Mcl-1 Expression Research Articles

Characterizing and Targeting of BCL-2 Family Members in Nasopharyngeal Carcinoma.

The success of BH3 mimetics in hematological malignancies has spurred interest in their application in solid tumors. We examined the expression of the BCL-2 family of molecules in NPC tumors and cell lines and explored the anticancer efficacy of BH3 mimetics invitro. Immunohistochemistry for BCL-2, MCL-1, BCL-xL, and transcriptomic analyses was conducted on NPC tumors. The efficacy of ABT-199, S63845, and ABT-737 were examined as monotherapy and in combination with cisplatin in NPC cell lines. RNA sequencing was performed to identify up and downregulated pathways in sensitive cell lines. One hundred and forty-nine EBV-positive NPC and 15 EBV-negative NPC were identified. Expression of BCL-2 was more frequent in EBV-positive NPC. BCL-2, MCL-1, and BCL-xL expression was not prognostic for overall survival. Marked sensitivity was seen with the combination of S63845 and cisplatin in NPC43. Our study demonstrates the therapeutic potential of combining cisplatin and S63845, which warrants further investigation.

Prolonged Survival of Neutrophils Induced by Tumor-Derived G-CSF/GM-CSF Promotes Immunosuppression and Progression in Laryngeal Squamous Cell Carcinoma.

Tumor-associated neutrophils (TANs) play a crucial role in tumor progression and exhibit prolonged survival. However, the mechanism underlying their extended lifespan and significance in laryngeal squamous cell carcinoma (LSCC) remains unclear. Herein, it is observed that apoptosis of TANs is significantly delayed owing to induction by tumor-derived G-CSF and GM-CSF through the activation of the PI3K-AKT signaling pathway, upregulation of anti-apoptotic Mcl-1 expression, and downregulation of activated Caspase-3 levels. It is found that prolonged survival of TANs leads to the accumulation of aged CXCR4+ neutrophils that exhibit potent immunosuppressive properties and are associated with poor patient prognosis. Furthermore, extended survival promotes the enhanced immunosuppressive function of CD8+ T cells by TANs, thereby facilitating the in vitro and in vivo progression and growth of human LSCC tumors. Importantly, this effect could be reversed by blocking G-CSF and GM-CSF stimulation of neutrophils. These findings elucidate the pivotal role of pathologically prolonged neutrophil survival in impairing CD8+ T cell immunity and suggest targeting it as a potential therapeutic strategy for tumors.

Phase I First-in-Human Dose Escalation Study of the oral Casein Kinase 1α and Cyclin Dependent Kinase 7/9 inhibitor BTX-A51 in advanced MDS and AML.

BTX-A51, a first-in-class oral small molecule inhibitor of casein kinase 1α (CK1α) and cyclin dependent kinase (CDK) 7 and 9, induces apoptosis of leukemic cells by activating p53 and inhibiting expression of Mcl1. Here, we report on the results of the phase 1 clinical trial of BTX-A51 in patients with relapsed or refractory AML and MDS. Thirty-one patients were enrolled into 8 dose-escalation cohorts at BTX-A51 doses ranging from 1mg to 42mg dosed three days/week for 21 or 28 days out a 28-day cycle. The recommended phase 2 dose was 21mg dosed three days/week for 4 weeks of a 28-day cycle. BTX-A51 increased expression of p53 and reduced expression of MCL1 and RNA polymerase II phosphorylation on pre- and post-treatment immunocytochemistry studies. Overall, 3 patients (10%) experienced complete remission with incomplete count recovery (CRi). All 3 responding patients had RUNX1 mutations and the CR/CRi rate for RUNX1 -mutated patients receiving BTX-A51 at efficacious doses (11mg or higher) was 30%. Ex-vivo studies confirmed higher efficacy of BTX-A51 on RUNX1 -mutated myeloblasts and demonstrate synergy with azacitidine and venetoclax. Although the overall efficacy was modest, this study lays the groundwork for future studies with improved patient selection and combination approaches.

