Tumor-Intrinsic and Immune Modulatory Roles of Receptor Interacting Protein Kinases

Abstract

Receptor-Interacting Protein Kinase 1 (RIPK1) and RIPK3 are signaling adaptors that critically regulate cell death and inflammation. Tumors have adapted to subvert RIPK-dependent cell death, suggesting that these processes play key roles in tumor regulation. Moreover, RIPK-driven cancer cell death may bolster durable anti-tumor immunity. On the other hand, there are examples in which RIPKs induce inflammation and aid tumor progression. Furthermore, the RIPKs can exert its effect on tumor growth through regulating the activity of immune effectors in the tumor microenvironment, thus highlighting the context-dependent roles of RIPKs. Here, we review recent advances on the regulation of RIPK activity in tumors and immune cells and how these processes coordinate with each other to control tumorigenesis.

Receptor Interacting Protein Kinases: Key regulators of cell death

Necrosis is cell death marked by rupture of the plasma membrane, and was once considered to be cell death caused by non-specific injury [1]. However, recent evidence indicates that necrosis can be regulated and triggered by specific receptors and signal adaptors. In particular, necroptosis is a form of regulated necrosis that is optimally induced when Caspase 8 is inhibited [2]. Similar to apoptosis, necroptosis resistance is widely observed across tumor types. Paradoxically, necroptosis signal adaptors can induce apoptosis or promote cell survival under certain conditions. Moreover, necroptosis signal adaptors have been shown to stimulate inflammatory gene transcription independent of cell death. The pro-inflammatory property of necroptosis renders it an attractive target in anti-tumor therapy.

Receptor interacting serine/threonine protein kinase 3 (RIPK3) is a critical kinase for necroptosis that is activated by one of three distinct upstream activators: the RIPK3-related kinase RIPK1, the toll-like receptor 3 adaptor TIR-domain-containing adapter-inducing interferon-β (TRIF), and the viral RNA sensor Z-DNA Binding Protein 1 (ZBP1). RIPK1 and RIPK3 form a complex termed the necrosome with the essential apoptosis signal adaptors FADD and Caspase 8. Inhibition of Caspase 8 proteolytic cleavage of RIPK1 allows stable interaction between RIPK1 and RIPK3 and downstream activation of mixed lineage kinase domain-like (MLKL); RIPK3 phosphorylates the effector molecule MLKL to trigger plasma membrane leakage [3]. In addition to tumor cell death, RIPK3 and RIPK1 also regulate innate inflammation by immune effectors in the tumor microenvironment (see below). Hence, the RIPKs can regulate tumor growth and anti-tumor immune responses through multiple mechanisms. Furthermore, the RIPKs have also been shown to aid tumor progression and metastasis in some contexts [4–6]. These seemingly conflicting roles highlight the complexity of RIPKs’ function in cancer. In this review, we will discuss recent findings on the role and regulation of RIPK1 and RIPK3 in cancer cell death, tumor-specific immune responses, and therapeutic opportunities that target RIPK signaling in cancer. The readers are referred to these excellent reviews for more in-depth review on necroptosis signaling [7, 8].

Necroptosis in cancer: Friend or foe

In certain tumor types, increased necrosis is associated with tumor progression and poor prognosis. Tumor cell necrosis is especially prominent in the inner core of the tumor mass and is throught to be the consequence of cellular stress, such as nutrient deprivation and hypoxia. Could necroptosis be responsible for tumor necrosis? Indeed, elevated RIPK3 expression correlates with increased mortality in glioma [9], implying that necroptosis might contribute to tumor necrosis. Additionally, strong RIPK3 and MLKL activation was detected in a mouse mammary tumor model [10], and CRISPR-mediated knockout of Mlkl reduced but did not fully rescue tumor necrosis, indicating that necroptosis contributes to, but is not the sole mechanism of tumor necrosis. Moreover, Mlkl knockout had minimal impact on tumor growth but significantly reduced metastasis to the lung [10], suggesting that tumor necroptosis primarily facilitates metastasis in mouse mammary tumor.

