Targeting Bcl-2 Research Articles

Abstract 2006: Preclinical evaluation of dual AURKA (LY3295668) and BCL2 (venetoclax) inhibition in neuroblastoma (NB)

Abstract Introduction: Deregulated expression of the MYCN oncogene is associated with the development of many neural-derived pediatric cancers including NB, medulloblastoma and atypical teratoid rhabdoid tumors. Amplification of the MYCN oncogene is present in over 40% of high-risk NBs that remain lethal in most patients, thus developing better therapies for these patients is essential. While direct therapeutic targeting of MYCN protein remains challenging, we previously demonstrated synergistic activity of dual targeting of BCL-2 (venetoclax) and AURKA (MLN8237) in MYCN-amplified NB (Ham Cancer Cell, 2016). With MLN8237 no longer being developed due to toxicities related to AURKB inhibition, testing of a highly selective AURKA inhibitor, LY3295668, is warranted. Methods: LY3295668 (Chemie Tek) and venetoclax (Selleck Chem) were tested alone and in combination in six NB PDX models using 2-3 mice/arm. Five Patient Derived Xenograft (PDX) models tested (COG-N-424x, COG-N-453x, NB-1643, Obelix-Rx and Obelix-2x) were MYCN-amplified and one xenograft model (SK-N-AS) was MYCN-non-amplified. First, a comprehensive toxicity study, testing LY3295668 at 30mg/kg with venetoclax at 100mg/kg, 75mg/kg, 50mg/kg, and 25mkg/kg, was performed. Based on tolerability of combination, an optimal dose of 30mg/kg for LY3295668 and 50mg/kg for venetoclax was selected and administered once daily, seven days a week, via oral gavage for 60 days. These were the maximum tolerated doses when tested in combination, that did not result in weight loss greater than 15% or other overt signs of toxicity. Results: LY3295668 (30mg/kg) and venetoclax (50mg/kg) were well tolerated as monotherapy, and when administered in combination showed a maximum weight loss of 13%. Single agent therapy resulted in minimal if any growth delay. Four of five MYCN-amplified PDX models demonstrated maintained complete responses and one showed significant tumor growth delay. No effect was observed in the MYCN-non-amplified model with single agent or combination treatment compared to vehicle. Conclusions: Dual targeting of AURKA and BCL2 with LY3295668 and venetoclax is tolerable and demonstrates compelling anti-tumor activity in MYCN-amplified NB PDX models, validating our earlier findings (Ham et al). These preclinical results provisionally support clinical development of this combination for NB patients with MYCN amplification. Assessment of additional PDX models is currently ongoing and will be reported. Citation Format: David Groff, Colleen Casey, Kateryna krytska, Matthew Tsang, Christina Acholla, John M. Maris, Yael P. Mosse. Preclinical evaluation of dual AURKA (LY3295668) and BCL2 (venetoclax) inhibition in neuroblastoma (NB) [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 2006.

Abstract 1490: Development of a 5-FU modified miR-129 mimic as a novel therapeutic for non-small cell lung cancer

Abstract Lung cancer is the leading cancer type of cancer-related deaths in the U.S., with non-small cell lung cancer (NSCLC) accounting for a majority of cases. Despite innovations in cancer therapeutics, due to resistance to these therapeutics, the 5-year survival rate for patients with metastatic lung cancer has remained poor. As a result, there is an urgent need to develop new and effective therapeutics, including the study of RNA-based therapeutics. Previous studies from our laboratory have shown that 5-fluorouracil (5-FU) modified tumor suppressor miRNAs significantly enhance the therapeutic efficacy, stability, and deliverability of miRNAs in colorectal cancer (CRC) and pancreatic cancer. In this study, we have characterized the therapeutic potential of miR-129 modified with 5-FU (5-FU-miR-129) in non-small cell lung cancer in vitro via Annexin V staining, cell cycle analysis, and Western Blot. 5-FU-miR-129’s therapeutic potential was also assessed in vivo via IVIS imaging of NSCLC xenografts. We have demonstrated that 5-FU-miR-129 can induce apoptosis (7.2-fold in A549 and 2.9-fold in Calu-1) and cell cycle arrest (12.7-fold change in G1/S phase ratio in A549) in NSCLC in vitro. Furthermore, 5-FU-miR-129 has been found to retain target specificity by downregulating miR-129 targets BCL2 and HMGB1 in a cell line-dependent manner. As was observed in previous studies, we have also demonstrated the ability of 5-FU-miR-129 to be delivered in cancer cells without the use of a transfection vehicle in vitro. Finally, we were also able to demonstrate the therapeutic potential of 5-FU-miR-129 in inhibiting NSCLC tumor growth in vivo, as seen by a significantly decreased change in tumor growth by 37 days post-treatment in the 5-FU-miR-129 treated group compared to the negative control group (79.8% reduction in tumor growth). In addition, we observed a significant increase in survival probability and a reduced hazard ratio in mice treated5-FU-miR-129 when compared to the negative control group. Furthermore, we did not observe any visible side effects (hair loss) of mice treated with 5-FU-miR-129 compared to chemotherapeutic drugs 5-FU and gemcitabine (Gem). 5-FU-miR-129’s therapeutic potential in NSCLC further demonstrates the potential for modified miRNAs as a platform technology in cancer therapeutics. Citation Format: Ga-Ram Hwang, John G. Yuen, Hannah Farley, Jingfang Ju. Development of a 5-FU modified miR-129 mimic as a novel therapeutic for non-small cell lung cancer [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 1490.

