Discovery of Dual MER/AXL Kinase Inhibitors as Bifunctional Small Molecules for Inhibiting Tumor Growth and Enhancing Tumor Immune Microenvironment.

A series of bifunctional compounds have been discovered for their dual functionality as MER/AXL inhibitors and immune modulators. The furanopyrimidine scaffold, renowned for its suitability in kinase inhibitor discovery, offers at least three distinct pharmacophore access points. Insights from molecular modeling studies guided hit-to-lead optimization, which revealed that the 1,3-diketone side chain hybridized with furanopyrimidine scaffold that respectively combined amino-type substituent and 1H-pyrazol-4-yl substituent on the top and bottom of the aryl regions to produce 22 and 33, exhibiting potent antitumor activities in various syngeneic and xenograft models. More importantly, 33 demonstrated remarkable immune-modulating activity by upregulating the expression of total T-cells, cytotoxic CD8+ T-cells, and helper CD4+ T-cells in the spleen. These findings underscored the bifunctional capabilities of 33 (BPR5K230) with excellent oral bioavailability (F = 54.6%), inhibiting both MER and AXL while modulating the tumor microenvironment and highlighting its diverse applicability for further studies to advance its therapeutic potential.

Pharmacological inhibition of MERTK induces in vivo retinal degeneration: a multimodal imaging ocular safety assessment.

The receptor tyrosine kinase, MERTK, plays an essential role in homeostasis of the retina via efferocytosis of shed outer nuclear segments of photoreceptors. The Royal College of Surgeons rat model of retinal degeneration has been linked to loss-of-function of MERTK, and together with the MERTK knock-out mouse, phenocopy retinitis pigmentosa in humans with MERTK mutations. Given recent efforts and interest in MERTK as a potential immuno-oncology target, development of a strategy to assess ocular safety at an early pre-clinical stage is critical. We have applied a state-of-the-art, multi-modal imaging platform to assess the in vivo effects of pharmacological inhibition of MERTK in mice. This involved the application of mass spectrometry imaging (MSI) to characterize the ocular spatial distribution of our highly selective MERTK inhibitor; AZ14145845, together with histopathology and transmission electron microscopy to characterize pathological and ultra-structural change in response to MERTK inhibition. In addition, we assessed the utility of a human retinal in vitro cell model to identify perturbation of phagocytosis post MERTK inhibition. We identified high localized total compound concentrations in the retinal pigment epithelium (RPE) and retinal lesions following 28 days of treatment with AZ14145845. These lesions were present in 4 of 8 treated animals, and were characterized by a thinning of the outer nuclear layer, loss of photoreceptors (PR) and accumulation of photoreceptor outer segments at the interface of the RPE and PRs. Furthermore, the lesions were very similar to that shown in the RCS rat and MERTK knock-out mouse, suggesting a MERTK-induced mechanism of PR cell death. This was further supported by the observation of reduced phagocytosis in the human retinal cell model following treatment with AZ14145845. Our study provides a viable, translational strategy to investigate the pre-clinical toxicity of MERTK inhibitors but is equally transferrable to novel chemotypes.

AXL/MERTK inhibitor ONO-7475 potently synergizes with venetoclax and overcomes venetoclax resistance to kill FLT3-ITD acute myeloid leukemia.

FMS-like Tyrosine Kinase 3 (FLT3) mutation is associated with poor survival in acute myeloid leukemia (AML). The specific Anexelekto/MER Tyrosine Kinase (AXL) inhibitor, ONO-7475, kills FLT3-mutant AML cells with targets including Extracellular- signal Regulated Kinase (ERK) and Myeloid Cell Leukemia 1 (MCL1). ERK and MCL1 are known resistance factors for Venetoclax (ABT-199), a popular drug for AML therapy, prompting the investigation of the efficacy of ONO-7475 in combination with ABT-199 in vitro and in vivo. ONO-7475 synergizes with ABT-199 to potently kill FLT3-mutant acute myeloid leukemia cell lines and primary cells. ONO-7475 is effective against ABT-199-resistant cells including cells that overexpress MCL1. Proteomic analyses revealed that ABT-199-resistant cells expressed elevated levels of pro-growth and anti-apoptotic proteins compared to parental cells, and that ONO-7475 reduced the expression of these proteins in both the parental and ABT-199-resistant cells. ONO-7475 treatment significantly extended survival as a single in vivo agent using acute myeloid leukemia cell lines and PDX models. Compared to ONO-7474 monotherapy, the combination of ONO-7475/ABT-199 was even more potent in reducing leukemic burden and prolonging the survival of mice in both model systems. These results suggest that the ONO-7475/ABT-199 combination may be effective for AML therapy.