Increased RIPK1-RIPK3-MLKL-mediated necroptosis was also observed in cholangiocarinoma. In this case however, necroptosis was associated with improved overall survival [11]. The correlation between RIPK3-dependent necroptosis and improved survival is widely observed in cancers across multiple tissue origins [12, 13]. Experimentally, serial passaging of human tumor xenografts in mice led to loss of RIPK3 expression [14], supporting the notion that necroptosis resistance confers selective advantage to tumor progression. It should be noted that RIPK3 expression is variable in different tissues, while expression of MLKL is in part controlled by interferon signaling [15, 16]. Hence, differences in the inflammatory milieu of the tumor microenvironment may impact sensitivity to necroptosis and whether the RIPKs and necroptosis function to inhibit or promote tumors in different tumor types and contexts.

Mechanisms of cancer resistance to necroptosis

Several distinct mechanisms have been shown to mediate cancer cell loss of RIPK3 expression (Figure 1). As early as in 2006, the RIPK3 gene promoter was reported to be hypermethylated in lung cancer [17]. This observation was later validated in other cancer types. For example, the demethylating agent 5-Aza-2′-deoxycytidine and the histone deacetylase inhibitor Trichostatin A restored RIPK3 expression in mesothelioma [18], non-small cell lung cancer [19], and breast cancer [20]. Small interfering RNA (siRNA)-mediated gene silencing of the DNA methylation mediator UHRF1 or the DNA methyltransferase DNMT1 restored RIPK3 expression [18, 21, 22]. These results provide strong evidence that epigenetic DNA modification is a key mechanism that enforces silencing of RIPK3 transcription in cancer (Figure 1b).

Figure 1. Inhibitory mechanisms of RIP kinases in cancer.

Several mechanisms by which tumors impair the expression or activity of RIP kinases have been described. (a) Hypoxia occurs in the tumor microenvironment due to poor vascularization as tumors outgrow their blood supply. To counter cell death activation in response to hypoxia, tumors downregulate transcription of RIPK1 and RIPK3. (b) Impaired expression of RIPK3 due to hypermethylation of the RIPK3 promoter has been observed in several tumor types. RIPK3 gene methylation is enforced by the coordinate action of the DNA methyltransferase DNMT1 or the methylation enhancer UHRF1. (c) Over-expression of the oncogenes AXL and BRAF activating mutants drives loss of RIPK3 expression in tumors through undefined mechanisms. (d) However, the oncogene Myc was reported to bind to RIPK3 to prevent RIPK3-RIPK1 interaction and necroptosis. (e) Increased acidity in the tumor microenvironment may also impair RIPK-dependent cell death through inhibition of the kinase activity of RIPK1.

Besides promoter methylation, RIPK3 expression is also regulated by certain oncogenes. By analyzing the gene expression and mutation profile of 941 cancer cell lines, Najafov and colleagues found that over-expression of the oncogenes AXL and TYRO3, and also gain-of-function mutations of BRAF, track with loss of RIPK3 expression; pharmacologic inhibition of AXL or BRAF restored RIPK3 expression and sensitivity to necroptosis in many RIPK3 negative cancer cell lines [14]. Since there is no association between DNMT1 expression and mutational status of AXL or BRAF [23], they likely represent distinct mechanisms that regulate RIPK3 expression. Paradoxically, AXL and the other TAM kinases TYRO3 and MerTK have also been shown to phosphorylate MLKL at Y376 to promote MLKL oligomerization and necroptosis in RIPK3-expressing tumor cells [24]. Since the TAM kinases are known regulators of immune responses, these opposing effects may reflect tumor-specific roles in regulating inflammation (Figure 1c). In addition to AXL and BRAF, the oncogene c-MYC was implicated to impair necroptosis by direct binding to RIPK3 to restrict RIPK1-RIPK3 interaction [25]. MYC-deficiency enhanced RIPK1-RIPK3 necrosome formation and tumor xenograft regression in response to treatment with the necroptosis sensitizers SMAC mimetic and caspase inhibitor [25] (Figure 1d).