Abstract 3959: Targeting BCL2 family protein phosphorylation in venetoclax resistant lymphoid malignancies

Abstract Introduction: BCL2 family protein dysfunction is an important mediator of chemoresistance in lymphoid malignancies. Venetoclax (VEN), a BH3 mimetic that selectively targets the anti-apoptotic protein BCL2, is FDA approved for treatment of chronic lymphocytic leukemia (CLL), with clinical trials underway for other lymphoid malignancies. Despite the clinical efficacy of VEN monotherapy, complete remission and progression free rates are still relatively low, thereby indicating the existence of inherent or acquired mechanisms of resistance. Preclinically, increases in anti-apoptotic BCL2 and MCL1 phosphorylation (BCL2-P/MCL1-P) and MCL1 protein level have been implicated in chemoresistance. We therefore question if phosphorylation of these proteins may drive VEN resistance and if removing this modification may re-sensitize resistant cells to VEN induced cell death. Methodology: BH3 profiling (a functional assay that assesses mitochondrial apoptotic priming and anti-apoptotic protein dependence), western blot and cell death assays were performed. Result: We report that VEN acquired resistant DLBCL cell line (OCI-Ly1R) has increased BCL2-P, MCL1-P and MCL1 protein levels as compared to sensitive parental cell line (OCI-Ly1S), while inherently resistant DHL cell line (Su-DHL4) possesses higher MCL1-P and MCL1 protein levels as compared to OCI-Ly1S. We further show that this increase in phosphorylation status contributes to the induction of VEN resistance. Using BH3 profiling, we demonstrate that anti-apoptotic protein phosphorylation-driven VEN resistance involves a change in sensitivity to pro-apoptotic proteins, with an increase in sensitivity to MCL1 inhibition and reciprocal decrease in sensitivity to BCL2 inhibition. We further show that BCL2 and MCL1 dephosphorylation by PP2A activating drugs (PADs) re-sensitizes resistant cells to VEN treatment. Mechanistically, PADs such as the fingolimod (FTY720) not only dephosphorylate BCL2-P and MCL1-P, but also dissociate BAX from BCL2 and destabilize MCL1 protein. These changes lead to the rewiring of cellular sensitivity to BCL2 inhibition, thereby re-sensitizing both VEN acquired and inherent resistant cells to VEN induced cell death. Importantly, the PP2A activating effects of FTY720 were recapitulated in primary cells from the peripheral blood of 16 CLL patients, where FTY720 treatment also reduced BCL2-P, MCL1-P and MCL1 protein levels and dissociated BAX from BCL2. This led to an increased sensitivity to BCL2 inhibition and enhanced cell death upon combined treatment with FTY720 and VEN. Conclusion: Our work defines a new targetable mechanism of VEN resistance in lymphoid malignancies due to the phosphorylation of anti-apoptotic BCL2 family proteins. PADs re-sensitize resistant malignant cells to VEN treatment via dephosphorylation of anti-apoptotic proteins, suggesting a promising combination approach to explore further. Citation Format: Stephen J. Chong, Fen Zhu, Jolin X. Lai, Liam Hackett, Mary C. Collins, Shazib Pervaiz, Jean-Philippe Coppe, Matthew S. Davids. Targeting BCL2 family protein phosphorylation in venetoclax resistant lymphoid malignancies [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 3959.

Cytotoxicity of Amphotericin B and AmBisome: In Silico and In Vivo Evaluation Employing the Chick Embryo Model.

