Jeko-1 Cells Research Articles

Novel gold-based complex GC7 suppresses cancer cell proliferation via impacting energy metabolism mediated by mitochondria

Due to their pivotal roles in regulating energy metabolism and apoptosis, mitochondria in cancer cells have been considered a vulnerable and feasible target. Many anticancer agents, e.g., metal-based compounds, are found to target and disturb mitochondria primarily, which may lead to the disturbance of energy metabolism and, more importantly, the initiation of apoptosis. In this work, a gold-based complex 7 (GC7) was synthesized and evaluated in a series of different cancer cell lines. The anticancer efficacies of GC7 on cell viability, apoptosis, and colony formation were determined. Cellular thioredoxin reductase (TrxR) activity, oxygen consumption rate (OCR), glucose uptake, and lactate production following GC7 treatment were evaluated and analyzed. The Jeko-1 and A549 xenograft models were used to assess GC7’s tumor-suppressing effects. The results showed that GC7 possessed a broad-spectrum anticancer effect, with IC50 values ranging from 0.43 to 1.2 μM in multiple cancer cell lines, which was more potent than gold-based auranofin (∼2–6 folds). GC7 (0.3 and 1 μM) efficiently induced apoptosis of Jeko-1, A549, and HCT116 cells, and it suppressed the sphere formation of cancer stem cells GSC11 and GSC23 cells at 0.1 μM, and it completely eliminated colony at 0.3 μM. The preliminary mechanistic study showed that GC7 inhibited cellular TrxR activity, suppressed mitochondrial OCR, reduced mitochondrial membrane potential (MMP), decreased glucose uptake, and possibly suppressed glycolysis to reduce lactate production. GC7 was predicted to have a similar yet slightly different pharmacokinetic profile as auranofin. Finally, GC7 (20 mg/kg, oral, 5/week, or 3 mg/kg, IP, 3/week) significantly inhibited tumor growth. In conclusion, GC7 showed great potential in suppressing cancer cell proliferation, probably via inhibiting TrxR and impacting mitochondria-mediated energy metabolism.

WNT16 as a promising biomarker for systemic lupus erythematosus and its role in regulating cell proliferation and apoptosis.

To investigate the expression and function of WNT16, a member of the WNT family protein, in the context of systemic lupus erythematosus (SLE). WNT16 expression was assessed in peripheral blood mononuclear cells (PBMCs) from 35 SLE patients and 25 healthy individuals using quantitative polymerase chain reaction. Additionally, serum WNT16 protein levels were quantified via enzyme-linked immunosorbent assay in 162 SLE patients, 96 healthy controls (HC), and disease controls comprised 154 individuals with rheumatoid arthritis (RA) and Sjögren's syndrome (SS). We investigated the associations between WNT16 protein levels and clinical manifestations, laboratory indices, and disease activity in SLE patients. Receiver operating characteristic (ROC) curve analysis was employed to evaluate the diagnostic potential of serum WNT16 for SLE. Furthermore, we performed a knockdown assay on Jeko-1 cells and assessed cell proliferation and apoptosis using Cell Counting Kit-8 and flow cytometry. WNT16 mRNA in SLE patients' PBMCs were significantly lower than those in HC. Furthermore, serum WNT16 in SLE patients were markedly reduced compared to HC, RA, and SS cohorts. ROC curve analysis indicated that plasma WNT16 levels could serve as a potential biomarker for SLE identification (AUC=0.809, SLE vs. HC; AUC=0.760, SLE vs. RA; AUC=0.710, SLE vs. SS). Notably, a weak positive correlation was observed between WNT16 protein and both alkaline phosphatase and lymphocyte percentages. Conversely, a weak negative correlation existed between WNT16 and low-density lipoprotein, neutrophil percentage, and the incidence of pleurisy and disease activity. Additionally, our study confirmed that WNT16 knockdown impairs cell proliferation and enhances apoptosis. Serum WNT16 levels effectively differentiate SLE patients from healthy controls and individuals with other autoimmune disorders. WNT16 serves as a potential biomarker with high sensitivity. The diminished expression of WNT16 in SLE may have a significant role in its pathogenesis through the regulation of cell proliferation and apoptosis.

Inhibition of Autophagy Augments the Anticancer Activity of KPT-330 in Mantle Cell Lymphoma Cells

To investigate the influence of novel CRM1 inhibitor KPT-330 on the autophagy of mantle cell lymphoma (MCL) cells, and effect of KPT-330 on the prolifiration of MCL cells in the presence or absence of autophagy inhibitor. CCK-8 assay was used to detect the effect of KPT-330 on MCL cell lines Z-138， Jeko-1， Granta-519， Rec-1. The effect of KPT-330 on autophagy features were determined by detecting acidic vesicular organelles (AVO) by MDC staining under fluorescence microscope and detecting protein expression of LC3B-II assessed by Western blot. Further combined application of lysosomal inhibitor Chloroquine (CQ) was used to observe its effect on the increase of LC3B-Ⅱ caused by KPT-330. CalcuSyn 2.0 software was used to detected the Combination index (CI) of KPT-330 combined with CQ. The proliferation of MCL cell lines （Z-138, Jeko-1, Grant-519, Rec-1） could be inhibited by KPT-330 in a dose-dependent manner (r =0.930, 0.946, 0.691, 0.968 respectively). The number of acidic vesicular organelles (AVO) and the expression of LC3B-II were increased in KPT-330 treated Jeko-1 and Granta-519 cells in a dose-dependent manner (r Jeko-1=0.993, r Granta-519=0.971). LC3B-II protein amounts still increased upon KPT-330 treatment with the existence of lysosomal inhibitor CQ in Jeko-1 and Granta-519 cells, which was higher than CQ or KPT-330 single drug group. The combination of KPT-330 and CQ produced the synergistic effects on cells proliferation inhibition with CalcuSyn 2.0 analysis. KPT-330 can inhibit MCL cell proliferation and induce autophagy. KPT-330 combined with autophagy inhibitor CQ could produce synergistic anti MCL effects, providing experimental basis for clinical combination therapy.

