TAM Receptors Research Articles

Abstract 1735: The potent and selective MERTK/AXL inhibitor PF-5807/ARRY-067 activates dendritic cells to cross-prime CD8+ T cells for anti-tumor activity

Abstract The TAM family of receptor tyrosine kinases (MERTK, AXL, and TYRO3) are expressed on DCs and macrophages, and they serve a critical role in efferocytosis, by which apoptotic bodies are cleared by engulfment. Their activation drives phagocytic activity concomitant to promoting an immuno-suppressive, tolerogenic state in DCs and the M2 polarization of macrophages. This dual signaling mechanism allows for the clearance of cells undergoing non-pathologic apoptotic turnover without inappropriately breaking self-tolerance and driving auto-immunity. Unlike most immuno-therapy targets, the KOs of the TAM kinases, PD-1, and CTLA-4 share common auto-immune disease phenotypes such as splenomegaly, immune infiltration of the liver, arthritis, and lupus. Furthermore, viruses have evolved to promote PD-1/CTLA-4 signaling to suppress the anti-viral immune response, and they similarly target the TAM receptors via apoptotic mimicry to reduce the inflammatory response. This confluence of immune phenotypes provided the biological rationale for the discovery of PF-5807/ARRY-067, a potent and selective inhibitor of MERTK/AXL as a DC-activating immuno-oncology therapeutic. Based on the IC50 for inhibition of phospho-MERTK/AXL in cell-based assays, our mouse PK and PK/PD studies determined that 50 mg/kg PF-5807 provided IC90 plasma coverage for 12 hours. This PK profile supported a twice-daily dosing schedule for maintaining continuous suppression of MERTK and AXL, which was used for mechanistic and efficacy studies in mouse models. To investigate DC and T cell responses to PF-5807, mice were adoptively-transferred with OT-I T cells and challenged with ovalbumin immunization; flow cytometry analysis indicated that PF-5807 treatment increased the fraction of activated, Class II MHC-positive DCs, as well as cross-primed IFNγ+ TNFα+ OT-I T cells. Also, we found that cytotoxic T lymphocytes were necessary for tumor control in the E0771 mouse syngeneic tumor model, as depletion of CD8+ T cells abrogated all activity by PF-5807 in inhibiting tumor growth. This anti-tumor activity was further demonstrated in the MC-38 and EMT6 tumor models, where single agent PF-5807 effected 57-88% TGI and a 13-38% cure rate, while combination with PD-1 blockade drove 99-100% TGI and a 75-100% cure rate. Finally, these complete responders rejected tumor re-challenge without further treatment, resulting in extended survival and durable cures that corroborate the development of immunologic memory. Therefore, we find that PF-5807 activates DCs to cross-prime CD8+ T cells, which are necessary for tumor control and are likely responsible for the establishment of tumor-specific memory T cells. PF-5807 is currently in an ongoing Phase I clinical trial in patients with advanced or metastatic solid tumors, with the intent to explore combination with anti-PD-1/anti-PD-L1 antibodies. Citation Format: Jim Wong, Jacqueline Harrison, Karyn Bouhana, Nhan Nguyen, Namir Shaabani, Vincent F. Vartabedian, LouAnn Cable, Robert Rieger, Lisa Pieti Opie, Shelley Allen, Adam Cook, Joshua Dahlke, Ronald Hinklin, David Chantry, Shannon Winski, John Teijaro. The potent and selective MERTK/AXL inhibitor PF-5807/ARRY-067 activates dendritic cells to cross-prime CD8+ T cells for anti-tumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1735.

Abstract CT035: Safety/tolerability and preliminary antitumor activity of sitravatinib plus tislelizumab in patients with PD-(L)1 refractory/resistant unresectable or metastatic melanoma from a phase 1b study

