Overexpression Of Mcl-1 Research Articles

Runx2 silencing sensitized human renal cell carcinoma cells to ABT-737 apoptosis

The prognostic value of Runt-related transcription factor 2 (Runx2) and its involvement in cell growth and motility have been reported in patients diagnosed with renal cell carcinoma (RCC). Since Runx2 may have the potential to be a target for the purpose of antitumor intervention, there is an urgent need to gain insight into its oncogenic properties. Using human 786-O, Caki-1 and ACHN RCC cells as models, the silencing of cellular Runx2 expression brought about a reduction in cyclin D1 and β-catenin expression, cell growth and migration without any significant cell death. Runx2-silenced cells turned into apoptosis vulnerable in the presence of ABT-737, a BH3 mimetic Bcl-2 inhibitor. Data from biochemical and molecular studies have revealed a positive correlation between Runx2 expression and Akt phosphorylation, Mcl-1 expression, and fibronectin expression. Results of genetic silencing studies have indicated the potential involvement of Mcl-1 and fibronectin in the decision of RCC cell ABT-737 resistance and sensitivity. The regulatory roles of the PI3K/Akt axis in the expression of Mcl-1 and fibronectin were suggested by means of the results taken from experiments involving pharmacological study of the PI3K/Akt. Since overexpression and prognostic roles of Runx2, activated Akt, Mcl-1, fibronectin, cyclin D1, and β-catenin have been revealed in RCC, it is important to explore the precise mechanisms underlying Runx2 oncogenic effects. Although the linking details between Runx2 and PI3K/Akt have yet to be identified, our findings suggest that Mcl-1 and fibronectin are downstream effectors of Runx2 via a regulatory axis of the PI3K/Akt and their promotion of cell growth, migration, and ABT-737 resistance in RCC cells.

The Protective Effects of Mcl-1 on Mitochondrial Damage and Oxidative Stress in Imiquimod-Induced Cancer Cell Death.

Mitochondria, vital organelles that generate ATP, determine cell fate. Dysfunctional and damaged mitochondria are fragmented and removed through mitophagy, a mitochondrial quality control mechanism. The FDA-approved drug IMQ, a synthetic agonist of Toll-like receptor 7, exhibits antitumor activity against various skin malignancies. We previously reported that IMQ promptly reduced the level of the antiapoptotic Mcl-1 protein and that Mcl-1 overexpression attenuated IMQ-triggered apoptosis in skin cancer cells. Furthermore, IMQ profoundly disrupted mitochondrial function, promoted mitochondrial fragmentation, induced mitophagy, and caused cell death by generating high levels of ROS. However, whether Mcl-1 protects mitochondria from IMQ treatment is still unknown. In this study, we demonstrated that Mcl-1 overexpression induced resistance to IMQ-induced apoptosis and reduced both IMQ-induced ROS generation and oxidative stress in cancer cells. Mcl-1 overexpression maintained mitochondrial function and integrity and prevented mitophagy in IMQ-treated cancer cells. Furthermore, IL-6 protected against IMQ-induced apoptosis by increasing Mcl-1 expression and attenuating IMQ-induced mitochondrial fragmentation. Mcl-1 overexpression ameliorates IMQ-induced ROS generation and mitochondrial fragmentation, thereby increasing mitochondrial stability and ultimately attenuating IMQ-induced cell death. Investigating the roles of Mcl-1 in mitochondria is a potential strategy for cancer therapy development.

Discovery of a Myeloid Cell Leukemia 1 (Mcl-1) Inhibitor That Demonstrates Potent In Vivo Activities in Mouse Models of Hematological and Solid Tumors.

Myeloid cell leukemia 1 (Mcl-1) is a key regulator of the intrinsic apoptosis pathway. Overexpression of Mcl-1 is correlated with high tumor grade, poor survival, and both intrinsic and acquired resistance to cancer therapies. Herein, we disclose the structure-guided design of a small molecule Mcl-1 inhibitor, compound 26, that binds to Mcl-1 with subnanomolar affinity, inhibits growth in cell culture assays, and possesses low clearance in mouse and dog pharmacokinetic (PK) experiments. Evaluation of 26 as a single agent in Mcl-1 sensitive hematological and solid tumor xenograft models resulted in regressions. Co-treatment of Mcl-1-sensitive and Mcl-1 insensitive lung cancer derived xenografts with 26 and docetaxel or topotecan, respectively, resulted in an enhanced tumor response. These findings support the premise that pro-apoptotic priming of tumor cells by other therapies in combination with Mcl-1 inhibition may significantly expand the subset of cancers in which Mcl-1 inhibitors may prove beneficial.

