Tumour DDR1 promotes collagen fibre alignment to instigate immune exclusion.

Immune exclusion predicts poor patient outcomes in multiple malignancies, including triple-negative breast cancer (TNBC)1. The extracellular matrix (ECM) contributes to immune exclusion2. However, strategies to reduce ECM abundance are largely ineffective or generate undesired outcomes3,4. Here we show that discoidin domain receptor 1 (DDR1), a collagen receptor with tyrosine kinase activity5, instigates immune exclusion by promoting collagen fibre alignment. Ablation of Ddr1 in tumours promotes the intratumoral penetration of T cells and obliterates tumour growth in mouse models of TNBC. Supporting this finding, in human TNBC the expression of DDR1 negatively correlates with the intratumoral abundance of anti-tumour T cells. The DDR1 extracellular domain (DDR1-ECD), but not its intracellular kinase domain, is required for immune exclusion. Membrane-untethered DDR1-ECD is sufficient to rescue the growth of Ddr1-knockout tumours in immunocompetent hosts. Mechanistically, the binding of DDR1-ECD to collagen enforces aligned collagen fibres and obstructs immune infiltration. ECD-neutralizing antibodies disrupt collagen fibre alignment, mitigate immune exclusion and inhibit tumour growth in immunocompetent hosts. Together, our findings identify a mechanism for immune exclusion and suggest an immunotherapeutic target for increasing immune accessibility through reconfiguration of the tumour ECM.

DDR1 (Discoidin Domain Receptor-1)-RhoA (Ras Homolog Family Member A) Axis Senses Matrix Stiffness to Promote Vascular Calcification.

OBJECTIVE: Vascular calcification is a pathology characterized by arterial mineralization, which is a common late-term complication of atherosclerosis that independently increases the risk of adverse cardiovascular events by fourfold. A major source of calcifying cells is transdifferentiating vascular smooth muscle cells (VSMCs). Previous studies showed that deletion of the collagen-binding receptor, DDR1 (discoidin domain receptor-1), attenuated VSMC calcification. Increased matrix stiffness drives osteogenesis, and DDR1 has been implicated in stiffness sensing in other cell types; however, the role of DDR1 as a mechanosensor in VSMCs has not been investigated. Here, we test the hypothesis that DDR1 senses increased matrix stiffness and promotes VSMC transdifferentiation and calcification. Approach and Results: Primary VSMCs isolated from Ddr1+/+ (wild-type) and Ddr1-/- (knockout) mice were studied on collagen-I-coated silicon substrates of varying stiffness, culturing in normal or calcifying medium. DDR1 expression and phosphorylation increased with increasing stiffness, as did in vitro calcification, nuclear localization of Runx2 (Runt-related transcription factor 2), and expression of other osteochondrocytic markers. By contrast, DDR1 deficient VSMCs were not responsive to stiffness and did not undergo transdifferentiation. DDR1 regulated stress fiber formation and RhoA (ras homolog family member A) activation through the RhoGEF (rho guanine nucleotide exchange factor), Vav2. Inhibition of actomyosin contractility reduced Runx2 activation and attenuated in vitro calcification in wild-type VSMCs. Finally, a novel positive feedforward loop was uncovered between DDR1 and actomyosin contractility, important in regulating DDR1 expression, clustering, and activation. CONCLUSIONS: This study provides mechanistic insights into DDR1 mechanosignaling and shows that DDR1 activity and actomyosin contractility are interdependent in mediating stiffness-dependent increases in VSMC calcification.

Diabetic Vascular Calcification Mediated by the Collagen Receptor Discoidin Domain Receptor 1 via the Phosphoinositide 3-Kinase/Akt/Runt-Related Transcription Factor 2 Signaling Axis.

Objective- Vascular calcification is a common and severe complication in patients with atherosclerosis which is exacerbated by type 2 diabetes mellitus. Our laboratory recently reported that the collagen receptor discoidin domain receptor 1 (DDR1) mediates vascular calcification in atherosclerosis; however, the underlying mechanisms are unknown. During calcification, vascular smooth muscle cells transdifferentiate into osteoblast-like cells, in a process driven by the transcription factor RUNX2 (runt-related transcription factor 2). DDR1 signals via the phosphoinositide 3-kinase/Akt pathway, which is also central to insulin signaling, and upstream of RUNX2, and this led us to investigate whether DDR1 promotes vascular calcification in diabetes mellitus via this pathway. Approach and Results- Ddr1+/+ ; Ldlr-/- (single knock-out) and Ddr1-/- ; Ldlr-/- (double knock-out) mice were placed on high-fat diet for 12 weeks to induce atherosclerosis and type 2 diabetes mellitus. Von Kossa staining revealed reduced vascular calcification in the aortic arch of double knock-out compared with single knock-out mice. Immunofluorescent staining for RUNX2 was present in calcified plaques of single knock-out but not double knock-out mice. Primary vascular smooth muscle cells obtained from Ddr1+/+ and Ddr1-/- mice were cultured in calcifying media. DDR1 deletion resulted in reduced calcification, a 74% reduction in p-Akt levels, and an 88% reduction in RUNX2 activity. Subcellular fractionation revealed a 77% reduction in nuclear RUNX2 levels in Ddr1-/- vascular smooth muscle cells. DDR1 associated with phosphoinositide 3-kinase, and treatment with the inhibitor wortmannin attenuated calcification. Finally, we show that DDR1 is important to maintain the microtubule cytoskeleton which is required for the nuclear localization of RUNX2. Conclusions- These novel findings demonstrate that DDR1 promotes RUNX2 activity and atherosclerotic vascular calcification in diabetes mellitus via phosphoinositide 3-kinase/Akt signaling.

Discoidin domain receptor 1 kinase activity is required for regulating collagen IV synthesis.

Discoidin domain receptor 1 (DDR1) is a receptor tyrosine kinase that binds to and is activated by collagens. DDR1 expression increases following kidney injury and accumulating evidence suggests that it contributes to the progression of injury. To this end, deletion of DDR1 is beneficial in ameliorating kidney injury induced by angiotensin infusion, unilateral ureteral obstruction, or nephrotoxic nephritis. Most of the beneficial effects observed in the DDR1-null mice are attributed to reduced inflammatory cell infiltration to the site of injury, suggesting that DDR1 plays a pro-inflammatory effect. The goal of this study was to determine whether, in addition to its pro-inflammatory effect, DDR1 plays a deleterious effect in kidney injury by directly regulating extracellular matrix production. We show that DDR1-null mice have reduced deposition of glomerular collagens I and IV as well as decreased proteinuria following the partial renal ablation model of kidney injury. Using mesangial cells isolated from DDR1-null mice, we show that these cells produce significantly less collagen compared to DDR1-null cells reconstituted with wild type DDR1. Moreover, mutagenesis analysis revealed that mutations in the collagen binding site or in the kinase domain significantly reduce DDR1-mediated collagen production. Finally, we provide evidence that blocking DDR1 kinase activity with an ATP-competitive small molecule inhibitor reduces collagen production. In conclusion, our studies indicate that the kinase activity of DDR1 plays a key role in DDR1-induced collagen synthesis and suggest that blocking collagen-mediated DDR1 activation may be beneficial in fibrotic diseases.