferative Activity in Mantle Cell Lymphoma

Background: Pyrazolopyrimidine scaffold is an important pharmacophore in drug discovery. This pharmacophore has been reported to produce numerous biological activities, of which anticancer is an important one. The development of novel pyrazolopyrimidine derivatives is of great importance for antitumor drug research. Objective: Compound 6, a pyrazolopyrimidine derivative reported by our group, showed weak antiproliferative activity with IC50 values of over 30 μM against mantle cell lymphoma (MCL) cell lines. In this study, we will further perform the structural optimization of compound 6 to screen highly active pyrazolopyrimidine derivatives. Methods: A novel series of 1,3,4-trisubstituted pyrazolopyrimidine derivatives were synthesized and their structures were elucidated by 1H-NMR, 13C-NMR, and HRMS. The antiproliferative activities of target compounds against MCL cell lines (Mino, Jeko-1, and Z138) were evaluated by the CellTiter-Glo luminescent cell viability assay. The effect of representative compounds to induce apoptosis was evaluated by Annexin V/Propidium Iodide (PI)-binding assay. Mitochondrial membrane potential and reactive oxygen species (ROS) levels in 15c-treated Z138 cells were tested by JC-1 and DCFH-DA probes, respectively. Results: Most compounds demonstrated improved antiproliferative activity against MCL cell lines compared to the lead compound 6, especially 15c, 15f, 15g, 15j, and 15o, with IC50 values at low micromolar levels. In addition, compound 15c could induce apoptosis in a dose-dependent manner in Z138 cells through reduction of mitochondrial membrane potential and enhancing reactive oxygen species production. Conclusion: The results showed that 1,3,4-trisubstituted pyrazolopyrimidine derivatives could be valuable lead compounds for the further development of anti-lymphoma agents.

Activating NSD2 Mutations Drive Oncogenic Reprogramming By Disturbing Epigenetic Landscape in Mantle Cell Lymphoma

Background: NSD2/MMSET/WHSC1, a histone lysine methyltransferase (HMT), is an oncoprotein first characterized by its overexpression in multiple myeloma (MM). NSD2 mutations within the catalytic SET domain are frequently identified in lymphoid malignancies including acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), and mantle cell lymphoma (MCL). We previously demonstrated that NSD2 p.E1099K mutation caused an imbalance of H3K36me2/H3K27me3 and drove glucocorticoid resistance in pediatric ALL. In MCL, NSD2 mutations, especially p.E1099K and p.T1150A, are found in 10-15% of cases of MCL and are enriched in MCL patients who relapse from targeted therapies such as ibrutinib, an inhibitor of B cell signaling. However, the activity of NSD2 mutations in MCL remains unexplored. We hypothesize that NSD2 mutations contribute to disease progression and therapy resistance due to aberrant chromatin modification. Aim: To demonstrate the role of NSD2 mutations in MCL progression due to aberrant chromatin modification. Methods: We created isogenic MCL cell lines by knock-in NSD2 p.E1099K and NSD2 p.T1150A mutation into Z138 and Jeko-1 cell lines using CRISPR/Cas9 gene editing. We then determined H3K36me2/H3K27me3/H3K36me3 using immunoblot and the biological activities including cell growth (IncuCyte), apoptosis (Annexin V/PI staining), cell cycle (BrdU incorporation), and in vivo experiments using luciferase-tagged cell lines in NOD-SCID mice. We determined alteration of gene transcriptome using RNA-Seq and histone modification using CUT&RUN (H3K36me2, H3K27me3 and H3K4me3) and ChIP-Seq (H3K27ac) in isogenic Z138 cell lines. Finally, we integrated multi-omics analysis of our MCL cell lines and human patient samples and to comprehensively disclose the epigenetic landscape in MCL with NSD2 mutations. Results: We successfully created isogenic Z138 cell lines with NSD2 p.E1099K mutation or p.T1150A mutation, and isogenic Jeko-1 cell line with NSD2 p.E1099K mutation. Insertion of either NSD2 p.E1099K mutation or p.T1150A mutation by CRISPR/Cas9 gene editing led to a striking increase of H3K36me2, a decrease of H3K27me3 but no change of H3K36me3. NSD2 p.E1099K mutation significantly increased cellular growth and enhanced S phase entry while decreased G0/G1 phase and apoptosis. In NOD-SCID mouse xenografts, NSD2 p.E1099K mutation resulted in a higher tumor burden and shorter survival lifespan in mice. RNA-Seq analysis demonstrated striking changes in gene expression with 1794 genes upregulated and 1492 genes downregulated in NSD2 mutant MCL cells. Among them, anti-apoptotic genes BCL2 and BCL2L2 were upregulated while pro-apoptotic genes BAX, BID, and BIK were downregulated in NSD2 mutant cells, which was consistent with the biological phenotypes. Surprisingly, CCND1, CCNE1, CCNE2, TP53, and CDKN2C ( p21) were downregulated while CDKN2D, CDKL3, and CDKL5 were upregulated in NSD2 mutant cells. Altered gene expression in isogenic Z138 cells was compared with patient expression profiles, with gene ontology revealing significant overlap and enrichment of cell adhesion, neural pathways, and evidence of activated signaling. NSD2 mutation drove these oncogenic reprogramming by disturbing epigenetic landscape as increased levels of H3K36me2, H3K4me3 and H3K27ac but decreased levels of H3K27me3, which contributed to aberrant gene expression patterns. Conclusions: The NSD2 mutation led to increased tumor cell growth but decreased apoptosis due to the dysregulation of epigenetic landscape and gene expression, suggesting an oncogenic reprogramming driven by activated NSD2 mutations in MCL.

