Anti-apoptotic Protein B-cell Lymphoma Research Articles

Venetoclax-Related Neutropenia in Leukemic Patients: A Comprehensive Review of the Underlying Causes, Risk Factors, and Management.

Venetoclax is a Bcl-2 homology domain 3 (BH3) mimetic currently approved for the treatment of chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML) that has proven to be highly effective in reinstating apoptosis in leukemic cells through the highly selective inhibition of the anti-apoptotic protein B-cell lymphoma-2 (Bcl-2). Clinically, venetoclax has provided lasting remissions through the inhibition of CLL and AML blasts. However, this activity has often come at the cost of grade III/IV neutropenia due to hematopoietic cells' dependence on Bcl-2 for survival. As life-threatening infections are an important complication in these patients, an effective management of neutropenia is indispensable to maximize patient outcomes. While there is general consensus over dose reduction and scheduling modifications to minimize the risk of neutropenia, the impact of these modifications on survival is uncertain. Moreover, guidelines do not yet adequately account for patient-specific and disease-specific risk factors that may predict toxicity, or the role combination treatment plays in exacerbating neutropenia. The objective of this review is to discuss the venetoclax-induced mechanism of hematological toxicity, the potential predictive risk factors that affect patient vulnerability to neutropenia, and the current consensus on practices for management of neutropenia.

Abstract 7227: Isatin analogs potentiate the cytotoxicity of venetoclax in acute myeloid leukemia cells

Abstract Acute Myeloid Leukemia (AML) is a rare but common hematological malignancy in children and adults. AML is a highly heterogeneous disease caused by the acquisition of several mutations affecting multiple cellular processes, thus requiring concomitant targeting of key survival nodes to treat effectively. As current first-line therapies for AML suffer with poor outcomes, drug resistance, and high relapse rates, there is an immediate need for new combinatorial therapies that are both safe and effective. Venetoclax (VEN), a selective inhibitor of antiapoptotic protein B-cell lymphoma-2 (BCL2), has been FDA-approved for treating adult AML and displayed potential in pediatric leukemia treatment. However, the activation of an antiapoptotic BCL2 family member, Myeloid Cell Leukemia 1 (MCL1), is often a primary mechanism of resistance to VEN therapy. Our group has developed Isatin analogs (ISA) that exhibited diverse biological activities, including anti-cancer activities alone or in combination with standard agents. ISA demonstrated anti-leukemic activity by targeting leukemic stem cells (LSCs) with high aldehyde dehydrogenase (ALDH) activity. In this study, we demonstrate ISA induces apoptosis by downregulating antiapoptotic MCL1 levels in pediatric and adult AML cell lines. Based on the inhibitory effects of Isatin analogs on key signaling pathways (PI3K/AKT, STAT3) and MCL1, we combined ISA with a BCL2 inhibitor, VEN, for effective AML treatment. The combination of ISA with VEN exhibited synergetic cytotoxicity in naïve (MOLM-13, MV4-11) and VEN-resistant (HL-60, OCI-AML3, U937, and KG-1) human AML cell lines by bliss analysis. ISA also showed significant synergetic cytotoxicity in combination with ABT-737, a BH3 mimetic inhibitor of Bcl-xL, Bcl-2, and Bcl-w. Combinatorial treatment of AML cell lines with sub-lethal doses of ISA and VEN showed a significant increase in cellular apoptosis as indicated by the levels of annexin V measured by flow cytometry. Immunoblotting analysis after co-treatment with ISA and VEN showed downregulation of MCL1 levels along with alteration of other key apoptosis regulators in both VEN-sensitive (MV4-11) and inherently VEN-resistant (U937) AML cell lines. Current and future studies will primarily focus on further exploiting the molecular mechanism of action for the combinatorial approach. Furthermore, our aim will be to demonstrate activity in primary patient cells along with preclinical efficacy in AML animal models. To summarize, in vitro findings showed synergetic cooperation between ISA and VEN in combination for effective induction of apoptosis and anti-leukemic activity in AML cells that are sensitive and resistant to VEN. Citation Format: Upendarrao Golla, Satyam Patel, Jeremy Hengst, Charyguly Annageldiyev, Joseph Schramm, Shantu Amin, Dhimant Desai, David Claxton, Sinisa Dovat, Arati Sharma. Isatin analogs potentiate the cytotoxicity of venetoclax in acute myeloid leukemia 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 7227.

