In addition to T-cells, the vast majority of tumor-associated leukocytes within the TME are myeloid cells, predominantly MDSCs, macrophages and neutrophils in varying stages of differentiation [31]. Subsets of these myeloid cells have been shown to promote, carcinogenesis, angiogenesis and metastasis [32]. MDSCs and TAMs are the two primary myeloid culprits that facilitate the immunosuppressive nature of the TME. Although both are derived from a common myeloid progenitor, there is significant heterogeneity among the myeloid cell populations of cancer, and it is now thought that myeloid cells in tumors exist within a spectrum of differentiation from monocytes/M-MDSCs towards TAMs [33]. MDSCs are classified as polymorphonuclear (PMN)-MDSC or monocytic (M)-MDSC, reflecting their similarities to neutrophils and monocytes, respectively. Current studies have shown that in general, M-MDSCs and PMN-MDSC are explicitly tumor-promoting, whereas TAM are duplicitous in their nature, exerting both anti- and pro-tumor effects [34, 35]. Not surprisingly, the presence and penetration of these cells within the tumor tissue are associated with poor prognosis [36, 37].

Understanding macrophage phenotype polarization is important to elucidating their role in malignancy. Within any tissue, particularly in tumors, macrophage activation can proceed along two vastly different macrophage phenotypes; where the “M1” phenotype is considered pro-inflammatory and “M2” is considered anti-inflammatory [38]. Phenotypic expression of macrophages is dependent on signals from their microenvironment, such as cytokine expression. In healthy tissue, macrophages exist in equilibrium between M1 and M2 phenotypes. However, in progressive cancers, the phenotype is driven towards M2 and skewed away from an M1 phenotype, and M1 phenotype has been noted in regressing tumors [39–41]. In pancreatic cancer, anti-inflammatory pro-tumor polarized macrophages, are associated with increased invasiveness secondary to elevated lymphatic vessel density and significantly poor prognosis [42].

Within the TME, TAMs and MDSCs are in a background of cytokines that lead to chronic inflammation as well as immune evasion. Inflammatory cytokines, such as tumor-necrosis factor-α (TNF-α), interleukin-6 (IL-6) and IL-8, are often upregulated and promote the invasive properties of cancer, such as angiogenesis and metastasis [43, 44]. Other cytokines, such as IL-4, IL-13 and IL-10, have been reported to be propagators of an anti-inflammatory environment and facilitators of adaptive immune response suppression [41]. Together, the chronic inflammatory milieu and the purveyors of immune evasion modulate the TAM and MDSCs towards promoting tumor proliferation, therapy resistance and metastatic growth [45, 46]. There is also significant crosstalk between MDSCs and other immunosuppressive cells, such as regulatory T-cells (Tregs), which further promotes immune silencing within the TME via cytotoxic CD8+ T-cell inactivation and anergy [47].

In multiple xenograft models, cytokines such as CSF-1 are not only attractants of myeloid cells, like MDSCs and TAMs, but also as promoters of the M2 phenotype [48, 49]. With its ability to muster M2 phenotype macrophages into the TME and increase metalloproteinase secretion to support metastases, the CSF-1-mediated pathway becomes an appealing therapeutic target for small molecule intervention [50].

The tumor ECM functions more than a simple scaffold in which the cells and the lymphatic and vascular system reside; it also plays a critical role in supporting the inflammatory milieu needed for tumor progression and metastasis [51, 52]. The ECM is a depot for cytokines, growth factors and other molecules, and their effects are communicated via the integrins that couple the ECM to the actin cytoskeleton. Interactions between TAMs and ECM proteins can promote metastasis, and in this regard, CSF-1 and FAK serve as important examples of how the interaction between the ECM and the inflammatory milieu leads to cancer progression (Fig. 2) [52]. CSF-1 signaling via CSF-1R leads to increased FAK phosphorylation in macrophages, and FAK then mediates cell adhesion turnover. Without FAK, macrophages cannot form stable protrusions (i.e. broad lamellipodia), nor form a leading edge for migration [53, 54]. Thus, chemotaxis by macrophages to a chemo-attractants such as CSF-1 is precluded, as is random migration, leading to decreased macrophages at sites of inflammation. In addition, ECM protein fibronectin’s interaction with integrins activates FAK and leads to ligand-independent phosphorylation of CSF-1R and subsequent myeloid cell migration [55].Fig2Fig. 2

Signaling pathways for CSF-1 and FAK. CSF-1R predominantly modulates differentiation, proliferation and survival via PI3K or the RAF/MEK/ERK pathway. For the regulation of cell adhesion and migration, the binding of CSF-1 to CSF-1R leads to phosphorylation of FAK, which in turn activates numerous signaling pathways that lead to actin polymerization/cytoskeleton remodeling, adhesion dynamics and migration (via ERK, N-WASP/CDC42, VCAM and Selectin). However, like CSF-1/CSF-1R, FAK is also involved in cell survival via the PI3/AKT pathway. Interaction of ECM protein (e.g. fibronectin) with integrins can also activate FAK, which leads to ligand-independent phosphorylation of CSF-1R, and thus cell migration (inset on left lower portion of Fig. 1)

