AIM:The purpose of this study is to evaluate safety, tolerability, pharmacokinetics (PK), food-effect (FE) and pharmacodynamics (PD) of an oral tyrosine kinase-2 (TYK2)/Janus kinase-1 (JAK1) inhibitor, SDC-1801, in healthy adult participants. METHODS:This first-in-human study randomized 95 male and female participants. Parts one and two were double-blind, placebo-controlled escalation designs, investigating single (5, 10, 30, 75, 150 mg) and multiple (30, 100, 75, 140 mg/day) doses of SDC-1801 or matching placebo. Part three evaluated the FE of 75 mg SDC-1801 in an open-label, crossover design. RESULTS:There were no deaths or treatment-related serious adverse events (AEs) following SDC-1801, up to 150 mg; no treatment-emergent AEs (TEAEs) were severe with non-related headache most frequently reported. PK showed low to moderate variability and no consistent dose-exposure relationship; Tmax occurred 3-5 h post-dose with a geometric mean (GM) t1/2 of 15.2-27.0 h. Bioavailability appeared solubility limited, with lower exposure from the higher strength capsule and modestly higher exposure in the fed state. No safety stopping criteria were met. PD assessments demonstrated anti-inflammatory activity, with IFN-gamma induced protein-10 (IP-10), high sensitivity C reactive protein (hsCRP), and signal transducer and activator of transcription (STAT3/5) phosphorylation reductions with increasing exposure giving greater inhibition. At the highest exposure, there was a clear reduction of IP-10 levels at pre-dose on day 9, which rebounded following drug withdrawal. CONCLUSIONS:SDC-1801 was well tolerated to 150 mg; all TEAEs were mild or moderate, with unrelated headache most frequently reported. The PK profile supports the potential for a once- or twice-daily regimen, with evidence of target engagement for both TYK2 and JAK1.

PDF file, 63KB, Kinase inhibitory profile of SAR-020106 at 10 μMa.

PDF file, 79K, Supplementary Table 3 - Mouse body weights for data in Figure 4A Body Weights expressed as a percentage of the weight on day 0 for mice bearing HT29 xenografts and treated with gemcitabine (100mg/kg iv), CCT244747 (75mg/kg po) or the combination. Values are meanSE, n= 3 to 6. Supplementary Table 4 - Mouse body weights for data in Figure 4C Body Weights expressed as a percentage of the weight on day 0 for mice bearing HT29 xenografts and treated with irinotecan (25mg/kg ip), CCT244747 (150mg/kg po) or the combination. Values are meanSE, n= 3 to 6. Supplementary Table 5 - Mouse body weights for data in Figure 4D Body Weights expressed as a percentage of the weight on day 0 for mice bearing Calu6 xenografts and treated with gemcitabine (100 mg/kg iv), CCT244747 (75mg/kg po) or the combination. Values are meanSE, n= 3 to 6. Supplementary Table 6 - Mouse body weights for data in Supplementary Table 2 Body Weights expressed as a percentage of the weight on day 0 for mice bearing SW620 xenografts and treated with gemcitabine (100 mg/kg iv), CCT244747 (75mg/kg po) or the combination. Values are meanSE, n= 3 to 6.

PDF file, 118K, Supplementary Table 2 Summary of Effects of Irinotecan, Gemcitabine and CCT244747 alone or in combination on human tumor xenografts growth delay.

PDF file, 76K, Supplementary Table 1 Kinome selectivity profile of CCT244747 CCT244747 was tested at 1 μM (140 enzymes) and 10 μM (121 enzymes) for inhibition of human kinases in a radiometric protein phosphorylation assay (MRC Protein Phosphorylation Unit, Dundee University, UK). Assays were conducted in the presence of ATP at the approximate Km,ATP for each kinase. Results are quoted as % control kinase activity remaining (100% = no inhibition, 0% = complete inhibition). Kinases inhibited by >50% at each concentration are highlighted. n.d. = not determined.

