Tyro3, Axl, And Mer Receptor Research Articles

STELLAR: A Phase II, Open-Label, Single-Arm, Prospective Clinical Study of Tislelizumab Combined with Sitravatinib as Consolidation Treatment after Chemoradiotherapy in Locally Advanced, Unresectable Non-Small-Cell Lung Cancer

<h3>Purpose/Objective(s)</h3> Consolidation durvalumab has become the standard of care in patients with unresectable, stage III non-small-cell lung cancer (NSCLC) without disease progression after concurrent chemoradiation therapy (cCRT) based on the PACIFIC study. However, there are still about 44% of patients experiencing disease progression or recurrence within one year. New regimens for consolidation treatment are worth exploring to further improve survival for this population. Tislelizumab, an anti-PD-1 antibody engineered to minimize binding to FcγR on macrophages to abrogate antibody-dependent phagocytosis (a potential mechanism of resistance), has shown improved efficacy in advanced NSCLC with a tolerable safety profile. Sitravatinib is a spectrum-selective tyrosine kinase inhibitor targeting TAM (Tyro3, Axl, and Mer) and split tyrosine-kinase domain-containing receptors (VEGFR2, KIT), which may potentially augment immune checkpoint blockade through reducing the number of myeloid-derived suppressor cells/regulatory T cells and increasing the ratio of M1/M2 polarized macrophages. An early trial of tislelizumab plus sitravatinib showed acceptable tolerability and promising antitumor activity in advanced NSCLC. STELLAR (NCT05176925) is evaluating tislelizumab plus sitravatinib as consolidation treatment in patients with unresectable, locally advanced, stage III NSCLC after cCRT. <h3>Materials/Methods</h3> This phase II, open-label, single-arm, prospective study is enrolling patients aged from 18-75 years with histologically/cytologically confirmed, locally advanced, unresectable, stage III NSCLC who have not progressed following definitive, platinum-based cCRT, and with ECOG PS of 0 or 1. Patients will be excluded if they have a mixed histological type of small cell lung cancer and NSCLC, or receive prior anti-VEGF mAb, VEGFR TKI or ICI therapy. Patients will receive tislelizumab (200 mg, IV, d1, Q3W) combined with sitravatinib (starting dose 70 mg, po, qd; escalating to 100 mg, qd, after 2 cycles) for up to 1 year until disease progression, intolerable toxicity, withdrawal of consent or death. The primary endpoint is 1-year progression-free survival (PFS) rate by investigator assessment per RECIST v1.1. Secondary end points are PFS, objective response rate, duration of response and disease control rate assessed by investigators per RECIST 1.1, overall survival, time to distant metastasis, and safety. AEs will be graded by National Cancer Institute Common Terminology Criteria for Adverse Events (NCI CTCAE) v5.0. Enrollment begins in January 2022, and approximately 36 patients will be enrolled. <h3>Results</h3> N/A <h3>Conclusion</h3> N/A

Red blood cell exchange for SARS-CoV-2: A Gemini of therapeutic opportunities

As of now, therapeutic strategies for the novel coronavirus (SARS-CoV-2) are limited and much focus has been placed on social distancing techniques to “flatten the curve”. Initial treatment efforts including ventilation and hydroxychloroquine garnered significant controversy and today, SARS-CoV-2 outbreaks are still occurring throughout the world. Needless to say, new therapeutic strategies are needed to combat this unprecedented pandemic. Nature Reviews Immunology recently published an article hypothesizing the pathogenesis of TAM (Tyro3, Axl, and Mer) receptor signaling in COVID-19. In it they expressed that hypercoagulation and immune hyper-reaction could occur secondary to decreased Protein S (PROS1). And hypoxia has been recently discovered to significantly decrease expression of PROS1. Regarding the cause of hypoxia in COVID-19; NIH funded research utilizing state-of-the-art topologies has recently demonstrated significant metabolomic, proteomic, and lipidomic structural aberrations in hemoglobin (Hb) secondary to infection with SARS-CoV-2. In this setting, Hb may be incapacitated and unable to respond to environmental variations, compromising RBCs and oxygen delivery to tissues. The use of red blood cell exchange would target hypoxia at its source; representing a Gemini of therapeutic opportunities.

Modulation of protein S and growth arrest specific 6 protein signaling inhibits pancreatic cancer cell survival and proliferation.

