The dissection of signaling networks that regulate cell growth and survival has led to the discovery of a number of oncogenic pathways that are hyperactive in cancer cells. Gain-of-function aberrations in oncogenes that drive these pathways and loss-of-function alterations to tumor suppressor genes that normally restrain their activity all contribute to cancer progression and ultimately define the molecular distinction between neoplastic versus normal cellular growth. Importantly, in addition to their pro-proliferative and pro-survival effects, many dysregulated oncogenic signaling pathways have also been found to promote tumor immune evasion (Fig. 2). Based on these findings, there is an emerging appreciation that therapeutically targeting oncogenic signaling pathways has the potential not only to interfere directly with cancer cell growth but also to support the efficacy of immune-based treatments for cancer.
Tumor-intrinsic Activation of the RAS/MAPK Pathway and Immune Resistance
Comprehensive genomic analyses of all cancer types catalogued in The Cancer Genome Atlas have revealed the RAS/MAPK pathway as the most frequently altered cell signaling pathway in cancer cells [115]. Gain-of-function mutations as well as copy number and epigenetic alterations in genes encoding receptor tyrosine kinases, RAS GTPases, and MAP kinases such as BRAF and MEK can all promote RAS/MAPK pathway activity, leading to uncontrolled cell division, enhanced cell survival, and cellular dedifferentiation [116]. Additionally, activation of this pathway has also been shown to mediate immune evasion by a number of mechanisms, including those associated with T lymphocyte recruitment to the TME, suppression of T lymphocyte effector function within the TME, and cancer cell susceptibility to CTL-mediated killing.
In terms of T lymphocyte recruitment, genomic/transcriptomic activation of RAS/MAPK signaling has been correlated with reduced numbers of tumor-infiltrating lymphocytes (TIL) in murine models as well as cancer patients [117–119]. These effects have been reported in the context of both endogenous anti-tumor responses as well as in adoptive transfer settings. One study found that MAPK pathway activation in BRAF-mutant melanomas reduced T cell infiltration of tumors by activating VEGF secretion [117], likely contributing to vascular abnormalities that preclude efficient T cell influx into tumors. Another study using KRAS-driven models of lung cancer found that this pathway limits cancer cell secretion of the T cell chemoattractant CXCL10 [119].
Multiple mechanisms by which RAS/MAPK pathway activation suppresses the activity of T lymphocytes that do infiltrate tumor tissue have also been described. Several studies have reported that this pathway promotes expression of the PD-L1 checkpoint ligand on cancer cells [118, 120, 121]. In tumor models engineered to enable inducible expression of constitutively active KRASG12V expression, oncogene activation triggers mitochondrial dysfunction and an increase in reactive oxygen species, which in turn promote PD-L1 expression by enhancing EGFR and FGFR1 growth factor receptor activity [120]. Work in a KRASG12D mutant tumor model has also shown that constitutive KRAS activation drives eIF4-mediated translation of ARF6 and AMAP1, which cooperate to recycle intracellular PD-L1 for re-expression on the cell surface [121]. Another cell surface inhibitory molecule whose expression has been linked with activating NRAS or BRAF mutations is the ectoenzyme CD73, which catabolizes extracellular ATP into the immunosuppressive oncometabolite adenosine [122]. In addition to these cell surface immunoregulators, tumor cell secretion of immunosuppressive molecules, including IL-6 and IL-10, is enhanced by MAPK pathway signaling as well, further contributing to the immunologically hostile nature of TMEs in which this pathway is hyperactive [123].
Finally, pathways associated with the recognition and response of anti-tumor T lymphocytes are also dysregulated by oncogenic RAS/MAPK signaling in cancer cells. Oncogenic signaling through BRAFV600E has been shown to limit tumor cell expression of class I and class II MHC molecules via multiple mechanisms. In the case of class I MHC, BRAFV600E signaling triggers rapid internalization and intracellular sequestration of class I MHC molecules within endolysosomal compartments [124]. At the same time, signaling mediated by this oncoprotein also blocks transcription of genes encoding class I and class II MHC [125, 126]. This latter mechanism is at least in part attributable to the fact that RAS/MAPK pathway activity also limits tumor cell responsiveness to both type I and type II IFN, blocking expression of several IFN pathway target genes with important anti-tumor functions [118, 125, 127–129].
