Preclinical characterization of pirtobrutinib, a highly selective, noncovalent (reversible) BTK inhibitor.

Bruton tyrosine kinase (BTK), a nonreceptor tyrosine kinase, is a major therapeutic target for B-cell-driven malignancies. However, approved covalent BTK inhibitors (cBTKis) are associated with treatment limitations because of off-target side effects, suboptimal oral pharmacology, and development of resistance mutations (eg, C481) that prevent inhibitor binding. Here, we describe the preclinical profile of pirtobrutinib, a potent, highly selective, noncovalent (reversible) BTK inhibitor. Pirtobrutinib binds BTK with an extensive network of interactions to BTK and water molecules in the adenosine triphosphate binding region and shows no direct interaction with C481. Consequently, pirtobrutinib inhibits both BTK and BTK C481 substitution mutants in enzymatic and cell-based assays with similar potencies. In differential scanning fluorimetry studies, BTK bound to pirtobrutinib exhibited a higher melting temperature than cBTKi-bound BTK. Pirtobrutinib, but not cBTKis, prevented Y551 phosphorylation in the activation loop. These data suggest that pirtobrutinib uniquely stabilizes BTK in a closed, inactive conformation. Pirtobrutinib inhibits BTK signaling and cell proliferation in multiple B-cell lymphoma cell lines, and significantly inhibits tumor growth in human lymphoma xenografts in vivo. Enzymatic profiling showed that pirtobrutinib was highly selective for BTK in >98% of the human kinome, and in follow-up cellular studies pirtobrutinib retained >100-fold selectivity over other tested kinases. Collectively, these findings suggest that pirtobrutinib represents a novel BTK inhibitor with improved selectivity and unique pharmacologic, biophysical, and structural attributes with the potential to treat B-cell-driven cancers with improved precision and tolerability. Pirtobrutinib is being tested in phase 3 clinical studies for a variety of B-cell malignancies.

Prostate tumor RON receptor signaling mediates macrophage recruitment to drive androgen deprivation therapy resistance through Gas6-mediated Axl and RON signaling.

BACKGROUND: Androgen deprivation therapy (ADT), or chemical castration, is the first-line therapy for prostate cancer; however, resistance leaves few treatment options. Prostatic tumor-associated macrophages (TAMs) have been shown to promote prostate cancer growth and are abundant in castration-resistant prostate cancer (CRPC), suggesting a role in promoting CRPC. We recently showed a tumor cell-intrinsic mechanism by which RON promotes CRPC. Given previous reports that RON alters prostate cancer cell chemokine production and RON-overexpressing tumors alter macrophage function, we hypothesized that a macrophage-dependent mechanism regulated by tumor cell intrinsic RON also promotes CRPC. METHODS: Using RON-modulated genetically engineered mouse models (GEMMs) and GEMM-derived cell lines and co-cultures with bone marrow-derived macrophages, we show functional and molecular characteristics of signaling pathways in supporting CRPC. Further, we used an unbiased phosphokinase array to identify pathway interactions regulated by RON. Finally, using human prostate cancer cell lines and prostate cancer patient data sets, we show the relevance of our findings to human prostate cancer. RESULTS: Studies herein show that macrophages recruited into the prostate tumor microenvironment (TME) serve as a source for Gas6 secretion which serves to further enhance RON and Axl receptor activation in prostate tumor cells thereby driving CRPC. Further, we show targeting RON and macrophages in a murine model promotes CRPC sensitization to ADT. CONCLUSIONS: We discovered a novel role for the RON receptor in prostate cancer cells in promoting CRPC through the recruitment of macrophages into the prostate TME. Macrophage-targeting agents in combination with RON/Axl inhibition are likely to provide clinical benefits for patients with CRPC.

Development of an Energy Efficient and Cost Effective Autonomous Vehicle Research Platform.

Commercialization of autonomous vehicle technology is a major goal of the automotive industry, thus research in this space is rapidly expanding across the world. However, despite this high level of research activity, literature detailing a straightforward and cost-effective approach to the development of an AV research platform is sparse. To address this need, we present the methodology and results regarding the AV instrumentation and controls of a 2019 Kia Niro which was developed for a local AV pilot program. This platform includes a drive-by-wire actuation kit, Aptiv electronically scanning radar, stereo camera, MobilEye computer vision system, LiDAR, inertial measurement unit, two global positioning system receivers to provide heading information, and an in-vehicle computer for driving environment perception and path planning. Robotic Operating System software is used as the system middleware between the instruments and the autonomous application algorithms. After selection, installation, and integration of these components, our results show successful utilization of all sensors, drive-by-wire functionality, a total additional power* consumption of 242.8 Watts (*Typical), and an overall cost of $118,189 USD, which is a significant saving compared to other commercially available systems with similar functionality. This vehicle continues to serve as our primary AV research and development platform.

Loss of Ron receptor signaling leads to reduced obesity, diabetic phenotypes and hepatic steatosis in response to high-fat diet in mice.