Thrombotic complications and hypercoagulopathies are commonly associated with the progression of pancreatic ductal adenocarcinoma (PDAC). Although the mechanistic link between the two phenomena is uncertain, there is evidently an increase in procoagulant proteins and a decrease in anticoagulants in PDAC patients. For example, the anticoagulant protein S (PS) is decreased during the progression of PDAC, a condition that possibly contributes to the hypercoagulopathies. PS is also an important signaling molecule that binds a family of tyrosine kinase receptors known as TAM (Tyro3, Axl and Mer) receptors; TAM receptors are often upregulated in different cancers. Growth Arrest Specific 6 or GAS6 protein, a homolog of PS, is also a TAM receptor family ligand. The downstream signaling pathways triggered by this ligand-receptor interaction perform diverse functions, such as cell survival, proliferation, efferocytosis, and apoptosis. Targeting the TAM receptors to treat cancer has had limited success; side effects are a significant obstacle due to the widespread numerous functions of TAM receptors. In the present study, it was revealed that PS-TAM interaction was pro-apoptotic, whereas GAS6-mediated TAM signaling promoted proliferation and survival in select PDAC cell lines. Furthermore, by regulating the balance between these two signaling pathways (by overexpressing PS or knocking down GAS6), the proliferative potential of the cells was decreased. Both long-term and short-term effects of natural PS overexpression were comparable to the treatment of the cells with the drug UNC2025, which inhibits the Mer-receptor. The present study lays the foundation for investigation of PS as a therapeutic agent to control cancer progression and to concurrently arrest thrombotic events.

AXL Targeting Overcomes Human Lung Cancer Cell Resistance to NK- and CTL-Mediated Cytotoxicity.

Immune resistance may arise from both genetic instability and tumor heterogeneity. Microenvironmental stresses such as hypoxia and various resistance mechanisms promote carcinoma cell plasticity. AXL, a member of the TAM (Tyro3, Axl, and Mer) receptor tyrosine kinase family, is widely expressed in human cancers and increasingly recognized for its role in cell plasticity and drug resistance. To investigate mechanisms of immune resistance, we studied multiple human lung cancer clones derived from a model of hypoxia-induced tumor plasticity that exhibited mesenchymal or epithelial features. We demonstrate that AXL expression is increased in mesenchymal lung cancer clones. Expression of AXL in the cells correlated with increased cancer cell-intrinsic resistance to both natural killer (NK)- and cytotoxic T lymphocyte (CTL)-mediated killing. A small-molecule targeting AXL sensitized mesenchymal lung cancer cells to cytotoxic lymphocyte-mediated killing. Mechanistically, we showed that attenuation of AXL-dependent immune resistance involved a molecular network comprising NF-κB activation, increased ICAM1 expression, and upregulation of ULBP1 expression coupled with MAPK inhibition. Higher ICAM1 and ULBP1 tumor expression correlated with improved patient survival in two non-small cell lung cancer (NSCLC) cohorts. These results reveal an AXL-mediated immune-escape regulatory pathway, suggest AXL as a candidate biomarker for tumor resistance to NK and CTL immunity, and support AXL targeting to optimize immune response in NSCLC.

TYRO3: A potential therapeutic target in cancer.

Cancer is among the leading causes of death worldwide. In 2016, 8.9 million people are estimated to have died from various forms of cancer. The current treatments, including surgery with chemotherapy and/or radiation therapy, are not effective enough to provide full protection from cancer, which highlights the need for developing novel therapy strategies. In this review, we summarize the molecular biology of a unique member of a subfamily of receptor tyrosine kinase, TYRO3 and discuss the new insights in TYRO3-targeted treatment for cancer therapy.

Activation of the Receptor Tyrosine Kinase AXL Regulates the Immune Microenvironment in Glioblastoma.

Glioblastoma (GBM) is a lethal disease with no effective therapies available. We previously observed upregulation of the TAM (Tyro-3, Axl, and Mer) receptor tyrosine kinase family member AXL in mesenchymal GBM and showed that knockdown of AXL induced apoptosis of mesenchymal, but not proneural, glioma sphere cultures (GSC). In this study, we report that BGB324, a novel small molecule inhibitor of AXL, prolongs the survival of immunocompromised mice bearing GSC-derived mesenchymal GBM-like tumors. We show that protein S (PROS1), a known ligand of other TAM receptors, was secreted by tumor-associated macrophages/microglia and subsequently physically associated with and activated AXL in mesenchymal GSC. PROS1-driven phosphorylation of AXL (pAXL) induced NFκB activation in mesenchymal GSC, which was inhibited by BGB324 treatment. We also found that treatment of GSC-derived mouse GBM tumors with nivolumab, a blocking antibody against the immune checkpoint protein PD-1, increased intratumoral macrophages/microglia and activation of AXL. Combinatorial therapy with nivolumab plus BGB324 effectively prolonged the survival of mice bearing GBM tumors. Clinically, expression of AXL or PROS1 was associated with poor prognosis for patients with GBM. Our results suggest that the PROS1-AXL pathway regulates intrinsic mesenchymal signaling and the extrinsic immune microenvironment, contributing to the growth of aggressive GBM tumors.Significance: These findings suggest that development of combination treatments of AXL and immune checkpoint inhibitors may provide benefit to patients with GBM. Cancer Res; 78(11); 3002-13. ©2018 AACR.

