Targeting cancer with kinase inhibitors

Abstract

Kinase inhibitors have played an increasingly prominent role in the treatment of cancer and other diseases. Currently, more than 25 oncology drugs that target kinases have been approved, and numerous additional therapeutics are in various stages of clinical evaluation. In this Review, we provide an in-depth analysis of activation mechanisms for kinases in cancer, highlight recent successes in drug discovery, and demonstrate the clinical impact of selective kinase inhibitors. We also describe the substantial progress that has been made in designing next-generation inhibitors to circumvent on-target resistance mechanisms, as well as ongoing strategies for combining kinase inhibitors in the clinic. Last, there are numerous prospects for the discovery of novel kinase targets, and we explore cancer immunotherapy as a new and promising research area for studying kinase biology.

Introduction

Protein phosphorylation is the most common form of reversible post-translational modification (1), with an estimated 50% of all proteins undergoing phosphorylation (2). The phosphorylation state of any given protein is controlled by the coordinated action of specific kinases and phosphatases that add and remove phosphate, respectively. There are at least 518 kinases (3) and 156 phosphatases (4) in the human genome, and despite intensive research in this area, the underlying biology of many of these enzymes remains to be fully elucidated. Notwithstanding, signaling networks that employ phosphorylation to modulate target activities have been shown to be critically involved in all aspects of cellular function, and in cancer, the abnormal activation of protein phosphorylation is frequently either a driver or direct consequence of the disease (5). For instance, kinase signaling pathways have been shown to drive many of the hallmark phenotypes of tumor biology (6), including proliferation, survival, motility, metabolism, angiogenesis, and evasion of antitumor immune responses (Figure 1).

Figure 1. Regulation of tumorigenic programming by activation of kinases.

Survival: Kinase pathways regulate programmed cell death by either directly modulating apoptosis regulators or altering their expression. Necroptosis is controlled by signaling pathways that intersect apoptosis regulators, including RIPK3. Motility: Cytoskeletal dynamics are mediated by RTKs and integrin clustering to promote the activation of FAK and GTPases. Effector kinases include ROCK1, MLCK (encoded by MYLK3), PAK1, and LIMK1. Evading antitumor immunity: Numerous cell types, including CTLs, Tregs, DCs, MDSCs, and macrophages, are actively involved in modulating the tumor immune response. MAP4K1, PI3K∂, MST1R, MERTK, and PKCη, represent potential targets for reversing the myriad mechanisms involved in tumor-mediated immunosuppression of the tumor microenvironment. Proliferation: MAPK pathway components increase levels of CCND1. CCND1 regulates the activity of CDK4/6 and, in turn, induces cell cycle progression via RB1 and E2F transcriptional regulators. DNA damage checkpoints: Following induction of double-strand DNA breaks by ionizing radiation or chemotherapeutic agents, ATM and ATR phosphorylate CHK1. Activated CHK1 subsequently prevents cell cycle progression and induces DNA repair mechanisms. Angiogenesis: Tumor cells modulate the microenvironment by secreting angiogenic factors (ligands for VEGFR, FGFRs, and the angiopoietin receptor TIE2 [encoded by TEK]) to stimulate endothelial cells and enhance vascularization. Pericytes are in turn stimulated by PDGF and TGF growth factors to contribute to endothelial cell proliferation and migration. Metabolism: PI3K/AKT pathway activity is directly correlated with increased glucose metabolism in cancer cells. Signaling is mediated through hexokinases, MTOR, and HIF1α (encoded by HIF1A) to increase oxidative phosphorylation, tricarboxylic acid cycle, and mitochondrial respiration.

