Interferon-γ primes macrophages for pathogen ligand-induced killing via a caspase-8 and mitochondrial cell death pathway.

Cell death plays an important role during pathogen infections. Here, we report that interferon-γ (IFNγ) sensitizes macrophages to Toll-like receptor (TLR)-induced death that requires macrophage-intrinsic death ligands and caspase-8 enzymatic activity, which trigger the mitochondrial apoptotic effectors, BAX and BAK. The pro-apoptotic caspase-8 substrate BID was dispensable for BAX and BAK activation. Instead, caspase-8 reduced pro-survival BCL-2 transcription and increased inducible nitric oxide synthase (iNOS), thus facilitating BAX and BAK signaling. IFNγ-primed, TLR-induced macrophage killing required iNOS, which licensed apoptotic caspase-8 activity and reduced the BAX and BAK inhibitors, A1 and MCL-1. The deletion of iNOS or caspase-8 limited SARS-CoV-2-induced disease in mice, while caspase-8 caused lethality independent of iNOS in a model of hemophagocytic lymphohistiocytosis. These findings reveal that iNOS selectively licenses programmed cell death, which may explain how nitric oxide impacts disease severity in SARS-CoV-2 infection and other iNOS-associated inflammatory conditions.

Loss-of-Function but Not Gain-of-Function Properties of Mutant TP53 Are Critical for the Proliferation, Survival, and Metastasis of a Broad Range of Cancer Cells.

Mutations in the tumor suppressor TP53 cause cancer and impart poor chemotherapeutic responses, reportedly through loss-of-function, dominant-negative effects and gain-of-function (GOF) activities. The relative contributions of these attributes is unknown. We found that removal of 12 different TP53 mutants with reported GOFs by CRISPR/Cas9 did not impact proliferation and response to chemotherapeutics of 15 human cancer cell lines and colon cancer-derived organoids in culture. Moreover, removal of mutant TP53/TRP53 did not impair growth or metastasis of human cancers in immune-deficient mice or growth of murine cancers in immune-competent mice. DepMap mining revealed that removal of 158 different TP53 mutants had no impact on the growth of 391 human cancer cell lines. In contrast, CRISPR-mediated restoration of wild-type TP53 extinguished the growth of human cancer cells in vitro. These findings demonstrate that LOF but not GOF effects of mutant TP53/TRP53 are critical to sustain expansion of many tumor types. SIGNIFICANCE: This study provides evidence that removal of mutant TP53, thereby deleting its reported GOF activities, does not impact the survival, proliferation, metastasis, or chemotherapy responses of cancer cells. Thus, approaches that abrogate expression of mutant TP53 or target its reported GOF activities are unlikely to exert therapeutic impact in cancer. See related commentary by Lane, p. 211 . This article is featured in Selected Articles from This Issue, p. 201.

Mitochondrial E3 ubiquitin ligase MARCHF5 controls BAK apoptotic activity independently of BH3-only proteins.

Intrinsic apoptosis is principally governed by the BCL-2 family of proteins, but some non-BCL-2 proteins are also critical to control this process. To identify novel apoptosis regulators, we performed a genome-wide CRISPR-Cas9 library screen, and it identified the mitochondrial E3 ubiquitin ligase MARCHF5/MITOL/RNF153 as an important regulator of BAK apoptotic function. Deleting MARCHF5 in diverse cell lines dependent on BAK conferred profound resistance to BH3-mimetic drugs. The loss of MARCHF5 or its E3 ubiquitin ligase activity surprisingly drove BAK to adopt an activated conformation, with resistance to BH3-mimetics afforded by the formation of inhibitory complexes with pro-survival proteins MCL-1 and BCL-XL. Importantly, these changes to BAK conformation and pro-survival association occurred independently of BH3-only proteins and influence on pro-survival proteins. This study identifies a new mechanism by which MARCHF5 regulates apoptotic cell death by restraining BAK activating conformation change and provides new insight into how cancer cells respond to BH3-mimetic drugs. These data also highlight the emerging role of ubiquitin signalling in apoptosis that may be exploited therapeutically.

MARCH5 requires MTCH2 to coordinate proteasomal turnover of the MCL1:NOXA complex.

MCL1, a BCL2 relative, is critical for the survival of many cells. Its turnover is often tightly controlled through both ubiquitin-dependent and -independent mechanisms of proteasomal degradation. Several cell stress signals, including DNA damage and cell cycle arrest, are known to elicit distinct E3 ligases to ubiquitinate and degrade MCL1. Another trigger that drives MCL1 degradation is engagement by NOXA, one of its BH3-only protein ligands, but the mechanism responsible has remained unclear. From an unbiased genome-wide CRISPR-Cas9 screen, we discovered that the ubiquitin E3 ligase MARCH5, the ubiquitin E2 conjugating enzyme UBE2K, and the mitochondrial outer membrane protein MTCH2 co-operate to mark MCL1 for degradation by the proteasome-specifically when MCL1 is engaged by NOXA. This mechanism of degradation also required the MCL1 transmembrane domain and distinct MCL1 lysine residues to proceed, suggesting that the components likely act on the MCL1:NOXA complex by associating with it in a specific orientation within the mitochondrial outer membrane. MTCH2 has not previously been reported to regulate protein stability, but is known to influence the mitochondrial localization of certain key apoptosis regulators and to impact metabolism. We have now pinpointed an essential but previously unappreciated role for MTCH2 in turnover of the MCL1:NOXA complex by MARCH5, further strengthening its links to BCL2-regulated apoptosis.