CO-DEPENDENCIES IN THE TUMOR IMMUNE MICROENVIRONMENT

SUMMARY

Activated oncogenes and disrupted tumor suppressor genes (TSGs) not only endow aspiring cancer cells with new biological capabilities but also influence the composition and function of host cells in the tumor microenvironment (TME). These non-cancer host cells can in turn provide cancer cells with growth support and protection from the anti-tumor immune response. In this ecosystem, geospatially heterogenous ‘subTME’ adds to the complexity of the ‘global’ TME which bestows tumors with increased tumorigenic ability and resistance to therapy. This review highlights how specific genetic alterations in cancer cells establish various symbiotic co-dependencies with surrounding host cells and details the cooperative role of the host cells in tumor biology. These essential interactions expand the repertoire of targets for the development of precision cancer treatments.

INTRODUCTION

The paradigm of cancer as a genetic disease is supported by a century of observations including the presence of abnormal mitoses in cancer cells [1], cancer-causing genes in retroviruses, and mutational activation of cellular proto-oncogene orthologs in mammalian cells [2–4], and mutational inactivation of tumor suppressor genes (TSGs) in cancer cells [5]. Accumulating oncogene and TSG alterations are known to commandeer cell regulatory networks and endow cells with new biological capabilities to drive tumor initiation and progression. Initially, these genetic aberrations were viewed as the sole primary drivers of tumorigenesis, whereas host components of the tumor microenvironment (TME), such as carcinoma-associated fibroblasts (CAFs) [6, 7] were considered to be passive albeit supportive subjugates to cancer cells.

In addition to CAFs, the role of immune cells has been a focus in tumor biology. The study of immunity in cancer began in 1890s with Coley’s use of bacterial toxin injections to activate anti-tumor immunity in sarcoma patients [8]. This approach failed to gain traction due to inconsistent patient benefit and lack of mechanistic understanding in tumor immunology. A rebirth of tumor immunology occurred in the late 1950s with the cancer immunosurveillance concept by Paul Ehrlich [9] and others [10], later refined by Robert Schreiber [11, 12]. This concept proposed that malignant cells emerge frequently but are eliminated by a vigilant immune response mediated primarily by lymphocytes. Indeed, increased cancer incidence is observed in those with weakened immunity such as the aged, organ transplant recipients, and individuals with human immunodeficiency virus (HIV) [13, 14]. It is now well understood that the immune system can act to constrain or facilitate tumor growth and progression, acting via three sequential phases of elimination, equilibrium and escape. More recently, a deeper understanding of the mechanisms governing immune activation and suppression has illuminated transformative therapeutic advances that harness the power of the immune system. Here, the most celebrated advances include the discovery of the T cell receptor leading to chimeric antigen receptor (CAR)-T therapy and the discovery of immune checkpoint mechanisms culminating in immune checkpoint inhibitors such as anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and anti-programmed cell death protein 1 (PD-1) [15–18]. Finally, there is a growing recognition that cancer genetic alterations and immunity are tightly linked through the creation of neoantigens from these genetic mutations, and the ability of oncogenic alterations to alter immunocyte composition and functions in specific ways [12]. Beyond the diverse cellular components of the TME, the extracellular matrix, circulating exosomes, cytokines, metabolic milieu, and microbiome act as mediators that govern interactions among various cell types of the TME.

This review focuses on how oncogenic alterations in cancer cells shape the tumor immune microenvironment, the role of host cell factors in supporting cancer cell growth and survival, and the growing recognition that heterogenous TME, called as ‘subTME’ impact anti-tumor immunity and immunotherapy. We discuss the therapeutic opportunities arising from these paracrine signaling insights. This review did not cover in detail the cancer associated fibroblasts (CAFs) and endothelial cells, as this crosstalk are extensively reviewed elsewhere [19, 20].

TISSUE-SPECIFIC GENETIC ABERRATIONS AND TUMOR MICROENVIRONMENT

In cancer, different genetic drivers can generate distinctive tumor immune profiles even within the same tumor type. However, we should note that the tumorigenesis and immunosuppressive TME are not triggered by a single genetic alteration, but by combined alterations of a series of oncogenes and TSGs as well as their interactions. For example, mesenchymal glioblastoma (GBM) is an aggressive type of brain cancer highly enriched with infiltrating myeloid cells and for mutational alterations in PTEN, TP53, NF1, and RB1 [21–23]. What follows are illustrative examples of how specific oncogenes and TSGs alterations can affect immune composition and function in various tumor types (Figure 1).