In addition to epigenetic control and oncogene activation, environmental cues that are frequently found in cancer also regulate necroptosis. For instance, hypoxia tunes cancer cell response to necroptosis by inhibiting RIPK1 and RIPK3 gene transcription [26] (Figure 1a). Moreover, the cell death-inducing RIPK1 kinase activity is inhibited in low pH environment, a condition that is commonly found in cancer [27] (Figure 1e). These many inhibitory mechanisms argues that necroptosis is primarily anti-tumor in nature.

In contrast to RIPK3, there is no apparent strong selection against RIPK1 or MLKL expression in cancer (see Outstanding Questions). In macrophages and dendritic cells, RIPK3 has been shown to promote inflammatory cytokine expression through NF-κB and inflammasome activation [28]. Although RIPK1 also stimulates cytokine expression, RIPK1 deficiency sensitizes many primary cell types to cell death [29, 30]. Moreover, loss of RIPK1 protein expression led to very early onset of inflammatory bowel disease, combined immunodeficiency and lower life expectancy in human [31]. As such, loss of RIPK1 may blunt inflammatory immune effectors and shorten the life span of the host to restrict tumorigenesis. Unlike RIPK1 and RIPK3, MLKL does not have major role in promoting inflammatory gene expression. These factors may explain why RIPK3 but not RIPK1 or MLKL is selectively lost in cancer.

Outstanding Questions.

Why is there strong selection against expression of RIPK3 but not other necroptosis adaptors in cancer?

What are the tumor-intrinsic and tumor-extrinsic factors that determine whether necroptosis promotes or inhibit tumorigenesis?

Does RIPK1 and RIPK3 inhibition contribute to kinase inhibitors used in current cancer therapy?

What are the roles of RIPK1 and RIPK3 in different immune effectors?

What are the effects of RIPK kinase inhibitors on immune cell functions?

Although they are not classical oncogenes or tumor suppressors, somatic mutations in RIPK1 and MLKL have been identified in many different tumor types (https://portal.gdc.cancer.gov/). Further, a genome-wide CRISPR screen identified RIPK1 as a factor that confers tumor resistance to immunotherapy [32]. In some populations, RIPK1 polymorphism correlates with cancer susceptibility [33, 34]. The selective loss of RIPK3 but not other necroptosis signal adaptors implies that RIPK3 may inhibit tumorigenesis through mechanisms beyond necroptosis.

Regulation of cancer cell death by RIPKs

In cancers with intact RIPK1 and RIPK3 expression, RIPK-mediated cell death largely appears to improve tumor control. For example, treatment with the death receptor ligands, CD95L or TNF-related apoptosis-inducing ligand (TRAIL), in addition to SMAC mimetic, enhanced RIPK1-dependent cell death in a subset of cancer cells from patients with acute myeloid leukemia [35]. In esophageal squamous cell carcinoma, increased RIPK1 expression increased the efficacy of tumor cell death in response to treatment with the chemotherapeutic agent Cisplatin [36]. Further, combining SMAC mimetic with the chemotherapeutic agent Doxorubicin bolstered tumor control through enhancing RIPK1-dependent apoptosis in a colorectal cancer xenograft mouse model [37]. RIPK1 was also required for SMAC mimetic induced cell death in cholangiocarcinoma and breast cancer cells [11, 38]. Recently, RIPK1 and other necrosome components such as Caspase 8, FADD, and cFLIP were found to form a complex that maintains chromosomal integrity during mitosis [39]. A similar complex was also found to be involved in sensing of proliferation-associated DNA damage in liver cancer [40]. Hence, the RIPKs may restrict the oncogenic process through mechanisms other than cell death.

