Jacob Valiyaveettil

Phosphodiesterase 10A is overexpressed in lung tumor cells and inhibitors selectively suppress growth by blocking β-catenin and MAPK signaling

Phosphodiesterase 10A (PDE10) is a cyclic nucleotide (e.g. cGMP) degrading enzyme highly expressed in the brain striatum where it plays an important role in dopaminergic neurotransmission, but has limited expression and no known physiological function outside the central nervous system. Here we report that PDE10 mRNA and protein levels are strongly elevated in human non-small cell lung cancer cells and lung tumors compared with normal human airway epithelial cells and lung tissue, respectively. Genetic silencing of PDE10 or inhibition by small molecules such as PQ10 was found to selectively inhibit the growth and colony formation of lung tumor cells. PQ10 treatment of lung tumor cells rapidly increased intracellular cGMP levels and activated cGMP-dependent protein kinase (PKG) at concentrations that inhibit lung tumor cell growth. PQ10 also increased the phosphorylation of β-catenin and reduced its levels, which paralleled the suppression of cyclin D1 and survivin but preceded the activation of PARP and caspase cleavage. PQ10 also suppressed RAS-activated RAF/MAPK signaling within the same concentration range and treatment period as required for cGMP elevation and PKG activation. These results show that PDE10 is overexpressed during lung cancer development and essential for lung tumor cell growth in which inhibitors can selectively induce apoptosis by increasing intracellular cGMP levels and activating PKG to suppress oncogenic β-catenin and MAPK signaling.

Abstract A30: Novel non-COX inhibitory sulindac derivative with PDE10 inhibitory activity reduces incidence and multiplicity of colorectal adenomas in the APC+/min-FCCC mouse model

Sulindac has been reported to reduce the number and size of precancerous colonic adenomas in patients with familial adenomatous polyposis (FAP), but is not recommended for long-term use as a cancer chemopreventive drug because of potentially fatal toxicities associated with cyclooxygenase (COX) inhibition. We report here a novel sulindac derivative, ADT-061, that lacks COX-1 and COX-2 inhibitory activity, yet potently and selectively inhibits colon tumor cells with IC 50 values of 0.3-0.5 μM with no significant effect on the growth of normal colonocytes. By comparison, sulindac sulfide inhibits colon tumor cell growth with IC 50 values of 40-60 μM, and has only modest tumor cell selectivity. ADT-061 inhibits phosphodiesterase 10A (PDE10), which increases intracellular cGMP levels, thus activates protein kinase G, suppresses nuclear levels of β-catenin, and induces cell cycle arrest and apoptosis, all within the same concentration range that inhibits tumor cell growth. ADT-061was evaluated for chemopreventive activity in the APC +/min-FCCC mouse model of colon cancer, which harbors colorectal adenomas that express PDE10. Colonoscopy exams were initially performed on all mice to ensure the absence of colorectal adenomas prior to treatment. At 7-8 weeks of age, male mice were treated with ADT-061 (1000ppm and 1500ppm) in the diet for 14 weeks. ADT-061 reduced the incidence of colorectal adenomas from 95.8% in the control group to 73.9% in the 1000ppm group (p=0.048), and 59.1% (p=0.004) in the 1500ppm group. Control untreated mice had an average of 2.98 colorectal adenomas per mouse, while mice treated with 1000ppm and 1500ppm ADT-061 had an average of 2.04 (p=0.102) and 1.91 (p=0.039) adenomas per mouse, respectively. These observations suggest that ADT-061, or analogs thereof, merit further drug development efforts for colorectal cancer chemoprevention. Citation Format: Kevin J. Lee, Xi Chen, Jacob Valiyaveettil, Ashley S. Lindsey, Joel Andrews, Veronica Ramirez-Alcantara, Adam B. Keeton, Gary A. Piazza, Margie L. Clapper, Wen-Chi L. Chang. Novel non-COX inhibitory sulindac derivative with PDE10 inhibitory activity reduces incidence and multiplicity of colorectal adenomas in the APC+/min-FCCC mouse model. [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer: From Initiation to Outcomes; 2016 Sep 17-20; Tampa, FL. Philadelphia (PA): AACR; Cancer Res 2017;77(3 Suppl):Abstract nr A30.