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

BackgroundCervical 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. PurposeTo investigate the oncogenic role of MCL1 and elucidate the antitumor activity of Sanggenon C, along with its molecular mechanisms, in cervical cancer. MethodsIn 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. ResultsOur 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. ConclusionOur 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.

The RNA helicases DDX19A/B modulate selinexor sensitivity by regulating MCL1 mRNA nuclear export in leukemia cells.

Selinexor, a first-in-class exportin1 (XPO1) inhibitor, is an attractive anti-tumor agent because of its unique mechanisms of action; however, its dose-dependent toxicity and lack of biomarkers preclude its wide use in clinical applications. To identify key molecules/pathways regulating selinexor sensitivity, we performed genome-wide CRISPR/Cas9 dropout screens using two B-ALL lines. We identified, for the first time, that paralogous DDX19A and DDX19B RNA helicases modulate selinexor sensitivity by regulating MCL1 mRNA nuclear export. While single depletion of either DDX19A or DDX19B barely altered MCL1 protein levels, depletion of both significantly attenuated MCL1 mRNA nuclear export, reducing MCL1 protein levels. Importantly, combining selinexor treatment with depletion of either DDX19A or DDX19B markedly induced intrinsic apoptosis of leukemia cells, an effect rescued by MCL1 overexpression. Analysis of Depmap datasets indicated that a subset of T-ALL lines expresses minimal DDX19B mRNA levels. Moreover, we found that either selinexor treatment or DDX19A depletion effectively induced apoptosis of T-ALL lines expressing low DDX19B levels. We conclude that XPO1 and DDX19A/B coordinately regulate cellular MCL1 levels and propose that DDX19A/B could serve as biomarkers for selinexor treatment. Moreover, pharmacological targeting of DDX19 paralogs may represent a potential strategy to induce intrinsic apoptosis in leukemia cells.

Discovery of Phenylpyrazole Derivatives as a New Class of Selective Inhibitors of MCL-1 with Antitumor Activity.

MCL-1, an antiapoptotic member of the BCL-2 family, is dysregulated and overexpressed in various tumors. In tumors with MCL-1 overexpression, selective inhibitors of MCL-1 are expected to overcome the drug resistance caused by BCL-2 inhibitors currently used in clinical treatment. Here, we employed docking-based virtual screening to identify an active hit, LC126, with binding affinity around 10 μM for MCL-1 and BCL-2. Under the guidance of structure-based design, we obtained a few selective inhibitors of MCL-1 after three rounds of structural optimization. The representative compound GQN-B37-E exhibited binding affinity for MCL-1 at the submicromolar range (K i = 0.6 μM) without apparent binding to BCL-2 or BCL-XL. 15N-heteronuclear single-quantum coherence NMR spectra suggested that this compound binds to the BH3-domain-binding pocket in the MCL-1 surface. Cellular assays revealed that GQN-B37-Me, the precursor of GQN-B37-E, is effective particularly on leukemia cells (such as H929 and MV-4-11) to induce caspase-dependent apoptosis. Its interaction with MCL-1 in cells was confirmed by coimmunoprecipitation. Administration of GQN-B37-Me to MV-4-11 xenograft mice at 50 mg/kg every 2 days for 20 days led to 43% tumor growth inhibition. GQN-B37-Me also exhibited reasonable in vitro stability in GSH and liver microsomes from several species. This new class of MCL-1 inhibitor may have potential to be further developed into a preclinical candidate for treating leukemia.