Amentoflavone mediated hepatoprotection to counteract paraquat instigated hepatotoxicity via modulating Nrf2/keap1 pathway: A biochemical, inflammatory, apoptotic and histopathological study

Paraquat (PQ) is a ubiquitous and water-soluble herbicide which has potential to cause systematic poisoning. PQ intoxication is known to be associated with various clinical complications including hepatotoxicity. Amentoflavone (AMF) is an active phenolic compound that exhibits a broad range of biological as well as pharmacological activities. This study was designed to determine the hepato-protective potential of AMF against PQ instigated hepatotoxicity in rats. Forty-eight rats were distributed into four groups such as control group, PQ-treated group (5 mg/kg), PQ (5 mg/kg) + AMF (40 mg/kg) exposed group and AMF (40 mg/kg) only supplemented group. It was revealed that PQ exposure reduced nuclear factor erythroid 2–related factor 2 (Nrf2) and antioxidative genes expression whereas increase the expression of Kelch-like ECH-associated protein 1(Keap1). Besides, PQ intoxication reduced the activities of superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GSR), glutathione peroxidase (GPx), Heme- oxygenase-1 (HO-1) & glutathione (GSH) content. Furthermore, the levels of reactive oxygen species (ROS) & malondialdehyde (MDA) were increased. In addition, PQ significantly increased the hepatic serum enzymes including alkaline phosphatase (ALP), aspartate transaminase (AST), & alanine transaminase (ALT) along with inflammatory biomarkers levels such as tumor necrosis- α (TNF- α), nuclear factor- κB (NF-κB), interleukin-6 (IL-6), interleukin 1beta (IL-1β), & cyclo‑oxygenase-2 (COX-2) activity. PQ intoxication increased the expressions of pro-apoptotic markers i.e., Bcl-2-associated X protein (Bax) & Cysteine-aspartic protease-3 (Caspase-3) while reducing the expression of anti-apoptotic protein B-cell lymphoma 2 (Bcl-2). Furthermore, PQ intoxication prompted various histopathological impairments. However, the co-administration of AMF significantly improved the abovementioned hepatic damages induced by PQ. The present study indicated that AMF may be an effective therapeutic candidate to mitigate PQ provoked hepatic impairments due to its anti-apoptotic, antioxidant & anti-inflammatory properties.

B-cell lymphoma-2 family proteins in the crosshairs: Small molecule inhibitors and activators for cancer therapy.

The B-cell lymphoma-2 (BCL-2) family of proteins plays a crucial role in the regulation of apoptosis, offering a dual mechanism for its control. Numerous studies have established a strong association between gene disorders of these proteins and the proliferation of diverse cancer cell types. Consequently, the identification and development of drugs targeting BCL-2 family proteins have emerged as a prominent area in antitumor therapy. Over the last two decades, several small-molecules have been designed to modulate the protein-protein interactions between anti- and proapoptotic BCL-2 proteins, effectively suppressing tumor growth and metastasis in vivo. The primary focus of research has been on developing BCL-2 homology 3 (BH3) mimetics to target antiapoptotic BCL-2 proteins, thereby competitively releasing proapoptotic BCL-2 proteins and restoring the blocked intrinsic apoptotic program. Additionally, for proapoptotic BCL-2 proteins, exogenous small molecules have been explored to activate cell apoptosis by directly interacting with executioner proteins such as BCL-2-associated X protein (BAX) or BCL-2 homologous antagonist/killer protein (BAK). In this comprehensive review, we summarize the inhibitors and activators (sensitizers) of BCL-2 family proteins developed over the past decades, highlighting their discovery, optimization, preclinical and clinical status, and providing an overall landscape of drug development targeting these proteins for therapeutic purposes.

Drug repurposing for the identification of new Bcl-2 inhibitors: In vitro, STD-NMR, molecular docking, and dynamic simulation studies

BackgroundThe anti-apoptotic protein B-Cell Lymphoma 2 (Bcl-2) is a key target for the development of anti-cancer agents, as its overexpression can render cancer cells resistant to chemotherapeutic treatments. Aims and objectivesThe current study has systematically evaluated a library of FDA-approved drugs for Bcl-2 inhibition using a drug repurposing strategy via in vitro, biophysical, and in-silico techniques. Materials and methodsIn vitro anticancer activity was performed, followed by apoptosis assay. The selected compounds were subjected to Saturation Transfer Difference Nuclear Magnetic Resonance (STD-NMR) spectroscopy, molecular docking, and molecular dynamic simulation for ligand-protein interactions. Key findingsIn the initial screening, seventy-five (75) drugs were evaluated against the HL-60 (human blood promyelocytic leukemia) cancer cell line. Among them, paroxetine HCl, carvedilol, clomipramine HCl, and clomifene citrate showed significant anti-proliferative activity (IC50 = 9.733 ± 0.524, 11.940 ± 0.079, 12.376 ± 1.242, and 6.155 ± 0.363 μM, respectively), in comparison to the reference drug venetoclax (IC50 = 7.086 ± 0.041 μM). This indicated that the test drugs have comparable IC50 values to the standard drug. Furthermore, the drugs were able to induce apoptosis in HL-60 cells. These drugs showed interactions with Bcl-2 protein in STD-NMR analysis. Docking and MD simulation studies further supported the interaction of these drugs with Bcl-2 protein, mainly via hydrophobic contacts leading to stable drug-Bcl-2 complexes. SignificanceThis study, identifies paroxetine HCl, carvedilol, clomipramine HCl, and clomifene citrate as significant Bcl-2 inhibitors and needs further pre-clinical and clinical studies for potential anti-cancer agents' evaluation.