Inhibitory mechanisms of necroptosis in cancer

In addition to inhibition of RIPK3 expression, tumors also develop other mechanisms to subvert RIPKs-dependent cell death. The kinases IKK, TAK1, and TBK1 directly phosphorylate RIPK1 to limit its activity and to dampen its death-inducing activity [41–44]. Inhibition of IKK or TAK1 reversed this inhibition and enhanced RIPK1-dependent cell death of adult T-cell leukemia [45]. High expression of TAK1 correlates with worsened disease free survival in melanoma; TAK1 kinase inhibitor relieved this inhibition and increased apoptosis of melanoma [46]. In contrast, genetic ablation of TAK1 in hepatocytes and cholangiocytes resulted in early development of hepatocellular carcinoma (HCC) due to chronic hepatocellular damage-induced proliferation and fibrosis. Deficiency in RIPK1 kinase activity or RIPK3 inhibited liver injury-induced inflammation and HCC development [4, 47]. The tumor suppressor cylindromatosis (CYLD), which promotes RIPK1-dependent cell death, is also controlled by inhibitory phosphorylation by the IKK and TBK1 kinases [45]. Thus, cancer evasion of necroptosis can be mediated through the action of the inhibitory kinases IKK, TAK1, or TBK1 (Table 1).

Table 1.

Post-Translational Modifications of RIP Kinases. H: human, M: Mouse.

Regulator	Modification	Target	Target Residue(s)	Effect	Reference
IKK	Phosphorylation	RIPK1	S25	Inhibits RIPK1 kinase activity	[43, 44]
TAK1/MK2	Phosphorylation	RIPK1	S321	Inhibits RIPK1-dependent cell death	[4, 41, 44, 46, 47]
TBK1	Phosphorylation	RIPK1	T189(H)/T190(M)	Inhibits RIPK1-dependent cell death	[42, 43]
cIAP1/2	Ubiquitination	RIPK1	K377 (H)/K376(M)	Inhibits RIPK1-dependent cell death	[48–50]
CYLD	De-ubiquitination	RIPK1	Unknown	Enhances RIPK1-dependent cell death	[45]
LUBAC	Ubiquitination	RIPK1	Unknown	Inhibits RIPK1-dependent cell death, promotes NF-κB activation	[51, 52]
PELI1	Ubiquitination	RIPK1, RIPK3	RIPK1: K115; RIPK3: Unknown	Activates RIPK1 but promotes RIPK3 ubiquitination and degradation	[56, 57]
A20	Ubiquination	RIPK3	RIPK3: K5	Promotes K63-linked RIPK3 ubiquitination to restrict necroptosis	[58–61, 94]
MIB2	Ubiquitination	RIPK1	K377 (H)/K376(M), K634	Inhibits RIPK1-dependent cell death	[62, 63]
Parkin	Ubiquitination	RIPK1	K377 (H)/K376(M)	Promotes NF-κB and MAPK activation and inhibits RIPK1-RIPK3 necrosome formation	[64, 65]
TRIM25	Ubiquitination	RIPK3	K501	Enhances proteasomal degradation of RIPK3	[67]

In addition to the inhibitory kinases, ubiquitination also has a key role in regulating RIPKs activity in cancer. For example, cellular inhibitor of apoptosis protein 1 (cIAP1) and cIAP2, E3 ligases that ubiquitinate RIPK1 to restrict its death-inducing activity, are often overexpressed in cancers. SMAC mimetics, which cause auto-ubiquitination and proteasomal degradation of the IAPs, can enhance cancer cell death through stimulating RIPK1 death-inducing activity [48–50]. The linear ubiquitin chain assembly complex (LUBAC) also restricts RIPK1-induced cell death, promotes NF-κB in breast cancer, and and prevents genotoxic stress-induced cell death in B cell lymphoma [51, 52]. The deubiquitinase OTU deubiquitinase with linear linkage specificity (OTULIN), which cleaves linear ubiquitin chains generated by LUBAC, has a protective role in HCC. Indeed, hepatocyte-specific deletion of OTULIN induced HCC through RIPK1 kinase activity and FADD-dependent apoptosis [53]. The tumor suppressor CYLD also promotes necroptosis by deubiquitination of RIPK1 [54, 55]. These results indicate that RIPK1 ubiquitination is a key event that controls its death-inducing activity in different types of tumor (Table 1).

