Development of a BCL-xL and BCL-2 dual degrader with improved anti-leukemic activity.
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ABSTRACT:
SUBMITTER: Lv D
PROVIDER: S-EPMC8617031 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Overexpression of BCL-xL and BCL-2 play key roles in tumorigenesis and cancer drug resistance. Advances in PROTAC technology facilitated recent development of the first BCL-xL/BCL-2 dual degrader, 753b, a VHL-based degrader with improved potency and reduced toxicity compared to previous small molecule inhibitors. Here, we determined crystal structures of VHL/753b/BCL-xL and VHL/753b/ BCL-2 ternary complexes. The two ternary complexes exhibit markedly different architectures that are accompanied by distinct networks of interactions at the VHL/PZ753b-linker/target interfaces. The importance of these interfacial contacts was validated via functional analysis and informed subsequent rational and structure-guided design focused on the 753b linker and BCL-2/BCL-xL warhead. This resulted in the design of a novel degrader, WH244, with enhanced potency to degrade BCL-xL/BCL-2 in cells. Using biophysical assays followed by in cell activities, we were able to explain the enhanced target degradation of BCL-2/BCL-xL in cells. Most PROTACs are empirically designed and lack structural studies, making it challenging to understand their modes of action and specificity. Our work presents a streamlined approach that combines rational design and structure-based insights backed with cell-based studies to develop effective PROTAC-based cancer therapeutics.
2024-03-12 | PXD049976 | Pride
Project description:Bcl-xL suppresses mitochondria-mediated apoptosis and is frequently overexpressed in cancer to promote cancer cell survival. Bcl-xL also promotes metastasis. However, it is unclear whether this metastatic function is dependent on its anti-apoptotic activity in the mitochondria. Here we demonstrate that Bcl-xL promotes metastasis independent of its anti-apoptotic activity. We show that apoptosis-defective Bcl-xL mutants and an engineered Bcl-xL targeted to the nucleus promote epithelial-mesenchymal transition, migration, invasion and stemness in pancreatic neuroendocrine tumour (panNET) and breast cancer cell lines. However, Bcl-xL proteins targeted to the mitochondria or outside of the nucleus do not have these functions. We confirm our findings in spontaneous and xenograft mouse models. Furthermore, Bcl-xL exerts metastatic function through epigenetic modification of the TGFβ promoter to increase TGFβ signalling. Consistent with these findings, we detect nuclear Bcl-xL in human metastatic panNETs. Taken together, the metastatic function of Bcl-xL is independent of its anti-apoptotic activity and its residence in the mitochondria.
| S-EPMC4735924 | biostudies-literature
Project description:Overexpression of BCL-xL and BCL-2 play key roles in tumorigenesis and cancer drug resistance. Advances in PROTAC technology facilitated recent development of the first BCL-xL/BCL-2 dual degrader, 753b, a VHL-based degrader with improved potency and reduced toxicity compared to previous small molecule inhibitors. Here, we determined crystal structures of VHL/753b/BCL-xL and VHL/753b/ BCL-2 ternary complexes. The two ternary complexes exhibit markedly different architectures that are accompanied by distinct networks of interactions at the VHL/PZ753b-linker/target interfaces. The importance of these interfacial contacts was validated via functional analysis and informed subsequent rational and structure-guided design focused on the 753b linker and BCL-2/BCL-xL warhead. This resulted in the design of a novel degrader, WH244, with enhanced potency to degrade BCL-xL/BCL-2 in cells. Using biophysical assays followed by in cell activities, we were able to explain the enhanced target degradation of BCL-2/BCL-xL in cells. Most PROTACs are empirically designed and lack structural studies, making it challenging to understand their modes of action and specificity. Our work presents a streamlined approach that combines rational design and structure-based insights backed with cell-based studies to develop effective PROTAC-based cancer therapeutics.
