AXL Activation Research Articles

Gas6 Promotes Microglia Efferocytosis and Suppresses Inflammation Through Activating Axl/Rac1 Signaling in Subarachnoid Hemorrhage Mice.

Early brain injury (EBI) following subarachnoid hemorrhage (SAH) is characterized byrapid development of neuron apoptosis and dysregulated inflammatory response. Microglia efferocytosis plays a critical role in the clearance of apoptotic cells, attenuation of inflammation, and minimizing brain injury in various pathological conditions. Here, using a mouse SAH model, we aim to investigate whether microglia efferocytosis is involved in post-SAH inflammation and to determine the underlying signaling pathway. We hypothesized that TAM receptors and their ligands regulate this process. To prove our hypothesis, the expression and cellular location of TAM (Tyro3, Axl, and Mertk) receptors and their ligands growth arrest-specific 6 (Gas6) and Protein S (ProS1) were examined by PCR, western blots, and fluorescence immunostaining. Thirty minutes after SAH, mice received an intraventricular injection of recombinant Gas6 (rGas6) or recombinant ProS1 (rPros1) and underwent evaluations of inflammatory mediator expression, neurological deficits, and blood-brain barrier integrity at 24h. Microglia efferocytosis of apoptotic neurons was analyzed in vivo and in vitro. The potential mechanism was determined by inhibiting or knocking down TAM receptors and Rac1 by specific inhibitors or siRNA. SAH induced upregulation of Axl and its ligand Gas6. The administration of rGas6 but not rPros1 promoted microglia efferocytosis, alleviated inflammation, and ameliorated SAH-induced BBB breakdown and neurological deficits. The beneficial effects of rGas6 were arrogated by inhibiting or knocking down Axl and Rac1. We concluded that rGas6 attenuated the development of early brain injury in mice after SAH by facilitating microglia efferocytosis and preventing inflammatory response, which is partly dependent on activation of Axl and Rac1.

290 (PB070) - Discovery and characterization of potent and selective AXL receptor tyrosine kinase inhibitor AB801

Background: AXL receptor tyrosine kinase (AXL) is a transmembrane protein that is overexpressed in a variety of tumors and correlates with poor prognosis in cancer patients. AXL is expressed in cancer and stromal cells and has been implicated in the development of resistance to chemotherapy, targeted therapies & immunotherapies. Activation of AXL by its ligand, growth arrest specific protein 6 (GAS6), or ligand-independent dimerization facilitates AXL phosphorylation, initiates signaling cascades that promote cancer cell proliferation, survival, and an immunosuppressive microenvironment.

309 (PB089) - AB801 is a potent and selective AXL inhibitor that demonstrates significant anti-tumor activity in combination with standard of care therapeutics

Background: The receptor tyrosine kinase AXL, expressed in cancer, stromal, and select immune cells, has been implicated in resistance to chemotherapy, targeted therapies & immunotherapies. AXL expression correlates with poor prognosis in cancer patients. Activation of AXL can occur in a ligand-dependent manner via its ligand, growth arrest specific protein 6 (Gas6), or in a ligand-independent manner, both of which facilitate AXL phosphorylation & initiation of signaling cascades promoting cancer cell proliferation, survival, and an immunosuppressive microenvironment. Here we present the preclinical evaluation of a novel, highly potent, and selective AXL inhibitor, AB801. Materials and Methods: Inhibition of AXL phosphorylation in cancer cells was evaluated by Western blot and levels of soluble and cell-surface AXL were assessed by ELISA and flow cytometry, respectively. Downstream signaling of AXL was evaluated by Western blot and qPCR. Pharmacokinetics (PK), pharmacodynamics (PD) and anti-tumor efficacy in combination with standard of care therapies were evaluated in murine cancer models. Results: AB801 inhibits both ligand-dependent and ligand-independent AXL phosphorylation. AXL phosphorylation and subsequent signaling leads to receptor internalization, thereby decreasing both surface AXL and soluble AXL levels. AB801 inhibits AXL activity in a concentration-dependent manner reducing AXL phosphorylation and increasing both surface AXL expression and soluble AXL levels. Moreover, AXL inhibition results in upregulation of cell surface proteins that mediate immune/cancer cell interaction, e.g., LFA-1. Significant anti-tumor efficacy is observed in combination with targeted therapies in several in vivo models. Importantly, AB801 treatment significantly reduces tumor growth after relapse to single-agent therapy. Conclusions: AB801 demonstrates significant anti-tumor activity in combination with standard of care therapeutics. Selective AXL inhibition is a promising therapeutic strategy to overcome resistance to therapy. Conflict of interest: Ownership: Arcus Biosciences Shareholder Corporate-sponsored Research: Employee of Arcus Biosciences.