PLX-3397 was one of the first small molecule inhibitors of the CSF-1 pathway, and not only is it a potent tyrosine kinase inhibitor of CSF-1R, but it also targets cKIT and FLT3. In preclinical lung adenocarcinoma mouse models, PLX-3397 was shown to modify TAM distribution in the TME and decrease tumor burden [79]. Similarly, in syngeneic mouse models of BRAF V600E-mutated melanoma, PLX-3397 combination therapy with adoptive cell transfer immunotherapy, showed reduction in TAMs and increase in tumor infiltrating lymphocytes leading to increased release of IFN-γ [80]. When combined with BRAF inhibitor, PLX4032, in similar melanoma mouse models, PLX-3397 was shown to substantially reduce M2 phenotype macrophage recruitment, leading to significant tumor growth suppression [81]. In this same study, expression of PD-1 and PD-L1 was increased on intratumoral CD11b + myeloid cells, suggesting an attenuating mechanism on the combination therapy of BRAF and CSF-1R inhibition. When PD-L1/PD-1 inhibitory therapy was added to PLX4032/PLX-3397-treated mice, outcomes improved. This suggested a role for PD-L1/PD-1 blockade as adjunctive therapy to PLX-3397.

In pancreatic cancer xenograft models, Zhu et al. demonstrated that CSF-1R blockade with PLX-3397 decreased CD206 TAMs (i.e. M2 phenotype macrophage) within the TME and reprogrammed the remaining TAMs towards an anti-tumor phenotype [24]. This study also reaffirmed that CSF-1/CSF-1R inhibition altered T-cell checkpoint signaling, as was previously shown in melanoma models treated with PLX-3397. Zhu et al. found that PD-1 and PD-L1 expression on TAMs and CTLA-4 expression on CD8+ T-cells were upregulated by CSF-1R inhibition. The addition of PD-1 or CTLA-4 antagonists in conjunction with PLX-3397 led to a more than 90% reduction in tumor progression. This study again suggested that small molecule inhibition with CSF-1R can enhance checkpoint blockade therapy.

Other small molecules targeting CSF-1R, such as BLZ945 and ARRY-382, have also been developed and have shown similar preclinical outcomes to PLX-3397. BLZ945 is a unique CSF-1R inhibitor with the ability to penetrate the central nervous system (CNS). For this reason, it was investigated in glioblastoma multiforme (GBM) mouse models [82]. Despite multiple tumor-specific factors in GBM that dampened TAM depletion, BLZ945 was found to reduce polarization towards an M2 macrophage phenotype [82]. BLZ945 ultimately inhibited tumor growth and led to increased survival in GBM [82]. CSF-1R inhibition and its anti-tumor effects are not limited to solid tumor subtypes, but have also been appreciated in hematologic malignancies, where CSF-1R expressing macrophages within the TME stimulate tumor survival. For instance, when two inhibitors of CSF-1R, GW-580 and ARRY-382, were added to the sera of chronic lymphocytic leukemia patients in vitro, it resulted in decreased tumor-supportive macrophages and depleted CD14+ monocytes in the TME [83].

Studies have also shown that CSF-1R inhibition may sensitize tumor cells to more traditional cytotoxic therapy [84]. In lung cancer preclinical models, CSF-1R inhibition has been shown to sensitize cisplatin-resistant lung cancer cell populations against platinum-based therapy, further supporting its roles as an adjunctive agent not only to immunotherapy but also chemotherapy [85].

Preclinical investigations of PLX-3397, BLZ945 and ARRY-382 have paved the way for clinical studies of CSF-1R inhibition via small molecules and mABs in diverse tumor types from GBM to pancreatic, ovarian and colorectal cancers (Table 1). Among these small molecule inhibitors of the CSF-1/CSF-1R pathway, PLX-3397 (Pexidartinib) currently has the most clinical data. PLX-3397 was evaluated in 37 patients with recurrent GBM, where it was tolerated well and with excellent CNS penetration. However it had minimal clinical efficacy, as only 8.6% had a progression free survival of 6 months, with no objective responses observed [86]. A phase I dose escalation study of PLX-3397, among multiple advanced tumor types (CRC, ovarian, breast, leiomyosarcoma, PDAC, lung) also noted a favorable safety profile and a marked reduction in a defined subset of circulating monocytes (CD14^dim/CD16⁺) [87]. In these studies, the most common side effects noted for PLX-3397 were fatigue, nausea, anemia, decreased appetite, rash, hair depigmentation, headache, constipation and transaminitis. Most recently, a pivotal phase III study (ENLIVEN) evaluating PLX-3397 was completed in 120 patients with advanced symptomatic tenosynovial giant cell tumors (TGCT), also known as pigmented villonodular synovitis, a malignancy in which surgical tumor resection often results in worsening functional status and morbidity [88]. Overexpression of CSF-1 is associated with this rare tumor type and the disease itself is linked to significant reactive inflammation in the tumor environment, suggesting a role of CSF-1 targeted therapy [89]. ENLIVEN showed that PLX-3397 significantly reduced the tumor size with a 39% overall tumor response, compared to no tumor response in patients treated with placebo [88].Tab1

