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Introduction and Objectives: To investigate the utility of combination approach for treating bladder cancer, mice bearing UMUC3 (human transitional cell carcinoma) and T24 (human urinary bladder) tumors were treated with HPPH (2-[1-hexyloxyethyl]-2-devinyl pyropheophorbide-a)-PDT alone and in combination with BCG. The HPPH, a multifunctional agent, was selected as a photosensitizer because of its higher tumor avidity, improved fluorescence imaging ability, and limited skin phototoxicity than Photofrin, a current clinical standard. Methods: Imaging: IVIS/Spectrum: A noninvasive in vivo, continuous wave fluorescence imaging apparatus used to assess the localization and distribution of the PS at variable time points. In vivo efficacy studies using UMUC3 and T24 tumors in SCID mice with PS HPPH in combination with BCG. Results: Imaging: Tumor uptake in UMUC3 and T24 tumors using IVIS Imaging. PS HPPH showed high tumor uptake and significant difference in their pharmacokinetic profiles in UMUC3 and T24 tumors implanted in SCID mice: The SCID mice bearing either UMUC3 or T24 tumors (4-5 mm size) were injected with the PS HPPH. The tumored mice (3 mice/group) at a dose of 0.47 u-mol/kg) and imaged at variable time points. The uptake of tumor, liver, and skin was measured by IVIS system and interesting pharmacokinetic profiles of the PS were observed. The optimal tumors uptake in UMUC3 and T24 were at 24 hours and 8-12 hours, respectively. In vivo efficacy studies: PS (HPPH) in combination with BCG shows enhanced PDT efficacy CR (Cures) Day 30 = 14/22 = 63.6% and RG (Re-Growth) 6/22 = 36.4% in bladder tumors (UMUC3). To investigate the utility of combination treatment approach for treating bladder cancer, mice bearing UMUC3 tumors were injected intravenously with HPPH (0.47 u-mol/kg) and the tumors were exposed to laser light (135 J/cm2, 75 mW/cm2) at 24 h post-injection. One hour after the PDT treatment, BCG was injected (1-1.5 × 106 CFU), SQ weekly x 3 doses, and at day 30 the results are very encouraging. Conclusions: The HPPH showed higher in vivo uptake (determined by fluorescence) in UMUC3 and T24 bladder cancer tumors with significant differences in the two tumor types. The treatment of HPPH-PDT in combination with BCG using SCID mice bearing UMUC3 tumors showed enhanced long-term tumor cure. Before initiating this approach to human phase I clinical trial, further studies are under way. Source of funding: Roswell Park Alliance Foundation. Citation Format: Farukh Durrani, Ahmed Aly, Khurshid Guru, Ravindra Pandey. Photodynamic therapy in combination with immunotherapy enhances the long-term cure of bladder cancer in tumored mice [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2019 May 18-21; Denver, CO. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(15_Suppl):Abstract nr A02.

Introduction: Focal adhesion kinase (FAK) and vascular endothelial growth factor receptor 3 (VEGFR3) are tyrosine kinases, which function as key modulators of survival and metastasis signals in cancer cells. We have previously shown that VEGFR3 binds to the FAT domain of FAK and this interaction promotes the survival of tumor cells. We developed novel small molecule compounds C9 and C10 which specifically bind and inhibit the FAK-VEGFR3 interaction. Both these compounds inhibit cancer cell proliferation, induce apoptosis, disrupt the FAK-VEGFR3 signaling axis, and significantly inhibit tumor growth in xenograft mouse models of breast and pancreatic cancer. Since FAK and VEGFR3 have been implicated in tumor angiogenesis, we investigated the potential anti-angiogenic effects of C9 and C10. Methods: Binding of C9 and C10 to the FAT domain of FAK was tested using fluorescence polarization. Effects of C9 and C10 were tested in vitro by MTS assay (viability) and tube formation assay using HUVEC cells. Directed in vivo angiogenesis assay (DIVAA) kit was used to determine anti-angiogenic effects of both compounds. Single agent efficacy of C9 and C10 was tested in a pancreatic xenograft mouse model for 30 days. Mouse tumor tissues were analyzed for markers of proliferation (Ki67), endothelial vessels (CD31), and lymphangiogenic vessels (LYVE1) using immunohistochemistry. Results: Fluorescence polarization confirmed that C9 and C10 effectively bind to the FAT domain of FAK. C9 and C10 reduced cell viability of HUVEC cells and dramatically decreased tube formation by reducing total branching points, total loops and total tube length in a dose and time dependent manner. DIVAA assay confirmed that C9 and C10 significantly reduced the formation of endothelial blood vessels as compared to untreated groups. In vivo, 30 days treatment with low doses of C9 and C10 as single agents led to 40% to 45% reduction in tumor volume (P Conclusions: We have shown that targeting the FAK-VEGFR3 protein protein interaction site allows for dual inhibition of tumor proliferation as well as tumor angiogenesis. This study highlights the importance of oral-based small molecule inhibitors that might impact the therapeutic management of solid tumors. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B3. Citation Format: Priyanka A. Agharkar, Manivannan Ethirajan, Jianqun Liao, Michael Yemma, Brian Buckley, Mikhail Chernov, William Cance, Ravindra Pandey, Elena Kurenova. Anti-angiogenic properties of FAK-VEGFR3 scaffold inhibitors, C9 and C10. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B3.