Abstract Background: While PD-(L)1 inhibitors have improved outcomes for melanoma, a substantial proportion of patients do not respond or develop resistance. Sitravatinib, a spectrum-selective TKI targeting TAM receptors (Tyro3/Axl/MerTK) and VEGFR2, reduces the number of myeloid-derived suppressor cells and regulatory T cells while increasing the ratio of M1/M2-polarized macrophages, which may overcome an immunosuppressive tumor microenvironment and augment antitumor immune responses. Tislelizumab, an anti-PD-1 antibody engineered to minimize binding to FcγR on macrophages to abrogate antibody-dependent phagocytosis, has shown clinical activity as a single agent and in combination with chemotherapy in patients with advanced solid tumors, including melanoma. Methods: We report results from the melanoma cohort of an ongoing multicohort phase 1b study (BGB-900-103; NCT03666143) assessing safety/tolerability and preliminary antitumor activity of sitravatinib + tislelizumab in advanced solid tumors. Eligible patients had unresectable or metastatic melanoma refractory/resistant to PD-(L)1 inhibitors and had not received other prior immunotherapy (eg, anti-CTLA-4, -OXO40, or -CD137) or anti-BRAF/MEK therapy. Patients received oral sitravatinib 120 mg once daily and tislelizumab 200 mg IV Q3W until a discontinuation criterion was met. The primary endpoint was safety/tolerability; key secondary endpoints included investigator-assessed objective response rate (ORR), disease control rate (DCR), and progression-free survival (PFS). Results: As of Oct 13, 2020, 25 patients were enrolled; 16 patients (64%) remained on treatment. All patients received 1 prior line of PD-(L)1 therapy, median age was 51 years (range: 23-79), and baseline histology included cutaneous (n=12; 48%), acral (n=7; 28%), and mucosal (n=4; 16%) subtypes. Median duration of study follow-up was 5.5 months (range: 1.5-13.3). Adverse events (AEs) were reported in 25 patients (100%); the most commonly reported grade ≥3 AE was hypertension (n=3; 12%). Serious AEs were reported in 4% (n=1/25) of patients. Dose reductions of sitravatinib due to AEs occurred in 13 patients. No AEs led to death. Six patients achieved a confirmed partial response. Confirmed ORR was 24.0% (95% CI: 9.36-45.13); DCR was 88.0% (95% CI: 68.78-97.45). Median PFS was 6.7 months (95% CI: 4.07, not evaluable). Conclusions: Sitravatinib + tislelizumab was generally well tolerated, had a manageable safety/tolerability profile, and demonstrated preliminary antitumor activity in patients with refractory/resistant unresectable or metastatic melanoma. Further investigation of sitravatinib + tislelizumab in these patients is warranted. Citation Format: Chuanliang Cui, Hongming Pan, Matteo Carlino, Jiuwei Cui, Xuan Wang, Cheng Chen, Xiao Xiang, Liu Yang, Jun Guo. Safety/tolerability and preliminary antitumor activity of sitravatinib plus tislelizumab in patients with PD-(L)1 refractory/resistant unresectable or metastatic melanoma from a phase 1b study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr CT035.

Abstract CT013: Safety/tolerability and preliminary antitumor activity of sitravatinib plus tislelizumab in patients with advanced platinum-resistant ovarian cancer (PROC)

Abstract Background: Combining a PD-1 inhibitor and an agent with immune modulatory and antitumor properties may enhance antitumor activity of either agent. Sitravatinib, a spectrum-selective TKI targeting TAM receptors (Tyro3/Axl/MerTK) and VEGFR2, reduces the number of myeloid-derived suppressor cells and regulatory T cells while increasing the ratio of M1/M2-polarized macrophages, which may overcome an immunosuppressive tumor microenvironment and augment antitumor immune responses. Tislelizumab, an anti-PD-1 antibody engineered to minimize binding to FcγR on macrophages and abrogate antibody-dependent phagocytosis, has shown single-agent clinical activity in patients (pts) with advanced solid tumors. This open-label, multicohort, phase 1b study assessed safety/tolerability and preliminary antitumor activity of sitravatinib + tislelizumab in advanced solid tumors (BGB-900-103; NCT03666143). We report results from the PROC cohort. Methods: Anti-PD-(L)1 antibody-naïve pts with histologically confirmed, advanced PROC (disease progression <6 mo after last platinum treatment) were enrolled. While platinum-resistant pts were included, pts with platinum-refractory disease were excluded. Patients received sitravatinib 120 mg PO QD and tislelizumab 200 mg IV Q3W. Primary endpoint was safety/tolerability of sitravatinib + tislelizumab. Key secondary endpoints were investigator-assessed objective response rate (ORR), disease control rate (DCR), and progression-free survival (PFS) per RECIST v1.1; overall survival (OS) was also assessed. PD-L1 IHC assay (Ventana SP263) and assessment of plasma VEGF/serum CXCL10 were retrospective. Results: As of Oct 13, 2020, 60 PROC pts were enrolled; 13 (22%) remained on treatment. Median age was 64 yrs (range 26-80); pts received a median of 4 (range 1-11) prior regimens. Median follow-up was 6.0 mo (range 0.2-23.4). Treatment-emergent adverse events (TEAEs) of any grade/grade ≥3 occurred in 97%/68% of pts; TEAEs led to sitravatinib dose reduction in 50% of pts. Nausea (33%), hypertension (18%), and abdominal pain (12%) were the most commonly reported grade ≥3 TEAEs. The 2 fatal AEs (malignant GI obstruction, dyspnea) were deemed unrelated to treatment. Confirmed ORR was 26.4% (95% CI, 15.3-40.3), with 14 pts achieving partial response; DCR was 77.4% (95% CI, 63.8-87.7). Median duration of response was 4.7 mo (95% CI, 2.8-not estimable). There was no clear association between PD-L1 expression and clinical response; plasma VEGF and serum CXCL10 increased after treatment (P<0.0001 for both). Median PFS was 4.1 mo (95% CI, 4.0-5.1); preliminary median OS was 12.9 mo (95% CI, 6.3-17.2). Conclusions: Sitravatinib + tislelizumab was tolerable and showed preliminary antitumor activity in pts with advanced PROC. Further investigation of sitravatinib + tislelizumab in PROC is warranted. Citation Format: Jeffrey Goh, Jermaine Coward, Bo Gao, Ines P. da Silva, Mark Voskoboynik, Daphne Day, Amy L. Body, Hui K. Gan, Cheng Chen, Xiao Xiang, Cong Fei, Liu Yang, Michael Millward. Safety/tolerability and preliminary antitumor activity of sitravatinib plus tislelizumab in patients with advanced platinum-resistant ovarian cancer (PROC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr CT013.