The role of PIWI proteins in glioma formation

This review explores the role of PIWI proteins, particularly PIWIL1, in glioma formation. The overexpression and dysregulation of PIWIL1 is seen in gliomas, impacting normal cell function and tumor suppressor genes. It is unknown if the overexpression of PIWIL1 causes tumor formation or if tumor formation causes the protein to be overexpressed. Dysregulation of tumor-suppressing genes like BTG2, FBXW7, and P27 by PIWIL1 contributes to aberrant cell cycle progression and growth. Elevated levels of CCND2, NESTIN, OLIG2, and MCL1 due to PIWIL1 dysregulation further promote tumor growth. Evidence reveals that reducing PIWIL1 leads to decreased tumor size and increased survival in animal models. Additionally, the overexpression of MCL-1, a protein regulated by PIWIL1, compromises chemotherapy efficacy. This research suggests that PIWIL1 overexpression is pivotal in glioma tumorigenesis.

A phase I study of pevonedistat, azacitidine, and venetoclax in patients with relapsed/refractory acute myeloid leukemia.

Azacitidine/venetoclax is an active regimen in patients with newly diagnosed acute myeloid leukemia (AML). However, primary or secondary resistance to azacitidine/venetoclax is an area of unmet need and overexpression of MCL1 is suggested to be a potential resistance mechanism. Pevonedistat inhibits MCL1 through activation of NOXA, and pevonedistat/azacitidine has previously shown activity in AML. To assess the tolerability and efficacy of adding pevonedistat to azacitidine/ venetoclax in relapsed/refractory AML, we conducted a phase I, multicenter, open-label study in 16 adults with relapsed/ refractory AML. Patients were treated with azacitidine, venetoclax along with pevonedistat intravenously on days 1, 3 and 5 of each 28-day cycle at doses of 10, 15 or 20 mg/m2 in successive cohorts in the dose escalation phase. The impact of treatment on protein neddylation as well as expression of pro-apoptotic BCL2 family members was assessed. The recommended phase II dose of pevonedistat was 20 mg/m2. Grade 3 or higher adverse events included neutropenia (31%), thrombocytopenia (13%), febrile neutropenia (19%), anemia (19%), hypertension (19%) and sepsis (19%). The overall response rate was 46.7% for the whole cohort including complete remission in five of seven (71.4%) patients who had not previously been treated with the hypomethylating agent/venetoclax. No measurable residual disease was detected in 80.0% of the patients who achieved complete remission. The median time to best response was 50 (range, 23-77) days. Four patients were bridged to allogeneic stem cell transplantation. The combination of azacitidine, venetoclax and pevonedistat is safe and shows encouraging preliminary activity in patients with relapsed/refractory AML. (NCT04172844).

Abstract 5720: Targeting CDK9 in cutaneous T-cell lymphoma using patient-derived xenograft models

Abstract Cutaneous T-cell lymphoma (CTCL) is a rare form of non-Hodgkin lymphoma. Mycosis fungoides and Sézary syndrome (MF/SS) are the most common subtypes in CTCL. Early-stage MF/SS has a favorable prognosis. The advanced-stage CTCL has limited treatment options and a median survival of <5 years, highlighting the urgent need to develop targeted therapies in CTCL. Preclinical studies of CDK9 inhibitors have shown anti-tumor activity in various subtypes of hematologic malignancies. Several CDK9 inhibitors have entered clinical trials for lymphomas and leukemias. However, whether targeting CDK9 in CTCL is a therapeutic option is little known and has resulted in CTCL not being included in ongoing clinical investigations of CDK9 inhibitors. AZD4573 is a highly selective and potent CDK9 inhibitor currently under clinical investigation for Hodgkin and non-Hodgkin lymphomas. It was shown that AZD4573 induces tumor cell death through depletion of MCL-1. Overexpression of MCL-1 has been shown in CTCL cell lines and skin biopsy specimens of CTCL patients. MCL-1 expression appears to be particularly elevated in patients with advanced stage disease. Here, we investigated the ex vivo anti-tumor activity of AZD4573 using patient-derived CTCL xenograft models (PDXs). Primary tumor cells harvested from CTCL PDX mice were activated with CD2/CD3/CD28 beads and incubated in hIL-2 and hIL-7 at an optimal cell density. Subsequently, cells were exposed to AZD4573 at various concentrations for 8 hr, then treated cells were harvested, washed, and placed in fresh media for another 48 hr. Growth inhibition was measured by the Real-Time Glo cell viability assay, and apoptotic activity was assessed by flow cytometry analysis of Caspase 3/7 activity and Annexin V + propidium iodide (PI) double staining. Additionally, we conducted RNA-seq analysis for MCL-1 expression in CTCL PDX primary tumor cells. AZD4573 inhibited cell proliferation in a dose-dependent manner in primary tumor cells from three CTCL PDX mice (PRS-1, PRS-2 PRS-3), with GI50 of 43.5 nM, 175.4 nM, and 113.5 nM, respectively. Furthermore, AZD4573 significantly increased caspase 3/7 activity and apoptosis in PRS-1 and PRS-3, but not PRS-2 primary tumor cells. In addition, our RNA-seq analysis showed that compared with normal controls (normal PBMC from healthy donors), the MCL-1 gene expression is increased 10-fold (P-value <0.0001) in PRS1 cells. The MCL-1 gene expression in PRS2 and PRS3 cells is the same as normal control. Furthermore, PRS-1, which has the highest MCL-1 expression level, is most sensitive to AZD4573. In conclusion, AZD4573 demonstrated dose-dependent growth inhibition and apoptosis induction in primary CTCL tumor cells. In addition, the expression level of MCL-1 appeared to be correlated with the ex vivo anti-tumor activity of AZD4573. Our findings provide a rationale for clinical investigation of targeting CDK9 in CTCL. Citation Format: Yan-Jin Liu, Chi-Heng Wu, Laura Pincus, Weiyun Z. Ai. Targeting CDK9 in cutaneous T-cell lymphoma using patient-derived xenograft models [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 5720.