Figure 1: Molecular circuitry underlying reciprocal interactions between cancer cells intrinsic genetic aberrations and its TME.

Tumor intrinsic genetics aberrations mediates the recruitment and activation of immunosuppressive immune cells. The genetic events increase or decrease expression and secretion of cytokines and chemokines that modulates the differential recruitment and/or inhibition of immune cells. Abbreviations. ILC2: Innate lymphoid cell 2, T_H2: T helper cell 2, MDSC: Myeloid derived suppressor cell, GM-CSF: granulocyte-macrophage colony-stimulating factor, PD-1: Programmed cell death protein 1 (CD279), PD-L1: Programmed death-ligand 1 (CD274), LOX: lysyl oxidase, IRF2: interferon regulatory factor 2.

KRAS.

Activating mutations in KRAS (designated as KRAS*) are among the most frequent genetic alterations in human cancer, such as pancreas, colon and lung cancer [24, 25]. KRAS* and its downstream signaling cascade are known to regulate many aspects of tumor biology such as proliferation and metabolism and play a particularly prominent role in blunting anti-tumor immunity. In colorectal cancer (CRC), KRAS* can down-regulate major histocompatibility complex class I (MHC-I) expression to diminish tumor antigen presentation and immune recognition [26] and can repress expression of interferon regulatory factor 2 (IRF2) [27]. IRF2 in turn directly represses C-X-C motif chemokine ligand (CXCL) 3 which binds C-X-C Motif Chemokine Receptor 2 (CXCR2) to recruit and activate myeloid derived suppressor cells (MDSCs). On another level, KRAS* can modulate the expression of key immunoregulatory molecules on cancer cells such as programmed death-ligand 1 (PD-L1) which fosters immune evasion via binding to PD-1 receptor on T cells and inhibiting T cell function [28]. In pancreatic cancer, KRAS* has been shown to upregulate interleukin (IL) receptors that bind cognate cytokines produced by infiltrating immune cells, thereby enabling a cancer-immune cell paracrine circuit that drives anaerobic glycolysis in cancer cells [29]. Disruption of this paracrine circuit impairs tumor growth and increases survival. Also, in pancreatic cancer, KRAS* can promote production and secretion of granulocyte-macrophage colony-stimulating factor/colony stimulating factor 1 (GM-CSF/CSF1) which recruits Gr1⁺CD11b⁺ (granulocytic) MDSCs from the bone marrow [30, 31]. Suppression of GM-CSF decreases mobilization of granulocytic MDSCs, increases infiltration of cytotoxic CD8⁺ T cells, and reduces tumor growth [32]. Finally, in lung cancer, KRAS*-MEK–ERK–AP-1 pathway promotes the expression and secretion of immunosuppressive cytokine IL-10 which promotes regulatory T (T_reg) cell infiltration and activation [33]. Moreover, KRAS* upregulates the expression of TGFβ which further promotes T_reg cell infiltration. Accordingly, KRAS inhibition reduces T_reg cell infiltration and promotes tumor regression. Together, these studies convey that the actions of KRAS* extend beyond cancer cell intrinsic functions of proliferation and survival to foster an immune-suppressive environment permissive for tumor growth. It is also notable that, even with cancer cell intrinsic hallmarks such as metabolic reprogramming, the immune system can serve an integral role. Knowledge of these circuits can provide novel therapeutic strategies.

PTEN.