Imatinib prevents dexamethasone-induced pancreatic β-cell apoptosis via decreased TRAIL and DR5.

Prolonged administration of dexamethasone, a potent anti-inflammatory drug, can lead to steroid-induced diabetes. Imatinib, a medication commonly prescribed for chronic myeloid leukemia (CML), has been shown to improve diabetes in CML patients. Our recent study demonstrated that dexamethasone induces pancreatic β-cell apoptosis by upregulating the expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptor, death receptor 5 (DR5). We hypothesized that imatinib may protect against dexamethasone-induced pancreatic β-cell apoptosis by reducing the expression of TRAIL and DR5, thereby favorably modulating downstream effectors in apoptotic pathways. We test this hypothesis by assessing the effects of imatinib on dexamethasone-induced apoptosis in rat insulinoma cell line cells. As anticipated, dexamethasone treatment led to increased TRAIL and DR5 expression, as well as an elevation in superoxide production. Conversely, expression of the TRAIL decoy receptor (DcR1) was decreased. Moreover, key effectors in the extrinsic and intrinsic apoptosis pathways, such as B-cell lymphoma 2 (BCL-2) associated X (BAX), nuclear factor kappa B (NF-κb), P73, caspase 8, and caspase 9, were upregulated, while the antiapoptotic protein BCL-2 was downregulated. Interestingly and importantly, imatinib at a concentration of 10 µM reversed the effect of dexamethasone on TRAIL, DR5, DcR1, superoxide production, BAX, BCL-2, NF-κB, P73, caspase 3, caspase 8, and caspase 9. Similar effects of imatinib on dexamethasone-induced TRAIL and DR5 expression were also observed in isolated mouse islets. Taken together, our findings suggest that imatinib protects against dexamethasone-induced pancreatic β-cell apoptosis by reducing TRAIL and DR5 expression and modulating downstream effectors in the extrinsic and intrinsic apoptosis pathways.

Cancer Cell Membrane Wrapped Nanoparticles for the Delivery of a Bcl-2 Inhibitor to Triple-Negative Breast Cancer.

Overexpression of the antiapoptotic protein B-cell lymphoma 2 (Bcl-2) is correlated with poor survival outcomes in triple-negative breast cancer (TNBC), making Bcl-2 inhibition a promising strategy to treat this aggressive disease. Unfortunately, Bcl-2 inhibitors developed to date have limited clinical success against solid tumors, owing to poor bioavailability, insufficient tumor delivery, and off-target toxicity. To circumvent these problems, we loaded the Bcl-2 inhibitor ABT-737 in poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) that were wrapped with phospholipid membranes derived from 4T1 murine mammary cancer cells, which mimic the growth and metastasis of human TNBC. We show that the biomimetic cancer cell membrane coating enabled the NPs to preferentially target 4T1 TNBC cells over noncancerous mammary epithelial cells in vitro and significantly increased NP accumulation in orthotopic 4T1 tumors in mice after intravenous injection by over 2-fold compared to poly(ethylene glycol)-poly(lactide-co-glycolic) (PEG-PLGA) copolymer NPs. Congruently, the ABT-737 loaded, cancer cell membrane-wrapped PLGA NPs (ABT CCNPs) induced higher levels of apoptosis in TNBC cells in vitro than ABT-737 delivered freely or in PEG-PLGA NPs. When tested in a syngeneic spontaneous metastasis model, the ABT CCNPs significantly increased apoptosis (evidenced by elevated active caspase-3 and decreased Bcl-2 staining) and decreased proliferation (denoted by reduced Ki67 staining) throughout tumors compared with saline or ABT-loaded PEG-PLGA NP controls. Moreover, the ABT CCNPs did not alter animal weight or blood composition, suggesting that the specificity afforded by the TNBC cell membrane coating mitigated the off-target adverse effects typically associated with ABT-737. Despite these promising results, the low dose of ABT CCNPs administered only modestly reduced primary tumor growth and metastatic nodule formation in the lungs relative to controls. We posit that increasing the dose of ABT CCNPs, altering the treatment schedule, or encapsulating a more potent Bcl-2 inhibitor may yield more robust effects on tumor growth and metastasis. With further development, drug-loaded biomimetic NPs may safely treat solid tumors such as TNBC that are characterized by Bcl-2 overexpression.