Active Recruiting Current Clinical Trials with CSF-1R Inhibitors in Various Malignancies

To enhance the clinical responses garnered by CSF-1R inhibition, numerous ongoing clinical trials are combining small molecules inhibitors or mABs of CSF-1R with immunotherapy and/or cytotoxic chemotherapy (Table 1). Recently, preliminary efficacy data from a phase 1 dose escalation and expansion trial by Wainberg et al. looking at a combination of anti-CSF-1R (cabiralizumab) and anti-PD-1 mABs reported an objective response rate of 13% (four patients) amongst a cohort of 31 patients with advanced pancreatic cancer and most of whom were heavily-pretreated. All four of these patients had microsatellite stable disease, which historically has been unresponsive to PD-1/PD-L1 blockade. Three of these patients experienced a partial response and one had stable disease, with two patients experiencing a reduction in target lesions of 50% or more [90]. Despite cabiralizumab being a mAb, this study provides evidence to support further investigation of small molecules targeting CSF-1R in combination with immunotherapy. Small molecule inhibition of CSF-1R with chemotherapy has also shown promising clinical results. For instance, ABT-869, another novel small molecule inhibitor of CSF-1R, in combination with weekly paclitaxel in a small phase I study, showed clinical activity in 2 of 5 patients [91].

In conclusion, preclinical and clinical studies have demonstrated the benefit of combining CSF-1R inhibitors with immunotherapy and/or chemotherapy. This is an active area of research where CSF-1R inhibitors are a novel class of immunomodulatory therapeutics that have the capacity to unlock the full potential of immunotherapy in advanced malignancies.

As such, the effect of FAK is not only limited to cells of tumor origin, but also to cells within or recruited to the TME. FAK signaling is intimately involved in various aspects of the TME, particularly immunosuppression and stromal alterations. Studies have shown that inhibition of FAK diminishes the recruitment and migration of CAFs [96]. CAFs are abundant in the tumor stromal environment and are implicated in tumor growth, angiogenesis, metastasis and drug resistance [97]. In pancreatic cancer, the stroma and TME are characterized by increased collagen deposition with an elevated fibrotic response and infiltration of CAFs [98]. In a study by Stokes et al., pancreatic tumors from animals treated with PF-562,271 (VS-6063, [defactinib] a small molecule inhibitor of FAK) led to a significant decrease in the number CAFs and a significant decrease in tumor cell proliferation [96]. Additionally, CAFs have been shown to suppress CD8+ T-cells, where those cells conditioned by CAFs had diminished cytotoxic capacity. Furthermore, CAFs are associated with T-cell dysfunction via PD-L2 and fas ligand engagement [99].

Beyond CAFs, many preclinical studies have revealed that FAK signaling is closely involved in the activity of MDSCs, TAMs and Tregs within the TME [64, 67]. In squamous cell carcinoma mouse models, small molecule FAK inhibitor, VS4718, was shown to decrease immunosuppressive MDSCs, TAMs and Tregs, which then led to increased CD8+ T-cells within the tumor and enhancement of CD8+ T-cell-mediated suppression of cancerous cells [66].

In many tumors, particularly pancreatic cancer, studies have shown that the efficacy of traditional cytotoxic chemotherapy and immunotherapy can be improved by decreasing the density of peri-tumor stroma and the infiltration of myeloid cells [100, 101]. Jiang et al. demonstrated that FAK inhibition can reduce both fibrosis and immune-inhibitory myeloid cells [67]. Using genetically modified KPC (p48-Cre/LSL-Kras^G12D/p53^Flox/Flox) mouse models, Jiang et al. found that FAK inhibitor, VS-4718, decreased the stromal density of the pancreatic tumors, and reduced MDSCs, TAMs and Tregs infiltration into the tumor. They also discovered that FAK inhibition potentiated anti-PD1 therapy, thereby decreasing tumor burden and improving survival. Mice treated with gemcitabine, anti-PD-1 therapy and FAK inhibition had a 2.5-fold increase in median survival compared to those treated without FAK inhibition. Tumors from mice treated with FAK inhibition, gemcitabine and anti-PD1 therapy also had a significantly increased number of tumor-infiltrating CD8+ T-cells compared to mice treated with gemcitabine and anti-PD1 therapy without FAK inhibition [67].

