Expression Of Myeloid Cell Leukemia-1 Protein Research Articles

Abstract 6001: Repurposing dihydroergotamine mesylate as a potential MCL-1 inhibitor and its antitumor effect in pancreatic cancer cells

Abstract Background: Myeloid cell leukemia 1 (MCL-1) is an anti-apoptotic member of the BCL-2 protein family which is involved in intrinsic (mitochondrial) pathway of apoptosis and is deregulated in several cancers. Current MCL-1 specific inhibitors have limitations in terms of efficiency, tolerability, and off-target effects and challenges associated with shallow binding groove of MCL-1. In this study, dihydroergotamine mesylate (DHE), an approved anti-migraine drug, was found as a potential ligand of the MCL-1 BH3-binding pocket and was further evaluated for its potential MCL-1 inhibition and effect on pancreatic cancer cells. Methods: PubChem Database was used for library preparation and virtual screening (OpenBabel and AutoDock Vina). The 55 crystal structures available for MCL-1 in Protein Data Bank (PDB) were clustered using principal component analysis with Bio3D R-package to select representative PDBs for further screening. Consensus voting and k-means clustering was used to shortlist compounds for Molecular dynamics (MD) simulations. MD simulations (100 nanoseconds) by GROMACS were employed to measure stability of ligands in MCL-1 binding pocket. DHE efficacy on cell proliferation was evaluated with MTS and clonogenic assays and on apoptosis using the FITC/Annexin V flow cytometry assay in MiaPaCa-2 and PANC-1 pancreatic cancer cell lines. Effect of DHE treatment on MCL-1 gene and protein expression was analyzed using qRT-PCR and western blotting, respectively. Results: Computational screening identified DHE (PubChem ID: 12308974) as a top hit exhibiting consistent interactions within the MCL-1 BH3-binding pocket. Notably, DHE displayed stable RMSD and formed four hydrogen bonds throughout MD simulations. In vitro results showed that DHE significantly reduced viability of MiaPaCa-2 and PANC-1 pancreatic cancer cells with IC50 values of 31 μM and 47 μM, respectively. DHE significantly (P<0.001) suppressed colony formation by pancreatic cancer cells in a dose-dependent (10μM, 20μM, and 40μM) manner. The percentage of Annexin V-positive and Propidium iodide-negative fraction in MiaPaCa-2 cells treated with DHE also increased in a dose-dependent manner. DHE did not have significant effect on MCL-1 mRNA expression. However, western blotting demonstrated MCL-1 protein levels were significantly reduced in both pancreatic cancer cell lines upon DHE treatment. Conclusion: Our findings showed DHE suppresses the pancreatic cancer cells viability and proliferation, and significantly increased early apoptotic cells. DHE treatment had no effect on MCL-1 at the transcription level but significantly reduced the protein, suggesting posttranslational modifications, and thus future mechanism of action studies is required. The present study reports that the anti-migraine drug DHE might be a novel therapeutic agent for pancreatic cancer and needs to be explored further. Citation Format: Pooja Mittal, Jae Ho Lo, Shivani Soni, Francesca Battaglin, Shradheya RR Gupta, Lesly Torres-Gonzalez, Yan Yang, Sandra Algaze, Priya Jayachandran, Karam Ashouri, Alexandra Wong, Wu Zhang, Jian Yu, Lin Zhang, Joshua Millstein, Indrakant Kumar Singh, Heinz Josef Lenz. Repurposing dihydroergotamine mesylate as a potential MCL-1 inhibitor and its antitumor effect in pancreatic cancer 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 6001.

MCL1 inhibition: a promising approach to augment the efficacy of sorafenib in NSCLC through ferroptosis induction