Although small molecule inhibitors targeting the RAS/MAPK pathway have shown efficacy against a range of cancer types, tumor-acquired resistance and disease relapse occur in a majority of patients [116]. Importantly, following discontinuation of targeted therapy, these patients also respond poorly to subsequent immunotherapy [130–133]. The development of such cross-resistance results from several factors associated with tumor evolution in response to prior MAPK pathway inhibition. In vitro studies with human melanoma cell lines harboring BRAFV600 mutations have demonstrated that prolonged treatment with BRAF inhibitors leads to loss of both shared melanocyte differentiation antigens as well as a unique neoantigen, compromising tumor recognition by autologous TIL and antigen-specific CTL clones [134]. Recent work in preclinical murine models has also shown that acquired resistance to BRAF inhibition, mediated by constitutive reactivation of MEK, triggers a change in the chromatin landscape and transcriptional output of tumor cells that confers resistance to both ICB and ACT therapies. This unique genetic program, not present in targeted therapy-naïve tumors with hyperactive MAPK signaling, involves a downregulation of type I IFN pathway genes and upregulation of immune evasion genes and could be reversed by subsequent inhibition of MEK, which in turn synergized with concomitant immunotherapy to promote durable tumor control [129]. An alternate mechanism for targeted therapy-induced cross-resistance to immunotherapy has also recently been reported by Terp et al., who found that sustained activation of p38 MAPK signaling compensated for MEK inhibition, leading to upregulation of CD73 in several murine tumor models. Targeting CD73 in conjunction with MEK also improved tumor control in this setting [135]. These findings likely explain the synergistic anti-tumor effects that have previously been observed when BRAF/MEK inhibition is combined with targeted inhibition of the A2A adenosine receptor in a murine melanoma model [136]. Finally, others have shown that prolonged exposure and resistance to BRAF or MEK1/2 inhibitors are associated with tumor-extrinsic shifts in infiltrating immune cell populations, marked by a notable accumulation of MDSC in the TME [137, 138]. This shift in the TME after prolonged exposure to targeted therapy coincides with a lack of response to anti-PD-1 treatment, which is otherwise efficacious when administered shortly after targeted therapy, at a time when tumor-infiltrating MDSC are reduced by MAPK pathway inhibition [138].
Based on the collective ways in which hyperactive RAS/MAPK signaling in cancer cells confers immune resistance, there is indeed strong rationale for coupling pathway-targeting interventions with immune-based treatment regimens. In considering these types of coupled approaches, insights into the immune cross-resistance that accompanies resistance to MAPK pathway inhibitors underscores the significance of therapeutic scheduling in the design of such strategies, arguing for combinatorial, rather than sequential, treatment regimens. Indeed, several preclinical studies have shown that MAPK pathway inhibitors support a range of immunotherapies, including ICB, ACT, and CAR-T regimens, when administered in conjunction with the particular immune-based therapy under investigation [118, 126, 129, 139, 140]. Early results from some of the clinical trials investigating combination front-line therapy with MAPK pathway inhibitors and ICB for BRAFV600 mutant melanoma have shown similar promise, though treatment-related adverse events have led to discontinuation of therapy in some patients [141, 142]. To maximize the safety and efficacy of such combinatorial regimens going forward, it will therefore be important to: 1) define the optimal duration of combination therapy administration and 2) identify prognostic biomarkers that predict those patients most likely to benefit from such regimens. Finally, in light of the suppressive effects that constitutive MAPK pathway signaling has on IFN responsiveness in cancer cells, it is also interesting to consider how oncolytic viruses might be leveraged as an alternative to targeted therapy for use as an immune-supporting agent against RAS/MAPK pathway hyperactive tumors. In this regard, loss of IFN responsiveness in such tumors increases their susceptibility to oncolytic viruses [127], which can ultimately trigger immunogenic cell death to support both natural and therapy-associated anti-tumor immune responses. Collectively, these approaches to target the tumor-intrinsic vulnerabilities manifested by hyperactive RAS/MAPK signaling have the potential to improve the reach of immunotherapy in the large percentage of cancer patients whose tumors are driven by this pathway.
