Kinase AXL Research Articles

DS-1205b, a novel selective inhibitor of AXL kinase, blocks resistance to EGFR-tyrosine kinase inhibitors in a non-small cell lung cancer xenograft model

The AXL receptor tyrosine kinase is involved in signal transduction in malignant cells. Recent studies have shown that the AXL upregulation underlies epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI) resistance in EGFR-mutant non-small cell lung cancer (NSCLC). In this study, we investigated the effect of DS-1205b, a novel and selective inhibitor of AXL, on tumor growth and resistance to EGFR TKIs. In AXL-overexpressing NIH3T3 cells, DS-1205b potently inhibited hGAS6 ligand-induced migration in vitro and exerted significant antitumor activity in vivo. AXL was upregulated by long-term erlotinib or osimertinib treatment in HCC827 EGFR-mutant NSCLC cells, and DS-1205b treatment in combination with osimertinib or erlotinib effectively inhibited signaling downstream of EGFR in a cell-based assay. In an HCC827 EGFR-mutant NSCLC xenograft mouse model, combination treatment with DS-1205b and erlotinib significantly delayed the onset of tumor resistance compared to erlotinib monotherapy, and DS-1205b restored the antitumor activity of erlotinib in erlotinib-resistant tumors. DS-1205b also delayed the onset of resistance when used in combination with osimertinib in the model. These findings strongly suggest that DS-1205b can prolong the therapeutic benefit of EGFR TKIs in nonclinical as well as clinical settings.

Abstract 4139: Q702, selective Axl, Mer and CSF1R triple kinase inhibitor with dual potentials leading to tumor regression: Immuno-oncology therapy and targeted cancer therapy

Abstract Functional composition of tumor micro-environment (TME) has been regarded as one of the most important factors in tumor progression. Cancer cells induce immune suppressive condition in TME in order to evade immune system as well as to promote metastasis. Axl, Mer and CSF1R play important roles in making tumor friendly TME by increasing regulatory T cells (Tregs), M2 macrophages, myeloid-derived suppression cells (MDSCs) and suppressing antigen presentation. On the other hand, in cancer cells, overexpression of Axl is reported to have correlation with poor prognosis of patients through promoting epithelial-to-mesenchymal transition (EMT) and inducing drug resistance to chemo or targeted therapy. Q702 is an orally available selective Axl, Mer and CSF1R triple kinase inhibitor under IND enabling studies. In this poster, we present dual potentials of Q702 leading to tumor regression through: 1) immune stimulating activities of Q702 through decreasing Tregs, M2 macrophages, MDSCs population and promoting antigen presentation, 2) direct cytotoxic activity in tumor models including acute myeloid leukemia (AML) and EGFR TKI resistant non-small cell lung cancer (NSCLC). Citation Format: Yeong-In Yang, Hwankyu Kang, Dongsik Park, Yeejin Jeon, Jeongjun Kim, Baejung Choi, Jaeseung Kim, Jinkyung Rho, Jaecheol Lee, Kiyean Nam. Q702, selective Axl, Mer and CSF1R triple kinase inhibitor with dual potentials leading to tumor regression: Immuno-oncology therapy and targeted cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4139.

Abstract 3804: The potent AXL kinase inhibitor, TP-0903, is active in pre-clinical models of EGFR positive non-small cell lung cancer

Abstract Lung cancer is the leading cause of cancer related death in the US (2017). Non-small cell lung cancer (NSCLC) accounts for more than 80% of all lung cancer cases. While EGFR inhibitors (EGFRi) have shown activity in NSCLC, single agent response rates often do not exceed 10%. EGFR mutation is a common oncogenic driver in NSCLC, appearing in between 13% and 47% of all NSCLC patients depending on ethnicity. Third generation EGFRis, such as osimertinib, were approved upon demonstrating improved progression-free survival (PFS) rates versus standard therapy. However, EGFR inhibitor resistance, including resistance to osimertinib, has been reported to result from the epithelial-to-mesenchymal transition (EMT) that is coincident with increased AXL kinase expression. The AXL RTK drives EMT and constitutes a bypass survival pathway for tumor cells under EGFRi treatment pressure. We have shown that treatment with TP-0903, our potent AXL inhibitor, leads to a reversal of the mesenchymal phenotype in multiple cancer models. We therefore hypothesized that TP-0903 treatment may potentiate EGFRi treatment in cancer, and in particular EGFR mutant NSCLC. To interrogate our hypothesis, we treated cells with TP-0903 and assessed changes in cell viability with the celltiter-glo assay, changes in mRNA expression using RT-qPCR, and protein expression changes, using standard immunoblotting. In cell viability assays in the H1650 NSCLC cell line, TP-0903 showed an EC50 of 39 nM, while osimertinib showed an EC50 of 2.2 µM. In mRNA and protein assays, we observed changes consistent with a reversal of the mesenchymal phenotype. Following treatment, Slug mRNA expression was inhibited as much as 3.8-fold. However, E-cadherin expression was increased by 1.6-fold. To assess the combination in vivo, we utilized the H1650 xenograft model for NSCLC. In pharmacodynamic assessment of EMT markers in vivo, Snail protein expression was reduced as much as 56% following a single dose of TP-0903 (40 mpk, at 24hrs). In assessment of treatment efficacy in vivo, and with TP-0903 treatment (40 mpk, qd), we observed 60% tumor growth inhibition (%TGI) over the course of a 21-day treatment regimen. With osimertinib treatment (20 mpk, qd), we observed 121 %TGI. However, with the combination, we observed 140 %TGI. Due to its ability to reverse the aggressive mesenchymal phenotype of cancer cells, TP-0903 is a promising agent with the potential to have single agent activity and combined synergy with targeted anti-cancer agents. A Phase I trial with this investigational agent is ongoing, which includes patients with EGFR positive non-small cell lung cancer (clincaltrials.gov, NCT02729298). Taken together, the current study supports continued development of AXL inhibitors in NSCLC, especially in combination with EGFRis. Citation Format: Ryan Mangelson, Ethika Tyagi, Peter Peterson, Adam Siddiqui-Jain, Clifford J. Whatcott, David J. Bearss, Steven L. Warner. The potent AXL kinase inhibitor, TP-0903, is active in pre-clinical models of EGFR positive non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3804.