Abstract 6813: Immunosuppressive monocytes suppress CART19 functions through modulation of the IL-1 pathway

Abstract CD19-directed chimeric antigen receptor T (CART19) cell therapy has shown remarkable outcomes in B cell malignancies and was FDA approved in multiple indications, but durable remissions are limited to ~40%. Inhibitory myeloid cells in the tumor microenvironment have been found to suppress T cell expansion and contribute to CART19 failure. Here, we studied interactions between monocytes, CART19, and tumor cells to understand the impact of monocytes on CART19 effector functions. First, CD28-costimulated CART19 (CART19-28ζ) generated in the lab from healthy donors were cocultured with JeKo-1, a CD19+ mantle cell lymphoma cell line, and freshly isolated monocytes or ex vivo differentiated M2-like macrophages. M2-like macrophages were generated by incubating fresh monocytes with rhGM-CSF followed by coculturing with JeKo-1. CART19 antigen-specific proliferation was significantly inhibited by M2-like macrophages but not fresh monocytes (JeKo-1+CART19 vs JeKo-1+M2+CART19, p=0.00503). Transwell assays indicated that suppression from M2-like macrophages was not contact-dependent. IL-1 receptor antagonist (IL-1ra) was significantly elevated in M2-like macrophage coculture supernatant (JeKo-1+Mono+CART19 vs JeKo-1+M2+CART19, p=0.0292) and intracellularly in M2-like macrophages after coculture (90%+). We therefore hypothesized that M2-like macrophages inhibit CART19 by secreting IL-1ra, which blocks IL-1 signaling in CART19. We interrogated the role of IL-1ra in CART19. JeKo-1 was cocultured with CART19, supplemented with rhIL-1β only, IL-1β + IL-1ra, or IL-1β +IL-1ra + IL-1ra neutralizing antibody (neuAb). CART19 antigen-specific proliferation was improved by IL-1β (PBS vs IL-1β, p<0.0001). IL-1ra inhibited IL-1β-dependent CART19 proliferation (IL-1β vs IL-1β+IL1ra p=0.0006), which was restored by IL-1ra neutralization (IL-1β+IL-1ra vs IL-1β+IL-1ra+IL-1ra neuAb, p=0.0215). Additionally, expression of IL-1RI, the receptor of IL-1β and IL-1ra, was measured on T cells after coculturing CART19, JeKo-1, and M2-like macrophages. M2-like macrophages downregulated IL-1RI expression on CART cells, which is a potential mechanism of blocking CART response to IL-1β, resulting in further suppression of CART proliferation (JeKo-1+M2+CART19 vs JeKo-1+CART19, p=0.001). Next, we assessed the impact of IL-1ra on CART cell functions in the presence of M2-like macrophage in a mantle cell lymphoma xenograft model. NOD-SCID-γ−/- (NSG) mice were subcutaneously injected with human macrophages and luciferase+ JeKo-1 cells. Upon tumor engraftment by bioluminescent imaging, mice were randomized to treatment with CART19 + IL-1ra neuAb or control IgG for 3 weeks. CART19 + IL-1ra neuAb led to improved antitumor activity (IL-1ra neuAb vs control IgG, p=0.0033). Overall, our study revealed a role of IL-1ra in macrophage-induced CART inhibition mediated by blocking IL-1β signaling through IL-1RI. Citation Format: Kun Yun, Reona Sakemura, Truc Huynh, Claudia Manriquez Roman, Olivia Sirpilla, Carli Stewart, James Girsch, Ekene Ogbodo, Ismail Can, Jennifer Feigin, Long Mai, Hong Xia, Brooke Kimball, Makena Rodriguez, Lionel Kankeu Fonkoua, Mehrdad Hefazi, Michael Ruff, Elizabeth Siegler, Saad Kenderian. Immunosuppressive monocytes suppress CART19 functions through modulation of the IL-1 pathway [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 6813.

Abstract 3165: Targeting angiogenesis for improved survival in mantle cell lymphoma

Abstract Mantle cell lymphoma (MCL) is a subtype comprising 6% of all NHL cases and is characterized by overexpression of cyclin D1 due to chromosomal translocation t(11;14) in the nodal MCL subtype. One of the best discriminatory genes between these conventional and indolent MCL tumors is SOX11; in addition, SOX11 has been shown to promote angiogenesis in MCL. Further SOX11 protein expression in MCL patients correlated with increased microvascular density and poor outcomes. Bulk transcriptomics analysis was performed on MCL patients (n=24) and CD19-purified B cells from healthy donors (HD,n=3). To understand the role of the angiogenic process in MCL, we queried 141 genes related to angiogenesis (GO:0001525). Our analysis showed that many genes involved in angiogenesis were predominantly overexpressed in MCL compared to HD, including FLT1 and KDR (p-value;0.05; FC>2). SOX11 has been shown to regulate PDGFA in MCL cell lines. Consistent with this observation, we also see upregulated PDGFA levels in MCL patients. However, PDGFA upregulation was not statistically significant in our analysis. Further expression of VEGFR2 is overexpressed in MCL Patients (n=122) compared to naïve or activated-B cells from HD. We validated that VEGFR2 protein levels were higher in MCL primary patients than in healthy donor PBMC. Further, in two independent cohorts, high VEGFR2 expression shows significant adverse effects on 5-year overall survival (OS). To explore the potential of targeting VEGFR, we utilized lucitanib, which potently and selectively inhibits VEGFRs. Treatment with lucitanib resulted in a dose-dependent decrease in cell viability and cellular death as determined by Annexin PI staining; further, MCL cells treated with lucitanib showed inhibition of autophosphorylation of VEGFR2 and demonstrated PARP cleavage associated with cytotoxicity in-vitro. To assess the role of VEGFR2 inhibitor lucitanib as a single agent in MCL growth in-vivo, we utilized two different murine models of MCLs. First, we used cell-line-derived xenograft (CDX) models of MCL where Jeko-1 and HS5 cells were injected together, followed by treatment (lucitanib;15mg/kg QOD; IP) and measured tumor volume until the ERC of any experimental mice was reached. Kaplan-Meier survival graphs were plotted, demonstrating that lucitanib treatment prolongs survival in-vivo. Further, to confirm if the lucitanib can provide an overall survival benefit in the systemic PDX MCL model, we injected the PDX cells via the tail vein in NSG mice and followed human-B2M levels for disease progression, and overall survival was the endpoint. Lucitanib treatment significantly prolonged median overall survival significantly in the systemic MCL model. Our data demonstrates that targeting angiogenesis in MCL can provide therapeutic benefits, as demonstrated in preclinical MCL models. Citation Format: Satishkumar Singh, Anuvrat sircar, Audrey Odier, Narendranath Epperla, Natarajan Muthusamy, Samir Parekh, Lalit Sehgal. Targeting angiogenesis for improved survival in mantle cell lymphoma [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 3165.