Leishmaniasis has been identified as a significant disease in tropical and subtropical regions of the world, with Iran being one of the disease-endemic areas. Various treatments have been applied for this disease, and amphotericin B (Amp B) is the second line of treatment. Side effects of this drug have been reported in various organs. The present study investigated the effects of different types of Amp B on fetal organs using in silico and in vivo assays (chicken embryos). In vivo analysis was done by checking pathological changes, angiogenesis, and apoptosis alterations on eggs treated by Amp B and AmBisome. In silico approach was employed to predict the affinity of Amp B and AmBisome to the vascular endothelial growth factor A (VEGF-A), its receptor (KDR1), apoptotic-regulator proteins (Bcl-2-associated X protein (Bax), B-cell lymphoma (Bcl-2), and Caspase-8. The ADME-toxicity prediction reveals that AmBisome possesses a superior pharmacological effect to Amp B. The best result of all the dockings in the Molegro Virtual Docker (MVD) was obtained between Bax, Bcl-2, Caspase-8, KDR1, and VEGF-A targets. Due to the lower Egap (HOMO–LUMO) of AmBisome, the chemical reactivity of AmBisome was higher than that of Amp B. In vivo analysis showed that embryos that received Amp B exhibited less vascular density than AmBisome. Amp B alone significantly increased the expression of apoptosis and decreased angiogenesis genes compared to AmBisome. The histopathology analysis of the treated embryos showed a reduction in the blood vessel collapse and an increase in degenerative and apoptotic–necrotic changes in the embryonic tissues. Overall, the results suggest the potential benefits of AmBisome over Amp B, which might be a better treatment strategy to treat leishmaniasis during pregnancy.

First-in-human study of pelcitoclax (APG-1252) in combination with paclitaxel in patients (pts) with relapsed/refractory small-cell lung cancer (R/R SCLC).

e20612 Background: Targeting BCL-2/BCL-xL proteins is considered an important approach for anticancer drug development. Investigational pelcitoclax (APG-1252) as a single agent is a novel BCL-2/BCL-xL dual inhibitor, which has demonstrated synergistic antitumor activity in combination with chemotherapy preclinically through targeting both BCL-xL and MCL-1 pathways. The phase I study of pelcitoclax suggested promising antitumor activity and a favorable safety profile. Methods: The primary aim of this study was to determine the safety and preliminary efficacy of pelcitoclax combined with paclitaxel in patients with R/R SCLC. Patients received pelcitoclax 160 mg or 240 mg IV infusion on Days 1, 8, and 15 plus paclitaxel 80 mg/m2 on Days 1 and 8 of a 21-day cycle. The primary endpoint of phase Ib was to characterize the safety and tolerability of the combination. The primary endpoint of phase 2 was the objective response rate (ORR) of pelcitoclax at the RP2D plus paclitaxel using RECIST v1.1. Results: On December 20, 2021, 28 pts (median age, 63 [range, 37-77] years) were enrolled. The median (range) duration of treatment was 84 (7-259) days. The RP2D of pelcitoclax was determined to be 240 mg. A total of 26 pts experienced any grade treatment-related adverse events (TRAEs), including anemia (32.1%); ALT or AST elevation (28.6% each); neutropenia (25%); fatigue, leukopenia, or thrombocytopenia (21.4% each); peripheral neuropathy, nausea, or alopecia (17.9% each); peripheral sensory neuropathy or decreased appetite (14.3% each); and lymphopenia, hyponatremia, or dizziness (10.7% each). A total of 15 pts had at least one ≥ grade 3-4 TRAE, and 11 (39.3%) had serious AEs. No dose-limiting toxicity was reported. A total of 5 of 20 evaluable pts (4 pts in the 240 mg cohort and 1 in the 160 mg cohort) reported PR (ORR 25%), with a median response duration of 83 days. The pharmacokinetics profile showed that pelcitoclax systemic exposure increased between 160 and 240 mg, with an average terminal half-life of 3.7 to 7.4 hours and without any drug-drug interaction. Conclusions: Pelcitoclax was well tolerated at doses of up to 240 mg/week. Treatment with pelcitoclax 240 mg and paclitaxel 80 mg/m2 demonstrated modest antitumor activity in pts with R/R SCLC. Further clinical exploration, preferably in selected patient populations with relevant biomarkers, is warranted to fully elucidate the clinical activity of pelcitoclax. Clinical trial information: NCT04210037.

Single-cell immune mapping of adenoid cystic carcinoma (ACC) reveals potential therapeutic targets for the aggressive solid subtype.