Co-Ultramicronized Palmitoylethanolamide/Luteolin-Induced Oligodendrocyte Precursor Cell Differentiation is Associated With Tyro3 Receptor Upregulation.

Remyelination in patients with multiple sclerosis frequently fails, especially in the chronic phase of the disease promoting axonal and neuronal degeneration and progressive disease disability. Drug-based therapies able to promote endogenous remyelination capability of oligodendrocytes are thus emerging as primary approaches to multiple sclerosis. We have recently reported that the co-ultramicronized composite of palmitoylethanolamide and the flavonoid luteolin (PEALut) promotes oligodendrocyte precursor cell (OPC) maturation without affecting proliferation. Since TAM receptor signaling has been reported to be important modulator of oligodendrocyte survival, we here evaluated the eventual involvement of TAM receptors in PEALut-induced OPC maturation. The mRNAs related to TAM receptors -Tyro3, Axl, and Mertk- were all present at day 2 in vitro. However, while Tyro3 gene expression significantly increased upon cell differentiation, Axl and Mertk did not change during the first week in vitro. Tyro3 gene expression developmental pattern resembled that of MBP myelin protein. In OPCs treated with PEALut the developmental increase of Tyro3 mRNA was significantly higher as compared to vehicle while was reduced gene expression related to Axl and Mertk. Rapamycin, an inhibitor of mTOR, prevented oligodendrocyte growth differentiation and myelination. PEALut, administered to the cultures 30 min after rapamycin, prevented the alteration of mRNA basal expression of the TAM receptors as well as the expression of myelin proteins MBP and CNPase. Altogether, data obtained confirm that PEALut promotes oligodendrocyte differentiation as shown by the increase of MBP and CNPase and Tyro3 mRNAs as well as CNPase and Tyro3 immunostainings. The finding that these effects are reduced when OPCs are exposed to rapamycin suggests an involvement of mTOR signaling in PEALut effects.

TAM kinase signaling is indispensable for proper skeletal muscle regeneration in mice

Skeletal muscle regeneration following injury results from the proliferation and differentiation of myogenic stem cells, called satellite cells, located beneath the basal lamina of the muscle fibers. Infiltrating macrophages play an essential role in the process partly by clearing the necrotic cell debris, partly by producing cytokines that guide myogenesis. Infiltrating macrophages are at the beginning pro-inflammatory, but phagocytosis of dead cells induces a phenotypic change to become healing macrophages that regulate inflammation, myoblast fusion and growth, fibrosis, vascularization and return to homeostasis. The TAM receptor kinases Mer and Axl are known efferocytosis receptors in macrophages functioning in tolerogenic or inflammatory conditions, respectively. Here we investigated their involvement in the muscle regeneration process by studying the muscle repair following cardiotoxin-induced injury in Mer−/− mice. We found that Axl was the only TAM kinase receptor expressed on the protein level by skeletal muscle and C2C12 myoblast cells, while Mer was the dominant TAM kinase receptor in the CD45+ cells, and its expression significantly increased during repair. Mer ablation did not affect the skeletal muscle weight or structure, but following injury it resulted in a delay in the clearance of necrotic muscle cell debris, in the healing phenotype conversion of macrophages and consequently in a significant delay in the full muscle regeneration. Administration of the TAM kinase inhibitor BMS-777607 to wild type mice mimicked the effect of Mer ablation on the muscle regeneration process, but in addition, it resulted in a long-persisting necrotic area. Finally, in vitro inhibition of TAM kinase signaling in C2C12 myoblasts resulted in decreased viability and in impaired myotube growth. Our work identifies Axl as a survival and growth receptor in the mouse myoblasts, and reveals the contribution of TAM kinase-mediated signaling to the skeletal muscle regeneration both in macrophages and in myoblasts.

Loss of the TAM receptor Mer contributes to Sjögren’s syndrome-like disease in mice

Abstract Sjögren’s syndrome (SjS) is a chronic autoimmune disease primarily involving the exocrine glands where the involvement of the innate immune system is largely uncharacterized. Mer signaling has been found to be protective in several autoimmune diseases but remains unstudied in SjS. Here we report that soluble Mer (sMer) levels were elevated in SjS patient sera and positively correlated with focus score, ocular staining scores, rheumatoid factor, and Ro60 autoantibody levels. Increased sMer was also detected in C57BL/6.NOD-Aec1Aec2 (SjSS) mouse sera, coinciding with higher ADAM17 activity, the enzyme responsible for cleavage of Mer, and inactivation of such. Mer signaling outcomes were observed to be diminished in SjSS mice as evidenced by impaired efferocytosis in SjSS mouse macrophages and decreased Socs1 and Socs3 expression in SjSS salivary gland (SG). SjS diagnostic criteria were examined in MerKO mice to evaluate the protective role of Mer in SjS disease pathogenesis. MerKO mice developed SG infiltrates of B and T lymphocytes, SG apoptotic cells, antinuclear antibodies, and reduced saliva flow. Together, our data suggest a sequence of events where enhanced ADAM17 activity increases Mer inactivation, depresses Mer signaling, and removes a protection against the loss of self-tolerance and onset of autoimmune disease in SjSS mice.

