Discoidin Domain Receptor 1 Inhibitor Research Articles

AI identifies drug candidate in weeks

The artificial intelligence start-up Insilico Medicine has used machine learning to find credible drug candidates in a matter of weeks (Nat. Biotechnol. 2019, DOI: 10.1038/s41587-019-0224-x). Experts say it’s an important demonstration of what machine learning can do in drug discovery, but it isn’t a revolution. Insilico Medicine has been showing off its progress in teaching computers to find new drugs since its founding in 2014. The company’s latest effort involves generative reinforcement learning, a technique that uses rewards to guide an algorithm as it searches for molecules that satisfy its goals. In this case, the algorithm was hunting for small molecules that are inhibitors of discoidin domain receptor 1 (DDR1), a kinase linked to fibrosis. The researchers trained their algorithm using databases of known DDR1 inhibitors, kinase inhibitors, nonkinase inhibitors, and patent-protected molecules. Using measures of novelty and DDR1 inhibition, the algorithm proposed 30,000 potential drugs. This group was filtered

Discoidin domain receptors: A promising target in melanoma.

The discoidin domain receptor 1 (DDR1) is a member of the receptor tyrosine kinase family that signals in response to collagen and that has been implicated in cancer progression. In the present study, we investigated the expression and role of DDR1 in human melanoma progression. Immunohistochemical staining of human melanoma specimens (n=52) shows high DDR1 expression in melanoma lesions that correlates with poor prognosis. DDR1 expression was associated with the clinical characteristics of Clark level and ulceration and with BRAF mutations. Downregulation of DDR1 by small interfering RNA (siRNA) in vitro inhibited melanoma cells malignant properties, migration, invasion, and survival in several human melanoma cell lines. A DDR tyrosine kinase inhibitor (DDR1-IN-1) significantly inhibited melanoma cell proliferation in vitro, and ex vivo and in tumor xenografts, underlining the promising potential of DDR1 inhibition in melanoma.

Interaction of Discoidin Domain Receptor 1 with a 14-3-3-Beclin-1-Akt1 Complex Modulates Glioblastoma Therapy Sensitivity

Glioblastoma (GBM) is highly refractory to therapy and associated with poor clinical outcome. Here, we reveal a critical function of the promitotic and adhesion-mediating discoidin domain receptor 1 (DDR1) in modulating GBM therapy resistance. In GBM cultures and clinical samples, we show a DDR1 and GBM stem cell marker co-expression that correlates with patient outcome. We demonstrate that inhibition of DDR1 in combination with radiochemotherapy with temozolomide in GBM models enhances sensitivity and prolongs survival superior to conventional therapy. We identify a 14-3-3-Beclin-1-Akt1 protein complex assembling with DDR1 to be required for prosurvival Akt and mTOR signaling and regulation of autophagy-associated therapy sensitivity. Our results uncover a mechanism driven by DDR1 that controls GBM therapy resistance and provide a rationale target for the development of therapy-sensitizing agents.

Epithelial polarization in 3D matrix requires DDR1 signaling to regulate actomyosin contractility.

Epithelial cells form sheets and tubules in various epithelial organs and establish apicobasal polarity and asymmetric vesicle transport to provide functionality in these structures. However, the molecular mechanisms that allow epithelial cells to establish polarity are not clearly understood. Here, we present evidence that the kinase activity of the receptor tyrosine kinase for collagen, discoidin domain receptor 1 (DDR1), is required for efficient establishment of epithelial polarity, proper asymmetric protein secretion, and execution of morphogenic programs. Lack of DDR1 protein or inhibition of DDR1 kinase activity disturbed tubulogenesis, cystogenesis, and the establishment of epithelial polarity and caused defects in the polarized localization of membrane-type 1 matrix metalloproteinase (MT1-MMP), GP135, primary cilia, laminin, and the Golgi apparatus. Disturbed epithelial polarity and cystogenesis upon DDR1 inhibition was caused by excess ROCK (rho-associated, coiled-coil-containing protein kinase)-driven actomyosin contractility, and pharmacological inhibition of ROCK was sufficient to correct these defects. Our data indicate that a DDR1-ROCK signaling axis is essential for the efficient establishment of epithelial polarity.