Abstract 4670: KS18, an Mcl-1 inhibitor, triggers cell death and enhances the therapeutic efficacy of venetoclax in bortezomib-resistant multiple myeloma cells

Abstract A critical clinical challenge in the management of multiple myeloma (MM) is the development of both innate and acquired resistance to the treatment with bortezomib. Our prior research showed that the overexpression of the anti-apoptotic Bcl-2 family protein Mcl-1 can result in resistance to the chemotherapy drugs bortezomib and venetoclax. In order to overcome the issues posed by resistance, this study investigates the possibility of a small chemical called KS18, a novel Mcl-1 inhibitor. KS18 outperforms other Mcl-1 inhibitors such as S63845, VU661013, and AZD5991, according to the findings of our research, which shows that it is extraordinarily effective against MM cells that have become resistant to bortezomib. We establish the selectivity of KS18 through tests employing Mcl-1 knock-down (KD), and we find that it is both effective and efficient. In addition, we used immunoprecipitation (IP) and protein-protein interaction analysis (PPIs) to investigate the dynamic connections that exist between Mcl-1 and pro-apoptotic proteins. The KS18 therapy exhibits an outstanding ability to cause complete apoptosis, as seen by the remarkable 88.8% apoptosis rate recorded in MM patient samples. Not only does KS18 restore apoptosis in bortezomib-resistant MM cells, but it also synergizes with venetoclax to enhance the apoptotic response. This is an important finding. It has been demonstrated through both in vitro and in vivo research that KS18 can re-sensitize MM cells to the effects of bortezomib, which represents a potential step forward in the treatment of the disease. In bortezomib-resistant MM cells, treatment with KS18 led to a remarkable 92% reduction in colony formation compared to untreated controls. In vivo experiments that were performed on mice models that were resistant to bortezomib provide additional evidence that KS18 is effective. The extraordinary reduction in tumor weight of 55% that occurred as a direct result of treatment with KS18 at a dose of 1 mg/kg for a period of 15 days exemplifies the powerful anti-resistance characteristics of this compound. These intriguing findings highlight the potential value of KS18 as an adjunct to therapies aimed at overcoming bortezomib resistance in MM and other associated cancers. Bortezomib-resistant malignancies provide a challenge for patients, but additional research into the clinical value of KS18 holds the potential to improve treatment outcomes and broaden the range of therapeutic alternatives available to these patients. Citation Format: Omar S. Al Odat, Osama Aloudat, Emily Nelson, Tulin Budak-Alpogan, Subash Jonnalagadda, Yong Chen, Manoj Pandey. KS18, an Mcl-1 inhibitor, triggers cell death and enhances the therapeutic efficacy of venetoclax in bortezomib-resistant multiple myeloma 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 4670.