Abstract 2840: Development, characterization, and validation of a systemic human mantle cell lymphoma model utilizing luciferase expressing Jeko-1 tumor cells

Abstract Mantle cell lymphoma (MCL) is an aggressive subtype of B-cell non-Hodgkin lymphoma that is clinically and biologically heterogenous. MCL murine models are scarce and reliable in vivo models are necessary for pre-clinical testing and identification of improved novel therapeutics. To address this need, we established and characterized a human MCL in vivo model by systemic implantation of the luciferase-expressing Jeko-1 cell line (Jeko-1-luc) into immunodeficient NCG mice. Tumor growth kinetics was evaluated by testing multiple cell inoculums, and disease progression was monitored by in vivo serial bioimaging, three-dimensional optical imaging (3D), and ex-vivo endpoint analyses. Jeko-1-luc tumor growth profile revealed moderate latency with a median survival of 40 days post implant. Subsequently, we utilized 3D optical imaging and ex-vivo imaging that confirmed the localization of tumor growth predominantly in lungs, spleen, and skeletal regions. Additionally, we determined the surface expression of selected B-cell lineage markers including CD19, CD20, and CD22. Next, we evaluated the efficacy and effect on survival in response to various standard of care agents including Vincristine, Rituximab, Palbociclib, Temsirolimus, and Cyclophosphamide. The quantification of tumor burden by in vivo and ex-vivo bioluminescent imaging demonstrated that the highest therapeutic activity and overall survival was achieved by Palbociclib and Vincristine. Flow cytometric analyses further demonstrated that tumor cells obtained from blood and selected tissues including spleen and bone marrow showed reduced tumor burden in treatment groups relative to the control group. Taken together, these results illustrate the translational value of the Jeko-1-luc model and provides a platform for evaluating novel treatment modalities for mantle cell lymphoma. Citation Format: David Harris, Andrew Wong, Elizabeth Rainbolt, Nicole Westrick, Justin Avery, Joseph Kolb, Aswani Vunnava, Zachary Ward, Chassidy Hall, Bincy John. Development, characterization, and validation of a systemic human mantle cell lymphoma model utilizing luciferase expressing Jeko-1 tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2840.

Effect of Chlorambucil Combined with Ibrutinib on Mantle Cell Lymphoma Cell Line Jeko-1 and Its Related Mechanism

To investigate the toxic effect of chlorambucil combined with ibrutinib on mantle cell lymphoma (MCL) cell line Jeko-1 and its related mechanism. The MCL cell line Jeko-1 was incubated with different concentrations of chlorambucil or ibrutinib or the combination of the two drugs, respectively. CCK-8 assay was used to detect the proliferation of the cells, and Western blot was used to measure the protein expression levels of BCL-2, caspase-3, PI3K, AKT and P-AKT. After Jeko-1 cells were treated with chlorambucil (3.125, 6.25, 12.5, 25, 50 μmol/L) and ibrutinib (3.125, 6.25, 12.5, 25, 50 μmol /L) alone for 24, 48, 72h respectively, the cell proliferation was inhibited in a time- and dose-dependent manner. Moreover, the two drugs were applied in combination at low doses (single drug inhibition rate<50%), and the results showed that the combination of two drugs had a more significant inhibitory effect (all P < 0.05). Compared with the control group, the apoptosis rate of the single drug group of chlorambucil (3.125, 6.25, 12.5, 25, 50 μmol/L) and ibutinib (3.125, 6.25, 12.5, 25, 50 μmol/L) was increased in a dose-dependent manner. The combination of the two drugs at low concentrations (3.125, 6.25, 12.5 μmol/L) could significantly increase the apoptosis rate compared with the corresponding concentration of single drug groups (all P < 0.05). Compared with control group, the protein expression levels of caspase-3 in Jeko-1 cells were upregulated, while the protein expression levels of BCL-2, PI3K, and p-AKT/AKT were downregulated after treatment with chlorambucil or ibrutinib alone. The combination of the two drugs could produce a synergistic effect on the expressions of the above-mentioned proteins, and the differences between the combination group and the single drug groups were statistically significant (all P < 0.05). Chlorambucil and ibrutinib can promote the apoptosis of MCL cell line Jeko-1, and combined application of the two drugs shows a synergistic effect, the mechanism may be associated with the AKT-related signaling pathways.

Jeduxiaoliu Formula can Induce Apoptosis of Lymphoma Cells In Vitro and In Vivo.

Lymphoma is the most common malignancy of the haematological system. Jeduxiaoliu formula (JDXLF) exerts good therapeutic effects against lymphoma, however, the mechanisms underlying these effects remain unclear. Therefore, this study aimed to investigate the mechanism of action of JDXLF. RNA-Seq was performed to examine the molecular mechanisms underlying the therapeutic effects of JDXLF against lymphoma. CCK-8 assay was performed to examine the effects of JDXLF on the proliferation of lymphoma cells. Electron microscopy was performed to examine the morphology of lymphoma cells. Flow cytometry was performed to examine the apoptosis and cell cycle of lymphoma cells. qPCR and Western blotting were performed to detect the expression of apoptotic genes and proteins. In vivo, the tumour-suppressive effect of JDXLF on lymphoma transplanted tumours was examined by establishing a subcutaneous transplantation tumour model in nude mice, and the expression of apoptotic proteins in tumour tissues was analysed via immunohistochemical staining. RNA-Seq revealed 71, 350 and 620 differentially expressed genes (DEGs) in the 1mg/mL, 4mg/mL and 8mg/mL JDXLF treatment groups, respectively. KEGG pathway analysis showed that the DEGs were significantly associated with apoptosis, TNF signalling and NF-κB signalling. In vitro experiments revealed that JDXLF inhibited the proliferation of lymphoma (Raji and Jeko-1) cells in a dose-dependent manner, induced apoptosis and upregulated the expression of Bax/Bcl-2 and caspase3. In vivo experiments revealed that JDXLF had a significant tumourshrinking effect on mice and increased the expression of the apoptosis-related proteins caspase3 and Bax/Bcl-2. This study indicates that JDXLF can induce apoptosis in lymphoma cells in vitro and in vivo. We suggest this may provide a direction for further research into lymphoma therapy.

Reduction of HIF-1α/PD-L1 by Catalytic Topoisomerase Inhibitor Induces Cell Death Through Caspase Activation in Cancer Cells Under Hypoxia.