DNA-Encoded Library-Derived DDR1 Inhibitor Prevents Fibrosis and Renal Function Loss in a Genetic Mouse Model of Alport Syndrome.

The importance of Discoidin Domain Receptor 1 (DDR1) in renal fibrosis has been shown via gene knockout and use of antisense oligonucleotides; however, these techniques act via a reduction of DDR1 protein, while we prove the therapeutic potential of inhibiting DDR1 phosphorylation with a small molecule. To date, efforts to generate a selective small-molecule to specifically modulate the activity of DDR1 in an in vivo model have been unsuccessful. We performed parallel DNA encoded library screens against DDR1 and DDR2, and discovered a chemical series that is highly selective for DDR1 over DDR2. Structure-guided optimization efforts yielded the potent DDR1 inhibitor 2.45, which possesses excellent kinome selectivity (including 64-fold selectivity over DDR2 in a biochemical assay), a clean in vitro safety profile, and favorable pharmacokinetic and physicochemical properties. As desired, compound 2.45 modulates DDR1 phosphorylation in vitro as well as prevents collagen-induced activation of renal epithelial cells expressing DDR1. Compound 2.45 preserves renal function and reduces tissue damage in Col4a3–/– mice (the preclinical mouse model of Alport syndrome) when employing a therapeutic dosing regime, indicating the real therapeutic value of selectively inhibiting DDR1 phosphorylation in vivo. Our results may have wider significance as Col4a3–/– mice also represent a model for chronic kidney disease, a disease which affects 10% of the global population.

Inhibition of EP300 and DDR1 synergistically alleviates pulmonary fibrosis in vitro and in vivo

ObjectivesPulmonary fibrosis is strongly correlated with inflammation factors, cytokine, and collagen secretion, whereby discoidin domain receptor 1 (DDR1) signaling plays an important role. EP300 is defined as an acetyltransferase that can acetylate histone and has been broadly studied in several chronic diseases, including cancer, inflammation and fibrosis. This study aimed to investigate the relationship between p300 and DDR1 in the pathological processes of pulmonary fibrosis. Materials and methodsTranscriptome analysis of single cell RNA-sequencing for idiopathic pulmonary fibrosis (IPF) bronchial epithelial cells demonstrated that both DDR1 and EP300 were up-regulated and involved in the regulation of autophagy, cellular response to organonitrogen compounds, and collagen metabolic pathways, respectively. The anti-fibrotic and anti-inflammation effects of Pim1 and DDR1 inhibitors in bleomycin-induced IPF murine models were estimated. ResultsWe discovered that overexpression of EP300 signaling induced MRC5 human fibroblast cells that up-regulated the expression of DDR1 and FN1; however, no effects on COL1 A1 and DDR1 phosphorylation were observed. Mechanistically, TGF-β1 activated FN1, collagen, and DDR1 signaling could be reversed by the combination of p300 siRNA and DDR1 inhibitors. Moreover, the EP300 inhibitor SGC-CBP30 displayed synergistic effects with DDR1 inhibitors in pathogenic scores, airway goblet cell counts in bronchoalveolar lavage fluid (BALF), IL-4, IFN-γ, FN1COL1 A1 secretion and α-SMA, a marker of myofibroblast. ConclusionsThe EP300 siRNA and inhibitors sensitized DDR1 inhibitors in our pulmonary fibrosis models in vitro and in vivo, implicating a combined inhibition of DDR1 with EP300 as potential therapies for IPF.

A putative role for Discoidin Domain Receptor 1 in cancer chemoresistance

ABSTRACTThe Discoidin Domain Receptor 1 (DDR1) receptor tyrosine kinase performs pleiotropic functions in the control of cell adhesion, proliferation, survival, migration, and invasion. Aberrant DDR1 function as a consequence of either mutations or increased expression has been associated with various human diseases including cancer. Pharmacological inhibition of DDR1 results in significant therapeutic benefit in several pre-clinical cancer models. Here, we discuss the potential implication of DDR1-dependent pro-survival functions in the development of cancer resistance to chemotherapeutic regimens and speculate on the molecular mechanisms that might mediate such important feature.

