Polyribonucleotide nucleotidyltransferase 1 participates in metabolic-associated fatty liver disease pathogenesis by affecting lipid metabolism and mitochondrial homeostasis.

OBJECTIVE: Metabolic-associated fatty liver disease (MAFLD) represents one of the most prevalent chronic liver conditions worldwide, but its precise pathogenesis remains unclear. This research endeavors to elucidate the involvement and molecular mechanisms of polyribonucleotide nucleotidyltransferase 1 (PNPT1) in the progression of MAFLD. METHODS: The study employed western blot and qRT-PCR to evaluate PNPT1 levels in liver specimens from individuals diagnosed with MAFLD and in mouse models subjected to a high-fat diet. Cellular studies investigated the effects of PNPT1 on lipid metabolism, apoptosis, and mitochondrial stability in hepatocytes. Immunofluorescence was utilized to track the subcellular movement of PNPT1 under high lipid conditions. RNA immunoprecipitation and functional assays were conducted to identify interactions between PNPT1 and Mcl-1 mRNA. The role of PPARα as an upstream transcriptional regulator of PNPT1 was investigated. Recombinant adenoviral vectors were utilized to modulate PNPT1 expression in vivo. RESULTS: PNPT1 was found to be markedly reduced in liver tissues from MAFLD patients and HFD mice. In vitro, PNPT1 directly regulated hepatic lipid metabolism, apoptosis, and mitochondrial stability. Under conditions of elevated lipids, PNPT1 relocated from mitochondria to cytoplasm, modifying its physiological functions. RNA immunoprecipitation revealed that the KH and S1 domains of PNPT1 bind to and degrade Mcl-1 mRNA, which in turn affects mitochondrial permeability. The transcriptional regulator PPARα was identified as a significant influencer of PNPT1, impacting both its expression and subsequent cellular functions. Alterations in PNPT1 expression were directly correlated with the progression of MAFLD in mice. CONCLUSIONS: The study confirms the pivotal function of PNPT1 in the development of MAFLD through its interactions with Mcl-1 and its regulatory effects on lipid metabolism and mitochondrial stability. These insights highlight the intricate association between PNPT1 and MAFLD, shedding light on its molecular pathways and presenting a potential new therapeutic avenue for MAFLD management.

Regulatory axis of circular RNA DTNB, microRNA-485-5p, and myeloid cell leukemia 1 attenuates inflammation and apoptosis in caerulein-treated AR42J cells.

Acute pancreatitis (AP) is an inflammatory disease of the pancreas and the main cause of hospital admissions for gastrointestinal diseases. Here, the work studied the circular RNA DTNB/microRNA-485-5p/MCL1 axis in AP and hoped to unravel the related mechanism. Caerulein exposure replicated an AP model in AR42J cells, and caerulein-mediated expression of circDTNB, miR-485-5p, and MCL1 was recorded. After exposure, cells were intervened with transfection plasmids and tested for LDH release, apoptosis, and inflammation. To determine the interwork of circDTNB, miR-485-5p, and MCL1, prediction results and verification experiments were conducted. Caerulein exposure reduced circDTNB and MCL1, while elevated miR-485-5p levels in AR42J cells. Upregulating circDTNB protected AR42J cells from caerulein-induced LDH cytotoxicity, apoptosis, and inflammation, but circDTNB upregulation-induced protections could be muffled by inhibiting MCL1. On the contrary, downregulating circDTNB further damaged AR42J cells under caerulein exposure, however, this phenomenon could be partially rescued after silencing miR-485-5p. miR-485-5p was mechanistically verified to be a target of circDTNB to mediate MCL1. Overall, the circDTNB/miR-485-5p/MCL1 axis protects inflammatory response and apoptosis in caerulein-exposed AR42J cells, promisingly identifying circDTNB as a novel molecule for AP treatment.

Clitocine enhances the drug sensitivity of colon cancer cells by promoting FBXW7-mediated MCL-1 degradation via inhibiting the A2B/cAMP/ERK axis.

