Anti-apoptotic protein BCL-XL as a therapeutic vulnerability in gastric cancer

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. Coimmunoprecipitation(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.

Myeloid peroxisome proliferator-activated receptor α deficiency accelerates liver regeneration via IL-6/STAT3 pathway after 2/3 partial hepatectomy in mice

Background:Liver regeneration is a fundamental process for sustained body homeostasis and liver function recovery after injury.Emerging evidence demonstrates that myeloid cells play a critical role in liver regeneration by secreting cytokines and growth factors.Peroxisome proliferator-activated receptorα(PPARα),the target of clinical lipid-lowering fibrate drugs,regulates cell metabolism,proliferation,and survival.However,the role of myeloid PPARαin partial hepatectomy(PHx)-induced liver regeneration remains unknown.Methods:Myeloid-specific PPARa-deficient(Ppara^(Mye−/−))mice and the littermate controls(Ppara^(fl/fl))were subjected to sham or 2/3 PHx to induce liver regeneration.Hepatocyte proliferation and mitosis were assessed by immunohistochemical(IHC)staining for 5-bromo-2'-deoxyuridine(BrdU)and Ki67 as well as hematoxylin and eosin(H&E)staining.Macrophage and neutrophil infiltration into livers were reflected by IHC staining for galectin-3 and myeloperoxidase(MPO)as well as flow cytometry analysis.Macrophage migration ability was evaluated by transwell assay.The mRNA levels for cell cycle or inflammation-related genes were measured by quantitative real-time RT-PCR(qPCR).The protein levels of cell proliferation related protein and phosphorylated signal transducer and activator of transcription 3(STAT3)were detected by Western blotting.Results:Ppara^(Mye−/−)mice showed enhanced hepatocyte proliferation and mitosis at 32 h after PHx compared with Ppara^(fl/fl)mice,which was consistent with increased proliferating cell nuclear antigen(Pcna)mRNA and cyclinD1(CYCD1)protein levels in Ppara^(Mye−/−)mice at 32 h after PHx,indicating an accelerated liver regeneration in Ppara^(Mye−/−)mice.IHC staining showed that macrophages and neutrophils were increased in Ppara^(Mye−/−)liver at 32 h after PHx.Livers of Ppara^(Mye−/−)mice also showed an enhanced infiltration of M1 macrophages at 32 h after PHx.In vitro,Ppara-deficient bone marrow-derived macrophages(BMDMs)exhibited markedly enhanced migratory capacity and upregulated M1 genes Il6 and Tnfa but downregulated M2 gene Arg1 expressions.Furthermore,the phosphorylation of STAT3,a key transcript factor mediating IL6-promoted hepatocyte survival and proliferation,was reinforced in the liver of Ppara^(Mye−/−)mice after PHx.Conclusions:This study provides evidence that myeloid PPARαdeficiency accelerates PHx-induced liver regeneration via macrophage polarization and consequent IL-6/STAT3 activation,thus providing a potential target for manipulating liver regeneration.