MiR-199a/b-5p Inhibits Lymphangiogenesis by Targeting Discoidin Domain Receptor 1 in Corneal Injury.

Discoidin domain receptor 1 (DDR1) is involved in tumorigenesis and angiogenesis. However, its role in lymphangiogenesis has been unknown. Here, we tested whether downregulation of DDR1 expression by miR-199a/b can suppress lymphangiogenesis. We also aimed to identify miRNA target site(s) in the 3′ untranslated region (UTR) of DDR1. Transfection with miR-199a/b-5p mimics reduced expression of DDR1 and tube formation in primary human dermal lymphatic endothelial cells, whereas miR-199a/b-5p inhibitors showed the opposite effects. Critically, injection of miR-199a/b-5p mimics suppressed DDR1 expression and lymphangiogenesis in a corneal alkali-burn rat model. The three well-conserved seed matched sites for miR-199a/b-5p in the DDR1 3′-UTR were targeted, and miRNA binding to at least two sites was required for DDR1 inhibition. Our data suggest that DDR1 promotes enhanced lymphangiogenesis during eye injury, and miR-199a/b-5p suppresses this activity by inhibiting DDR1 expression. Thus, this miRNA may be useful for the treatment of lymphangiogenesis-related eye diseases.

Inhibition of Discoidin Domain Receptor 1 Reduces Collagen-mediated Tumorigenicity in Pancreatic Ductal Adenocarcinoma.

The extracellular matrix (ECM), a principal component of pancreatic ductal adenocarcinoma (PDA), is rich in fibrillar collagens that facilitate tumor cell survival and chemoresistance. Discoidin domain receptor 1 (DDR1) is a receptor tyrosine kinase that specifically binds fibrillar collagens and has been implicated in promoting cell proliferation, migration, adhesion, ECM remodeling, and response to growth factors. We found that collagen-induced activation of DDR1 stimulated protumorigenic signaling through protein tyrosine kinase 2 (PYK2) and pseudopodium-enriched atypical kinase 1 (PEAK1) in pancreatic cancer cells. Pharmacologic inhibition of DDR1 with an ATP-competitive orally available small-molecule kinase inhibitor (7rh) abrogated collagen-induced DDR1 signaling in pancreatic tumor cells and consequently reduced colony formation and migration. Furthermore, the inhibition of DDR1 with 7rh showed striking efficacy in combination with chemotherapy in orthotopic xenografts and autochthonous pancreatic tumors where it significantly reduced DDR1 activation and downstream signaling, reduced primary tumor burden, and improved chemoresponse. These data demonstrate that targeting collagen signaling in conjunction with conventional cytotoxic chemotherapy has the potential to improve outcome for pancreatic cancer patients. Mol Cancer Ther; 16(11); 2473-85. ©2017 AACR.