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.

Enitociclib, a selective CDK9 inhibitor: in vitro and in vivo preclinical studies in multiple myeloma

Multiple myeloma (MM) is a cancer of the plasma cells that remains incurable despite advances in treatment options. In this study, a library of 216 clinically feasible small-molecule inhibitors was screened to identify agents that selectively inhibit MM cell proliferation. Enitociclib, a CDK9-specific small-molecule inhibitor, was found to be highly effective at decreasing cell viability and inducing apoptosis in four MM cell lines. Enitociclib inhibited the phosphorylation of the CTD of RNA Pol II at Ser2/Ser5 and repressed the protein expression of oncogenes c-Myc, Mcl-1, and PCNA in MM cells. Additionally, enitociclib demonstrated synergistic effects with several anti-MM agents, including bortezomib, lenalidomide, pomalidomide, and venetoclax. These results suggest that enitociclib may represent a promising therapeutic option for the treatment of MM, either as a single agent or in combination with other anti-MM agents.

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.

Mcl-1 expression is a predictive marker of response to gemcitabine plus nab-paclitaxel for metastatic pancreatic cancer

Antiapoptotic protein, including Mcl-1, expression is frequently observed in pancreatic cancer. Gemcitabine plus nabpaclitaxel (GnP) is the standard chemotherapy for metastatic pancreatic cancer (MPC); however, predictive markers for its efficacy remain unestablished. This study evaluated the association between GnP’s therapeutic effects and Mcl-1 expression in tissue samples obtained using endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) for pancreatic tumor or percutaneous ultrasound-guided biopsy for metastatic liver tumor. We retrospectively reviewed 38 patients with histologically diagnosed MPC who received GnP as the first-line chemotherapy at our institute between December 2014 and July 2018. Post-immunohistochemistry analysis for Mcl-1 expression detection, patients were divided to into two groups based on the cell proportion showing Mcl-1 immunoreactivity: positive (> 20%; 23 [60.5%] patients) and negative (≤ 20%; 15 [39.5%] patients) groups. Clinical characteristics did not differ between the two groups. The Mcl-1 positive group showed a significantly higher disease control rate (95.7% vs. 73.3%; P = 0.046), longer progressionfree survival (PFS) (7.2 months vs. 4.9 months; P = 0.018) and longer overall survival (OS) (14.9 months vs. 9.2 months; P = 0.008) than the Mcl-1 negative group. Multivariate analysis showed that Mcl-1 expression was an independent predictive marker for PFS and OS. Mcl-1 expression could be a predictive marker for favorable response to GnP.

Flow cytometric expression of Bcl-2, Mcl-1, and their ratios correlates with primary and secondary cytogenetic changes and their combinations in multiple myeloma.

Response to BH3 mimetics in multiple myeloma (MM) correlates with CCND1-rearrangement or expression of anti-apoptotic molecules, particularly Bcl-2 and Mcl-1. Our study investigates the relationship between cytogenetic abnormalities (CGAs) and intracellular Bcl-2 and Mcl-1 expression in myeloma plasma cells(MPCs) using flow cytometry (FCM). We measured median fluorescence intensity (MFI) of Bcl-2 and Mcl-1 in 163 bone marrow samples (143 MM, 20 controls) across various cell types. Both Bcl-2MFI and Mcl-1MFI were significantly higher in MPCs compared to other cells, with Bcl-2 MFI exceeding Mcl-1 MFI in MPCs. Bcl-2 expression peaked in CCND1-rearranged cases, while Mcl-1 expression was highest in cases with 1q21 gain/amplification. Notably, 65-74% of cases with other CGAs exhibited moderate to strong Bcl-2 or Mcl-1 expression, indicating potential utility of BH3 mimetics in this group, while 25% showed dim to absent expression of one or both markers, suggesting potential futility in these patients. Our study highlights FCM's potential for rapid Bcl-2 and Mcl-1 quantification, surpassing traditional methods. We propose that direct measurement of Bcl-2 and Mcl-1 expression in PCs by FCM, combined with cytogenetic characterization, could improve therapeutic decision-making regarding the use of BH3 mimetics in MM, potentially enhancing outcomes and overcoming resistance.