Circulating Small Extracellular Vesicles Activate TYRO3 to Drive Cancer Metastasis and Chemoresistance.

Extracellular vesicles (EV) in the tumor microenvironment have emerged as crucial mediators that promote proliferation, metastasis, and chemoresistance. However, the role of circulating small EVs (csEV) in cancer progression remains poorly understood. In this study, we report that csEV facilitate cancer progression and determine its molecular mechanism. csEVs strongly promoted the migration of cancer cells via interaction with phosphatidylserine of csEVs. Among the three TAM receptors, TYRO3, AXL, and MerTK, TYRO3 mainly interacted with csEVs. csEV-mediated TYRO3 activation promoted migration and metastasis via the epithelial-mesenchymal transition and stimulation of RhoA in invasive cancer cells. Additionally, csEV-TYRO3 interaction induced YAP activation, which led to increased cell proliferation and chemoresistance. Combination treatment with gefitinib and KRCT-6j, a selective TYRO3 inhibitor, significantly reduced tumor volume in xenografts implanted with gefitinib-resistant non-small cell lung cancer cells. The results of this study show that TYRO3 activation by csEVs facilitates cancer cell migration and chemoresistance by activation of RhoA or YAP, indicating that the csEV/TYRO3 interaction may serve as a potential therapeutic target for aggressive cancers in the clinic. SIGNIFICANCE: These findings demonstrate that circulating extracellular vesicles are a novel driver in migration and survival of aggressive cancer cells via TYRO3 activation. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/13/3539/F1.large.jpg.

Microglia use TAM receptors to detect and engulf amyloid β plaques.

Two microglial TAM receptor tyrosine kinases - Axl and Mer - have been linked to Alzheimer’s disease, but their roles in disease have not been tested experimentally. We find that in Alzheimer’s disease and its mouse models, induced expression of Axl and Mer in amyloid plaque-associated microglia was coupled to induced plaque decoration by the TAM ligand Gas6 and its co-ligand phosphatidylserine. In the APP/PS1 mouse model of Alzheimer’s disease, genetic ablation of Axl and Mer resulted in microglia that were unable to normally detect, respond to, organize, or phagocytose amyloid beta plaques. These major deficits notwithstanding, TAM-deficient APP/PS1 mice developed fewer dense-core plaques than APP/PS1 mice with normal microglia. Our findings reveal that the TAM system is an essential mediator of microglial recognition and engulfment of amyloid plaques, and that TAM-driven microglial phagocytosis does not inhibit, but rather promotes, dense-core plaque development.

Macrophage AXL receptor tyrosine kinase inflames the heart after reperfused myocardial infarction.

Tyro3, AXL, and MerTK (TAM) receptors are activated in macrophages in response to tissue injury and as such have been proposed as therapeutic targets to promote inflammation resolution during sterile wound healing, including myocardial infarction. Although the role of MerTK in cardioprotection is well characterized, the unique role of the other structurally similar TAMs, and particularly AXL, in clinically relevant models of myocardial ischemia/reperfusion infarction (IRI) is comparatively unknown. Utilizing complementary approaches, validated by flow cytometric analysis of human and murine macrophage subsets and conditional genetic loss and gain of function, we uncover a maladaptive role for myeloid AXL during IRI in the heart. Cross signaling between AXL and TLR4 in cardiac macrophages directed a switch to glycolytic metabolism and secretion of proinflammatory IL-1β, leading to increased intramyocardial inflammation, adverse ventricular remodeling, and impaired contractile function. AXL functioned independently of cardioprotective MerTK to reduce the efficacy of cardiac repair, but like MerTK, was proteolytically cleaved. Administration of a selective small molecule AXL inhibitor alone improved cardiac healing, which was further enhanced in combination with blockade of MerTK cleavage. These data support further exploration of macrophage TAM receptors as therapeutic targets for myocardial infarction.

A phase II study of sitravatinib (Sitra) in combination with nivolumab (Nivo) in patients (Pts) undergoing nephrectomy for locally-advanced clear cell renal cell carcinoma (accRCC).