Under severe hypoxia, cellular apoptosis is induced through hypoxia-inducible factor 1, alpha subunit (HIF-1α)-dependent P53 accumulation and P53 phosphorylation via ataxia telangiectasia mutated and ataxia telangiectasia and RAD3-related (ATR) activation via replication stress-induced DNA damage response (DDR) activation. We previously demonstrated that the topoisomerase I catalytic inhibitor, 3-O-(2'E,4'Z-decadienoyl)-20-O-acetylingenol (3EZ,20Ac-ingenol), induced apoptosis in Jeko-1 and Panc-1 cells, both of which show cyclin D1 overexpression. After progression to the S phase facilitated by nuclear cyclin D1, an intra S phase checkpoint was induced in the presence of 3EZ,20Ac-ingenol, by ATR activation in response to replication stress-induced DDR. In this study, we examined whether 3-O-(2'E,4'E-decadienoyl)-20-O-acetylingenol (3EE,20Ac-ingenol) might induce a higher degree of P53 phosphorylation and additional HIF-1α and P53 accumulation in response to replication stress-induced DDR activation under hypoxic conditions than under normoxic conditions, by controlling ATR activation. In the Panc-1 cells, 3EE,20Ac-ingenol induced P53 activation and HIF-1α-dependent P53 accumulation through cooperative ATR activation via hypoxia-induced DDR activation. Jeko-1 cells showed slight HIF-1α accumulation under hypoxia, but HIF-1α-dependent 53 accumulation was not observed in the presence of 3EE,20Ac-ingenol, so that the cells remained resistant to hypoxia. 3EE,20Ac-ingenol induces an intricate interplay between P53 and HIF-1α accumulation via ATR activation that results in a high P53 accumulation, which promoted transient expression and early disappearance of HIF-1α, accelerating cell death. Strong P53 accumulation and consequent phosphatase and tensin homolog deleted on chromosome 10 activation in Panc-1 cells also reduced HIF-1α accumulation and programmed death-ligand 1 expression, which resulted in intense apoptosis.

Synthesis and biological evaluation of novel bi-gold mitocans in lung cancer cells.

Mitochondria are promising drug target for cancer treatment. We previously demonstrated that a bi-gold compound BGC2a was more potent than the mono-gold drug auranofin in suppressing cancer cells due to increased gold atom number that led to higher drug accumulation in and thereby inhibition of mitochondria. To exploit the potential of this new strategy, we further designed and synthesized a series of bi-gold mitocans, the compounds targeting mitochondria. The results showed that most of the newly synthesized mitocans exhibited obviously lower IC50 than auranofin, an old drug that is repurposed in clinical trials for cancer treatment. The best mitocan C3P4 was nearly 2-fold more potent than BGC2a in human non-small cell lung cancer A549 cells and mantle cell lymphoma Jeko-1 cells, exhibiting substantial colony formation-suppressing and tumor-suppressing effects in A549 cells xenograft model. C3P4 induced apoptosis in a dose-dependent manner and arrested cell cycle at G0/G1 phase. The mechanistic study showed that C3P4 significantly increased the global reactive oxygen species and mitochondrial superoxide level, and reduced the mitochondrial membrane potential. C3P4 preferentially accumulated in mitochondria as measured by the gold content and substantially inhibited oxygen consumption rate and ATP production. These results further validated our hypothesis that targeting mitochondria would be promising to develop more potent anticancer agents. C3P4 may be further evaluated as a drug candidate for lung cancer treatment.

The Novel CDK9/CDK4/6/PI3K Triple Inhibitor LCI139 for the Treatment of MYC-Driven Mantle Cell Lymphoma

Mantle cell lymphoma (MCL) is an aggressive B cell malignancy with characteristic t(11;14)(q13;q32) translocation juxtaposing IGH and CCND1 gene loci. This chromosomal rearrangement leads to Cyclin D1 overexpression and cell cycle dysregulation at the G1/S phase transition. MCL is generally diagnosed at advanced stages and follows an aggressive course of disease. Although chemotherapy-based combination regimens are initially effective, most patients relapse with a median survival of only 2.5-5 years. While novel targeted therapeutics have shown promising activity in relapsed/refractory patients, inevitable relapse associated with progressively declining efficacy and increasing resistance to single agent targeted therapy is a major concern. To overcome this barrier, we designed in-silico based a novel multitarget small molecule inhibitor LCI139 which simultaneously blocks three oncogenic targets: cyclin-dependent kinase 9 (CDK9), CDK4/6, and phosphatidylinositol-3 kinase (PI3K). LCI139 is a potent CDK9/CDK4/6/PI3K inhibitor with IC 50s of 0.000039µM (CDK9), 0.0015µM/0.0036µM (CDK4/6) 0.070µM (p110α), 0.461µM (p110γ), and 0.214 (p110δ), as determined by cell free assay alpha screens. Dose-dependent screening of LCI139 against a panel of MCL cell lines revealed robust cytotoxic effects with maximal IC 50 of 86.67 nM in JeKo-1 cells, 64.8 nM in Mino cells, and 202.5 nM in Rec-1 cells. Compared with clinical single agent CDK9 inhibitor AZD4573, LCI139 is less toxic to normal human stromal cells (HS-5), whereas both LCI139 and AZD4573 have no significant cytotoxic effect on normal human foreskin fibroblast cells (Hs68). LCI139 treatment at various concentrations for 24hrs induced significant apoptotic response in two MCL cell lines, JeKo-1 (p&amp;lt;0.001 DMSO vs LCI139 100 nM, p&amp;lt;0.0001 DMSO vs LCI139 200 nM) and Mino (p&amp;lt;0.05 DMSO vs LCI139 100 nM, p&amp;lt;0.0001 DMSO vs LCI139 200 nM) as determined by Annexin V/7-AAD apoptosis staining. Similarly, LCI139-induced apoptotic cell death was further confirmed by increasing cl-PARP protein levels compared to control as assessed by Western blot analysis. LCI139 induced robust G2/M cell cycle arrest in JeKo-1 cells and G1 arrest in Mino cells. The mechanism of action of pan-CDK inhibitors has been associated with downregulation of the anti-apoptotic protein MCL-1 through inhibition of transcriptional CDKs. Our results show that short-term exposure of LCI139 (1hr and 3hr) significantly suppressed MCL-1 and MYC mRNA expression in JeKo-1 (p&amp;lt;0.00001 DMSO vs LCI139) and Mino cells (p&amp;lt;0.00001 DMSO vs LCI139). LCI139 suppressed pRpb1 (Ser2), MCL-1 and cMYC protein expression in IgM-stimulated JeKo-1 and Mino cells in a dose-dependent manner. Treatment with LCI139 had no significant effect on pAKT (Ser473) in Jeko-1 cells, whereas pAKT (Ser473) was completely abolished in Mino cells at 500nM of LCI139. Mechanistically, our findings also demonstrate that LCI139 decreased MCL-1 and cMYC protein stability and reduced half-life as accessed by cycloheximide chase assay. This decrease in cMYC stability is associated with decreased of cMYC phosphorylation at Ser62. Adaptive kinome remodeling via drug-induced transcriptional reprogramming is a known contributor to ibrutinib resistant (IR) MCL. RNA-sequencing analysis of JeKo-1 vs Jeko-1 IR cells revealed a total of 3468 differentially expressed genes (FC &amp;gt; 1.5 and FDR &amp;lt; 0.05) where 110 kinases display altered expression in JeKo-1 IR cells. Finally, we show that LCI139 overcomes chronic ibrutinib resistance by decreasing viability in JeKO-1 IR and Mino IR cells with an IC 50 of 79 nM and 89 nM, respectively. Therefore, the multitarget small molecule inhibitor LCI139 has superior potency against IR MCL cell lines and overcomes ibrutinib-resistance at nanomolar doses.

