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Background New therapeutic targets are needed to improve the outcomes for gastric cancer (GC) patients with advanced disease. Evasion of programmed cell death (apoptosis) is a hallmark of cancer cells and direct induction of apoptosis by targeting the pro‐survival BCL2 family proteins represents a promising therapeutic strategy for cancer treatment. Therefore, understanding the molecular mechanisms underpinning cancer cell survival could provide a molecular basis for potential therapeutic interventions. Method Here we explored the role of BCL2L1 and the encoded anti‐apoptotic BCL‐XL in GC. Using Droplet Digital PCR (ddPCR) technology to investigate the DNA amplification of BCL2L1 in GC samples and GC cell lines, the sensitivity of GC cell lines to selective BCL‐XL inhibitors A1155463 and A1331852, pan‐inhibitor ABT‐263, and VHL‐based PROTAC‐BCL‐XL was analyzed using (CellTiter‐Glo) CTG assay in vitro. Western Blot (WB) was used to detect the protein expression of BCL2 family members in GC cell lines and the manner in which PROTAC‐BCL‐XL kills GC cells. Co‐immunoprecipitation (Co‐IP) was used to investigate the mechanism of A1331852 and ABT‐263 kills GC cell lines. DDPCR, WB, and real‐time PCR (RTPCR) were used to investigate the correlation between DNA, RNA, protein levels, and drug activity. Results The functional assay showed that a subset of GC cell lines relies on BCL‐XL for survival. In gastric cancer cell lines, BCL‐XL inhibitors A1155463 and A1331852 are more sensitive than the pan BCL2 family inhibitor ABT‐263, indicating that ABT‐263 is not an optimal inhibitor of BCL‐XL. VHL‐based PROTAC‐BCL‐XL DT2216 appears to be active in GC cells. DT2216 induces apoptosis of gastric cancer cells in a time‐ and dose‐dependent manner through the proteasome pathway. Statistical analysis showed that the BCL‐XL protein level predicts the response of GC cells to BCL‐XL targeting therapy and BCL2L1 gene CNVs do not reliably predict BCL‐XL expression. Conclusion We identified BCL‐XL as a promising therapeutic target in a subset of GC cases with high levels of BCL‐XL protein expression. Functionally, we demonstrated that both selective BCL‐XL inhibitors and VHL‐based PROTAC BCL‐XL can potently kill GC cells that are reliant on BCL‐XL for survival. However, we found that BCL2L1 copy number variations (CNVs) cannot reliably predict BCL‐XL expression, but the BCL‐XL protein level serves as a useful biomarker for predicting the sensitivity of GC cells to BCL‐XL‐targeting compounds. Taken together, our study pinpointed BCL‐XL as potential druggable target for specific subsets of GC. We demonstrated that both selective BCL‐XL inhibitors and VHL‐based PROTAC BCL‐XL can potently kill GC cells that reliant on BCL‐XL for survival. We also confirmed that the effect caused by DT2216 or selective BCL‐XL inhibitors is predominantly BAX/BAK dependent.

Purpose: Doxorubicin is administrated as a single agent in first-line therapy of breast cancer to induce apoptosis in tumor cells. Bax, Bcl-xL, Caspase-8 and 9 proteins are involved in induction of apoptosis. The present study describes Bax, Bcl-xL gene expression and Caspase-8 and 9 protein levels in MCF-7 cells incubated with doxorubicin at different doses an incubation times. Methods: The cytotoxic effects of doxorubicin were studied using MTT assay. MCF-7 cells were treated with three concentrations of doxorubicin (0.1, 0.5, 1 μM) and incubated for 24, 48 and 72 hours then expression levels of Bax and Bcl-xL genes were elucidated by Real-time RT-PCR technique and protein levels of caspase-8 and caspase-9 proteins were measured using ELISA method. Morphological modifications of the cells were also monitored via light microscopic images. Results: Doxorubicin decreased the anti-apoptotic Bcl-xL and increased pro-apoptotic Bax mRNA levels. Doxorubicin induced a significant increase in Bax /Bcl-xL ratio in all doses and incubation times (p<0.05). Highest (more than 10 fold) increase in Bax /Bcl-xL ratio was revealed after 48 h incubation of the cells with in all doses of doxorubicin. Doxorubicin also increased caspase-9 level in a time and dose-dependent manner, while caspase-8 level didn't follow time and dose dependency pattern. Conclusion: Our results confirm that doxorubicin induces mitochondrial-dependent apoptosis by down-regulation of Bcl-xL and up- regulation of Bax and caspase-9 expressions.