The phosphatase and tensin homolog (PTEN) tumor suppressor negatively regulates the phosphoinositide 3-kinases/protein kinase B (PKB), also known as Akt (PI3K/AKT) cascade and loss of PTEN and/or mutational activation of PI3K signaling components are observed in virtually all cancers [34–37]. In addition to promoting survival and anabolic growth of cancer cells, PTEN loss can upregulate cytokines that drive infiltration of immune-suppressive myeloid cells (e.g., MDSCs, macrophages and neutrophils) and T cells. In prostate cancer, PTEN loss increases infiltration of myeloid cells, especially MDSCs, which can be further enhanced by concomitant loss of the SMAD4 tumor suppressor [38]. Mechanistically, PTEN loss results in Glycogen synthase kinase-3 beta (GSK-3β)-mediated stabilization of chromatin helicase DNA-binding protein 1 (CHD1) [39]. CHD1 upregulates IL-6 to recruit and activate granulocytic MDSCs infiltration; correspondingly, CHD1 depletion or anti-IL-6 mAb treatment inhibits tumor growth specifically in PTEN-null prostate cancers [40]. Finally, in prostate cancer, PTEN loss upregulates M-CSF (mouse CSF) and IL-1β to further promote MDSC infiltration [41] and can increase CXCL8 secretion to promote macrophage infiltration [42] and increase CXCL2 to drive an immune suppressive ‘M2’-like phenotype [43]. Inhibition of CXCL2 receptor, CXCR2, reprograms ‘M2’-like macrophages towards an anti-cancer ‘M1’-like phenotype [43]. Consistent with these mechanistic insights, anti-myeloid therapy such as CXCR2 inhibitors can sensitize castration resistant prostate cancer to immune checkpoint blockade therapy [44]. In other tumor types, PTEN loss can operate via entirely different factors to repress anti-tumor immunity via myeloid mechanisms. For example, in glioma, PTEN loss activates the YAP1 transcription factor to upregulate lysyl oxidase (LOX) which recruits immune-suppressive macrophages via LOX binding and activation of the β1 integrin receptor and activation ofpProtein tyrosine kinase 2 beta (also known as PYK2) to promote migration [45]. Interestingly, these invading macrophages secrete osteopontin which spurs angiogenesis and promotes glioma cell survival, providing another example of symbiotic signaling between cancer cells and host cells in the TME [45]. Thus, while PTEN loss is not druggable, knowledge of it’s signaling circuits in the TME provides genotype-specific vulnerabilities in cancer.

WNT/APC/β-catenin.

Aberrant WNT/ adenomatous polyposis coli (APC)/β-catenin signaling is common in many tumor types including CRC, hepatocellular carcinoma (HCC) and melanoma [46, 47]. Typically, mutational inactivation of the APC tumor suppressor activates T cell factor (TCF) 4/β-catenin transcriptional activity to drive many genes governing cancer cell metabolism and stemness [48]. The WNT/APC/β-catenin pathway also plays a major role in suppressing anti-tumor immunity. In APC-deficient CRC, cancer cells upregulate chemokine CXCL5 resulting in recruitment of macrophages which are not only immunosuppressive but also secret GAS6 to activates AXL on CRC cancer cells [49]. In an APC-driven adenoma mouse model, CCL2 has been shown to enhance the accumulation of MDSCs and foster their immune-suppressive properties. The neutralization of CCL2 decreases MDSC infiltration and function; and inhibits tumor growth [50]. In an APC-deficient HCC mouse model, oncogenic β-catenin signaling activates the NF-κB inflammatory program which promotes infiltration of macrophages, neutrophils, natural killer (NK) cells and B cells [51]. In melanoma, activated WNT/β-catenin signaling inhibits the recruitment of CD103⁺ dendritic cells (DCs) by reducing the expression of CCL4 and CXCL10, leading to T cell exclusion [52, 53]. In a study of 9,244 cancer patients, mutation of β-catenin signaling molecules are three-fold more frequent in non-T cell-inflamed tumors relative to T cell-inflamed tumors [54, 55]. Thus, in addition to its classical functions in cancer cells, aberrant activation of the WNT/β-catenin signaling pathway can suppress anti-tumor immunity via regulation of many aspects of DCs, T_reg cells, T helper cells and effector T cells [56–58]. Given the central role of the WNT/APC/β-catenin pathway in cancer, the development of effective therapies targeting this pathway would be expected to impact many cancer hallmarks and be especially useful in overcoming primary, acquired and adaptive resistance to cancer immunotherapy [59].

MYC.

The MYC family of transcription factors can impact virtually all cancer hallmarks particularly tumor metabolism and immunity [60, 61]. In a Kras^G12D-driven lung adenocarcinoma model, c-Myc upregulates CCL9 expression which recruits macrophages. The recruited macrophages via the production of vascular endothelial growth factor (VEGF) promote neo-angiogenesis and via upregulation of PD-L1 induce T cell exhaustion [62]. In pancreatic cancer, c-Myc promotes the expression of multiple cytokines (e.g., IL-19, IL-3 and IL-11) and chemokines (e.g., CCL7, CCL8 and CCL12), which increases the recruitment of immune-suppressive macrophages and neutrophils [63]. Moreover, patient tumor exhibiting high c-Myc and NOTCH2 expression and deletion/mutation of cyclin-dependent kinase inhibitor 2A/B (CDKN2A/B) correlate negatively with lower intratumoral cytotoxic T cell activity [64]. Similarly, in neuroblastoma, high N-Myc levels are associated with a low T cell infiltration due to reduced interferon pathway activity and chemokine expression [65]. Moreover, in primary human neuroblastoma cell lines, higher N-Myc expression correlates negatively with low NK cell activating factors such as NKG2D [66]. In acute T cell lymphoblastic leukemia, c-MYC is frequently overexpressed, which, in turn, promotes macrophage recruitment [67].

p53.