Immunologic Consequences of Tumor-intrinsic WNT Signaling
Another oncogenic pathway that has frequently been linked with tumor immune resistance is the WNT/β-catenin pathway. Hyperactivation of this pathway in cancer cells may be achieved in a variety of ways, including overexpression of autocrine-acting WNT family growth factors, silencing of genes encoding WNT antagonists, gain-of-function mutations in β-catenin, and loss-of-function mutations in the APC tumor suppressor or other components of the β-catenin destruction complex that otherwise limit pathway activity by targeting β-catenin for proteasomal degradation in the absence of WNT signaling [143, 144]. In addition to these and other genetic aberrations that activate mediators of canonical WNT signaling, genetic dysregulation of non-canonical, β-catenin-independent WNT pathways also contribute to tumor progression and immune evasion [145]. Importantly, these effects are mediated not only by tumor-intrinsic consequences of WNT pathway activation in cancer cells themselves but also by extrinsic changes to the TME that are triggered by cancer cells in which WNT pathways are dysregulated.
As with dysregulated RAS/MAPK signaling, tumor-intrinsic WNT signaling can promote immune evasion in several ways. Non-canonical signaling mediated by WNT5A has been shown to reduce CTL recognition of cancer cells by activating PKC- and STAT3-dependent downregulation of tumor-associated antigen gene expression [146]. At the same time, activation of the canonical WNT/β-catenin pathway induces expression of immune checkpoint ligands, including PD-L1 and CD47, the latter of which negatively regulates innate immunity by transmitting a so-called “don’t eat me” signal through SIRPα expressed on phagocytes [147–149]. While these negative regulators are easily targeted with checkpoint inhibitors, several studies have shown that WNT signaling also confers resistance to ICB therapy [150–156], highlighting checkpoint-independent immune evasion functions for this pathway as well. Indeed, the majority of work on WNT pathway activity in the context of cancer immune evasion has revealed that WNT signaling promotes tumor immune exclusion [157], a phenomenon that of course precludes responsiveness to checkpoint inhibitors whose efficacy relies on tumor-infiltration by T lymphocytes. This mechanism of WNT-associated immune evasion was first reported by Spranger et al., who found that WNT/β-catenin signaling blocked tumor cell secretion of CCL4 and CXCL1, impeding tumor-infiltration by CD103 + DC needed to prime anti-tumor CD8 + T cell responses [150]. Others have since shown that tumor-intrinsic β-catenin also dampens expression of CXCL9, CXCL10, and CXCL11, directly interfering with recruitment of CXCR3 + T lymphocytes into the TME [158]. Not only does this effect have consequences for endogenous anti-tumor T cells that might be supported by ICB therapy, but it likely also contributes to the exclusion of adoptively transferred T cells that has been observed in ACT therapy-resistant tumors [159].
In addition to driving immune exclusion, WNT signaling can act in both autocrine and paracrine manners to promote immunosuppressive TME dynamics. For example, WNT/β-catenin pathway activity compromises the immunogenicity of DC that do infiltrate the TME. Xenograft studies with human melanoma cell lines expressing a hyperactive β-catenin mutant have shown that the β-catenin/TCF complex directly drives expression of IL-10, which suppresses the immunostimulatory capacity of both tumor-infiltrating and splenic DC [160]. Tumor-derived WNT5A can also promote β-catenin-dependent metabolic reprogramming in DC to support other immunoregulatory, tolerogenic functions. Specifically, this pathway increases lipid uptake and fatty acid oxidation by DC, leading to IDO production and tryptophan metabolism within the TME. Elevated levels of the kynurenine byproduct of tryptophan catabolism in turn supports intratumoral Treg accumulation and activity [161, 162]. Tumor-intrinsic WNT signaling can also activate CXCL5-dependent MDSC recruitment to the TME, driving tumor-acquired resistance to ICB therapy. This phenomenon was recently reported by Theivanthiran et al., who demonstrated that CD8 + T cell activation following PD-1 blockade drives IFNγ- and PD-L1-dependent activation of the NLRP3 inflammasome, leading to HSP70 release and autocrine signaling through TLR4. This axis promoted expression of WTN5A, which in turn activated non-canonical, YAP-dependent CXCL5 expression, MDSC recruitment to the TME, and tumor progression [163].