Besides the IAPs and LUBAC, other E3 ligases have also been shown recently to regulate RIPKs activity. Reduced expression of the E3 ligase PELI1 correlates with poor survival in non-small cell lung carcinoma [13]. However, the effect of the E3 ligase PELI1 on necroptosis is controversial: while PELI1 was reported to ubiquitinate RIPK1 to promote necroptosis in primary mouse embryonic fibroblasts [56], another group found that PELI1 inhibits necroptosis by targeting RIPK3 for proteasomal degradation in transformed cells [57]. Thus, the same E3 ligase might exert opposing effects on necroptosis in primary versus transformed cells. A20, which possesses both ubiquitinase and de-ubiquitanase functions, is another key adaptor that restricts RIPK1-dependent cell death through stabilizing the pro-survival ubiquitin scaffold of complex I [58]. However, elevated A20, a condition observed in human inflammatory bowel disease patients, stimulated RIPK1 and intestinal epithelial cell apoptosis [59]. The A20- and ubiquitin-binding protein ABIN-1 promotes A20 and RIPK1 interaction [60]. In the absence of ABIN-1, kinase activity of RIPK1 was enhanced, leading to increased chemotherapy-induced necroptosis and impaired tumor growth of colorectal cancer xenografts in mice [61].

Additionally, the E3 ligase Mind Bomb-2 (MIB2) was shown to ubiquitinate RIPK1 to inhibit its death-inducing kinase activity [62, 63], and the Parkinson’s Disease-associated E3 ligase Parkin, which also functions as a tumor suppressor, was reported to ubiquitinate RIPK1 to promote NF-κB and MAPK activation [64]. Parkin deficiency enhanced RIPK1-RIPK3 interaction, necroptosis-induced inflammation, and tumorgenesis in an inflammation-induced colorectal cancer model [65]. Finally, the E3 ligase TRIM25, which regulates innate inflammation [66], was found to ubiquitinate RIPK3 to promote its proteasomal degradation [67]. TRIM25 has been implicated to promote tumorigenesis [68], although it is not clear at present if the tumorigenic activity of TRIM25 requires its degradative activity towards RIPK3. It is noteworthy that different ubiquitin acceptor sites have been identified for RIPK1 and RIPK3 [69–71]. In many cases, RIPK1 ubiquitination at different sites are mediated by distinct ubiquitinating enzymes. Thus, it is plausible that these ubiquitinating enzymes regulate RIPK1 and RIPK3 activities in a context and cell type-specific manner (Table 1).

Pro-survival function of RIPK1 in cancer

Although most studies indicate that RIPK1 exerts cytostatic effects on tumors, it can also promote tumorigenesis in certain situations (see Outstanding Questions). For instance, in a murine orthotopic gallbladder cancer model, RIPK1 promotes tumor proliferation and metastasis through NF-κB and AP-1-driven upregulation of vascular endothelial growth factor c (VEGF-C) [5]. This result is consistent with the role of RIPK1 in NF-κB and MAPK activation [72]. In human colorectal cancer, RIPK1 interacts with the Mitochondrial Calcium Uniporter (MCU) to increase mitochondrial respiration, leading to increased tumor burden in mouse xenografts [73]. In contrast to deficiency of upstream regulators of RIPK1, genetic deletion of RIPK1 in liver parenchymal cells prompted the proteasomal degradation of the survival factor TRAF2, which led to enhanced Caspase 8 activation, hepatocyte apoptosis, and subsequent HCC development [74].