Enhancement of the Sensitivity of the Acute Lymphoblastic Leukemia Cells to ABT-737 by Formononetin.

Overexpression of (myeloid leukemia cell differentiation protein 1) Mcl-1 is associated with the reduction of ABT-737 toxicity and secondary resistance. In this study, the effect of formononetin (biochanin B) on Mcl-1 expression, cell growth, apoptosis, and ABT-737 sensitivity of the acute lymphoblastic leukemia (ALL) cells was investigated. In this experimental study, the cell proliferation and MTT assays were used to investigate the effect of formononetin on cell growth and survival. qRT-PCR was performed for the measurement of gene expression. Hoechst 33342 staining and caspase-3 activity assay were used for the determination of apoptosis. Our data showed that formononetin and ABT-737 both led to a significant reduction in the IC50 value and synergistically reduced the cell growth and survival relative to single treatment. Overexpression of Mcl-1 was found after the treatment with ABT-737. Formononetin decreased the expression of B-cell lymphoma 2 (Bcl-2) and Mcl-1 and increased the Bcl-2-associated protein x (Bax) and P21 expression. Moreover, formononetin enhanced the apoptotic effect of ABT-737 in ALL cells. In summary, formononetin showed anti-carcinogenic activities in human ALL cells via suppression of cell growth and survival. Formononetin enhanced the apoptotic effect of ABT-737, with contribution by inhibition of the Mcl-1 expression.

Small molecule Mcl-1 inhibitor for triple negative breast cancer therapy.

Apoptosis is an evolutionarily conserved cell death pathway that plays a crucial role in maintaining tissue homeostasis, orchestrating organismal development, and eliminating damaged cells. Dysregulation of apoptosis can contribute to the pathogenesis of malignant tumors and neurodegenerative diseases. Anticancer drugs typically possess the capacity to induce apoptosis in tumor cells. The Bcl-2 protein family, consisting of 27 members in humans, serves as the key regulator of mitochondrial function. This family can be divided into two functional groups: anti-apoptotic proteins (e.g., Bcl-2, Bcl-xl, Mcl-1) and pro-apoptotic proteins (e.g., Bad, Bax). Mcl-1 exerts its function by binding pro-apoptotic Bcl-2 proteins thereby preventing apoptosis induction. Overexpression of Mcl-1 not only correlates closely with tumorigenesis but also associates significantly with resistance towards targeted therapy and conventional chemotherapy. Effective induction of apoptosis can be achieved through inhibition or interference with Mcl-1. Thus, this mini review discusses existing Mcl-1 inhibitors.

The Role of Bcl-2 Family Proteins and Sorafenib Resistance in Hepatocellular Carcinoma.

Liver cancer has been reported to be one of the most malignant diseases in the world. It is late diagnosis consequently leads to a difficult treatment, as the cancer reached an advanced stage. Hepatocellular carcinoma (HCC) is the primary type of cancer diagnosed in the liver, with deadly characteristics and a poor prognosis. The first-in-line treatment for advanced HCC is sorafenib. Sorafenib acts by inhibiting cell proliferation and by inducing apoptosis as well as blocks receptors associated with these mechanisms. Due to its constant use, sorafenib resistance has been described, especially to proteins of the Bcl-2 family, and their overexpression of Bcl-XL and Mcl-1. This review focuses on the role of the Bcl-2 proteins in relation to sorafenib resistance as a consequence of first-in-line treatment in HCC.

Genome-Wide CRISPR/Cas9 Screens Identify DDX19A/DDX19B As a Critical Regulator of Intrinsic Apoptosis By Regulating MCL1 mRNA Cellular Localization