2024-02-21 | MSV000094144 | MassIVE
Project description:Abstract OBJECTIVE Repurposing represents a promising approach to safely accelerate the clinical application of therapeutics with anti-cancer activity. In this study, we examined whether inhibition of the anti-apoptotic Bcl-2 family proteins Bcl-2 and Bcl-xL enhances the biological effects of the repurposed CUSP9 regimen in an in vitro setting of glioblastoma. METHODS We applied MTT assays to assess cellular proliferation. Annexin V/PI and TMRE staining were used to examine apoptosis. Western blotting, RT-PCR and specific knockdown experiments using siRNA were employed to examine molecular mechanisms of action. RESULTS Bcl-2/Bcl-xL inhibition by the BH3 mimetic ABT263, yielded synergistic anti-proliferative effects across a wide panel of established and primary cultured glioblastoma cells when combined with CUSP9 which had been reduced to only one tenth of its original concentration (CUSP9 1/10). The combination treatment also led to enhanced apoptosis with loss of mitochondrial membrane potential and activation of caspases. On the molecular level, CUSP9 1/10 counteracted ABT263-mediated upregulation of Mcl-1. Silencing of Mcl-1 enhanced ABT263-mediated apoptosis, indicating that Mcl-1 is crucial for the induction of cell death conveyed by the combination treatment. Levels of Mcl-1 mRNA were not decreased following combination therapy, and co-treatment with cycloheximide showed reduced protein stability, pointing towards a post-translational mechanism of action. CONCLUSION These data suggest that Bcl-2/Bcl-xL inhibition enhances the susceptibility of glioblastoma cells towards CUSP9, allowing dramatic dose reduction and potentially decreased toxicity when applied clinically. A clinical trial involving the original CUSP doses (CUSP9v3) is currently ongoing in our institution (NCT02770378). The Bcl-2/Bcl-xL inhibitor ABT263 is in clinical trials and might represent a valuable adjunct to the original CUSP.
| S-EPMC6217449 | biostudies-literature
Project description:Glioblastoma (GBM) is one of the cancers with the worst prognosis, despite huge efforts to understand its unusual heterogeneity and aggressiveness. This is mainly due to glioblastoma stem cells (GSCs), which are also responsible for the frequent tumor recurrence following surgery, chemotherapy or radiotherapy. In this study, we investigate the expression pattern of the anti-apoptotic BCL-xL protein in several GBM cell lines and the role it might play in GSC-enriched tumorspheres. We report that several GBM cell lines have an increased BCL-xL expression in tumorspheres compared to differentiated cells. Moreover, by artificially modulating BCL-xL expression, we unravel a correlation between BCL-xL and tumorsphere size. In addition, BCL-xL upregulation appears to sensitize GBM tumorspheres to newly developed BH3 mimetics, opening promising therapeutic perspectives for treating GBM patients.
| S-EPMC7599739 | biostudies-literature
Project description:XPO1 has recently emerged as a viable treatment target for solid malignancies, including glioblastoma (GBM), the most common primary malignant brain tumor in adults. However, given that tumors become commonly resistant to single treatments, the identification of combination therapies is critical. Therefore, we tested the hypothesis that inhibition of anti-apoptotic Bcl-2 family members and XPO1 are synthetically lethal. To this purpose, two clinically validated drug compounds, the BH3-mimetic, ABT263, and the XPO1 inhibitor, Selinexor, were used in preclinical GBM model systems. Our results show that inhibition of XPO1 reduces cellular viability in glioblastoma cell cultures. Moreover, addition of ABT263 significantly enhances the efficacy of XPO1 inhibition on the reduction of cellular viability, which occurs in a synergistic manner. While selinexor inhibits the proliferation of glioblastoma cells, the combination treatment of ABT263 and selinexor results in substantial induction of cell death, which is accompanied by activation of effector- initiator caspases and cleavage of PARP. Mechanistically we find that XPO1 inhibition results in down-regulation of anti-apoptotic Mcl-1 and attenuates ABT263 driven Mcl-1 up-regulation. Consistently, siRNA mediated silencing of Mcl-1 sensitizes for ABT263 mediated cell death and partially for the combination treatment. By using a human patient-derived xenograft model of glioblastoma in mice, we demonstrate that the combination treatment of ABT263 and Selinexor reduces tumor growth significantly more than each compound alone. Collectively, these results suggest that inhibition of XPO1 and Bcl-2/Bcl-xL might be a potential strategy for the treatment of malignant glial tumors.