Antiphospholipid Antibodies Inhibit Trophoblast Toll-Like Receptor and Inflammasome Negative Regulators.

Women with antiphospholipid antibodies (aPL) are at risk for pregnancy complications associated with poor placentation and placental inflammation. Although these antibodies are heterogeneous, some anti-β2 -glycoprotein I (anti-β2 GPI) antibodies can activate Toll-like receptor 4 (TLR-4) and NLRP3 in human first-trimester trophoblasts. The objective of this study was to determine the role of negative regulators of TLR and inflammasome function in aPL-induced trophoblast inflammation. Human trophoblasts were not treated or were treated with anti-β2 GPI aPL or control IgG in the presence or absence of the common TAM (TYRO3, AXL, and Mer tyrosine kinase [MERTK]) receptor ligand growth arrest-specific protein 6 (GAS6) or the autophagy-inducer rapamycin. The expression and function of the TAM receptor pathway and autophagy were measured by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), Western blotting, and enzyme-linked immunosorbent assay (ELISA). Antiphospholipid antibody-induced trophoblast inflammation was measured by qRT-PCR, activity assays, and ELISA. Anti-β2 GPI aPL inhibited trophoblast TAM receptor function by reducing cellular expression of the receptor tyrosine kinases AXL and MERTK and the ligand GAS6. The addition of GAS6 blocked the effects of aPL on the TLR-4-mediated interleukin-8 (IL-8) response. However, the NLRP3 inflammasome-mediated IL-1β response was not affected by GAS6, suggesting that another regulatory pathway was involved. Indeed, anti-β2 GPI aPL inhibited basal trophoblast autophagy, and reversing this with rapamycin inhibited aPL-induced inflammasome function and IL-1β secretion. Basal TAM receptor function and autophagy may serve to inhibit trophoblast TLR and inflammasome function, respectively. Impairment of TAM receptor signaling and autophagy by anti-β2 GPI aPL may allow subsequent TLR and inflammasome activity, leading to a robust inflammatory response.

Elevated Plasma Growth Arrest-Specific 6 Protein Levels Are Associated with the Severity of Disease During Hantaan Virus Infection in Humans.

Hemorrhagic fever with renal syndrome (HFRS) is caused by Hantaan virus (HTNV) and Seoul virus infection in Asia. The clinical manifestation of HFRS is characterized by the rapid loss of renal function (acute kidney injury) and thrombocytopenia. The specific immune mechanisms that cause thrombocytopenia in HFRS are not well described. The growth arrest-specific 6 (Gas6) protein and TAM (Tyro3, Axl and Mer) receptors have been recently shown to play prominent roles in immune regulation, and high plasma levels of Gas6 may predict the severity of diseases. The association of TAM receptors with several autoimmune diseases has been investigated, although the relationship between TAM receptors and these diseases remains unclear in HFRS. Therefore, the aim of this study was to evaluate the clinical significance of Gas6 and TAM receptor expression in HFRS. The concentrations of Gas6 in the plasma from 144 patients and the expression of TAM receptors on monocytes from 117 patients were quantified. The relationship between Gas6 levels and disease course, severity, and clinical parameters was analyzed. We first found that the plasma Gas6 levels were significantly higher in HFRS patients, whereby they were positively correlated with white blood cell counts and negatively correlated with platelet counts. The expression of Tyro3 was increased on monocytes in HFRS patients compared with that in controls. Taken together, our data indicate that elevated plasma Gas6 levels is associated with the severity of disease during HTNV infection in humans, suggesting that Gas6 may play an important role by binding with Tyro3 on monocytes, which will be assessed in future studies.