Chemotherapy resistance to colon cancer is an unavoidable obstacle in the clinical management of the disease. Clitocine, an adenosine analog, played a significant role in the chemosensitivity of human colon cancer cells by promoting MCL-1 protein degradation. However, the detailed mechanism remains to be further elucidated. We found that clitocine up-regulates the expression of FBXW7, a ubiquitin ligase involved in the MCL-1 degradation. Transcriptome sequencing analysis revealed that clitocine significantly inhibits the cAMP and ERK downstream signaling pathways in colon cancer cells, thereby enhancing FBXW7 expression and subsequently promoting the ubiquitination degradation of MCL-1 protein. We verified that clitocine regulated intracellular cAMP levels by competitive binding with the adenosine receptor A2B. Molecular docking assay also verified the binding relationship. By decreasing intracellular cAMP levels, clitocine blocks the activation of downstream signaling pathways, which ultimately enhances the drug sensitivity of colon cancer cells through increased FBXW7 expression due to the inhibition of its promoter DNA methylation. Both knock-out of adenosine receptor A2B and Br-cAMP treatment can effectively attenuate the function of clitocine in vitro and in vivo. This study clarified that clitocine enhanced the drug sensitivity of colon cancer cells by promoting FBXW7-mediated MCL-1 degradation via inhibiting the A2B/cAMP/ERK axis, providing further knowledge of the clinical application for clitocine.

Targeting MCL1 with Sanggenon C overcomes MCL1-driven adaptive chemoresistance via dysregulation of autophagy and endoplasmic reticulum stress in cervical cancer.

BACKGROUND: Cervical cancer ranks as one of the most prevalent malignancies among women worldwide and poses a significant threat to health and quality of life. MCL1 is an antiapoptotic protein closely linked to tumorigenesis, drug-resistance and poor prognosis in various cancers. Sanggenon C, a natural flavonoid derived from Morus albal., exhibits multiple activities, including anti-oxidant, anti-inflammatory, antivirus, and antitumor properties. However, the molecular mechanisms by which Sanggenon C exerts antitumor effects on in cervical cancer remain unclear. PURPOSE: To investigate the oncogenic role of MCL1 and elucidate the antitumor activity of Sanggenon C, along with its molecular mechanisms, in cervical cancer. METHODS: In vitro, the effects of Sanggenon C on proliferation, the cell cycle, apoptosis, and autophagy were explored. Transcriptome sequencing was employed to analyze critical genes and pathways. The expression of genes or proteins was evaluated via immunofluorescence, qRT-PCR, immunohistochemistry, and Western blotting. To identify targets of Sanggenon C, various techniques such as clinical database analysis, molecular docking, cellular thermal shift assays, co-immunoprecipitation, and ubiquitination assays were utilized. Additionally, Xenograft mouse models were established to further investigate Sanggenon C as a novel MCL1 inhibitor and its anti-tumor activity in vivo. RESULTS: Our investigation reveals that Sanggenon C effectively inhibits cervical cancer cell proliferation both in vitro and in vivo. Furthermore, Sanggenon C induces endoplasmic reticulum stress and triggers protective autophagy via activation of the ATF4-DDIT3-TRIB3-AKT-MTOR signaling axis. Furthermore, Sanggenon C specifically targets MCL1 to exert its antitumor effects by modulating MCL1 protein stability through SYVN1-mediated ubiquitination. Notably, MCL1 overexpression attenuates the Sanggenon C-induced decrease in cell viability and apoptosis. Our study further characterizes the role of MCL1 in cisplatin resistance and identifies MCL1 as a promising target for Sanggenon C, which effectively inhibits proliferation and induces apoptosis in cisplatin-resistant cervical cancer cells. Importantly, combining Sanggenon C with an autophagy inhibitor represents a promising strategy to enhance therapeutic outcomes in cisplatin-resistant cervical cancer cells. CONCLUSION: Our findings demonstrates that Sanggenon C induces endoplasmic reticulum stress and highlights the potential of targeting MCL1 to exploit vulnerabilities in drug-resistant cervical cancer cells. Sanggenon C emerges as a promising therapeutic agent against MCL1-driven adaptive chemoresistance through disruption of autophagy and endoplasmic reticulum stress in cervical cancer.

Discovery and development of thiazolidine-2,4-dione derivatives as Bcl-2/Mcl-1 dual inhibitors.