Loss of p53 tumor suppressor function is another frequent genetic aberration in cancer. p53 is a master transcriptional regulator governing metabolism and autophagy, proliferation and senescence, DNA damage response and repair, and cell death [68, 69]. p53 also regulates many aspects of tumor immunity at the level of cancer cells and the TME. In a Pten-deficient prostate cancer model, p53 deficiency upregulates the infiltration of immature myeloid cells, monocytes and neutrophils via upregulation of CXCL17 [70]. In breast cancer, loss of p53 triggers the production of WNT ligands that stimulate tumor-associated macrophages (TAMs) to secrete IL-1β, which, in turn, drives neutrophil-mediated systemic inflammation [71]. In glioma, loss of p53 activates the NF-κB pathway, which in turn upregulates CCL7 expression to promote infiltration of immune-suppressive microglia and bone marrow-derived macrophages [72]. In addition, p53 loss can activate the NF-κB and Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) pathway in cancer cells of colorectal [73, 74] and pancreatic cancer [75], which in turn promotes the infiltration of macrophages, MDSCs and neutrophils. In epidermal growth factor receptor (EGFR) overexpressing lung adenocarcinoma and pancreatic cancer, p53 loss enhances cancer cell secretion of chemokines CCL11, CXCL1, CXCL5, CCL3 and M-CSF to promote macrophage infiltration [76]. Finally, in addition to promoting myeloid cell migration, p53 loss can affect the biological state of myeloid cells, particularly macrophages. For example, in HCC, p53-expressing senescent stellate cells polarize macrophages toward an ‘M1’-like phenotype, while proliferating p53-suppressed stellate cells promote an ‘M2’-like phenotype via secretion of several cytokines, such as IL-13, IL-4, and IL-5 [77]. Analogously, p53 loss in CRC cancer cells produces exosome containing miR-1246, which can be uptaken by macrophages, and then foster TAMs towards an ‘M2’-like phenotype [78]. The relationship between p53 loss and suppressive myeloid cells is apparent on several levels in various human cancers where p53 loss correlates positively with (i) CSF1 response signtures in breast cancer [79], (ii) chemokine CXCL7 expression and myeloid cell density in prostate cancer [70], and (iii) ‘M2’-like macrophage density in CRC [78]. In pancreas and lung cancers, p53 loss-induced infiltration of suppressive myeloid cells results in robust inhibition of CD4⁺ and CD8⁺ T cell-mediated immune response. Moreover, p53 loss promotes a suppressive T_reg cell population to further dampen T cell-mediated anti-tumor immunity [76]. In addition to loss-of-function, the point mutation of p53 also affects the immunosuppressive TME. For example, P53 missense mutations increases the expression of mutant p53 proteins, which, in turn, decreases the infiltration of cytotoxic CD8⁺ T cells in pancreatic cancer [80]. Similarly, gain-of-function p53^R172H mutation in mouse pancreatic ductal epithelial cells results in an influx of CD11b⁺Ly6G⁺ neutrophils via upregulation of CXCL2, and a decreases of T cells (e.g., CD3⁺ T cells, CD8⁺ T cells, and CD4⁺ T helper 1 cells) in tumors [81]. As a result, depletion of neutrophils in mice improves the anti-tumor efficiency of CD40 immunotherapy in p53 mutated pancreatic cancer [81]. Together, these observations support the combined testing of myeloid inhibitor and T cell activator therapies in p53-deficient and mutated cancers.

EGFR.

EGFR is a receptor tyrosine kinase that plays an essential role in epithelial tissue homeostasis and tumorigenesis via both autocrine and paracrine mechanisms [82]. In a mouse model of lung cancer, EGFR mutation induces a robust infiltration of macrophages and neutrophils but reduces the infiltration of CD8⁺ T cells [83–85]. Treatment with EGFR-inhibitors (e.g., erlotinib and cetuximab) results in a significant decrease in macrophage infiltration [86]. Although the mechanism underlying macrophage and neutrophil infiltration is unknown, the downregulation of CD8⁺ T cells is due to EGFR-induced upregulation of PD-L1 and downregulation of CCL5 and CXCL10 in cancer cells [84, 85]. On the other hand, EGFR mutation upregulates the expression of c-Jun N-terminal kinases (JNK)/cJUN- and Interferon regulatory factor 1 (IRF1)-mediated CCL2 expression, which in turn promotes the infiltration of T_reg cells in lung cancer [85]. The effect of EGFR in macrophage infiltration is also observed in GBM, where EGFR and its truncation mutant EGFRvIII cooperate to promote macrophage infiltration via upregulation of CCL2 [87].