Ferroptosis, an iron-dependent form of regulated cell death, plays a crucial role in modulating the therapeutic response in non-small cell lung cancer (NSCLC) patients. Studies have identified the signal transducer and activator of transcription 3 (STAT3) and myeloid cell leukemia-1 (MCL1) as potential targets for sorafenib, which exhibits activities in inducing ferroptosis. However, the role of STAT3-MCL1 axis in sorafenib-induced ferroptosis in NSCLC is still unclear. This study provided evidence that ferroptosis is a critical driver of sorafenib-induced cell death in NSCLC, supported by the accumulation of lipid peroxidation products, indicative of oxidative stress-induced cell death. Additionally, both in vitro and in vivo experiments showed that ferroptosis contributed to a significant portion of the anti-cancer effects elicited by sorafenib in NSCLC. The noticeable accumulation of lipid peroxidation products in sorafenib-treated mice underscored the significance of ferroptosis as a contributing factor to the therapeutic response of sorafenib in NSCLC. Furthermore, we identified the involvement of the STAT3/MCL1 axis in sorafenib-induced antitumor activity in NSCLC. Mechanistically, sorafenib inhibited endogenous STAT3 activation and downregulated MCL1 protein expression, consequently unleashing the ferroptosis driver BECN1 from the BECN1-MCL1 complex. Conversely, there is an augmented association of BECN1 with the catalytic subunit of system Xc−, SLC7A11, whose activity to import cystine and alleviate lipid peroxidation is hindered upon its binding with BECN1. Notably, we found that MCL1 upregulation correlated with ferroptosis resistance in NSCLC upon sorafenib treatment. Our findings highlight the importance of sorafenib-triggered ferroptosis in NSCLC and offer a novel strategy to treat advanced NSCLC patients: by downregulating MCL1 and, in turn, predispose NSCLC cells to ferroptosis.

Abstract 5813: Pharmacodynamic biomarker strategies for CDK9 inhibition

Abstract Cyclin-dependent kinase 9 (CDK9) is a promising target for cancer therapy due to its regulation of RNA polymerase II (RPB1) and key downstream anti-apoptosis proteins such as myeloid cell leukemia-1 (MCL-1). TP-1287 is a novel oral prodrug of alvocidib, a potent CDK9 inhibitor, and is currently under clinical investigation in patients with advanced solid tumors (clinicaltrials.gov, NCT03604783). TP-1287 is converted to alvocidib by alkaline phosphatases. CDK9 mediates phosphorylation of RPB1 at Ser2 and subsequently drives transcription of key oncogenic signaling genes, such as MCL-1. MCL-1 is a critical factor for survival of tumor types including acute myeloid leukemia (AML), multiple myeloma (MM) and myelodysplastic syndromes (MDS). MCL-1 is also expressed in normal peripheral blood mononuclear cells (PBMCs), thus it was hypothesized that CDK9 inhibition can be assessed clinically using PBMCs as a surrogate biomarker at multiple time points. We hypothesized that alvocidib, the pharmaceutically active form of TP-1287, would modulate CDK9 signaling pathways in cancer cells, resulting in tumor growth inhibition. In cell viability assays using CellTiter-Glo to evaluate the in vitro anti-tumor activity of alvocidib, we observed an IC50 of around 100 nM in AML and MM cell lines: 53-78 nM in MOLM-13, MV-4-11, and HL-60 AML cell lines and 31-223 nM in NCI-H929 and RPMI-8226 MM cell lines. In flow cytometry analysis, alvocidib reduced phosphorylation of RPB1 (p-RPB1) and MCL-1 protein expression in RPMI-8226 and MV-4-11 cells in a dose and time-dependent manner. In a pharmacodynamic study using the RPMI-8226 xenograft model after a single oral administration of TP-1287, we observed a substantial decrease of p-RPB1 and MCL-1 in tumor tissues followed by induction of cleaved caspase-3, an indicator of apoptosis. Furthermore, in an in vivo efficacy study using the RPMI-8226 model, TP-1287 achieved tumor growth inhibition (%TGI), ranging from 17.3% at 0.5 mg/kg (QD) to 86.6% at 15 mg/kg (Q7D) at day 22 after treatment. We hypothesized that PBMCs could serve as a surrogate tissue to measure CDK9 inhibition. To assess this approach in PBMCs, we performed ex vivo assays using human PBMCs from multiple healthy donors. Alvocidib demonstrated dose-dependent p-RPB1 and MCL-1 inhibition after 24 hr treatment. Preliminary data from the Phase 1 trial with TP-1287 showed p-RPB1 suppression in PBMCs from multiple patients, suggesting the successful execution of this assay and evidence of target engagement in the clinical setting. Taken together, TP-1287 demonstrated potent cell and tumor growth inhibition in multiple hematological cell lines, including AML and MM. Furthermore, a newly established flow cytometry system for p-RPB1 and MCL-1 to evaluate CDK9 inhibition in human PBMCs was developed, which could be useful as a surrogate biomarker for TP-1287 in clinical trials and warrants further investigation. Citation Format: Yuta Matsumura, Ethika Tyagi, Clifford J. Whatcott, Jason M. Foulks, Adam Siddiqui-Jain, David J. Bearss, Steven L. Warner. Pharmacodynamic biomarker strategies for CDK9 inhibition [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5813.