Increasing the levels of antiapoptotic Bcl-2 proteins is an important way that cancer cells utilize to get out of apoptosis, underscoring their significance as promising targets for anticancer therapies. Lately, a primary compound 1 bearing thiazolidine-2,4-dione was discovered to exhibit comparable Mcl-1 inhibitory activity in comparison to WL-276. Herein, thirty-nine thiazolidine-2,4-dione analogs were yielded through incorporating different biphenyl moieties (R1), amino acid side chains (R2) and sulfonamides (R3) on 1. The findings indicated that certain compounds exhibited favorable inhibitory effects against Bcl-2/Mcl-1, while demonstrating limited or negligible binding affinity towards Bcl-xL. In particular, compounds 16 and 20 exhibited greater Bcl-2/Mcl-1 inhibition compared to AT-101, WL-276 and 1. Moreover, they demonstrated notable antiproliferative effects and significantly induced apoptosis in U937 cells. The western blot and co-immunoprecipitation assays confirmed that 20 could induce alterations in the expression of apoptosis-associated proteins to result in apoptosis through on-target Bcl-2 and Mcl-1 inhibition. In addition, 20 exhibited favorable stability profiles in both rat plasma and rat liver microsomes. In total, 20 could be used as a promising compound to discover Bcl-2/Mcl-1 dual inhibitors with favorable therapeutic properties.

Initial AXL and MCL-1 inhibition contributes to abolishing lazertinib tolerance in EGFR-mutant lung cancer cells.

Lazertinib, a novel third-generation epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), demonstrates marked efficacy in EGFR-mutant lung cancer. However, resistance commonly develops, prompting consideration of therapeutic strategies to overcome initial drug resistance mechanisms. This study aimed to elucidate the adaptive resistance to lazertinib and advocate novel combination treatments that demonstrate efficacy in preventing resistance as a first-line treatment for EGFR mutation-positive NSCLC. We found that AXL knockdown significantly inhibited lung cancer cell viability in the presence of lazertinib, indicating that AXL activation contributes to lazertinib resistance. However, long-term culture with a combination of lazertinib and AXL inhibitors led to residual cell proliferation and increased the MCL-1 expression level, which was mediated by the nuclear translocation of the transcription factor YAP. Triple therapy with an MCL-1 or YAP inhibitor in combination with lazertinib and an AXL inhibitor significantly reduced cell viability and increased the apoptosis rate. These results demonstrate that AXL and YAP/MCL-1 signals contribute to adaptive lazertinib resistance in EGFR-mutant lung cancer cells, suggesting that the initial dual inhibition of AXL and YAP/MCL-1 might be a highly effective strategy in eliminating lazertinib-resistant cells.

Capivasertib reverses chemotherapy-induced esophageal cancer resistance via inhibiting Akt-associated Mcl-1 upregulation.

The development of resistance to chemotherapy in esophageal cancer represents a significant challenge in cancer treatment. Therefore, our study aimed to identify effective therapeutic strategies by examining the molecules involved in this chemoresistance. We consistently observed an increase in the expression of Mcl-1 in cells exposed to both short and long-term treatment with cisplatin, a drug commonly used in esophageal cancer therapy. Functional analysis showed that Mcl-1 regulates esophageal cancer cell response to cisplatin treatment. Notably, this upregulation of Mcl-1 was not dependent on eukaryotic initiation factor 4E (eIF4E). Instead, it was associated with increased stability due to the activation of Akt. Capivasertib, a potent pan-Akt kinase drug, significantly decreased Mcl-1 level via inhibiting Akt signaling pathway in chemo-resistant cells. In addition, capivasertib not only decreased the viability of chemo-resistant esophageal cancer cells but also synergistically enhanced the effects of cisplatin. In multiple mouse models, representing both chemo-resistant and chemo-sensitive esophageal cancer, capivasertib administered at non-toxic doses demonstrated remarkable efficacy. It significantly extended the overall survival of the mice. Our research underscores the pivotal role of Akt-associated Mcl-1 upregulation in the development of chemo-resistance in esophageal cancer cells. Furthermore, it highlights the potential of capivasertib to reverse this resistance mechanism.

The CDK9 inhibitor enitociclib overcomes resistance to BTK inhibition and CAR-T therapy in mantle cell lymphoma.