6090 Background: ACC is a common salivary gland malignancy for which there is no FDA approved therapies. Despite a quiet genome, ACC has significant tumoral heterogeneity, which may facilitate the metastatic relapse. Studies focusing on the deep profile of ACC tumor microenvironment (TME) are lacking. Here we explored the TME of ACC using imaging mass cytometry (IMC) and assessed TME attributes and its association with histology and clinical outcomes. Methods: Two tissue microarrays from 62 ACC patients (pts) were built and stained with 37 metal-tagged markers. IMC was performed with the Fluidigm Helios CyTOF instrument utilizing the Hyperion Imaging System Laser ablation. Tissue and cell segmentation and multiplex imaging analysis were performed with Visiopharm software using pre-trained artificial intelligence algorithms. Comparison of cell types and markers densities among histology sub-groups were analyzed with Wilcoxon signed-rank test and its association with overall survival (OS) with log-rank and Cox Proportional Hazards. Results: Of 62 pts, 37% (23/62) had solid histology, 19% (12/62) were metastatic at diagnosis, and 55% (30/54) had disease recurrence during follow-up. With a median follow-up of 7.9 y, the median OS was 9.3y (CI 95%, 7.8-NR). Pts with solid subtype had poorer OS than non-solid histology (5.2 vs 14.6 y, p = 0.004). The IMC final dataset comprised 507,524 single-cells. No significant differences were found in cell subpopulations density between solid and non-solid histology. The most represented cell population in the stroma were macrophages, followed by CD8+ T cells, and fibroblasts. A higher percentage of M2-macrophage was associated with poor survival (p = 0.001), whereas, a higher percentage of M1-macrophage (M1:M2 ratio) was associated with better prognosis. No other immune cell type, fibroblasts or immune cell functional markers (TIGIT, TIM3, granzyme B, and PD-L1) correlated with survival. Regarding tumor markers, a higher expression of BCL-2, B7-H4, and Ki67 in the tumor cells were associated with worst survival; and remained statistically associated after adjustment for histology and staging (all p < 0.001). Solid histology had a significantly higher density of tumor cells expressing B7-H4, BCL-2, and Ki67 compared to cribriform and tubular histology. A higher expression of myoepithelial marker (p63+) were associated with a better survival when compared with tumors with low p63 expression. Conclusions: ACC’s TME is composed mainly of macrophages. Despite having no significant differences in cellular composition, a higher density of tumor cells expressing BCL-2, B7-H4 and Ki67 were found in the solid histology and these markers were independent predictors of poor prognosis. The overexpression of BCL-2 and B7-H4 in the solid histology provides a scientific rationale for BCL-2 and/or B7-H4 targeting for the most aggressive ACC.

Puerarin: A Potential Therapeutic for Colon Adenocarcinoma (COAD) Patients Suffering From SARS-CoV-2 Infection.

Patients with colonic adenocarcinoma (COAD) are at relatively high risk of SARS-CoV-2 infection. However, there is a lack of medical strategies to treat COVID-19/COAD comorbidity. Puerarin, a natural product, is a known antiviral, antitumor, and immunomodulatory effect. Therefore, we hypothesised that puerarin could be used to treat COVID-19/COAD patients. Based on network pharmacology and bioinformatics analysis, the potential targets and pharmacological mechanisms of puerarin in COVID-19/COAD were identified. By intersecting therapeutic target genes for puerarin, COVID-19-related genes and COAD-related genes, 42 target genes of puerarin that could potentially treat COVID-19/COAD comorbidity were obtained. By using the 42 potential target genes to construct the protein-protein interaction (PPI) network, we obtained five core target genes, namely RELA, BCL2, JUN, FOS, and MAPK1. The results of bioinformatics analysis revealed that puerarin could be able to treat COVID-19/COAD comorbidity through apoptosis, antiviral, antioxidant, NF-κB signaling pathway, MAPK signaling pathway, IL-17 signaling pathway, TNF signaling pathway, and HIF-1 signaling pathway etc. This study found that puerarin has the potential to treat COVID-19/COAD patients and that the therapeutic target genes obtained in the study may provide clues for the treatment of COVID19/COAD comorbidity.