Abstract 2351: AXL and c-MET cross-talk in the regulation of PD-L1 expression on renal cancer cells

Abstract Purpose: The purpose of this research is to study the cross-talk between receptor tyrosine kinases (RTKs) c-MET and AXL in renal cancer cells; and to explore if the blockade of AXL-c-MET pathways can restrict PD-L1-mediated growth and immune escape of renal cancer cells. Background: c-MET and AXL are overexpressed in renal cell carcinoma (RCC). Upon binding with their respective ligands hepatocyte growth factor (HGF) and GAS6, c-MET and AXL activate a wide range of pathways, including tumor promoting events. Renal tumors show increased T cell infiltration, yet the T cells are rendered incapable of tumor rejection. The co-inhibitory molecules PD-1 and PD-L1 belong to cell surface glycoprotein B7 family that governs T cell function and proliferation. In RCC, tumor-infiltrating T cells express PD-1 and are associated with poor outcomes for patients. Similarly, PD-L1 expression on renal cancer cells hinders prognosis and indicates tumor aggressiveness. Therefore, PD-L1 in renal tumors may play a significant role in down-regulating host anti-tumor immune responses. Our recent report suggests that c-MET activation induces overexpression of PD-L1 in renal cancer cells; and the blockade of PD-L1 promotes immune-mediated killing of renal cancer cells. However, the cross talk between AXL and c-MET in the regulation of PD-L1 has not been studied in RCC. Results: We have confirmed that both AXL and c-MET are overexpressed in human renal cancer cells (786-O and ACHN) when compared with normal renal tubular epithelial cells. We performed co-immunoprecipitation assays with c-MET antibody and found that c-MET forms a complex with AXL in renal cancer cells. We utilized siRNA-mediated gene silencing approach and found that the knock-down of AXL increased the sensitivity of 786-O and ACHN cells to apoptosis following treatment with XL-184 (a small molecule inhibitor of c-MET, AXL and VGFRs, obtained from Exelixis) as measured by flow cytometry in Annexin V/PI stained cells. We also found that GAS6 treatment enhanced HGF-induced expression of cytoprotective molecule Heme oxygenase-1 (HO-1) and anti-apoptotic molecules Bcl-2 and Bcl-xL in renal cancer cells. In both 786-O and ACHN cells, c-MET activation increased the cell surface expression of PD-L1 as measured by flow cytometry. Interestingly, GAS6 treatment further increased the expression of PD-L1, suggesting a synergistic action of RTKs AXL and c-MET in renal cancer cells. We also utilized a PD-L1 promoter-luciferase plasmid to measure PD-L1 promoter activity and found that GAS6 treatment enhanced HGF-induced effects on PD-L1 transcription. In mouse renal cancer cells (RENCA), which express very high levels of PD-L1, we observed a significant decrease in the expression of PD-L1 when AXL was knocked down in cells. Conclusion: Together, our results demonstrate that AXL-mediated signaling enhances c MET-induced cytoprotection and PD-L1 expression on renal cancer cells. Citation Format: Murugabaskar Balan, Toni Choueiri, Soumitro Pal. AXL and c-MET cross-talk in the regulation of PD-L1 expression on renal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2351.