PDF file, 810K, Supplementary Figure 3 Characterisation of the effects of minimally toxic concentrations of CCT244747 alone or in combination with genotoxic agents (+) in HT29 and SW620 colon cancer cell lines. A, HT29 cells were treated with SN38 (100nM) or CCT244747 alone or in combination for 24h. B, SW620 cells were treated with gemcitabine (200nM) or CCT244747 alone or in combination for 24h. Cells were pre-treated with CCT244747 alone 1h prior to cytotoxic exposure. Protein expression was assessed by western blotting (40μg per lane) as described in Figure 2 and Materials and Methods.

Endothelial homeostasis is a central feature of vascular health. The vascular endothelium is under constant mechanical stress resulting from blood flow and, therefore, requires a high degree of resilience to adapt to stresses and resist development of disease. In this review, we discuss the molecular mechanisms by which the endothelium maintains proteostasis in response to haemodynamic forces by regulating three key areas: protein synthesis, recycling and degradation.

The repertoire of extratranslational functions of components of the protein synthesis apparatus is expanding to include control of key cell signaling networks. However, very little is known about noncanonical functions of members of the protein synthesis machinery in regulating cellular mechanics. We demonstrate that the eukaryotic initiation factor 6 (eIF6) modulates cellular mechanobiology. eIF6-depleted endothelial cells, under basal conditions, exhibit unchanged nascent protein synthesis, polysome profiles, and cytoskeleton protein expression, with minimal effects on ribosomal biogenesis. In contrast, using traction force and atomic force microscopy, we show that loss of eIF6 leads to reduced stiffness and force generation accompanied by cytoskeletal and focal adhesion defects. Mechanistically, we show that eIF6 is required for the correct spatial mechanoactivation of ERK1/2 via stabilization of an eIF6-RACK1-ERK1/2-FAK mechanocomplex, which is necessary for force-induced remodeling. These results reveal an extratranslational function for eIF6 and a novel paradigm for how mechanotransduction, the cellular cytoskeleton, and protein translation constituents are linked.

Shear stress on arteries produced by blood flow is important for vascular development and homeostasis but can also initiate atherosclerosis1. Endothelial cells that line the vasculature use molecular mechanosensors to directly detect shear stress profiles that will ultimately lead to atheroprotective or atherogenic responses2. Plexins are key cell-surface receptors of the semaphorin family of cell-guidance signalling proteins and can regulate cellular patterning by modulating the cytoskeleton and focal adhesion structures3–5. However, a role for plexin proteins in mechanotransduction has not been examined. Here we show that plexin D1 (PLXND1) has a role in mechanosensation and mechanically induced disease pathogenesis. PLXND1 is required for the response of endothelial cells to shear stress in vitro and in vivo and regulates the site-specific distribution of atherosclerotic lesions. In endothelial cells, PLXND1 is a direct force sensor and forms a mechanocomplex with neuropilin-1 and VEGFR2 that is necessary and sufficient for conferring mechanosensitivity upstream of the junctional complex and integrins. PLXND1 achieves its binary functions as either a ligand or a force receptor by adopting two distinct molecular conformations. Our results establish a previously undescribed mechanosensor in endothelial cells that regulates cardiovascular pathophysiology, and provide a mechanism by which a single receptor can exhibit a binary biochemical nature. PLXND1 is a mechanosensor that is required for endothelial cells to respond to shear stress both in vitro and in vivo by regulating the site-specific distribution of atherosclerotic lesions.