| S-EPMC6194027 | biostudies-literature
Project description:Background and purposeDrug repurposing represents a promising approach to safely accelerate the clinical application of therapeutics with anti-cancer activity. In this study, we examined whether inhibition of the anti-apoptotic Bcl-2 family proteins Bcl-2 and Bcl-xL enhances the biological effects of the repurposed CUSP9 regimen in an in vitro setting of glioblastoma.Experimental approachWe applied 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assays to assess cellular proliferation. Annexin V/propidium iodide and tetramethylrhodamine, ethyl ester staining were used to examine apoptosis. Western blotting, RT-PCR, and specific knockdown experiments using siRNA were employed to examine molecular mechanisms of action.Key resultsBcl-2/Bcl-xL inhibition exerted synergistic anti-proliferative effects across established, primary cultured, and stem-like glioblastoma cells when combined with CUSP9 which had been reduced to only one tenth of its proposed original concentration (CUSP9-LD). The combination treatment also led to enhanced apoptosis with loss of mitochondrial membrane potential and activation of caspases. On the molecular level, CUSP9-LD counteracted ABT263-mediated up-regulation of Mcl-1. Silencing of Mcl-1 enhanced ABT263-mediated apoptosis which indicates that down-regulation of Mcl-1 is crucial for the induction of cell death by the combination treatment.Conclusion and implicationsThese data suggest that Bcl-2/Bcl-xL inhibition enhances the susceptibility of glioblastoma cells towards CUSP9, allowing dramatic dose reduction and potentially decreased toxicity when applied clinically. A clinical trial involving the original CUSP doses (CUSP9v3) is currently ongoing in our institution (NCT02770378). The Bcl-2/Bcl-xL inhibitor ABT263 is in clinical trials and might represent a valuable adjunct to the original CUSP.
| S-EPMC6715605 | biostudies-literature
Project description:PURPOSE:Uveal melanoma (UM) is associated with a high risk of metastases and lack of efficient therapies. Reduced capacity for apoptosis induction by chemotherapies is one obstacle to efficient treatments. Human UM is characterized by high expression of the anti-apoptotic protein Bcl-2. Consequently, regulators of apoptosis such as Bcl-2 family inhibitors may constitute an attractive approach to UM therapeutics. In this aim, we have investigated the efficacy of the Bcl-2/Bcl-XL inhibitor S44563 on 4 UM Patient-Derived Xenografts (PDXs) and derived-cell lines. EXPERIMENTAL DESIGN:Four well characterized UM PDXs were used for in vivo experiments. S44563 was administered alone or combined with fotemustine either concomitantly or after the alkylating agent. Bcl-2, Bcl-XL, and Mcl-1 expressions after S44563 administration were evaluated by immunohistochemistry (IHC). RESULTS:S44563 administered alone by at 50 and 100 mg/kg i.p. induced a significant tumour growth inhibition in only one xenograft model with a clear dose effect. However, when S44563 was concomitantly administered with fotemustine, we observed a synergistic activity in 3 out of the 4 tested models. In addition, S44563 administered after fotemustine induced a tumour growth delay in 2 out of 3 tested xenografts. Finally, IHC analyses showed that Bcl-2, Bcl-XL, and Mcl-1 expression were not modified after S44563 administration. CONCLUSION:The novel anti-apoptotic experimental compound S44563, despite a relative low efficacy when administered alone, increased the efficacy of fotemustine in either concomitant or sequential combinations or indeed subsequent to fotemustine. These data support further exploration of potential therapeutic effect of Bcl-2/Bcl-xl inhibition in human UM.
| S-EPMC3890263 | biostudies-literature
Project description:Bcl-xL is an oncogene of which the survival functions are finely tuned by post-translational modifications (PTM). Within the Bcl-2 family of proteins, Bcl-xL shows unique eligibility to deamidation, a time-related spontaneous reaction. Deamidation is still a largely overlooked PTM due to a lack of easy techniques to monitor Asn?Asp/IsoAsp conversions or Glu?Gln conversions. Being able to detect PTMs is essential to achieve a comprehensive description of all the regulatory mechanisms and functions a protein can carry out. Here, we report a gel composition improving the electrophoretic separation of deamidated forms of Bcl-xL generated either by mutagenesis or by alkaline treatment. Importantly, this new gel formulation proved efficient to provide the long-sought evidence that even doubly-deamidated Bcl-xL remains eligible for regulation by phosphorylation.
| S-EPMC6888115 | biostudies-literature
Project description:Cyclin-dependent kinase 2 (CDK2) is a potential therapeutic target for the treatment of cancer. Development of CDK2 inhibitors has been extremely challenging as its ATP-binding site shares high similarity with CDK1, a related kinase whose inhibition causes toxic effects. Here, we report the development of TMX-2172, a heterobifunctional CDK2 degrader with degradation selectivity for CDK2 and CDK5 over not only CDK1, but transcriptional CDKs (CDK7 and CDK9) and cell cycle CDKs (CDK4 and CDK6) as well. In addition, we demonstrate that antiproliferative activity in ovarian cancer cells (OVCAR8) depends on CDK2 degradation and correlates with high expression of cyclin E1 (CCNE1), which functions as a regulatory subunit of CDK2. Collectively, our work provides evidence that TMX-2172 represents a lead for further development and that CDK2 degradation is a potentially valuable therapeutic strategy in ovarian and other cancers that overexpress CCNE1.
| S-EPMC7703486 | biostudies-literature