312 Background: Sitra is a spectrum-selective receptor tyrosine kinase inhibitor (TKI) that targets TAM receptors (TYRO3, AXL, MERTK), VEGFR2, c-Kit, and MET. These receptors regulate several immune suppressive cell types in the tumor microenvironment, including M2-polarized macrophages, MDSCs, and T regulatory cells, which are implicated in resistance to checkpoint inhibitors. ccRCC is characterized by upregulation of VEGF and overexpression of MET and AXL. Sitra may combine effectively with immune checkpoint inhibition to augment antitumor activity in ccRCC. About 39% of patients with accRCC who receive surgery with curative intent relapse representing an unmet need in this setting. Together these data support the evaluation of neoadjuvant sitra with nivo in accRCC. Methods: This phase II study (NCT03680521) evaluated sitra and nivo in pts with locally- advanced ccRCC who were candidates for curative nephrectomy. Single-agent sitra (120 mg) was administered daily (QD) for 2 weeks, with nivo (240 mg intravenously Q2W) added to sitra for 4-6 weeks. A plan for potential dose de-escalation was implemented using a modified toxicity probability interval method with a maximum toxicity of 20% at the tolerated dose. Pts underwent pathology/tissue evaluation at 3 timepoints: biopsy prior to treatment, biopsy prior to the addition of nivo, and nephrectomy specimen evaluation at time of nephrectomy. The primary endpoint was objective response (RECIST 1.1); secondary endpoints included safety, PK, and correlative immune effects (selected protein and gene expression and immune cell populations). Results: A total of 20 pts were evaluated for safety (95% had T3 or higher stage tumors, 65% with baseline hypertension). Dose-limiting toxicities (DLTs) led to a dose de-escalation, resulting in 7 pts treated at 120 mg QD sitra and 13 pts treated at 80 mg QD. DLTs included grade 3 (Gr3) hypertension (n=6); deep vein thrombosis and pulmonary embolism (Gr3) were observed in 1 additional pt. Median duration of sitra treatment was 6.3 weeks at the 80 mg dose and 7.1 weeks at the 120 mg dose. With a median follow-up of 9.4 months after initiation of systemic therapy, no pts have relapsed. In 17 pts evaluable for efficacy, the investigator-assessed confirmed ORR was 11.8%, including 2 PRs (33.3% ORR in pts who received 120 mg sitra). No pts experienced progressive disease while on therapy. Median DFS was not reached. There was 1 delayed surgery due to nivo-related thyroiditis that resolved. Reported TRAEs: Gr1/Gr2 in 55% of pts (dysphonia 50%, fatigue 45%, diarrhea 40%, hypertension 30%, increased ALT 30%), Gr3 in 45% of pts (hypertension 30%). There were no Gr4/Gr5 TRAEs. Correlative blood and tissue analyses will be presented. Conclusions: The combination of sitra and nivo is clinically active with a manageable safety profile as a neoadjuvant therapy for accRCC. Clinical trial information: NCT03680521 .

Phase I/IB trial of sitravatinib (Sitra) + nivolumab (Nivo) + ipilimumab (Ipi) in patients (pts) with advanced clear cell renal cell carcinoma (accRCC) or other solid malignancies.

TPS365 Background: Sitra is a spectrum-selective receptor tyrosine kinase inhibitor (TKI) that targets TAM receptors (TYRO3, AXL, MERTK), VEGFR2, c-Kit, and c-MET. Sitra has shown antitumor activity in accRCC, and reduces type 2 tumor-associated macrophages, regulatory T-cells and myeloid-derived suppressor cells and enhances T cell–mediated antitumor immune responses. The combination of sitra with anti-PD1 immune checkpoint inhibitors (ICIs), such as nivo, has an acceptable safety profile and demonstrates efficacy across multiple tumor types, including accRCC (Msaouel et al. J Clin Oncol, 2020, abstr 612). Ipi enhances the efficacy of nivo through distinct but complementary pathways to those targeted by sitra. We hypothesize that the triple combination of sitra + nivo + ipi will lead to more potent antitumor responses. This study is designed to determine the optimal dose of sitra when combined with nivo + ipi, and assess the safety as well as the preliminary efficacy of this combination in accRCC, and potentially other cancers, that have shown favorable responses to nivo + ipi. Methods: This phase I/Ib study (NCT04518046) is evaluating sitra + nivo + ipi in frontline advanced or metastatic ccRCC or other solid malignancies. The primary endpoint of the trial is safety and tolerability of this regimen. Secondary endpoints include: objective response rate (RECIST 1.1), duration of response, progression-free survival, overall survival, and pharmacokinetic measurements. The phase I dose escalation portion of the trial will enroll approximately 30 pts with intermediate- or poor-risk accRCC by International Metastatic RCC Database Consortium (IMDC) criteria, who will receive escalating doses of sitra (starting dose 35 mg orally QD) following the Time-to-Event Bayesian Optimal Interval (TITE-BOIN) design (Yuan et al. Clin Cancer Res, 2018), combined with nivo 3 mg/kg and ipi 1 mg/kg (NIVO3/IPI1) every 3 weeks (wks) for up to 4 doses, followed by maintenance nivo (240 mg every 2 wks or 480 mg every 4 wks). The phase Ib dose expansion cohorts will be initiated following identification of the recommended dose of sitra in combination with NIVO3/IPI1 and will enroll up to 31 pts for each of 2 cohorts: pts with intermediate-/poor-risk accRCC (Cohort A) and pts with favorable-risk accRCC (Cohort B). Pts will receive study treatment until disease progression, unacceptable adverse events, or pt withdrawal of consent. Future dose expansion cohorts may be added to include other solid tumors in which favorable activity has been previously demonstrated with nivo + ipi, such as metastatic urothelial carcinoma, melanoma, non–small-cell lung cancer, hepatocellular carcinoma, and colorectal cancer. The study is currently enrolling at 1 US site and additional sites may be added at dose expansion. At the time of the abstract submission, 3 pts have been enrolled. Clinical trial information: NCT04518046 .