Inhibitors of Bruton's tyrosine kinase (BTKi) and chimeric antigen receptor T-cell (CAR-T) therapy targeting CD19 are paradigm-shifting advances in treating patients with aggressive mantle cell lymphoma (MCL). However, clinical relapses following BTKi and CD19-directed CAR-T treatments are a fast-growing medical challenge. Development of novel therapies to overcome BTKi resistance (BTKi-R) and BTKi-CAR-T dual resistance (Dual-R) are urgently needed. Our single-cell RNA sequencing data revealed major transcriptomic reprogramming, with great enrichment of MYC-targets evolving as resistance to these therapies developed. Interestingly, cyclin-dependent kinase 9 (CDK9), a critical component of the positive transcription elongation factor-b complex, was among the top upregulated genes in Dual-R vs. BTKi-R samples. We therefore hypothesized that targeting CDK9 may turn off MYC-driven tumor survival and drug resistance. Enitociclib (formerly VIP152) is a selective CDK9 inhibitor whose potency against MCL has not been assessed. In this study, we found that enitociclib was highly potent in targeting lymphoma cells, with the half-maximal inhibitory concentration (IC50) ranging from 32 to 172 nM in MCL and diffuse large B-cell lymphoma cell lines. It inhibited CDK9 phosphorylation and downstream events including de novo synthesis of the short-lived proteins c-MYC, MCL-1, and cyclin D1, and induced apoptosis in a caspase-3-dependent manner. Enitociclib potently inhibited in vivo tumor growth of cell line-derived and patient-derived xenografts having therapeutic resistance. Our data demonstrate the potency of enitociclib in overcoming therapeutic resistance in MCL models and provide evidence in favor of its clinical investigation.

Gypenosides exert cardioprotective effects by promoting mitophagy and activating PI3K/Akt/GSK-3ß/Mcl-1 signaling.

Background: Gynostemma pentaphyllum (Thunb.) Makino, a well-known edible and medicinal plant, has anti-aging properties and is used to treataging-associated conditions such as diabetes, metabolic syndrome, and cardiovascular diseases. Gypenosides (GYPs) are the primary constituents of G. pentaphyllum. Increasing evidence indicates that GYPs are effective at preserving mitochondrial homeostasis and preventing heart failure (HF). This study aimed to uncover the cardioprotective mechanisms of GYPs related to mitochondrial regulation. Methods: The bioactive components in GYPs and the potential targets in treating HF were obtained and screened using the network pharmacology approach, followed by drug-disease target prediction and enrichment analyses. The pharmacological effects of GYPs in cardioprotection, mitochondrial function, mitochondrial quality control, and underlying mechanisms were further investigated in Doxorubicin (Dox)-stimulated H9c2 cardiomyocytes. Results: A total of 88 bioactive compounds of GYPs and their respective 71 drug-disease targets were identified. The hub targets covered MAPK, EGFR, PI3KCA, and Mcl-1. Enrichment analysis revealed that the pathways primarily contained PI3K/Akt, MAPK, and FoxO signalings, as well as calcium regulation, protein phosphorylation, apoptosis, and mitophagy process. In Dox-stimulated H9c2 rat cardiomyocytes, pretreatment with GYPs increased cell viability, enhanced cellular ATP content, restored basal oxygen consumption rate (OCR), and improved mitochondrial membrane potential (MMP). Furthermore, GYPs improved PINK1/parkin-mediated mitophagy without influencing mitochondrial fission/fusion proteins and the autophagic LC3 levels. Mechanistically, the phosphorylation of PI3K, Akt, GSK-3ß, and the protein level of Mcl-1 was upregulated by GYP treatment. Conclusion: Our findings reveal that GYPs exert cardioprotective effects by rescuing the defective mitophagy, and PI3K/Akt/GSK-3ß/Mcl-1 signaling is potentially involved in this process.

The HTLV-I oncoprotein Tax inactivates the tumor suppressor FBXW7.