Despite recent advancements, the prognosis of relapsed/refractory acute lymphoblastic leukemia (ALL) remains dismal, necessitating novel therapies. Selinexor, an XPO1 (Exportin-1) inhibitor, shows effectiveness in multiple myeloma (MM), diffuse large B-cell lymphoma (DLBCL), and ALL mouse models. However, its broad target spectrum could lead to off-target toxicities, limiting its clinical utility. To elucidate Selinexor's molecular mechanisms in ALL we used CRISPR-Cas9 screens in the presence of Selinexor on ALL cell lines NALM-6 and KOPN-49. Among 18 genes displaying synthetic lethality in both cell lines with Selinexor, we focused on paralogous genes DDX19A and DDX19B, critical in nucleocytoplasmic transport of mRNA and translation initiation by dissociating protein complexes bound to mRNA. Competition assays showed prominent growth inhibitions when DDX19B was knocked out prior to DDX19A depletion in both NALM-6 and KOPN-49, while single KO of either DDX19A or DDX19B did not remarkably inhibit cell growth. Additionally, in MOLT-3 with low DDX19B expression, significant growth inhibitions were observed upon single KO of DDX19A and these phenotypes were canceled by the overexpression of DDX19B, suggesting functional redundancy between DDX19A and DDX19B. Further, we generated MOLT-3 that stably express HA-FKBP12 F36V-tagged DDX19A and knocked out endogenous DDX19A. Western blot analysis showed depletion of DDX19A by dTAG V-1 led to upregulate cleaved caspase-3 expression level within 4 hours, accompanied by a significant decrease in MCL1 protein levels in a BAX/BAK dependent manner. These phenotypes were rescued by the overexpression of MCL1, suggesting that the loss of DDX19A/19B induced intrinsic apoptosis through the downregulation of MCL1 expression. Given that the depletion of DDX19A/19B led to the mislocalization of polyadenylated mRNA and that neither the stability of the MCL1 protein nor the translation of MCL1 mRNA was affected by this depletion, as confirmed by RNA-FISH, and Actinomycin-D and Cycloheximide chase assays respectively, we concluded that DDX19A/19B are crucial for the nucleocytoplasmic transport of MCL1 mRNA. Furthermore, in exploring the relationship between the loss of DDX19A/19B and sensitivity to Selinexor, we found that combined treatment with Selinexor and the depletion of either DDX19A or DDX19B leads to a reduction in MCL1 protein, which can be rescued by overexpressing MCL1. Through CRISPR-Cas9 screens, we identified DDX19A/19B, which are in a paralogous relationship, as potential therapeutic targets for ALL. Mechanistically, the loss of DDX19A/19B leads to impaired cytoplasmic mRNA transport, leading to the inhibition of novel synthesis and reduced expression of MCL1 protein, thereby inducing intrinsic apoptosis. Furthermore, we found that DDX19A/19B depletion exerts synergistic effects with Selinexor through the downregulation of MCL1 expression, suggesting that DDX19A/19B levels could serve as a biomarker for Selinexor treatment in ALL.

Drug Resistance Can be Overcome in Multiple Myeloma and Acute Myeloid Leukemia By Targeting Mcl-1 with the Small Chemical KS18

Drug resistance and patient recurrence are still key clinical difficulties in multiple myeloma (MM) treatment, despite the tremendous improvements provided by bortezomib therapy. In many cancers, Mcl-1, a member of the Bcl-2 protein family, helps keep cells alive by preventing their natural death process, apoptosis. The anti-cancer therapeutic promise of Mcl-1 inhibitors is highlighted by the fact that Mcl-1 overexpression increases carcinogenesis and medication resistance. Here, we provide KS18, a powerful pyoluteorin derivative with excellent effectiveness towards Mcl-1-dependent cancer cells, to address this unmet therapeutic need. Based on our results, we conclude that KS18 causes apoptosis in cancer cells through activating the mitochondrial outer membrane permeabilization (MOMP) pathway, with the participation of Bak and Bax, in particular in MM and acute myeloid leukemia (AML). With IC 50 values below 1.5µM, KS18 demonstrated efficacy against bortezomib-resistant MM cells, outperforming other Mcl-1 inhibitors in clinical trials such as S63845, VU661013, and AZD5991. Total apoptosis in MM patient samples treated with KS18 was an impressive 88.8 percent. After four weeks of treatment with a single tolerable dose each week, a model of bortezomib-resistant MM showed considerable tumor shrinkage, demonstrating the powerful anti-tumor efficacy of KS18. According to these results, KS18 may be able to overcome resistance by concentrating on Mcl-1. These encouraging findings have prompted the development of many models of drug-resistant MM and AML for in vivo evaluation of this potential chemical.

