Necroptosis, a cell death pathway mediated by the RIPK1-RIPK3-MLKL signaling cascade downstream of tumor necrosis factor α (TNF-α), has been implicated in many inflammatory diseases. Members of the TAM (Tyro3, Axl, and Mer) family of receptor tyrosine kinases are known for their anti-apoptotic, oncogenic, and anti-inflammatory roles. Here, we identify an unexpected role of TAM kinases as promoters of necroptosis, a pro-inflammatory necrotic cell death. Pharmacologic or genetic targeting of TAM kinases results in a potent inhibition of necroptotic death in various cellular models. We identify phosphorylation of MLKL Tyr376 as a direct point of input from TAM kinases into the necroptosis signaling. The oligomerization of MLKL, but not its membranal translocation or phosphorylation by RIPK3, is controlled by TAM kinases. Importantly, both knockout and inhibition of TAM kinases protect mice from systemic inflammatory response syndrome. In conclusion, this study discovers that immunosuppressant TAM kinases are promoters of pro-inflammatory necroptosis, shedding light on the biological complexity of the regulation of inflammation.

During emergency hematopoiesis, hematopoietic stem cells (HSCs) rapidly proliferate to produce myeloid and lymphoid effector cells, a response that is critical against infection or tissue injury. If unresolved, this process leads to sustained inflammation, which can cause life-threatening diseases and cancer. Here, we identify a role of double PHD fingers 2 (DPF2) in modulating inflammation. DPF2 is a defining subunit of the hematopoiesis-specific BAF (SWI/SNF) chromatin-remodeling complex, and it is mutated in multiple cancers and neurological disorders. We uncovered that hematopoiesis-specific Dpf2-KO mice developed leukopenia, severe anemia, and lethal systemic inflammation characterized by histiocytic and fibrotic tissue infiltration resembling a clinical hyperinflammatory state. Dpf2 loss impaired the polarization of macrophages responsible for tissue repair, induced the unrestrained activation of Th cells, and generated an emergency-like state of HSC hyperproliferation and myeloid cell-biased differentiation. Mechanistically, Dpf2 deficiency resulted in the loss of the BAF catalytic subunit BRG1 from nuclear factor erythroid 2-like 2-controlled (NRF2-controlled) enhancers, impairing the antioxidant and antiinflammatory transcriptional response needed to modulate inflammation. Finally, pharmacological reactivation of NRF2 suppressed the inflammation-mediated phenotypes and lethality of Dpf2Δ/Δ mice. Our work establishes an essential role of the DPF2-BAF complex in licensing NRF2-dependent gene expression in HSCs and immune effector cells to prevent chronic inflammation.

Ubiquitylation of the TNFR1 signalling complex (TNF-RSC) controls the activation of RIPK1, a kinase critically involved in mediating multiple TNFα-activated deleterious events. However, the molecular mechanism that coordinates different types of ubiquitylation modification to regulate the activation of RIPK1 kinase remains unclear. Here, we show that ABIN-1/NAF-1, a ubiquitin-binding protein, is recruited rapidly into TNF-RSC in a manner dependent on the Met1-ubiquitylating complex LUBAC to regulate the recruitment of A20 to control Lys63 deubiquitylation of RIPK1. ABIN-1 deficiency reduces the recruitment of A20 and licenses cells to die through necroptosis by promoting Lys63 ubiquitylation and activation of RIPK1 with TNFα stimulation under conditions that would otherwise exclusively activate apoptosis in wild-type cells. Inhibition of RIPK1 kinase and RIPK3 deficiency block the embryonic lethality of Abin-1 -/- mice. We propose that ABIN-1 provides a critical link between Met1 ubiquitylation mediated by the LUBAC complex and Lys63 deubiquitylation by phospho-A20 to modulate the activation of RIPK1.