Highlights of This Issue

Cytotoxic chemotherapy is known to induce pro-metastatic changes resulting in poor survival. Harney, Karagiannis and colleagues show that the TIE2 kinase inhibitor rebastinib reduces primary tumor growth and metastasis in a preclinical breast cancer model, and is synergistic in combination with taxane chemotherapy. They demonstrate that taxane chemotherapy is associated with metastasis through recruitment of tumor-infiltrating TIE2+ macrophages that promote angiogenesis, TMEM-dependent tumor cell intravasation and lung metastasis, events reversed by rebastinib. In an adjuvant study, rebastinib in combination with eribulin led to a significant improvement in survival compared to single agent eribulin. Thus, rebastinib is a promising therapy for inhibiting pro-metastatic microenvironments in cancer patients.Epithelial-mesenchymal transition (EMT) plays an important role in cancer progression, relapse, and metastasis. By using the myxobacterial natural compound Archazolid A as a tool, Merk and colleagues discovered the V-ATPase, a multimeric proton pump that regulates lysosomal acidification, as a crucial player in EMT and Archazolid A as a promising strategy to block EMT through the inhibition of VATPase. Mechanistic studies found that Archazolid A disrupts the process of EMT, the generation of tumor-initiating cells and the turnover of E-cadherin. These evidences suggest that Archazolid A is a promising cancer therapeutic agent that is worth further investigation.The extracellular matrix limits the efficacy of chemotherapy in pancreatic ductal adenocarcinoma (PDA) in part by active cell signaling. Discoidin domain receptor 1 (DDR1) is a receptor tyrosine kinase that binds fibrillar collagens and is expressed by PDA tumor cells. Aguilera, Huang and colleagues demonstrate that pharmacologic inhibition of DDR1 with the small molecule 7rh substantially improves the efficacy of standard chemotherapy in robust preclinical models of PDA in the absence of additional normal tissue toxicity. These results suggest that inhibition of collagen signaling with 7rh has the potential to improve efficacy of conventional chemotherapy in PDA patients.High tumor mutational burden (TMB) predicts response to checkpoint blockade in lung cancer and melanoma. Our study demonstrates that TMB can be used to predict response to checkpoint blockade independent of tumor histology. Higher TMB was independently and linearly associated with improved response rates and progression-free survival on multivariate analysis. Response rates were 67% with very high TMB (>50 mutations/mb). Further, the median TMB was significantly higher for patients who responded to anti-PD-1/PD-L1 monotherapy compared to those who did not. Interestingly, benefit from dual checkpoint blockade did not show a similarly strong dependence on TMB.

Abstract LB-216: Stroma-induced up-regulation of discoidin domain receptor 1 enhances peritoneal metastasis of gastric carcinomas

Abstract Introduction: Discoidin domain receptor-1 (DDR1), a receptor tyrosine kinase, is expressed by about 50% of gastric tumor cells in previous our study and is activated by fibrillar collagens, which are major components of tumor stroma. Further, it has been proposed that peritoneal metastasis is associated with high stroma content in gastric carcinomas (GCs). In this study we investigated the contribution of DDR1 activity to peritoneal metastasis of GCs. Methods: We performed the immunohistochemistry for DDR1 and Masson’s trichrome staining for accumulation of collagen bundles in 202 human GC tissues. The human GC cell lines (MKN-28 and KATO-III) were co-cultured with GCs-associated fibroblasts (CAFs), and the expression of DDR1 and signal transduction pathways were investigated by Western blot. We evaluated CAF-induced tumorigenesis of GCs cell lines and the effects of DDR1 specific inhibitor in 3D co-culture system and peritoneal seeding in xenograft animal models. Results: High stromal collagen correlated with peritoneal recurrence (P=0.004) and was positively associated with expression of DDR1 in GC tissues (P<0.001). We confirmed that co-culture with CAFs elevated the expression of DDR1 in GC cell lines. CAFs also enhanced GC cell line spheroid formation in vitro in a tumor cell DDR1-dependent manner. Co-implantation of CAFs with GC cells enhanced peritoneal tumor formation in vivo, an effect that was sensitive to pharmacologic inhibition of DDR1. Conclusion: Our findings suggest that CAF-induced elevation of tumor cells DDR1 enhances peritoneal tumorigenesis of GCs and the inhibition of DDR1 signaling is an attractive strategy for the treatment of GC peritoneal metastasis. Citation Format: Hyejin Jin, In-Hye Ham, Hye Jung Oh, Dakeun Lee, Sang-Uk Han, Rolf A. Brekken, Hoon Hur. Stroma-induced up-regulation of discoidin domain receptor 1 enhances peritoneal metastasis of gastric carcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-216. doi:10.1158/1538-7445.AM2017-LB-216

Abstract 1987: Inhibition of collagen receptor discoidin domain receptor-1 (DDR1) reduces gastric cancer cell motility and metastasis