MiR-665 overexpression inhibits the apoptosis of luteal cells in small ruminants suppressing HPGDS

Evidence has shown that microRNA-665 (miR-665) is highly expressed in the mid-luteal phase compared with the early and end-luteal phase of the corpus luteum (CL) life cycle. However, whether miR-665 is a positive regulator of the life span of the CL is still unknown. The objective of this study is to explore the effect of miR-665 on the structural luteolysis in the ovarian CL. In this study, the targeting relationship between miR-665 and hematopoietic prostaglandin synthase (HPGDS) was firstly verified by dual luciferase reporter assay. Then, quantitative real-time PCR (qRT-PCR) was used to detect the expression of miR-665 and HPGDS in luteal cells. Following miR-665 overexpression, the apoptosis rate of the luteal cells was determined using flow cytometry; B-cell lymphoma-2 (BCL-2) and caspase-3 mRNA and protein were measured using qRT-PCR and Western blot (WB) analysis. Finally, the DP1 and CRTH2 receptors of PGD2, a synthetic product of HPGDS, were localized using immunofluorescence. Results confirmed that HPGDS was a direct target gene of miR-665, and miR-665 expression was negatively correlated with HPGDS mRNA expression in luteal cells. Meanwhile, after miR-665 was overexpressed, the apoptotic rate of the luteal cells showed a significant decrease (P < 0.05) and this was accompanied by elevated expression levels of anti-apoptotic factor BCL-2 mRNA and protein and decreased expression levels of apoptotic factor caspase-3 mRNA and protein (P < 0.01). Moreover, the immune fluorescence staining results showed that the DP1 receptor was also significantly decreased (P < 0.05), but the CRTH2 receptor was significantly increased (P < 0.05) in luteal cells. Overall, these results indicate that miR-665 reduces the apoptosis of luteal cells via inhibiting caspase-3 expression and promoting BCL-2 expression, and the biological function of miR-665 may be attributed to its target gene HPGDS which regulates the balance of DP1 and CRTH2 receptors expression in luteal cells. As a consequence, this study suggests that miR-665 might be a positive regulator of the life span of the CL rather than destroy the integrity of CL in small ruminants.

Iron deficiency in hepatocellular carcinoma cells induced sorafenib resistance by upregulating HIF-1α to inhibit apoptosis

Sorafenib is the first-line therapeutic agent for hepatocellular carcinoma (HCC), but the drug resistance has become a major impediment. Previously we found that the abnormal iron metabolism in HCC led to iron deficiency, whether it induces sorafenib resistance during the treatment of HCC is not yet disclosed. In this study, we observed the effects of iron deficiency on sorafenib resistance and explored the underlying mechanisms. The results revealed that the killing effects of sorafenib on HCC cells were weakened by iron deficiency but effectively restored by iron re-supplementation. The ferroptosis indicators, including the contents of lipid hydroperoxide (LPO) and malondialdehyde (MDA), the level of intracellular reactive oxygen species (ROS), and the expression of glutathione peroxidase 4 (GPX4), were not significantly changed by iron deficiency in sorafenib-treated HCC cells. However, the sorafenib-induced apoptosis of HCC cells was inhibited by iron deficiency. Notably, the expression of anti-apoptotic protein B-cell lymphoma-2 (BCL-2) was elevated, and the expressions of other apoptotic proteins, BCL2-associated X (Bax), caspase-3, and caspase-9, were inhibited by iron deficiency. Mechanistically, iron deficiency upregulated hypoxia-inducible factor 1 alpha (HIF-1α) to increase BCL-2. Inhibition of HIF-1α suppressed the iron deficiency-induced BCL-2 and sorafenib resistance. In summary, iron deficiency in HCC cells generated sorafenib resistance by increasing HIF-1α and BCL-2, which therefore inhibited the sorafenib-induced apoptosis of HCC cells. These results identified iron deficiency as a new factor of sorafenib resistance in HCC cells, which would be an effective target to alleviate sorafenib resistance.

Doxorubicin-conjugated siRNA lipid nanoparticles for combination cancer therapy.