Protein arginine methyltransferase 5 (PRMT5) catalyzes the symmetric di-methylation of arginine residues in histones H3 and H4, marks that are generally associated with transcriptional repression. However, we found that PRMT5 inhibition or depletion led to more genes being downregulated than upregulated, indicating that PRMT5 can also act as a transcriptional activator. Indeed, the global level of histone H3K27me3 increases in PRMT5 deficient cells. Although PRMT5 does not directly affect PRC2 enzymatic activity, methylation of histone H3 by PRMT5 abrogates its subsequent methylation by PRC2. Treating AML cells with an EZH2 inhibitor partially restored the expression of approximately 50% of the genes that are initially downregulated by PRMT5 inhibition, suggesting that the increased H3K27me3 could directly or indirectly contribute to the transcription repression of these genes. Indeed, ChIP-sequencing analysis confirmed an increase in the H3K27me3 level at the promoter region of a quarter of these genes in PRMT5-inhibited cells. Interestingly, the anti-proliferative effect of PRMT5 inhibition was also partially rescued by treatment with an EZH2 inhibitor in several leukemia cell lines. Thus, PRMT5-mediated crosstalk between histone marks contributes to its functional effects.

The mechanisms of cellular energy sensing and AMPK-mediated mTORC1 inhibition are not fully delineated. Here, we discover that RIPK1 promotes mTORC1 inhibition during energetic stress. RIPK1 is involved in mediating the interaction between AMPK and TSC2 and facilitate TSC2 phosphorylation at Ser1387. RIPK1 loss results in a high basal mTORC1 activity that drives defective lysosomes in cells and mice, leading to accumulation of RIPK3 and CASP8 and sensitization to cell death. RIPK1-deficient cells are unable to cope with energetic stress and are vulnerable to low glucose levels and metformin. Inhibition of mTORC1 rescues the lysosomal defects and vulnerability to energetic stress and prolongs the survival of RIPK1-deficient neonatal mice. Thus, RIPK1 plays an important role in the cellular response to low energy levels and mediates AMPK-mTORC1 signaling. These findings shed light on the regulation of mTORC1 during energetic stress and unveil a point of crosstalk between pro-survival and pro-death pathways.

Protein arginine methyltransferase 5 (PRMT5) is overexpressed in many cancer types and is a promising therapeutic target for several of them, including leukemia and lymphoma. However, we and others have reported that PRMT5 is essential for normal physiology. This dependence may become dose limiting in a therapeutic setting, warranting the search for combinatorial approaches. Here, we report that PRMT5 depletion or inhibition impairs homologous recombination (HR) DNA repair, leading to DNA-damage accumulation, p53 activation, cell-cycle arrest, and cell death. PRMT5 symmetrically dimethylates histone and non-histone substrates, including several components of the RNA splicing machinery. We find that PRMT5 depletion or inhibition induces aberrant splicing of the multifunctional histone-modifying and DNA-repair factor TIP60/KAT5, which selectively affects its lysine acetyltransferase activity and leads to impaired HR. As HR deficiency sensitizes cells to PARP inhibitors, we demonstrate here that PRMT5 and PARP inhibitors have synergistic effects on acute myeloid leukemia cells.

Necroptosis is a lytic programmed cell death mediated by the RIPK1-RIPK3-MLKL pathway. The loss of Receptor-interacting serine/threonine-protein kinase 3 (RIPK3) expression and necroptotic potential have been previously reported in several cancer cell lines; however, the extent of this loss across cancer types, as well as its mutational drivers, were unknown. Here, we show that RIPK3 expression loss occurs progressively during tumor growth both in patient tumor biopsies and tumor xenograft models. Using a cell-based necroptosis sensitivity screen of 941 cancer cell lines, we find that escape from necroptosis is prevalent across cancer types, with an incidence rate of 83%. Genome-wide bioinformatics analysis of this differential necroptosis sensitivity data in the context of differential gene expression and mutation data across the cell lines identified various factors that correlate with resistance to necroptosis and loss of RIPK3 expression, including oncogenes BRAF and AXL. Inhibition of these oncogenes can rescue the RIPK3 expression loss and regain of necroptosis sensitivity. This genome-wide analysis also identifies that the loss of RIPK3 expression is the primary factor correlating with escape from necroptosis. Thus, we conclude that necroptosis resistance of cancer cells is common and is oncogene driven, suggesting that escape from necroptosis could be a potential hallmark of cancer, similar to escape from apoptosis.