Abstract Accumulating evidences suggest that a unique set of receptor tyrosine kinases, known as discoidin domain receptors (DDRs), play a role in cancer progression by interaction with their surrounding collagen matrix. Although abnormal expression of DDRs is reported in some human cancers, little is known about the expression and function of DDRs in gastric cancer progression. In this study, we investigated expression and the role of DDR1 in the tumor progression of human gastric cancer. The expression and distribution of DDR1 was analyzed by immunohistochemistry in 127 human gastric cancer cases, and the expression level of DDR1 in 7 human gastric cancer cell lines was analyzed by RT-PCR and western blotting. Proliferation, migration, invasion, and tube formation assays were conducted in DDR1 silenced gastric cancer cells. The effect of DDR1 on the tumor growth and metastasis were examined in orthotopically implanted nude mice model and a model of liver metastasis. DDR1 was expressed in human gastric cancer cell lines, and expression of DDR1 in human gastric tumor was associated with poor prognosis. Among 7 gastric cancer cell lines, MKN74 expressed DDR1 at highest level. DDR1-silenced MKN74 cells showed unaltered proliferation activity. In contrast, migration, invasion, and tube formation were significantly reduced. When examined in an orthotopic nude mouse model, DDR1-silenced implanted tumor significantly decreased angiogenesis and lymphangiogensesis, thereby leading to reduction of lymph node metastasis and liver metastasis. We showed that treatment of DDR1 inhibitor also inhibited lymph node metastasis in an orthotopic nude mouse model These results suggest that DDR1 is involved in GC tumor progression and metastasis. DDR1 could be not only an independent prognostic classifier, but also a promising therapeutic target in patients with GC. Citation Format: Ryo Yuge, Yasuhiko Kitadai, Hidehiko Takigawa, Shinji Tanaka, Kazuaki Chayama, Wataru Yasui. Inhibition of collagen receptor discoidin domain receptor-1 (DDR1) reduces gastric cancer cell motility and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1987. doi:10.1158/1538-7445.AM2017-1987

Effects of discoidin domain receptor 1 mediated phosphorylation of protein Tau on hypoxic-ischemic brain damage in neonatal rats

Objective To study the effects of discoidin domain receptor 1 (DDR1) mediated phosphorylation of protein Tau on hypoxic-ischemic brain damage (HIBD) in neonatal rats and its possible mechanism. Methods Sixty-four seven-day-old male specific-pathogen-free Wistar rats were randomly divided into four groups with sixteen in each: Sham, HIBD, HIBD with normal saline (HIBD+NS) and HIBD with DDR1 inhibitor (HIBD+DI) groups. A rat model of HIBD was established by subjecting the rats to left common carotid artery ligation, followed by exposing them to hypoxia for two hours. In HIBD+DI group, the inhibitor of DDR1 was immediately injected into lateral cerebroventricles of the rats following modeling. Forty-eight hours after injection, tissues of left cerebral cortex were collected from each rat to evaluate histopathological changes with HE staining. Western-blotting was used to assess the phosphorylation levels of DDR1 and protein Tau. Enzyme-linked immunosorbent assay was performed to detect the concentrations of acetylcholine. Analysis of variance or t test were used for statistical analysis. Results (1) Damages in cerebral cortex: Percentages of abnormal neurons in the rats of HIBD group were higher than those in Sham group [(80.28±4.51)% vs (10.40±2.17)%, t=39.491, P<0.01]. Pyknotic or necrotic neurons in the rats of HIBD+DI group were less than those in HIBD+NS group [(31.91±3.05)% vs (82.01±7.20)%, t=18.123, P<0.01]. (2) Phosphorylation of DDR1 and protein Tau: Levels of phosphorylated DDR1 in the cerebral cortexes of rats in HIBD group were higher than those in Sham group (0.922±0.199 vs 0.095±0.023, t=10.379, P<0.01), and those levels in HIBD+NS group were higher than those in HIBD+DI group (1.200±0.171 vs 0.255±0.111, t=11.901, P<0.01). The phosphorylation of protein Tau was similar to that of DDR1 (0.919±0.228 vs 0.194±0.224 in HIBD and Sham groups, t=7.347; 1.100±0.167 vs 0.291±0.210 in HIBD+NS and HIBD+DI groups, t=9.447; both P<0.01). (3) Levels of acetylcholine: Levels of acetylcholine in cerebral cortexes of rats in HIBD group were lower than those in Sham group [(3.685±0.472) vs (7.429±0.861) ng/g protein, t=10.781, P<0.01], and that levels in HIBD+DI group were higher than those in HIBD+NS group [(7.058±0.915) vs (2.521±0.723) ng/g protein, t=10.989, P<0.01]. Conclusions Activation of DDR1 plays a key role in enhancing the phosphorylation of protein Tau and in reducing the secretion of acetylcholine in cerebral cortexes of rats with HIBD. Inhibitor of DDR1 could protect neonatal rats from HIBD through the decreasing of protein Tau phosphorylation and increasing of acetylcholine release by inhibiting the activation of DDR1. Key words: Hypoxia-ischemia,brain; Discoidin domain receptor 1; Tau proteins; Phosphorylation; Animals, newborn; Rats