MCL1 gene silencing promotes senescence and apoptosis of glioma cells via inhibition of the PI3K/Akt signaling pathway.

Glioma is known to be the most prevalent primary brain tumor. In recent years, there has been evidence indicating myeloid cell leukemia-1 (MCL1) plays a role in brain glioblastoma. Therefore, the present study was conducted with aims of exploring the ability of MCL1 silencing to influence glioma cell senescence and apoptosis through the mediation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway. Glioma and tumor-adjacent tissues were collected in order to detect the presence of higher levels of MCL1 protein expression. Next, the mRNA and protein expression of MCL1, PI3K, Akt, B cell lymphoma 2 (Bcl2), Bcl2-associated X (Bax), B lymphoma Mo-MLV insertion region 1 homolog (Bmi-1), and phosphatase and tensin homolog (PTEN) were determined. Cell counting kit-8 assay was applied to detect cell proliferation, β-galactosidase staining for cell senescence, and flow cytometry for cell cycle entry and apoptosis. Initially, the results revealed higher positive expression rate of MCL1 protein, increased mRNA and protein expression of MCL1, PI3K, Akt, Bmi-1, and Bcl-2 and decreased that of Bax and PTEN in human glioma tissues. The silencing of MCL1 resulted in a decrease in mRNA and protein expression of PI3K, Akt, Bmi-1, and Bcl-2 and an increase in Bax and PTEN expressions in glioma cells. Moreover, silencing of MCL1 also inhibited cell proliferation and cell cycle entry in glioma cells, and promoted glioma cell senescence and apoptosis. In conclusion, the aforementioned results collectively suggested that the silencing of MCL1 promotes senescence and apoptosis in glioma cells through inhibiting the PI3K/Akt signaling pathway. Thus, decreasing the expression of MCL1 might have therapeutic functions in glioma. © 2018 IUBMB Life, 71(1):81-92, 2019.

Myeloid cell leukemia-1 protein expression and myeloid cell leukemia-1 gene amplification in non small cell lung cancer.

Myeloid cell leukemia-1 (Mcl-1) is a member of the B-cell lymphoma 2 family known to play a significant role in the regulation of apoptosis. Mcl-1 expression has been studied in nonsmall cell lung cancer (NSCLC) cell lines but has not been previously evaluated as a prognostic factor in clinical samples. Formalin-fixed, paraffin-embedded sections from 119 NSCLC, including 33 squamous cell carcinomas (SCC), 55 adenocarcinomas (AC), and 31 either pure adenocarcinoma in situ (AIS) or AC with lepidic features were immunostained by an automated method with rabbit polyclonal Mcl-1. Cytoplasmic Mcl-1 (cMcl-1) immunoreactivity was scored based on intensity and percentage of positive tumor cells in both tumor and adjacent benign epithelium in each case. MCL1 amplification was determined by hybrid capture-based comprehensive genomic profiling (CGP) on a separate cohort of 9393 NSCLC samples. Intense diffuse cMcl-1 overexpression was noted in 35/119 (29%) tumors overall and correlated with tumor type (52% AIS vs. 31% AC vs. 6% SCC, P < 0.0001), tumor grade (48% grade 1 vs. 14% grade 2 vs. 31% grade 3, P = 0.007), small tumor size (36% ≤3.0 cm vs. 16% >3.0 cm, P = 0.016), and lengthened survival within the AIS subgroup (100% alive vs. 42% expired, P = 0.018) while showing a trend toward correlation with nonrecurrent disease overall (32% nonrecurrent vs. 11% recurrent, P = 0.072) and within the AC subgroup (33% nonrecurrent vs. 0% recurrent, P = 0.092). MCL1 amplification was identified in 569 (6%) of 9393 NSCLC by CGP. cMcl-1 overexpression appears to occur independently from MCL1 gene amplification in NSCLC and correlates with AIS histologic type, lower tumor grade, smaller tumor size, nonrecurrent disease, and increased survival.