The TAM receptors Axl and Mer play a joint-specific protective role in experimental arthritis

Abstract Introduction Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by an unrestrained inflammatory response in synovial joints. One family of tyrosine kinase receptors involved in anti-inflammatory feedback mechanism are Tyro3, Axl and Mer (TAM). The plasma level of the TAM receptor ligand Gas6 is significantly reduced in RA patients suggesting this feedback mechanism is impaired in RA. We investigated the individual role of each TAM receptor in experimental arthritis. Methods The KRN serum transfer model of arthritis was induced by two intraperitoneal injections of arthritic K/BxN serum in TAM receptor single gene knock-out mice (Tyro3−/−, Axl−/−, Mer−/−) and wild-type (WT) mice. Ankle joints were macroscopically scored for 7 days and knee and ankle joints were taken at the end of the experiment for histology and immunohistochemistry. Results Mer−/− mice had an increased macroscopic ankle score until day 4 whereas Axl−/− mice had an enhanced macroscopic score from day 4 until the end of the experiment. Histology of the ankle joints showed significantly increased arthritis pathology in Axl−/− mice compared to WT. In contrast, enhanced macroscopic score and arthritis pathology in the knee joints of Mer−/− mice was observed. Both in ankle and knee joints, no protective or aggravating role of Tyro3 was seen. To explain the discrepancy of Axl involvement between ankle and knee, we looked for Axl expression in synovium of naïve mice. The lining layer of ankle synovium was completely Axl-positive whereas only mild expression in the knee joints was observed. Conclusion These findings identify the TAM receptors Axl and Mer as important protective players in arthritis in addition to being expressed in a joint-specific manner.

Opposing roles of tyrosine kinase receptors Mer and Axl determine clinical outcome in a mouse model of nephritis

Abstract Systemic lupus erythematosus (SLE) is an autoimmune disorder with unknown etiology. Lupus nephritis remains one of the most severe manifestations of SLE, with considerable morbidity and mortality. There remains a major void in the successful management of lupus nephritis. TAM (Tyro-3, Axl and Mer) receptor tyrosine kinases play an important role in the maintenance of immune homeostasis. Mer is constitutively highly expressed on glomerular endothelial cells; Axl expression is inducible in kidney under inflammatory conditions. To investigate the functions of Axl and Mer in lupus nephritis, we induced glomerulonephritis in WT, Axl-KO, Mer-KO, and A/M-KO (Axl/Mer-double knockout) mice by injection of nephrotoxic serum (NTS). Mer-KO mice developed severe glomerulonephritis, with significantly decreased survival rate and increased blood urea nitrogen (BUN) levels as compared to WT mice given the same treatment. Two weeks after injection, necrotic cell death was evident in the glomeruli of Mer-KO mice. However, Axl-KO presented with significantly increased survival rate and reduced BUN as compared to the WT, Mer-KO and A/M-KO mice injected with the same serum. Interestingly, the A/M-KO mice developed kidney inflammation comparable to WT mice. Western blot analysis revealed significantly increased Stat3 phosphorylation and caspase-1 activation in the kidney of Mer-KO mice after NTS injection. In contrast, Axl deficient NTS-injected mice developed decreased Akt phosphorylation and Bcl-xl upregulation. The reciprocal activation of Axl and Mer receptor tyrosine kinases may have a major impact on renal inflammation. Results from these studies may lead to novel therapeutic drug development targeted to lupus nephritis.

Axl Receptor Axis: A New Therapeutic Target in Lung Cancer

Abstract The Axl receptor tyrosine kinase belongs to the TAM (Tyro3, Axl, Mer) family of proteins and is upregulated in multiple types of cancers, including non-small cell lung cancer. In normal tissues, TAM receptor tyrosine kinases contribute to immune regulation. In cancer, Axl expression is associated with poor outcomes and is controlled through various transcriptional and epigenetic mechanisms. Preclinical studies have shown that Axl is involved in cancer cell migration, growth, survival and resistance to chemotherapy through activation of multiple downstream signaling pathways, such as Ras/MAPK, PI3K and Rac1. Axl is also expressed on endothelial cells and plays a role in angiogenesis. In preclinical models, Axl inhibition decreases cancer growth and impedes lung cancer metastases. Small molecule Axl inhibitors have been developed and are currently being used as monotherapy or in combination with cytotoxic chemotherapy or anti-EGFR therapy in early clinical trials. Here, we review Axl structure, functions, regulation, and preclinical and clinical studies in lung cancer.

Overexpression of Tyro3 and its implications on hepatocellular carcinoma progression.

