Concurrent inhibition of oncogenic and wild-type RAS-GTP for cancer therapy.

RAS oncogenes (collectively NRAS, HRAS and especially KRAS) are among the most frequently mutated genes in cancer, with common driver mutations occurring at codons 12, 13 and 611. Small molecule inhibitors of the KRAS(G12C) oncoprotein have demonstrated clinical efficacy in patients with multiple cancer types and have led to regulatory approvals for the treatment of non-small cell lung cancer2,3. Nevertheless, KRASG12C mutations account for only around 15% of KRAS-mutated cancers4,5, and there are no approved KRAS inhibitors for the majority of patients with tumours containing other common KRAS mutations. Here we describe RMC-7977, a reversible, tri-complex RAS inhibitor with broad-spectrum activity for the active state of both mutant and wild-type KRAS, NRAS and HRAS variants (a RAS(ON) multi-selective inhibitor). Preclinically, RMC-7977 demonstrated potent activity against RAS-addicted tumours carrying various RAS genotypes, particularly against cancer models with KRAS codon 12 mutations (KRASG12X). Treatment with RMC-7977 led to tumour regression and was well tolerated in diverse RAS-addicted preclinical cancer models. Additionally, RMC-7977 inhibited the growth of KRASG12C cancer models that are resistant to KRAS(G12C) inhibitors owing to restoration of RAS pathway signalling. Thus, RAS(ON) multi-selective inhibitors can target multiple oncogenic and wild-type RAS isoforms and have the potential to treat a wide range of RAS-addicted cancers with high unmet clinical need. A related RAS(ON) multi-selective inhibitor, RMC-6236, is currently under clinical evaluation in patients with KRAS-mutant solid tumours (ClinicalTrials.gov identifier: NCT05379985).

Conditional Deletion of Pdcd1 Identifies the Cell-Intrinsic Action of PD-1 on Functional CD8 T Cell Subsets for Antitumor Efficacy.

Programmed cell death-1 (PD-1) blockade has a profound effect on the ability of the immune system to eliminate tumors, but many questions remain about the cell types involved and the underlying mechanisms of immune activation. To shed some light on this, the cellular and molecular events following inhibition of PD-1 signaling was investigated in the MC-38 colon carcinoma model using constitutive (PD-1 KO) and conditional (PD1cKO) mice and in wild-type mice treated with PD-1 antibody. The impact on both tumor growth and the development of tumor immunity was assessed. In the PD-1cKO mice, a complete deletion of Pdcd1 in tumor-infiltrating T cells (TILs) after tamoxifen treatment led to the inhibition of tumor growth of both small and large tumors. Extensive immune phenotypic analysis of the TILs by flow and mass cytometry identified 20-different T cell subsets of which specifically 5-CD8 positive ones expanded in all three models after PD-1 blockade. All five subsets expressed granzyme B and interferon gamma (IFNγ). Gene expression analysis of the tumor further supported the phenotypic analysis in both PD-1cKO- and PD-1 Ab-treated mice and showed an upregulation of pathways related to CD4 and CD8 T-cell activation, enhanced signaling through costimulatory molecules and IFNγ, and non-T-cell processes. Altogether, using PD-1cKO mice, we define the intrinsic nature of PD-1 suppression of CD8 T-cell responses in tumor immunity.

Allosteric Inhibition of SHP2 Stimulates Antitumor Immunity by Transforming the Immunosuppressive Environment.