Tetrahydroisoquinoline-7-carboxamide Derivatives as New Selective Discoidin Domain Receptor 1 (DDR1) Inhibitors.

Acute lung injury (ALI) is a deadly symptom for serious lung inflammation. Discoidin Domain Receptor 1 (DDR1) is a new potential target for anti-inflammatory drug discovery. A new selective tetrahydroisoquinoline-7-carboxamide based DDR1 inhibitor 7ae was discovered to tightly bind the DDR1 protein and potently inhibit its kinase function with a Kd value of 2.2 nM and an IC50 value of 6.6 nM, respectively. The compound dose-dependently inhibited lipopolysaccharide (LPS)-induced interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) release in mouse primary peritoneal macrophages (MPMs). In addition, 7ae also exhibited promising in vivo anti-inflammatory effects in a LPS-induced mouse ALI model. To the best of our knowledge, this is the first "proof of concept" investigation on the potential application of a small molecule DDR1 inhibitor to treat ALI.

Abstract B14: Discoidin domain receptor 1 activity drives an aggressive phenotype in gastric adenocarcinoma

Abstract Purpose: Stromal content in gastric cancer patients inversely correlates with outcome. However, underlying molecular mechanisms for these clinical observations have not been determined. Here, we investigated the contribution of the collagen receptor, discoidin domain receptor-1 (DDR1), in the aggressive phenotype of gastric cancer induced by tumor stroma. Experimental Design: We conducted immunohistochemical analyses with a tissue microarray of 217 gastric cancer specimens. We examined the effect of collagen-induced activation of DDR1 on cell signaling, tumorigenesis and cell migration in gastric cancer, and evaluated the effects of DDR1 inhibition in organotypic culture and xenograft animal models of gastric cancer. Results: High DDR1 expression in gastric cancer tissue significantly correlated with poor prognosis. Gastric cancer patients were classified into two groups based on the stromal collagen and the expression of DDR1 was significantly associated with cancer invasion and lymph node metastasis in the collagen-positive group. Decreased expression of E-cadherin was associated with cancer progression in the collagen and DDR1 positive groups. We also demonstrated that collagen activated DDR1 and increased clonogenicity and cell migration. Importantly, we demonstrated that a DDR1 inhibitor, 7rh benzamide, reduced the invasiveness of gastric cancer in organotypic culture system and suppressed tumor growth in gastric cancer xenografts. Conclusions: Taken together, these findings suggest that collagen enhances the aggressive phenotype of gastric cancer through the DDR1 signaling pathway and that inhibition of DDR1 is an attractive candidate for the therapy of aggressive gastric cancer. Citation Format: Hoon Hur, In-hye Ham, Da Keun Lee, Hye-Jin Jin, Aguilera Kristina Y., Hae Jeong Oh, Sang-Uk Han, Ji-Eun Kwon, Young Bae Kim, Ke Ding, Rolf Brekken. Discoidin domain receptor 1 activity drives an aggressive phenotype in gastric adenocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr B14.