Given this opportunity for therapeutic intervention, there has been considerable effort in developing pharmacological tools to inhibit protein phosphorylation and interrogate signaling (7). Currently, there are multiple examples of small molecule kinase inhibitors with both selectivity and suitable pharmaceutical properties that have produced meaningful clinical benefit. For instance, imatinib is utilized to inhibit BCR-ABL1 in chronic myelogenous leukemia (CML) and acute lymphoblastic leukemia with the Philadelphia chromosome (8); crizotinib and other ALK kinase inhibitors for cancers driven by ALK fusions (9); lapatinib for ERBB2/HER2-amplified tumors (10); gefitinib and erlotinib for EGFR mutated tumors (10); and vemurafenib for BRAF mutant tumors (11). In each of these cases, superior clinical benefit was observed for the targeted agent in molecularly selected patients compared with prior standard-of-care regimens. Our evolving ability to genomically characterize tumors heralds a new era in which selective kinase inhibitors can be utilized to inactivate molecular drivers of the malignant state.

Despite these successes (12), the field is still just beginning to develop an understanding of how to fully exploit kinase inhibitors for therapeutic benefit. In this Review, we examine new trends in targeting protein and lipid kinases employing next-generation small molecule inhibitors and focus on four areas: (a) identification of novel, putative driver kinases as a result of advances in deep sequencing technologies and genomic analyses; (b) the use of increasingly potent and selective kinase inhibitors to further our understanding of resistance mechanisms; (c) potential combination strategies to improve antitumor efficacy and disrupt resistance mechanisms; and (d) novel kinase targets in the regulation of the tumor microenvironment and immune responses.

Kinase activation by genomic events

The abnormal oncogenic activation of protein and lipid kinases derives from multiple types of genetic and epigenetic changes (12, 13). These alterations result in increased specific activity of the kinase itself, its overexpression, or the loss of negative regulation. Most frequently, tumor cells harbor somatic point mutations at structurally conserved residues, or mutation hotspots, which constitutively upregulate kinase activity (14). Examples of these hotspots include M918T in RET (15) and M1268T in MET (16), which occur at a structurally equivalent position within the kinase domain. Another prevalent mutation hotspot conserved across several oncogenic kinases is BRAF V600 (17), which corresponds to KIT D816 (18) and FLT3 D835 (19). Recent efforts from large-scale consortia such as The Cancer Genome Atlas (TCGA) and the International Cancer Genome Consortium (ICGC) have uncovered many new mutations in kinases and enabled a robust delineation of the spectrum of activating kinase mutations in cancer through careful statistical analysis (refs. 20, 21, Figure 2, and Table 1). In addition, genomic instability, a hallmark of cancer cells (6, 22), can also result in elevated kinase activity that enhances signaling through a number of distinct mechanisms. Defects in the surveillance pathways that maintain genomic integrity can produce amplifications of large chromosomal regions or complex chromosomal rearrangements, which in turn result in the mis-expression of a kinase or the expression of a constitutively activated chimeric form (kinase fusions) (Figure 2 and Table 1).

Figure 2. Activating genomic alterations of protein and lipid kinases.

(A) Activating point mutations in genes coding for kinases lead to the expression of a constitutively activated kinase. Such mutations either lead to an amino acid substitution in the catalytic site, rendering it active; or change the general properties of the protein, for instance by disrupting the interaction with negative regulators, by releasing a mechanism of autoinhibition within the kinase itself, or by inducing constitutive dimerization. Last, they can cause changes in the splicing of the mRNA. Point mutations are the most common mechanism of kinase activation. (B) Chromosomal amplification of a region containing a kinase leads to its increased transcription and the production of an increased amount of protein in the cell. Consequently, the downstream pathway becomes overactivated. (C) Chromosomal alterations such as translocations or deletions can localize a kinase gene in proximity to another gene and lead to the expression of a constitutively activated chimeric or truncated kinase, or deregulate the expression of the kinase by putting it under the control of another promoter.

Table 1. Examples of known mechanisms of kinase activation in cancer.

Although the target gene(s) within an amplification that confers a growth advantage can be difficult to identify — as these genomic regions often include multiple genes — studies of large cohorts of tumors have facilitated the identification of several driver kinases from genomically amplified loci (refs. 23, 24, Figure 2, and Table 1). In particular, the receptor tyrosine kinases (RTKs) EGFR, ERBB2/HER2, and MET and the serine/threonine kinases CDK4 and CDK6 are most frequently overexpressed as a consequence of gene amplification.