Immunoglobulin Light Chains Control Permissiveness to Malignant B-Cell Transformation By RAS-Pathway Lesions

Background: RAS-pathway activating mutations (e.g. NRAS, KRAS, BRAF) cause constitutive ERK-activation which can lead to divergent outcomes. ERK-activation not only drives early stages of pre-B cell differentiation (Yasuda et al., 2008) but also induces negative selection and cell death to eliminate aberrantly activated B-cells at later stages of development (Limnander et al., 2011). Germline mutations in this pathway cause lymphoproliferative disorder and autoimmunity but not leukemia. Furthermore, activating lesions are very common in B-cell malignancies derived from the earliest (e.g. B-ALL) and the latest (e.g. multiple myeloma) stages of B-cell development, but they are rarely found in B-cell receptor (BCR)-expressing mature B-cell derived tumors ( Figure 1). These findings are consistent with previous observations that transgenic mouse models for RAS-oncogene were able to transform BCR-negative B-cell precursors to develop B-cell acute lymphoblastic leukemia (Chan et al., 2020). In contrast, transgenic expression of these oncogenes fails to transform BCR-positive mature B-cells (Mullins et al., 2013; Chung et al., 2014). Signals from the BCR, consisting of immunoglobulin (Ig) heavy chains (μ HC) and conventional light chains (kappa LC or lambda LC), and its precursor the pre-BCR, consisting of μ HC and surrogate light chains (SLC; λ5 and VpreB), regulate the development and function of B-cells. Here, we investigated whether structural elements of the (pre-) BCR determine the outcome of oncogenic RAS activation and how oncogenic RAS in B-ALL avoids cell death to promote leukemogenesis. Results: We first studied the impact of oncogenic RAS-activation in BCR-positive mantle cell lymphoma cells. Expression of NRAS G12D in BCR-positive mantle cell lymphoma (JeKo-1) cells transiently reduced the surface expression of μ HC and eventually resulted in depletion of cells from culture in growth competition assays. We then examined whether CRISPR/Cas9-based genetic ablation of BCR components will enable permissiveness to oncogenic RAS signaling. Despite loss of μ HC function, oncogenic RAS caused depletion of JeKo-1 cells from culture. In contrast, this effect was partially reversed by genetic ablation of the constant region of immunoglobulin kappa LC ( IGKC). We next examined whether reconstitution of surrogate light chain (SLC) components of the pre-BCR will confer permissiveness to oncogenic RAS signaling in JeKo-1 cells. We engineered JeKo-1 cells to express Dox-inducible expression of λ5 (Igll1) and VpreB. In the presence of kappa LC, overexpression of SLC alone did not reverse the deleterious impact of oncogenic RAS expression. Notably, reconstitution of SLC components following genetic deletion of IGKC amplified the effect of IGKC-deletion alone and largely rescued cells from oncogenic RAS-induced survival disadvantage. Taken together, our results showed that immunoglobulin light chains control the consequences of oncogenic RAS activation in B-cells - while SLC of the pre-BCR plays a productive role in enabling oncogenic RAS-signaling, conventional light chain of the BCR plays a counterproductive role. Supporting this scenario, we found that SLC enabled oncogenic RAS to massively induce the proto-oncogene Bcl6 at the expense of the tumor suppressor Prdm1 to avoid cell death, promoting RAS-driven transformation of B-cell precursors. Upon genetic ablation of λ5, upregulation of Bcl6 was abrogated while Prdm1 was de-repressed. Consequently, RAS-driven malignant transformation of B-cell precursors was subverted. Reflecting the critical role of SLC in enabling RAS-BCL6 signaling, mature B-cells, which express BCR and lack SLC, failed to induce Bcl6 upon oncogenic RAS-activation, leading to upregulation of Prdm1. This observation may explain why persistent activation of the RAS-pathway in mature B-cells can lead to negative selection and cell death (Limnander et al., 2011). Conclusions: Our results demonstrated that immunoglobulin light chains determine permissiveness to RAS-driven malignant transformation of B-cells. Expression of SLC enables RAS-BCL6 activation, allowing B-cell precursors to avoid PRDM1-mediated tumor suppression to promote leukemogenesis. In contrast, conventional LC opposes oncogenic RAS signaling, providing a mechanistic basis of the low frequency of RAS-pathway activating mutations in BCR-expressing mature B-cell lymphomas.

Depletion of the Mitochondrial E3 Ligase MARCH5 Induces Synthetic Lethality to BCL2 Inhibitor (Venetoclax) Therapy in Cell Lines Representative of Diverse Blood Cancers