Myeloproliferative neoplasms are divided into essential thrombocythemia (ET), polycythemia vera (PV) and primary myelofibrosis (PMF). Although ruxolitinib was proven to be effective in reducing symptoms, patients rarely achieve complete molecular remission. Therefore, it is relevant to identify new therapeutic targets to improve the clinical outcome of patients. Bcl‐xL protein, the long isoform encoded by alternative splicing of the Bcl‐x gene, acts as an anti‐apoptotic regulator. Our study investigated the role of Bcl‐xL as a marker of severity of MPN and the possibility to target Bcl‐xL in patients. 129 MPN patients and 21 healthy patients were enrolled in the study. We analysed Bcl‐xL expression in leucocytes and in enriched CD34+ and CD235a+ cells. Furthermore, ABT‐737, a Bcl‐xL inhibitor, was tested in HEL cells and in leucocytes from MPN patients. Bcl‐xL was found progressively over‐expressed in cells from ET, PV and PMF patients, independently by JAK2 mutational status. Moreover, our data indicated that the combination of ABT‐737 and ruxolitinib resulted in a significantly higher apoptotic rate than the individual drug. Our study suggests that Bcl‐xL plays an important role in MPN independently from JAK2 V617F mutation. Furthermore, data demonstrate that targeting simultaneously JAK2 and Bcl‐xL might represent an interesting new approach.

Restoring apoptosis in malignant cells represents a central goal of anticancer therapy. Tumour cells often escape cell death by overexpressing anti‐apoptotic members of the BCL‐2 protein family, particularly BCL‐2, BCL‐xL, and MCL1. These proteins inhibit the intrinsic mitochondrial apoptotic pathway through intricate interactions with pro‐apoptotic partners and direct modulation of the mitochondrial outer membrane. Their pivotal role in cell survival has established them as attractive therapeutic targets. Over the past two decades, significant efforts have been devoted to developing selective small‐molecule inhibitors capable of neutralising these proteins and reactivating apoptosis. A first milestone was the discovery of ABT‐263 (navitoclax), a dual BCL‐2/BCL‐xL inhibitor. Building on this achievement, the development of venetoclax, a highly selective BCL‐2 inhibitor, marked a major breakthrough, demonstrating potent pro‐apoptotic activity and clinical efficacy in several leukaemia subtypes. Despite these advances, the design of inhibitors of BCL‐2 family members remains challenging, largely due to the structural characteristics of the BH3‐binding groove, which is both shallow and hydrophobic, complicating the identification of molecules with optimal binding affinity and selectivity. PROTACs targeting BCL‐xL may represent a promising future strategy, potentially overcoming the intrinsic limitations of small molecule inhibitors.

Senescent tumor cells can be selectively eliminated by the BH3 mimetic, ABT‐263 (navitoclax), via senolysis. We show that ABT‐263 inhibits the interaction between anti‐apoptotic BCL‐2 family member, BCL‐XL, and pro‐apoptotic effector, BAX. This results in senescent tumor cell death in vitro and reduced tumor volume in vivo. This work highlights the utilization of senolytic agents to enhance efficacy of anticancer therapy. Tumor cells undergo senescence in response to both conventional and targeted cancer therapies. The induction of senescence in response to cancer therapy can contribute to unfavorable patient outcomes, potentially including disease relapse. This possibiliy is supported by our findings that tumor cells induced into senescence by doxorubicin or etoposide can give rise to viable tumors in vivo. We further demonstrate sensitivity of these senescent tumor cells to the senolytic ABT‐263 (navitoclax), therefore providing a “two‐hit” approach to eliminate senescent tumor cells that persist after exposure to chemotherapy or radiation. The sequential combination of therapy‐induced senescence and ABT‐263 could shift the response to therapy toward apoptosis by interfering with the interaction between BCL‐XL and BAX. The administration of ABT‐263 after either etoposide or doxorubicin also resulted in marked, prolonged tumor suppression in tumor‐bearing animals. These findings support the premise that senolytic therapy following conventional cancer therapy may improve therapeutic outcomes and delay disease recurrence.