Abstract PO015: Intrinsic and extrinsic role of Axl in models of hepatocellular carcinoma

Abstract The activation of the receptor tyrosine kinase Axl by binding to its high affinity ligand Gas6 is a major driver of tumor progression and metastasis in various cancer entities. In hepatocellular carcinoma (HCC), about 40% of patients show upregulation of Axl expression correlating with vascular invasion and poor survival of patients. In pre-malignant stages of HCC, Axl is essentially involved in developing hepatic fibrosis and cirrhosis by activation of hepatic stellate cells. Despite recent insights, the dichotomy of intrinsic and extrinsic Axl functions in HCC is poorly understood. Therefore, we aimed at analyzing cell-specific aspects of Axl in liver tumorigenesis using advanced mouse tumor models. To particularly address the tumor-cell specific role of Axl, we employed a transplantation-based mouse models. In order to investigate both the tumor-intrinsic and extrinsic impact of Axl, we employed an autochthonous model of diethylnitrosamine (DEN)-induced liver tumor formation in systemic Axl-deficient mice. We found that tumor-intrinsic Axl expression in HCC cells did not affect proliferation in vitro and in vivo, however increased the number of pulmonary metastatic colonies and reduced overall survival of mice. In line, Gas6/Axl signaling enhanced the invasive abilities of tumor cells in vitro as observed in 3-dimensional cell culture settings. The Axl-driven invasive phenotype of HCC cells associated with the upregulated expression of transcription factors Snai1/Snai2 and downregulation of E-Cadherin indicating changes in epithelial plasticity. Intriguingly, the tumor burden was enhanced in DEN-treated Axl−/− mice in the background of CCl4-induced inflammation by showing higher proliferative rates of malignant hepatocytes. Interestingly, these tumors were less infiltrated with cytotoxic CD8+ T cells and granzyme B+ cells. Additionally, we observed an increased infiltration of FoxP3+ regulatory T cells and F4/80+ macrophages as well as elevated expression of M2-macrophage-specific Mrc-1, Fizz-1 and TGF-β1 suggesting that Axl−/− tumors shift towards an immunosuppressive environment. Remarkably, PD-L1 levels did not vary between Axl+/+ and Axl−/− tumors, suggesting alternative immune escape mechanisms. In accordance, analysis of HCC patient datasets showed a positive correlation of Axl expression with cytotoxic immune gene signatures. From these data we conclude that tumor intrinsic Gas6/Axl signaling triggers the metastatic phenotype of HCC cells while Axl deficiency escapes immune surveillance and therefore promotes liver tumorigenesis. Together, our findings show that Axl plays a prominent role in HCC dissemination and highlight the importance of Axl expression in the tumor microenvironment. Citation Format: Kristina Breitenecker, Denise Heiden, Gerhard Weber, Wolfgang Mikulits. Intrinsic and extrinsic role of Axl in models of hepatocellular carcinoma [abstract]. In: Proceedings of the AACR Special Conference: Advances in the Pathogenesis and Molecular Therapies of Liver Cancer; 2022 May 5-8; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(17_Suppl):Abstract nr PO015.

Abstract 3968: AXL is responsible for much of TKI-mediated adaptive resistance in FLT3/ITD AML cells

Abstract FMS-like tyrosine kinase-3 (FLT3) is the most frequently mutated receptor tyrosine kinase (RTK) in acute myeloid leukemia (AML). Internal tandem duplication mutations (ITD) confer a poor prognosis on FLT3/ITD patients, making FLT3/ITD AML susceptible to treatment with tyrosine kinase inhibitors (TKI). However, FLT3 TKI success has been hindered by mechanisms of resistance including adaptive resistance, in which downstream signaling pathways are rapidly reactivated after initial inhibition with targeted therapies. Past work has shown that FLT3/ITD cells undergo adaptive resistance through the reactivation of ERK signaling within 24 hours of sustained FLT3 inhibition. We are interested in finding the mechanisms responsible for this ERK reactivation. AXL is a RTK that is implicated in acquired resistance for many cancers, including against targeted therapy in FLT3/ITD AML. However, AXL signaling has not been implicated in early adaptive resistance in the context of FLT3/ITD AML. Thus, we sought to determine if AXL plays a role in the ERK reactivation found in TKI-treated AML cells. To address this, we first treated FLT3/ITD cell lines (Molm14 and MV4;11) with FLT3 TKI for 24 hours and saw an increase in AXL protein and mRNA levels as early as 4 hours after starting treatment. AXL phosphorylation occurred as early as 16 hours, which coincides with the beginning of the ERK phosphorylation (pERK) rebound. Further characterization revealed AXL upregulation, like ERK reactivation, is at least partially serum-dependent and that the ERK reactivation itself is not causing the AXL induction. These results established that TKI-induced AXL activation is associated with the adaptive ERK reactivation. To determine if AXL signaling actually plays a role in ERK reactivation in FLT3/ITD AML, we inhibited AXL activation using both small-molecule inhibitors (TP-0903 and R428) and selective inhibition (shRNA knockdown and ligand trapping with an AXL chimeric protein) concurrently with FLT3 TKI treatment. Each method of AXL inhibition diminished the pERK rebound, decreased cell proliferation and increased apoptosis of FLT3 TKI-treated FLT3/ITD cells. Combined AXL and FLT3 inhibition also suppressed leukemic cell recovery even after brief treatment exposure. Furthermore, 24-hour FLT3 and AXL TKI treatments of both relapsed and newly-diagnosed FLT3/ITD patient leukemic samples yielded the same upregulation of AXL protein and decreased pERK rebound observed in the cell lines. In summation, FLT3 TKI treatment causes rapid AXL upregulation resulting in adaptive ERK reactivation. Concurrent FLT3 and AXL inhibition diminishes pERK rebound and sensitives FLT3/ITD cells to TKI treatment. This data suggests that AXL plays a role in adaptive resistance in FLT3/ITD AML, and the addition of AXL inhibition to FLT3 TKI treatment has the potential to improve FLT3/ITD patient outcomes. Citation Format: Tessa Seale, Li Li, Jaesung Seo, Bao Nguyen, Mark Levis, Christine Pratilas, Donald Small. AXL is responsible for much of TKI-mediated adaptive resistance in FLT3/ITD AML cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3968.