While various tyrosine kinases have been associated with the pathogenesis of hepatocellular carcinoma (HCC), the identification of a dominant therapeutic target among them remains a challenge. Here, we investigated the role of Tyro3, a relatively uncharacterized member of the TAM (Tyro3, Axl and Mer) receptor family. The present study aimed to profile and identify potential association between Tyro3 expression in HCC and cancer phenotypes. RNAs obtained from 55 HCC patients were quantified for Tyro3 expression in both cancerous tissue and the adjacent normal tissue. Expression profile was correlated with clinical data. These observations were further substantiated with invitro HCC cell culture investigations.Tyro3 was strongly upregulated (>2-fold elevation) in the tumor tissue of ~42% of the patients. It was shown that higher expression level of Tyro3 was associated with the key tumor marker AFP, and the tumor diameter and liver injury marker ALT. Subsequent cell culture models indicated high expression in various HCC cell lines, in particular Hep3B. Gene silencing of Tyro3 in Hep3B effectively reduced cell proliferation, ERK phosphorylation and cyclinD1 expression, indicating a key in maintaining the proliferative state of these cells. Notably, silencing also suppressed the transcriptional and translational expression of HCC tumor marker AFP. Overall, these data suggest that Tyro3 contributes significantly to tumor growth, aggressiveness and liver dysfunction. Inhibition of Tyro3 and its aberrant signaling in tumors with high expression could present new opportunities for HCC treatment.

Mer tyrosine kinase promotes the survival of t(1;19)-positive acute lymphoblastic leukemia (ALL) in the central nervous system (CNS)

AbstractPatients with t(1;19)-positive acute lymphoblastic leukemia (ALL) are prone to central nervous system (CNS) relapses, and expression of the TAM (Tyro3, Axl, and Mer) receptor Mer is upregulated in these leukemias. We examined the functional role of Mer in the CNS in preclinical models and performed correlative studies in 64 t(1;19)-positive and 93 control pediatric ALL patients. ALL cells were analyzed in coculture with human glioma cells and normal rat astrocytes: CNS coculture caused quiescence and protection from methotrexate toxicity in Merhigh ALL cell lines, which was antagonized by short hairpin RNA–mediated knockdown of Mer. Mer expression was upregulated, prosurvival Akt and mitogen-activated protein kinase signaling were activated, and secretion of the Mer ligand Galectin-3 was stimulated. Merhigh t(1;19) primary cells caused CNS involvement to a larger extent in murine xenografts than in their Merlow counterparts. Leukemic cells from Merhigh xenografts showed enhanced survival in coculture. Treatment of Merhigh patient cells with the Mer-specific inhibitor UNC-569 in vivo delayed leukemia onset, reduced CNS infiltration, and prolonged survival of mice. Finally, a correlation between high Mer expression and CNS positivity upon initial diagnosis was observed in t(1;19) patients. Our data provide evidence that Mer is associated with survival in the CNS in t(1;19)-positive ALL, suggesting a role as a diagnostic marker and therapeutic target.

The Involvement of GAS6 Signaling in the Development of Obesity and Associated Inflammation.

Growth arrest-specific 6 (GAS6), a vitamin K-dependent protein, plays a role in the survival, proliferation, migration, differentiation, adhesion, and apoptosis of cells. GAS6 is highly expressed during growth arrest, followed by a sharp decrease during differentiation in adipocytes. The functions of GAS6 signaling are limited to TAM (Tyro3, Axl, and Mer) receptors and are dependent on the cell type. While many studies have focused on the role of GAS6 in inflammation and cancer, only few studies focused on its roles of GAS6 in obesity. Accordingly, the participation of GAS6 in the progression of obesity remains controversial. In this review, we summarize the results of current studies from clinical and basic research to elucidate the possible role of GAS6 signaling in obesity and associated disorders. In addition, this summary may offer a direction to develop clinical therapeutic strategies for the prevention and treatment of obesity and related complications.

Preclinical Testing Of a Novel Axl-Kinase Inhibitor In Chronic Lymphocytic Leukemia