Hydroquinone-selected chronic myelogenous leukemia cells are sensitive to chloroquine-induced cytotoxicity via MCL1 suppression and glycolysis inhibition

Previous studies have provided evidence that repeated exposure to the benzene metabolite hydroquinone (HQ) induces malignant transformation and increases basal autophagy in the chronic myeloid leukemia (CML) cell line K562. This study explored the cytotoxicity of the autophagy inhibitor chloroquine (CQ) on parental and HQ-selected K562 (K562/HQ) cells. CQ triggered apoptosis in these cells independently of inhibiting autophagic flux; however, in K562/HQ cells, CQ-induced cytotoxicity was higher than in K562 cells. Mechanistically, CQ-induced NOXA upregulation led to MCL1 downregulation and mitochondrial depolarization in K562/HQ cells. MCL1 overexpression or NOXA silencing attenuated CQ-mediated cytotoxicity in K562/HQ cells. CQ triggered ERK inactivation to increase Sp1, NFκB, and p300 expression, and co-assembly of Sp1, NFκB, and p300 in the miR-29a promoter region coordinately upregulated miR-29a transcription. CQ-induced miR-29a expression destabilized tristetraprolin (TTP) mRNA, which in turn reduced TTP-mediated NOXA mRNA decay, thereby increasing NOXA protein expression. A similar mechanism explained the CQ-induced downregulation of MCL1 in K562 cells. K562/HQ cells relied more on glycolysis for ATP production than K562 cells, whereas inhibition of glycolysis by CQ was greater in K562/HQ cells than in K562 cells. Likewise, CQ-induced MCL1 suppression and glycolysis inhibition resulted in higher cytotoxicity in CML KU812/HQ cells than in KU812 cells. Taken together, our data confirm that CQ inhibits MCL1 expression through the ERK/miR-29a/TTP/NOXA pathway, and that inhibition of glycolysis is positively correlated to higher cytotoxicity of CQ on HQ-selected CML cells.

Dihydroartemisinin Synergistically Promotes the Antileukemic Activity of Venetoclax By Downregulating Mcl-1 in Acute Myeloid Leukemia Cells

Background: Venetoclax is a BH3 mimetic, which selectively inhibits Bcl-2. For older persons with newly diagnosed AML, the FDA has approved the use of venetoclax in conjunction with hypomethylating drugs or low-dose cytarabine. However, the selective inhibition of Bcl-2 in AML often results in the overexpression of Mcl-1, another dominant anti-apoptotic Bcl-2 family protein conferring venetoclax resistance. Dihydroartemisinin (DHA) is a herbal monomer that is taken orally to cure malaria. Studies have shown that DHA downregulates MCL-1 protein expression in AML cells. In the present study, we investigated whether the combination of venetoclax and DHA efficiently promotes apoptosis in AML cells. Methods: We assessed the combined effect of venetoclax and DHA on cell viability, apoptosis, combination index, and cell cycle in AML cells from AML cell lines and deno AML patients. Western blot analysis was used to validate the mechanisms of venetoclax and DHA. Results: Venetoclax efficiently impaired cell viability and dose-dependently promoted apoptosis when combined with DHA; their synergism was aptly represented by the combination index. Venetoclax and DHA had significant synergistic inhibition of cell viability both in cells from AML cell lines and in deno AML patients. The combination of venetoclax and DHA impaired cell cycle progression by arresting the cell cycle in S-phase and inducing apoptosis. Mechanistically, DHA mitigated venetoclax-induced upregulation of Mcl-1 and BCL-XL. Moreover, the combination inhibited the expression of C-MYC. Conclusions: Our findings demonstrate the synergistic, preferential antileukemic effects of venetoclax and DHA on AML cells, providing a rationale for preclinical and clinical trials by combining these agents already being used in clinical practice to treat AML.