Kinase fusions represent ideal targets for the development of cancer drugs because they often confer oncogenic dependency in hematopoietic and solid malignancies, as demonstrated by the success of several kinase inhibitors (refs. 25, 26, and Table 1). For example, imatinib induces remission in leukemia patients who are positive for BCR-ABL1 fusions (8), and crizotinib and ceritinib have produced significant clinical benefit in patients with lung adenocarcinomas and mesenchymal tumors harboring ALK fusions (9, 27). In certain solid tumors, such as thyroid carcinomas or glioblastoma, kinase fusions represent an important driver mechanism of the disease (occurring in 12% and 6% of patients, respectively; ref. 26). In other solid tumor types genomically profiled in TCGA, fusions in ALK, BRAF, EGFR, FGFR1–3, NTRK1–3, PDGFRA, RAF1, RET, and ROS1 collectively occur in 1%–3% of patients and constitute strong driver events of the disease. Moreover, oncogenes such as MET and PIK3CA, known to be activated by mutations or amplifications, have been recently shown to harbor fusions as well (26). In some instances, novel kinase fusions have been implicated as the sole drivers of tumorigenesis, as has been recently shown with PRKACA in fibrolamellar hepatocellular carcinoma (28). The mechanisms of kinase activation can be diverse depending on the fusions; however, for most RTKs, the acquisition of a dimerization domain contributed by the fusion partner promotes trans-autophosphorylation. Loss of a kinase autoinhibitory domain and overexpression of the kinase (such as observed in the context of gene amplification) have also been described, especially for non-RTKs (26).

Other mechanisms of kinase activation include overexpression as a consequence of epigenetic changes (29), activation of a kinase transcription factor (30), inactivating mutations of negative regulators (31, 32), modulation of regulatory proteases (33), alternative splicing (34), splice site mutations causing exon skipping (35), and the overexpression of a ligand or an upstream positive regulator by genetic or epigenetic regulation (ref. 36 and Figure 1). Taken together, the multitude of direct and indirect events that result in the activation of kinase pathways in tumors represent important and tractable opportunities for therapeutic intervention.

Overcoming therapeutic resistance

The genomically informed application of selective kinase inhibitors in clinical oncology has shown tremendous promise in improving patient outcomes (37). For example, prior to the development of imatinib, CML was treated with a combination of hydroxyurea and interferon, a regimen that allowed disease control but did not prevent its progression to more advanced stages (38). In contrast, imatinib therapy has produced remarkably durable responses, with an 8-year survival rate of 85% (39). Despite the significant clinical benefit of some agents, patients who initially respond to targeted therapeutics commonly relapse. Efforts to elucidate the molecular mechanisms underlying this process have been greatly aided by the genomic characterization of paired samples of pre-treatment and post-relapse tumors. These studies have revealed that resistance to kinase inhibitors can occur through a variety of processes: (a) “on-target” resistance via mutations, amplifications, or altered splicing of the target kinase (40, 41); (b) “on-pathway” resistance via activation of downstream components or disruption of negative feedback loops; (c) bypass mechanisms that upregulate parallel signaling pathways (42, 43); (d) epigenetic alterations and histological transformations that modify a cell’s phenotypic state (44, 45); and (e) modulation of cellular processes that influence drug transport, distribution, or stability (40, 46, 47).

On-target mutations that reactivate a kinase are a predominant mechanism of resistance to small molecule inhibitors. For example, resistance mutations in the molecular targets of imatinib, BCR-ABL1 and KIT, have been described in both indications for which this agent is approved as therapy, namely CML and gastrointestinal stromal tumors (GISTs) (48–51). Similarly, on-target resistance mutations have been documented in the kinases FLT3 (52) ALK (53, 54), ROS1 (55), EGFR (56, 57), KIT (51), and PDGFRA (58), as well as in kinases that are not directly genetically deregulated, such as BTK (59, 60) and MTOR (61). In terms of molecular mechanisms, BCR-ABL1 mutations conferring resistance to imatinib provide a useful paradigm to understand the structural basis for diminished inhibitor potency by kinase mutations. By reducing the inherent affinity of the compound for BCR-ABL1 either by elimination of critical interactions, such as mutation of the active site gatekeeper residue, or by induction of a conformational change in the kinase, these mutations reduce imatinib binding to BCR-ABL1 and therefore its clinical efficacy (62). For other kinases, such as EGFR, resistance mutations can increase the kinase’s affinity for ATP and thereby lower the relative cellular potency of ATP-competitive inhibitors (63). Mutational reactivation of kinases in relapsed tumors firmly establishes their roles as disease drivers and highlights the prominent role of this mechanism in mediating on-target resistance.