Free fatty acid-induced miR-181a-5p stimulates apoptosis by targeting XIAP and Bcl2 in hepatic cells

AimsNon-alcoholic fatty liver disease is one of the major health concerns in the World. The dietary free fatty acids (FFAs) affect the metabolic status of the hepatocytes by modulating cellular pathways. In this study, we showed that free fatty acids stimulate apoptosis by upregulating miR-181a-5p expression, which in turn targets XIAP and Bcl2. MethodsHuh7 cells were incubated with FFAs for 72 h and the expression of XIAP, Bcl2, bax, pAkt, Akt, PTEN and β-actin were determined by Western blots, and miR-181a-5p expression was determined using real-time RT-PCR. The Huh7 cells were transfected with either miR-181a-5p pre-miRs or anti-miR-181a-5p and the regulation of apoptosis and proliferation was studied. Three groups of C57BL/6 mice (n = 6 per group) were fed with standard diet, CSAA or CDAA diet for 6, 18, 32 and 54 weeks. Total protein and RNA were isolated from the liver tissues and used for Western blots and real-time RT-PCR respectively. Key findingsFFAs inhibited Akt phosphorylation, expression of XIAP and Bcl2, while upregulating the expression of PTEN, bax, and miR-181a-5p in Huh7 cells. Similar results were observed when the Huh7 cells were transfected with miR-181a-5p premiRs, while these changes were reversed in anti-miR-181a-5p-transfected, FFA-treated Huh7 cells. The CDAA-fed mice showed a significant inhibition of Akt phosphorylation, XIAP and Bcl2, whereas PTEN and bax expression were upregulated. The expression of miR-181a-5p was also significantly higher in CDAA-fed mice. SignificanceThese findings showed that free fatty acids induced apoptosis via upregulating miR-181a-5p in hepatic cells.

Targeting MYC and BCL2 by a natural compound for "double-hit" lymphoma.

Concurrent translocations of MYC and BCL2 lead to abnormal expression of both oncoproteins, which contribute to the aggressive clinical characteristics of double-hit lymphoma (DHL). An effective therapy for DHL remains an unmet clinical need. In this study, we showed that both Ca2+/calmodulin-dependent protein kinase II δ (CAMKIIδ) and γ (CAMKIIγ) were highly expressed in DHL. Both isoforms of CAMKII stabilize c-Myc protein by phosphorylating it at Ser62, increase BCL2 expression, and promote DHL tumor growth. Inhibition of CAMKIIδ and CAMKIIγ by either berbamine (BBM) or one of its derivatives (PA4) led to the down regulation of c-Myc and BCL2 proteins. BBM/PA4 also exhibited anti-tumor efficacy in DHL cell lines and NSG xenograft models. Altogether, CAMKIIδ and CAMKIIγ appear to be critical for DHL tumor development and are promising therapeutic targets for DHL.

Rhomboid pseudoproteases: An Achilles heel's for BCL-2/IP3R-dependent resistance to ER stress-induced cell death

Anti-apoptotic BCL-2 targets and inhibits IP3 receptors (IP3Rs), thereby preventing apoptosis by limiting ER-mitochondrial Ca2+ flux. Recently, Dulloo et al. (2022), Nat. Comm. 13:1257[6] revealed a novel role for rhomboid pseudoproteases in ER stress-induced apoptosis by stripping BCL-2 from IP3Rs, thereby enabling pro-apoptotic Ca2+ signaling.

Redefining CD56 as a Biomarker and Therapeutic Target in Multiple Myeloma.

Multiple myeloma is an incurable, genetically heterogeneous disease, without available tailored therapeutic approaches. CD56 signaling promotes multiple myeloma growth and adhesion, by activating CREB1 target genes, MCL1 and BCL2. Inhibition of CREB1 alone or in combination with lenalidomide is an unexplored synthetic lethal approach in CD56-expressing patients with multiple myeloma.

Effect of lentivirus-mediated peroxiredoxins 6 gene silencing on the phenotype of human gastric cancer BGC-823 cells.

Peroxiredoxins (PRDX6) regulates the occurrence and progression of cancer. The aim of this study is to investigate the effect of PRDX6 knockdown on the biological behavior of human gastric cancer cell line BGC-823 cells. Research article. The differential expression of PRDX6 in gastric cancer and normal gastric tissues was tested by immunohistochemistry. Ribonucleic acid plasmid of PRDX6 gene was packaged using a lentivirus, and BGC-823 cells were transfected with the lentivirus to obtain a BGC-823 cell line in which the expression of PRDX6 was stably silenced. The proliferation activity of BGC-823 cells was detected using the cell counting kit-8 method. The effect of PRDX6 on the migration and invasion of BGC-823 cells was evaluated using the scratch test and Transwell assay, and the expression of related proteins was detected by western blot. The expression of PRDX6 in gastric cancer was significantly increased (P < 0.05). Compared with those in the untransfected and negative control groups. The proliferation, migration, and invasion of gastric cancer BGC-823 cells were significantly inhibited, and the apoptotic rates were significantly increased in the lentivirus-transfected (short hairpin-PRDX6) group. Western blot analysis showed that the expression of Bax protein increased, whereas that of proliferating cell nuclear antigen, Bcl-2, PI3K, phospho (p-Akt), and phosphorylated-mammalian target of rapamycin (mTOR) decreased significantly compared with that in WT and vector groups (P < 0.05). The knockdown of PRDX6 gene expression in BGC-823 cells can inhibit the proliferation, migration, and invasion of gastric cancer cells and promote apoptosis, thereby affecting gastric cancer cells.