Abstract LB-034: Myc instructs and maintains pancreatic adenocarcinoma phenotype

Abstract Pancreatic ductal adenocarcinoma (PDAC) sporadically develops from pre-cancerous pancreatic intraepithelial neoplasia (PanIN). It has a very poor prognosis and lacks effective therapies. Expression of the pleiotropic transcription factor Myc is deregulated in the majority of human cancers, including PDAC. Using a rapidly and reversibly switchable genetic mouse model to toggle Myc activity, we show that Myc drives immediate transition of indolent KRasG12D-induced PanINs into aggressive pancreatic adenocarcinomas that share all the signature features of spontaneous human PDAC. Myc induces a distinct set of instructive signals, some hitherto unknown, that together orchestrate coordinated changes in the multiple, distinct stromal and inflammatory cell types that generate the characteristic PDAC stroma. We also demonstrate that continued Myc activity is required for PDAC maintenance - Myc deactivation immediately triggers meticulous disassembly of PDAC, including its attendant stroma. Thus, both the formation and deconstruction of the complex PDAC phenotype can be mediated by a single, reversible molecular switch. We have also identified a previously unknown Myc-dependent GAS6-AXL pathway that is required for stromal changes characteristic of PDAC. Using the same pre-clinical mouse PDAC model, we show that TP-0903, a specific inhibitor of AXL kinase, profoundly blocks Myc-induced influx of neutrophils into PanINs and B220+ lymphoid cells into the PanIN periphery. This significantly retards Myc-driven stellate cell activation and proliferation and impedes PDAC growth. Hence, the GAS6-AXL-mediated signaling pathway is a promising therapeutic target for pancreatic ductal adenocarcinoma. TP-0903 is currently in a Phase I/Ib clinical study in in patients with solid tumors, including PDAC. Citation Format: Nicole M. Sodir, Roderik M. Kortlever, Valentin J.A. Barthet, Luca Pellegrinet, Tania Campos, Steven Kupczak, Laura Soucek, Mark J. Arends, Trevor D. Littlewood, Gerard I. Evan. Myc instructs and maintains pancreatic adenocarcinoma phenotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-034.

Abstract 4543: Proteomic characterization of AXL kinase inhibitors and signaling pathways

Abstract AXL is an attractive drug target because of its role in EMT-mediated resistance to EGFR tyrosine kinase inhibitor (TKI) in lung cancer (LC). Lack of genetic alterations and the role of stroma-mediated AXL activation in cancer cells, underscore the need to better characterize AXL TKIs, understand their effects on signaling and phenotype of cells, and develop assays to visualize active AXL signaling complexes. For this, 25 LC cells were analyzed for total (t) and phosphorylated (p) AXL expression. AXL TKIs, RXDX106, R428 and Cabozantinib, were profiled using western blotting (WB), viability assay and activity-based protein profiling (ABPP). Phosphoproteins (pSTY) altered by RXDX106 were identified using mass spectrometry. Effects of RXDX106 on signaling, viability and migration of LC cells were also evaluated. Cell line models of EMT-mediated acquired drug resistance, treated with a combination of AXL and EGFR TKIs, were analyzed for changes in signaling, cell viability and EMT. Immunoprecipitation (IP) identified adaptors of AXL signaling, and Proximity Ligation Assays (PLA) were developed to detect these active complexes in situ. H1299 cells, expressing highest levels of p and t AXL among the LC lines screened, was used in this study. RXDX106 and Cabozantinib potently inhibited pAXL in H1299 cells, but did not affect cell viability at these doses. R428 reduced cell viability at doses that did not efficiently inhibit pAXL, suggesting AXL independent phenotypic effects. Our ABPP data shows that apart from AXL, these TKIs target other overlapping and distinct subsets of proteins. R428 has the highest number of off targets and its unique ability to inhibit the FoxO pathway may explain the AXL independent phenotypic effects of R428. The pSTY data shows that RXDX106 deregulates phosphorylation of proteins involved in PI3K signaling, receptor endocytosis and cell migration pathways in H1299 cells. WB and phenotypic assays support these results by showing that RXDX106 inhibits pAXL, downstream pAKT but not pERK, and migration/invasion in these cells. In EGFR TKI resistant cells, EGFR and AXL TKI combination fails to alter downstream signaling, cell viability or EMT. Consistent with the WB and pSTY analyses, IP identifies PI3KR1 as an AXL interactor. PLAs to detect active AXL:PI3KR1 and AXL:pY100 signaling complexes show high basal PLA foci in H1299 and Calu1 cells that are abrogated by AXL TKI. HCC827 cells, which lack ligand independent pAXL, do not show significant labeling by either PLA. Overall, we demonstrate that different AXL TKIs have distinct target profiles and that inhibition of AXL suppresses downstream PI3K/AKT signaling and migration/ invasion of LC cells. We also show that AXL TKI fails to suppress downstream signaling, cell viability or EMT in EGFR TKI resistant cell lines. We have also established a PLA to annotate AXL adaptor foci that could be developed as a tool to measure drug-targetable active AXL complexes in patient tissues. Citation Format: Anurima Majumder, Guolin Zhang, Emma Adhikari, Bin Fang, Eric A. Welsh, John M. Koomen, Eric B. Haura. Proteomic characterization of AXL kinase inhibitors and signaling pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4543.