Combination therapies incorporating the BCL2 inhibitor, venetoclax (VEN), are standards of care in chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML), and display promising activity against mantle cell lymphoma (MCL), multiple myeloma (MM) and many other hematologic malignancies. However, primary resistance is observed in some diseases and even across responsive malignancies, enduring cells typically persist, ultimately driving relapse and precluding cure. Genome wide CRISPR/Cas9 screens have the potential to identify novel mediators of venetoclax resistance. However, conventional knock-out/drop out (KO/DO) screens have several limitations including the frequent identification of common essential genes as false positives and the large scale required to detect DO with high statistical confidence. We devised a novel augmentation on the conventional CRISPR/Cas9 KO/DO screen utilizing two MCL cell lines (Jeko1, Z138) which were both resistant to apoptosis induced by VEN +/- ibrutinib (IBR)(Fig 1). Rather than detecting sensitizing hits through DO, we positively enriched for sensitizing hits by flow-sorting the rare (~10%) population of cells initiating apoptosis as indicated by AnnexinV positivity after brief (6hr) BH3-mimetic exposure. At this early time point, cells whose KO had led to a lowered apoptotic threshold could be identified without sufficient time for DNA degradation. DNA yield from dying cells approached 100% efficiency. sgRNA frequencies among the control cells and those undergoing early apoptosis were analyzed using MAGecKE-MLE. This method was anticipated to reduce common essential gene contamination, increase statistical power to detect sensitizing hits due to enrichment rather than DO of relevant genes, and identify targets likely to exhibit synthetic lethality (i.e. neither VEN nor KO alone are lethal, but combination achieves potent killing). The screens in both cell lines demonstrated significant depletion of common essential genes defined by DepMap by D14 (prior to treatment). Consistent with the increased power expected from a positive selection screen, both yielded &amp;gt;50 statistically significant VEN-sensitizing hits, after correction for multiple hypothesis testing. These included the expected KOs of MCL1 and BCL2L1 (BCL-X L), confirming the validity of the method. sgRNAs resulting in KO of the E3 ligase MARCH5 were among the most enriched within dying (VEN-sensitized) cells in both Jeko1 and Z138 screens, with either VEN only or VEN+IBR treatment (Fig 1). The screen method was repeated in Jeko1 cells using the MCL1 inhibitor AMG176 and BCL-X L inhibitor A1331853, and again yielded BCL2 as well as novel hits, including MARCH5 KO as a sensitizing hit to BCL-X L inhibition. KO of MARCH5 markedly sensitized multiple human blood cancer cell lines to venetoclax (Table 1), including sensitive and resistant lines, with and without functional TP53. In Jeko1 (MCL) cells, which are highly resistant to BCL2 or BCL-X L inhibition when wildtype (10-20% apoptosis at 6µM), MARCH5 KO induced marked sensitization to single-agent VEN (LC50 8nM) or A1331853 (LC50 375nM).By introducing a degron dTAG MARCH5 construct into MARCH5 KO Jeko1 cells, we demonstrated dose-dependent sensitisation to VEN with MARCH5 depletion, marked synergy (mean Bliss score: 22.97) and synthetic lethality. Degron dTAG-mediated depletion of MARCH5 demonstrated synergy with venetoclax in MOLM13 (AML) and Z138 (MCL) cell lines, and enhanced in vitro killing of MOLM13 by VEN + azacytidine, and killing of Jeko1 and Z138 by VEN + IBR. CRISPR “death” represents a powerful modification to conventional CRISPR KO screens, with reduced common essential gene false positives, increased statistical power and enrichment for synthetic lethal hits. We demonstrate the pan-hematologic, dose-dependent VEN-sensitization achieved by MARCH5 depletion across multiple blood cancer lines with and without normal TP53 function. MARCH5 depletion also sensitizes blood cancer cell lines to BCL-X L inhibition. Addition of MARCH5 depletion to VEN-combination treatment improved MCL and AML killing in vitro and this target should be prioritized for accelerated drug development.

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

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

Humanized Monoclonal Antibody IMM47, Targeting CD24, Exhibits Exceptional Anti-Tumor Efficacy By Blocking the CD24/Siglec-10 Interaction and Can be Used As Monotherapy or in Combination with Anti-PD1 Antibodies for Cancer Immunotherapy

Background: To evaluate the anti-tumor mechanism of IMM47 by blocking the interaction of CD24/Siglec-10 through macrophage antigen presentation. Methods: IMM47 was constructed and produced using an in-house-developed CHO-K1 cell expression system. BLI, ELSIA, and flow cytometry methods were used to measure the EC50 of IMM47 binding, ADCC, ADCP, ADCT and CDC activities. hSiglec-10 Tg C57BL/6 mice congenitally transplanted with MC38-hCD24 were used to analyze the in vivo efficacy. IMM47 monotherapy or in combination with SIRP-Fc fusion protein (IMM01) or anti-PD-1 antibodies were used to test IMM47's anti-tumor efficacy in SCID mice with Jeko-1 or MCF-7 tumor cell xenotransplantation and hPD-1 Tg C57BL/6 mice with MC38-hCD24/hPD-L1 tumor cell homologous transplantation models. Results: The BLI assay results showed that the affinity between IMM47 and CD24 was 0.876 nM. ELISA results showed that the EC50s of IMM47 binding to CD24 were 0.2847 nM. The EC50 of IMM47 binding to REH, Hela, MCF-7, and HCC1954 cells were 14.09 nM, 7.14 nM, 7.54 nM and 54.89 nM, respectively. IMM47 can specifically bind to CD24-positive cells but not to CD24-negative cells. IMM47 binding to CD24 is independent of the glycosylation modification of the extracellular domain. IMM47 can only bind to CD24 in humans and chimpanzees but not in other species, including mice, rats, pigs, dogs, and cynomolgus. IMM47 does not bind to human erythrocytes but strongly binds to granulocytes. Mechanism of action study results showed that IMM47 blocks the binding of CD24 to Siglec-10, with ADCC EC50 values of 0.030 nM, 0.162 nM, 0.017 nM, and 0.202 nM on REH, Hela, MCF-7, and HCC1954, respectively. IMM47 has strong ADCT activity with an EC50 of 0.333 nM against REH. IMM47 has an ADCP EC50 of 0.067 nM and 0.099 nM on REH and MCF-7, respectively. The phagocytic activity of IMM47 was significantly stronger than that of IMM01, a SIRPα-Fc fusion protein. The CDC activity results showed that the EC50 of IMM47 on REH cells was 27.29 nM. With hSiglec-10 Tg C57BL/6 mice congenitally transplanted with MC38-hCD24 cells, in vivo efficacy assays showed that IMM47 has strong anti-tumor activity, with higher spleen M1 and M2 immune cells. After the IMM47 treatment, when tumor cells were reinoculated into mice, they did not form tumors, suggesting a memory immune response had been established. After the IMM47 treatment, tumors in 5 of 6 mice were completely cured at a dose of 3 mg/kg, with a dose-dependent relationship being observed. Pharmacodynamic in vivo study in the Jeko-1 tumor cell heterologous transplantation SCID mouse model, IMM47 exhibits strong anti-tumor activity and a dose-dependent relationship. The combination of IMM47 and IMM01 has significantly better efficacy than that of any single drug in the model of MCF-7 xenograft SCID mice. The combination of IMM47 and the PD-1 antibody Tislelizumab in the MC38-hCD24/hPD-L1 tumor cells in the homologously transplanted PD-1 transgenic C57BL/6 mouse model showed the best efficacy, and tumors in all mice were eliminated. The efficacy of IMM47 combined with either the PD-1 antibody Opdivo or Keytruda is better than that of monotherapy, and tumors in 5/6 and 6/6 mice were eliminated when combined with Opdivo and Keytruda, respectively. Four weeks after drug withdrawal, the re-inoculated cells could not form tumors and were eventually eliminated, while the re-inoculated cells in the control group grew normally and formed tumors. Conclusion: Our research showed that the humanized anti-CD24 mAb IMM47 had excellent anti-tumor effect. The extracellular domain's N-glycosylation alteration has no effect on IMM47's ability to bind to CD24. The in vitro assays revealed that IMM47 exhibits significant ADCC, ADCP, ADCT, and CDC activities. IMM47 displays strong anti-tumor activity in transgenic mouse models, and a memory immune response is generated after treatment. Additionally, an in vivo pharmacodynamics assay of IMM47 in combination with different PD-1 antibodies revealed that IMM47 exhibits synergistic therapeutic efficacy when combined with Tislelizumab, Opdivo, and Keytruda. This suggests that IMM47 has significant potential for use in the clinical setting for cancer immunotherapy, either alone or in combination with other immune checkpoint inhibitors. Finally, IMM47 avoids the adverse effects displayed by some anti-CD47 antibodies because it does not bind to human red blood cells and has a high safety profile.