Several strategies for targeting WNT signaling pathways have been explored in the context of combinatorial regimens with cancer immunotherapy. In an autochthonous melanoma model, WNT ligand antagonism synergized with PD-1 blockade to suppress both primary tumor growth and metastatic progression. In this same study, preliminary data from a Phase I study of the PORCN inhibitor ETC-159 also demonstrated that this WNT antagonist could remodel the tumor immune microenvironment of distinct solid tumors, suggesting that this intervention is likely to support a more favorable response to ICB therapy in patients as well [152]. Likewise, a pharmacologic inhibitor of β-catenin (iCRT14) that interferes with TCF complex formation and transcriptional activity augmented the efficacy of a tumor vaccine in a murine model of colorectal cancer [158]. Another β-catenin inhibitor, RX-5902, was similarly shown to potentiate ICB therapies directed against either CTLA4 or PD-1 in a mouse model of triple negative breast cancer [155]. Biomimetic nanoclusters that target β-catenin for degradation have been used as an alternative approach to enhance PD-1/PD-L1 blockade in experimental models as well [164]. Finally, in addition to these direct methods of WNT pathway inhibition, interventions to disrupt immunosuppressive networks that arise in WNT pathway-hyperactive tumors also have the potential to reshape the TME, creating T cell-inflamed conditions that are more conducive to immunotherapy [162].
Dysregulated PI3K/AKT/mTOR Signaling and Tumor Immune Resistance
First recognized as a pro-survival pathway in cancer cells, the PI3K/AKT/mTOR pathway is now appreciated as a driver of several hallmarks of cancer progression, including immune evasion. This pathway may become hyperactive in cancer cells as a result of excessive hormone and growth factor receptor signaling or by various genetic aberrations in its various pathway components, which have been found to occur at a high frequency across a wide range of cancer types. Indeed, one study of nearly 20,000 solid tumors reported that 30% exhibited loss of the PTEN tumor suppressor gene that negatively regulates this pathway, and another 6% were found to harbor PTEN mutations, including those known to confer loss of function. Activating mutations in the gene encoding the PIK3CA catalytic subunit of PI3K were also reported at a high frequency, as were mutations in AKT1, though these were less common [165]. Altogether, these aberrations mark the PI3K/AKT/mTOR pathway as one of the most significant oncogenic drivers of cancer progression.
Loss of the PTEN tumor suppressor gene was first linked with immune evasion when it was shown to enable PI3K/AKT-mediated induction of PD-L1 on glioma cells, conferring resistance to T cell-mediated lysis [166]. Shortly thereafter, experimental work in models of immune susceptible versus immune resistant tumor cell lines found that AKT activation could also confer resistance to T cell killing [167]. More recently, AKT and its upstream activator, PIK3CA, were shown to reduce tumor immunogenicity by downregulating expression of class I MHC and costimulatory molecules on cancer cells [168]. Much like tumor-intrinsic WNT/β-catenin pathway activation, in addition to these mechanisms of immune escape and suppression, PI3K/AKT hyperactivity in cancer cells also correlates with: 1) T cell exclusion from the TME [169, 170] and 2) an increase in the tumor-infiltrating immunosuppressive myeloid compartment [171].