Abstract 2010: SKI-G-801, an AXL kinase inhibitor, blocks metastasis and induces anti-tumor immune responses in various syngeneic cancer models

Abstract Introduction: Aberrant AXL expression plays a critical role in cancer cell migration, metastasis and drug resistance. Researchers have revealed that AXL signaling is also over expressed on cells associated with tumor microenvironment. These findings highlight AXL as an attractive drug candidate for targeting tumor evasion and metastasis. Here we present SKI-G-801, a small molecule inhibitor that targets phosphorylation of AXL (IC50 = 20 nM) and its downstream signals. Methods: Inhibitory effects of SKI-G-801 on cancer viability (MTT and colony formation assay), invasion and migration (trans-well invasion assay) were examined in AXL high-expressing lung cancer cells in vitro. LLC2 lung and 4T1 breast cancer bearing mouse models were established. in addition, C57BL/6 mice were injected intravenously with B16F10 melanoma cells to establish lung metastasis model. Mice were administrated with 30 mg/kg of SKI-G-801 orally before (metastasis model) or after (syngeneic model) tumor injection. To elucidate the involvement of AXL inhibitor on tumor microenvironment, the population of T cells and myeloid cells was analyzed by flow cytometry from the LLC2 tumor. Results: Treatment of SKI-G-801 showed strong inhibition of cancer migration and invasion, when its direct killing effect on cancer cells was modest. These results were reproduced in vivo test that pretreatment of SKI-G-801 significantly reduced metastatic burden in B16F10 model (p < 0.05). LLC2 and 4T1 tumors were decreased in SKI-G-801 treatment group (p < 0.05), but not in anthemic nude mice. CD3+CD8+ T cell population and memory Tc cells were increased in SKI- G-801 treatment group (p < 0.05). Especially, granzyme B+ Tc and gp70+ tumor specific Tc were increased (p < 0.05). SKI-G-801 increase the helper T cell population; CD3+CD4+ (p < 0.05), and CD44+ memory Th cells (p < 0.01). Conclusion: SKI-G-801 demonstrates great potential in anti-cancer activity though immune responses. The anti-cancer effects lead to a reversal of the metastatic phenotype in animal model. Our results suggest that SKI-G-801 is a promising drug for prevention against metastatic cancer. Citation Format: Chun-Feng Xin, Sung Eun Kim, Kyoung-Ho Pyo, Ha Ni Jo, Jae Seok Cho, Jae Hwan Kim, Wongeun Lee, Hee Kyu Lee, Jung-Ho Kim, Ho-Juhn Song, Jong Sung Koh, Byoung Chul Cho. SKI-G-801, an AXL kinase inhibitor, blocks metastasis and induces anti-tumor immune responses in various syngeneic cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2010.

Abstract 2006: Targeting AXL kinase with TP-0903 successfully reverses the mesenchymal phenotype and extends survival in preclinical models of advanced ovarian cancer

Abstract Epithelial-to-mesenchymal transition (EMT) is a critical step in the progression of ovarian cancer (OVCA), which drives local invasion, metastasis and drug resistance leading to poor survival. AXL, a receptor tyrosine kinase, functions as a molecular driver of EMT in multiple cancers, including OVCA. The high mortality of this tumor is explained by the fact that the majority (75%) of patients present at an advanced stage, with widely metastatic disease within the peritoneal cavity often associated with ascites. The extent of ascites is a prognostic factor for survival, impacts quality of life, and correlates with the EMT phenotype. Approaches to block or reverse EMT are needed to improve the prognosis of OVCA patients, particularly those with refractory or early relapse (<6 months) disease following debulking surgery and conventional adjuvant treatments. TP-0903 is a potent, small molecule AXL inhibitor that has activity in reversing EMT in multiple models of disease and embryonic development. The objective of this study was to explore the anti-tumor activity in preclinical mesenchymal OVCA-derived models, which may provide rationale to evaluate TP-0903 in treatment-resistant or refractory OVCA patients. TP-0903 showed potent effects on cell viability in multiple OVCA-derived cell lines. The IC50 values in cell viability studies ranged from 33 nM (OVTOKO cells) to 840 nM (OVMANA cells). Kuramochi cells represent the most common ovarian cancer subtype, high-grade serous ovarian cancer and was particularly platinum-resistant (>5 μM), yet sensitive to TP-0903 (210 nM). Baseline phospho-AXL (Y702) was exceptionally high in Kuramochi cells and TP-0903 inhibited this phosphorylation at concentrations as low as 100 nM. TP-0903 reversed EMT in OVCA cells as evident by the down-regulation of mesenchymal markers including Snail and Slug. Consistent with the reversal of the EMT, TP-0903 inhibited the migration of ES-2 OVCA cells in a scratch assay. TP-0903 suppressed ascites development in preclinical models of OVCA and inhibited metastatic intraperitoneal tumor dissemination. Treatment suppressed ascites development (ascites volume, vehicle: 3.34 mL, TP-0903: 0.28 mL; abdominal circumference, vehicle: 7.96 cm (day 15), TP-0903: 6.82 cm (day 15), p<0.05) in an orthotopic ES-2 model and extended overall survival compared to vehicle or cisplatin alone. Phospho-AXL modulation and changes in other mechanism-validating biomarkers were observed in the ES-2 model. Similar preclinical activities, including decreased ascites volume, were observed in A2780cis and OVCAR-3 xenograft models. These results support further clinical evaluation of TP-0903 in either single-agent or combination regimens treating recurrent or refractory OVCA. TP-0903 is investigated in a Phase I trial that includes an expansion cohort of this patient population (NCT02729298). Citation Format: Nozomi Tomimatsu, Ken Fujimura, Yuta Matsumura, Hiroki Umehara, Lars Mouritsen, Steven L. Warner, David J. Bearss. Targeting AXL kinase with TP-0903 successfully reverses the mesenchymal phenotype and extends survival in preclinical models of advanced ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2006.