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&amp;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.

Discovery of benzamide-based PI3K/HDAC dual inhibitors with marked pro-apoptosis activity in lymphoma cells

Inhibition of PI3K and histone deacetylase (HDAC) activity simultaneously using a single molecule appears to be a promising approach for cancer treatment. Current PI3K/HDAC dual inhibitors commonly use hydroxamate moiety as zinc binding group, which lack HDAC isoform selectivity and have potential genotoxicity. In this study, a novel series of benzamide-based PI3K/HDAC dual inhibitors were rationally designed and synthesized. Representative compound PH14 showed potent inhibitory activity toward PI3Kα and HDAC3, with IC50 values of 20.3 nM and 24.5 nM, respectively. This was further supported by the blockage of AKT phosphorylation and an increase in acetylated histone H3 levels in Western blot study. The advantage of simultaneously targeting PI3Kα and HDAC is not only reflected in the significant antiproliferative activity, but also in its ability to promote the apoptosis in Jeko-1 cells. Moreover, PH14 had weak inhibitory effects on CYP450 enzymes and hERG. In the pharmacokinetic study, the administration of 1 mg/kg of PH14 the administration of 1 mg/kg of PH14 resulted in a t1/2 of 10 h and an AUC (0-∞) of 2772 h ng/mL. Our study may provide ideas for the further development of novel HDAC/PI3K dual inhibitors.

Human Single Domain Antibody-Based CAR-T Cells Targeting BAFF-R Demonstrate Promising Preclinical Activity in B Cell Malignancies

Background: B-cell activating factor-receptor (BAFF-R) is a potential B-cell specific target, highly expressed in B cell malignancies and regulates B cell proliferation and survival. Single-chain antibody-based CAR-T cells targeting BAFF-R (PMB101) has demonstrated antitumor effects against human B cell malignancies and can overcome CD19 antigen loss (Qin et al. Sci Transl Med. 2019;11(511)). However, CARs built on antigen-specific single chain antibody variable fragment (scFv) may have some immunological risks. Recently, single variable domain of heavy chain antibodies are becoming an alternative to scFv to construct CARs. To improve the function of BAFF-R CAR-T cells, we designed CARs with only a fully human heavy-chain variable domain. Methods: A fully human antibody phage display library (IMARS, IASO Biotherapeutics) was used to select anti-BAFF-R clones using optimal protein/cell alternate panning. The enzyme-linked immunosorbent assay (ELISA) and flow cytometry were applied to evaluate the specificity of clones. The screened clones were grafted into a second-generation CAR with CD8α hinge, 4-1BB costimulatory and CD3ζ intracellular signaling domains to generate CAR-T cells. PMB101 and CAR-T cells constructed with VAY736 were used as positive controls. Non-transduced T cells from the same donor were used as a negative control. Results: Four candidate clones including clone 1, 5, 77 and 80 were finally screened by in vitro functional evaluation, among which clone 5, 77 and 80 were variable domain heavy chain antibodies. The expression levels of the degranulation marker CD107a on CAR-T cells constructed with the four candidate clones were significantly upregulated after coculture with BAFF-R positive tumor cells, while BAFF-R negative tumor cells could not effectively activate CAR-T cells (data not shown). The four clones exhibited similar cytotoxicity in CAR-T cells compared with the PMB101 and VAY736 CAR-T cells using the luciferase-based cytotoxicity assay (data not shown). Xenografts were established in NPG mice following intravenous injection of 1 x 10 6 jeko-1 cells expressing the firefly luciferase gene on day - 5, and a single dose of 4 x 10 6 CAR T cells were infused IV on day 0. Compared to the other groups, no progressive tumor growth was discovered in mice treated with clone 5 CAR-T cells until day 49. Clone 5 CAR-T cells showed excellent in vivo antitumor activity to PMB101 and conferred long-term survival (data not shown). Conclusion: We developed a novel BAFF-R CAR-T cell product structured on single-domain antibody. This product demonstrated promising preclinical activity, and it may provide an alternative choice for patients with B cell malignancies.