Introduction: FAK functions as both, a critical signaling kinase and a scaffolding protein to collectively mediate proliferation, survival, cell adhesion and migration signals. VEGFR3 is a receptor tyrosine kinase that acts as a key modulator of survival signals and lymphangiogenesis. Upregulation of FAK and VEGFR3 in multiple tumor types, with low levels in normal tissues make them attractive anti-cancer targets. Previously we have shown that VEGFR3 binds to FAK and the physical interaction between the two proteins confers a significant survival advantage to tumor cells. Subsequently we discovered small molecule C4 that specifically targets the VEGFR3 binding site on the FAT domain of FAK and exerts anti-tumor effects at moderate concentrations. In this study we designed, synthesized and characterized novel C4 analogs which show enhanced potency and specificity for the target site. Methods: Modifications were made on the C4 moiety and the resulting analogs were screened for anti-cancer inhibitory effects in different cancer cell lines by MTS assay. Effects of C4 analogs on cancer cell lines were determined by MTS assay (viability), Clonogenecity assay, Western blot (phosphorylation, apoptosis), Immunofluorescence staining (apoptosis) and Flow cytometry (apoptosis). Computer modeling, target specificity (immunoprecipitation) and binding confirmation (Forte-Bio's Octet Red assay) of all analogs were tested. In vitro wound healing and cell invasion assays were also performed. In vivo anti-tumor activity of lead analogs was done using a subcutaneous xenograft mouse model of pancreatic cancer. Results: Based on the preliminary screening results in several cancer cell lines, we selected analogs C9 and C10 for further analyses. Both analogs inhibit colony formation of pancreatic cancer cells and show target specificity by disrupting FAK-VEGFR3 interaction at ≤10μM. Binding of C9 and C10 to the FAT domain of FAK was confirmed with Octet method. Induction of apoptosis after treatment with C9 and C10 was confirmed via caspase 3/7 activation, caspase-3 and PARP cleavage. Down-regulation of phosphorylated forms of Akt, Erk1/2, Jnk, Grb2 and paxillin was seen in a dose and time dependent manner after 24h treatment with C9 and C10. These drugs also inhibited migration and invasion of pancreatic cancer cells at ≤10μM. In vivo, 30 days treatment with low doses of C9 and C10 led to massive reduction in tumor growth when treated as single agents (P Conclusions: FAK-VEGFR3 targeted therapeutics represents a novel approach towards anti-cancer treatment options. Our promising results thereby form the basis for future clinical applications of FAK-VEGFR3 small molecule inhibitors for cancer therapy. Citation Format: Priyanka A. Agharkar, Manivannan Ethirajan, Andrew Magis, Jian Liao, Michael Yemma, William Cance, Ravindra Pandey, Elena Kurenova. Novel small molecule inhibitors C9 and C10 specifically disrupt the FAK-VEGFR3 signaling axis in cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3247. doi:10.1158/1538-7445.AM2013-3247

Introduction: FAK and VEGFR3 have stirred considerable interest in recent years as attractive anti-cancer targets, owing to their high expression across several tumor types and relatively low levels in normal tissues. Our lab has shown that VEGFR3 binds to FAK and the physical interaction between the two proteins confers a significant survival advantage to tumor cells. We discovered small molecule C4 that specifically targets and disrupts FAK-VEGFR3 interaction to exert anti-tumor effects, but does so at moderate concentrations. This prompted us to design C4 analogs that display enhanced potency, minimum toxicity and high specificity for the target site. Here, we have shown that some C4 analogs disrupt FAK-VEGFR3 interaction and dramatically inhibit pancreatic tumor growth at low concentrations as single agents as well as in combination with Gemcitabine. Methods: The structure of C4 was modified at various positions by substituting alkyl and aryl moieties. The resulting analogs were screened for anti-cancer inhibitory effects in different cancer cell lines by MTS assay. Effects of C4 analogs on pancreatic cancer cell lines