The receptor tyrosine kinase Axl belongs to the TAM (Tyro-3, Axl and Mer) family and is involved in the progression of several human malignancies including chronic lymphocytic leukemia (CLL), where it is has been found to be overexpressed in comparison to normal B-cells. An increasing body of evidence suggests that Axl acts as an oncogene which increases the survival, proliferation, metastatic potential and chemotherapy resistance of tumor cells. Hence, it has been recently identified as a potential therapeutic target in a wide range of tumor entities with deregulated Axl expression including prostate cancer, glioma, lung cancer and CLL. Here, we investigated two different Axl inhibitors for their ability to inhibit the migratory capacity and survival of leukemic cells in preclinical CLL models.In vitro studies: We measured soluble Axl plasma concentrations by enzyme-linked immunosorbent assay (ELISA) in 71 CLL patients and 24 healthy donors. Soluble Axl levels were not significantly higher in CLL patients compared to healthy donors (p=0. 11). However, in CLL patients high sAxl plasma concentrations were differentially expressed with some patients exhibiting normal and others elevated plasma concentrations. The latter showed an association with shorter time to first treatment (p=0.0005) and several poor prognostic markers (e.g. CD38, FISH cytogentics, Binet stage).Freshly isolated PBMC (>90% CD5+CD19+) from CLL patients were incubated in serum free medium for 48h containing concentrations series of 2 different Axl inhibitors: BMS777607, a previously published inhibitor of the MET kinase family, and LDC2636, a novel inhibitor of the TAM receptor tyrosine kinase (RTK) family with high affinity to Axl. Viability of CLL cells was assessed by trypan blue staining and flow cytometry employing annexin V staining. Cellular polarization was analyzed by time-lapse microscopy. We detected cytotoxic effects in a patient-dependent manner that were more prevalent in LDC2636 as compared to BMS777607 treated cells (IC50= 0.21 µM vs. 2.88 µM, p<0.05, n=5). The cellular polarization of the remaining viable cells was significantly reduced in a dose dependent fashion in comparison to vehicle only controls (LDC2636 IC50 = 7.2 µM, p<0.00001; BMS777607: IC50=6.2µM; p=0.0004). Of note, both Axl inhibitors exhibited significantly weaker effects on both, the viability and polarization of normal PBMC over the whole concentration range tested (p<0.05, n=5).In vivo studies To verify our hypothesis that reduced cell polarization results in decreased homing of leukemic cells in vivo we employed a recently developed adoptive transfer model of CLL. In this model NOD/SCID/IL2Rgcnull(NSG) mice were pre-treated with a single intraperitoneal (i.p.) bolus of LDC2636 or BMS777607 (20 mg/kg) and subsequently transplanted with primary CLL cells. Both Axl inhibitors significantly reduced the homing capacity of CLL cells to the BM of NSG mice by 46% and 59%, respectively, compared to vehicle treated controls (LDC2636: p=0.0063, BMS777607 p=0.0007; n=4).To evaluate if LDC2636 also exhibits effects in a disease-relevant CLL model, we applied a MEC-1 xenograft model which causes a lethal leukemia in NSG mice. We pretreated the mice with 40mg/kg Axl inhibitor or vehicle-only control i.p. and subsequently transplanted the CLL cell line MEC-1 intravenously. The following four days the mice were injected again with 40mg/kg LDC2636 or vehicle-only i.p. We evaluated the survival time and found that mice treated with LDC2636 lived significantly longer than vehicle-only controls (24 vs. 18 days median survival, p=0.0016, n=15). Mice that received only LDC2636 and no Mec-1 cells did not show any effect.These data demonstrate that Axl inhibitors exert potent in vitro and in vivo activity against human CLL cells, which is caused at least in part by the suppression of CLL homing to their supportive stromal niches. Disclosures:Schultz-Fademrecht:Lead Discovery Center GmbH: Employment. Unger:Lead Discovery Center GmbH: Employment. Klebl:Lead Discovery Center GmbH: Employment. Choidas:Lead Discovery Center GmbH: Employment.