Glycerolipid Metabolism Via the MGA-MYC-NME1-PGP Axis As a Key Regulator of Oxidative Phosphorylation in Richter's Transformation

Despite advances in targeted therapies for chronic lymphocytic leukemia (CLL), Richter's transformation (RT) remains a clinical challenge. RT is an aggressive shift from CLL to lymphoma, associated with metabolic changes and aberrations in the MYC transcriptional network. Genomic analysis of paired CLL and RT cases revealed recurrent mutations or deletions in the MYC suppressor Max-gene-associated (MGA) increase from 5% (CLL) to 30% (RT) cases, highlighting its critical yet unexplored role as a driver event. We recently developed a murine RT model using B cell-restricted in vitro editing of LSK progenitors from Cd19Cre-Cas9-del(13q)-Sf3b1 ( Sf3b1-K700E) with Mga-targeting sgRNAs. This murine model demonstrated upregulation of Mga targets Myc and Nme1 (nucleoside diphosphate kinase), a distinct OXPHOS-based transcription signature, increased oxygen consumption rate, elevated ROS levels, suggesting a significant link between the Mga-Myc-Nme1 axis and OXPHOS dysregulation in RT. Given the crucial role of metabolic reprogramming in cancer, we seek to understand the molecular mechanisms of metabolites driving OXPHOS and accelerating CLL transformation. Unraveling these processes could revolutionize therapeutic approaches for managing RT in clinical settings. To identify metabolites driving dysregulated OXPHOS in RT, we first conducted paired RNA-seq and unlabeled metabolomic profiling on normal and RT mouse splenic B cells (n=3/group). To translate our discovery from the murine RT model to human disease, we performed metabolomic profiling using MEC1 and Nalm6 cell lines with MGA deletion. Our metabolomic analysis of murine RT cells revealed 44 upregulated metabolites, including TCA cycle substrates (glutamine fold change (FC)=9.6, p=0.001;asparagine FC= 4.0, p=0.006), fatty-acid intermediates (glycerol-3P FC=11.7, p=0.003; carnitine FC=4.3, p=0.02; palmitate FC= 4.1, p= 0.08), and glycolysis intermediates (3-phosphoglycerate FC= 4.0, p=0.03; UDP-glucose FC=12.7, p=0.02; glycine FC=4.7, p=0.03). Moreover, integrated metabolite analysis (MetaboAnalyst v5.0) identified 63 common upregulated metabolites in MGA KO (knockout) human cell lines and murine RT, enriching glycerolipid, and fatty acid oxidation, associated pathways, indicating a potential role for MGA deletion in driving dysregulated fatty acid metabolism. Pathway enrichment analysis using integrated RNA-seq and metabolite analyses showed that the glycerolipid pathway was one of the most enriched pathways among mouse RT and B-cell lines (Nalm6 and MEC1) with MGA KO. Additionally, two key fatty-acid enzymes, fatty acid synthetase (FASN) and phosphoglycerate phosphatase (PGP), displayed significant mRNA and protein changes, as validated by qPCR and immunoblotting. Furthermore, elevated lipid accumulation and increased free glycerol were confirmed based on lipi-green flow cytometry and glycerol-bioluminescent assays, suggesting an important role of glycerolipid metabolism in MGA KO-associated OXPHOS. PGP, a glycerol-3-phosphatase, regulates glycerol-3-P levels, a key intermediate in glucose, lipid, and energy metabolism. Overexpression of MYC and NME1 increases PGP levels in B-cell lines (MEC1, Nalm6, and Jeko), confirming PGP as a direct target of the MGA-MYC-NME1 axis. To determine the role of PGP in MGA KO (knock-out) cells, we used CRISPR/Cas9 to KO PGP in MEC1, Nalm6, and JeKo cell lines. KO of PGP decreased cell growth and reduced OXPHOS compared to controls based on CCK8 absorbance and Seahorse Mitostress assays respectively. Moreover, PGP KO downregulated the mTOR (p4E-BP1) pathway, emphasizing the significance of dysregulated glycerol and fatty acid metabolism in CLL to RT-driven OXPHOS. Our study reveals key metabolites driving RT and highlights the crucial role of the MGA-MYC-NME1-PGP axis in regulating OXPHOS in Mga KO RT cells. Our results thus suggest targeting glycerolipid metabolism and related pathways may offer effective strategies to conquer RT

Abstract 4098: Chimeric antigen receptor (CAR) T cells overexpressing Bcl-xL increase proliferation and antitumor activity alone and in combination with BH3 mimetics

Abstract Background: Chimeric antigen receptor (CAR) T cells have become a well-established treatment option for patients, with six products approved for different hematologic diseases and new approvals allowing for their therapeutic use as early as second line. However, relapse rates of around 50% have been observed in all patient subsets, with one major mechanism associated with CAR T failure being cancer cell resistance to apoptosis. A form of cancer therapeutic named BH3-mimetics has been designed to inhibit members of the anti-apoptotic B cell lymphoma-2 (Bcl-2) family and, therefore, directly activate the apoptotic machinery in malignant cells. We hypothesized that integration of these anti-apoptotic molecules into CAR T cells would induce resistance towards the BH3 mimetics and allow combinational therapeutic approaches. Methods: 4-1BB and CD28 CAR constructs were designed to overexpress one of four anti-apoptotic proteins: wildtype Bcl-2, a Venetoclax-resistant Bcl-2 variant (G101V), B cell extra-large (Bcl-xL), or myeloid cell leukemia-1 (Mcl-1). CAR T cells made from these constructs were tested against leukemia (Nalm6) and lymphoma (JeKo-1) cell lines in combination with three different BH-3 mimetics: Venetoclax (ABT-199, an FDA-approved Bcl-2 inhibitor), Navitoclax (ABT-263, a Bcl-2/Bcl-xL inhibitor), and AZD5991 (an Mcl-1 inhibitor). Results: CAR T cells with a 4-1BB costimulatory domain tended to have increased killing over CARs with CD28 and were less susceptible to apoptosis; therefore, 4-1BB CARs were used for all further testing. Integration of Bcl-2 family proteins into CAR T cells didn’t impair or even increase tumor cell clearance in vitro; however, in combination with Venetoclax, Navitoclax, or AZD5991, killing capacity significantly increased compared to control CAR T cells. Even without combination with drugs, CAR T cells overexpressing Bcl-xL and Bcl-2 (both wildtype and mutant) provided higher anti-tumor activity and prolonged survival against JeKo-1 cells in vivo, whereas only Bcl-xL overexpression showed increased tumor control compared to regular 4-1BB CARs against Nalm6 cells. Conclusion: Of the tested antiapoptotic proteins, Bcl-xL overexpressing CAR T cells proved superior, having higher proliferation and increased anti-tumor activity in combination with or without BH3 mimetics, providing a new strategy to optimize CAR T cell function for the treatment of leukemia and lymphoma. Citation Format: Felix Korell, Michael Olson, Diego Salas-Benito, Mark B. Leick, Rebecca C. Larson, Harrison Silva, Alessandro Gasparetto, Trisha R. Berger, Amanda Bouffard, Michael C. Kann, Markus Mergen, Tamina Kienka, Marc Wehrli, Stefanie R. Bailey, Anthony Letai, Marcela V. Maus. Chimeric antigen receptor (CAR) T cells overexpressing Bcl-xL increase proliferation and antitumor activity alone and in combination with BH3 mimetics. [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 4098.