Evasion of apoptosis is a hallmark of cancer, attributed in part to overexpression of the anti-apoptotic protein B-cell lymphoma 2 (Bcl-2). In a variety of cancer types, including lymphoma, Bcl-2 is overexpressed. Therapeutic targeting of Bcl-2 has demonstrated efficacy in the clinic and is the subject of extensive clinical testing in combination with chemotherapy. Therefore, the development of co-delivery systems for Bcl-2 targeting agents, such as small interfering RNA (siRNA), and chemotherapeutics, such as doxorubicin (DOX), holds promise for enabling combination cancer therapies. Lipid nanoparticles (LNPs) are a clinically advanced nucleic acid delivery system with a compact structure suitable for siRNA encapsulation and delivery. Inspired by ongoing clinical trials of albumin-hitchhiking doxorubicin prodrugs, here we developed a DOX-siRNA co-delivery strategy via conjugation of doxorubicin to the surface of siRNA-loaded LNPs. Our optimized LNPs enabled potent knockdown of Bcl-2 and efficient delivery of DOX into the nucleus of Burkitts' lymphoma (Raji) cells, leading to effective inhibition of tumor growth in a mouse model of lymphoma. Based on these results, our LNPs may provide a platform for the co-delivery of various nucleic acids and DOX for the development of new combination cancer therapies.

Inhibition of antiapoptotic BCL-2 proteins with ABT-263 induces fibroblast apoptosis, reversing persistent pulmonary fibrosis.

Patients with progressive fibrosing interstitial lung diseases (PF-ILDs) carry a poor prognosis and have limited therapeutic options. A hallmark feature is fibroblast resistance to apoptosis, leading to their persistence, accumulation, and excessive deposition of extracellular matrix. A complex balance of the B cell lymphoma 2 (BCL-2) protein family controlling the intrinsic pathway of apoptosis and fibroblast reliance on antiapoptotic proteins has been hypothesized to contribute to this resistant phenotype. Examination of lung tissue from patients with PF-ILD (idiopathic pulmonary fibrosis and silicosis) and mice with PF-ILD (repetitive bleomycin and silicosis) showed increased expression of antiapoptotic BCL-2 family members in α-smooth muscle actin-positive fibroblasts, suggesting that fibroblasts from fibrotic lungs may exhibit increased susceptibility to inhibition of antiapoptotic BCL-2 family members BCL-2, BCL-XL, and BCL-W with the BH3 mimetic ABT-263. We used 2 murine models of PF-ILD to test the efficacy of ABT-263 in reversing established persistent pulmonary fibrosis. Treatment with ABT-263 induced fibroblast apoptosis, decreased fibroblast numbers, and reduced lung collagen levels, radiographic disease, and histologically evident fibrosis. Our studies provide insight into how fibroblasts gain resistance to apoptosis and become sensitive to the therapeutic inhibition of antiapoptotic proteins. By targeting profibrotic fibroblasts, ABT-263 offers a promising therapeutic option for PF-ILDs.

Efficacy and safety of venetoclax-based regimens for the treatment of relapsed/refractory multiple myeloma: a systemic review and meta-analysis.

Patients with relapsed/refractory multiple myeloma (RRMM) usually have dismal prognostic outcomes. Venetoclax, a selective inhibitor of antiapoptotic protein B-cell lymphoma-2 (BCL-2), demonstrates antimyeloma activity in plasma cells with t(11;14) or high BCL-2 expression. This meta-analysis aimed to investigate the efficacy and safety of venetoclax-based therapy in RRMM. This is a meta-analysis study. PubMed, Embase, and Cochrane were searched for studies published up to 20 December 2021. Overall response rate (ORR), rate of very good partial response or better (≧VGPR), and complete response (CR) rate were pooled with the random-effects model. Safety was evaluated by the incidences of grade ≧3 adverse events. Subgroup analysis and meta-regression were performed to identify the causes of heterogeneities. All the analyses were conducted by STATA 15.0 software. A total of 14 studies with 713 patients were included for analysis. The pooled ORR, rate of ≧VGPR, and CR for all patients were 59% [95% confidence interval (CI) = 45-71%], 38% (95% CI = 26-51%), and 17% (95% CI = 10-26%), respectively. The median progression-free survival (PFS) ranged from 2.0 months to not reached (NR), and the median overall survival (OS) ranged from 12.0 months to NR. Meta-regression showed that patients treated with more drugs combined or less heavily pretreated had higher response rates. Patients with t(11;14) had superior ORR [relative risk (RR) = 1.47, 95% CI = 1.05-2.07], ≧VGPR (RR = 1.71, 95% CI = 1.12-2.60), CR (RR = 1.86, 95% CI = 1.34-2.57), PFS [hazard ratio (HR) = 0.47, 95% CI = 0.30-0.65], and OS (HR = 0.30, 95% CI = 0.08-0.52) compared with patients without t(11;14). Most grade ≧3 adverse events were hematologic, gastrointestinal, and infectious related and were manageable. Venetoclax-based therapy is an effective and safe option for RRMM patients, especially those with t(11;14).