Background Haemodynamic forces play a critical role in proper development of the heart, however much less is known about the mechanisms that regulate cardiac remodelling and function in response to haemodynamic stress in the adult. Platelet endothelial cell adhesion molecule-1 (PECAM-1) is a cell adhesion and signalling molecule that has important roles in regulation of junctional integrity, transendothelial migration and mechanotransduction in response to fluid shear stress. Our previous work identified a role for PECAM-1 in regulating baseline cardiac function via regulation of endothelial-cardiomyocyte communication. Methods This study investigates the role of PECAM-1 in cardiac remodelling in response to biomechanical stress due to pressure overload induced by transaortic constriction (TAC). Results Our data reveal that loss of PECAM-1 is associated with systolic dysfunction that is further accentuated following TAC. Adaptive increases in cardiomyocyte cross-sectional area, capillary density and hypertrophic gene expression were all affected with loss of PECAM-1. In control mice, maintained cardiac function was associated with activation of the c-Jun NH([2][1])-terminal kinase (JNK) pathway, whereas PECAM-1 deletion significantly decreased JNK activation after pressure overload. Our data suggest that in the absence of PECAM-1 signalling, inadequate remodelling of the heart under increased mechanical strain leads to further deterioration of cardiac function, characterized by reduced cardiomyocyte hypertrophy, capillary density and defects in the JNK signalling pathway. Conclusions Our study reveals a role for PECAM-1 in preservation of cardiac function in response to biomechanical stress induced by pressure overload. ### Competing Interest Statement The authors have declared no competing interest. [1]: #ref-2

The response of endothelial cells to mechanical forces is a critical determinant of vascular health. Vascular pathologies, such as atherosclerosis, characterized by abnormal mechanical forces are frequently accompanied by endothelial-to-mesenchymal transition (EndMT). However, how forces affect the mechanotransduction pathways controlling cellular plasticity, inflammation, and, ultimately, vessel pathology is poorly understood. Here, we identify a mechanoreceptor that is sui generis for EndMT and unveil a molecular Alk5-Shc pathway that leads to EndMT and atherosclerosis. Depletion of Alk5 abrogates shear stress-induced EndMT responses, and genetic targeting of endothelial Shc reduces EndMT and atherosclerosis in areas of disturbed flow. Tensional force and reconstitution experiments reveal a mechanosensory function for Alk5 in EndMT signaling that is unique and independent of other mechanosensors. Our findings are of fundamental importance for understanding how mechanical forces regulate biochemical signaling, cell plasticity, and vascular disease.

The cardiovascular system can sense and adapt to changes in mechanical stimuli by remodeling the physical properties of the heart and blood vessels in order to maintain homeostasis. Imbalances in mechanical forces and/or impaired sensing are now not only implicated but are, in some cases, considered to be drivers for the development and progression of cardiovascular disease. There is now growing evidence to highlight the role of mechanical forces in the regulation of protein translation pathways. The canonical mechanism of protein synthesis typically involves transcription and translation. Protein translation occurs globally throughout the cell to maintain general function but localized protein synthesis allows for precise spatiotemporal control of protein translation. This Review will cover studies on the role of biomechanical stress -induced translational control in the heart (often in the context of physiological and pathological hypertrophy). We will also discuss the much less studied effects of mechanical forces in regulating protein translation in the vasculature. Understanding how the mechanical environment influences protein translational mechanisms in the cardiovascular system, will help to inform disease pathogenesis and potential areas of therapeutic intervention.

The authors have requested that this preprint be removed from Research Square.

Mechanical forces acting on biological systems, at both the macroscopic and microscopic levels, play an important part in shaping cellular phenotypes. There is a growing realization that biomolecules that respond to force directly applied to them, or via mechano-sensitive signalling pathways, can produce profound changes to not only transcriptional pathways, but also in protein translation. Forces naturally occurring at the molecular level can impact the rate at which the bacterial ribosome translates messenger RNA (mRNA) transcripts and influence processes such as co-translational folding of a nascent protein as it exits the ribosome. In eukaryotes, force can also be transduced at the cellular level by the cytoskeleton, the cell's internal filamentous network. The cytoskeleton closely associates with components of the translational machinery such as ribosomes and elongation factors and, as such, is a crucial determinant of localized protein translation. In this review we will give (1) a brief overview of protein translation in bacteria and eukaryotes and then discuss (2) how mechanical forces are directly involved with ribosomes during active protein synthesis and (3) how eukaryotic ribosomes and other protein translation machinery intimately associates with the mechanosensitive cytoskeleton network.