Mer Receptor Tyrosine Kinase Is a Novel Therapeutic Target In Multiple Myeloma

IntroductionAlthough many novel therapeutic agents have been explored, multiple myeloma (MM) remains an incurable hematopoietic malignancy. Mer receptor tyrosine kinase (Mer TK) is a member of the TAM (Tyro3, Axl and Mer) family and is involved in the progression of several human malignancies. Inhibition of Mer expression reduces pro-survival signaling, increases chemosensitivity in vitro, and delays tumor progression in vivo. These observations make Mer TK inhibitors to excellent candidates for targeted therapies. We show here for the first time that Mer TK is ectopically expressed in MM cells and test the response of MM cell lines to our previously described small molecule Mer inhibitor, UNC1062, a substituted pyrazolopyrimidine. Here we demonstrate the biochemical and biologic effects of treatment with UNC1062, which suggests efficacy of Mer inhibitors as a novel strategy for the treatment of MM. Methods: Western blot analysis was used to determine expression of the TAM receptors and to evaluate inhibition of Mer TK activation and downstream signaling by UNC1062 in MM cell lines. UNC1062-mediated anti-myeloma activity with or without bortezomib was determined using short-term (MTT) and long-term (colony-formation) assays. Cleaved PARP and caspase 3 were detected by western blot analysis as indicators of apoptosis. Results: Western blot analysis of protein extract from MM cell lines showed that four (RPMI8226, U226, AMO-1 and KMS-12BM) out of six lines (66.6%) express Mer TK protein at varying levels, with AMO-1 expressing the least and U226 expressing the most. The MM cell lines OPM-2 and NCI-H929 did not express Mer TK. UNC1062 potently inhibits Mer TK activity in vitro (Mer IC50= 1.1 nM, Morrison Ki = 0.33 nM). In cell-based assays, treatment with UNC1062 inhibited accumulation of phospho-Mer and downstream signaling through ERK and Stat5 in U226 cells. UNC1062 also reduced proliferation and/or survival in Mer TK-expressing RPMI8226 cells (IC50 = 0.98 ± 0.14 μM, n=7) and U226 (IC50 = 0.28 ± 0.09 μM, n=3) cells. Treatment with UNC1062 did not lead to a meaningful reduction of metabolically active cells in Mer-negative NCI-H929 and OPM-2 cells. In contrast, UNC1062-treated Mer-positive MM cells exhibited increased levels of cleaved caspase 3 and cleaved PARP confirming apoptosis induction. Treatment with UNC1062 (100 nM) resulted in a statistically significant, dose-dependent decrease in colony-formation in methylcellulose compared to control cultures in Mer-positive RPMI8226 cells (418 ± 23 vs. 291 ± 37 colonies, p = 0.04, n = 3). No significant reduction of colony formation was observed in Mer-negative OPM-2 cells. Treatment of U226 cells with UNC1062 (800 nM) in combination with bortezomib resulted in a statistically significant reduction in relative cell number compared with bortezomib alone (0.61 ± 0.06 vs 0.41 ± 0.06, p = 0.04, n =3). Conclusion: Mer TK is ectopically expressed in the majority of investigated MM cell lines. UNC1062 treatment of Mer TK positive MM cells inhibits Mer TK activity and downstream signaling, mediates anti-neoplastic activity in liquid culture, and decreases colony-formation in methylcellulose. In addition, UNC1062 treatment sensitizes MM cells to bortezomib. Taken together, these data support further development of Mer TK inhibitors as novel MM therapy alone and in combination with bortezomib. Disclosures:No relevant conflicts of interest to declare.

Breakdown of immune homeostasis in the testis of mice lacking Tyro3, Axl and Mer receptor tyrosine kinases

Tyro3, Axl and Mer (TAM) receptor tyrosine kinases triple knockout (TAM(-/-)) mice are male infertile due to impaired spermatogenesis. However, the mechanism by which TAM receptors regulate spermatogenesis remains unclear. In this study, we demonstrate that the testicular immune homeostasis was impaired in TAM(-/-) mice. As development after the onset of sexual maturity, germ cells were progressively degenerated. Macrophages and lymphocytes infiltrated into the testis as TAM(-/-) mice aged. Moreover, the integrity of blood-testis barrier was impaired, and the autoantibodies against germ cell antigens were produced. Major inflammatory cytokines, including tumor necrosis factor-α, interleukin-6 and monocyte chemotactic protein 1 were upregulated in the testis of TAM(-/-) mice, and predominantly located in Sertoli cells (SCs). In vitro assays showed that TAM(-/-) SCs secrete significantly high levels of inflammatory cytokines compared with wild-type SCs after coculture with apoptotic germ cells. These results suggest that TAM receptors are important in the maintenance of the immune homeostasis in the testis.

Apoptotic cell recognition receptors, Tyro3, Axl and Mer, demonstrate distinct patterns of ligand-inducible and ligand-independent receptor activation (P2016)

Abstract Proper recognition and removal of apoptotic cells by phagocytes lead to the active suppression of inflammatory responses and the induction of tolerance. Tyro3, Axl and Mer (TAM) receptor tyrosine kinases recognize apoptotic cells through their ligands, Protein S (ProS) and Growth-Arrest-Specific Gene 6 (GAS6), which in turn interact with phosphatidylserine (PS) exposed on the surface of apoptotic cells. Although TAMs share significant similarity, very little is known about the specificity of interaction between TAMs and their ligands, in the context of apoptotic cells, and about downstream signaling cascades triggered through TAMs. To study ligand-receptor interaction, we generated a series of reporter cell lines expressing chimeric TAM receptors that allowed us to demonstrate that each TAM has a unique pattern of interaction with GAS6 and ProS, which is also differentially affected by the presence of apoptotic cells. We also developed another set of chimeric receptors that triggered ligand-independent activation of the TAM intracellular domains, leading to distinct patterns of phosphorylated signaling molecules, as well as different levels of cytokine induction. Overall, these studies suggest that despite their similarity, Tyro3, Axl and Mer are functionally unique in the recognition of apoptotic cells, activation of downstream signaling cascades and gene expression, and therefore, contribute differently to cell survival and the induction of tolerance during tumorigenesis.