Despite many reports that support a role for RIPK1 in regulating tumor growth, there are examples in which RIPK1 is dispensable for tumor development. For example, continuous treatment with RIPK1 kinase inhibitor failed to impair tumor growth in multiple genetically engineered pancreatic cancer mouse models [75]. A similar observation was made in a mouse metastatic melanoma model using mice that express a kinase inactive RIPK1 mutant [75]. Thus, RIPK1 appears to regulate tumor growth through its scaffolding function rather than its kinase activity. Further, in mouse mammary tumor, the viral RNA sensor ZBP1 induced RIPK3-MLKL-dependent tumor cell necroptosis [76]; knockout of Zbp1 or Mlkl, but not Ripk1, reduced lung metastasis without affecting tumor growth, suggesting RIPK1, not just its kinase activity, is dispensable in some cases. This situation is analogous to necroptosis induction in virus infection in which viral Z-form nucleic acid binds to ZBP1 to activate RIPK3 [77]. The induction of RIPK1-independent necroptosis in certain tumors might explain the discrepant roles of RIPK1 in different tumor models. Further investigation is required to determine whether RIPK1-independent necroptosis is restricted to cancers driven by oncogenic viruses.

RIPKs in anti-tumor inflammation

Necroptosis is widely considered to be a form of immunogenic cell death due to the release of damage-associated molecular patterns (DAMPs). However, early studies showed that overexpression of RIPK1 and RIPK3 could both stimulate the inflammatory transcription factor NF-κB (reviewed in [78]), indicating that the RIPKs can also directly stimulate pro-inflammatory cytokines expression independent of cell death [79] (Figure 2). In support of this, RNA-seq analysis of the colorectal cancer cell line HT-29 revealed that the RIPK1-RIPK3-MLKL axis facilitates NF-κB and p38-dependent inflammatory cytokine expression [80]. In fact, a separate study showed that inflammatory gene expression can continue even after necroptosis-induced plasma membrane leakage [81]. Mechanistically, the RIPK1-RIPK3-MLKL axis promotes degradation of IκBα and nuclear translocation of NF-κB [80]. Since inflammatory cytokine expression was greatly diminished when necroptosis was induced through forced dimerization of MLKL [80], maximal inflammation required synergism between DAMPs release and inflammatory cytokine expression.

Figure 2. The multi-faceted functions of RIPKs in anti-tumor immunity.

Tumor cell necroptosis can be induced by cytokines or direct killing by dendritic cells or cytotoxic CD8+ T lymphocytes. Tumor necroptosis results in DAMP release and cytokine secretion, which enhance dendritic cell activation and cross-priming of tumor-specific T cells in a feed-forward manner to bolster anti-tumor immunity. RIPK1 and RIPK3 expression in myeloid cells infiltrating the tumor, including dendritic cells and macrophages, also directly enhances inflammatory cytokine expression to further strengthen anti-tumor T cell responses. Moreover, tumor RIPK3 expression can also promote anti-tumor immunity through immunosuppression of inhibitory effectors such as MDSCs.

Another recent study suggests that the kinase activity of RIPK3 enhances cytokine expression by phosphorylation and inhibition of the NF-κB transcriptional repressor Tripartite Motif Protein 28 (TRIM28) [82]. Ultimately, these pro-inflammatory signals converge to activate dendritic cells, CD8⁺ T cell cross-priming, and induction of anti-tumor T cell responses. Human dendritic cells have been shown to directly induce tumor cell necroptosis [83], suggesting that a feed-forward amplification loop between tumor cell necroptosis and dendritic cell activation may further boost anti-tumor T cell immunity (Figure 2).