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.

On the mechanism of miR-29b enhancement of etoposide toxicity in vitro

MicroRNA hsa-miR-29 was connected to a number of malignancies. Its target genes are many, among them Mcl-1 that is expressed in three possible isoforms, one of which is anti-apoptotic and another one pro-apoptotic. Ratio of these two isoforms appears to affect cell response to external stimuli. We have demonstrated that miR-29b enhanced etoposide toxicity in HeLa cell line by modulating this ratio of Mcl-1 isoforms. However, it is not known whether the described miR-29 effect is common to various cancer types or even have the opposite effect. This represents a significant problem for possible future applications. In this report, we demonstrate that miR-29b affects toxicity of 60 μM etoposide in cell lines derived from selected malignancies. The mechanism, however, differs among the cell lines tested. Hep G2 cells demonstrated similar effect of miR-29b on etoposide toxicity as was described in HeLa cells, i.e. modulation of Mcl-1 expression. Target protein down-regulated by miR-29b resulting in enhanced etoposide toxicity in Caco-2 cells was, however, Bcl-2 protein. Moreover, H9c2, Hek-293 and ARPE-19 cell lines selected as a representatives of non-malignant cells, showed no effect of miR-29b on etoposide toxicity. Our data suggest that miR-29b could be a common enhancer of etoposide toxicity in malignant cells due to its modulation of Bcl family proteins.

Innovative Therapeutic Delivery of Metastasis-Associated in Colon Cancer 1-Suppressing miRNA Using High Transmembrane 4 L6 Family Member 5-Targeting Exosomes in Colorectal Cancer Mouse Models.

Elevated metastasis-associated in colon cancer 1 (MACC1) expression in colorectal cancer patients, and high transmembrane 4 L6 family member 5 (TM4SF5) protein expressed on various solid tumors' surface, are linked to aggressive cancer behavior and progression. In this study, adipose-derived stem cells (ASCs) were engineered to produce exosomes (Ex) that target the TM4SF5 protein on tumors. Moreover, MACC1-targeting microRNA was encapsulated within the Ex, resulting in TM4SF5-targeting Ex (MACC1-suppressing miRNA; miR-143). The anticancer effects of these Ex were investigated in vitro using the human colorectal cell line HCT116 and in vivo using colorectal cancer mouse xenograft models. In the in vivo assessment, administration of TM4SF5-targeting Ex[miR-143], referred to as tEx[miR-143] herein, resulted in the smallest tumor size, the lowest tumor growth rate, and the lightest excised tumors compared to other treatments (p < 0.05). It also led to the decreased expression of MACC-1 and anti-apoptotic markers MCL-1 and Bcl-xL while inducing the highest expression of pro-apoptotic markers BAX and BIM. These results were consistent with in vitro findings, where t Ex[miR-143] demonstrated the highest inhibition of HCT116 cell migration and invasion. These findings highlight the potential of tEx[miR-143] as an effective therapeutic strategy for colorectal cancer, demonstrating promising results in both targetability and anti-tumor effects in vitro and in vivo, warranting further investigation in clinical settings.