LKB1.

The liver kinase B1 (LKB1) also known as Serine/threonine kinase 11 (STK11) is a potent TSG in endometrial cancer, where the loss of LBK1 in cancer cells induces the secretion of CCL2, which recruits macrophages into tumors. Consequently, inhibition of CCL2 in the LKB1-driven endometrial cancer mouse model impairs tumor growth and increases survival [88]. In a Kras-driven non-small-cell lung carcinoma (NSCLC) mouse model, deficiency of LKB1 upregulates CXCL7, G-CSF and IL-6, which in turn enhance the recruitment of neutrophils. As a result, these neutrophils suppress T cell function and activity via secretion of IL-10, galectin-9, arginase 1, milk fat globulin EGF factor 8 protein (MFGE8) and IL-6. Depletion of neutrophils using anti-Ly-6G/Gr-1 antibodies or inhibition of IL-6 using neutralizing antibodies in LKB1-deficient lung cancer mouse models yields significant therapeutic benefits associated with reduced neutrophil infiltration and enhanced cytotoxic CD8⁺ T cells [89]. In lung squamous cell carcinoma, loss of LKB1 and PTEN induces predominantly neutrophil infiltration in the TME [90]. Together, these findings highlight the contribution of genetic aberrations of LBK1 in shaping the tumor immune landscape.

Geospatial differences within the TME

Although the tumor intrinsic genetic changes shape the overall TME, there is a growing realization of the importance of extratumoral factors working in concert with classical genetic events to create a heterogenous TME, with implications on disease progression and therapy resistance. This heterogenous TME is a complex microcosm that is not uniformly stimulatory or inhibitory making it difficult to assign distinct functions and characteristics to the overall TME. Using cutting-edge technologies such as geospatial transcriptomics, digital pathology, sequential fluorescence in situ hybridization (seqFISH+) and slide-seq, recent studies have highlighted the significance of understanding this heterogeneity, within individual tumors, where distinct regional programs act as an individual unit with varied proliferation, metastatic and immunogenic characteristics [91–93]. Spatial transcriptomic analysis in human PDAC evaluated single-cell populations in situ which revealed a remarkable cellular complexity of the TME, comprising numerous stromal cell subpopulations, especially in the regional histological pattern in the tumors across patients, stages, and sites [94]. This heterogenous TME is aptly named ‘subTME’, where the tumor exhibit distinct immune phenotypes and CAF differentiation states, and has been classified into three phenotypic groups-(i) deserted regions with thin, spindle-shaped fibroblasts and loose matured fibers, (ii) reactive regions containing fibroblasts with enlarged nuclei, few acellular components, often rich in the inflammatory infiltrate and (iii) regions with intermediate levels of these features [94]. Further, co-culture studies of CAFs and PDAC cell lines in different ratios identified shifts in the signaling pattern in cancer cells towards a more invasive epithelial-to-mesenchymal transition (EMT) and proliferative (PRO) phenotypes. The phenotypes are linked to molecular changes in mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 3 (STAT3) signaling [95]. Moreover, this study has identified and validated TGF-β1 as the main cytokine released by CAFs that drives the EMT and PRO phenotypes. A similar study in lung cancer combined multiregion exome, RNA-seq data with spatial histology and deep learning that identified cancer subclones in immune cold regions, which are closely related in mutation space and are relatively more recent than subclones from immune hot regions [96]. Furthermore, tumors with more than one immune cold region had a higher risk of relapse, independent of tumor size, stage and number of samples per patient, illuminating the clinical significance of immune cold subregions which might evade the immune response and immunotherapy treatment. Overall, these provocative findings provide a glimpse of the complex nature of the TME, however it warrants further investigation to understand the mechanisms that contribute to the spatial variability within the TME.

TARGETING SYMBIOTIC CO-DEPENDENCIES TO MAXIMIZE THE EFFECT OF IMMUNOTHERAPY

As discussed above, genetic alterations in cancer cells are critical for regulating the infiltration and activation of immunosuppressive myeloid cells, as well as the function and activity of T cells. These findings not only support the concept that genetic events in cancer cells are a determinant for the “cancer-immunity cycle” [97] but also highlight the effectiveness of personalized immunotherapies in cancer patients with specific genetic backgrounds. Therefore, these findings raise the possibility that to maximize the effectiveness of immunotherapy, strategies targeting genetic alterations in cancer cells and their related immune landscape in the TME should be considered (Figure 2).