Abstract 743: Tyrosine kinase discoidin domain receptor-1 (DDR1) regulates cytotoxicity of recombinant immunotoxin for cancer therapy

Abstract Discoidin domain receptor 1 (DDR1) is a collagen-activated tyrosine kinase that facilitates cell adhesion, migration, proliferation and matrix remodeling. The collagen/DDR1 axis is also thought to modulate tumor-stromal interaction and potentially affects tumor response to therapy. Mesothelin is a cell-surface protein over-expressed in several human cancers including mesothelioma, ovarian, lung, breast, and pancreatic cancers with limited expression on normal cells. RG7787 is a clinically optimized recombinant immunotoxin (RIT) consisting of a humanized anti-mesothelin Fab fused to domain III of Pseudomonas exotoxin A in which immunogenic B cell epitopes are silenced. To enhance therapeutic efficacy of RITs we conducted a kinome RNAi sensitization screen which identified DDR1 as a potential target. We hypothesized that DDR1 regulates RIT activity and its inhibition might enhance cell killing by RG7787. Knockdown of DDR1 by siRNA or treatment with inhibitor, ‘7rh’, greatly enhanced the cytotoxic activity of RG7787 in several cancer cell lines. Investigation into the mechanism of action showed DDR1 silencing was associated with a decrease in several ribosomal proteins and enhanced inhibition of protein synthesis. Induction of DDR1 expression or stimulation of DDR1 activity by collagen protected cancer cells from RG7787 killing, while DDR1 inhibition enhanced killing by RG7787. Moreover, the combination of immunotoxin and DDR1 inhibitor caused significantly more shrinkage of epithelial A431/H9 and pancreatic KLM1 tumor xenografts than either agent alone. These data demonstrate that DDR1 has a critical role in modulating immunotoxin activity. Inhibition of DDR1 represents a novel strategy to enhance therapeutic efficacy of RITs targeting mesothelin-expressing cancers. Citation Format: Fatima Ali-Rahmani, David Fitzgerald, Scott Martin, Paresma Patel, Marco Prunotto, Pinar Ormanoglu, Craig Thomas, Ira Pastan. Tyrosine kinase discoidin domain receptor-1 (DDR1) regulates cytotoxicity of recombinant immunotoxin for cancer therapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 743.

Structure-Based Design of Tetrahydroisoquinoline-7-carboxamides as Selective Discoidin Domain Receptor 1 (DDR1) Inhibitors

The structure-based design of 1, 2, 3, 4-tetrahydroisoquinoline derivatives as selective DDR1 inhibitors is reported. One of the representative compounds, 6j, binds to DDR1 with a Kd value of 4.7 nM and suppresses its kinase activity with an IC50 value of 9.4 nM, but it is significantly less potent for a panel of 400 nonmutated kinases. 6j also demonstrated reasonable pharmacokinetic properties and a promising oral therapeutic effect in a bleomycin-induced mouse pulmonary fibrosis model.

Anticancer Effects of Mesothelin-Targeted Immunotoxin Therapy Are Regulated by Tyrosine Kinase DDR1.

Recombinant immunotoxins (RIT) have been highly successful in cancer therapy due, in part, to the high cancer-specific expression of cell surface antigens such as mesothelin, which is overexpressed in mesothelioma, ovarian, lung, breast, and pancreatic cancers, but is limited in normal cells. RG7787 is a clinically optimized RIT consisting of a humanized anti-mesothelin Fab fused to domain III of Pseudomonas exotoxin A, in which immunogenic B-cell epitopes are silenced. To enhance the therapeutic efficacy of RITs, we conducted a kinome RNAi sensitization screen, which identified discoidin domain receptor 1 (DDR1), a collagen-activated tyrosine kinase, as a potential target. The collagen/DDR1 axis is implicated in tumor-stromal interactions and potentially affects tumor response to therapy. Therefore, we investigated the effects of DDR1 on RIT. Knockdown of DDR1 by siRNA or treatment with inhibitor, 7rh, greatly enhanced the cytotoxic activity of RG7787 in several cancer cell lines. Investigation into the mechanism of action showed DDR1 silencing was associated with decreased expression of several ribosomal proteins and enhanced inhibition of protein synthesis. Conversely, induction of DDR1 expression or collagen-stimulated DDR1 activity protected cancer cells from RG7787 killing. Moreover, the combination of RG7787 and DDR1 inhibitor caused greater shrinkage of tumor xenografts than either agent alone. These data demonstrate that DDR1 is a key modulator of RIT activity and represents a novel therapeutic strategy to improve targeting of mesothelin-expressing cancers.