Axl and Mertk Receptors Cooperate to Promote Breast Cancer Progression by Combined Oncogenic Signaling and Evasion of Host Antitumor Immunity.

Despite the promising clinical benefit of targeted and immune checkpoint blocking therapeutics, current strategies have limited success in breast cancer, indicating that additional inhibitory pathways are required to complement existing therapeutics. TAM receptors (Tyro-3, Axl, and Mertk) are often correlated with poor prognosis because of their capacities to sustain an immunosuppressive environment. Here, we ablate Axl on tumor cells using CRISPR/Cas9 gene editing, and by targeting Mertk in the tumor microenvironment (TME), we observed distinct functions of TAM as oncogenic kinases, as well as inhibitory immune receptors. Depletion of Axl suppressed cell intrinsic oncogenic properties, decreased tumor growth, reduced the incidence of lung metastasis and increased overall survival of mice when injected into mammary fat pad of syngeneic mice, and demonstrated synergy when combined with anti-PD-1 therapy. Blockade of Mertk function on macrophages decreased efferocytosis, altered the cytokine milieu, and resulted in suppressed macrophage gene expression patterns. Mertk-knockout mice or treatment with anti-Mertk-neutralizing mAb also altered the cellular immune profile, resulting in a more inflamed tumor environment with enhanced T-cell infiltration into tumors and T-cell-mediated cytotoxicity. The antitumor activity from Mertk inhibition was abrogated by depletion of cytotoxic CD8α T cells by using anti-CD8α mAb or by transplantation of tumor cells into B6.CB17-Prkdc SCID mice. Our data indicate that targeting Axl expressed on tumor cells and Mertk in the TME is predicted to have a combinatorial benefit to enhance current immunotherapies and that Axl and Mertk have distinct functional activities that impair host antitumor response. SIGNIFICANCE: This study demonstrates how TAM receptors act both as oncogenic tyrosine kinases and as receptors that mediate immune evasion in cancer progression.

Tyro3, Axl, Mertk receptor-mediated efferocytosis and immune regulation in the tumor environment.

Three structurally related tyrosine receptor cell surface kinases, Tyro3, Axl, and Mertk (TAM) have been recognized to modulate immune function, tissue homeostasis, cardiovasculature, and cancer. The TAM receptor family appears to operate in adult mammals across multiple cell types, suggesting both widespread and specific regulation of cell functions and immune niches. TAM family members regulate tissue homeostasis by monitoring the presence of phosphatidylserine expressed on stressed or apoptotic cells. The detection of phosphatidylserine on apoptotic cells requires intermediary molecules that opsonize the dying cells and tether them to TAM receptors on phagocytes. This complex promotes the engulfment of apoptotic cells, also known as efferocytosis, that leads to the resolution of inflammation and tissue healing. The immune mechanisms dictating these processes appear to fall upon specific family members or may involve a complex of different receptors acting cooperatively to resolve and repair damaged tissues. Here, we focus on the role of TAM receptors in triggering efferocytosis and its consequences in the regulation of immune responses in the context of inflammation and cancer.

Embryonic Microglia Interact with Hypothalamic Radial Glia during Development and Upregulate the TAM Receptors MERTK and AXL following an Insult

Despite a growing appreciation for microglial influences on the developing brain, the responsiveness of microglia to insults during gestation remains less well characterized, especially in the embryo when microglia themselves are still maturing. Here, we asked if fetal microglia could coordinate an innate immune response to an exogenous insult. Using time-lapse imaging, we showed that hypothalamic microglia actively surveyed their environment by near-constant "touching" of radial glia projections. However, following an insult (i.e., IUE or AAV transduction), this seemingly passive touching became more intimate and long lasting, ultimately resulting in the retraction of radial glial projections and degeneration into small pieces. Mechanistically, the TAM receptors MERTK and AXL were upregulated in microglia following the insult, and Annexin V treatment inhibited radial glia breakage and engulfment by microglia. These data demonstrate a remarkable responsiveness of embryonic microglia to insults during gestation, a critical window for neurodevelopment.