The specific mechanisms of resistance that arise in a tumor are influenced by several factors, including the molecular features of the compound, the kinase target itself, and the underlying genomic characteristics of the tumor. For instance, imatinib-resistant CML patients who have progressed on second-line dasatinib treatment harbor mutations that are distinct from those patients having relapsed following second-line treatment with nilotinib (64). This phenomenon has also been documented in KIT-driven GISTs, where primary and secondary KIT mutations have been observed upon relapse to either first- or second-line treatments, respectively (65, 66). In this regard, in vitro mutagenesis or in vivo resistance screens combined with structural analysis and genomic characterization of resistant cells can delineate the mechanisms that tumors utilize to evade pharmacological interventions. These approaches have tremendous value in designing second-generation therapies that can resensitize tumors to targeted agents. Moreover, the occurrence of secondary or tertiary mutations in a kinase following multiple rounds of targeted therapy suggests that some tumors exhibit a near-obligate dependence on a driver kinase. In support of this notion, in CML clones from patients who have undergone multiple rounds of kinase inhibitor therapies, the number of silent mutations in BCR-ABL1 increased disproportionately compared with the total number of coding substitutions (64). This finding suggests there may be a fundamental limit to the number of drug-resistance mutations that BCR-ABL1 can tolerate while retaining its catalytic activity (64) and supports the development of a repertoire of selective kinase inhibitors — including both ATP-competitive and allosteric inhibitors (67) — to target all substitutions that disrupt drug binding to a given oncogenic kinase.

Combination therapies

Drug combinations within a single kinase pathway.

Preclinical and clinical data demonstrate that therapeutic combinations enhance primary antitumor responses and delay the onset of resistance (68), and combinations of kinase inhibitors are actively being explored for the treatment of cancer. Prioritization of kinase inhibitor combinations has frequently been based on either robust synthetic lethality in model systems (69) or identification of putative resistance mechanisms in tumor tissues following relapse in patients on monotherapy (10). The multipronged approaches to targeting the MAPK pathway in melanoma represent an emerging example of the effective use of combination therapies within a single pathway to circumvent compensatory signaling as well as genetic resistance mechanisms. Activating, oncogenic mutations of BRAF occur in 50% of malignant melanomas, and catalytic inhibition using vemurafenib or dabrafenib has elicited positive clinical responses (70). However, patient benefit is generally short lived (11), and there is a need to understand the molecular events driving patient relapse. To date, the majority of putative resistance mechanisms involve reactivation or hyperactivation of the MAPK pathway itself (on-pathway resistance). The molecular mechanisms include amplification, mutation, or gene fusions of upstream (NRAS), parallel (CRAF; encoded by RAF1), or downstream (MEK1 and MEK2; MAP2K1 and MAP2K2, respectively) pathway components (71–73). As such, combined blockade of BRAF together with its immediate effectors, MEK1 and MEK2, has been examined in advanced clinical trials (74, 75). For example, patients receiving dabrafenib and trametinib combination treatment had improved progression-free survival (PFS) compared with those receiving dabrafenib monotherapy (76). These results are consistent with preclinical studies showing that sustained >80% pathway inhibition and relief of ERK1- and ERK2-dependent (encoded by MAPK3 and MAPK1, respectively) feedback results in enhanced antitumor efficacy (77–79). Furthermore, preclinical and clinical evidence suggests that additional MAPK pathway alterations, including activating mutations in MEK1 and MEK2, confer resistance to combined BRAF and MEK1/2 inhibition (80–82). The emergence of these additional mechanisms of resistance supports the development of novel inhibitors of multiple upstream and downstream kinase components for the next generation of combination therapy treatments.