Figure 2: Targeting symbiotic co-dependencies to maximize the effect of immunotherapy.

The bidirectional crosstalk observed between cancer cells and the TME is manifested on several levels, that creates an opportunity to target these interactions for cancer therapy. Abbreviations. CRC: colorectal cancer, PDAC: pancreatic ductal adenocarcinoma, LAC: lung adenocarcinoma, PCa: prostate cancer, FMT: Fecal microbiota transplantation, TKI: tyrosine kinase inhibitor, TGF-β: Transforming growth factor beta.

1. Targeting genetic lesions to alter TME and enhance immunotherapy response.

One important mechanism for how cancer cell genetic aberrations shape the immune landscape and affect immunotherapies is related to their function in modulating immunosuppressive myeloid cells, including macrophages, MDSCs and neutrophils, in the TME. Myeloid cells are the most abundant immune cells of the TME, which contribute to many cancer hallmarks including cancer cell growth and survival, metastasis and angiogenesis via distinct mechanisms by secretion of a variety of factors [98–101]. For example, TAMs can secrete osteopontin to promote cancer cell survival and stimulate angiogenesis [45], and MDSCs are able to produce VEGF, bFGF, MMPs and TGF-β to stimulate angiogenesis and metastasis [102]. Neutrophils can release neutrophil extracellular traps (NETs) containing DNA (NET-DNA) to promote cancer metastasis via its receptor CCDC25 on cancer cells [103]. In addition to supporting cancer cell growth and metastasis, [101] these myeloid cells display a potent inhibitory effect on T cells by producing a large number of immune modulatory factors [104, 105]. Indeed, as noted above, many genetic aberrations can trigger the infiltration of macrophages, MDSCs and neutrophils, which, in turn, influence the function and activity of T cells, and the effectiveness of immunotherapies. Thus, these findings highlight a potential to develop novel therapeutic strategies combining immunotherapies and myeloid cell-targeted therapies in a specific tumor genetic background. For example, in PTEN-deficient prostate cancer and melanoma mouse models, targeted therapies against MDSCs using IL-6 neutralizing antibodies [40], multikinase inhibitors such as cabozantinib and BEZ235 [44], NLRP3 inhibitor MCC950 [106], or phenformin [107], show robust synergistic effects with immune checkpoint inhibitors (ICIs), including anti-PD-1/PD-L1 and anti-CTLA-4 therapies. In addition, PTEN loss leads to defective T cell migration and a decrease in the efficacy of anti-PD-1 therapy [108]. Loss of PTEN increases the expression of immune-suppressive cytokines such as CCL2 and VEGF, resulting in a decrease in T cell infiltration. Moreover, studies in melanoma patients receiving anti-CTLA-4, and anti-PD-1 response better in the presence of PTEN expression, suggesting PTEN can be used as a biomarker for patient selection eligible for anti-CTLA-4 and anti-PD-1 therapy [109]. These studies hint towards a PTEN mediated pathway that is amenable for combination therapy with anti-PI3K and immunotherapy for prostate cancer.

In Kras-mutated CRC [27], or p53-mutated rhabdomyosarcoma [110] mouse models, the anti-PD-1 therapy resistance is overcome by blocking MDSC infiltration via enforcing IRF2 expression or inhibition of CXCR2. Similar to targeting MDSCs, depletion (e.g., CSF1/CSF1R inhibition) or repolarization (e.g., HDAC inhibition) of macrophages have been shown to improve the effectiveness of ICIs (including anti-PD-1/PD-L1 or anti-CTLA-4) in a variety of mouse models, including Kras-mutated PDAC [111], Braf-mutated melanoma [112, 113], MMTV-PyMT breast cancer [114]. Finally, depletion of neutrophils using CXCR1/2 inhibitor, anti-Ly6G or anti-Gr1 antibodies also produce a synergistic effect with anti-PD-1 therapy in lung cancer, including Pten^fl/fl;Lkb1^fl/fl [115] and Kras^LSL-G12D/WT;p53^fl/fl [116] mouse models. A study in lung cancer found mutant KRAS is correlated with PD-L1 upregulation by inhibition of tristetetrapolin (TTP), a negative regulator of PD-L1 expression, through KRAS-induced MEK signaling [117, 118]. However, this increased PD-L1 expression provides opportunities for ICI therapy, with anti-PD-1/L1 study in NSCLC show clinical benefits [119]. A modest clinical benefit is also observed in mismatch repair (MMR) deficient CRC (representing ~2–4% of all CRC patients) which shows response to anti-PD-1/PD-L1 therapy [120]. However, the clinical benefits of ICI cannot be extended to all KRAS mutant tumors, especially in PDAC, where most ICI trials have resulted in limited clinical benefits. The ineffectiveness of ICIs in PDAC is attributed to the complex TME, however combinatorial approach targeting multiple pathways (e.g., PD-L1 and colony stimulating factor receptor 1(CSFR1), chemokine C-X-C receptor 4 (CXCR4)) are under clinical investigations [121]. LKB1 mutant tumors also express low amount of PD-L1 therefore respond poorly to ICI therapies. Mechanistically, LKB1 loss is associated with suppression of stimulator of interferon genes (STING) leading to downregulation of PD-L1. Ectopic expression of LKB1 or STING pathway increase responses to anti-PD-1 therapy [122].