Schisandra chinensis (Turcz.) Baill neutral polysaccharides alleviate Parkinson's disease via effectively activating MCL-1 expression regulation of autophagy signaling

The purified neutral polysaccharide fraction, namely SBP-1, was isolated and characterized from Schisandra chinensis (Turcz.) Baill crude polysaccharides, which have anti-Parkinson's disease activity were investigated in vivo and in vitro. Experiments have shown that the main chain of SBP-1 was Glcp-(1→, →4)-Glcp-(1→ and →4,6)-Glcp-(1→. We also revealed the effect of SBP-1 on the PD mice model and the potential underlying molecular mechanism. The results showed that SBP-1 administration improved behavioral deficits, increased tyrosine hydroxylase-positive cells, attenuated loss of dopaminergic neurons in MPTP-exposed mice, and reduced cell death induced by MPP+. The MCL-1 was identified as the target of SBP-1 by the combination of docking-SPR-ITC, WB, and IF experiments. Subsequently, the study showed that SBP-1 could target MCL-1 to enhance autophagy with a change in the apoptotic response, which was further demonstrated by a change in LC3/P62, PI3K/AKT/mTOR, and possesses a change in the expression of BCL2/BAX/Caspase3. These results demonstrate that SBP-1 may protect neurons against MPP+ or MPTP-induced damage in vitro and in vivo through enhancing autophagy. In summary, these findings indicate that SBP-1 and S. chinensis show potential as effective candidates for further investigation in the prevention and treatment of PD or associated illnesses, specifically through autophagy apoptotic-based mechanisms.

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.

Functional overlap of inborn errors of immunity and metabolism genes defines T cell metabolic vulnerabilities.

Inborn errors of metabolism (IEMs) and immunity (IEIs) are Mendelian diseases in which complex phenotypes and patient rarity have limited clinical understanding. Whereas few genes have been annotated as contributing to both IEMs and IEIs, immunometabolic demands suggested greater functional overlap. Here, CRISPR screens tested IEM genes for immunologic roles and IEI genes for metabolic effects and found considerable previously unappreciated crossover. Analysis of IEMs showed that N-linked glycosylation and the hexosamine pathway enzyme Gfpt1 are critical for T cell expansion and function. Further, T helper (TH1) cells synthesized uridine diphosphate N-acetylglucosamine more rapidly and were more impaired by Gfpt1 deficiency than TH17 cells. Screening IEI genes found that Bcl11b promotes the CD4 T cell mitochondrial activity and Mcl1 expression necessary to prevent metabolic stress. Thus, a high degree of functional overlap exists between IEM and IEI genes, and immunometabolic mechanisms may underlie a previously underappreciated intersection of these disorders.

Integrative single-cell RNA-seq and spatial transcriptomics analyses reveal diverse apoptosis-related gene expression profiles in EGFR-mutated lung cancer

In EGFR-mutated lung cancer, the duration of response to tyrosine kinase inhibitors (TKIs) is limited by the development of acquired drug resistance. Despite the crucial role played by apoptosis-related genes in tumor cell survival, how their expression changes as resistance to EGFR-TKIs emerges remains unclear. Here, we conduct a comprehensive analysis of apoptosis-related genes, including BCL-2 and IAP family members, using single-cell RNA sequence (scRNA-seq) and spatial transcriptomics (ST). scRNA-seq of EGFR-mutated lung cancer cell lines captures changes in apoptosis-related gene expression following EGFR-TKI treatment, most notably BCL2L1 upregulation. scRNA-seq of EGFR-mutated lung cancer patient samples also reveals high BCL2L1 expression, specifically in tumor cells, while MCL1 expression is lower in tumors compared to non-tumor cells. ST analysis of specimens from transgenic mice with EGFR-driven lung cancer indicates spatial heterogeneity of tumors and corroborates scRNA-seq findings. Genetic ablation and pharmacological inhibition of BCL2L1/BCL-XL overcome or delay EGFR-TKI resistance. Overall, our findings indicate that BCL2L1/BCL-XL expression is important for tumor cell survival as EGFR-TKI resistance emerges.