Drug combinations targeting parallel kinase pathways.

Kinase inhibitor sensitivity can also be influenced by the broader network of signaling events, including crosstalk and feedback to parallel pathways. For instance, several factors that have been implicated in conferring resistance to MAPK pathway therapeutics fall within the PI3K pathway (Figure 3). These events include the mutation of PIK3CA, loss of the PTEN and INPP4B tumor suppressors, increased basal activity of the PI3K effector kinase AKT, induction of AKT activation in response to MAPK inhibition, and signaling via TORC1 (83–86). Given that the MAPK and PI3K signaling pathways also act on key regulators of proliferation and survival in normal cells, intermittent scheduling regimens and the targeting of different nodes within the signaling network are being evaluated to improve tolerability and efficacy in the clinic (87, 88). In addition to direct activation of the PI3K pathway, increased expression of RTKs, such as EGFR, PDGFRB, and IGF1R, has also been proposed as a mediator of resistance to MAPK pathway inhibitors (89–92). In the case of EGFR-mediated resistance to MAPK pathway inhibition, elevated EGFR expression has been proposed to occur via an epigenetic adaptive response (89). The non-genetic nature of this mechanism suggests that resistance may be readily reversible, and there is early evidence that melanoma patients who have developed resistance to BRAF inhibitors can regain sensitivity following a prolonged drug holiday (93). Taken together, these results suggest that combinations of kinase inhibitors that target multiple nodes within a complex signaling network will require extensive preclinical and clinical characterization to identify the critical pathway interdependencies and reveal the best targets and optimal dosing regimens to maximize efficacy and tolerability for patients.

Figure 3. Potential therapeutic combinations to overcome resistance to BRAF inhibitors.

Dual blockade within the MAPK pathway or between the MAPK and parallel PI3K pathways improves clinical outcome by reducing compensatory signaling and delaying the onset of resistance. Mut, mutation; OE, overexpression; LOH, loss of heterozygosity; ↓Exp, decreased expression.

Drug combinations converging on a hallmark characteristic of tumors.

Kinase inhibitors can also be used in combination to regulate a hallmark phenotype of tumorigenesis (Figure 1). For instance, even though many kinase inhibitors function as cytostatic agents, kinase inhibition has been utilized to modulate the activity of cytotoxic agents; however, these combinations have often been opportunistic, with kinase inhibitors simply added to standard-of-care chemotherapeutic regimens approved for a particular tumor indication (94). An emerging rational strategy for improving the clinical benefit of DNA-damaging agents is the combined inhibition of kinases that regulate cell cycle checkpoints and phase transitions (95). For instance, the serine/threonine kinase CHK1 is activated via ATR-mediated phosphorylation during DNA repair and is required for the S- and G₂/M-phase DNA damage checkpoints (96). Accordingly, CHK1 inhibition potentiates antitumor efficacy of chemotherapies via inactivation of these checkpoints, resulting in premature entry into mitosis with damaged DNA and subsequent mitotic catastrophe and cell death (97, 98). Kinase inhibitors can also be used to mitigate the effects of cytotoxic agents on rapidly proliferating normal tissue. For example, preclinical studies have utilized palbociclib to inhibit the G₁/S-phase checkpoint kinases CDK4 and CDK6 and ameliorate the dose-limiting toxicities of chemotherapies (99). Inhibition of CDK4/6 induced G₁ arrest and pharmacological quiescence of hematopoietic cells to mitigate apoptosis and myelosuppression (100). Moreover, treatment of genetically engineered mouse models with palbociclib reduced irradiation-induced toxicity without limiting therapeutic responses (101). Taken together, these approaches demonstrate that cell cycle kinases can be selectively targeted to both enhance antitumor efficacy and reduce certain normal tissue toxicities induced by standard-of-care chemotherapeutics.