2. Genetic alterations as predictive biomarkers for ICI response and combination immunotherapy.

Another mechanism by which cancer cell genetic aberrations may affect immunotherapy response is via regulation of T cell function and immune checkpoint molecule expression. Among the genetic aberrations, PTEN mutation/deletion is a common event across many cancer types, which displays a potent effect to induce ICI resistance. For example, in melanoma, PTEN loss reduces T cell infiltration and inhibits T cell-mediated tumor killing effect by reducing autophagic activity. As a result, inhibition of the PI3K pathway using PI3Kβ inhibitor improves the anti-tumor response of ICIs in PTEN-deficient melanoma mouse models [108]. Similarly, anti-PD-1 and/or anti-CTLA-4 therapies show better clinical benefits in PTEN-intact melanoma patients compared to PTEN-deficient patients [109]. In glioblastoma, loss of PTEN increases PD-L1 expression, thus inducing immune-resistance [123]. A recent retrospective study in glioblastoma patients revealed a significant enrichment of PTEN mutations in anti-PD-1 non-responders and found that anti-PD-1 therapy resistance is related to the infiltration of macrophages, microglia and neutrophils in PTEN-mutated patients [124]. Similar to melanoma and glioblastoma, loss of PTEN is also associated with resistance to anti-PD-1 therapy in metastatic uterine leiomyosarcoma [125]. In lung cancer, bi-allelic loss of PTEN and LKB1 can stimulate PD-L1 expression in cancer cells [90]. However, it should be noted that conflicting results have been reported in lung cancer, where PTEN loss downregulates PD-L1 expression [126, 127]. These results suggest that in addition to PTEN, there are additional genetic alterations that may have a positive or negative effects on PD-L1 expression [128]. For example, PD-L1 levels have been shown to be elevated in p53-mutated [129] and EGFR-mutated [130, 131], but reduced in PIK3CA-mutated [129, 132] lung adenocarcinoma. Functionally, EGFR mutation upregulates the recruitment of T_reg cells but downregulates the infiltration of CD8⁺ effector T cells in lung cancer [85]. Inhibition of EGFR reduces the infiltration of T_reg cells and synergizes with anti-PD-1 therapy in EGFR-mutated, but not EGFR-WT, lung cancer [103]. Encouraged by the experimental findings, clinical trials are underway for treatments combining EGFR-Tyrosine kinase inhibitors (TKIs) with various ICIs in lung cancer [128, 133]. In addition to the mutations of PTEN and EGFR, loss of LKB1 has been identified as the prevalent genomic driver for anti-PD-1 therapy resistance in Kras-mutated lung adenocarcinoma mouse model and patients [134]. Together, these findings reveal the molecular mechanisms for how specific genetic aberrations induce ICI resistance and highlight the potential therapeutic strategies for overcoming therapy resistance to ICs.