As a result of these diverse immunologic consequences, genetic aberrations in PI3K/AKT pathway components, particularly PTEN, have emerged as significant correlates of immune therapy resistance, underscoring the relevance of this pathway to tumor immune evasion [151, 167, 169, 171–174]. Specifically, clinical analyses of triple negative breast cancer patients have shown that PTEN alterations correlate negatively with response to PD-1 or PD-L1 checkpoint inhibitors [174], and PTEN loss similarly correlates with poor response of melanoma patients to ICB therapy [173]. The lack of response to PD-1-targeting checkpoint inhibitors (nivolumab or pembrolizumab) observed in this latter study is likely explained by reduced CD8 + T cell infiltration into tumors exhibiting PTEN loss. Of note, although CD8 + TIL could still be recovered at low levels from PTEN-deficient tumors, TIL expansion from these tumors was also compromised, a finding that has important implications for ACT-based therapies as well. Biallelic PTEN loss has also been observed in longitudinal analyses of tumor biopsy specimens from patients whose cancers developed acquired resistance to either anti-PD-1 monotherapy [172] or anti-CTLA-4/anti-PD-1 combination therapy [151], further highlighting the role of this pathway in tumor immune escape.
Several strategies for manipulating PI3K/AKT pathway activity have been investigated for their potential to enhance the outcome of cancer immunotherapy. In murine models of Pten-mutant or Pten-null tumors, immune resistance can be overcome by administering PTEN-encoding mRNA-encased polymeric nanoparticles to restore PTEN activity in tumors [175]. In addition to promoting immunogenic tumor cell death, restoration of PTEN function in this way enhanced tumor infiltration by both TH1 helper T cells and CD8 + T cells while decreasing Treg and MDSC infiltration. Importantly, this maneuver also synergized with PD-1 blockade to enhance tumor immune control. Alternatively, the availability of small molecule inhibitors targeting PI3K/AKT pathway components has also spearheaded efforts to pharmacologically disrupt this pathway in cancer cells. While several pathway inhibitors have been developed, the role of PI3K/AKT signaling in the activation and function of T lymphocytes and other immune cell populations is a complicating factor that must be considered when implementing such strategies as anti-cancer regimens. One way to limit deleterious effects of PI3K/AKT pathway inhibition in T lymphocytes while still achieving anti-tumor activity is to administer drugs targeting this pathway on an intermittent, rather than a daily, dosing schedule [176]. In this regard, ongoing use of the PI3Kα/β/δ inhibitor BAY1082439 was recently found to reduce the number of both CD4 + and CD8 + TIL in Pten-null prostate tumors, whereas intermittent drug cycling had no effect on the number of intratumoral CD4 + T cells and actually increased the number of CD8 + TIL. This drug also exhibited tumor cell-intrinsic effects associated with improved immunogenicity, including activation of type I and type II IFN pathway genes that promote antigen presentation (B2M) and T cell recruitment (CCL5 and CXCL10), and its intermittent use drove intratumoral expansion of CD8 + T cells as well. By creating a T cell-inflamed TME, this intervention also overcame primary resistance to PD-1 blockade [176]. Another attractive approach to avoid potential negative immunologic consequences of PI3K pathway inhibition is to selectively target specific PI3K isoforms that drive cancer progression, rather than administering pan-PI3K inhibitors. A dual-targeting PI3Kα/δ inhibitor targeting isoforms known to activate tumor-intrinsic signaling (PI3Kα) as well as tumor-extrinsic signaling in Tregs (PI3Kδ) has been shown to enhance the anti-tumor activity of CD8 + T cells [177]. Interestingly, a selective PI3Kδ inhibitor has also been shown to restrain CD8 + T cell differentiation ex vivo, improving engraftment and anti-tumor efficacy of adoptively transferred T cells [178]. These data suggest that inhibitors targeting this particular PI3K isoform might also prevent terminal differentiation and exhaustion of endogenous CD8 + T cells, further contributing to the anti-tumor and pro-immune effects of such regimens. Specific targeting of PI3K isoforms has indeed led to synergism with various forms of ICB therapy in murine models [173, 179], and promising results have been reported from an early phase clinical trial testing a PI3Kδ inhibitor in combination with pembrolizumab [180]. Finally, targeting downstream mediators of the PI3K pathway such as AKT and mTOR has also been shown to enhance the efficacy of immunotherapy in murine systems [167, 181, 182], highlighting the potential for these combinatorial strategies to improve therapeutic outcomes in clinical settings as well.