Erythroid/megakaryocytic differentiation confers BCL-XL dependency and venetoclax resistance in acute myeloid leukemia

AbstractMyeloid neoplasms with erythroid or megakaryocytic differentiation include pure erythroid leukemia, myelodysplastic syndrome with erythroid features, and acute megakaryoblastic leukemia (FAB M7) and are characterized by poor prognosis and limited treatment options. Here, we investigate the drug sensitivity landscape of these rare malignancies. We show that acute myeloid leukemia (AML) cells with erythroid or megakaryocytic differentiation depend on the antiapoptotic protein B-cell lymphoma (BCL)-XL, rather than BCL-2, using combined ex vivo drug sensitivity testing, genetic perturbation, and transcriptomic profiling. High-throughput screening of >500 compounds identified the BCL-XL–selective inhibitor A-1331852 and navitoclax as highly effective against erythroid/megakaryoblastic leukemia cell lines. In contrast, these AML subtypes were resistant to the BCL-2 inhibitor venetoclax, which is used clinically in the treatment of AML. Consistently, genome-scale CRISPR-Cas9 and RNAi screening data demonstrated the striking essentiality of BCL-XL-encoding BCL2L1 but not BCL2 or MCL1, for the survival of erythroid/megakaryoblastic leukemia cell lines. Single-cell and bulk transcriptomics of patient samples with erythroid and megakaryoblastic leukemias identified high BCL2L1 expression compared with other subtypes of AML and other hematological malignancies, where BCL2 and MCL1 were more prominent. BCL-XL inhibition effectively killed blasts in samples from patients with AML with erythroid or megakaryocytic differentiation ex vivo and reduced tumor burden in a mouse erythroleukemia xenograft model. Combining the BCL-XL inhibitor with the JAK inhibitor ruxolitinib showed synergistic and durable responses in cell lines. Our results suggest targeting BCL-XL as a potential therapy option in erythroid/megakaryoblastic leukemias and highlight an AML subgroup with potentially reduced sensitivity to venetoclax-based treatments.

Therapeutic Targeting of MERTK and BCL-2 in T-Cell and Early T-Precursor Acute Lymphoblastic Leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) accounts for 15% of childhood ALL. The early T-precursor (ETP-ALL) subset is characterized by an immature T-cell phenotype, chemoresistance, and high rates of induction failure. MERTK receptor tyrosine kinase is ectopically expressed in half of T-ALLs, particularly those with an immature T-cell phenotype, suggesting a role in ETP-ALL. The anti-apoptotic protein B-cell lymphoma-2 (BCL-2) is essential for ETP-ALL cell survival. Here, we show that MERTK and BCL-2 mRNA and protein are preferentially expressed in ETP-ALL patient samples. The dual MERTK/FLT3 inhibitor MRX-2843 decreased MERTK activation and downstream signaling, inhibited cell expansion, and induced cell death in ETP-ALL cell lines. Further, 54% (21/39) of primary T-ALL patient samples were sensitive to MERTK inhibition. Treatment with MRX-2843 significantly reduced leukemia burden and prolonged survival in cell-line-derived T-ALL and ETP-ALL xenograft models. In a patient-derived ETP-ALL xenograft model, treatment with MRX-2843 markedly reduced peripheral blood leukemia and spleen weight compared to vehicle-treated mice and prolonged survival. MRX-2843 also synergized with venetoclax to provide enhanced anti-leukemia activity in ETP-ALL cell cultures, with a dose ratio of 1:20 MRX-2843:venetoclax providing optimal synergy. These data demonstrate the therapeutic potential of MRX-2843 in patients with T-ALL and provide rationale for clinical development. MRX-2843 monotherapy is currently being tested in patients with relapsed leukemia (NCT04872478). Further, our data indicate that combined MERTK and BCL-2 inhibition may be particularly effective for treatment of ETP-ALL.

Lactobacillus spp. inhibits the growth of HCT-116 and reduces IL-8 secretion by Salmonella typhimurium-infected HCT-116 colorectal carcinoma cells