An additional benefit of FAK inhibition is its ability to decrease CSCs. CSCs are unique cells within a tumor that are capable of self-renewal, able to generate more cancer cells with heterogeneous differentiation and typically resistant to standard therapies, leading to tumor resistance, recurrence and metastasis [102, 103]. In preclinical malignant mesothelioma models, standard cytotoxic therapies such as pemetrexed, cisplatin, gemcitabine and vinorelbine have been shown to increase CSCs, but when FAK inhibition is added, CSCs decrease [104]. CSCs do not exist in isolation, but are influenced by critical factors within the TME such as cytokines, small RNAs, TAMs and fibroblasts, which impact their unique niche [105, 106]. These factors regulate the invasiveness, metastatic potential and differentiation of CSCs, as well as confer a tumor-protective phenotype.

Based on these promising preclinical studies elucidating the role of FAK inhibition in modulating the immune milieu and fibrosis within the TME, clinical trials are investigating combination therapy of FAK inhibitors with cytotoxic chemotherapy and/or immunotherapy (Table 2). FAK overexpression has been noted in many tumor types, with associated negative prognostic factors, including HCC, NSCLC, colon, breast, pancreatic and ovarian cancers [26]. One study found that 68% of invasive ovarian cancers overexpressed FAK, which was associated with significantly higher tumor stages and tumor grades, positive lymph nodes and distant metastasis, and supported investigation of FAK inhibitor in advanced ovarian cancer [107].Tab2

Active Recruiting Current Clinical Trials with FAK Inhibitors in Various Malignancies

Preliminary data from a phase 1 dose escalation study of Defactinib, anti-PD1 therapy pembrolizumab and gemcitabine in patients with advanced solid tumors, with an expansion cohort for patients with advanced PDAC, have already shown that the combination therapy is well-tolerated (NCT02546531) [19]. Defactinib (VS-6063) is a selective adenosine triphosphate (ATP) that is a competitive and reversible inhibitor of human FAK and one of many FAK inhibitors in development. In addition, the study also reported that biopsies in patients with PDAC have decreased p-FAK and changes in T-cells infiltration following treatment [19]. The most common side effects noted with FAK inhibition were nausea, vomiting, pruritus, fevers and myalgias. The expansion cohort is currently ongoing with pending correlative and efficacy data. This phase I study and preclinical work with FAK have led to a phase II clinical trial (NCT03727880) combining neoadjuvant and adjuvant pembrolizumab and defactinib following neoadjuvant standard of care chemotherapy in subjects with high-risk resectable PDAC. This study will evaluate if reprograming the TME following chemotherapy by modulating TAMs and MDSCs with FAK inhibition can potentiate anti-PD-1 antibody therapy, and thus lead to improved effector T-cell infiltration and pathologic response.

Defactinib was also studied in malignant pleural mesothelioma in a phase II study with 30 participants. Objective partial response was observed in 13%, stable disease in 67% and progression in 17% of patients. This study also investigated the biological and immune implications of FAK inhibitor therapy on the TME, and showed that treatment with defactinib in malignant pleural mesothelioma resulted in a 75% reduction in p-FAK. Within the TME of treated subjects, there was increased naïve CD4+ and CD8+ T-cells, reduction of myeloid and Treg immuno-suppressive cells and reduction of exhausted T-cells and peripheral MDSCs. This study showed that defactintib has both therapeutic and immunomodulatory effects in patients with an aggressive malignancy, such as malignant pleural mesothelioma [108]. Currently there is a dose escalation study is underway in Europe, where defactinib is being combined with pembrolizumab in refractory advanced solid tumors and expansion cohorts in NSCLC, mesothelioma and pancreatic neoplasms (NCT02758587).

Defactinib has also shown clinical promise in combination with chemotherapy. Based on evidence showing elevated FAK expression in ovarian cancer, defactinib has also been studied in 18 patients with advanced ovarian cancer in combination with weekly paclitaxel, where a decrease of p-FAK was observed in all 3 patients who underwent paired biopsies. One patient had a complete response by RECIST, one patient an ongoing partial response of > 6 months and one patient with ongoing stable disease of > 8 months [109].

FAK has tremendous potential as a small molecular target, as it is implicated in modulating the immunosuppressive components of the TME, as well as the resistant and aggressive phenotype of CSCs. FAK inhibition leads to anti-tumor activity and when used in combination therapy, has the potential to increase the effectiveness of traditional cytotoxic chemotherapy and immunotherapy, particularly for aggressive and refractory malignancies.