Inhibition of CDC20 Suppressed the Development and Progression of Mantle Cell Lymphoma

Background: Cell division cycle 20 homologue (CDC20) is an important factor regulating the cell mitosis phase. CDC20 is highly expressed in several types of cancers, which is related to the clinicopathological characteristics and prognosis of cancer patients. This study aimed to explore whether CDC20 could affect the development and progression of mantle cell lymphoma (MCL) and the underlying mechanism. Methods: The expression level of CDC20 was detected in peripheral blood mononuclear cells (PBMCs), bone marrow mononuclear cells (BMNCs) and pathological tissues of MCL patients and MCL cell lines (Z138, Mino, Rec1). The CDC20 inhibitor apcin was used to evaluate the effect of CDC20 on the cell phenotypes of MCL cells. The changes in cell proliferation, apoptosis, cell cycle, cell migration and invasion were determined using CCK-8, flow cytometry, and Transwell assays, respectively. The anti-tumor effect of apcin were investigated in vivo in the Z138-driven xenograft tumor model. RNA-seq was performed to explore the pathways that changed in MCL cells after apcin treatment, and then these possible changes were verified in vitro and in vivo by western blot. Results: CDC20 was overexpressed in PBMCs, BMNCs, and pathological tissues of MCL patients and MCL cells compared with their respective control group. In Z138, Mino, and Rec1 cells, apcin could inhibit cell proliferation, migration, and invasion, promote cell apoptosis, and induce cell cycle arrest. Moreover, apcin confirmed its safety and anti-tumor activity in vivo. In apcin-treated MCL cells and mice, the PI3K/AKT pathway was significantly inhibited (Figure 1). Conclusions: This study confirms the essential role of CDC20 in MCL tumorigenesis. Anti-tumor therapy targeting CDC20 is expected to be a powerful treatment strategy for MCL patients.

Translational development of BAFF-R-specific chimeric antigen receptor T-cell therapy targeting B-cell lymphoid malignancies.

e19501 Background: Relapsed and/or refractory (R/R) disease is observed in patients who receive CD19 targeting chimeric antigen receptor (CAR)-T cells to treat B-cell lymphoid leukemias and lymphomas. We generated a novel CAR, MC10029 that targets B-cell activating factor receptor (BAFF-R) to address this unmet medical need. Methods: We engineered a second-generation BAFF-R CAR (MC10029 CAR). The construct includes the single chain variable fragment (scFv) of our newly developed BAFF-R antibody with CD28 costimulatory and CD3ζ signaling domains. We confirmed MC10029 CAR-T cell antigen-specific activation with in vitro degranulation assays and a direct cytolytic assay. We used BAFF-R positive leukemia and lymphoma cell lines in both in vitro and in vivo models to further characterize MC10029 CAR-T cells, and finally, we enriched tumor primary cells from chronic lymphocytic leukemia (CLL) patients as a target for MC10029 CAR-T cells. In preparation for our Phase I clinical trial, we have manufactured three batches of CAR-T cells using a MC10029-encoding lentiviral vector produced under GMP conditions. The production batches of MC10029 CAR-T cells were evaluated for product quality with Viability, Identity and Potency as well as Safety from adventitious viral agents. Antigen specific cytotoxicity of the MC10029 CAR-T cells was confirmed using our standard degranulation assay. Results: MC10029 CAR-T cells elicited in vitro antigen-specific cytotoxicity against Nalm-6 cells, Z138 cells, MEC-1 cells, and a CD19 knock out Nalm-6 model designed to mimic CD19-negative R/R disease. We demonstrated in vivo antitumor effects of MC10029 CAR-T cells in NSG mice challenged with these same cell lines. We showed cytotoxicity of MC10029 CAR-T cells against CLL patients’ tumors. We also showed our quality control results to highlight our clinical trial trajectory. Conclusions: Novel MC10029 CAR-T cells showed broad utility against BAFF-R positive B-cell lymphoid malignancies in a series of in vitro, in vivo, and ex vivo models. With these robust data, we have moved forward to generate clinical grade MC10029 CAR-T cells using cGMP manufacturing and quality protocols in preparation of a MC10029 CAR-T cell Phase I clinical trial.

Abstract 2655: Anti-CD19 antibody-drug conjugate therapy in B cell non-Hodgkin lymphoma

Abstract Previously, we have shown that overexpression of Polo-like kinase (PLK)-1 is associated with poor clinical outcomes in patients with aggressive B cell lymphomas. PLK-1 selective inhibitor, Volasertib (BI 6727), has a potent anti-tumor effect in aggressive B cell lymphomas. Unfortunately, Volasertib was withdrawn in Phase III clinical trials due to severe myelosuppression. In this study, we developed a Volasertib antibody-drug conjugate (V-ADC) using -CD19 antibody Inebilizumab to increase the targeting specificity for B-cell lymphoma cells and minimize the side effects of Volasertib. However, we found that V-ADC treatment exhibited little cytotoxic effects, comparing to free Volasertib treatment in Z138 cells, a mantle cell lymphoma (MCL) cell line. We hypothesized that the expression level/density of CD19 on B-lymphoma cells as well as its gene mutations may affect V-ADC binding affinity required for its endocytosis. We, therefore, established Z138 cell lines with overexpression of either wild-type (WT) or mutant (Mut) CD19. The cell viability at 48h post 10nM V-ADC treatment was determined to be 90%, 61%, and 69% in parent, WT, and Mut CD19-overexpressing Z138 cells, respectively. In comparison, the cell viability with 10nM free volasertib treatment ranged from 3-30% in these cell lines. Therefore, other factors, in addition to CD19 expression, may have also played an important role in V-ADC induced cytotoxicity. Upon endocytosis, the V-ADC will enter the endolysosome where cathepsin, a lysosomal enzyme, cleaves the linker between Volasertib and Inebilizumab and releases free Volasertib into the cells. Our previous works have shown that a starvation condition (culture media with 1% FBS) increased lysosomal cathepsin activity, comparing to the culture media with 10% FBS. Herein, we found that upon treatment with 10nM V-ADC, CD19-overexpressing Z138 cells cultured in media with 1% FBS exhibited markedly increased apoptotic cell death comparing to cells in media with 10% FBS (64% vs 41%, respectively). Furthermore, WT and Mut CD19-overexpressing Z138 cells exhibited 2.8- and 2.4-folds of apoptotic cell death compared to control Z138 cells at 48h post 10nM V-ADC treatment with 1% FBS. The cell apoptosis occurred via upregulation of caspase 3, but not caspase 9, with induction of cleavage of poly (ADP-ribose) polymerase (PARP) and reduction of mitochondria membrane potential. In conclusion, starvation and lysosomal cathepsin activation increased V-ADC-induced apoptotic cell death in CD19 overexpression Z138 cell lines. We are actively investigating the in vivo therapeutic effects of V-ADC in patient-derived xenograft (PDX) animal models of MCL and other aggressive B-cell lymphomas. Citation Format: Chieko Saito, Chengfeng Bi, Kai Fu. Anti-CD19 antibody-drug conjugate therapy in B cell non-Hodgkin lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2655.