Finally, the mode of necroptosis induction also crucially dictates the role of NF-κB-driven inflammatory cytokine production in necroptosis-induced anti-tumor CD8⁺ T cell responses. For example, while chemical-induced dimerization of RIPK3 stimulates anti-tumor immunity, inclusion of the death domain from FADD in the dimerization cassette obviates the dependence on NF-κB for optiml induction of necroptosis-induced anti-tumor immune responses [84, 85]. These studies provide valuable information for future design of necroptosis-based therapeutic strategies.

RIPKs signaling in immune effectors

Immune cells in the tumor microenvironment play critical roles in tumor growth and metastasis. Besides regulating tumor cell survival and cell death, RIPKs can also impact tumor growth and outcome through tuning the activity of immune effectors. For example, Kupffer cells, which function as macrophage-like cells in the liver, undergo RIPK3-dependent necroptosis to promote monocyte recruitment, TNF production, and colorectal cancer metastasis to the liver [86]. Tumor-specific knockdown of RIPK3 by small hairpin RNA (shRNA) promotes Myeloid-derived Suppressor Cell (MDSC) accumulation and limits CD8+ T cell recruitment to the tumor in human and mouse hepatocarcinoma mouse implant model [87]. However, in Apc^Min/+ mice and MC38 tumor implant models, inhibition of RIPK3 kinase activity with pharmacologic inhibitor reduced the MDSC compartment [88] (Figure 2). The distinct effects achieved with knockdown of RIPK3 in tumor cells alone versus RIPK3 inhibition in both tumor and immune cells with the kinase inhibitor indicate that RIPK3 exerts its effects on tumor growth through multiple cell types (see Outstanding Questions). Indeed, this postulate is supported by recent evidence that some tumors exhibit differential growth in hosts that lack RIPK3 expression [6].

Therapeutic opportunities

The above discussions of the roles of RIPKs in cancer progression and cell death lead to the question of whether the RIPKs are viable therapeutic targets in cancer. Recent studies suggest that this is indeed a possibility. For instance, in a CRISPR screen of immune checkpoint blockade resistant melanoma, the TNF receptor signal adaptor TNF receptor-associated factor 2 (TRAF2) was identified as a factor that contributes to tumor resistance to RIPK1-dependent TNF-induced cytotoxicity [89]. This effect appeared to involve cIAPs degradation and sensitization to RIPK1-dependent cell death, since TRAF2 is required for stabilization of the cIAPs. As such, SMAC mimetics and checkpoint inhibitors synergized with each other to restrict tumor growth by lowering the threshold of autocrine TNF- and RIPK1-dependent tumor cell death and enhancing anti-tumor CD8+ T cell response [89]. Similarly, in a rat sarcoma model, bolstering RIPK1-dependent cell death through the use of SMAC mimetic in addition to the standard of care treatment TNF and melphalan increased tumor T and natural killers (NK) cell infiltration, response to checkpoint inhibitors, prolonged survival, and conferred greater protection against tumor rechallenge [90]. In addition, SMAC mimetics were also suggested to enhance the cytoxicity of chimeric antigen receptor (CAR)-T cells treatment of B cell acute lymphoblastic leukemia [91]. It is also noteworthy that many of the clinical kinase inhibitors also target RIPK1 and RIPK3. These include many of the inhibitors that target Bcr-Abl [92, 93], which raises the intriguing possibility that inhibition of RIPKs may contribute to the efficacy of current cancer target therapies.

Concluding remarks

Our understanding of RIPKs biology have advanced substantially in recent years. Experimental models provide strong rationale that maniupulating necroptosis and other forms of immunogenic cell death is a viable anti-tumor strategy, especially when applied together with other cancer therapies. Recent results also reveal key functions of the RIPKs in immune effectors. Application of RIPKs-targeted therapy hinges on maximizing the anti-tumor effects without compromising key immune functions, which we argue should be the focus of the research community in the near future (see Outstanding Questions).

Highlights.