Deregulated cellular apoptosis is a hallmark of cancer and chemotherapy resistance. The B-cell lymphoma 2 (BCL-2) protein family members are sentinel molecules that regulate the mitochondrial apoptosis machinery and arbitrate cell fate through a delicate balance between pro- and anti-apoptotic factors. The recognition of the anti-apoptotic BCL2 gene as an oncogenic driver in hematological malignancies has directed attention toward unraveling the biological significance of each of the BCL-2 superfamily members in cancer progression and garnered interest in the targeting of apoptosis in cancer therapy. Accordingly, the approval of venetoclax (ABT-199), a small molecule BCL-2 inhibitor, in patients with chronic lymphocytic leukemia and acute myeloid leukemia has become the proverbial torchbearer for novel candidate drug approaches selectively targeting the BCL-2 superfamily. Despite the inspiring advances in this field, much remains to be learned regarding the optimal therapeutic context for BCL-2 targeting. Functional assays, such as through BH3 profiling, may facilitate prediction of treatment response, development of drug resistance and shed light on rational combinations of BCL-2 inhibitors with other branches of cancer therapy. This review summarizes the pathological roles of the BCL-2 family members in cancer, discusses the current landscape of their targeting in clinical practice, and highlights the potential for future therapeutic inroads in this important area.

The Aurora‐A kinase inhibitor MLN8237 has shown efficacy in clinical trials for advanced breast cancer; however, its use as a monotherapy is limited by significant side effects and modest efficacy. Therefore, combining MLN8237 with other agents at lower doses may provide a viable alternative. In this study, we evaluated the combination of MLN8237 with the BH3 mimetic ABT263 for the treatment of triple‐negative breast cancer (TNBC). We found that this combination significantly suppressed tumor growth and metastasis in immunocompetent syngeneic mouse models, whereas its efficacy was attenuated in immunodeficient xenograft models. Mechanistic studies revealed that the combination enhanced anti‐tumor immunity by increasing the presence of CD8+ T cells and NK cells, while reducing the number of immunosuppressive cells in the tumor microenvironment. This shift resulted in elevated levels of IFN‐γ and granzyme B, which activated the extrinsic apoptotic pathways in cancer cells. Notably, the combination treatment did not affect tumor cell proliferation but promoted apoptosis with minimal toxicity. Furthermore, the synergistic effect of MLN8237 and ABT263 in inducing intrinsic apoptosis was primarily driven by the inhibition of the AKT‐Mcl‐1 and Bcl‐xL survival pathways in cultured tumor cells. Together, these findings support the MLN8237‐ABT263 combination as an effective treatment strategy for TNBC, promoting both immune‐mediated extrinsic apoptosis and inactivation of Bcl‐xL/Mcl‐1‐dependent intrinsic anti‐apoptotic pathways. The combination of the Aurora‐A kinase inhibitor MLN8237 and the BH3 mimetic ABT263 enhanced anti‐tumor immunity by boosting CD8+ T cells and natural killer cells, while reducing immunosuppressive cells in the tumor microenvironment. It induced apoptosis via extrinsic and intrinsic pathways with minimal toxicity, offering a promising therapeutic strategy for TNBC.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with poor survival and lacks effective treatments. MRTX1133, a new investigational drug targeting the KRAS G12D mutation, which is common in PDAC, shows promise in preclinical studies but is unlikely to be effective as a single agent. In this study, we combined MRTX1133 with a BCL-xL-targeting degrader (DT2216) and the mTOR inhibitor everolimus for enhanced therapeutic efficacy. The triple combination more effectively killed KRAS G12D-mutant PDAC cells in culture and slowed tumor growth in mice than MRTX1133 alone, which was associated with simultaneously strengthening pro-death signals and blocking survival mechanisms in cancer cells. Notably, the triple combination also showed efficacy against mouse tumors that developed resistance to MRTX1133. These findings suggest that combining MRTX1133 with DT2216 and everolimus could be a more effective treatment strategy for patients with KRAS G12D-mutant PDAC.