Abstract 2197: The AXL kinase inhibitor, TP-0903, demonstrates efficacy in preclinical models of colorectal cancer independent of KRAS mutation status

Abstract Colorectal cancer (CRC) is the third leading cause of cancer-related mortality in the US (2017). Most patients present with stage II or III disease at diagnosis, with the 5-year survival rate between 53-89%. Survival in patients with stage IV CRC, however, is a discouraging 11%. Metastasis in CRC is linked to a mesenchymal phenotype, which is associated with chemoresistance. AXL, a receptor tyrosine kinase, promotes the mesenchymal phenotype in cancer cells and its expression is associated with drug resistance and poor outcomes. TP-0903, a clinical-stage, investigational small molecule inhibitor of AXL has been shown to reverse the mesenchymal phenotype and restore drug sensitivity in cells that no longer respond to standard agents in preclinical models. TP-0903 is hypothesized to be an active agent in CRC through reversal of the mesenchymal phenotype. In cell viability assays of CRC lines, TP-0903 treatment resulted in IC50 values ranging from 4.5 – 123 nM. Notably, cell growth inhibition by TP-0903 was independent of KRAS mutation status; the KRAS mutant HCT-116 line was the most sensitive CRC cell line tested. Mesenchymal markers, including Snail, were suppressed by 7.6-fold (mRNA) and 4.9-fold (protein) in the HCT-116 line at 500 nM. TP-0903 activity was also assessed in vivo using two KRAS mutant CRC models: HCT-116 and a patient-derived xenograft (PDX) model. In the HCT-116 xenograft model, single agent TP-0903 treatment achieved 69% tumor growth inhibition (%TGI) with an oral dosing schedule at 40 mg/kg. In a KRAS-mutant PDX model, TP-0903 achieved 44% TGI when mice were dosed at 40 mg/kg. Pharmacodynamic analyses were performed on tissues from the HCT-116 and PDX models. The ligand for AXL, GAS6, was significantly upregulated in tissues after TP-0903 treatment in both CRC in vivo models while soluble AXL and GAS6 were significantly downregulated in plasma in the PDX model. Furthermore, Axin2, a Wnt/β-catenin regulated gene, was downregulated by TP-0903 in tumor tissue from the PDX model, suggesting inhibition of the Wnt/βcatenin pathway. These data support a potential role for AXL in the promotion of the mesenchymal phenotype in CRC, and showed that AXL inhibition by TP-0903 suppressed the mesenchymal phenotype and was effective against CRC cells regardless of KRAS mutation status. These observations support further clinical investigation of TP-0903 as a potential therapeutic agent in metastatic CRC. A Phase I trial with this investigational agent is ongoing, including patients with KRAS mutant CRC (clincaltrials.gov, NCT02729298). Citation Format: Ryan Mangelson, Peter Peterson, Jason M. Foulks, Yuta Matsumura, Lars Mouritsen, Clifford J. Whatcott, David J. Bearss, Steven L. Warner. The AXL kinase inhibitor, TP-0903, demonstrates efficacy in preclinical models of colorectal cancer independent of KRAS mutation status [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2197.

Curcumin-Synthetic Analogs Library Screening by Docking and Quantitative Structure-Activity Relationship Studies for AXL Tyrosine Kinase Inhibition in Cancers.

AXL is an important drug target for cancers. Two-dimensional quantitative structure-activity relationship (2D-QSAR) tests were performed to elucidate a relationship between molecular structures and the activity of a series of 400 curcumin derivatives subjected to AXL kinase by ATP competition in the catalytic site. The partial least square regression method implanted in molecular operating environment software was applied to develop QSAR models, which were further validated for statistical significance by internal and external validation. The best model has proven to be statistically robust with a good predictive correlation of R 2 = 0.996 and a significant cross-validation correlation coefficient of q 2 = 0.707. Docking analysis reveled that three curcumin derivatives have the best affinity for AXL and formed a hydrogen bond with the important amino acid residues in the binding pocket. As treated in this article, the docking studies and 2D-QSAR approach will pave the way for the development of new drugs while highlighting curcumin and its derivatives.

Targeting of the AXL receptor tyrosine kinase by small molecule inhibitor leads to AXL cell surface accumulation by impairing the ubiquitin-dependent receptor degradation