Abstract 2087: AXL inhibition enhances Type 1 interferon (IFN) response and potentiates chemo-immunotherapy

Abstract Chemotherapy elicits anti-tumor immune responses by inducing immunogenic tumor cell death, targeting suppressive immune cells and activating Type 1 interferon (IFN) responses (1, 2, 3). Chemotherapeutic agents lead to cGAS - STING cytosolic DNA sensing pathway activation that results in Type 1 IFN release (4). IFN receptor (IFNAR1, IFNAR2) signaling mimics viral immune responses, is associated with clinical benefit (5, 6) and is exploited by chemo-immunotherapies (7, 8). The receptor tyrosine kinase AXL is associated with immune evasion and immunotherapy resistance (8). AXL serves as a critical regulatory checkpoint for TLR-induced IFN responses in dendritic cells, macrophages and natural killer cells (9, 10). IFN receptor signaling induces AXL expression (11) and AXL activation on dendritic cells is targeted by viruses (e.g. Zika) to abrogate IFN responses and inhibit anti-viral immunity. AXL serves as an IFN-response checkpoint by blocking IFNAR1 and IFNAR2 signaling. We hypothesized that tumor cells exploit AXL signaling to abrogate Type 1 IFN responses and inhibit antitumor immunity. We evaluated whether AXL inhibition promotes Type 1 IFN signalling and enhances immune checkpoint inhibitor efficacy. AXL kinase inhibition (bemcentinib) in combination with chemotherapy (doxorubicin) increased IFN response gene expression in mammary carcinoma and melanoma cell lines. In vivo, bemcentinib treatment potentiated the efficacy of immune checkpoint inhibitor treatment in combination with intratumoral doxorubicin injection (i.t) in the syngeneic myeloid derived suppressor cell (MDSC)- rich refractory mammary adenocarcinoma 4T1 model. In addition, bemcentinib treatment in conjunction with i.t. doxorubicin enhanced Type 1 IFN response, reduced cancer stemness and epithelial to mesenchymal (EMT) gene expression in this model. Furthermore, this combination treatment regimen sensitized the immune checkpoint inhibitor refractory Braf-mutant melanoma (YUMM1.7) by enhancing the type 1 IFN response resulting in significantly improved median overall survival. In conclusion, this study provides evidence that bemcentinib potentiates chemo-immunotherapy by enhancing tumor Type 1 IFN response and dampening EMT.

Abstract 406: Mechanism of AXL regulation in chemotherapy resistance

Abstract Constitutive activation of receptor tyrosine kinase AXL is an important phenomenon in chemotherapy resistance and is highly expressed in several malignancies. Although studies over the past decade revealed that AXL is upregulated during treatment, the biological significance and the mechanism by which AXL is activated upon chemotherapy are yet to be determined. In this study, we aim to understand the biology of AXL mediated chemo-resistance, and the consequences of AXL activation. To investigate the biological significance of AXL, we generated iRFP-luciferase-expressing cell lines which facilitate live monitoring of cancer progression during treatment in an in vivo setting. In parallel, by knocking down AXL using shRNA, we observed inhibitory effect on cell migration and tumor forming efficiency in vitro. Moreover, using in vitro cell based models, we showed that while AXL expression was upregulated upon treatment with chemotherapeutic agents, pharmacological inhibition of AXL caused synergistic effect between selective AXL inhibitors and cisplatin. Additionally, our data suggest that upregulation of AXL and its tyrosine phosphorylation may mediate resistance to chemotherapy. Using phosphoproteomics, we identified a link between CDK9 activation and drug-induced AXL expression. We showed that while AXL expression was upregulated upon treatment with tyrosine kinase inhibitor, pharmacological inhibition of CDK9 completely blocked AXL expression. Collectively, our data suggest that activation of kinase signaling might be required for AXL repression, and thereby, for targeting AXL-axis in chemotherapy resistance. Citation Format: Mehreen Ahmed, Julhash Uddin Kazi. Mechanism of AXL regulation in chemotherapy resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 406.

Abstract 3913: Quantitative phosphoproteomic analysis of AXL signaling network in breast cancer