P53 directly downregulates the expression of CDC20 to exert anti-tumor activity in mantle cell lymphoma

BackgroundCell cycle dysregulation characterized by cyclin D1 overexpression is common in mantle cell lymphoma (MCL), while mitotic disorder was less studied. Cell division cycle 20 homologue (CDC20), an essential mitotic regulator, was highly expressed in various tumors. Another common abnormality in MCL is p53 inactivation. Little was known about the role of CDC20 in MCL tumorigenesis and the regulatory relationship between p53 and CDC20 in MCL.MethodsCDC20 expression was detected in MCL patients and MCL cell lines harboring mutant p53 (Jeko and Mino cells) and wild-type p53 (Z138 and JVM2 cells). Z138 and JVM2 cells were treated with CDC20 inhibitor apcin, p53 agonist nutlin-3a, or in combination, and then cell proliferation, cell apoptosis, cell cycle, cell migration and invasion were determined by CCK-8, flow cytometry and Transwell assays. The regulatory mechanism between p53 and CDC20 was revealed by dual-luciferase reporter gene assay and CUT&Tag technology. The anti-tumor effect, safety and tolerability of nutlin-3a and apcin were investigated in vivo in the Z138-driven xenograft tumor model.ResultsCDC20 was overexpressed in MCL patients and cell lines compared with their respective controls. The typical immunohistochemical marker of MCL patients, cyclin D1, was positively correlated with CDC20 expression. CDC20 high expression indicated unfavorable clinicopathological features and poor prognosis in MCL patients. In Z138 and JVM2 cells, either apcin or nutlin-3a treatment could inhibit cell proliferation, migration and invasion, and induce cell apoptosis and cell cycle arrest. GEO analysis, RT-qPCR and WB results showed that p53 expression was negatively correlated with CDC20 expression in MCL patients, Z138 and JVM2 cells, while this relationship was not observed in p53-mutant cells. Dual-luciferase reporter gene assay and CUT&Tag assay revealed mechanistically that CDC20 was transcriptionally repressed by p53 through directly binding p53 to CDC20 promoter from − 492 to + 101 bp. Moreover, combined treatment of nutlin-3a and apcin showed better anti-tumor effect than single treatment in Z138 and JVM2 cells. Administration of nutlin-3a/apcin alone or in combination confirmed their efficacy and safety in tumor-bearing mice.ConclusionsOur study validates the essential role of p53 and CDC20 in MCL tumorigenesis, and provides a new insight for MCL therapeutics through dual-targeting p53 and CDC20.

Design, Synthesis, and Biological Evaluation of 2-Aminothiazole Derivatives as Novel Checkpoint Kinase 1 (CHK1) Inhibitors.

A series of 2-aminothiazole derivatives were designed, synthesized on the basis of bioisosterism strategy and evaluated for their CHK1 inhibitory activity. Most of them exhibited potent CHK1 inhibition, and excellent antiproliferative activity against MV-4-11 and Z-138 cell lines. Systematic structure-activity relationship (SAR) efforts led to the discovery of a promising compound 8 n, which showed potent CHK1 inhibitory activity with IC50 value of 4.25±0.10 nM, excellent antiproliferative activity against MV-4-11 and Z-138 cells with IC50 value of 42.10±5.77 nM and 24.16±6.67 nM, respectively, as well as moderate oral exposure (AUC(0-t) =1076.25 h ⋅ ng/mL) in mice. Additionally, treatment of MV-4-11 cells with compound 8 n for 2 h led to robust inhibition of CHK1 autophosphorylation on serine 296. Furthermore, kinase selectivity assay revealed that 8 n displayed acceptable selectivity toward 15 kinases. These results demonstrated that compound 8 n may be a promising potential anticancer agent for further development.

Structure-activity relationship study of a series of nucleoside derivatives bearing sulfonamide scaffold as potent and selective PRMT5 inhibitors

Protein arginine methyltransferase 5 (PRMT5) is a promising target for the treatment of malignant tumors. The discovery of nucleoside-derived inhibitors against PRMT5 with novel scaffold has been challenging. Herein, we report our effort on the design and synthesis of nucleoside derivatives bearing sulfonamide scaffold as potent PRMT5 inhibitors. The representative compound 23n was identified as a potent and selective PRMT5 inhibitor with an IC50 value of 8 nM. Molecular docking study demonstrated the binding mode of compound 23n and illustrated its inhibitory activity to PRMT5. The Trimethyl Lock prodrug strategy was used to afford prodrug 36 with lower polarity which could rapidly release the active compound 23n after entering the tumor cells. Cell-based assays revealed that the prodrug 36 restrained the proliferation of Z-138 and MOLM-13 cells and suppressed methylation of PRMT5 substrate more potently than 23n. Additionally, both compound 23n and 36 exerted antiproliferative effects against Z-138 cells mainly by inducing apoptosis effectively rather than arresting cell cycle. Thus, compounds 23n and 36 represent a series of potent PRMT5 inhibitor with novel scaffold.