Human T-cell leukemia virus type 1 (HTLV-I) is the etiological agent of adult T-cell leukemia (ATL). Mutational analysis has demonstrated that the tumor suppressor, F-box and WD repeat domain containing 7 (FBXW7/FBW7/CDC4), is mutated in primary ATL patients. However, even in the absence of genetic mutations, FBXW7 substrates are stabilized in ATL cells, suggesting additional mechanisms can prevent FBXW7 functions. Here, we report that the viral oncoprotein Tax represses FBXW7 activity, resulting in the stabilization of activated Notch intracellular domain, c-MYC, Cyclin E, and myeloid cell leukemia sequence 1 (BCL2-related) (Mcl-1). Mechanistically, we demonstrate that Tax directly binds to FBXW7 in the nucleus, effectively outcompeting other targets for binding to FBXW7, resulting in decreased ubiquitination and degradation of FBXW7 substrates. In support of the nuclear role of Tax, a non-degradable form of the nuclear factor kappa B subunit 2 (NFκB2/p100) was found to delocalize Tax to the cytoplasm, thereby preventing Tax interactions with FBXW7 and Tax-mediated inhibition of FBXW7. Finally, we characterize a Tax mutant that is unable to interact with FBXW7, unable to block FBXW7 tumor suppressor functions, and unable to effectively transform fibroblasts. These results demonstrate that HTLV-I Tax can inhibit FBXW7 functions without genetic mutations to promote an oncogenic state. These results suggest that Tax-mediated inhibition of FBXW7 is likely critical during the early stages of the cellular transformation process. IMPORTANCE: F-box and WD repeat domain containing 7 (FBXW7), a critical tumor suppressor of human cancers, is frequently mutated or epigenetically suppressed. Loss of FBXW7 functions is associated with stabilization and increased expression of oncogenic factors such as Cyclin E, c-Myc, Mcl-1, mTOR, Jun, and Notch. In this study, we demonstrate that the human retrovirus human T-cell leukemia virus type 1 oncoprotein Tax directly interacts with FBXW7, effectively outcompeting other targets for binding to FBXW7, resulting in decreased ubiquitination and degradation of FBXW7 cellular substrates. We further demonstrate that a Tax mutant unable to interact with and inactivate FBXW7 loses its ability to transform primary fibroblasts. Collectively, our results describe a novel mechanism used by a human tumor virus to promote cellular transformation.

Modeling the Binding of Anticancer Peptides and Mcl-1.

Mcl-1 (myeloid cell leukemia 1), a member of the Bcl-2 family, is upregulated in various types of cancer. Peptides representing the BH3 (Bcl-2 homology 3) region of pro-apoptotic proteins have been demonstrated to bind the hydrophobic groove of anti-apoptotic Mcl-1, and this interaction is responsible for regulating apoptosis. Structural studies have shown that, while there is high overall structural conservation among the anti-apoptotic Bcl-2 (B-cell lymphoma 2) proteins, differences in the surface groove of these proteins facilitates binding specificity. This binding specificity is crucial for the mechanism of action of the Bcl-2 family in regulating apoptosis. Bim-based peptides bind specifically to the hydrophobic groove of Mcl-1, emphasizing the importance of these interactions in the regulation of cell death. Molecular docking was performed with BH3-like peptides derived from Bim to identify high affinity peptides that bind to Mcl-1 and to understand the molecular mechanism of their interactions. The interactions of three identified peptides, E2gY, E2gI, and XXA1_F3dI, were further evaluated using 250 ns molecular dynamics simulations. Conserved hydrophobic residues of the peptides play an important role in their binding and the structural stability of the complexes. Understanding the molecular basis of interaction of these peptides will assist in the development of more effective Mcl-1 specific inhibitors.

Novel markers of MCL1 inhibitor sensitivity in triple-negative breast cancer cells.

Triple-negative breast cancer (TNBC) is an aggressive breast cancer sub-type with limited treatment options and poor prognosis. Currently, standard treatments for TNBC include surgery, chemotherapy, and anti-PDL1 therapy. These therapies have limited efficacy in advanced stages. Myeloid-cell leukemia 1 (MCL1) is an anti-apoptotic BCL2 family protein. High expression of MCL1 contributes to chemotherapy resistance and is associated with a worse prognosis in TNBC. MCL1 inhibitors are in clinical trials for TNBC, but response rates to these inhibitors can vary and predictive markers are lacking. Currently, we identified a 4-member (AXL, ETS1, IL6, EFEMP1) gene signature (GS) that predicts MCL1 inhibitor sensitivity in TNBC cells. Factors encoded by these genes regulate signaling pathways to promote MCL1 inhibitor resistance. Small molecule inhibitors of the GS factors can overcome resistance and sensitize otherwise resistant TNBC cells to MCL1 inhibitor treatment. These findings offer insights into potential therapeutic strategies and tumor stratification for MCL1 inhibitor use in TNBC.

Targeting MCL1-driven anti-apoptotic pathways overcomes blast progression after hypomethylating agent failure in chronic myelomonocytic leukemia.