Acquired resistance to venetoclax plus azacitidine in acute myeloid leukemia: In vitro models and mechanisms

The combination of venetoclax (VEN) and azacitidine (AZA) has become the standard of care for acute myeloid leukemia (AML) patients who are ≥ 75 years or unfit for intensive chemotherapy. Though initially promising, resistance to the combination therapy is an issue and VEN + AZA-relapsed/refractory patients have dismal outcomes. To better understand the mechanisms of resistance, we developed VEN + AZA-resistant AML cell lines, MV4-11/VEN + AZA-R and ML-2/VEN + AZA-R, which show > 300-fold persistent resistance compared to the parental lines. We demonstrate that these cells have unique metabolic profiles, including significantly increased levels of cytidine triphosphate (CTP) and deoxycytidine triphosphate (dCTP), changes in fatty acid and amino acid metabolism and increased utilization and reliance on glycolysis. Furthermore, fatty acid transporter CD36 is increased in the resistant cells compared to the parental cells. Inhibition of glycolysis with 2-Deoxy-D-glucose re-sensitized the resistant cells to VEN + AZA. In addition, the VEN + AZA-R cells have increased levels of the antiapoptotic protein Mcl-1 and decreased levels of the pro-apoptotic protein Bax. Overexpression of Mcl-1 or knockdown of Bax result in resistance to VEN + AZA. Our results provide insight into the molecular mechanisms contributing to VEN + AZA resistance and assist in the development of novel therapeutics to overcome this resistance in AML patients.

5,6-Dichloro-1-β-D-ribofuranosylbenzimidazole (DRB) induces apoptosis in breast cancer cells through inhibiting of Mcl-1 expression

The effective treatment of breast cancer remains a profound clinical challenge, especially due to drug resistance and metastasis which unfortunately arise in many patients. The transcription inhibitor 5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole (DRB), as a selective inhibitor of cyclin-dependent kinase 9, was shown to be effective in inducing apoptosis in various hematopoietic malignancies. However, the anticancer efficacy of DRB against breast cancer is still unclear. Herein, we demonstrated that administration of DRB to the breast cancer cell line led to the inhibition of cellular proliferation and induction of the typical signs of apoptotic cells, including the increases in Annexin V-positive cells, DNA fragmentation, and activation of caspase-7, caspase-9, and poly (ADP ribose) polymerase (PARP). Treatment of DRB resulted in a rapid decline in the myeloid cell leukemia 1 (Mcl-1) protein, whereas levels of other antiapoptotic proteins did not change. Overexpression of Mcl-1 decreased the DRB-induced PARP cleavage, whereas knockdown of Mcl-1 enhanced the effects of DRB on PARP activation, indicating that loss of Mcl-1 accounts for the DRB-mediated apoptosis in MCF-7 cells, but not in T-47D. Furthermore, we found that co-treatment of MCF-7 cells with an inhibitor of AKT (LY294002) or an inhibitor of the proteasome (MG-132) significantly augmented the DRB-induced apoptosis. These data suggested that DRB in combination with LY294002 or MG-132 may have a greater therapeutic potency against breast cancer cells.

Osimertinib is a dual inhibitor of hepatocellular carcinoma and angiogenesis in an EGFR-independent manner, and synergizes with venetoclax.

To investigate the effects of osimertinib on hepatocellular carcinoma (HCC) and angiogenesis, and its combinatory effects with venetoclax in HCC. Viability was assessed by flow cytometry of Annexin V in multiple HCC cell lines after drug treatment. In vitro angiogenesis assay was performed using primary human liver tumor associated endothelial cell (HLTEC). HCC-bearing model was generated by subcutaneous implantation of Hep3B cells to investigate the efficacy of osimertinib alone and its combination with venetoclax. Osimertinib significantly induced apoptosis in a panel of HCC cell lines regardless of EGFR expression level. It inhibited capillary network formation and induced apoptosis in HLTEC. Using HCC xenograft mouse model, we further showed that osimertinib at non-toxic dose inhibited tumor growth by ~ 50% and remarkably decreased blood vessel in tumor. Mechanism studies demonstrated that osimertinib acted on HCC cells in an EGFR-independent manner. It decreased level of VEGF and Mcl-1 in HCC cells via suppressed phosphorylation of eIF4E, thus leading to inhibition of eIF4E-mediated translation. Mcl-1 overexpression reversed pro-apoptotic effect of osimertinib, suggesting an important role of Mcl-1 in osimertinib's action in HCC cells. Of note, the combination of osimertinib and venetoclax achieved approximately complete HCC cell death and tumor growth in mice. We provide pre-clinical evidence that osimertinib is a promising candidate for the treatment of HCC via targeting tumor cells and angiogenesis. The combination of osimertinib and venetoclax is synergistic in inhibiting HCC.