The Ron receptor tyrosine kinase is a heterodimeric, membrane-spanning glycoprotein that participates in divergent processes, including proliferation, motility, and modulation of inflammatory responses. We observed male C57BL/6 mice with a global deletion of the Ron tyrosine kinase signaling domain (TK(-/-)) to be leaner compared with control (TK(+/+)) mice under a standard diet. When fed a high-fat diet (HFD), TK(-/-) mice gained 50% less weight and were more insulin sensitive and glucose tolerant than controls. Livers from HFD TK(-/-) mice were considerably less steatotic and weighed significantly less than TK(+/+) livers. Serum cytokine levels of HFD TK(-/-) mice were also significantly altered compared with TK(+/+) mice. Fewer and smaller adipocytes were present in the TK(-/-) mice on both control and HFD and were accompanied by diminished adiponectin and peroxisome proliferator-activated receptor-γ expression. In vitro adipogenesis experiments suggested reduced differentiation in TK(-/-) embryonic fibroblasts (MEFs) that was rescued by Ron reconstitution. Likewise, signal transducer and activator of transcription (STAT)-3 phosphorylation was diminished in TK(-/-) MEFs but was increased after Ron reconstitution. The adipogenic inhibitors, preadipocyte factor 1 and Sox9, were elevated in TK(-/-) MEFs and increased in both groups after STAT3 silencing. In total, these studies document a previously unknown function for the Ron receptor in mediating HFD-induced obesity and metabolic dysregulation.

Prostate Epithelial RON Signaling Promotes M2 Macrophage Activation to Drive Prostate Tumor Growth and Progression.

Effective treatment of advanced prostate cancer persists as a significant clinical need as only 30% of patients with distant disease survive to 5 years after diagnosis. Targeting signaling and tumor cell-immune cell interactions in the tumor microenvironment has led to the development of powerful immunotherapeutic agents, however, the prostate tumor milieu remains impermeable to these strategies highlighting the need for novel therapeutic targets. In this study, we provide compelling evidence to support the role of the RON receptor tyrosine kinase as a major regulator of macrophages in the prostate tumor microenvironment. We show that loss of RON selectively in prostate epithelial cells leads to significantly reduced prostate tumor growth and metastasis and is associated with increased intratumor infiltration of macrophages. We further demonstrate that prostate epithelial RON loss induces transcriptional reprogramming of macrophages to support expression of classical M1 markers and suppress expression of alternative M2 markers. Interestingly, our results show epithelial RON activation drives upregulation of RON expression in macrophages as a positive feed-forward mechanism to support prostate tumor growth. Using 3D coculture assays, we provide additional evidence that epithelial RON expression coordinates interactions between prostate tumor cells and macrophages to promote macrophage-mediated tumor cell growth. Taken together, our results suggest that RON receptor signaling in prostate tumor cells directs the functions of macrophages in the prostate tumor microenvironment to promote prostate cancer. IMPLICATIONS: Epithelial RON is a novel immunotherapeutic target that is responsible for directing the macrophage antitumor immune response to support prostate tumor growth and progression.

Therapeutic Considerations for Ron Receptor Expression in Prostate Cancer.

INTRODUCTION: The Ron receptor tyrosine kinase was initially discovered as a protein which played a critical role in regulating inflammatory responses. This effect was primarily determined through studies in various macrophage populations. Since its initial discovery, a role has emerged for Ron as a driver of cancer within epithelial cells. After numerous publications have detailed a role for Ron in promoting tumor initiation, growth, and metastasis, Ron has been designated as an emerging therapeutic option in a variety of cancers. AREAS COVERED: This review discusses the current literature regarding the role of Ron in prostate cancer and places special emphasis on the role of Ron in both epithelial cells and macrophages. Whole body loss of Ron signaling initially exposed a variety of prostate cancer growth mechanisms regulated by Ron. With the knowledge that Ron plays an integral part in regulating the function of epithelial cells and macrophages, studies commenced to discern the cell type specific functions for Ron in prostate cancer. A novel role for Ron in promoting Castration Resistant Prostate Cancer has recently been uncovered, and the results of these studies are summarized herein. Furthermore, this review gives a summary of several currently available compounds which show promise at targeting Ron in both epithelial and macrophage populations. OUTLOOK: Sufficient evidence has been provided for the initiation of clinical trials focused on targeting Ron in both macrophage and epithelial compartments for the treatment of prostate cancer. A number of therapeutic avenues for targeting Ron in prostate cancer are currently available; however, special consideration will need to take place knowing that Ron signaling impacts multiple cell types. Further understanding of the cell type specific functions of Ron in prostate cancer will help inform and shape future clinical research and therapeutic strategies.

Tumor Cell Autonomous RON Receptor Expression Promotes Prostate Cancer Growth Under Conditions of Androgen Deprivation.

Current treatment strategies provide minimal results for patients with castration-resistant prostate cancer (CRPC). Attempts to target the androgen receptor have shown promise, but resistance ultimately develops, often due to androgen receptor reactivation. Understanding mechanisms of resistance, including androgen receptor reactivation, is crucial for development of more efficacious CRPC therapies. Here, we report that the RON receptor tyrosine kinase is highly expressed in the majority of human hormone-refractory prostate cancers. Further, we show that exogenous expression of RON in human and murine prostate cancer cells circumvents sensitivity to androgen deprivation and promotes prostate cancer cell growth in both in vivo and in vitro settings. Conversely, RON loss induces sensitivity of CRPC cells to androgen deprivation. Mechanistically, we demonstrate that RON overexpression leads to activation of multiple oncogenic transcription factors (namely, ß-catenin and NF-κB), which are sufficient to drive androgen receptor nuclear localization and activation of AR responsive genes under conditions of androgen deprivation and support castration-resistant growth. In total, this study demonstrates the functional significance of RON during prostate cancer progression and provides a strong rationale for targeting RON signaling in prostate cancer as a means to limit resistance to androgen deprivation therapy.