Opposite trends of GAS6 and GAS6-AS expressions in breast cancer tissues

Growth arrest-specific gene 6 (GAS6) is a growth factor-like cytokine whose function is related with vitamin K. This protein interacts with receptor tyrosine kinase proteins such as Tyro3, Axl, and TAM Receptor family, therefore affecting the tumorigenic processes via different mechanisms. GAS6-antisense 1 (GAS6-AS1) is a long non-coding RNAs (lncRNAs) that is transcribed from a genomic regions nearby GAS6. This lncRNA is also implicated in the pathobiology of cancer. We intended to judge the role of GAS6 and GAS6-AS1 in the pathogenesis of breast cancer through appraisal of their expression levels in breast cancer tissues and their paired neighboring non-cancerous samples. Expression of GAS6 was up-regulated in breast cancer tissues compared with neighboring tissues (Ratio of Mean Expressions = 2.18, P value = 4.98E-02). On the other hand, expression of GAS6-AS1 was down-regulated in breast tumor tissues compared with controls (Ratio of Mean Expressions = 0.37, P value = 4.26E-03). There were substantial correlations between expression levels GAS6 and GAS6-AS1 in non-cancerous tissues (r = 0.74, P value = 1.47e-13) and cancer tissues (r = 0.85, P value = 2.28e-20). Expression of GAS6-AS was associated with progesterone receptor status (P value = 1.36E-02). However, expressions of this gene and the sense transcript were not linked with any other clinical or demographic variable. Taken together, GAS6 and GAS6-AS1 might partake in the development of breast cancer.

GAS6 signaling tempers Th17 development in patients with multiple sclerosis and helminth infection.

Multiple sclerosis (MS) is a highly disabling neurodegenerative autoimmune condition in which an unbalanced immune response plays a critical role. Although the mechanisms remain poorly defined, helminth infections are known to modulate the severity and progression of chronic inflammatory diseases. The tyrosine kinase receptors TYRO3, AXL, and MERTK (TAM) have been described as inhibitors of the immune response in various inflammatory settings. We show here that patients with concurrent natural helminth infections and MS condition (HIMS) had an increased expression of the negative regulatory TAM receptors in antigen-presenting cells and their agonist GAS6 in circulating CD11bhigh and CD4+ T cells compared to patients with only MS. The Th17 subset was reduced in patients with HIMS with a subsequent downregulation of its pathogenic genetic program. Moreover, these CD4+ T cells promoted lower levels of the co-stimulatory molecules CD80, CD86, and CD40 on dendritic cells compared with CD4+ T cells from patients with MS, an effect that was GAS6-dependent. IL-10+ cells from patients with HIMS showed higher GAS6 expression levels than Th17 cells, and inhibition of phosphatidylserine/GAS6 binding led to an expansion of Th17 effector genes. The addition of GAS6 on activated CD4+ T cells from patients with MS restrains the Th17 gene expression signature. This cohort of patients with HIMS unravels a promising regulatory mechanism to dampen the Th17 inflammatory response in autoimmunity.

AXL activation leads to reduced amyloid plaque deposition in APP/PS‐1 mice

AbstractBackgroundMicroglia, the immune cells of the brain, play seemingly antagonistic roles in the Alzheimer’s disease (AD) brain. On one hand, microglia contribute to clearance of amyloid beta (Aβ) plaques via phagocytosis. On the other hand, activated microglia produce inflammatory cytokines that contribute to a hostile neuronal environment, exacerbating AD pathogenesis. Axl, a member of the TAM receptor family, is increased on microglia that surround Aβ plaques. Gas6, the primary ligand for Axl, promotes phagocytosis and suppresses inflammation in peripheral immune cells.MethodTo test the hypothesis that manipulation of the Axl‐Gas6 pathway contributes to Aβ clearance, we carried out intrahippocampal injections of an AAV‐Gas6 vector in male and female APP/PS‐1 mice at 4 or 7 months of age.ResultHippocampal overexpression of Gas6 for one or six months led to decreased Aβ plaque load when compared to the opposite hippocampus injected with a control AAV‐Gas6(delta) vector that lacks the Gla and EGF domains required for Axl phosphorylation. We are currently quantifying microglial responses in these brains as well as carrying out in vitro studies with microglia from Axl knockout mice to better determine the roles of the Gas6‐Axl signaling pathway in mediating Aβ phagocytosis and microglial inflammatory responses.ConclusionAxl activation in vivo leads to reduced Aβ plaque load. Results from this work will provide insight regarding potential mechanisms that may be harnessed to reduce AD‐related pathology.

Inhibition of adjuvant-induced TAM receptors potentiates cancer vaccine immunogenicity and therapeutic efficacy.