Abstract Background: As a key receptor tyrosine kinase (RTK) regulating tumorigenesis, AXL is overexpressed in multiple different types of cancers. AXL can be activated by homodimerization after binding with its ligand, growth arrest specific 6 protein (Gas6) or by heterodimerization with other RTKs. AXL overexpression and/or activation have been reported to promote cancer cell proliferation, migration/invasion and to induce resistance to targeted therapy. However, the AXL downstream signaling network has not yet been fully elucidated. Methods: We employed a CRISPR-CAS9-based knockout technology to create AXL inducible knockout cells in a human triple negative breast cancer (TNBC) cell line, HCC1395. In order to explore the downstream signaling events driven by AXL, we induced AXL knockout for 72 hours and used a 6-plex TMT-labeling-based quantitative proteomic approach to characterize the protein phosphorylation alterations induced by AXL knockout. IMAC and anti-phosphotyrosine antibody (pY1000)-based phosphopeptide enrichment methods were used to comprehensively quantify the phosphoproteome changes induced by AXL knockout. Results: In this study, we identified 38,525 phospho serine/threonine sites and 1,427 phosphotyrosine sites. In response to AXL knockout, 1,581 phospho serine/threonine sites and 40 phosphotyrosine sites showed significant differences between AXL-KO and AXL-WT groups with >1.5-fold change and p<0.05 as the cutoff. Among them, 982 phosphoserine/threonine sites and 30 phosphotyrosine sites were decreased, while 599 phospho serine/threonine sites and 10 phosphotyrosine sites were increased in AXL-KO cells. Signaling pathway enrichment analysis revealed that suppressing AXL expression could inhibit multiple important cancer related signaling pathways, including cell cycle, focal adhesion, MAPK signaling pathway, p53 signaling pathway and mTOR signaling pathway. Conclusion: Quantitative analysis of the phosphoproteome driven by AXL demonstrated that AXL plays a pivotal role in regulating canonical oncogenic signaling but also signaling pathways involved in RNA processing and DNA repair. Our data also suggest that targeting AXL could suppress these oncogenic signaling pathways and have therapeutic potential to improve clinical outcomes for patients with AXL overexpression. Citation Format: Li Wang, Nicole Pearson, Yuning Xiong, Santosh Renuse, Ran Cheng, Jodi M. Carter, Akhilesh Pandey, Xinyan Wu. Quantitative phosphoproteomic analysis of AXL signaling network in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3913.

Abstract 2011: Targeting CDK4/6 inhibitor resistance in relapsed RB-proficient osteosarcoma patient-derived xenografts via PI3 Kinase/mTOR inhibition

Abstract In children, adolescents, and young adults (AYA), osteosarcoma (OS) is the most common type of bone cancer and ~35% patients relapse following frontline cytotoxic therapy. Thus, there is a critical need to identify therapies targeting specific molecular signatures in OS. Hyperactivation of cyclin-dependent kinases 4 and 6 (CDK4/6) has been identified by us and others as a top actionable marker in OS. CDK4/6 binds to cyclin D resulting in a complex that mediates RB phosphorylation leading to cell cycle progression. While CDK4/6 inhibitors (CDK4/6i) have shown promise clinically, one drawback is that CDK4/6i induces cell cycle arrest rather than cell death. Furthermore, prolonged CDK4/6 inhibitor therapy can confer therapeutic resistance in RB1-proficient (RB+) tumors where compensatory pathways such as PI3K/mTOR are activated. To mitigate such CDK4/6i resistance in OS, we hypothesized that dual inhibition of CDK4/6 and PI3K pathways will promote cytotoxicity in hyperactivated CDK4/6 OS models. RB+ OS cell lines and a TT2-77 xenoline were evaluated in vitro. Combination index and Bliss independence analyses indicated that inhibition of OS growth by exposure to CDK4/6i (Palbociclib or Abemaciclib) and PI3K/mTOR inhibitor (PI3K/mTORi-Voxtalisib or LY3023414) was additive-to-synergistic and lead to increased apoptosis at clinically relevant concentrations. Short-term pharmacodynamic study of vehicle- versus Palbociclib-treated TT2-77 patient-derived xenograft (PDX) was analyzed by global/phospho-proteomics and kinome profiling. RB1 and MKI67 phosphopeptides as well as the total protein levels of CDK1 were reduced by Palbociclib, thus, confirming modulation of the cell cycle. Kinome profiling analysis of PDX from Palbociclib-treated mice indicated increased activity of AXL, a receptor tyrosine kinase linked to PI3K pathway activation. Increased activity of the autophagy marker PIK3C3 was also evident. OS PDX models TT2-77 and HT96 (RB+, CDKN2Anull, CCND3 amplified) were treated with Palbociclib (50 mg/kg), Voxtalisib (50 mg/kg) or Palbociclib + Voxtalisib. In TT2-77 PDXs treated for four weeks, tumor growth was significantly reduced in single-agent and combo groups compared to vehicle (p<0.05, Two-way ANOVA; Holm-Sidak). We observed a trend that the combo was more efficacious than single agent, but statistical differences were not evident. Increasing the dosing timeline to six weeks may be beneficial. In HT96 PDXs, tumor growth was significantly decreased in single-agent and combo groups compared to vehicle. Notably, Palbociclib + Voxtalisib was more efficacious than single-agent (p<0.05, Two-way ANOVA; Holm-Sidak). These data highlight the need to optimize CDK4/6i+PI3K/mTORi dosing schedules and provide evidence that Palbociclib + Voxtalisib therapy is safe, efficacious, and can decrease CDK4/6i resistance in aggressive PDX models of OS. Citation Format: Farinaz Barghi, Pankita H Pandya, M. Reza Saadatzadeh, Khadijeh Bijangi-Vishehsaraei, Barbara J. Bailey, Erika A. Dobrota, Courtney Young, Melissa A. Trowbridge, Kathryn L. Coy, Henry Mang, Reagan K. Wohlford, Anthony L. Sinn, Emily C. Sims, Matt J. Repass, Nuri Damayanti, Niknam Riyahi, Harlan E. Shannon, Steve P Angus, Michael J Ferguson, Jamie L. Renbarger, Karen E. Pollok. Targeting CDK4/6 inhibitor resistance in relapsed RB-proficient osteosarcoma patient-derived xenografts via PI3 Kinase/mTOR inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2011.

Activated AXL Protects Against Hepatic Ischemia-reperfusion Injury by Upregulating SOCS-1 Expression.