High-resolution simulations of chromatin folding at genomic rearrangements in malignant B cells provide mechanistic insights into proto-oncogene deregulation.

Genomic rearrangements are known to result in proto-oncogene deregulation in many cancers, but the link to 3D genome structure remains poorly understood. Here, we used the highly predictive heteromorphic polymer (HiP-HoP) model to predict chromatin conformations at the proto-oncogene CCND1 in healthy and malignant B cells. After confirming that the model gives good predictions of Hi-C data for the nonmalignant human B cell–derived cell line GM12878, we generated predictions for two cancer cell lines, U266 and Z-138. These possess genome rearrangements involving CCND1 and the immunoglobulin heavy locus (IGH), which we mapped using targeted genome sequencing. Our simulations showed that a rearrangement in U266 cells where a single IGH super-enhancer is inserted next to CCND1 leaves the local topologically associated domain (TAD) structure intact. We also observed extensive changes in enhancer-promoter interactions within the TAD, suggesting that it is the downstream chromatin remodeling which gives rise to the oncogene activation, rather than the presence of the inserted super-enhancer DNA sequence per se. Simulations of the IGH-CCND1 reciprocal translocation in Z-138 cells revealed that an oncogenic fusion TAD is created, encompassing CCND1 and the IGH super-enhancers. We predicted how the structure and expression of CCND1 changes in these different cell lines, validating this using qPCR and fluorescence in situ hybridization microscopy. Our work demonstrates the power of polymer simulations to predict differences in chromatin interactions and gene expression for different translocation breakpoints.

Abstract 2157: SCR-6277, a potent and high selective arginine methyltransferase 5 (PRMT5) inhibitor with high tumor/plasma distribution ratio demonstrated robust anti-tumor activities and decreased hematological toxicities

Abstract Protein arginine methyltransferase 5 (PRMT5) is observed dysregulation in both solid and hematological tumors. Increasing evidences demonstrated that PRMT5 is a potential therapeutic target. Given the essential role in maintenance of adult hematopoietic cells, inhibition of PRMT5 leads to hematological toxicity. Anemia is the most common treatment related adverse event observed by PRMT5 inhibitor, such as GSK3326595, which limited the dose escalation. Therefore, developing a PRMT5 inhibitor with tumor-biased distribution could be an effective approach to avoid these target related toxicities. In this study, a novel and high selective PRMT5 inhibitor, SCR-6277 was identified. SCR-6277 inhibited the PRMT5 enzyme with an IC50 of 1.5 nM, and showed a high selectivity against other five methyltransferase isoforms (selectivity folds >1000). SCR-6277 potently inhibited the PRMT5-mediated SDMA production in Z-138 cells (IC50, 0.6 nM) and the cell proliferation (IC50, 3 nM). SCR-6277 also demonstrated robustly anti-proliferation activities on multiple PRMT5 proficient solid tumor cell lines (IC50 values ranging from 20 - 130 nM) and no inhibitory effects on PRMT5 deficient cell lines. SCR-6277 had good oral bioavailability and favorable pharmacokinetic properties. In Z-138 and 5637 xenograft models, SCR-6277 robustly inhibited tumor growth. In contrast to GSK3326595, SCR-6277 showed tumor biased distribution. Exposure of SCR-6277 in tumors were 50 folds higher than that in plasma. The extremely high T/P ratio made SCR-6277 achieve the better anti-tumor activities at lower blood exposure (Table 1). In conclusion, SCR-6277 is a novel, potent and high selective PRMT5 inhibitor. And higher tumor/plasma distribution ratio predicts SCR-6277 is a more tolerant and effective PRMT5 inhibitor in human. Table 1. Distribution and anti-tumor activity of SCR-6277 Dose TGI Plasma Conc. (ng/mL) * Tumor Conc. (ng/g) * T/P ratio GSK3326595 50 mpk,BID 87% 45 106 2 SCR-6277 15 mpk,BID 117% 20 668 50 Citation Format: Feng Zhou, Guimei Yang, Feng Tang, Liting Xue, Wenqing Yang, Ping Chen, Renhong Tang. SCR-6277, a potent and high selective arginine methyltransferase 5 (PRMT5) inhibitor with high tumor/plasma distribution ratio demonstrated robust anti-tumor activities and decreased hematological toxicities [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2157.

Design and synthesis of novel pyrazolopyrimidine-based derivatives as reversible BTK inhibitors with potent antiproliferative activity in mantle cell lymphoma

Development of Bruton’s tyrosine kinase (BTK) inhibitors is of great value and significance in the treatment of B-cell malignancies and autoimmune diseases. Herein, a novel class of pyrazolopyrimidine-based BTK inhibitors were designed and evaluated in mantle cell lymphoma (MCL) cell lines. We demonstrated that target compounds had made great progress in improvement of antiproliferative activity compared to lead compound. Compounds 13c, 13g, 13h, 13l, 13n and 13o demonstrated effectively antiproliferative activity in MCL cells lines with single-digit micromolar potency. Furthermore, compound 13l specifically disturbed mitochondrial membrane potential and increased reactive oxygen species level in Z138 cells in a dose-dependent manner. 13l induced cell apoptosis through the caspase 3- mediated apoptotic pathway in Z138 cells. Overall, this study provides valuable lead compounds for developing antitumor agents. Graphical abstract

Pirtobrutinib Overcomes Ibrutinib and Venetoclax Resistance in Mantle Cell Lymphoma