RAS pathway mutations, which are present in 30% of patients with chronic myelomonocytic leukemia (CMML) at diagnosis, confer a high risk of resistance to and progression after hypomethylating agent (HMA) therapy, the current standard of care for the disease. Here, using single-cell, multi-omics technologies, we seek to dissect the biological mechanisms underlying the initiation and progression of RAS pathway-mutated CMML. We identify that RAS pathway mutations induce transcriptional reprogramming of hematopoietic stem and progenitor cells (HSPCs) and downstream monocytic populations in response to cell-intrinsic and -extrinsic inflammatory signaling that also impair the functions of immune cells. HSPCs expand at disease progression after therapy with HMA or the BCL2 inhibitor venetoclax and rely on the NF-κB pathway effector MCL1 to maintain survival. Our study has implications for the development of therapies to improve the survival of patients with RAS pathway-mutated CMML.

Butein inhibits oral squamous cell carcinoma growth via promoting MCL-1 ubiquitination.

Oral squamous cell carcinoma (OSCC) is the most common malignant head and neck carcinoma type. Myeloid cell leukemia-1 (MCL-1), an anti-apoptotic BCL-1 protein, has been verified to be among the most highly upregulated pathologic proteins in human cancers linked to tumor relapse, poor prognosis and therapeutic resistance. Herein, therapeutic targeting MCL-1 is an attractive focus for cancer treatment. The present study found that butein, a potential phytochemical compound, exerted profound antitumor effects on OSCC cells. Butein treatment significantly inhibited cell viability, proliferation capacity and colony formation ability, and activated cell apoptotic process. Further potential mechanism investigation showed that promoting MCL-1 ubiquitination and degradation is the major reason for butein-mediated OSCC cell cytotoxicity. Our results uncovered that butein could facilitate E3 ligase FBW7 combined with MCL-1, which contributed to an increase in the ubiquitination of MCL-1 Ub-K48 and degradation. The results of both in vitro cell experiments and in vivo xenograft models imply a critical antitumor function of butein with the well-tolerated feature, and it might be an attractive and promising agent for OSCC treatment.

Chitosan-TPP encapsulated quercetin nanoparticles: amplified protection mechanisms unveiled against Ethion-induced developmental toxicity through comprehensive in-vivo and in-silico elucidation.

Aim: The study investigated Ethion-induced developmental toxicity in Wistar albino rats and the potential ameliorative effects of quercetin and nano-quercetin co-administration. Further, In-silico docking of Ethion and quercetin with MCL-1 was conducted. Methodology: Quercetin nanoparticles were synthesized by ionic-gelation method. The encapsulated quercetin nanoparticles were characterized for Zeta size, UV-Vis spectroscopy, encapsulation efficiency, and TEM studies. Male rats were administered Ethion (high/low dose), quercetin, and nano-quercetin alone or in combination for 60 days. Female rats were introduced for mating on the 61st day, and pregnant females were observed for 20 gestational days. On GD 20, rats were sacrificed and evaluated for body/organ weight, reproductive indices, fetal morphology, skeletal, and visceral deformities.In silico binding energies of ethion and quercetin with MCL-1 were determined. Results: Nanoparticle size was 363.2 ± 1.23 nm on day 0 and 385.63 ± 1.53 nm on day 60, with PDI of 0.247 and charge of 22.9 mV. Absorbance maxima were at 374 nm, with encapsulation efficacy of 85.16 ± 0.33%. EHD male crossed females showed decreased body/organ weights, reduced fertility, hematoma, cleft palate, tail curling, and absence of extremity. Nano-quercetin co-administration normalized parameters comparable to controls. Both Ethion and quercetin interacted with MCL-1, with quercetin exhibiting stronger binding energy. Conclusion: Nano-quercetin demonstrated stronger antioxidant properties than quercetin, counteracting ethion-induced maternal/fetal abnormalities.

A Novel Approach for Glioblastoma Treatment by Combining Apoptosis Inducers (TMZ, MTX, and Cytarabine) with E.V.A. (Eltanexor, Venetoclax, and A1210477) Inhibiting XPO1, Bcl-2, and Mcl-1.