Background.Hepatic ischemia-reperfusion (I/R) injury is the main factor affecting the morbidity and mortality associated with perioperative complications of liver transplantation and major hepatectomy. AXL is a member of the TYRO3, AXL, MERTK family and is involved in immune and apoptosis processes in multiple organs. However, the role of AXL in hepatic I/R injury remains to be elucidated.Methods.Mice pretreated with rmGas6 or R428 and mice tail vein injected with adeno-associated virus knockdown suppressor of cytokine signaling protein-1 (SOCS-1) underwent liver I/R surgery to detect the function of activated AXL in vivo. Primary hepatocytes undergo hypoxic reoxygenation injury in vitro.Results.AXL expression was significantly upregulated, and phosphorylated-AXL was substantially downregulated in liver transplantation patients and hepatic I/R surgery mice. A mouse model of hepatic I/R injury showed that AXL activation reduced liver inflammation and liver cells apoptosis. The inhibition of AXL activation (AXL-specific inhibitor R428) aggravated hepatic I/R injury, resulted in larger areas of liver injury, aggravated inflammatory response, and increased apoptosis of liver cells. In addition, activated AXL promotes the expression level of SOCS-1 and inhibits toll-like receptor 4 and its downstream signaling pathways. Finally, SOCS-1 was knocked down with an adeno-associated virus, and activated AXL failed to protect against hepatic I/R injury.Conclusions.AXL activation protects the liver from I/R injury by upregulating SOCS-1 and inhibiting the toll-like receptor 4/myeloid differentiation factor-88/nuclear factor kappa-B signaling axis. Targeting AXL may be a new therapeutic option for ameliorating hepatic I/R injury.

Tobacco smoke activated fibrogenic MARCKS/AXL complex promotes macrophage reprogramming and pulmonary fibrosis

Abstract Macrophages and tobacco smoke (TS) exposure have been demonstrated to play significant roles in modulating pulmonary fibrosis (PF). However, the mechanisms of how TS exposure modulates pro-fibrotic macrophage polarization and drives lung fibrosis is unclear. In our study, we investigated how TS modulates macrophage polarization and the functional consequences of this polarization. Multicolor flow cytometric data indicated that markers of M2 macrophage polarization were elevated in both human and mouse macrophage cells and tissues upon TS exposure. In addition, multiple primary lung fibroblast cells demonstrated elevated pro-fibrotic markers and aggressive phenotypes upon interacting with TS-exposed macrophage cells in a co-culture system. Elucidation of the signaling pathways activated by TS exposure through a receptor tyrosine kinase array screen revealed AXL receptor as a novel smoke-responsive molecule in macrophage cells. We noted elevated secretion of AXL ligand, Gas6, and AXL activity in TS exposed cells and tissues. Prior work had demonstrated an interaction between MARCKS, a smoke-responsive protein, and AXL in promoting a pro-fibrotic phenotype in lung fibroblasts. Similarly, we observed AXL activity positively correlated with MARCKS phosphorylation in macrophage cells. Pharmacologic and genetic targeting of the MARCKS/AXL signaling complex reduced M2 markers and profibrotic cytokine production in macrophages and reduced fibrotic changes in the co-culture model and in an animal model of smoke-mediated lung fibrosis. In all, our work suggests that the MARCKS/AXL fibrogenic complex is a potential target in attenuating macrophage activity in TS-mediated fibrosis. Supported by grants from NIH/NHLBI (R01HL146802), DOD DCMRP/PRMRP (PR202411), and UCOP TRDRP (28IR-0061 and T31DT1849).

MARCKS mediates the AXL/PD-L1 signaling axis and contributes to an immunosuppressive environment in lung cancer

Abstract The advent of immunotherapy with immune checkpoint blockade (ICB) has led to significant advances in lung cancer management; unfortunately, many issues remain to be resolved, especially regarding a low objective response rate. Through integrated large-scale omics data and biomarkers from published immune ICB trials, we identified the PIP2-binding protein, myristoylated alanine-rich C-kinase substrate (MARCKS), and the AXL receptor as top-ranked therapeutic targets in mediating immunotherapy resistance. Both immunofluorescence and immunoprecipitation data demonstrated that MARCKS forms a molecular complex with AXL in aggressive lung cancer cells. In a screening of 200 patients using immunohistochemistry staining, we observed a positive correlation between AXL phosphorylation and MARCKS expression in advanced lung cancer. MARCKS knockdown abated the activity of AXL and its downstream pathways including PI3K/AKT and STAT3/Src signaling. Data from our cytokine arrays showed upregulation of anti-tumoral cytokines including GM-CSF and CXCL10 in MARCKS-knockout cells. We further discovered that MARCKS acts in accordance with PD-L1 to modulate T cell activity and killing. Upon co-culture with MARCKS-expressing lung cancer cells, cytotoxic T cells expressed higher PD-1 levels on cell surfaces, whereas T cell killing activity was potentiated in the MARCKS-knockout group. In mouse xenograft models, mice bearing MARCKS-deficient tumors displayed increased sensitivity to PD-L1 treatment. Our data suggest an oncogenic role of the AXL-MARCKS axis in modulating the tumor immune microenvironment. Therapeutic targeting of MARCKS may warm up an immunologically “cold” microenvironment, sensitizing tumors to immunotherapy. This work was supported by grants funded by the Department of Defense Kidney Cancer Research Program (KCRP) W81XWH1910831 (log# KC180170) and the California UCOP Tobacco-Related Disease Research Program (TRDRP) T29IR0704.