Salmonella typhimurium causes symptoms resembling typhoid fever and gastroenteritis in humans. Its toxicity is due to an outer membrane consisting largely of lipopolysaccharides (LPS) which is responsible for the host immune response. The aim of this study is to evaluate the antimicrobial, anti-apoptotic ability of Lactobacillus plantarum and reduce Salmonella-induced pro-inflammatory cytokine IL-8 secretion. Adhesive tests were performed using lactobacilli co-cultured with the colon cancer cell line HCT-116 for 2 hours. The strains displaying the highest adhesion were selected for downstream 3- (4, 5- Dimethylthiazol -2-yl) -2, 5- diphenyltetrazolium bromide (MTT) tests to assess cytotoxicity. The supernatants of Lactobacillus cultured with HCT-116 cells for 24 and 48 h to evaluate the inhibitory effect. To determine Interleukin 8 (IL-8) secretion in colon cancer induced by S. typhimurium, we stimulated HCT-116 cells with S. typhimurium and co-cultured with lactobacilli for 24 h. Lactobacilli had the most significant inhibitory effects on cell growth, and their inhibitory effects were time-dependent. Strain No. 03-03-026 caused cancer cell deoxyribonucleic acid (DNA) fragmentation, and the anti-apoptosis protein (B-cell lymphoma 2) was reduced in the HCT-116 cells as determined. IL-8 production in colon cancer cells was significantly reduced by these lactobacilli. Our results suggested that lactobacilli maybe effectively reduce the numbers of S. typhimurium, IL-8 levels and the anti-apoptotic phosphorylated-p38 mitogen-activated protein kinase and B-cell lymphoma 2 proteins. Lactobacillus can be added to the diet as a food additive to prevent colorectal cancer and used to be the prophylactic agent against S. typhimurium.

Lisaftoclax in Combination with Alrizomadlin Overcomes Venetoclax Resistance in Acute Myeloid Leukemia and Acute Lymphoblastic Leukemia: Preclinical Studies.

Despite approval of B-cell lymphoma (BCL)-2 inhibitor venetoclax for certain hematologic malignancies, its broader clinical benefit is curtailed by resistance. Our study aimed to determine if treatment with novel anticancer agents targeting BCL-2 and mouse double minute 2 (MDM2) could overcome venetoclax resistance in preclinical models. Venetoclax-sensitive and venetoclax-resistant acute myeloid leukemia (AML) and acute lymphoblastic leukemia cells and xenograft models were used to evaluate antitumor effects and underlying mechanisms associated with combined BCL-2 inhibitor lisaftoclax (APG-2575) and MDM2 inhibitor alrizomadlin (APG-115). The combination exhibited synergistic antiproliferative and apoptogenic activities in TP53 wild-type AML cell lines in vitro. This synergy was further exemplified by deep antitumor responses and prolonged survival in AML cell line-derived and patient-derived xenograft models. Interestingly, the combination treatment resensitized (to apoptosis) venetoclax-resistant cellular and mouse models established via chronic drug exposure or genetically engineered with clinically relevant BCL-2 gene mutations. Synergistic effects in reducing cellular viability and proliferation were also demonstrated in primary samples of patients with venetoclax-resistant AML treated with lisaftoclax and alrizomadlin ex vivo. Mechanistically, alrizomadlin likely primes cancer cells to BCL-2 inhibition-induced cellular apoptosis by downregulating expression of antiapoptotic proteins myeloid cell leukemia-1 and BCL-extra-large and upregulating pro-death BCL-2-associated X protein. Lisaftoclax in combination with alrizomadlin overcomes venetoclax resistance mediated by various mechanisms, including BCL-2 mutations. In addition, we posit further, putative molecular mechanisms. Our data rationalize clinical development of this treatment combination in patients with diseases that are insensitive or resistant to venetoclax.

Diterpenoid DGA induces apoptosis via endoplasmic reticulum stress caused by changes in glycosphingolipid composition and inhibition of STAT3 in glioma cells

Glioma is one of the most common malignant primary brain tumors, with poor prognosis and high recurrence. There are currently few drugs approved for brain tumors; thus, it is necessary to develop new effective drugs. Natural diterpenoids have important biological activities, including antiinflammatory, antioxidative, and antitumor effects. In this study, 7α,14β-dihydroxy-ent-kaur-17-dimethylamino-3,15-dione (DGA), a diterpenoid compound modified from glaucocalyxin A, inhibited the proliferation of many tumor cells, especially glioma. Flow cytometry analysis showed that DGA induced apoptosis in glioma cells. DGA also inhibited xenograft tumors in nude mice. It affected the expression of ceramide synthases (CerS) in glioma cells; CerS1 decreased, and CerS2 and CerS5 increased, resulting in a change in the composition of glycosphingolipids containing varying acyl chain lengths. In glioma cells treated with DGA, the gene transcription of activating transcription factor 4 (ATF4), X-box binding protein-1 (XBP1), and C/EBP-homologous protein (CHOP) in unfolded protein response pathways was upregulated. Meanwhile, the ratio of proapoptotic protein Bcl-2–associated X protein (BAX) to antiapoptotic protein B-cell lymphoma 2 (Bcl-2) also increased. This suggested that an imbalance of glycosphingolipids caused by DGA induced severe endoplasmic reticulum stress and triggered cell apoptosis. Moreover, Western blotting showed DGA inhibited the signal transducers and activators of transcription 3 (STAT3) signaling pathway by reducing the phosphorylation of STAT3 and its upstream kinases, which also promoted the apoptosis of glioma cells. Together, these results explored the anticancer activities of DGA and highlighted it as a potential candidate for treating glioma.