Combination of kinase inhibitors with other therapies is also being explored to more effectively abrogate tumor immune evasion, which has recently emerged as a cancer hallmark in its own right, as well as an important therapeutic modality (Figure 1 and ref. 6). The cancer cells’ propensity to incur and accumulate genetic alterations results in the synthesis of mutant polypeptides, ultimately leading to the generation of neo-epitopes that enable the host’s immune system to recognize these cells as foreign and eliminate them (102). However, tumor cells can exert a substantial influence on their local environment to suppress these antitumor immune responses and escape this critical defense mechanism. Accordingly, therapeutic agents that interfere with these suppressive effects have elicited profound and durable clinical responses, with this line of research culminating in the approval of ipilimumab, an antibody that targets a negative regulator of T cell activation (CTLA4) and, more recently pembrolizumab, an antibody that inhibits an alternative T cell–negative regulator programmed death–1 (PD-1; encoded by PDCD1), for the treatment of advanced melanoma (103). Despite these successes, there remains a significant opportunity to improve upon the often-restricted response rates of these therapies, and a number of kinase inhibitors already approved for specific oncology indications are being clinically evaluated in combination with antitumor immune activators. In fact, there are currently more than 20 active or planned clinical trials exploring the combination and scheduling of various kinase inhibitors with ipilimumab, pembrolizumab, or agents targeting alternative negative immune regulators (103, 104). The overarching rationale behind the application of kinase inhibitors in this context is to directly target the tumor and reduce its immune-suppressive influences in order to shift the local microenvironment toward a proinflammatory state and thereby enhance the activity of the immune activators (105). For example, there are a number of combination trials assessing the relative benefit of BRAF and MEK1/2 inhibition in patients with BRAF-mutant melanoma in combination with ipilimumab or anti–PD-1 antibodies (106). While inhibition of these kinases alone has been shown to yield significant clinical responses and enhance progression-free and overall survival (74, 75), combination with agents that enhance antitumor immune activation reduction in tumor burden is expected to produce therapeutic benefit in addition to a reduction in tumor burden. However, as in the case of MEK, these kinases often play important roles in immune cell function that can abrogate or limit any therapeutic benefit of the combination. As a consequence, adjustments to clinical studies such as dosing holidays or staggered dosing regimens may be necessary (106). Accordingly, while the current deployment of kinase inhibitors in combination with immune activators is an important opportunistic approach to rapidly expand the utility of immune modulation in oncology, much more needs to be elucidated about the numerous pathways and processes involved before additional kinase inhibitors can be developed and appropriately leveraged to maximize their impact in this setting.

Future directions

Kinase inhibitor selectivity.

The discovery of small molecule kinase inhibitors was first reported more than 30 years ago (107). These early compounds bound in the ATP-binding pocket residing in the cleft between the N- and C-lobes of kinases. Even today, the majority of kinase inhibitors bind via a similar mechanism and mimic the hydrogen bond interactions normally formed by the adenosine ring of ATP (7). Of the current generation of small molecules, kinase inhibitors can vary with respect to certain properties, including the activation loop conformation that is preferentially recognized (type 1/DFG-in or type 2/DFG-out) (108), ATP displacement (competitive or non-competitive/allosteric), and the mechanism of inhibitor binding (reversible or irreversible/covalent). Significant clinical benefit can then be achieved by treating tumors harboring specific genetic alterations with highly selective targeted agents (8, 10, 109). One of the prominent themes of the past decade has therefore been to improve the overall kinome selectivity of small molecule inhibitors. There are a number of advantages to this approach, including (a) improvements in tolerability and therapeutic index, as well as enabling (b) the development of drug combination regimens, (c) validation of new targets and molecular dependencies of tumors, and (d) assessment of mechanisms of action. For instance, next-generation EGFR inhibitors that selectively target the inhibitor-sensitizing and resistance (T790M) mutations exhibit a dramatically improved safety profile (with reduced rash and diarrhea) by sparing wild-type EGFR activity in normal cells (110). In addition, concomitant inhibition of structurally related kinases using less-selective inhibitors, such as sunitinib or dasatinib, can have opposing and confounding effects in modulating the immune system (111), which could limit antitumor efficacy and complicate the interpretation of molecular mechanisms. Despite the considerable technical challenges in developing highly selective kinase inhibitors, recent efforts have been aided by using specialized chemical libraries and structure-­informed design (7, 112). Importantly, these efforts are meeting with overall success, such as for ATP-competitive small molecules that specifically target PAK4 (113), as well as cysteine-targeted covalent inhibitors of FGFR4 and CDK7 that selectively perturb proliferation and transcriptional regulation (114, 115). Additional highly selective kinase inhibitors are expected to be available in the near future to test new clinical hypotheses in the new era of precision medicine.