Abstract PD4-07: Targeting latent residual HER2+ breast cancer brain metastatic cells

Abstract Approximately fifty percent of breast cancer patients with brain metastasis are positive for human epidermal growth factor receptor 2 (HER2+). Systemically effective cytotoxic drugs, and HER2 targeted therapies are ineffective on brain metastases. Current understanding of HER2 brain metastatic disease is limited as biopsying brain metastatic lesions and detecting micro-metastasis from asymptomatic patients with residual disease remains a challenge. Therefore, to isolate disseminated tumor cells that stay latent in the brain, while retaining their metastasis-initiating capabilities, we performed a phenotypic screen in mouse employing human breast cancer cell lines. Using this approach, we isolated isogenic latent residual cells (Lat) and metachronous brain metastatic outbreaks (M-BM). Phenotypically stable latent cells display stem cell-like characteristics, downregulate immune activating sensors and survive in equilibrium with innate immune surveillance, while M-BM escape and metastasize. Brain-tropic Lat and M-BM cells are resistant to HER2 targeted therapies - lapatinib, neratinib and tucatinib. Transcriptomic analysis identified AXL, a receptor tyrosine kinase from the TAM receptor family, to be highly enriched in brain-tropic Lat and M-BM. Moreover, AXL is over expressed in metachronous brain metastasis samples compared to matched primary tumors from HER2+ breast cancer patients. AXL activation and overexpression has been associated with chemoresitance mechanisms, dormancy maintenance, invasion, tumor angiogenesis, immune regulation, metastasis and poor prognosis in cancer patients. Therefore, we hypothesized AXL promotes residual/latent breast cancer survival and brain metastatic relapse in HER2+ breast cancer. shRNA depletion of AXL in Lat and M-BM resulted in reduced oncosphere formation, an assay used to measure tumor or metastasis initiating capacity. Brain metastatic incidence was significantly attenuated in mice bearing AXL depleted M-BM compared to their controls. Bemcentinib, an AXL inhibitor currently in clinical trials for non-small cell lung cancer, triple negative breast cancer, pancreatic cancer and melanoma, alone or in combination with lapatinib, neratinib or tucatinib, reduced oncosphere formation in HER2 therapy resistant Lat and M-BM cells. Ongoing experiments are designed to assess the impact of bemcentinib on brain metastasis in vivo and to mechanistically define how AXL promotes survival of latent residual and brain metastatic HER2+ breast cancer cells. Citation Format: Mauricio Marquez-Palencia, Srinivas Malladi. Targeting latent residual HER2+ breast cancer brain metastatic cells [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD4-07.

AXL Receptor Tyrosine Kinase as a Promising Therapeutic Target Directing Multiple Aspects of Cancer Progression and Metastasis.

Simple SummaryMetastasis is a complex process that requires the acquisition of certain traits by cancer cells as well as the cooperation of several non-neoplastic cells that populate the stroma. Cancer-related deaths are predominantly associated with complications arising from metastases. Limiting metastasis therefore represents an important clinical challenge. The receptor tyrosine kinase AXL is required at many steps of the metastatic cascade and contributes to tumor microenvironment deregulation. In this review, we describe how AXL contributes to metastatic progression by governing various biological processes in cancer cells and in stromal cells, highlighting the potential of its inhibition.The receptor tyrosine kinase AXL is emerging as a key player in tumor progression and metastasis and its expression correlates with poor survival in a plethora of cancers. While studies have shown the benefits of AXL inhibition for the treatment of metastatic cancers, additional roles for AXL in cancer progression are still being explored. This review discusses recent advances in understanding AXL’s functions in different tumor compartments including cancer, vascular, and immune cells. AXL is required at multiple steps of the metastatic cascade where its activation in cancer cells leads to EMT, invasion, survival, proliferation and therapy resistance. AXL activation in cancer cells and various stromal cells also results in tumor microenvironment deregulation, leading to modulation of angiogenesis, fibrosis, immune response and hypoxia. A better understanding of AXL’s role in these processes could lead to new therapeutic approaches that would benefit patients suffering from metastatic diseases.

AXL Inhibitor TP-0903 Reduces Metastasis and Therapy Resistance in Pancreatic Cancer.

Pancreatic cancer is the third leading cause of cancer-related deaths in the United States with a 5-year survival less than 5%. Resistance to standard therapy and limited response to immune checkpoint blockade due to the immunosuppressive and stroma-rich microenvironment remain major challenges in the treatment of pancreatic cancer. A key cellular program involved in therapy resistance is epithelial plasticity, which is also associated with invasion, metastasis, and evasion of immune surveillance. The receptor tyrosine kinase AXL is a key driver of tumor cell epithelial plasticity. High expression and activity of AXL is associated with poor prognosis, metastasis, and therapy resistance in multiple types of cancer including pancreatic. Here, we show that an AXL inhibitor (TP-0903), has antitumor and therapy sensitizing effects in preclinical models of pancreatic ductal adenocarcinoma (PDA). We demonstrate that TP-0903 as a single agent or in combination with gemcitabine and/or anti-programmed cell death protein 1 (PD1) antibody has anti-metastatic and anti-tumor effects in PDA tumor bearing mice, leading to increased survival. In addition, gene expression analysis of tumors demonstrated upregulation of pro-inflammatory and immune activation genes in tumors from TP-0903-treated animals compared with the vehicle, indicating pharmacologic inhibition of AXL activation leads to an immunostimulatory microenvironment. This effect was augmented when TP-0903 was combined with gemcitabine and anti-PD1 antibody. These results provide clear rationale for evaluating TP-0903 in the treatment of pancreatic cancer.