Similarly, the involvement of KRAS in indirectly regulating the tumor microenvironment creates a vulnerability that can be potentially targeted. Especially, Kras mutation is associated with higher CD45⁺ cell infiltration, albeit immunosuppressive types such as TAMs and MDSCs in the lung, colon, and pancreatic cancer. In human CRC there is a higher TAM infiltration mediated by Kras driven production of CSF2 and higher lactate [135]. Further in CRC, Kras driven CXCL3 overexpression which recruits CXCR2⁺ MDSC can be overcome by utilizing CXCR2 inhibitors [27]. In PDAC, Kras promotes a type 2 (T_H2, innate lymphoid cell 2 [ILC2]) immune cell program by secreting IL-33 [136]. The anti-IL-33 antibody which is currently being developed for asthma and other respiratory diseases can be repurposed for PDAC (Clinical Trial #NCT03736967). Kras mutation in lung cancer is associated with a 3–5-fold increase in total infiltrating CD45+ cells compared to normal lung which provides an opportunity to combine conventional therapies with ICI such as PD-L1 which is highly expressed by the infiltrating MDSCs and TAMs. Recently, several studies have identified cancer cell intrinsic molecular pathways that are linked to T cell exclusion and function. For example, mutant KRAS and PTEN deficient tumors express CXCL chemokines that attract myeloid cells by binding to CXCR2 receptors. So, inhibitors for CXCR2 or its ligands such as CXCLs is now being tested for PDAC and metastatic castration resistant prostate [104, 110, 113, 137–139]. The CXCR2 inhibitors as a single agent may not provide the necessary benefit but create an opportunity for ICI therapy by increasing T cell infiltration, so a combination of CXCR2 along with PD-1/L1 should be tested for a better patient outcome [140]. Also, PI3K inhibitors are tested for PTEN deficient tumors (Clinical Trial# NCT02646748). Here, careful dosing needs to be considered because immune cells need PI3K pathways for activation so a global inhibition of PI3K pathway might eliminate the immune cell function. Other identified pathways such as JAK-STAT which are often upregulated in multiple cancers but are difficult to target in solid tumors, such as pancreatic cancer [141]. However, a specific Jak1/2 inhibitor is approved for hematological tumors such as polycythemia vera [142]. Similarly, WNT–β-catenin pathway is identified as a target in various other cancer types including melanoma, however, it remains challenging to target because of the essential role of this pathway in normal tissues, such as in gastrointestinal tract [143]. In conclusion, other than the combination therapy of ICIs and chemotherapy which have provided tremendous benefit new combinations that will target the cancer-cell intrinsic molecular pathways should be considered.

CONCLUSION AND FUTURE DIRECTIONS

Over the last decade, ICI treatment has provided dramatic antitumor responses with durable regression of the disease and even cures observed in a subset of patients. However, there are numerous challenges and hurdles that needs to be overcome to make immunotherapy beneficial to an even greater number of patients. Accumulating evidence suggests that tumor-intrinsic genetic aberrations determine the immune cell repertoire that supports immune evasion and resistance to ICI. Understanding the molecular mechanism that drives the effector T cell exclusion will allow the development of novel agents that will improve the efficacy of ICI. In addition, understanding the circuits that enable T cell exclusion will allow targeting of undruggable oncogenes or tumor suppressor deficiencies at the level of these circuits, for example Kras-IRF2-CXCL3 axis paradigm. This review specifically has cataloged the molecular circuitry underlying reciprocal interactions between cancer cells’ intrinsic genetic events and its TME that includes immune cells. This bidirectional crosstalk is manifested on several levels, including cancer cell intrinsic mutations that create a permissive environment to recruit protumorigenic immune cells, at the same time creating an opportunity to target these immune cells for cancer therapy.

Although encouraging, we need to remain cautious about the clinical takeaways of these studies as most current studies provide a mere correlation between tumor intrinsic genetic aberrations and TME. A more robust study should consider systemic immune response which will enable the use of the currently available T cell-based therapies. A case-in-point is the recent pre-clinical and clinical studies in melanoma showing that systemic immune response can be an important indicator of anti-tumor immune response and ICI efficacy. Further, the use of FMT, where stool from healthy or ICI responder patients is transferred to non-responder patients to improve ICI response [144]. Although it remains to be determined if the gut microbiota of ICI responders is uniquely manipulated by the tumor intrinsic genetic aberrations or the environmental factors that shape the gut microbiome, FMT provide a huge promise to sensitize ICI response.

Technological advances such as single-cell RNA sequencing, cytometry by time of flight (CyTOF), spatial transcriptomics and proteomics, metagenomic sequencing and big data analysis will provide the tools necessary to analyze the antitumor immune responses and what drives immune-suppressive environment and therapeutic resistance in the tumor. Further, integration of cancer cell intrinsic genomic data and immunology from high throughput analysis from preclinical and clinical studies will drive the cancer immunology field forward and will translate to new therapies. The combination of in vivo models and high throughput technology will help understand the cause-and-effect relationship between tumor intrinsic genetic perturbation and immune response. The discovery of new immune cell hallmarks which sustain tumor growth can be leveraged by rational drug combinations that includes ICI and cytokine/chemokine axis. We also need to identify therapy response markers to enlist the right patients into the right therapeutic protocol. Finally, we need to have a better understanding of toxicities associated with ICI combinations which will make the ICI therapy more accessible to older patients and patients with co-morbidities.