BackgroundMantle cell lymphoma (MCL) is a rare and aggressive B-cell lymphoma characterized by poor prognosis. Although remarkable therapeutic advances have been made by covalent Bruton's tyrosine kinase (BTK) inhibition and CAR T cell therapy, therapeutic resistance inevitably occurs and leads to dismal clinical outcome. Pirtobrutinib (LOXO-305) is a next-generation, highly selective and non-covalent BTK inhibitor. A phase 1/2 BRUIN study showed that pirtobrutinib demonstrated promising efficacy in heavily pretreated MCL patients with or without prior covalent BTK inhibition. Here, we investigated the mechanism of action of pirtobrutinib in MCL cells in vitro and proposed the potential combination therapy in a venetoclax-resistant xenograft model.MethodsMCL cell proliferation was monitored by trypan blue exclusion assay after 24-, 48- and 72-hour treatment with pirtobrutinib and ibrutinib. We performed Annexin V/PI staining to measure the apoptosis inductive effects. Cell cycle analysis using propidium iodide (PI) DNA staining was conducted to compare cell cycle progression kinetics between pirtobrutinib and ibrutinib. We performed RNAseq analysis in Z138 cells to compare differentially expressed genes (DEGs) between pirtobrutinib and ibrutinib treatment. Western blotting was utilized to detect specific signaling proteins. Mino-venetoclax-R cells were inoculated subcutaneously into NSG mice and used for in vivo drug efficacy determination.ResultsCompared to covalent BTK inhibitor ibrutinib, the novel non-covalent BTK inhibitor pirtobrutinib was more potent in inhibiting MCL cell proliferation in a panel of MCL cell lines, especially in ibrutinib/venetoclax resistant cell lines (pirtobrutinib vs. ibrutinib, p<0.01). Treatment with pirtobrutinib (10μM) for 24 hours induced higher levels of apoptosis than that by ibrutinib in all the MCL cell lines tested (p<0.05), which was also confirmed at the molecular level by stronger caspase-3 activation and PARP cleavage. To understand the mechanism of action, we performed whole transcriptomic profiling by RNAseq analysis using Z138 cells treated with/without pirtobrutinib or ibrutinib. Pirtobrutinib treatment resulted in upregulation of 137 genes and downregulation of 97 genes compared to the ibrutinib treatment (adjusted p<0.05). In addition to the downregulated MYC targets and PI3K/Akt pathway, gene set enrichment analysis (GSEA) revealed a significant enrichment for G2/M checkpoints and E2F targets signatures (key genes: PLK1, CDKN1A and CCNB1) in pirtobrutinib treated cells. Consistently, follow-up studies showed that γH2AX level was highly increased upon pirtobrutinib treatment. Pirtobrutinib treatment but not ibrutinib treatment resulted in G2/M cell cycle arrest. The blockade of cell cycle progression is positively correlated with decreased protein levels of critical regulators of S and G2/M phase transition such as cyclin B and CDC25C. BTK inhibitor (ibrutinib) in combination with venetoclax has shown great efficacy in preclinical models and in MCL patients. Therefore, here we assessed the in vivo efficacy of pirtobrutinib in combination with venetoclax with side-by-side comparison to ibrutinib & venetoclax in the Mino-venetoclax-R mouse model. Pirtobrutinib & venetoclax combination enhanced the efficacy of pirtobrutinib in restraining the tumor size (p<0.001) in the xenograft model. Notably, this novel combinatorial treatment exerted much higher potency than ibrutinib and venetoclax combination therapy (p<0.001). In addition, the pirtobrutinib & venetoclax combination was well tolerated and did not reduce overall mouse body weights compared with the vehicle treated mice.ConclusionsPirtobrutinib overcame both ibrutinib and venetoclax resistance in MCL cells in vitro and in vivo. G2/M checkpoints and E2F targets pathways were significantly enriched in both cases. Pirtobrutinib & venetoclax showed better in vivo efficacy in MCL models than combination of ibrutinib & venetoclax. [Display omitted] DisclosuresWang: Genentech: Consultancy; Juno: Consultancy, Research Funding; Kite Pharma: Consultancy, Honoraria, Research Funding; Clinical Care Options: Honoraria; CAHON: Honoraria; InnoCare: Consultancy, Research Funding; Moffit Cancer Center: Honoraria; Molecular Templates: Research Funding; Oncternal: Consultancy, Research Funding; DTRM Biopharma (Cayman) Limited: Consultancy; Hebei Cancer Prevention Federation: Honoraria; Lilly: Research Funding; Loxo Oncology: Consultancy, Research Funding; BioInvent: Research Funding; OMI: Honoraria; Miltenyi Biomedicine GmbH: Consultancy, Honoraria; Imedex: Honoraria; Physicians Education Resources (PER): Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Bayer Healthcare: Consultancy; Chinese Medical Association: Honoraria; Dava Oncology: Honoraria; Celgene: Research Funding; Mumbai Hematology Group: Honoraria; Acerta Pharma: Consultancy, Honoraria, Research Funding; BeiGene: Consultancy, Honoraria, Research Funding; Newbridge Pharmaceuticals: Honoraria; CStone: Consultancy; BGICS: Honoraria; The First Afflicted Hospital of Zhejiang University: Honoraria; Scripps: Honoraria; Epizyme: Consultancy, Honoraria; Pharmacyclics: Consultancy, Research Funding; AstraZeneca: Consultancy, Honoraria, Research Funding; VelosBio: Consultancy, Research Funding; Anticancer Association: Honoraria.

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.