BackgroundOverexpression of AXL receptor tyrosine kinase (AXL) in various human cancers correlates with reduced patients overall survival and resistance to first line therapies. Therefore, several AXL tyrosine kinase inhibitors (TKIs) are currently under clinical evaluation.ResultsAXL TKI BMS777607 treatment increased AXL protein levels after 24 h as observed by Western blot and flow cytometry analysis. Mechanistically, this inhibition-induced AXL cell surface accumulation was neither associated with epigenetic modifications, nor altered transcriptional and translational regulation. Further, we saw no impact on glycosylation and receptor shedding by α-secretases. However, we observed that BMS777607 increased the glycosylated 140 kDa AXL protein abundance, which was impaired in the kinase dead mutant AXL (K567R). We demonstrated that AXL kinase activity and subsequent kinase phosphorylation is necessary for GAS6-dependent receptor internalization and degradation. Blocking of kinase function by BMS777607 resulted in ubiquitination prohibition, impaired internalization and subsequent cell surface accumulation. Subsequently, AXL cell surface accumulation was accompanied by increased proliferation of 3D-Speroids induced by low μM levels of BMS777607 treatment.ConclusionOur data suggest a re-evaluation of anti-AXL clinical protocols due to possible feedback loops and resistance formation to targeted AXL therapy. An alternative strategy to circumvent feedback loops for AXL targeting therapies may exist in linkage of AXL TKIs to a degradation machinery recruiting unit, as already demonstrated with PROTACs for EGFR, HER2, and c-Met. This might result in a sustained inhibition and depletion of the AXL from tumor cell surface and enhance the efficacy of targeted anti-AXL therapies in the clinic.

Molecular modelling studies for 4-oxo-1,4-dihydroquinoline-3-carboxamide derivatives as anticancer agents

The ligand library reporting anticancer activities was taken from literature containing forty-two 4-oxo-1,4-dihydroquinoline-3-carboxamides derivatives and was thoroughly investigated against cancer target (pdb id-5U6B) using docking protocol. In order to design future analogues, we carried out our research to develop the Atom based- and Field based-3D QSAR (Quantitative structure–activity relationship models) models.PHASE was employed for the common pharmacophore generation and 3D-QSAR studies. The protein target (pdb id-5U6B) was selected and docking was used in order to check better binding modes between target and ligands. The crystal structure of Axl kinase domain in complex with a macrocyclic inhibitor complex (pdb id-5U6B) had a resolution of 2.84 Å. The free energy was calculated by using PRIME-Molecular mechanics with generalized Born and surface area solvation (MM/GBSA). The molecular properties and ADME parameter were calculated using Qikprop utility.Out of 12 developed pharmacophore hypothesis using the PHASE module, a six point AAHRRR_1 hypothesis found best with good scoring parameters involving the two acceptor features, one hydrophobic feature and three ring features. For the atom based-3D QSAR model, a good cross-validation correlation coefficient i.e. Q2 value as 0.5715 for test set and R2 (a correlation coefficient) as 0.8704 for training set molecules. In case of field based QSAR, a cross-validation correlation coefficient (Q2) for test set as 0.5697 and R2 (a correlation coefficient) as 0.817 for training set molecules. Both the models show good statistical significance for future designing. The compound 9L observed as best docked for target 5U6B having docking score of −4.085 kcal/mol and PRIME MM/GBSA (Prime/Molecular Mechanics Generalized Born Surface Area) dg bind energy as-63.302 kcal/mol. The values for rule of five for all dataset compounds were found within range. Percentage human oral absorption values were also >80% except few compounds. Finally, our current study will helpful for researchers in order to develop new 4-oxo-1,4-dihydroquinoline-3-carboxamides derivatives as more potent anticancer agents.

Osimertinib and dihydroartemisinin: A novel drug combination targeting head and neck squamous cell carcinoma.

e17526 Background: Recurrent and metastatic head and neck squamous cell carcinoma (HNSCC) has a dismal prognosis with limited progression-free survival and overall survival, even when treated with different combinations of chemotherapy, targeted therapies and immunotherapy. We explored in vitro and in vivo the effect of the epidermal growth factor (EGFR) inhibitor, osimertinib, alone and in combination with dihydroartemisinin (DHA) in HNSCC. Methods: The combination of osimertinib with DHA was tested in the FaDu and CAL27 HNSCC cell lines. Tumor cell proliferation assays were conducted in cultured cells and mouse xenografts. Western blotting analysis of related signal pathways was performed to investigate the molecular mechanisms of the inhibitory effect of DHA and the combination. Other compounds, which inhibit signal transducer and activator of transcription 3 (STAT3), Src-family kinases (SFKs), sphingosine kinase 1 (SPHK1), or the receptor tyrosine kinase (RTK) AXL were also combined with osimertinib in vitro. Results: Osimertinib exerted synergistic cytotoxicity toward FaDu and CAL27 HNSCC cells when combined with DHA. DHA reversed the osimertinib-induced STAT3 and Src, phosphorylation. The double combination inhibited AXL expression. The anticancer potential of osimertinib plus DHA combination was validated in vivo on FaDu and CAL27 xenografts in mice without notable side effects. Conclusions: The results illustrate that the combinatory therapy of osimertinib and DHA, as a repurposing anticancer drug, could be a novel therapeutic strategy for recurrent and/or metastatic HNSCC patients. The findings indicate that a clinical trial is warranted to confirm the benefit of the combination.