Cell Cycle Pathway Dysregulation and Tumor-intrinsic Immune Resistance
While loss of cell cycle regulation has long been recognized as a core tenet of the uncontrolled proliferation that is a hallmark of cancer progression, data has emerged in recent years demonstrating that cell cycle regulators can also confer tumor immune resistance. In particular, aberrations in several genes encoding cyclin and cyclin-dependent kinase family members have been linked with tumor resistance to immunotherapy. In lung adenocarcinoma, elevated expression of CDC25C is associated with reduced CD8 + T cell infiltration, increased Treg infiltration, and poor response to PD-1 checkpoint blockade [183]. Likewise, CDK4 gene amplification, CCND1 gene amplification, and copy number loss of the CDKN2A gene encoding a cyclin-dependent kinase inhibitor all correlate with primary resistance to PD-1 inhibition in patients with various melanoma subtypes [184]. Similar observations have been made for other solid tumor types harboring CDKN2A deletions or loss of function mutations as well [185, 186]. In this regard, it is worth noting that a recent gene set enrichment analysis of urothelial carcinomas harboring loss-of-function alterations in CDKN2A revealed that expression signatures for antigen processing, IFNγ signaling, and T cell receptor signaling pathways were all reduced in CDKN2A-altered tumors as compared with CDKN2A wild type tumors [185], effects that may at least be partially explained by concomitant loss of the chromosomally proximal JAK2 gene in cases of genomic deletions [187]. At the same time, however, experimental gene rescue of CDKN2A-null melanoma cells enhances tumor cell secretion of T cell-attracting chemokines [188], suggesting that direct influences of CDKN2A on the tumor immune microenvironment are also likely. Moreover, IFNγ pathway response genes are downregulated in tumors with CDK4 copy gain, an anomaly that is completely independent of a situation in which JAK deletion might potentially be coupled to loss of a nearby cell cycle-regulating gene [184]. Together with studies demonstrating that CDK inhibitors can: 1) convert immunologically “cold” tumors to T cell-inflamed tumors [189, 190] and 2) enhance CD8 + T cell effector function [191], these data provide convincing evidence that cell cycle regulators play active roles in immune evasion.
In light of these observations, several studies have investigated therapeutic strategies for targeting cell cycle regulators in the context of cancer immunotherapy. Importantly, the pleiotropic effects of CDK4/6 inhibition have been found to support anti-tumor immunity in a number of ways. First, by activating tumor-intrinsic expression of endogenous retroviral elements, CDK4/6 inhibitors promote dsRNA sensing, which in turn stimulates type III IFN production and tumor antigen presentation [189]. Second, by inducing metabolic stress in cancer cells, CDK4/6 inhibitors trigger expression of chemokines such as CCL5 and CXCL9/10/11 that recruit T lymphocytes to the TME [192]. These inhibitors also suppress Treg proliferation, an effect that correlates with reduced circulating and intratumoral Tregs in inhibitor-treated animals [189]. While CDK4/6 inhibitors also impact CD8 + T cell proliferation, this tumor-extrinsic action is offset by improved T cell activation, memory formation, and infiltration into tumor-bearing lung tissue [190, 192, 193]. Importantly, in these and related studies, the immune-potentiating effects of CDK4/6 inhibition have been shown to augment anti-tumor responses achieved by ACT/CAR-T therapy, OX-40/4-1BB agonist therapy, and ICB therapy [184, 189, 190, 192–195]. Similar results have also recently been reported in studies of ICB therapy in combination with CDK7 inhibitors [191, 196], a CDK12/13 inhibitor [197], and a broad-spectrum CDK1/2/5/9/12 inhibitor [198]. Collectively, these studies highlight the potential of targeting cell cycle regulators in the context of immunotherapy, providing strong rationale for the pursuit of such combination regimens in clinical settings.