Analyzing immunomodulatory elements operating during antitumor vaccination in prostate cancer patients and murine models we identified IL-10-producing DC as a subset with poorer immunogenicity and clinical efficacy. Inhibitory TAM receptors MER and AXL were upregulated on murine IL-10+ DC. Thus, we analyzed conditions inducing these molecules and the potential benefit of their blockade during vaccination. MER and AXL upregulation was more efficiently induced by a vaccine containing Imiquimod than by a poly(I:C)-containing vaccine. Interestingly, MER expression was found on monocyte-derived DC, and was dependent on IL-10. TAM blockade improved Imiquimod-induced DC activation in vitro and in vivo, resulting in increased vaccine-induced T-cell responses, which were further reinforced by concomitant IL-10 inhibition. In different tumor models, a triple therapy (including vaccination, TAM inhibition and IL-10 blockade) provided the strongest therapeutic effect, associated with enhanced T-cell immunity and enhanced CD8+ T cell tumor infiltration. Finally, MER levels in DC used for vaccination in cancer patients correlated with IL-10 expression, showing an inverse association with vaccine-induced clinical response. These results suggest that TAM receptors upregulated during vaccination may constitute an additional target in combinatorial therapeutic vaccination strategies.

Proof of Concept: Protein S As an Immune Modulatory Agent to Control Pancreatic Cancer

Background: Pancreatic cancer (PC) remains one of the most difficult tumors to treat. PC is nearly always diagnosed late with 80% of PC patients having Stage 4 disease and an average life expectancy of a mere 4-6 months. It affects men and women equally. In spite of our understanding of KRAS as the driver mutation in the overwhelming majority of PC patients, drugs to target KRAS remain elusive so far. Chemotherapy and radiation remain the standard of care for PC patients, many of whom are unresectable at diagnosis. This bleak prognosis encourages scientists and drug developers to propose bold, creative treatment strategies to better manage PC. Protein S (PS) is a 75 KDa vitamin K-dependent potent natural anticoagulant. PC patients have much higher risk of thrombosis than the general population. In 2018, we have shown that HIF1α, commonly upregulated in hypoxic cancer tissues, suppresses PS expression. PS has coagulation-independent, receptor-mediated effects through the TAM family of receptor tyrosine kinases (TYRO3, AXL, and MERTK). We recently published (in press) data that indicate that PS suppresses the growth of PC cells in vitro and that PROS1 (PS) gene expression is associated with better survival in PC. Survival analysis based on public domain RNASeq data shows that PROS1 mRNA expression in PC is significantly correlated with better overall survival (OS) up to 18 months (Figure 1). Additionally, very recent data indicate that PS receptor MERTK is a late costimulatory signal in CD8 T-cells, which is required for the establishment of immunological memory and anti-tumor immune responses in melanoma. Our proof of concept (POC) shown in the schematic is PS directly suppresses PC cell growth and promotes CD8-mediated tumor immunity against PC. Results & Discussion: PS and its properties as a signaling molecule in the context of cancer progression and cellular aggressiveness have not been studied in-depth. The expression levels of the individual TAM receptors showed less than 1-fold variation among the cell lines tested; whereas, PS and GAS6 levels were significantly different. PS/GAS6 ratio directly correlates with the aggressiveness of PC cell lines. We found that the lower the ratio, the higher was the aggressiveness. We used three cell lines, MIA PaCa-2, PANC-1, and BXPC-3 with population doubling times of 40 hours, 52 hours and 72 hours, respectively (per information from ATCC). The basal levels of the three TAM receptors were relatively uniform among the three cell lines, i.e., less than 5% difference in expression level. Interestingly, although PANC-1 cells, with an intermediate degree of aggressiveness, had a PS/GAS6 ratio of 1, MIA-PaCa-2 cells had a significantly lower PS/GAS6 ratio indicating that more aggressive cell line has more GAS6. The least aggressive cell line BXPC-3 cells had a much higher PS/GAS6 ratio. PS overexpression reduced survival and proliferation of PANC-1 and MIA PaCa-2 cells. To further confirm that PS overexpression accelerated apoptosis of PC cells, we generated flow data using Annexin V/7AAD (7-Aminoactinomycin D) staining. Notably, PS gene knockdown by SiRNA or GAS6 overexpression resulted in increase in aggressiveness of those cell lines tested in our published data (In press). Conversely, GAS6 gene knockdown attenuated the aggressive phenotypes of these cell lines tested. Conclusion: We propose to test PS infusion in PC to downregulate HIF1α and modulate the tumor microenvironment (TME) to promote sustained tumor immunity. Our preliminary data and publicly available gene expression profiles suggest that PS may have therapeutic value in PC. Disclosures No relevant conflicts of interest to declare.

Dual targeting of TAM receptors Tyro3, Axl, and MerTK: Role in tumors and the tumor immune microenvironment.

In both normal and tumor tissues, receptor tyrosine kinases (RTKs) may be pleiotropically expressed. The RTKs not only regulate ordinary cellular processes, including proliferation, survival, adhesion, and migration, but also have a critical role in the development of many types of cancer. The Tyro3, Axl, and MerTK (TAM) family of RTKs (Tyro3, Axl, and MerTK) plays a pleiotropic role in phagocytosis, inflammation, and normal cellular processes. In this article, we highlight the cellular activities of TAM receptors and discuss their roles in cancer and immune cells. We also discuss cancer therapies that target TAM receptors. Further research is needed to elucidate the function of TAM receptors in immune cells toward the development of new targeted immunotherapies for cancer.