Abstract Background: TYK2 (tyrosine kinase 2) is a member of the Janus family of non-receptor tyrosine kinases and has been shown to play an important role in the signalling of type I interferons, as well as IL-12 and IL-23, via phosphorylation of downstream STATs. The TYK2/STAT1/BCL-2 pathway is implicated in the survival of leukemic cells in a proportion of T-ALL cases. It has been reported that STAT3 signalling in both the tumor, and microenvironment, is critical in shifting the balance from IL-12, a central cytokine in antitumor and antiviral immunity, to potentially pro-carcinogenic IL-23 production. Furthermore there is increasing evidence that chronic tumor interferon signalling leads to multigenic T cell exhaustion and resistance to immune checkpoint blockade. Additionally, TYK2 has been suggested to play a key role in CTLA-4 STAT3 signal transduction in B cell lymphomas and in melanoma associated B cells. We have previously reported on SAR-20347, a 1,3-oxazole-4 carboxamide, which is an orally bioavailable potent and selective inhibitor of TYK2, which causes tumor regression in in vivo models of T-ALL, and which has shown striking reductions in STAT phosphorylation downstream of IFNα signalling, and IFNγ production in response to IL-12 both in vitro and in vivo. Here we report the effects of SAR-20351, an orally bioavailable optimised analog of SAR-20347, on tumor cell viability and components of the tumor microenvironment in immunocompetent mouse models. Methods: A range of syngeneic tumor models were used to establish SAR-20351 efficacy as both a monotherapy and in combination with standards of care. FACS analysis was used to identify immune cell sub-populations within tumor tissue and measure PD-1 and PD-L1 expression levels on appropriate cell types. Results: Reduced tumor growth was observed following SAR-20351 treatment as a monotherapy in the Panc02, CT26, MC38, B16F10, Renca and A20 models, and in combination with 5-FU or anti-CTLA-4 in the colon CT26 model. Similar effects were seen in the MC38 model when SAR-20351 was combined with 5-FU and in the Renca model when SAR-20351 was combined with everolimus. To elucidate mechanism of action in these models, tumors were grown in immunocompetent and immunodeficient animals and efficacy compared. Increased efficacy of SAR-20351 in an immunocompetent background compared to immunodeficient animals indicated immunotherapy as a mechanism of action. FACS analysis identified reduced myeloid and Treg cell infiltration in tumor tissue and reduced PD-1 expression was observed on TIL and TAMs, indicative of a less-exhausted phenotype. SAR-20351 was seen to reduce levels of PD-L1 expression by tumor cells, and reversed the increase in PD-L1 levels induced by certain chemotherapy or targeted agents. Reduction of pTYK2, pSTAT3 and cMYC was observed in B cells derived from the A20 model. No changes in animal bodyweight or behavior, and no significant differences in complete blood counts and blood chemistry parameters following treatment with SAR-20351 demonstrated that treatment was well-tolerated during these studies. Conclusions: The TYK2 inhibitor, SAR-20351 results in significant control of solid tumor growth and the mechanism of action involves immunotherapy, with reductions in myeloid and Treg cell infiltration in a range of tumor models. Citation Format: John Reader, Nicole Williams, James Bojdo, Jenny Worthington, Tim Mitchell. Immunotherapeutic effects of the TYK2 inhibitor SAR-20351 in syngeneic tumor models [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C086. doi:10.1158/1535-7163.TARG-19-C086

Fibronectin (FN) assembly and fibrillogenesis are critically important in both development and the adult organism, but their importance in vascular functions is not fully understood. Here we identify a novel pathway by which haemodynamic forces regulate FN assembly and fibrillogenesis during vascular remodelling. Induction of disturbed shear stress in vivo and in vitro resulted in complex FN fibril assembly that was dependent on the mechanosensor PECAM. Loss of PECAM also inhibited the cell-intrinsic ability to remodel FN. Gain- and loss-of-function experiments revealed that PECAM-dependent RhoA activation is required for FN assembly. Furthermore, PECAM−/− mice exhibited reduced levels of active β1 integrin that were responsible for reduced RhoA activation and downstream FN assembly. These data identify a new pathway by which endothelial mechanotransduction regulates FN assembly and flow-mediated vascular remodelling.