Inhibition of microRNA-665 Alleviates Septic Acute Kidney Injury by Targeting Bcl-2.

Sepsis can easily cause acute kidney injury (AKI) and seriously endanger human health. This article aims to investigate and study the role of microRNA-665 (miR-665) in septic AKI and the underlying molecular mechanism. Lipopolysaccharide (LPS) was used to construct cell and animal models of septic AKI. The expression of miR-665 in cells and kidney tissues was detected by quantitative reverse-transcription polymerase chain reaction (RT-PCR). The contents of inflammatory factors (TNF-α, IL-1β, and IL-6) in the cell supernatant were detected using commercial kits. Renal tissue damage was observed by hematoxylin-eosin (HE) staining. Kidney function was assessed by serum Cr, serum BUN, and urine NAG levels. The apoptosis of HK-2 cells was analyzed by flow cytometry and TUNEL staining. Luciferase activity assay was performed for the verification of the target of miR-665. The expression of miR-665 was increased in the cell model and animal model of septic AKI constructed by LPS. By transfecting miR-665 inhibitor in HK-2 cells and injecting miR-665 antagomir (antagomiR-665) through the tail vein of rats, the expression of miR-665 in HK-2 cells and rat kidneys was remarkably reduced. Silencing miR-665 dramatically inhibited the expression of inflammatory factors (TNF-α, IL-1β, and IL-6) in LPS-induced HK-2 cells and reduced LPS-induced apoptosis in HK-2 cells. At the same time, the levels of serum Cr, serum BUN, and urine NAG decreased markedly, and the damage of the kidney was also alleviated. Finally, luciferase reporter experiments demonstrated that miR-665 directly targets Bcl-2. We revealed that miR-665 expression was increased in septic AKI, and silencing miR-665 could inhibit LPS-induced inflammation and apoptosis of the kidney by targeting Bcl-2, thereby improving renal function.

Dual BTK/SYK inhibition with CG-806 (luxeptinib) disrupts B-cell receptor and Bcl-2 signaling networks in mantle cell lymphoma

Aberrant B-cell receptor (BCR) signaling is a key driver in lymphoid malignancies. Bruton tyrosine kinase (BTK) inhibitors that disrupt BCR signaling have received regulatory approvals in therapy of mantle cell lymphoma (MCL). However, responses are incomplete and patients who experience BTK inhibitor therapy failure have dire outcomes. CG-806 (luxeptinib) is a dual BTK/SYK inhibitor in clinical development in hematologic malignancies. Here we investigated the pre-clinical activity of CG-806 in MCL. In vitro treatment with CG-806 thwarted survival of MCL cell lines and patient-derived MCL cells in a dose-dependent manner. CG-806 blocked BTK and SYK activation and abrogated BCR signaling. Contrary to ibrutinib, CG-806 downmodulated the anti-apoptotic proteins Mcl-1 and Bcl-xL, abrogated survival of ibrutinib-resistant MCL cell lines, and partially reversed the pro-survival effects of stromal microenvironment-mimicking conditions in primary MCL cells. Dual BTK/SYK inhibition led to mitochondrial membrane depolarization accompanied by mitophagy and metabolic reprogramming toward glycolysis. In vivo studies of CG-806 demonstrated improved survival in one of the two tested aggressive MCL PDX models. While suppression of the anti-apoptotic Bcl-2 family proteins and NFκB signaling correlated with in vivo drug sensitivity, OxPhos and MYC transcriptional programs were upregulated in the resistant model following treatment with CG-806. BAX and NFKBIA were implicated in susceptibility to CG-806 in a whole-genome CRISPR-Cas9 library screen (in a diffuse large B-cell lymphoma cell line). A high-throughput in vitro functional drug screen demonstrated synergy between CG-806 and Bcl-2 inhibitors. In sum, dual BTK/SYK inhibitor CG-806 disrupts BCR signaling and induces metabolic reprogramming and apoptosis in MCL. The Bcl-2 network is a key mediator of sensitivity to CG-806 and combined targeting of Bcl-2 demonstrates synergy with CG-806 warranting continued exploration in lymphoid malignancies.