Efficacy and Synergy of Small Molecule Inhibitors Targeting FLT3-ITD+ Acute Myeloid Leukemia.

Simple SummaryFLT3-ITD mutations belong to the most frequent yet most detrimental genetic alterations in AML. Next-generation FLT3 inhibitors are potent therapeutics and often effective in AML patients carrying the FLT3-ITD driver kinase. However, AML cells are particularly quick in acquiring resistance to FLT3 kinase blockers. We aimed to identify novel therapeutic options for FLT3-ITD+ AML, to investigate possible emerging resistance mechanisms to FLT3 inhibitors and to explore alternative targeting strategies. We applied a kinase-focused drug screen to find alternative therapeutics. We identified ispinesib, a kinesin spindle blocker, and kinase blockers WS6, ponatinib and cabozantinib, as very efficacious agents against FLT3-ITD+ AML cells. Importantly, we identify the combination of cabozantinib and ispinesib as particularly potent against FLT3-ITD+ AML. We suggest that a combinatorial treatment with these drugs could overcome resistance mechanisms and kill FLT3-ITD+ AML blasts.Constitutive activation of FLT3 by ITD mutations is one of the most common genetic aberrations in AML, present in ~1/3 of cases. Patients harboring FLT3-ITD display worse clinical outcomes. The integration and advancement of FLT3 TKI in AML treatment provided significant therapeutic improvement. However, due to the emergence of resistance mechanisms, FLT3-ITD+ AML remains a clinical challenge. We performed an unbiased drug screen to identify 18 compounds as particularly efficacious against FLT3-ITD+ AML. Among these, we characterized two investigational compounds, WS6 and ispinesib, and two approved drugs, ponatinib and cabozantinib, in depth. We found that WS6, although not yet investigated in oncology, shows a similar mechanism and potency as ponatinib and cabozantinib. Interestingly, ispinesib and cabozantinib prevent activation of AXL, a key driver and mechanism of drug resistance in FLT3-ITD+ AML patients. We further investigated synergies between the selected compounds and found that combination treatment with ispinesib and cabozantinib or ponatinib shows high synergy in FLT3-ITD+ AML cell lines and patient samples. Together, we suggest WS6, ispinesib, ponatinib and cabozantinib as novel options for targeting FLT3-ITD+ AML. Whether combinatorial tyrosine kinase and kinesin spindle blockade is effective in eradicating neoplastic (stem) cells in FLT3-ITD+ AML remains to be determined in clinical trials.

Abstract P253: Potent and selective AXL tyrosine kinase inhibition demonstrates significant anti-tumor efficacy in combination with standard of care therapeutics in preclinical models

Abstract Background. AXL receptor tyrosine kinase (AXL) is a transmembrane protein that is overexpressed in a variety of tumors and correlates with poor prognosis in cancer patients. AXL is expressed in cancer and stromal cells and has been implicated in the development of resistance to chemotherapy, targeted therapies & immunotherapies. Activation of AXL by its ligand, growth arrest specific protein 6 (Gas6), or ligand-independent dimerization facilitates AXL phosphorylation, initiates signaling cascades that promote cancer cell proliferation, survival, and an immunosuppressive microenvironment. Here we present the discovery and characterization of a novel, highly potent and selective novel AXL inhibitor. Materials and Methods. The potency and specificity of the novel Arcus inhibitor against AXL and other kinases was determined using a panel of HTRF KinEASE-TK assays. Intracellular target engagement was evaluated by monitoring displacement of a competitive fluorescent tracer using an AXL NanoBRETTM intracellular kinase assay. To further assess the inhibitory effect of the novel Arcus inhibitor on AXL kinase activity, AXL autophosphorylation induced SH2 domain translocation was measured in cells using a PathHunterÒ U2OS AXL Functional assay system. Inhibition of AXL phosphorylation in cancer cells was evaluated by Western blot and levels of soluble and surface AXL were assessed by ELISA and flow cytometry, respectively. Pharmacokinetics (PK), pharmacodynamics (PD) and anti-tumor efficacy were evaluated in murine models. Results. A novel, potent, reversible, and highly selective AXL kinase inhibitor has been generated by Arcus. The novel inhibitor exhibits single-digit nanomolar potency in both biochemical and cell-based assays, retains significant activity in 100% human serum and does not show significant inhibition of the major CYP450 isoforms nor the hERG potassium channel. The novel Arcus molecule inhibits AXL phosphorylation mediated by both ligand-dependent Gas6 stimulation as well as ligand-independent autophosphorylation. AXL phosphorylation and subsequent signaling leads to receptor internalization, thereby decreasing both surface AXL expression and soluble AXL levels. AXL activity is inhibited in a concentration dependent manner significantly increasing both surface AXL expression and soluble AXL levels. More importantly, significant anti-tumor efficacy is observed in combination with targeted therapies in several in vivo models. Furthermore, AXL inhibition significantly reduces tumor growth after relapse to single-agent targeted therapy. Conclusions. A novel selective inhibitor of AXL tyrosine kinase activity has been developed that demonstrates single-digit nanomolar potency and inhibition of both ligand-dependent and ligand-independent AXL phosphorylation. Significant anti-tumor activity is observed in combination with targeted therapy and upon acquired resistance in xenograft models. Selective AXL inhibition is a promising therapeutic strategy to overcome resistance to chemotherapy, targeted therapy, and/or immunotherapy. Citation Format: Susan L. Paprcka, Subhasree Sridhar, Irene M. Luu, Salema Jafri, Dillon H. Miles, Suan Liu, Ruben Flores, Shiwei Qu, Manjunath Lamani, Sriivas Paladugu, Cesar Meleza, James Wu, Hema Singh, Yu Chen, Sean Cho, Akshata Udyavar, Angelo Kaplan, Enzo Stargnaro, Xiaoning Zhao, Lixia Jin, Manmohan R. Leleti, Stephen W. Young, Jay P. Powers, Matthew J. Walters, Ester Fernandez-Salas. Potent and selective AXL tyrosine kinase inhibition demonstrates significant anti-tumor efficacy in combination with standard of care therapeutics in preclinical models [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P253.