Adjuvant treatment for Glioblastoma Grade 4 with Temozolomide (TMZ) inevitably fails due to therapeutic resistance, necessitating new approaches. Apoptosis induction in GB cells is inefficient, due to an excess of anti-apoptotic XPO1/Bcl-2-family proteins. We assessed TMZ, Methotrexate (MTX), and Cytarabine (Ara-C) (apoptosis inducers) combined with XPO1/Bcl-2/Mcl-1-inhibitors (apoptosis rescue) in GB cell lines and primary GB stem-like cells (GSCs). Using CellTiter-Glo® and Caspase-3 activity assays, we generated dose-response curves and analyzed the gene and protein regulation of anti-apoptotic proteins via PCR and Western blots. Optimal drug combinations were examined for their impact on the cell cycle and apoptosis induction via FACS analysis, paralleled by the assessment of potential toxicity in healthy mouse brain slices. Ara-C and MTX proved to be 150- to 10,000-fold more potent in inducing apoptosis than TMZ. In response to inhibitors Eltanexor (XPO1; E), Venetoclax (Bcl-2; V), and A1210477 (Mcl-1; A), genes encoding for the corresponding proteins were upregulated in a compensatory manner. TMZ, MTX, and Ara-C combined with E, V, and A evidenced highly lethal effects when combined. As no significant cell death induction in mouse brain slices was observed, we conclude that this drug combination is effective in vitro and expected to have low side effects in vivo.

Co-operation of MCL-1 and BCL-XL anti-apoptotic proteins in stromal protection of MM cells from carfilzomib mediated cytotoxicity.

Introduction: BCL-2 family proteins are important for tumour cell survival and drug resistance in multiple myeloma (MM). Although proteasome inhibitors are effective anti-myeloma drugs, some patients are resistant and almost all eventually relapse. We examined the function of BCL-2 family proteins in stromal-mediated resistance to carfilzomib-induced cytotoxicity in MM cells. Methods: Co-cultures employing HS5 stromal cells were used to model the interaction with stroma. MM cells were exposed to CFZ in a 1-hour pulse method. The expression of BCL-2 family proteins was assessed by flow cytometry and WB. Pro-survival proteins: MCL-1, BCL-2 and BCL-XL were inhibited using S63845, ABT-199 and A-1331852 respectively. Changes in BIM binding partners were examined by immunoprecipitation and WB. Results: CFZ induced dose-dependent cell death of MM cells, primarily mediated by apoptosis. Culture of MM cells on HS-5 stromal cells resulted in reduced cytotoxicity to CFZ in a cell contact-dependent manner, upregulated expression of MCL-1 and increased dependency on BCL-XL. Inhibiting BCL-XL or MCL-1 with BH-3 mimetics abrogated stromal-mediated protection only at high doses, which may not be achievable in vivo. However, combining BH-3 mimetics at sub-therapeutic doses, which alone were without effect, significantly enhanced CFZ-mediated cytotoxicity even in the presence of stroma. Furthermore, MCL-1 inhibition led to enhanced binding between BCL-XL and BIM, while blocking BCL-XL increased MCL-1/BIM complex formation, indicating the cooperative role of these proteins. Conclusion: Stromal interactions alter the dependence on BCL-2 family members, providing a rationale for dual inhibition to abrogate the protective effect of stroma and restore sensitivity to CFZ.

FLT3 and IRAK4 Inhibitor Emavusertib in Combination with BH3-Mimetics in the Treatment of Acute Myeloid Leukemia.

Targeting the FLT3 receptor and the IL-1R associated kinase 4 as well as the anti-apoptotic proteins MCL1 and BCL2 may be a promising novel approach in the treatment of acute myeloid leukemia (AML). The FLT3 and IRAK4 inhibitor emavusertib (CA4948), the MCL1 inhibitor S63845, the BCL2 inhibitor venetoclax, and the HSP90 inhibitor PU-H71 were assessed as single agents and in combination for their ability to induce apoptosis and cell death in leukemic cells in vitro. AML cells represented all major morphologic and molecular subtypes, including FLT3-ITD and NPM1 mutant AML cell lines and a variety of patient-derived AML cells. Emavusertib in combination with MCL1 inhibitor S63845 or BCL2 inhibitor venetoclax induced cell cycle arrest and apoptosis in MOLM-13 cells. In primary AML cells, the response to emavusertib was associated with the presence of the FLT3 gene mutation with an allelic ratio >0.5 and the presence of NPM1 gene mutations. S63845 was effective in all tested AML cell lines and primary AML samples. Blast cell percentage was positively associated with the response to CA4948, S63845, and venetoclax, with elevated susceptibility of primary AML with blast cell fraction >80%. Biomarkers of the response to venetoclax included the blast cell percentage and bone marrow infiltration rate, as well as the expression levels of CD11b, CD64, and CD117. Elevated susceptibility to CA4948 combination treatments with S63845 or PU-H71 was associated with FLT3-mutated AML and CD34 < 30%. The combination of CA4948 and BH3-mimetics may be effective in the treatment in FLT3-mutated AML with differential target specificity for MCL1 and BCL2 inhibitors. Moreover, the combination of CA4948 and PU-H71 may be a candidate combination treatment in FLT3-mutated AML.