Cancer stem cells (CSCs) drive tumor initiation, metastasis, and therapy resistance. The role of cytoplasmic cyclin‐dependent kinase inhibitor 1A (CDKN1A, p21) in CSC biology remains unclear. Since cytoplasmic p21 correlated with advanced stage and metastasis in colorectal cancer (CRC) patients, we investigated its causal role in CSC features in vitro and in vivo. Cytoplasmic p21 increased spheroid formation and CD133 expression in a mechanism partly dependent on AKT activation. Phosphomimetic p21 (p21T145D) enhanced spheroid growth, CD133, and stemness factors (Oct3/4, Nanog, Sox2), whereas nuclear p21 (p21T145A) reduced them. Immunoprecipitation, proximity ligation assays, and in silico modeling demonstrated that cytoplasmic p21 interacts with the NFκB–IκB complex, promoting NFκB release and activation. Consequently, NFκB targets BCL‐xL and COX2 were upregulated in p21T145D‐ and AKTT308D,S473D CRC cells in vitro and in a chorioallantoic membrane (CAM) model, supporting their role as downstream effectors of cytoplasmic p21. Our findings uncover a new function of cytoplasmic p21 in regulating CSC properties through NFκB modulation. Screening p21 subcellular localization may stratify CRC patients with high metastatic risk providing a basis for CSC‐targeted therapeutic strategies. Cytoplasmic p21 promotes colorectal cancer stem cell (CSC) features by destabilizing the NFκB–IκB complex, activating NFκB signaling, and upregulating BCL‐xL and COX2. In contrast to nuclear p21, cytoplasmic p21 enhances spheroid formation and stemness transcription factor CD133. Our findings identify cytoplasmic p21 as a driver of CSC traits and a potential predictor of metastasis risk.

BH3 mimetics are small-molecule inhibitors of the antiapoptotic Bcl-2 family and have therapeutic efficacy against hematological malignancies. BH3 mimetic A-1331852 suppresses colorectal cancer cell proliferation. Progressive resistance to the widely used anticancer agent fluorouracil (5-FU) is a key reason for colorectal cancer recurrence; therefore, the present study tested if A-1331852 can suppress the proliferation of 5-FU-resistant colorectal cancer cells. A 5-FU-resistant colorectal cancer cell line was derived from HCT116 cells and compared with the parental line. Expression levels of the antiapoptotic Bcl-2 proteins Bcl-xL and myeloid cell leukemia 1 (Mcl-1) were determined via western blotting, proliferation in the presence of 5-FU and following small interfering (si)RNA-mediated Bcl-xL or Mcl-1 knockdown was assessed by WST-1 assay and sensitivity to A-1331852-induced apoptosis was assessed via western blotting and DNA fragmentation assay. In addition, a xenograft mouse model of 5-FU-resistant colorectal cancer was established via subcutaneous inoculation of 5-FU-resistant HCT116 cells to examine the in vivo antitumor efficacy of A-1331852. Compared with the parental line, 5-FU-resistant cells overexpressed Bcl-xL. Knockdown of Bcl-xL by siRNA and treatment with A-1331852 suppressed proliferation and induced the apoptosis of both 5-FU-resistant and parental HCT116 cells, but the potency of both effects was stronger in 5-FU-resistant than parental HCT116 cells. Furthermore, A-1331852 suppressed the growth of xenograft tumors derived from 5-FU-resistant cells by inducing apoptosis. Overall, the present findings suggested that Bcl-xL upregulation contributes to 5-FU resistance of colorectal cancer and targeted inhibition by A-1331852 may be an effective treatment strategy.