AXL targeting by a specific small molecule or monoclonal antibody inhibits renal cell carcinoma progression in an orthotopic mice model.

AXL tyrosine kinase activation enhances cancer cell survival, migration, invasiveness, and promotes drug resistance. AXL overexpression is typically detected in a high percentage of renal cell carcinomas (RCCs) and is strongly associated with poor prognosis. Therefore, AXL inhibition represents an attractive treatment option in these cancers. In this preclinical study, we investigated the antitumor role of a highly selective small molecule AXL inhibitor bemcentinib (BGB324, BerGenBio), and a newly developed humanized anti‐AXL monoclonal function blocking antibody tilvestamab, (BGB149, BerGenBio), in vitro and an orthotopic RCC mice model. The 786‐0‐Luc human RCC cells showed high AXL expression. Both bemcentinib and tilvestamab significantly inhibited AXL activation induced by Gas6 stimulation in vitro. Furthermore, tilvestamab inhibited the downstream AKT phosphorylation in these cells. The 786‐0‐Luc human RCC cells generated tumors with high Ki67 and vimentin expression upon orthotopic implantation in athymic BALB/c nude mice. Most importantly, both bemcentinib and tilvestamab inhibited the progression of tumors induced by the orthotopically implanted 786‐0 RCC cells. Remarkably, their in vivo antitumor effectiveness was not significantly enhanced by concomitant administration of a multi‐target tyrosine kinase inhibitor. Bemcentinib and tilvestamab qualify as compounds of potentially high clinical interest in AXL overexpressing RCC.

A unique role of p53 haploinsufficiency or loss in the development of acute myeloid leukemia with FLT3-ITD mutation

With an incidence of ~50%, the absence or reduced protein level of p53 is much more common than TP53 mutations in acute myeloid leukemia (AML). AML with FLT3-ITD (internal tandem duplication) mutations has an unfavorable prognosis and is highly associated with wt-p53 dysfunction. While TP53 mutation in the presence of FLT3-ITD does not induce AML in mice, it is not clear whether p53 haploinsufficiency or loss cooperates with FLT3-ITD in the induction of AML. Here, we generated FLT3-ITD knock-in; p53 knockout (heterozygous and homozygous) double-transgenic mice and found that both alterations strongly cooperated in the induction of cytogenetically normal AML without increasing the self-renewal potential. At the molecular level, we found the strong upregulation of Htra3 and the downregulation of Lin28a, leading to enhanced proliferation and the inhibition of apoptosis and differentiation. The co-occurrence of Htra3 overexpression and Lin28a knockdown, in the presence of FLT3-ITD, induced AML with similar morphology as leukemic cells from double-transgenic mice. These leukemic cells were highly sensitive to the proteasome inhibitor carfilzomib. Carfilzomib strongly enhanced the activity of targeting AXL (upstream of FLT3) against murine and human leukemic cells. Our results unravel a unique role of p53 haploinsufficiency or loss in the development of FLT3-ITD + AML.

AXL Inhibition Represents a Novel Therapeutic Approach in BCR-ABL Negative Myeloproliferative Neoplasms.

BCR-ABL negative myeloproliferative neoplasms (MPNs) consist of essential thrombocythemia, polycythemia vera, and myelofibrosis. The majority of patients harbor the JAK2-activating mutation V617F. JAK2 inhibitors were shown to reduce symptom burden and splenomegaly in MPN patients. However, treatment options are limited after failure of JAK2 inhibitors. AXL, a member of the TAM family of receptor tyrosine kinases, mediates survival and therapy resistance of different myeloid cancers including acute myeloid leukemia and chronic myeloid leukemia. We studied the relevance of AXL as a target in MPN using primary patient cells and preclinical disease models. We found that AXL is abundantly activated in MPN cells and that its ligand growth arrest-specific gene 6 is upregulated in MPN patients. Pharmacologic and genetic blockade of AXL impaired viability, decreased proliferation and increased apoptosis of MPN cells. Interestingly, ruxolitinib treatment induced increased phosphorylation of AXL indicating that activation of AXL might mediate resistance to ruxolitinib. Consistently, the AXL inhibitor bemcentinib exerted additive effects with ruxolitinib via impaired STAT3, STAT5, and AKT signaling. Both agents had activity when employed alone and exerted an additive effect on survival and splenomegaly in vivo. Moreover, bemcentinib treatment normalized red blood cell count and hemoglobin levels in vivo. Thus, our data indicate that AXL inhibition represents a novel treatment option in MPN warranting clinical investigation.