Preclinical Testing of a Novel Axl-Kinase Inhibitor in Chronic Lymphocytic Leukemia

Abstract 1799The receptor tyrosine kinase Axl belongs to the TAM (Tyro-3, Axl and Mer) family and is involved in the progression of several human malignancies including chronic lymphocytic leukemia (CLL), where it is has been found to be overexpressed in comparison to normal B-cells. An increasing body of evidence suggests that Axl acts as an oncogene which increases the survival, proliferation, metastatic potential and chemotherapy resistance of tumor cells. Hence, it has been recently identified as a potential therapeutic target in a wide range of tumor entities with deregulated Axl expression including prostate cancer, glioma, lung cancer and CLL. Here, we investigated two different Axl inhibitors for their potential to inhibit the migratory capacity and survival of leukemic cells in preclinical CLL models.In vitro studies: Freshly isolated PBMC (>90% CD5+CD19+) from CLL patients were incubated in serum free medium for 48h containing concentrations series of 2 different Axl inhibitors: BMS777607, a previously published inhibitor of the MET kinase family, and LDC2636, a novel inhibitor of the TAM receptor tyrosine kinase (RTK) family with high affinity to Axl. Viability of CLL cells was assessed by trypan blue staining and flow cytometry employing annexin V staining. Since a polarized phenotype is required for migration, cell polarization was analyzed by time-lapse video-microscopy. We detected cytotoxic effects in a patient dependent manner that were more prevalent in LDC2636 as compared to BMS777607 treated cells (LD50= 1.4 μM vs. 5.2 μM, p<0.004, n=5). Cell polarization of the remaining viable cells was significantly reduced in a dose dependent fashion in comparison to vehicle only controls (LDC2636 IC50 = 7.2 μM, p<0.00001; BMS777607: IC50=6.2μM; p=0.0004). Of note, both Axl inhibitors exhibited significantly weaker effects on both, the viability and cell polarization of normal PBMC over the whole concentration range tested (p<0.05, n=5).In vivo studies: To verify our hypothesis that reduced cell polarization results in decreased homing of leukemic cells in vivo we employed a recently developed adoptive transfer model of CLL. In this model NOD/SCID/gcnull(NSG) mice were pre-treated with a single intraperitoneal bolus of LDC2636 or BMS777607 (20 mg/kg) and subsequently transplanted with primary CLL cells. Both Axl inhibitors significantly reduced the homing capacity of CLL cells to the bone marrow of NSG mice by 43% and 59%, respectively, compared to vehicle treated controls (LDC2636: p=0.046, BMS777607 p=0.0077; n=3).These data demonstrate that Axl inhibitors exert potent in vitro and in vivo activity against human CLL cells, which is caused at least in part by the suppression of CLL homing to their supportive stromal niches. Disclosures:No relevant conflicts of interest to declare.

Identification of Axl as a downstream effector of TGF-β1 during Langerhans cell differentiation and epidermal homeostasis

Transforming growth factor-β1 (TGF-β1) is a fundamental regulator of immune cell development and function. In this study, we investigated the effects of TGF-β1 on the differentiation of human Langerhans cells (LCs) and identified Axl as a key TGF-β1 effector. Axl belongs to the TAM (Tyro3, Axl, and Mer) receptor tyrosine kinase family, whose members function as inhibitors of innate inflammatory responses in dendritic cells and are essential to the prevention of lupus-like autoimmunity. We found that Axl expression is induced by TGF-β1 during LC differentiation and that LC precursors acquire Axl early during differentiation. We also describe prominent steady-state expression as well as inflammation-induced activation of Axl in human epidermal keratinocytes and LCs. TGF-β1-induced Axl enhances apoptotic cell (AC) uptake and blocks proinflammatory cytokine production. The antiinflammatory role of Axl in the skin is reflected in a marked impairment of the LC network preceding spontaneous skin inflammation in mutant mice that lack all three TAM receptors. Our findings highlight the importance of constitutive Axl expression to tolerogenic barrier immunity in the epidermis and define a mechanism by which TGF-β1 enables silent homeostatic clearing of ACs to maintain long-term self-tolerance.