Showing papers by "Emad S. Alnemri published in 2022"
TL;DR: It is shown that the caspase-3 activator, raptinal, induces pyroptosis in both human and mouse melanoma cell line models and delays tumor growth in vivo.
Abstract: Lack of response and acquired resistance continue to be limitations of targeted and immune based therapies. Pyroptosis is an inflammatory form of cell death characterized by the release of inflammatory damage associated molecular patterns (DAMPs) and cytokines via gasdermin (GSDM) protein pores in the plasma membrane. Induction of pyroptosis has implications for treatment strategies in both therapy-responsive, as well as resistance forms of melanoma. We show that the caspase-3 activator, raptinal, induces pyroptosis in both human and mouse melanoma cell line models and delays tumor growth in vivo. Release of DAMPs and inflammatory cytokines was dependent on caspase activity and GSDME expression. Furthermore, raptinal stimulated pyroptosis in melanoma models that have acquired resistance to BRAF and MEK inhibitor therapy. These findings add support to efforts to induce pyroptosis in both the treatment naive and resistant settings. Implications: Raptinal can rapidly induce pyroptosis in naive and BRAFi plus MEKi resistant melanoma, which may be beneficial for patients who have developed acquired resistance to targeted therapies.
6 citations
TL;DR: In this paper , the role of GSDME-associated cell death in human papillomavirus-positive (HPV+) head and neck squamous cell carcinoma (HNSCC) mouse cell lines was evaluated.
Abstract: Purpose/Objective(s) Gasdermin-E (GSDME) is known to potentiate immunogenic cell death in many tumor types after exposure to anti-cancer agents in a caspase-3 dependent manner. This GSDME-mediated release of immunostimulatory proteins, such as HMGB1, has been shown to increase tumor immunogenicity in mouse model systems of melanoma and colon cancer, resulting in reduced tumor load and lower distal metastasis. We sought to evaluate the role of GSDME-mediated cell death in human papillomavirus-positive (HPV+) head and neck squamous cell carcinoma (HNSCC) mouse cell lines. Materials/Methods To test whether HNSCC exhibits a GSDME-associated anti-tumor response, two HPV+ HNSCC mouse cell lines, MTEC and TC-1, were screened for GSDME expression by Western blot and then treated with apoptotic agents (raptinal, cisplatin, etoposide, and UV). Primary outcome measures were HMGB1 release (a potent immunostimulatory factor), percent propidium iodide (%PI) uptake (an indicator of plasma membrane damage and pyroptosis) and cell morphology. Using retroviral transduction, MTEC cells were reconstituted with GSDME, and single cell clones expressing comparable levels of recombinant mouse wild type (WT) GSDME and a caspase-3-uncleavable inactive mutant (Aspartate 270/Glutamate) D270A GSDME were selected. Control MTEC transduced with empty vector (EV) were also generated. Single cell clones were tested in vitro with apoptotic agents and evaluated for HMGB1 release, %PI uptake, and cell morphology. Results Western blot analysis demonstrated GSDME expression in TC-1 but not MTEC cells. Following treatment with apoptotic agents, TC-1 cells demonstrated HMGB1 release and increased %PI uptake as well as a distinct secondary necrotic phenotype characterized by ballooning of the plasma membrane on morphological analysis. This was not seen in MTEC cells. To examine whether the absence of GSDME in MTEC cells correlated with reduced release of immune-stimulatory molecules, MTEC cells stably reconstituted with GSDME were treated with apoptotic agents. Unlike MTEC cells expressing the uncleavable D270A GSDME or EV control, MTEC cells expressing WT GSDME demonstrated a significant release of HMGB1 and increased %PI uptake. Morphological analysis demonstrated significant ballooning of the plasma membrane only in the WT GSDME MTEC cells. Conclusion Treatment with apoptotic agents induces a GSDME-dependent pyroptotic cell death in MTEC and TC-1 cells as evidenced by HMGB1 release, increased %PI uptake, and cell morphology. As pyroptotic cell death of cancer cells induces anti-tumor immunity through the release of HMGB1 and other immunostimulatory factors, promoting GSDME-dependent pyroptosis might be a strategy to overcome treatment resistance in some HNSCC. Gasdermin-E (GSDME) is