MCL-1 is modulated in Crohn\u2019s disease fibrosis by miR-29b via IL-6 and IL-8

The miR-29 family is involved in fibrosis in multiple organs, including the intestine where miR-29b facilitates TGF-β-mediated up-regulation of collagen in mucosal fibroblasts from Crohn’s disease (CD) patients. Myeloid cell leukemia-1 (MCL-1), a member of the B-cell CLL/Lymphoma 2 (BCL-2) apoptosis family, is involved in liver fibrosis and is targeted by miR-29b via its 3’-UTR in cultured cell lines. We investigate the role of MCL-1 and miR-29b in primary intestinal fibroblasts and tissue from stricturing CD patients. Transfection of CD intestinal fibroblasts with pre-miR-29b resulted in a significant increase in the mRNA expression of MCL-1 isoforms [MCL-1Long (L)/Extra Short (ES) and MCL-1Short (S)], although MCL-1S was expressed at significantly lower levels. Western blotting predominantly detected the anti-apoptotic MCL-1L isoform, and immunofluorescence showed that staining was localised in discrete nuclear foci. Transfection with pre-miR-29b or anti-miR-29b resulted in a significant increase or decrease, respectively, in MCL-1L foci. CD fibroblasts treated with IL-6 and IL-8, inflammatory cytokines upstream of MCL-1, increased the total mass of MCL-1L-positive foci. Furthermore, transfection of intestinal fibroblasts with pre-miR-29b resulted in an increase in mRNA and protein levels of IL-6 and IL-8. Finally, immunohistochemistry showed reduced MCL-1 protein expression in fibrotic CD samples compared to non-stricturing controls. Together, our findings suggest that induction of MCL-1 by IL-6/IL-8 may surmount any direct down-regulation by miR-29b via its 3’-UTR. We propose that an anti-fibrotic miR-29b/IL-6 IL-8/MCL-1L axis may influence intestinal fibrosis in CD. In the future, therapeutic modulation of this pathway might contribute to the management of fibrosis in CD.

Enhanced Stabilization of MCL1 by the Human T-Cell Leukemia Virus Type 1 bZIP Factor Is Modulated by Blocking the Recruitment of Cullin 1 to the SCF Complex.

Human T-cell leukemia virus type 1 (HTLV-1) is an oncogenic retrovirus that is the etiological agent of adult T-cell leukemia (ATL). The HTLV-1 basic leucine zipper factor (HBZ), which is encoded by the minus strand of the provirus, is constitutively expressed in all ATL patient cells and likely contributes to the development and maintenance of ATL. Furthermore, the overexpression of the myeloid cell leukemia 1 (MCL1) protein is frequently observed in hematological cancers as well as several other types of cancers. Here, we found that the expression of HBZ in cells stabilized MCL1 protein expression and suppressed the MCL1-mediated release of cytochrome c from the mitochondria. This effect was mediated by inhibition of the ubiquitin-dependent degradation of MCL1. In a serial binding assay, HBZ interacted with cullin 1 (CUL1) through a head-to-tail interaction. The association between CUL1 and Skp1, which serves as the molecular scaffold for the components of SCF ubiquitin ligase complexes, was markedly repressed in the presence of HBZ. Mechanistic analysis indicated that HBZ abrogated the CUL1 association with Skp1, which in turn promoted the cellular expression of MCL1. This novel function of HBZ likely plays a role in the viral pathogenesis of HTLV-1 and provides important insights into our understanding of the development of ATL.

MCL1 is deregulated in subgroups of diffuse large B-cell lymphoma

Myeloid cell leukemia-1 (MCL1) is an anti-apoptotic member of the BCL2 family that is deregulated in various solid and hematological malignancies. However, its role in the molecular pathogenesis of diffuse large B-cell lymphoma (DLBCL) is unclear. We analyzed gene expression profiling data from 350 DLBCL patient samples and detected that activated B-cell-like (ABC) DLBCLs express MCL1 at significantly higher levels compared with germinal center B-cell-like DLBCL patient samples (P=2.7 × 10(-10)). Immunohistochemistry confirmed high MCL1 protein expression predominantly in ABC DLBCL in an independent patient cohort (n=249; P=0.001). To elucidate molecular mechanisms leading to aberrant MCL1 expression, we analyzed array comparative genomic hybridization data of 203 DLBCL samples and identified recurrent chromosomal gains/amplifications of the MCL1 locus that occurred in 26% of ABC DLBCLs. In addition, aberrant STAT3 signaling contributed to high MCL1 expression in this subtype. Knockdown of MCL1 as well as treatment with the BH3-mimetic obatoclax induced apoptotic cell death in MCL1-positive DLBCL cell lines. In summary, MCL1 is deregulated in a significant fraction of ABC DLBCLs and contributes to therapy resistance. These data suggest that specific inhibition of MCL1 might be utilized therapeutically in a subset of DLBCLs.