Targeting Bcl6 in the TREX1 D18N murine model ameliorates autoimmunity by modulating T-follicular helper cells and germinal center B cells.

The Three Prime Repair EXonuclease I (TREX1) is critical for degrading post‐apoptosis DNA. Mice expressing catalytically inactive TREX1 (TREX1 D18N) develop lupus‐like autoimmunity due to chronic sensing of undegraded TREX1 DNA substrates, production of the inflammatory cytokines, and the inappropriate activation of innate and adaptive immunity. This study aimed to investigate Thelper (Th) dysregulation in the TREX1 D18N model system as a potential mechanism for lupus‐like autoimmunity. Comparison of immune cells in secondary lymphoid organs, spleen and peripheral lymph nodes (LNs) between TREX1 D18N mice and the TREX1 null mice revealed that the TREX1 D18N mice exhibit a Th1 bias. Additionally, the T‐follicular helper cells (Tfh) and the germinal celter (GC) B cells were also elevated in the TREX1 D18N mice. Targeting Bcl6, a lineage‐defining transcription factor for Tfh and GC B cells, with a commercially available Bcl6 inhibitor, FX1, attenuated Tfh, GC, and Th1 responses, and rescued TREX1 D18N mice from autoimmunity. The study presents Tfh and GC B‐cell responses as potential targets in autoimmunity and that Bcl6 inhibitors may offer therapeutic approach in TREX1‐associated or other lupus‐like diseases.

MiR-30b-5p inhibits osteoblast differentiation through targeting BCL6

ABSTRACT Human bone marrow mesenchymal stem cells (hBMSCs) are attractive candidates for new therapies to improve bone regeneration and repair. This study was to identify the function of the miR-30b-5p/BCL6 axis in osteogenic differentiation of hBMSCs. Realtime-quantitative PCR (RT-qPCR) and Western blotting were used to measure the relative expression of ALP, OCN, RUNX2, miR-30b-5p, and BCL6 during osteogenic differentiation of hBMSCs. The relationship between miR-30b-5p and BCL6 in hBMSCs was identified using dual-luciferase reporter system and RNA pull-down assay. Alizarin red S staining (ARS) was used to detect the calcium nodules in hBMSCs. We found that the expression of miR-30b-5p was downregulated, whereas that of BCL6 was upregulated during osteogenic differentiation of hBMSCs. Downregulating miR-30b-5p enhanced the expression of OCN, RUNX2, and ALP, and promoted calcium deposition. Conversely, transfection with si-BCL6 had the opposite effect that it inhibited osteogenic differentiation. However, the inhibitory effect of si-BCL6 was abrogated by miR-30b-5p inhibitor. miR-30b-5p inhibits the osteogenic differentiation of hBMSCs by targeting BCL6.

Associating drug sensitivity with differentiation status identifies effective combinations for acute myeloid leukemia

AbstractUsing ex vivo drug screening of primary patient specimens, we identified the combination of the p38 MAPK inhibitor doramapimod (DORA) with the BCL2 inhibitor venetoclax (VEN) as demonstrating broad, enhanced efficacy compared with each single agent across 335 acute myeloid leukemia (AML) patient samples while sparing primary stromal cells. Single-agent DORA and VEN sensitivity was associated with distinct, nonoverlapping tumor cell differentiation states. In particular, increased monocytes, M4/M5 French-American-British classification, and CD14+ immunophenotype tracked with sensitivity to DORA and resistance to VEN but were mitigated with the combination. Increased expression of MAPK14 and BCL2, the respective primary targets of DORA and VEN, were observed in monocytic and undifferentiated leukemias, respectively. Enrichment for DORA and VEN sensitivities was observed in AML with monocyte-like and progenitor-like transcriptomic signatures, respectively, and these associations diminished with the combination. The mechanism underlying the combination's enhanced efficacy may result from inhibition of p38 MAPK-mediated phosphorylation of BCL2, which in turn enhances sensitivity to VEN. These findings suggest exploiting complementary drug sensitivity profiles with respect to leukemic differentiation state, such as dual targeting of p38 MAPK and BCL2, offers opportunity for broad, enhanced efficacy across the clinically challenging heterogeneous landscape of AML.