Development of [18F]MIPS15692, a radiotracer with in vitro proof-of-concept for the imaging of MER tyrosine kinase (MERTK) in neuroinflammatory disease.

MER tyrosine kinase (MERTK) upregulation is associated with M2 polarization of microglia, which plays a vital role in neuroregeneration following damage induced by neuroinflammatory diseases such as multiple sclerosis (MS). Therefore, a radiotracer specific for MERTK could be of great utility in the clinical management of MS, for the detection and differentiation of neuroregenerative and neurodegenerative processes. This study aimed to develop an [18F] ligand with high affinity and selectivity for MERTK as a potential positron emission tomography (PET) radiotracer. MIPS15691 and MIPS15692 were synthesized and kinase assays were utilized to determine potency and selectivity for MERTK. Both compounds were shown to be potent against MERTK, with respective IC50 values of 4.6 nM and 4.0 nM, and were also MERTK-selective. Plasma and brain pharmacokinetics were measured in mice and led to selection of MIPS15692 over MIPS15691. X-ray crystallography was used to visualize how MIPS15692 is recognized by the enzyme. [18F]MIPS15692 was synthesized using an automated iPHASE FlexLab module, with a molar activity (Am) of 49 ± 26 GBq/µmol. The radiochemical purity of [18F]MIPS15692 was >99% and the decay-corrected radiochemical yields (RCYs) were determined as 2.45 ± 0.85%. Brain MERTK protein density was measured by a saturation binding assay in the brain slices of a cuprizone mouse model of MS. High levels of specific binding of [18F]MIPS15692 to MERTK were found, especially in the corpus callosum/hippocampus (CC/HC). The in vivo PET imaging study of [18F]MIPS15692 suggested that its neuroPK is sub-optimal for clinical use. Current efforts are underway to optimize the neuroPK of our next generation PET radiotracers for maximal in vivo utility.

Discovery of 5,6,7,8-tetrahydropyrido[3,4-d]pyrimidine derivatives as novel selective Axl inhibitors.

Axl and Mer are members of the TAM (Tyro3-Axl-Mer) family of receptor tyrosine kinases. Previously, we reported that enzyme-mediated inhibition of Mer by an Axl/Mer dual inhibitor led to retinal toxicity in mice, whereas selective Axl inhibition by compound 1 did not. On the other hand, compound 1 showed low membrane permeability. Here, we designed and synthesized a novel series of 5,6,7,8-tetrahydropyrido[3,4-d]pyrimidine derivatives and evaluated their Axl and Mer inhibitory activities, leading to identification of ER-001259851-000 as a potent and selective Axl inhibitor with drug-likeness and a promising pharmacokinetic profile in mice.

UNC5293, a potent, orally available and highly MERTK-selective inhibitor.

Inhibition of MER receptor tyrosine kinase (MERTK) causes direct tumor cell killing and stimulation of the innate immune response. Therefore, MERTK has been identified as a therapeutic target in a wide variety of human tumors. Clinical trials targeting MERTK have recently been initiated, however, none of these drugs are MERTK-specific. Herein, we present the discovery of a highly MERTK-selective inhibitor UNC5293 (24). UNC5293 has subnanomolar activity against MERTK with an excellent Ambit selectivity score (S50 (100 nM) = 0.041). It mediated potent and selective inhibition of MERTK in cell-based assays. Furthermore, it has excellent mouse PK properties (7.8 h half-life and 58% oral bioavailability) and was active in bone marrow leukemia cells in a murine model.

MerTK inhibits the activation of the NLRP3 inflammasome after subarachnoid hemorrhage by inducing autophagy.

The NLR family pyrin domain-containing 3 (NLRP3) multiprotein complex is associated with neuroinflammation and poor prognosis after subarachnoid hemorrhage (SAH). Accumulating evidence shows that Mer tyrosine kinase (MerTK) alleviates inflammatory responses via a negative feedback mechanism. However, the contribution and function of MerTK in SAH remain to be determined. In this study, we explored the role of MerTK during microglial NLRP3 inflammasome activation and evaluated its contribution to the outcome of SAH in mice. Activating MerTK with growth arrest-specific 6 (Gas6) alleviated brain edema, neuronal degeneration and neurological deficits after SAH by regulating neuroinflammation. Gas6 did not change the mRNA levels of Nlrp3 or Casp1 but decreased the protein expression of NLRP3, cleaved caspase1 (p20), interleukin-1ß and interleukin-18. Furthermore, Gas6 increased the expression of Beclin1, the ratio of LC3-II/LC3-I and the level of autophagic flux. Inhibiting autophagy with 3-MA reversed the inhibition of NLRP3 inflammasome activation and diminished the neuroprotective effects of Gas6. Thus, MerTK activation may exert protective effects by limiting neuroinflammation and promoting neurological recovery after SAH via autophagy induction.