MCL-363: Overexpression of CDC20 Promotes Cancer Progression in Mantle Cell Lymphoma

Context Mantle cell lymphoma (MCL) is an aggressive B-cell non-Hodgkin lymphoma with poor prognosis and high recurrence rates in patients. The development of MCL is largely attributed to cell cycle dysregulation. Cell division cycle 20 (CDC20) is a key member of cell cycle checkpoints, and high expression of CDC20 was reported in a variety of tumors. Few studies have explored the role of CDC20 in MCL. Objective This study was aimed to investigate whether CDC20 could promote cancer progression and the potential of CDC20 to be a therapeutic target in MCL. Design Peripheral blood mononuclear cells (PBMCs) from 18 MCL patients and 7 healthy donors and 5 MCL cell lines (Jeko, Mino, Rec1, Z138, and JVM2) were acquired to detect CDC20 expression. CDC20 inhibitor apcin was used in Z138 to evaluate its antitumor effects in vitro, including cell viability, cell apoptosis, cell cycle, cell migration, and invasion. Possible changes of some related genes were assessed after apcin treatment. Results CDC20 was overexpressed in 94.4% (17/18) of MCL patients and 5 MCL cell lines compared with healthy controls. Cell growth was inhibited, and cell apoptosis was induced in apcin-treated Z138 cells. After 48 h apcin treatment, cell cycle was arrested in the G2/M phase, characterized by an increased number of Z138 cells in this period. The abilities of cell migration and invasion were significantly attenuated with apcin treatment. The expression of cyclinB1, a marker of the G2/M phase, was significantly increased, together with increased Bim expression and decreased Bcl-xL expression in apcin-treated Z138 cells. Conclusions CDC20 might contribute to tumorigenesis in MCL. Further studies should be carried out to determine whether CDC20 inhibitors could impede tumor progression in MCL patients. This work was supported by grants from the Natural Science Foundation of Beijing Municipality [7182178].

The Proto-Oncogene Mer Is a Novel Therapeutic Target in Mantle Cell Lymphoma

Mantle cell lymphoma (MCL) is B-cell derived malignant tumor of the hematologic and lymphatic system, which is an incurable disease with most patients eventually relapsing and needing rescue therapy, so new therapeutic targets are emerging quickly nowadays. Mer tyrosine kinase (MerTK) is one of the TAM (Tyro-3, Axl, and MerTK) receptor tyrosine kinase family, and is often overexpressed or ectopic expressed in various malignant tumors, such as melanoma, gastric cancer, leukemia, lung cancer et.al, and is not expressed in normal B or T lymphocytes.To determine the role of MerTK in MCL, firstly, we analyzed MerTK expression in MCL cell lines by western-blot, and MerTK expression in MCL tumor tissues by Immunohistochemistry (IHC). Three (Z-138, Mino, JVM-2) of 6 (Z-138, Mino, JVM-2, Granta519, JVM-13, JeKo-1) MCL cell lines showed strong MerTK expression. Out of 132 MCL patients, 65 patients (48.9%) showed MerTK expression (positive percentage>10%). The correlation between clinical features and MerTK expression in MCL patients is currently under analysis.Lentivirus-mediated knockdown of MerTK in Z-138 and Mino cells decreased activation of AKT, ERK 1/2 and P38. Moreover, knockdown of MerTK resulted in inhibition of proliferation and invasion ability in Z-138, Mino and JVM-2 cells, as well as promoted chemosensitization in Z-138 and Mino cells. UNC2250, a MerTK-selective small molecular tyrosine kinase inhibitor, inhibited proliferation and invasion in Z-138, Mino and JVM-2 cells. In cell cycle analysis, UNC2250 induced apparently G2 phase arrest in Z-138, Mino and JVM-2 cells, and also induced polyploidy in JVM-2 cells. Furthermore, western-blot analysis revealed that the G2/M arrest was associated with decrease in pCdc2 and Cyclin B1 protein levels. Meanwhile, UNC2250 showed a dose- and time-dependent apoptotic death in Z-138, Mino and JVM-2 cells, which was associated with increase in Bax, cleaved forms of Caspase 3 and poly (ADP-ribose) polymerase (PARP) protein levels, and decrease in Bcl-2, Mcl-1 and Bcl-xL protein levels. UNC2250 also sensitized Z-138 and Mino cells to treatment with Vincristine and Doxorubicin, which may be a convincing evidence for UNC2250 or other new UNC2250-like MerTK inhibitor being applied in clinical therapy for MCL, allowing for dose reduction in combination with common cytotoxic chemotherapy. Further studies need to be completed in protein assay associated with MerTK inhibition in MCL cells, and the effect of UNC2250 in murine MCL xenograft models are ongoing.Our data proves that MerTK is highly expressed in MCL patients, and ectopic MerTK expression may be a novel therapeutic target in MCL. Our data also support further pre-clinical or even clinical research about UNC2250 or new MerTK inhibitor. DisclosuresNo relevant conflicts of interest to declare.

Preclinical Activity of Novel BET Protein Proteolysis Targeting Chimeras (BETP-PROTACs) and Their Combination with Venetoclax or CDK4/6 Inhibitor Against Human Mantle Cell Lymphoma (MCL) Cells