Development linear and non-linear QSAR models for predicting AXL kinase inhibitory activity of N-[4-(quinolin-4-yloxy)phenyl]benzenesulfonamides

In this research, we used CoMFA, LSSVM and FFANN for creating QSAR models for predicting AXL Kinase inhibitory activity of N-[4-(Quinolin-4-yloxy)phenyl]benzenesulfonamides. A CoMFA model with three components was developed and CoMFA contour maps were interpreted to extract chemical features that influence the inhibitory activity of these molecules. R2 for train and test set of CoMFA model were 0.8900 and 0.8171, respectively. Model created by five Dragon descriptors and LSSVM model showed slightly better predictive power with respect to CoMFA model. R2 for train, test set of created LSSVM model were 0.0.8477 and 0.8218, respectively. Also, a FFANN model, using the same five descriptors, was developed with 2 neurons in its hidden layer and R2 for its train and test sets were 0.8314 and 0.8522, respectively. All created models were validated by calculating several statistical parameters and their applicability domain were investigated by calculating leverage. Furthermore, a homology model was built for Axl structure and molecules with the lowest and the greatest activity were docked to it and their interactions with Axl were investigated.

Axl promotes ZIKV entry and modulates antiviral state of human Sertoli cells

Abstract Zika virus (ZIKV) is unique among mosquito-borne flaviviruses in its ability to be sexually transmitted. The ability of ZIKV to establish persistent infection in the testes clearly suggests involvement of unique immune suppressive pathways, however the underlying mechanisms are not defined. We demonstrated that human Sertoli cells (SC), the major cell type of seminiferous tubules, an immune privileged compartment of the testes, support prolonged ZIKV replication without compromising cell survival. Our recent study also provides evidence that human testicular organoids (HTO) can be used as a tool to study ZIKV immunopathogenesis. Recent reports have identified the TAM (Tyro3, Axl, Mer) receptor tyrosine kinase Axl, a critical regulator of innate immunity and apoptosis, as an entry receptor and/or immune modulator for ZIKV infection depending on cell type. Interestingly Axl receptor is highly expressed in the testes, however, the role of Axl in ZIKV infection of SC has not been determined. In this study, we show that Axl was highly expressed in SC as compared to Leydig cells that correlated with robust infection of ZIKV only in SC. Further, neutralizing Axl receptor and its ligand Gas6 strongly attenuated virus entry and replication in SC. Blocking of Axl kinase and downstream signaling was associated with decrease in the negative regulators of antiviral immunity including pSTAT1 and SOCS1 that led to increased expression of IFIT1 and MxA, and reduced virus replication in the SC and HTO. Together, our data demonstrates that Axl promotes ZIKV entry and negatively modulates antiviral state of SC to augment long-term infection of the testes and provides new insights into testis immunology in the context of ZIKV persistence.

Abstract P2-06-05: TP-0903, an AXL kinase inhibitor, reduces inflammatory breast cancer aggressiveness and macrophage polarization through additional mechanisms that may include JAK2 and Aurora B

Abstract Background: Inflammatory breast cancer (IBC) is the most lethal and aggressive type of breast cancer; it accounts for 2-4% of breast cancer cases but causes 8-10% of breast cancer deaths. Novel targeted therapy to improve the outcomes of patients with IBC is urgently needed. The receptor tyrosine kinase AXL is a driver for metastasis and drug resistance in various cancers, including breast cancer. Our previous work showed that AXL signaling contributes to the aggressiveness of IBC. In addition, emerging evidence indicates that the tumor microenvironment components, particularly tumor-associated macrophages, are critical drivers of the IBC clinical phenotype and promote IBC metastasis. AXL signaling has been shown to modulate the tumor microenvironment. In the present study, we investigated the impact of TP-0903, a small-molecule AXL kinase inhibitor, with additional activity against Aurora B and Janus kinase 2 (JAK2), on IBC cells and macrophage polarization. Methods: The effects of TP-0903 on IBC cell proliferation, migration/invasion, and mammosphere formation were analyzed. The effects of TP-0903 on the polarization of human monocytic cells THP-1 were tested in vitro. In addition, the signaling pathways involved in TP-0903-regulated M2 macrophage polarization were investigated using Western blotting. Results: The half-maximal inhibitory concentration (IC50) of TP-0903 in an array of IBC cells (including SUM149, SUM190, BCX010, FC-IBC-02, MDA-IBC-3, and KPL4) ranged from 66 nM to 346 nM, suggesting a strong cell growth inhibitory effect. TP-0903 treatment decreased the migration, invasion, and mammosphere formation of IBC cells. In addition, TP-0903 inhibited both AXL signaling and Aurora B activation, which induced a G2/M cell cycle arrest in IBC cells. Based on the importance of AXL and JAK2 in the regulation of the tumor microenvironment, we showed that TP-0903 decreased expression of CD163/CD206 and the CCL17/CCL18 cytokine, and markers of M2 macrophages, suggesting that TP-0903 treatment inhibits the polarization of THP-1 cells to M2 macrophages in vitro. We also found that TP-0903 treatment decreased the phosphorylation of STAT6, a critical molecule in M2 polarization, and that knockdown of STAT6 expression decreased M2 macrophage polarization, indicating that TP-0903 may regulate macrophage polarization via STAT6 signaling. Conclusion: Our results demonstrated the dual functions of TP-0903 targeting of both IBC cells and macrophages, possibly via the targeting of multiple kinases, including AXL and Aurora B. Examinations of the impact of TP-0903 on the cross-talk between IBC cells and macrophages in vitro and in vivo and the related mechanisms are ongoing and will be presented at the meeting. Citation Format: Cheng Y, Funakoshi Y, Wang X, Warner SL, Bearss DJ, Ueno NT. TP-0903, an AXL kinase inhibitor, reduces inflammatory breast cancer aggressiveness and macrophage polarization through additional mechanisms that may include JAK2 and Aurora B [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-06-05.