Study of Co1 and BIK BCL2 Gene Analysis in Gorontalo Local Chicken

Analysis studies of Co1 and BIK BCL2 genes in local Gorontalo chickens were carried out using real-time PCR. This study aims to detect the presence of Co1 and BIK BCL2 genes in local Gorontalo chickens so that they can be used as information on gene diversity that can be used as genetic markers to study the diversity of local chicken species in Indonesia. The method used in this study is the SYBR green real-time PCR method. The research conducted found that the concentration values of the extracted samples were in the range of 68.15-68.30 ng/µL. Meanwhile, the purity values measured at wavelength A260/A280 were obtained with purity ranges between 1.867-1.923. The amplification results of the Co1 target gene show that the Ct value is in the range of 20.90-21.20, while for Tm, it is 84.10-84.20. In the BIK BCL2 target gene analysis, the Ct value was in the range of 16.15-16.20, while for Tm, it was 91.10-91.20. From these results, it can be concluded that all research samples were sampled from six different regions detected with CT and Tm values from the real-time analysis carried out obtained homogeneous values and no more than 2 degrees of difference between samples. The research novelty was using Nisi chicken as the native, one of the local chickens in Indonesia as the object of study. Besides, we used a BIK BCL 2 gene as the marker to determine the genetic diversity of the local chicken. A suggestion for further research is to explore more diverse genes to obtain more data on genetic diversity.

Synergistic activity of combined inhibition of anti-apoptotic molecules in B-cell precursor ALL

Targeting BCL-2, a key regulator of survival in B-cell malignancies including precursor B-cell acute lymphoblastic leukemia, has become a promising treatment strategy. However, given the redundancy of anti-apoptotic BCL-2 family proteins (BCL-2, BCL-XL, MCL-1), single targeting may not be sufficient. When analyzing the effects of BH3-mimetics selectively targeting BCL-XL and MCL-1 alone or in combination with the BCL-2 inhibitor venetoclax, heterogeneous sensitivity to either of these inhibitors was found in ALL cell lines and in patient-derived xenografts. Interestingly, some venetoclax-resistant leukemias were sensitive to the MCL-1-selective antagonist S63845 and/or BCL-XL-selective A-1331852 suggesting functional mutual substitution. Consequently, co-inhibition of BCL-2 and MCL-1 or BCL-XL resulted in synergistic apoptosis induction. Functional analysis by BH3-profiling and analysis of protein complexes revealed that venetoclax-treated ALL cells are dependent on MCL-1 and BCL-XL, indicating that MCL-1 or BCL-XL provide an Achilles heel in BCL-2-inhibited cells. The effect of combining BCL-2 and MCL-1 inhibition by venetoclax and S63845 was evaluated in vivo and strongly enhanced anti-leukemia activity was found in a pre-clinical patient-derived xenograft model. Our study offers in-depth molecular analysis of mutual substitution of BCL-2 family proteins in acute lymphoblastic leukemia and provides targets for combination treatment in vivo and in ongoing clinical studies.

Deletion of murine Rhoh leads to de-repression of Bcl-6 via decreased KAISO levels and accelerates a malignancy phenotype in a murine model of lymphoma

ABSTRACT RHOH/TFF, a member of the RAS GTPase super family, has important functions in lymphopoiesis and proximal T cell receptor signalling and has been implicated in a variety of leukaemias and lymphomas. RHOH was initially identified as a translocation partner with BCL-6 in non-Hodgkin lymphoma (NHL), and aberrant somatic hypermutation (SHM) in the 5ʹ untranslated region of the RHOH gene has also been detected in Diffuse Large B-Cell Lymphoma (DLBCL). Recent data suggest a correlation between RhoH expression and disease progression in Acute Myeloid Leukaemia (AML). However, the effects of RHOH mutations and translocations on RhoH expression and malignant transformation remain unknown. We found that aged Rhoh−/− (KO) mice had shortened lifespans and developed B cell derived splenomegaly with an increased Bcl-6 expression profile in splenocytes. We utilized a murine model of Bcl-6 driven DLBCL to further explore the role of RhoH in malignant behaviour by crossing RhohKO mice with Iµ-HABcl-6 transgenic (Bcl-6Tg) mice. The loss of Rhoh in Bcl-6Tg mice led to a more rapid disease progression. Mechanistically, we demonstrated that deletion of Rhoh in these murine lymphoma cells was associated with decreased levels of the RhoH binding partner KAISO, a dual-specific Zinc finger transcription factor, de-repression of KAISO target Bcl-6, and downregulation of the BCL-6 target Blimp-1. Re-expression of RhoH in RhohKOBcl-6Tg lymphoma cell lines reversed these changes in expression profile and reduced proliferation of lymphoma cells in vitro. These findings suggest a previously unidentified regulatory role of RhoH in the proliferation of tumour cells via altered BCL-6 expression. (250)