Zearalenone Promotes LPS-Induced Oxidative Stress, Endoplasmic Reticulum Stress, and Accelerates Bovine Mammary Epithelial Cell Apoptosis.

Both zearalenone (ZEA) and lipopolysaccharide (LPS) can induce oxidative stress, and even apoptosis in bovine mammary epithelial cells (MAC-T), but not much attention has been given to the synergistic effect of ZEA and LPS. In this study, we treated MAC-T cells with different concentrations of LPS (1, 10, 50, and 100 μg/mL) and ZEA (5, 15, and 30 μM) to induce cell damage. Previous results show that MAC-T cell viability decreases with increasing LPS concentration. Meanwhile, 1 µg/mL LPS and ZEA were selected for combined treatment in subsequent studies. It was found that co-treatment with ZEA and LPS increases the accumulation of reactive oxygen species (ROS) and malondialdehyde (MDA), decreases mitochondrial membrane potential (MMP), and superoxide dismutase (SOD), and reduces glutathione (GSH). ZEA and LPS are found to activate endoplasmic reticulum (ER) stress by increasing the expression of glucose-regulated protein 78 kDa (GRP78), activating transcription factor 6 (ATF6) and C/EBP homologous protein (CHOP). It increases cell apoptosis by suppressing the expression of the anti-apoptotic protein B-cell lymphoma-2 (Bcl-2), indicated by up-regulation of Bcl2-associated X protein (Bax) and Cysteinyl aspartate-specific proteinases 3 (caspase-3) expression. The above results suggest that the synergistic effect of ZEA and LPS aggravate cytotoxicity.

Selective inhibition of MCL1 overcomes venetoclax resistance in a murine model of myelodysplastic syndromes.

Treatment for myelodysplastic syndromes (MDS) remains insufficient due to clonal heterogeneity and lack of effective clinical therapies. Dysregulation of apoptosis is observed across MDS subtypes regardless of mutations and represents an attractive therapeutic opportunity. Venetoclax (VEN), a selective inhibitor of anti-apoptotic protein B-cell lymphoma- 2 (BCL2), has yielded impressive responses in older patients with acute myeloid leukemia (AML) and high risk MDS. BCL2 family anti-apoptotic proteins BCL-XL and induced myeloid cell leukemia 1 (MCL1) are implicated in leukemia survival, and upregulation of MCL1 is seen in VEN-resistant AML and MDS. We determined in vitro sensitivity of MDS patient samples to selective inhibitors of BCL2, BCL-XL and MCL1. While VEN response positively correlated with MDS with excess blasts, all MDS subtypes responded to MCL1 inhibition. Treatment with combined VEN + MCL1 inhibtion was synergistic in all MDS subtypes without significant injury to normal hematopoiesis and reduced MDS engraftment in MISTRG6 mice, supporting the pursuit of clinical trials with combined BCL2 + MCL1 inhibition in MDS.

Lysine demethylase KDM1A promotes cell growth via FKBP8–BCL2 axis in hepatocellular carcinoma

Advanced hepatocellular carcinoma (HCC) has a dismal prognosis. KDM1A (lysine demethylase 1A), overexpressed in multiple cancer types, is a lysine demethylase that targets both histone and nonhistone proteins. However, it is unclear how KDM1A expression affects HCC etiology. Here, we show that KDM1A can interact with and demethylate FKBP8 (FKBP prolyl isomerase 8), a cytoplasmic protein that regulates cell survival through the antiapoptotic protein BCL2 (B-cell lymphoma-2). We show that demethylation of FKBP8 enhances its ability to stabilize BCL2. Consistently, we observed positive correlation between KDM1A and BCL2 protein levels in liver cancer patients. Functionally, we reveal that FKBP8 demethylation by KDM1A is critical for liver cancer cell growth in vitro and in vivo. We went on to explore the mechanisms that might regulate KDM1A cytoplasmic localization. We found that the cytoplasmic localization and protein stability of KDM1A were promoted by acetylation at lysine-117 by the acetyl transferase KAT8 (lysine acetyltransferase 8). In agreement with this, we show that KDM1A–K117 (lysine 117) acetylation promotes demethylation of FKBP8 and level of BCL2. Finally, it has been shown that the efficacy of sorafenib, a first-line treatment for advanced HCC, is limited by clinical resistance. We show that KDM1A and BCL2 protein levels are increased during acquired sorafenib resistance, whereas inhibiting KDM1A can antagonize sorafenib resistance. Collectively, these results define a functional KDM1A–FKBP8–BCL2 axis in HCC.