Generating Selective Leads for Mer Kinase Inhibitors-Example of a Comprehensive Lead-Generation Strategy.

Mer is a member of the TAM (Tyro3, Axl, Mer) kinase family that has been associated with cancer progression, metastasis, and drug resistance. Their essential function in immune homeostasis has prompted an interest in their role as modulators of antitumor immune response in the tumor microenvironment. Here we illustrate the outcomes of an extensive lead-generation campaign for identification of Mer inhibitors, focusing on the results from concurrent, orthogonal high-throughput screening approaches. Data mining, HT (high-throughput), and DECL (DNA-encoded chemical library) screens offered means to evaluate large numbers of compounds. We discuss campaign strategy and screening outcomes, and exemplify series resulting from prioritization of hits that were identified. Concurrent execution of HT and DECL screening successfully yielded a large number of potent, selective, and novel starting points, covering a range of selectivity profiles across the TAM family members and modes of kinase binding, and offered excellent start points for lead development.

MerTK activity is not necessary for the proliferation of glioblastoma stem cells.

MerTK has been identified as a promising target for therapeutic intervention in glioblastoma. Genetic studies documented a range of oncogenic processes that MerTK targeting could influence, however robust pharmacological validation has been missing. The aim of this study was to assess therapeutic potential of MerTK inhibitors in glioblastoma therapy. Unlike previous studies, our work provides several lines of evidence that MerTK activity is dispensable for glioblastoma growth. We observed heterogeneous responses to MerTK inhibitors that could not be correlated to MerTK inhibition or MerTK expression in cells. The more selective MerTK inhibitors UNC2250 and UNC2580A lack the anti-proliferative potency of less-selective inhibitors exemplified by UNC2025. Functional assays in MerTK-high and MerTK-deficient cells further demonstrate that the anti-cancer efficacy of UNC2025 is MerTK-independent. However, despite its efficacy in vitro, UNC2025 failed to attenuate glioblastoma growth in vivo. Gene expression analysis from cohorts of glioblastoma patients identified that MerTK expression correlates negatively with proliferation and positively with quiescence genes, suggesting that MerTK regulates dormancy rather than proliferation in glioblastoma. In summary, this study demonstrates the importance of orthogonal inhibitors and disease-relevant models in target validation studies and raises a possibility that MerTK inhibitors could be used to target dormant glioblastoma cells.

TAM kinase inhibition and immune checkpoint blockade- a winning combination in cancer treatment?

Introduction: Immune checkpoint inhibitors (ICI) have shown great promise in a wide spectrum of malignancies. However, responses are not always durable, and this mode of treatment is only effective in a subset of patients. As such, there exists an unmet need for novel approaches to bolster ICI efficacy.Areas covered: We review the role of the Tyro3, Axl, and Mer (TAM) receptor tyrosine kinases in promoting tumor-induced immune suppression and discuss the benefits that may be derived from combining ICI with TAM kinase-targeted tyrosine kinase inhibitors. We searched the MEDLINE Public Library of Medicine (PubMed) and EMBASE databases and referred to ClinicalTrials.gov for relevant ongoing studies.Expert opinion: Targeting of TAM kinases may improve the efficacy of immune checkpoint blockade. However, it remains to be determined whether this effect will be better achieved by the selective targeting of each TAM receptor, depending on the context, or by multi-receptor TAM inhibitors. Triple inhibition of all TAM receptors is more likely to be associated with an increased risk for adverse events. Clinical trial designs should use high-resolution clinical endpoints and proper control arms to determine the synergistic effects of combining TAM inhibition with immune checkpoint blockade.

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

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

A-loop interactions in Mer tyrosine kinase give rise to inhibitors with two-step mechanism and long residence time of binding.