Among the genetic alterations described in Mantle Cell Lymphoma (MCL) are those involving cyclin D1, deletion of INK4a/ARF, as well as copy number gains of MYC, CDK4 and BCL2 genes. Activated B-cell receptor (BCR) signaling, and the ensuing downstream pro-growth and pro-survival NFkB activity, is also a notable feature of MCL. Collectively, the genetic alterations and the ensuing deregulated signaling and activity of transcription factors, including c-MYC and NFkB, creates the MCL-specific ‘transcriptome’ that promotes growth and survival of MCL cells. Ibrutinib, a covalent inhibitor of Bruton's tyrosine kinase (BTK), exhibits unprecedented single-agent activity in relapsed/refractory MCL, however approximately 40% of patients demonstrate primary resistance and secondary resistance is common, with a poor one-year survival rate of only 22%. Mutations in CARD11/IKBKB/TRAF2/BIRC3/NIK or the C481S mutation in BTK, sustain alternative or classical NFkB signaling despite ibrutinib treatment and confer resistance to ibrutinib in MCL. We hypothesized that the resistance to ibrutinib in MCL cells would be governed by a dysregulated transcriptome dependent on the activity of the chromatin reader BET proteins (BETPs), including BRD4. Therefore, we determined the lethal preclinical activity of BETP PROTACs (proteolysis targeting chimeras) ARV-825, and its pharmacologically superior version ARV-771, that proteasomally degrade BETPs, against the following cellular models of ibrutinib resistance (MCL-IR): in vitro generated cultured MCL Mino-persister/resistant (Mino-P/R) cells (~4-fold resistant compared to parental Mino cells), endogenously resistant cultured Z138 and Maver-1 cells, as well as patient-derived xenograft (PDX) PDX1 and PDX2 cells that had exhibited in vivo resistance to ibrutinib. Protein expression evaluations by RPPA and/or Western analyses demonstrated that MCL-IR cells expressed relatively high levels of p-AKT over AKT, p-S6, p-4EBP1, MYC, BCL2, BCl-xL, XIAP and MCL1. Treatment with BETP-PROTAC dose-dependently (20 to 200 nM) induced significantly more growth inhibition and apoptosis than the BETP inhibitor BETi OTX015 against the cellular models of MCL-IR (p < 0.001), which was associated with marked depletion of BRD4, BRD2, MYC, BCL2, BCL-xL, cyclin D1, CDK4 and pRB (S807/811) levels, as well as of NFkB target gene expressions, including cIAP2, XIAP, cFLIP, TNFAIP3, BCl-xL, IL10, TNFa and BTK. Concomitantly, BETP-PROTAC increased expression of HEXIM1 and p21 levels in the MCL-IR cells. Treatment with the BCL2 inhibitor venetoclax or the CDK4/6 inhibitor abemaciclib dose-dependently induced apoptosis of MCL-IR cell types (p< 0.01). Notably, co-treatment with BETP-PROTAC ARV-771 and venetoclax or abemaciclib synergistically induced apoptosis of Mino-P/R, Maver-1 and PDX1 and PDX2 cells (combination indices < 1.0). Additionally, compared to treatment with vehicle control, treatment of luciferase-transduced PDX2 cells infused by the tail vein and engrafted in immune-depleted NSG mice with ARV-771 (30 mg/kg SQ daily) and venetoclax (15 mg/kg PO daily) for 3 weeks, significantly reduced the MCL burden (at 2 weeks of treatment) and improved survival of the mice. These findings demonstrate that BETP-PROTACs deplete BETPs and important MCL-relevant oncogene levels, including MYC, cyclin D1, CDK4, BCL2, Bcl-xL, as well as NFkB target gene expressions, leading to synergistic lethality in vitro in combination with venetoclax or abemaciclib, as well as exert in vivo activity in combination with venetoclax against PDX model of MCL-IR cells. DisclosuresRaina:4Arvinas, LLC. New Haven, CT: Employment. Qian:4Arvinas, LLC. New Haven, CT: Employment. Crew:Arvinas, Inc: Employment.

Design and synthesis of novel substituted benzyl pyrrolopyrimidine derivatives as selective BTK inhibitors for treating mantle cell lymphoma

Ibrutinib, a potent irreversible Bruton’s tyrosine kinase (BTK) inhibitor, was approved by the FDA for treating mantle cell lymphoma (MCL). Although ibrutinib exhibited excellent antitumor activity, it was associated with certain adverse reactions, with off-target effects against EGFR, Itk and Src family kinases. Our studies yielded a novel series of substituted benzyl pyrrolopyrimidine derivatives capable of potent inhibition of BTK. Compared with ibrutinib, compound 15c exhibited potent BTK inhibitory activity and enhanced antiproliferative activity, a 12–24-fold increase, against MCL cell lines, with IC50 values lower than 1 μM. Low micromolar doses of 15c inhibited the BCR signaling pathway and strongly induced the apoptosis of Z138 cells. Ibrutinib and 15c induced autophagy in a dose-dependent manner in Z138 cells. Moreover, compound 15c induced the production of reactive oxygen species (ROS), which may be a reason for its potent antiproliferative activity. Importantly, compound 15c showed greater BTK selectivity than ibrutinib, indicating a potentially safer treatment of MCL.

Targeting Glutamine Metabolism Overcomes Resistance to Targeted Therapies in Refractory Mantle Cell Lymphoma