Polymeric micellar nanoparticles for effective CRISPR/Cas9 genome editing in cancer.

The clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (Cas9) gene editing has attracted extensive attentions in various fields, however, its clinical application is hindered by the lack of effective and safe delivery system. Herein, we reported a cationic micelle nanoparticle composed of cholesterol-modified branched small molecular PEI (PEI-CHO) and biodegradable PEG-b-polycarbonate block copolymer (PEG-PC), denoted as PEG-PC/PEI-CHO/pCas9, for the CRISPR/Cas9 delivery to realize genomic editing in cancer. Specifically, PEI-CHO condensed pCas9 into nanocomplexes, which were further encapsulated into PEG-PC nanoparticles (PEG-PC/PEI-CHO/pCas9). PEG-PC/PEI-CHO/pCas9 had a PEG shell, protecting DNA from degradation by nucleases. Enhanced cellular uptake of PEG-PC/PEI-CHO/pCas9 nanoparticles was observed as compared to that mediated by Lipo2k/pCas9 nanoparticles, thus leading to significantly elevated transfection efficiency after escaping from endosomes via the proton sponge effect of PEI. In addition, the presence of PEG shell greatly improved biocompatibility, and significantly enhanced the in vivo tumor retention of pCas9 compared to PEI-CHO/pCas9. Notably, apparent downregulation of GFP expression could be achieved both in vitro and in vivo by using PEG-PC/PEI-CHO/pCas9-sgGFP nanoparticles. Furthermore, PEG-PC/PEI-CHO/pCas9-sgMcl1 induced effective apoptosis and tumor suppression in a HeLa tumor xenograft mouse model by downregulating Mcl1 expression. This work may provide an alternative paradigm for the efficient and safe genome editing in cancer.

ONC212 enhances YM155 cytotoxicity by triggering SLC35F2 expression and NOXA-dependent MCL1 degradation in acute myeloid leukemia cells.

Although the anticancer activity of ONC212 has been reported, the precise mechanism underlying its apoptotic effects remains unclear. In this study, we investigated the apoptotic mechanism of ONC212 in acute myeloid leukemia (AML) cells. ONC212 induces apoptosis, MCL1 downregulation, and mitochondrial depolarization in AML U937 cells. Ectopic MCL1 expression alleviates mitochondria-mediated apoptosis in ONC212-treated U937 cells. ONC212 triggers AKT phosphorylation, inducing NOX4-dependent ROS production and promoting HuR transcription. HuR-mediated ATF4 mRNA stabilization stimulates NOXA and SLC35F2 expression; ONC212-induced upregulation of NOXA leads to MCL1 degradation. The synergistic effect of ONC212 on YM155 cytotoxicity was dependent on increased SLC35F2 expression. In addition, YM155 feedback facilitated the activation of the ONC212-induced signaling pathway. A similar mechanism explains ONC212- and ONC212/YM155-induced AML HL-60 cell death. The continuous treatment of U937 cells with the benzene metabolite hydroquinone (HQ) generated U937/HQ cells, exhibiting enhanced responsiveness to the cytotoxic effects of ONC212. In U937/HQ cells, ONC212 triggered apoptosis through NOXA-mediated MCL1 downregulation, enhancing YM155 cytotoxicity. Collectively, our data suggested that ONC212 upregulated SLC35F2 expression and triggered NOXA-mediated MCL1 degradation in U937, U937/HQ, and HL-60 cells by activating the AKT/NOX4/HuR/ATF4 pathway. The ONC212-induced signaling pathway showed anti-AML activity and enhanced YM155 cytotoxicity.