The protein tyrosine phosphatase SHP2 binds to phosphorylated signaling motifs on regulatory immunoreceptors including PD-1, but its functional role in tumor immunity is unclear. Using preclinical models, we show that RMC-4550, an allosteric inhibitor of SHP2, induces antitumor immunity, with effects equivalent to or greater than those resulting from checkpoint blockade. In the tumor microenvironment, inhibition of SHP2 modulated T-cell infiltrates similar to checkpoint blockade. In addition, RMC-4550 drove direct, selective depletion of protumorigenic M2 macrophages via attenuation of CSF1 receptor signaling and increased M1 macrophages via a mechanism independent of CD8+ T cells or IFNγ. These dramatic shifts in polarized macrophage populations in favor of antitumor immunity were not seen with checkpoint blockade. Consistent with a pleiotropic mechanism of action, RMC-4550 in combination with either checkpoint or CSF1R blockade caused additive antitumor activity with complete tumor regressions in some mice; tumors intrinsically sensitive to SHP2 inhibition or checkpoint blockade were particularly susceptible. Our preclinical findings demonstrate that SHP2 thus plays a multifaceted role in inducing immune suppression in the tumor microenvironment, through both targeted inhibition of RAS pathway-dependent tumor growth and liberation of antitumor immune responses. Furthermore, these data suggest that inhibition of SHP2 is a promising investigational therapeutic approach. SIGNIFICANCE: Inhibition of SHP2 causes direct and selective depletion of protumorigenic M2 macrophages and promotes antitumor immunity, highlighting an investigational therapeutic approach for some RAS pathway-driven cancers.

Differential Role of Hematopoietic and Nonhematopoietic Cell Types in the Regulation of NK Cell Tolerance and Responsiveness.

Many NK cells express inhibitory receptors that bind self-MHC class I (MHC I) molecules and prevent killing of self-cells, while enabling killing of MHC I-deficient cells. But tolerance also occurs for NK cells that lack inhibitory receptors for self-MHC I, and for all NK cells in MHC I-deficient animals. In both cases, NK cells are unresponsive to MHC I-deficient cells and hyporesponsive when stimulated through activating receptors, suggesting that hyporesponsiveness is responsible for self-tolerance. We generated irradiation chimeras, or carried out adoptive transfers, with wild-type (WT) and/or MHC I-deficient hematopoietic cells in WT or MHC I-deficient C57BL/6 host mice. Unexpectedly, in WT hosts, donor MHC I-deficient hematopoietic cells failed to induce hyporesponsiveness to activating receptor stimulation, but did induce tolerance to MHC I-deficient grafts. Therefore, these two properties of NK cells are separable. Both tolerance and hyporesponsiveness occurred when the host was MHC I deficient. Interestingly, infections of mice or exposure to inflammatory cytokines reversed the tolerance of NK cells that was induced by MHC I-deficient hematopoietic cells, but not the tolerance induced by MHC I-deficient nonhematopoietic cells. These data have implications for successful bone marrow transplantation, and suggest that tolerance induced by hematopoietic cells versus nonhematopoietic cells may be imposed by distinct mechanisms.

NK cell self tolerance, responsiveness and missing self recognition.

Natural killer (NK) cells represent a first line of defense against pathogens and tumor cells. The activation of NK cells is regulated by the integration of signals deriving from activating and inhibitory receptors expressed on their surface. However, different NK cells respond differently to the same stimulus, be it target cells or agents that crosslink activating receptors. The processes that determine the level of NK cell responsiveness have been referred to collectively as NK cell education. NK cell education plays an important role in steady state conditions, where potentially auto-reactive NK cells are rendered tolerant to the surrounding environment. According to the "tuning" concept, the responsiveness of each NK cell is quantitatively adjusted to ensure self tolerance while at the same time ensuring useful reactivity against potential threats. MHC-specific inhibitory receptors displayed by NK cells play a major role in tuning NK cell responsiveness, but recent studies indicate that signaling from activating receptors is also important, suggesting that the critical determinant is an integrated signal from both types of receptors. An important and still unresolved question is whether NK cell education involves interactions with a specific cell population in the environment. Whether hematopoietic and/or non-hematopoietic cells play a role is still under debate. Recent results demonstrated that NK cell tuning exhibits plasticity in steady state conditions, meaning that it can be re-set if the MHC environment changes. Other evidence suggests, however, that inflammatory conditions accompanying infections may favor high responsiveness, indicating that inflammatory agents can over-ride the natural tendency of NK cells to adjust to the steady state environment. These findings raise many questions such as whether viruses and tumor cells manipulate NK cell responsiveness to evade immune-recognition. As knowledge of the underlying processes grows, the possibility of modulating NK cell responsiveness for therapeutic purposes is becoming increasingly attractive, and is now under serious investigation in clinical studies.