Combined inhibition of KRASG12C and mTORC1 kinase is synergistic in non-small cell lung cancer

Current KRASG12C (OFF) inhibitors that target inactive GDP-bound KRASG12C cause responses in less than half of patients and these responses are not durable. A class of RASG12C (ON) inhibitors that targets active GTP-bound KRASG12C blocks ERK signaling more potently than the inactive-state inhibitors. Sensitivity to either class of agents is strongly correlated with inhibition of mTORC1 activity. We have previously shown that PI3K/mTOR and ERK-signaling pathways converge on key cellular processes and that inhibition of both pathways is required for inhibition of these processes and for significant antitumor activity. We find here that the combination of a KRASG12C inhibitor with a selective mTORC1 kinase inhibitor causes synergistic inhibition of Cyclin D1 expression and cap-dependent translation. Moreover, BIM upregulation by KRASG12C inhibition and inhibition of MCL-1 expression by the mTORC1 inhibitor are both required to induce significant cell death. In vivo, this combination causes deep, durable tumor regressions and is well tolerated. This study suggests that the ERK and PI3K/mTOR pathways each mitigate the effects of inhibition of the other and that combinatorial inhibition is a potential strategy for treating KRASG12C-dependent lung cancer.

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.

Targeted inhibition of DHODH is synergistic with BCL2 blockade in HGBCL with concurrent MYC and BCL2 rearrangement

High-grade B-cell lymphoma (HGBCL), the subtype of non-Hodgkin lymphoma, to be relapsed or refractory in patients after initial therapy or salvage chemotherapy. Dual dysregulation of MYC and BCL2 is one of the important pathogenic mechanisms. Thus, combined targeting of MYC and BCL2 appears to be a promising strategy. Dihydroorotate dehydrogenase (DHODH) is the fourth rate-limiting enzyme for the de novo biosynthesis of pyrimidine. It has been shown to be a potential therapeutic target for multiple diseases. In this study, the DHODH inhibitor brequinar exhibited growth inhibition, cell cycle blockade, and apoptosis promotion in HGBCL cell lines with MYC and BCL2 rearrangements. The combination of brequinar and BCL2 inhibitors venetoclax had a synergistic inhibitory effect on the survival of DHL cells through different pathways. Venetoclax could upregulate MCL-1 and MYC expression, which has been reported as a resistance mechanism of BCL2 inhibitors. Brequinar downregulated MCL-1 and MYC, which could potentially overcome drug resistance to venetoclax in HGBCL cells. Furthermore, brequinar could downregulate a broad range of genes, including ribosome biosynthesis genes, which might contribute to its anti-tumor effects. In vivo studies demonstrated synergetic tumor growth inhibition in xenograft models with brequinar and venetoclax combination treatment. These results provide preliminary evidence for the rational combination of DHODH and BCL2 blockade in HGBCL with abnormal MYC and BCL2.

Antitumor activity of afatinib in EGFR T790M-negative human oral cancer therapeutically targets mTOR/Mcl-1 signaling axis.

This study investigates the role and effectiveness of the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) in oral cancer, focusing on the clinical relevance of EGFR and myeloid cell leukemia-1 (Mcl-1) in head and neck cancers (HNCs). It aims to explore the molecular mechanism of afatinib, a TKI, in treating human oral cancer. We conducted an in silico analysis using databases like The Cancer Genome Atlas, Gene Expression Omnibus, and Clinical Proteomic Tumor Analysis Consortium, along with immunohistochemistry staining, to study EGFR and Mcl-1 expression in HNCs. For investigating afatinib's anticancer properties, we performed various in vitro and in vivo analyses, including trypan blue exclusion assay, Western blotting, 4'-6-diamidino-2-phenylindole staining, flow cytometry, quantitative real-time PCR, Mitochondrial membrane potential assay, overexpression vector construction, transient transfection, and a tumor xenograft model. Higher expression levels of EGFR and Mcl-1 were observed in HNC patient tissues compared to normal tissues, with their co-expression significantly linked to poor prognosis. There was a strong correlation between EGFR and Mcl-1 expressions in oral cancer patients. Afatinib treatment induced apoptosis and suppressed Mcl-1 in oral cancer cell lines without the EGFR T790M mutation. The mechanism of afatinib-induced apoptosis involved the EGFR/mTOR/Mcl-1 axis, as shown by the effects of mTOR activator MHY1485 and inhibitor rapamycin. Afatinib also increased Bim expression, mitochondrial membrane permeabilization, and cytochrome c release. It significantly lowered tumor volume without affecting body, liver, and kidney weights. Afatinib, targeting the EGFR/mTOR/Mcl-1 axis, shows promise as a therapeutic strategy for oral cancer, especially in patients with high EGFR and Mcl-1 expressions.