The activation loop (A-loop) plays a key role in regulating the catalytic activity of protein kinases. Phosphorylation in this region enhances the phosphoryl transfer rate of the kinase domain and increases its affinity for ATP. Furthermore, the A-loop possesses autoinhibitory functions in some kinases, where it collapses onto the protein surface and blocks substrate binding when unphosphorylated. Due to its flexible nature, the A-loop is usually disordered and untraceable in kinase domain crystal structures. The resulting lack of structural information is regrettable as it impedes the design of drug A-loop contacts, which have proven favourable in multiple cases. Here, we characterize the binding with A-loop engagement between type 1.5 kinase inhibitor 'example 172' (EX172) and Mer tyrosine kinase (MerTK). With the help of crystal structures and binding kinetics, we portray how the recruitment of the A-loop elicits a two-step binding mechanism which results in a drug-target complex characterized by high affinity and long residence time. In addition, the type 1.5 compound possesses excellent kinome selectivity and a remarkable preference for the phosphorylated over the dephosphorylated form of MerTK. We discuss these unique characteristics in the context of known type 1 and type 2 inhibitors and highlight opportunities for future kinase inhibitor design.

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 ligand­receptor 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 PS­TAM interaction was pro­apoptotic, whereas GAS6­mediated 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 long­term and short­term effects of natural PS overexpression were comparable to the treatment of the cells with the drug UNC2025, which inhibits the Mer­receptor. The present study lays the foundation for investigation of PS as a therapeutic agent to control cancer progression and to concurrently arrest thrombotic events.

MERTK in cancer therapy: Targeting the receptor tyrosine kinase in tumor cells and the immune system.

The receptor tyrosine kinase MERTK is aberrantly expressed in numerous human malignancies, and is a novel target in cancer therapeutics. Physiologic roles of MERTK include regulation of tissue homeostasis and repair, innate immune control, and platelet aggregation. However, aberrant expression in a wide range of liquid and solid malignancies promotes neoplasia via growth factor independence, cell cycle progression, proliferation and tumor growth, resistance to apoptosis, and promotion of tumor metastases. Additionally, MERTK signaling contributes to an immunosuppressive tumor microenvironment via induction of an anti-inflammatory cytokine profile and regulation of the PD-1 axis, as well as regulation of macrophage, myeloid-derived suppressor cell, natural killer cell and T cell functions. Various MERTK-directed therapies are in preclinical development, and clinical trials are underway. In this review we discuss MERTK inhibition as an emerging strategy for cancer therapy, focusing on MERTK expression and function in neoplasia and its role in mediating resistance to cytotoxic and targeted therapies as well as in suppressing anti-tumor immunity. Additionally, we review preclinical and clinical pharmacological strategies to target MERTK.

Triple Therapy with MerTK and PD1 Inhibition Plus Radiotherapy Promotes Abscopal Antitumor Immune Responses.

PURPOSE: Radiotherapy (RT) traditionally has been used for local tumor control in the treatment of cancer. The recent discovery that radiotherapy can have anticancer effects on the immune system has led to recognition of its ability to sensitize the tumor microenvironment to immunotherapy. However, radiation can also prompt adverse immunosuppressive effects that block aspects of systemic response at other tumor sites. Our hypothesis was that inhibition of the MER proto-oncogene tyrosine kinase (MerTK) in combination with anti-programmed cell death-1 (α-PD1) checkpoint blockade will enhance immune-mediated responses to radiotherapy. EXPERIMENTAL DESIGN: We tested the efficacy of this triple therapy (Radiation + α-PD1 + α-MerTK mAbs) in 129Sv/Ev mice with bilateral lung adenocarcinoma xenografts. Primary tumors were treated with stereotactic radiotherapy (36 Gy in 3 12-Gy fractions), and tumors were monitored for response. RESULTS: The triple therapy significantly delayed abscopal tumor growth, improved survival rates, and reduced numbers of lung metastases. We further found that the triple therapy increased the activated CD8+ and NK cells populations measured by granzyme B expression with upregulation of CD8+CD103+ tissue-resident memory cells (TRM) within the abscopal tumor microenvironment relative to radiation only. CONCLUSIONS: The addition of α-PD1 + α-MerTK mAbs to radiotherapy could alter the cell death to be more immunogenic and generate adaptive immune response via increasing the retention of TRM cells in the tumor islets of the abscopal tumors which was proven to play a major role in survival of non-small cell lung cancer patients.

A flexible high content imaging assay for profiling macrophage efferocytosis.

Macrophages are a diverse population of cells originating from the myeloid lineage, which form an important component of the innate immune system, helping to regulate immune response through secretion of pro/anti-inflammatory cytokines. However they also have an important homeostatic role - helping to remove cellular debris and apoptotic cells from the body (a phagocytic process known as efferocytosis). Here we describe a robust 384 well microplate based imaging assay, using apoptotic target cells for the specific quantification of efferocytosis in human primary monocyte derived macrophages. The methodology described allows for the assay to run in either fixed end-point or live-cell format (the former offering multiple morphological and intensity-based readouts, whilst the latter opens the possibility for future expansion of the methodology to encompass kinetic profiling). Within the methodology described we couple high content image acquisition (on the Cell Voyager 7000S) with multi-parametric image analysis - using Perkin Elmer Columbus combined with GeneData Screener.