Recognition of tumors by the innate immune system and natural killer cells.

In recent years, roles of the immune system in immune surveillance of cancer have been explored using a variety of approaches. The roles of the adaptive immune system have been a major emphasis, but increasing evidence supports a role for innate immune effector cells such as natural killer (NK) cells in tumor surveillance. Here, we discuss some of the evidence for roles in tumor surveillance of innate immune cells. In particular, we focus on NK cells and other immune cells that express germline-encoded receptors, often labeled NK receptors. The impact of these receptors and the cells that express them on tumor suppression is summarized. We discuss in detail some of the pathways and events in tumor cells that induce or upregulate cell-surface expression of the ligands for these receptors, and the logic of how those pathways serve to identify malignant, or potentially malignant cells. How tumors often evade tumor suppression mediated by innate killer cells is another major subject of the review. We end with a discussion on some of the implications of the various findings with respect to possible therapeutic approaches.

Cutting edge: ABIN-1 protects against psoriasis by restricting MyD88 signals in dendritic cells.

Psoriasis is a chronic, inflammatory skin disease caused by a combination of environmental and genetic factors. The Tnip1 gene encodes A20 binding and inhibitor of NF-κB-1 (ABIN-1) protein and is strongly associated with susceptibility to psoriasis in humans. ABIN-1, a widely expressed ubiquitin-binding protein, restricts TNF- and TLR-induced signals. In this study, we report that mice lacking ABIN-1 specifically in dendritic cells (DCs), ABIN-1(fl) CD11c-Cre mice, exhibit perturbed immune homeostasis. ABIN-1-deficient DCs display exaggerated NF-κB and MAPK signaling and produce more IL-23 than do normal cells in response to TLR ligands. Challenge of ABIN-1(fl) CD11c-Cre mice with topical TLR7 ligand leads to greater numbers of Th17 and TCRγδ T cells and exacerbated development of psoriaform lesions. These phenotypes are reversed by DC-specific deletion of the TLR adaptor MyD88. These studies link ABIN-1 with IL-23 and IL-17, and they provide cellular and molecular mechanisms by which ABIN-1 regulates susceptibility to psoriasis.

Dimerization and ubiquitin mediated recruitment of A20, a complex deubiquitinating enzyme.

A20 is an anti-inflammatory protein linked to multiple human autoimmune diseases and lymphomas. A20 possesses a deubiquitinating motif and a zinc finger, ZF4, that binds ubiquitin and supports its E3 ubiquitin ligase activity. To understand how these activities mediate A20's physiological functions, we generated two lines of gene-targeted mice, abrogating either A20's deubiquitinating activity (Tnfaip3(OTU) mice) or A20's ZF4 (Tnfaip3(ZF4) mice). Both Tnfaip3(OTU) and Tnfaip3(ZF4) mice exhibited increased responses to TNF and sensitivity to colitis. A20's C103 deubiquitinating motif restricted both K48- and K63-linked ubiquitination of receptor interacting protein 1 (RIP1). A20's ZF4 was required for recruiting A20 to ubiquitinated RIP1. A20(OTU) proteins and A20(ZF4) proteins complemented each other to regulate RIP1 ubiquitination and NFκB signaling normally in compound mutant Tnfaip3(OTU/ZF4) cells. This complementation involved homodimerization of A20 proteins, and we have defined an extensive dimerization interface in A20. These studies reveal how A20 proteins collaborate to restrict TNF signaling.

Mature natural killer cells reset their responsiveness when exposed to an altered MHC environment.