Background: Mantle cell lymphoma (MCL) is an incurable B cell non-Hodgkin's lymphoma characterized by high refractory occurrence following drug treatment. Despite the encouraging initial MCL tumor response to ibrutinib (IBN), relapse occurs only after few months of treatment due to multiple resistance mechanisms. Thus, the novel therapeutic strategies targeting resistant mechanisms are crucial. Our group has recently shown that among the highly proliferative MCL population, a subpopulation of IBN-R cells exhibits increased OXPHOS activity that is fueled by increased glutaminolysis and rely more on mitochondrial respiration for their grow and survival. The aim of this work was to uncover potential targets responsible for the upregulation of OXPHOS pathway in the refractory/relapsed (R/R) MCL by using multiple biochemical and biological strategies. We focused the present study on glutaminase (GLS), the enzyme that converts glutamine to glutamate, a precursor of α-ketoglutarate (α-KG) that links glutamate to the TCA cycle. Incorporation of α-KG into the TCA cycle is a major anaplerotic step in proliferating cells and is critical for the maintenance of TCA cycle function. To further demonstrate the reliance of OXPHOS on glutamine anaplerosis, we have further tested the combinatory effects of targeting GLS and OXPHOS using their respective inhibitors, CB-839 and IACS-010759, on tumor killing activity in R/R MCL. Methods:Primary MCL cells from patient leukapheresis or whole blood specimens, as well as established MCL cell lines were used as experimental models of MCL. Metabolomic profiling was used to determine intracellular metabolite fluxes and levels. Cell Titer Glo assay was used to measure cell proliferation/viability after treatment with inhibitors. Annexin V and propidium iodide were used to measure cell apoptosis and cell cycle arrestviaflow cytometry analysis. Magnetic microbeads-based B-cell isolation method were used for the purification of malignant B cells from patient samples. Western blot analysis was used to evaluate protein level expression. Patient-derived Xenograft (PDX) mouse model created from patients with MCL was used to evaluate the in vivo anti-tumor activity and potential clinical value of GLS and OXPHOS inhibitors. Results:Our recent metabolomic profiling studies have demonstrated that glutaminolysis and OXPHOS are upregulated in IBN-R MCL, manifested by increased glutamine uptake in the ibrutinib-resistant MCL cell lines (p=0.03).Inhibition of glutamine metabolism with the allosteric GLS1-selective inhibitor BPTES resulted in inhibition of cell viability (0.2381uM-9.98uM), indicating that MCL cells are dependent on glutamine metabolism for their proliferation. To corroborate with the above finding, we also presented evidence that GLS1 is highly increased in IBN-R and CART-R MCL patient samples and cell lines confirmed by immunoblotting. Inhibiting of GLS would lead to significant reduction in OXPHOS, mitochondria membrane potential and ATP production, as either single drug or in combination with other targeting agents. To identify a clinical actionable GLS inhibitor for the treatment of MCL, we chose a GLS1 specific inhibitor CB-839 (Selleckchem), currently under several phase II and III clinical trials investigation on solid tumors. Inhibiting GLS1 with CB-839 leads to the decreased cell viability in MCL (0.5626nM-308.4nM). Of note, the treatment with CB-839 to MCL cell lines induces S phase reduction in both Jeko-1 (17.23%) and Z-138 (14.01%), as well as induces significant apoptosis (p=0.013 and p=0.002 in Jeko-1 and Z-138 cells). GLS inhibition will be further explored in the context of mitochondria defect or hypoxia, where OXPHOS maybe deficient. Importantly, while CB-839 is continuing its validation in several solid tumor models, this is the first study providing data on its efficacy in preclinical models of MCL. Conclusion:In conclusion, we report that glutaminolysis and OXPHOS are upregulated in IBN-R MCL that could be partially due to high expression of GLS1. Our preliminary results revealed that the new GLS inhibitor, GCB-839, may present a clinical potential for a new indication and warrants more in-depth investigation. Deciphering the mechanisms involved in MCL metabolic heterogeneity and adaptability during drug resistance development would be crucial to identify key actors enabling MCL cells to escape from therapy. Disclosures Wang: Acerta Pharma:Research Funding;Molecular Templates:Research Funding;InnoCare:Consultancy;Oncternal:Consultancy, Research Funding;Celgene:Consultancy, Other: Travel, accommodation, expenses, Research Funding;Targeted Oncology:Honoraria;MoreHealth:Consultancy;Kite Pharma:Consultancy, Other: Travel, accommodation, expenses, Research Funding;Lu Daopei Medical Group:Honoraria;OMI:Honoraria, Other: Travel, accommodation, expenses;Verastem:Research Funding;Nobel Insights:Consultancy;BioInvent:Research Funding;Guidepoint Global:Consultancy;AstraZeneca:Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding;Pharmacyclics:Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding;Janssen:Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding;Juno:Consultancy, Research Funding;Dava Oncology:Honoraria;Loxo Oncology:Consultancy, Research Funding;Pulse Biosciences:Consultancy;OncLive:Honoraria;Beijing Medical Award Foundation:Honoraria;VelosBio:Research Funding.

TOSO interacts with SYK and enhances BCR pathway activation in chronic lymphocytic leukemia.

BackgroundTOSO, also named Fas inhibitory molecule 3 (FAIM3), has recently been identified as an immunoglobulin M (IgM) Fc receptor (FcμR). Previous studies have shown that TOSO is specifically over-expressed in chronic lymphocytic leukemia (CLL). However, the functions of TOSO in CLL remain unknown. The B-cell receptor (BCR) signaling pathway has been reported to be constitutively activated in CLL. Here, we aimed to investigate the functions of TOSO in the BCR signaling pathway and the pathogenesis of CLL.MethodsWe over-expressed TOSO in B-cell lymphoma cell lines (Granta-519 and Z138) by lentiviral transduction and knocked down TOSO by siRNA in primary CLL cells. The over-expression and knockdown of TOSO were confirmed at the RNA level by polymerase chain reaction and protein level by Western blotting. Co-immunoprecipitation with TOSO antibody followed by liquid chromatography coupled with tandem mass spectrometry (IP/LCMS) was used to identify TOSO interacting proteins. Western blotting was performed to detect the activation status of BCR signaling pathways as well as B-cell lymphoma 2 (BCL-2). Flow cytometry was used to examine the apoptosis of TOSO-over-expressing B lymphoma cell lines and TOSO-down-regulated CLL cells via the staining of Annexin V and 7-AAD. One-way analyses of variance were used for intergroup comparisons, while independent samples t tests were used for two-sample comparisons.ResultsFrom IP/LCMS, we identified spleen tyrosine kinase (SYK) as a crucial candidate of TOSO-interacting protein and confirmed it by co-immunoprecipitation. After stimulation with anti-IgM, TOSO over-expression increased the phosphorylation of SYK, and subsequently activated the BCR signaling pathway, which could be reversed by a SYK inhibitor. TOSO knockdown in primary CLL cells resulted in reduced SYK phosphorylation as well as attenuated BCR signaling pathway. The apoptosis rates of the Granta-519 and Z138 cells expressing TOSO were (8.46 ± 2.90)% and (4.20 ± 1.21)%, respectively, significantly lower than the rates of the control groups, which were (25.20 ± 4.60)% and (19.72 ± 1.10)%, respectively (P < 0.05 for both). The apoptosis rate was reduced after knocking down TOSO in the primary CLL cells. In addition, we also found that TOSO down-regulation in primary cells from CLL patients led to decreased expression of BCL-2 as well as lower apoptosis, and vice versa in the cell line.ConclusionsTOSO might be involved in the pathogenesis of CLL by interacting with SYK, enhancing the BCR signaling pathway, and inducing apoptosis resistance.