- Necroptosis is an inflammatory cell death mode that regulates tumor growth through cell death-dependent and independent mechanisms.

- Tumors employ multiple mechanisms such as epigenetic silencing of the RIPK3 gene and phosphorylation or ubiquitination of RIPK1 and/or RIPK3 to suppress tumor cell necroptosis and to reduce survival of cancer patients.

- RIPK1 and RIPK3 promote inflammation in the tumor microenvironment and anti-tumor immunity through cell death-induced DAMP release and cell death-independent inflammatory cytokine transcription.

- Although RIPKs often inhibits tumor growth, there are examples in which they aid in tumor progression and metastasis through promoting tumor cell survival and metastasis.

Glossary

CD95L (Fas ligand)

a death ligand in the TNF superfamily that binds to Fas (aka CD95 or APO-1) to induce apoptosis. Under conditions of caspase 8 inhibition, CD95L and CD95 can also induce necroptosis

Complex I

a short-lived membrane-bound signal complex which forms in response to TNF stimulation of TNF receptor 1 (TNFR1) whose major function is to facilitate NF-κB and MAP kinase activation

Damage associated molecular patterns (DAMPs)

intracellular components that are released during lytic cell death to serve as adjuvants to stimulate the innate inflammation

Death receptors

transmembrane receptors of the TNF superfamily that contain a death domain in the cytoplasmic tails. These include Fas/CD95, TRAIL receptors, TNF receptor 1, Death receptor 3 (DR3), DR6, EDAR and the p75 NGF receptor. However, cell death signaling is not a major function for DR3, DR6, EDAR and p75 NGF receptor

DNA-methyltransferase 1 (DNMT1)

an enzyme that catalyzes the transfer of methyl groups onto specific CpG motifs in DNA

Hypoxia

cell stress induced by low tissue oxygen content

Inhibitor of apoptosis (IAP) proteins

E3 ligases that ubiquitinate many TNF receptor signal adaptors to promote cell survival and NF-κB activation

Myeloid-derived Suppressor Cells (MDSCs)

a heterogenous population of myeloid cells which expand in response to cancer, inflammation, and infection and act to suppress T cell responses

Necrosome

a cytosolic signaling complex that promotes necroptosis. RIPK1 and RIPK3 are the major components of this complex, although other adaptors such as FADD, Caspase 8 and MLKL may also be present

SMAC mimetic (aka IAP antagonist)

small molecule mimetics of the mitochondrial protein SMAC (aka DIABLO). They bind to cellular IAP proteins to trigger their proteasomal degradation and are often used to sensitize cells to RIPK1-regulated cell death

TAM kinases

consists of the receptor tyrosine kinases TYRO3, AXL, and MERTK. In the context of cancers, these kinases have largely been shown to promote tumor survival, chemoresistance, and motility

TIR-domain-containing adapter-inducing interferon-β (TRIF)

a RHIM-containing signaling adaptor downstream of Toll-like receptor 3 (TLR3) and TLR4 that can induce RIPK3-dependent necroptosis

TNF-related apoptosis-inducing ligand (TRAIL)

a death ligand in the TNF superfamily that can bind to the death receptors TRAIL-R1 and TRAIL-R2 to stimulate apoptosis. Under conditions of caspase 8 inhibition, TRAIL can also induce necroptosis

Ubiquitin-like, containing PHD and RING finger domains, 1 (UHRF1)

an E3 ubiquitin ligase that binds to hemi-methylated DNA and regulates gene expression through enhancing recruitment of the DNA methyltransferase DNMT1

Xenograft

tissue or organ derived from a donor of a species that differs from the recipient

Z-DNA Binding Protein 1 (ZBP1)

an interferon induced protein and Z-form nucleic acid sensor that interacts with RIPK3 via the “RIP homotypic interaction motif” (RHIM) to induce necroptosis in response to viral infections. ZBP1 activity is inhibited by RIPK1 in certain situations