Computational Design of Druglike Allosteric Inhibitors of AXL and MET Receptor Tyrosine Kinases

We report on the computational design of drug-like small molecules that could allosterically bind both the AXL and MET kinases. Such binding would inhibit the kinases and potentially promote apoptosis in cancer cells. AXL and MET are receptor tyrosine kinases (RTKs) that play important roles in healthy cells. However, both AXL and MET are overexpressed in many cancers where they have been linked to drug resistance and metastasis. Several inhibitors of AXL and MET are in various stages of clinical trials. However, generally, they bind to the active site of the kinases. The structure of the active site is similar in many kinases, making them difficult to target selectively. Molecules that broadly inhibit several kinases may damage healthy tissue and lead to undesirable side effects in patients. Efforts in drug discovery have been shifting toward inhibiting the allosteric sites of kinases. Allosteric sites differ among kinases, allowing for selective targeting. The small molecules designed here showed comparable or better drug-like properties than the known AXL and MET active-site inhibitors. Also, they had no implied toxic risks, showed good bioavailability, and had promising overall drug potential. The molecules bonded AXL and MET allosterically with high binding affinities. In the process, the kinase's active site conformation changed, preventing the ATP binding. Our findings, together with previous experimental investigations of active-site AXL and MET inhibitors, strongly imply that the molecules designed here may be effective allosteric inhibitors of both kinases and may lead to the development of novel, more effective cancer treatments.

Gilteritinib: First Global Approval.

Gilteritinib (Xospata®) is an orally available small molecule receptor tyrosine kinase inhibitor developed by Astellas Pharma in collaboration with Kotobuki Pharmaceutical for the treatment of acute myeloid leukaemia (AML) harbouring FMS-like tyrosine kinase 3 (FLT3) mutations. Gilteritinib inhibits FLT3 (STK1 or FLK2), AXL (UFO or JTK11) and anaplastic lymphoma kinase (ALK or CD246). Gilteritinib inhibits FLT3 signalling in cells expressing FLT3 internal tandem duplication (ITD), tyrosine kinase domain mutation FLT3-D835Y and the double mutant FLT3-ITD-D835Y, thereby inducing apoptosis. Gilteritinib also binds to and inhibits the wild-type and mutated forms of ALK, resulting in reduced tumour cell proliferation in cancer cell types that overexpress the mutation. Gilteritinib is approved in Japan for the treatment of relapsed or refractory AML with FLT3 mutation. Recently, it was also approved in the USA for the treatment of adult patients who have relapsed or refractory AML with a FLT3 mutation, as detected by an FDA-approved test. Clinical development of gilteritinib is underway in several countries worldwide. Development for non-small cell lung cancer and solid tumours has been discontinued.

Quinolone antibiotic derivatives as new selective Axl kinase inhibitors

Axl is a new promising molecular target for antineoplastic therapies. A series of quinolone antibiotic derivatives were designed and synthesized as new selective Axl inhibitors. One of the most promising compound 8i bound tightly to Axl with a Kd value of 1.1 nM, and inhibited its kinase activity with an IC50 value of 26 nM. The compound also significantly inhibited the phosphorylation of Axl and dose dependently inhibited cell invasion and migration in TGF-β1 induced MDA-MD-231 breast cancer cells. In addition, 8i demonstrated reasonable pharmacokinetic properties and exhibited extraordinary target selectivity over 468 kinases except for Flt3 (IC50 = 50 nM)), with a S(10) and S(35) value of 0.022 and 0.42 at 1.0 μM, respectively. Compound 8i may serve as a new valuable lead compound for future anticancer drug discovery.

Leptospira species promote a pro-inflammatory phenotype in human neutrophils.

Leptospirosis is a global zoonosis caused by pathogenic Leptospira. Neutrophils are key cells against bacterial pathogens but can also contribute to tissue damage. Because the information regarding the role of human neutrophils in leptospirosis is scant, we comparatively analysed the human neutrophil's response to saprophytic Leptospira biflexa serovar Patoc (Patoc) and the pathogenic Leptospira interrogans serovar Copenhageni (LIC). Both species triggered neutrophil responses involved in migration, including the upregulation of CD11b expression, adhesion to collagen, and the release of IL-8. In addition, both species increased levels of pro-inflammatory IL-1β and IL-6 associated with the inflammasome and NFκB pathway activation and delayed neutrophil apoptosis. LIC was observed on the neutrophil surface and not phagocytized. In contrast, Patoc generated intracellular ROS associated with its uptake. Neutrophils express the TYRO3, AXL, and MER receptor protein tyrosine kinases (TAM), but only LIC selectively increased the level of AXL. TLR2 but not TLR4-blocking antibodies abrogated the IL-8 secretion triggered by both Leptospira species. In summary, we demonstrate that Leptospira species trigger a robust neutrophil activation and pro-inflammatory response. These findings may be useful to find new diagnostic markers and therapeutic strategies against leptospirosis.