Some mature natural killer (NK) cells cannot be inhibited by major histocompatibility complex (MHC) I molecules, either because they lack corresponding inhibitory receptors or because the host lacks the corresponding MHC I ligands for the receptors. Such NK cells nevertheless remain self-tolerant and exhibit a generalized hyporesponsiveness to stimulation through activating receptors. To address whether NK cell responsiveness is set only during the NK cell differentiation process, we transferred mature NK cells from wild-type (WT) to MHC I-deficient hosts or vice versa. Remarkably, mature responsive NK cells from WT mice became hyporesponsive after transfer to MHC I-deficient mice, whereas mature hyporesponsive NK cells from MHC I-deficient mice became responsive after transfer to WT mice. Altered responsiveness was evident among mature NK cells that had not divided in the recipient animals, indicating that the cells were mature before transfer and that alterations in activity did not require cell division. Furthermore, the percentages of NK cells expressing KLRG1, CD11b, CD27, and Ly49 receptors specific for H-2(b) were not markedly altered after transfer. Thus, the functional activity of mature NK cells can be reset when the cells are exposed to a changed MHC environment. These findings have important implications for how NK cell functions may be curtailed or enhanced in the context of disease.

ABIN-1 is a ubiquitin sensor that restricts cell death and sustains embryonic development.

Proteins that directly regulate tumour necrosis factor receptor (TNFR) signalling have critical roles in regulating cellular activation and survival. ABIN-1 (A20 binding and inhibitor of NF-kappaB) is a novel protein that is thought to inhibit NF-kappaB signalling. Here we show that mice deficient for ABIN-1 die during embryogenesis with fetal liver apoptosis, anaemia and hypoplasia. ABIN-1 deficient cells are hypersensitive to tumour necrosis factor (TNF)-induced programmed cell death, and TNF deficiency rescues ABIN-1 deficient embryos. ABIN-1 inhibits caspase 8 recruitment to FADD (Fas-associated death domain-containing protein) in TNF-induced signalling complexes, preventing caspase 8 cleavage and programmed cell death. Moreover, ABIN-1 directly binds polyubiquitin chains and this ubiquitin sensing activity is required for ABIN-1's anti-apoptotic activity. These studies provide insights into how ubiquitination and ubiquitin sensing proteins regulate cellular and organismal survival.

Homeostatic MyD88-dependent signals cause lethal inflamMation in the absence of A20.

Toll-like receptors (TLRs) on host cells are chronically engaged by microbial ligands during homeostatic conditions. These signals do not cause inflammatory immune responses in unperturbed mice, even though they drive innate and adaptive immune responses when combating microbial infections. A20 is a ubiquitin-modifying enzyme that restricts exogenous TLR-induced signals. We show that MyD88-dependent TLR signals drive the spontaneous T cell and myeloid cell activation, cachexia, and premature lethality seen in A20-deficient mice. We have used broad spectrum antibiotics to demonstrate that these constitutive TLR signals are driven by commensal intestinal flora. A20 restricts TLR signals by restricting ubiquitylation of the E3 ligase tumor necrosis factor receptor-associated factor 6. These results reveal both the severe proinflammatory pathophysiology that can arise from homeostatic TLR signals as well as the critical role of A20 in restricting these signals in vivo. In addition, A20 restricts MyD88-independent TLR signals by inhibiting Toll/interleukin 1 receptor domain-containing adaptor inducing interferon (IFN) beta-dependent nuclear factor kappaB signals but not IFN response factor 3 signaling. These findings provide novel insights into how physiological TLR signals are regulated.

Multiple polymorphisms in the TNFAIP3 region are independently associated with systemic lupus erythematosus.

The TNFAIP3 (tumor necrosis factor alpha-induced protein 3) gene encodes a ubiquitin editing enzyme, A20, that restricts NF-kappaB-dependent signaling and prevents inflammation. We show that three independent SNPs in the TNFAIP3 region (rs13192841, rs2230926 and rs6922466) are associated with systemic lupus erythematosus (SLE) among individuals of European ancestry. These findings provide critical links between A20 and the etiology of SLE.