In silico design of novel multitarget small molecule inhibitors to treat PTEN-mutant endometrial carcinoma.

e15119 Background: Previously we described triple CDK4/6-CDK9 inhibitor, LCI133 for the treatment of endometrial carcinoma (EC). We used in silico modeling to optimize LCI133’s physicochemical properties to increase PI3K p110α potency/ADME to better target epithelial-derived cancers, resulting in nanomolar potent LCI139. We characterized LCI139’s in vitro potency, target specificity, activity in in vitro EC models, safety in normal cell lines. Methods: LCI139 + analogs were designed using computational chemistry based on X-ray crystal structures of each target protein + validated chemistry using nuclear magnetic resonance + high-resolution mass spectrometry. Specific target inhibition was assessed by cell-free kinase hotspot/lipid kinase assay + selectivity profile. LCI139 (0.001-10μM) was screened against EC cell lines AN3CA, RL95-2, Ishikawa, KLE, HEC-1-A and -B for 48 hours; IC50 values determined by CellTiter-Glo Assay. EC cells were treated with LCI139 or single agent control for 24 hours + stained with Annexin V/7-AAD to assess apoptosis. Representative PTEN wildtype (HEC-1A) + mutant (AN3CA) EC cell lines were chosen for mechanistic studies + treated 6 to 24 hours with multitarget or single agent inhibitor. Cells were stained with Vybrant DyeCycle Violet to determine cell cycle arrest by cytometer. Cellular lysates were prepared for Western blot (WB). Results: LCI139’s has a molecular weight of 502.57Da + is a highly potent PI3Kα-CDK4/6-CDK9 inhibitor by cell-free kinase assays: 0.070µM (PI3Kα), 0.461µM (PI3Kγ), 0.214µM (PI3Kδ), 0.0015µM (CDK4), 0.0036µM (CDK6), 0.000039µM (CDK9). KINOMEscan revealed a favorable S(35) selectivity score of 0.221 screened against 403 non-mutant kinases. PTEN mutant AN3CA (IC50 216.0nM), RL95-2 (IC50 22.8nM), Ishikawa (IC50 2.5nM) had nanomolar LCI139 IC50 values. PTEN-wild type cell lines were insensitive up to 10μM; confirmed by dose-dependent apoptosis + colony-forming assays. WB revealed LCI139 treatment ablated MCL-1 expression, decreased phosphorylation of AKT, Rb, Rpb1 (Ser2), induced significant amounts of cPARP in PTEN mutant AN3CA. In PTEN-wildtype HEC1A WB, dose-dependent expression of pAKT in PTEN siRNA was noted. LCI139 generated heterogenous cell line specific effects on cell cycle progression + arrest. LCI139 displayed favorable toxicity against normal human kidney cells (HEK-293) compared to single agent controls. Conclusions: Triple PI3K-CDK4/6-CDK9 inhibitor, LCI139, is nanomolar potent against PTEN mutant EC cell lines + displays decreased toxicity compared with CDK9 inhibitor reference compounds, AZD4573 + flavopiridol. Our results merit mechanistic dissection of PTEN interaction with PI3K + CDK9 signaling pathways, cell cycle machinery, CDK9-mediated transcriptional control. These data support extensive in vivo potency, toxicity, PK/PD studies to assess LCI139’s clinical utility in treating EC.