were determined by MTS assay (viability), Clonogenecity assay, Western blot (phosphorylation, apoptosis) and TUNEL assay. Target specificity (immunoprecipitation) and binding confirmation (Forte-Bio's Octet Red assay) of all analogs were performed. In vivo anti-tumor activity of lead analogs was done using a subcutaneous xenograft mouse model of pancreatic cancer. Results: Twenty C4 analogs were screened and four were selected for further analyses. These four analogs strikingly decreased survival of six pancreatic cancer cell lines. Analog C9 showed highest cytotoxic effects, inhibited colony formation of Panc-1 cells at 1µM, induced dose dependent cleavage of caspase-3 and PARP and decreased phosphorylated forms of Akt and Erk within 24h treatment. Disruption of FAK-VEGFR3 interaction was seen with 1µM C9. Octet method revealed that 20µM C9, KD (M) = 1.29×10-7. In vivo, 30 days treatment with low doses of the four analogs led to massive reduction in tumor growth when treated as single agents (60%) and in combination with Gemcitabine (80%)(P Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3756. doi:1538-7445.AM2012-3756

Introduction: FAK and VEGFR-3 have stirred considerable interest in recent years as attractive anti-cancer targets, owing to their high expression across several tumor types and relatively low levels in normal tissues. Our lab has shown that VEGFR-3 binds to the FAT domain of FAK and the physical interaction between the two proteins confers a significant survival advantage to tumor cells. We developed a platform for the discovery of small molecules that specifically target the FAK-VEGFR-3 interaction site, and identified small molecule C4. Although in vitro and in vivo studies with C4 are promising, it exerts anti-tumor effects at moderate concentrations. We next sought to discover C4 analogs that display enhanced potency, minimum toxicity and high specificity for the target site. By using C4 as the parent moiety, we synthesized a series of C4 analogs on the basis of structure activity relationship (SAR). In this study, we have shown that some C4 analogs disrupt FAK-VEGFR3 interaction and inhibit pancreatic tumor growth at low concentrations as single agents. Methods: The structure of C4 was modified at various positions by substituting alkyl and aryl moieties. The resulting analogs were screened for anti-cancer inhibitory effects in different cancer cell lines by MTT assay. Effects of C4 analogs on six pancreatic cancer cell lines were determined by MTT assay (viability and IC50), Clonogenecity assay (colony formation) and Western blot (phosphorylation and apoptosis). Target specificity and binding confirmation of C4 analogs was done using immunoprecipitation and Forte-Bio's Octet Red Technology. Subcutaneous xenograft mouse model of pancreatic cancer was used to determine anti-tumor effects of C4 analogs in vivo. Results: After in vitro screening of C4 analogs, C9, C10, C9A and C9B was selected for further analyses. All four analogs revealed dramatic decrease in cell viability in a panel of six pancreatic cancer cell lines. Analog C9 showed highest cytotoxic effects in pancreatic cancer cell lines and inhibited colony formation of Panc-1 cells at 1μM concentration. Treatment with C9 leads to apoptosis with dose dependent cleavage of caspase-3 and PARP. It caused decrease in phosphorylation levels of Akt and Erk, which are critical downstream targets of FAK that mediate survival signaling. Disruption of FAK-VEGFR3 interaction was seen after 24h treatment with 1μM C9. With Octet method we determined that at 20μM C9, KD (M) = 1.29×10(−7). In vivo, 30 days treatment with low doses of C9, C10 and C9A as single agents led to 40% to 45% reduction in tumor volume (P Conclusions: Our results demonstrate that targeting the FAK-VEGFR-3 interaction site inhibited the survival function of these two tyrosine kinases, thus representing a unique approach for molecular-targeted highly specific cancer therapeutics. The significance of this study lies in the discovery of an oral-based small molecule inhibitor that might impact the therapeutic management for pancreatic cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C124.