Kinases in cancer immunotherapy.

Much of past and current efforts to discover kinase inhibitors for oncology indications have focused on targets with well-established roles in the regulation of cell proliferation and survival or tumor angiogenesis, three of the fundamental hallmarks of cancer. However, potential kinase targets involved in the regulation of the remaining tumor hallmarks (Figure 1) have yet to be thoroughly elucidated, either preclinically or clinically. One such opportunity may exist in the realm of tumor immunity, which, as mentioned above, represents a key mechanism in the development of cancer and an important therapeutic strategy that has recently demonstrated significant promise across a range of diverse tumor types (103, 116). As outlined in greater detail elsewhere (117), tumor immunity is initiated by antigen-presenting cells (APCs; DCs and macrophages) displaying neo-epitopes on their surface derived from their engulfment and/or proteolytic processing of cancer cells. The APCs engage naive T cells that recognize these neo-epitopes via their T cell receptor (TCR), driving their expansion and maturation to cytotoxic T lymphocytes (CTLs), which can recognize and eradicate the cancerous cells. Given their critical role in tumor immunity and potential for triggering autoimmunity (118), CTL functions are tightly regulated to enable a robust, targeted, and limited immune response. Tumors often subvert the immune system’s attack by engaging the negative regulatory mechanisms that limit CTL function (118). These mechanisms are not entirely CTL autonomous, but involve other cell types, including Tregs (119, 120), regulatory or immature DCs (121, 122), M2 macrophages (123), and myeloid-derived suppressor cells (MDSCs) (124, 125), which are all present within the tumor microenvironment. As detailed below, kinases play a role in essentially all processes that constitute tumor immunity and tumor immune evasion. Accordingly, significant opportunities exist for the development of kinase inhibitors to reactivate the immune system to eradicate the tumor via mechanisms that are far beyond the current trials evaluating their use in combination with the antibody-based therapies.

Kinases are known to play pivotal roles in the resolution of an immune response and could therefore serve as relevant therapeutic intervention points. For example, multiple kinases are involved in limiting the extent and duration of TCR-mediated signaling in CTLs. One pathway involves the kinase PKA (encoded by PRKACA), which, when activated by cAMP (126), phosphorylates and activates c-SRC kinase (CSK), a negative regulator of SRC family kinases (127). CSK then phosphorylates and inhibits lymphocyte-specific protein tyrosine kinase (LCK), a kinase critical for transducing proximal TCR signals. LCK also limits the duration of its own signaling through activation of another non-RTK, MAP4K1, which phosphorylates the key adapter protein SLP76 (encoded by LCP2), thereby blocking LCK’s ability to recruit downstream signaling molecules (128). As an additional example, the α and ξ diacylglycerol kinases (DGKs; encoded by DGKA and DGKE, respectively) have been shown to be involved in limiting the activation of PKCθ (PRKCQ), another positive effector of TCR signaling, thereby blunting CTL proliferation and effector function (129). With the exception of PKA and CSK, the expression of these kinases is predominantly restricted to immune cells, suggesting selective roles in immune modulation. Accordingly, genetically engineered mouse models enabling the abrogation of PKA, CSK, MAP4K1, or DGKξ functions exhibit hallmarks of excessive immune activation and/or TCR hypersensitivity (128, 130–132). At least in the case of Dgka^–/–, Dgke^–/–, and Map4k1^–/– mice, the role of these kinases in tumor immune surveillance has already been established, confirming the viability of these approaches for restoring antitumor immunity (131, 133). In contrast, ubiquitous expression and pleiotropic functions of CSK and PKA may limit the viability of these targets in this context.