Abstract 5070: Inhibition of collagen receptor discoidin domain receptor-1 (DDR1) reduces colon cancer cell migration and metastasis

Abstract Accumulating evidence suggests that a unique set of receptor tyrosine kinases, known as discoidin domain receptors (DDRs), plays a role in cancer progression by regulating the interactions of tumor cells with their surrounding collagen matrix. We investigated the role of DDR1 in the tumor progression of colorectal cancer. We examined the DDR1 mRNA and protein expression levels in a series of human colorectal cancer cell lines by RT-PCR and Western blotting. We also analyzed the DDR1 protein expression in 27 surgical specimens of colorectal cancer by immunohistochemistry. Migration, invasion, and proliferation assays were conducted with a high-content time-lapse assay system in a series of colon cancer cell lines. Nilotinib, a well-known DDR1 inhibitor, and DDR1-silenced colon cancer cells were used for inhibition of DDR1 tyrosine kinase activity. In an in vivo study, we orthotopically implanted KM12SM human colon cancer cells into the cecum of nude mice and administered nilotinib (100 mg/kg/day) by daily oral gavage. After 4 weeks of treatment, primary tumors and metastatic lesions were resected, and specimens were analyzed histologically. DDR1 mRNA and protein expression was observed in the human colon cancer cell lines and in cancer cells of most of the surgical specimens. Colon cancer cell lines KM12SM, KM12c, LoVo, and HT29 exposed to nilotinib (20 μM) showed reduced migration. When incubated with type I collagen, however, these cells showed increased migratory activity. Nilotinib treatment significantly inhibited this effect. The migratory activity of DDR1-silenced cells was not affected by nilotinib treatment. The orthotopic xenografts grew invasively as aggressive tumors with a stromal reaction, but under nilotinib treatment, the stromal reaction was reduced, and tumors grew expansively. In addition, the numbers of lymph node metastases were significantly reduced. Disruption of DDR1 attenuated colon cancer cell migration and led to altered primary tumor morphology and to impaired lymph node metastasis. DDR1 may be a promising therapeutic target in patients with colon cancer. Citation Format: Ryo Yuge, Yasuhiko Kitadai, Kei Shinagawa, Mieko Onoyama, Shinji Tanaka, Wataru Yasui, Kazuaki Chayama. Inhibition of collagen receptor discoidin domain receptor-1 (DDR1) reduces colon cancer cell migration and metastasis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5070. doi:10.1158/1538-7445.AM2015-5070

The EBV Oncogene LMP1 Protects Lymphoma Cells From Cell Death Through The Collagen-Mediated Activation Of Discoidin Domain Receptor 1

The malignant Hodgkin/Reed-Sternberg (HRS) cells of Hodgkin lymphoma are surrounded by a tumour microenvironment which is composed of a variety of cell types, as well as non-cellular components such as collagen. Although HRS cells harbour oncogenic Epstein-Barr virus (EBV) in approximately 50% of cases, it is not known if the tumour microenvironment contributes to EBV-driven lymphomagenesis. We show that expression of the EBV-encoded latent membrane protein-1 (LMP1) in primary human germinal centre B cells, the presumed progenitors of HRS cells, up-regulates discoidin domain receptor 1 (DDR1), a receptor tyrosine kinase activated by collagen. We show that HRS cells intimately associated with collagen frequently over-express DDR1 and that short-term exposure to collagen is sufficient to activate DDR1 in Hodgkin lymphoma-derived cell lines. We also show that ectopic expression of DDR1 significantly increased the survival of collagen-treated DG75, Burkitt lymphoma cells, following etoposide treatment. Conversely, inhibition of DDR1, by RNA interference, significantly decreased the survival of collagen-treated L428 Hodgkin lymphoma cells in the absence of specific apoptotic stimulus suggesting that DDR1 also influences baseline survival. Our results identify a hitherto unknown function for collagen in protecting Hodgkin lymphoma from apoptosis and suggest an important contribution of the tumour microenvironment in promoting the oncogenic effects of EBV. Disclosures:No relevant conflicts of interest to declare.