Immuno-oncological Efficacy of RXDX-106, a Novel TAM (TYRO3, AXL, MER) Family Small-Molecule Kinase Inhibitor.

Expression of the TAM (TYRO3, AXL, MER) family of receptor tyrosine kinases (RTK) has been associated with cancer progression, metastasis, and drug resistance. In immune cells, TAM RTKs can dampen inflammation in favor of homeostatic wound-healing responses, thus potentially contributing to the evasion of cancer cells from immune surveillance. Here we characterize the small-molecule RXDX-106 as a selective and potent pan-TAM RTK inhibitor with slow dissociation kinetics and significant antitumor activity in multiple syngeneic tumor models. Expression of AXL and MER on both immune and tumor cells increased during tumor progression. Tumor growth inhibition (TGI) following treatment with RXDX-106 was observed in wild-type mice and was abrogated in immunodeficient mice, suggesting that the antitumor activity of RXDX-106 is, in part, due to the presence of immune cells. RXDX-106-mediated TGI was associated with increased tumor-infiltrating leukocytes, M1-polarized intratumoral macrophages, and activation of natural killer cells. RXDX-106 proportionally increased intratumoral CD8+ T cells and T-cell function as indicated by both IFNγ production and LCK phosphorylation (pY393). RXDX-106 exhibited its effects via direct actions on TAM RTKs expressed on intratumoral macrophages and dendritic cells, leading to indirect activation of other immune cells in the tumor. RXDX-106 also potentiated the effects of an immune checkpoint inhibitor, α-PD-1 Ab, resulting in enhanced antitumor efficacy and survival. Collectively, these results demonstrate the capacity of RXDX-106 to inhibit tumor growth and progression and suggest it may serve as an effective therapy against multiple tumor types. SIGNIFICANCE: The pan-TAM small-molecule kinase inhibitor RXDX-106 activates both innate and adaptive immunity to inhibit tumor growth and progression, indicating its clinical potential to treat a wide variety of cancers.

MERTK inhibition alters the PD-1 axis and promotes anti-leukemia immunity.

MERTK is ectopically expressed and promotes survival in acute lymphoblastic leukemia (ALL) cells and is thus a potential therapeutic target. Here we demonstrate both direct therapeutic effects of MERTK inhibition on leukemia cells and induction of anti-leukemia immunity via suppression of the coinhibitory PD-1 axis. A MERTK-selective tyrosine kinase inhibitor, MRX-2843, mediated therapeutic anti-leukemia effects in immunocompromised mice bearing a MERTK-expressing human leukemia xenograft. In addition, inhibition of host MERTK by genetic deletion (Mertk-/- mice) or treatment with MRX-2843 significantly decreased tumor burden and prolonged survival in immune-competent mice inoculated with a MERTK-negative ALL, suggesting immune-mediated therapeutic activity. In this context, MERTK inhibition led to significant decreases in expression of the coinhibitory ligands PD-L1 and PD-L2 on CD11b+ monocytes/macrophages in the leukemia microenvironment. Furthermore, although T cells do not express MERTK, inhibition of MERTK indirectly decreased PD-1 expression on CD4+ and CD8+ T cells and decreased the incidence of splenic FOXP3+ Tregs at sites of leukemic infiltration, leading to increased T cell activation. These data demonstrate direct and immune-mediated therapeutic activities in response to MERTK inhibition in ALL models and provide validation of a translational agent targeting MERTK for modulation of tumor immunity.

Design, synthesis, biological evaluation and cellular imaging of imidazo[4,5-b]pyridine derivatives as potent and selective TAM inhibitors.

The TAM kinase family arises as a new effective and attractive therapeutic target for cancer therapy, autoimmune and viral diseases. A series of 2,6-disubstituted imidazo[4,5-b]pyridines were designed, synthesized and identified as highly potent TAM inhibitors. Despite remarkable structural similarities within the TAM family, compounds 28 and 25 demonstrated high activity and selectivity in vitro against AXL and MER, with IC50 value of 0.77 nM and 9 nM respectively and a 120- to 900-fold selectivity. We also observed an unexpected nuclear localization for compound 10Bb, thanks to nanoSIMS technology, which could be correlated to the absence of cytotoxicity on three different cancer cell lines being sensitive to TAM inhibition.