known to potentiate immunogenic cell death in many tumor types after exposure to anti-cancer agents in a caspase-3 dependent manner. This GSDME-mediated release of immunostimulatory proteins, such as HMGB1, has been shown to increase tumor immunogenicity in mouse model systems of melanoma and colon cancer, resulting in reduced tumor load and lower distal metastasis. We sought to evaluate the role of GSDME-mediated cell death in human papillomavirus-positive (HPV+) head and neck squamous cell carcinoma (HNSCC) mouse cell lines. To test whether HNSCC exhibits a GSDME-associated anti-tumor response, two HPV+ HNSCC mouse cell lines, MTEC and TC-1, were screened for GSDME expression by Western blot and then treated with apoptotic agents (raptinal, cisplatin, etoposide, and UV). Primary outcome measures were HMGB1 release (a potent immunostimulatory factor), percent propidium iodide (%PI) uptake (an indicator of plasma membrane damage and pyroptosis) and cell morphology. Using retroviral transduction, MTEC cells were reconstituted with GSDME, and single cell clones expressing comparable levels of recombinant mouse wild type (WT) GSDME and a caspase-3-uncleavable inactive mutant (Aspartate 270/Glutamate) D270A GSDME were selected. Control MTEC transduced with empty vector (EV) were also generated. Single cell clones were tested in vitro with apoptotic agents and evaluated for HMGB1 release, %PI uptake, and cell morphology. Western blot analysis demonstrated GSDME expression in TC-1 but not MTEC cells. Following treatment with apoptotic agents, TC-1 cells demonstrated HMGB1 release and increased %PI uptake as well as a distinct secondary necrotic phenotype characterized by ballooning of the plasma membrane on morphological analysis. This was not seen in MTEC cells. To examine whether the absence of GSDME in MTEC cells correlated with reduced release of immune-stimulatory molecules, MTEC cells stably reconstituted with GSDME were treated with apoptotic agents. Unlike MTEC cells expressing the uncleavable D270A GSDME or EV control, MTEC cells expressing WT GSDME demonstrated a significant release of HMGB1 and increased %PI uptake. Morphological analysis demonstrated significant ballooning of the plasma membrane only in the WT GSDME MTEC cells. Treatment with apoptotic agents induces a GSDME-dependent pyroptotic cell death in MTEC and TC-1 cells as evidenced by HMGB1 release, increased %PI uptake, and cell morphology. As pyroptotic cell death of cancer cells induces anti-tumor immunity through the release of HMGB1 and other immunostimulatory factors, promoting GSDME-dependent pyroptosis might be a strategy to overcome treatment resistance in some HNSCC.
TL;DR: In this article , the role of ERK in mediating GRK2 mitochondrial translocation and pyroptotic signaling was investigated using a novel PDHα KI CRISPR Cas HEK cell line, and the hypothesize that PDH phosphorylation of this novel site was important for PDH activity.
Abstract: G protein-coupled receptors (GPCRs) are important regulators of cellular functions where agonist binding leads to receptor conformational changes and downstream activation of g-protein-mediated signaling cascades. Signal transduction is terminated by receptor phosphorylation mediated by G-protein coupled receptor kinases (GRKs). GPCR kinase 2 (GRK2) is a main GRK in the heart and is upregulated in heart failure (HF) patients. Recently, we and others have shown that GRK2 can translocate to cardiac mitochondria where it regulates metabolism. Notably, we found that phosphorylated GRK2 at S670 post- ischemia/reperfusion (IR) decreases glucose mitochondrial utilization and pyruvate dehydrogenase (PDH) activity. Furthermore, 2D-SDS PAGE followed by LC/MS/MS revealed a potential link between mitochondrial GRK2 and PDH phosphorylation. Using a novel PDHα KI CRISPR Cas HEK cell line, we tested the hypothesize that PDH phosphorylation of this novel site was important for PDH activity. Utilizing cytosolic and mitochondrially-targeted pyruvate indicators we measured pyruvate levels in WT and KI HEK cells. In parallel, using a novel mouse model GRK2-S670A, we investigated the role of ERK in mediating GRK2 mitochondrial translocation and pyroptotic signaling. Further experiments will determine long-term impact of mitochondrial GRK2 in cell survival in cardiac pathological conditions.