As mentioned above, a number of immune cell types act on CTLs to limit their capacity to carry out their effector functions, especially Tregs. This specialized T cell subtype acts via several mechanisms, including secretion of suppressive signaling molecules, titration of CTL activating factors (such as IL-2), and direct cell-cell interactions with CTLs and APCs, to blunt antitumor immune responses (119, 134). Notably, several kinases have been implicated in either the regulation of Treg development and homeostasis or their suppressor functions. In addition to suppressing CTL responses, the PKA/CSK pathway has been shown to be important for both the development and suppressive activity of Tregs (135). Likewise, the lipid kinase PI3Kδ (encoded by PIK3CD) has been shown to play a crucial role in the maturation and homeostasis of Tregs (136). In particular, a recent report demonstrated that loss of PI3Kδ activity, either by genetic knockout or small molecule inhibition, markedly decreased growth and reduced metastasis in immune-competent syngeneic mouse tumor models (137). This positive activity was associated with a substantial increase in CTL proliferation and activation in the tumor microenvironment. Another report implicates PKCη (encoded by PRKCH) and PAK2 as important modulators of Treg suppressor functions via modulation of Treg-APC interactions (138). The authors demonstrated that efficient Treg-mediated suppression of APC-driven CTL activation was dependent on these kinases. Upon Treg-APC engagement, CTLA4 and PKCη were shown to facilitate the disassembly of the structural complexes in Tregs that mediate these cell-cell interactions, thereby enabling the Tregs to release the APC to re-engage other cognate APCs in a serial process of immune suppression. One important unanswered question concerns the absolute requirement of the catalytic activity of either PKCη or PAK2 versus a scaffolding function for these essential Treg functions.

In addition to Tregs, macrophages, MDSCs and DCs play key roles in the suppression of antitumor immunity, and numerous kinases are critically involved in the modulation of these processes. The RTKs CSF1R and KIT regulate the proliferation, survival, and differentiation of myeloid precursor cells, including monocytes and macrophages (123, 139). Small molecule inhibition of CSF1R alone or in combination with adoptive cell therapy, gemcitabine, or antibodies against the T cell regulatory receptors PD-1 or CTLA4 has been shown to markedly inhibit tumor growth and metastasis in syngeneic mouse tumor models, with a corresponding improvement in overall survival (140–143). Inhibition of KIT activity has also been shown to both reduce MDSC numbers and activate DCs in the tumor microenvironment, resulting in enhanced tumor immunity (139, 144). As another example, MST1 (encoded by RON), an RTK predominantly expressed on macrophages, is involved in the resolution of macrophage proinflammatory responses, and a number of tumor types express this receptor’s ligand (MSP; MSMB), thereby contributing to an immunosuppressive tumor microenvironment (145). Small molecule inhibition of MST1 was shown to profoundly limit outgrowth in a syngeneic mouse tumor model of metastasis (146). Finally, as a member of the TAM RTK family, MERTK is important for both macrophage and DC suppressive activity (147). These cells are known to express the type I interferon receptor to which the TAMs heterodimerize and transduce signals to dampen inflammatory signaling. Accordingly, a recent report employing the Mertk^–/– mouse demonstrated that loss of this receptor significantly enhanced the latency and reduced the growth and metastasis of various syngeneic mouse tumor models (148).

Beyond the illustrative examples provided here, numerous other cell types and signaling pathways outside the scope of this Review can influence the tumor immune response. While modulation of tumor immunity via immune checkpoint modulation has been clinically validated, this field is still in its infancy, and much remains to be elucidated around the various regulatory mechanisms controlling the processes involved in activating and resolving immune responses to the tumor. Accordingly, it is anticipated that additional kinase targets will be identified, providing further therapeutic entry points and unique mechanisms to modulate this important cancer hallmark.

Conclusions

Kinases represent a large proportion of actionable, cancer gene alterations, and in the past decade there has been significant progress in generating novel therapeutic inhibitors. Given the recent advances described here in genomics and tumor biology, there is a vast landscape of new kinase targets and emerging inhibitors that warrant further investigation to benefit patients (20, 26). These exciting developments are generating increased optimism within the oncology community that selective targeted therapies will continue to improve response rates and significantly extend the lives of molecularly selected patient populations.