Abstract 5243: Novel non-COX inhibitory sulindac derivative with β-catenin suppressing activity reduces the formation of colorectal adenomas and adenocarcinomas in theAPC+/min-FCCCmouse model

Sulindac can reduce the number and size of precancerous colonic adenomas in patients with familial adenomatous polyposis (FAP), but is not recommended for long-term use as a cancer chemopreventive drug because of potentially fatal toxicities associated with cyclooxygenase (COX) inhibition. Here we characterize the antineoplastic properties of a novel sulindac derivative, ADT-061, that lacks COX-1 and COX-2 inhibitory activity. ADT-061 potently and selectively inhibited colon tumor cell growth in vitro with IC50 values of 0.3-0.5 μM by inducing apoptosis without affecting the growth of normal colonocytes at concentrations 100x higher. By comparison, sulindac sulfide (SS) inhibited colon tumor cell growth with IC50 values of 40-60 μM, and had only modest tumor cell selectivity. While SS non-selectively inhibits multiple cyclic GMP phosphodiesteraes (PDE) isozymes, previous studies have suggested an important role for PDE10 in colon cancer (Li et al, Oncogene, 2014; Lee et al, Oncotarget, 2015). At concentrations comparable to its IC50 values, ADT-061 selectively inhibits PDE10 to induce an increase in intracellular cGMP levels and activate protein kinase G (PKG). ADT-061 also suppressed nuclear levels of β-catenin and induced cell cycle arrest and apoptosis, within the same concentration range that inhibits tumor cell growth. With attractive drug-like properties, the chemopreventive activity ADT-061was assessed using the APC+/min-FCCC mouse model of spontaneous colorectal tumorigenesis. Prior to treatment, colonoscopy exams were performed on all APC+/min-FCCC mice (males, 6-7 weeks of age) to ensure the absence of colorectal adenomas. At 7-8 weeks of age, mice were assigned (19 per group) to receive either unsupplemented chow (control) or chow supplemented with ADT-061 (1500ppm) for 14 weeks. No discernible toxicity was observed during the experimental period. Double-blinded pathological analysis of fixed colon tissues revealed that ADT-061 not only reduced the multiplicity of colonic adenomas by 50% (Mean ± SEM, control vs. ADT-061, 3.95 ± 0.81 vs. 1.95 ± 0.58, P Citation Format: Kevin J. Lee, Xi Chen, Jacob Valiyaveettil, Ashley S. Lindsey, Joel Andrews, Veronica Ramirez-Alcantara, Adam B. Keeton, Gary A. Piazza, Harry Cooper, Margie Clapper, Wen-Chi L. Chang. Novel non-COX inhibitory sulindac derivative with β-catenin suppressing activity reduces the formation of colorectal adenomas and adenocarcinomas in the APC+/min-FCCC mouse model [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 5243. doi:10.1158/1538-7445.AM2017-5243

Abstract 5159: Characterization of a novel class of RAS inhibitory compounds with potent anti-tumor activity

Introduction: Over 30% of all human cancers harbor activating RAS mutations which induce deregulation of the cell cycle, uncontrolled proliferation, and decreased apoptosis. Through a phenotypic screening strategy, we have identified a series of indene derivatives which potently and selectively inhibit growth of tumor cells harboring activated RAS. A development candidate from this series, DC070-547, disrupts RAS-RAF binding, inhibits RAS signaling, causes cell cycle arrest and induces apoptosis, and exhibits strong anti-tumor activity in a mouse KRAS mutant tumor model. Methods: Viable cell number was measured using a luminescent indicator of ATP. RAS activation was measured by GST-RAF1-RBD pull-down and western blotting using an anti-RAS antibody. Disruption of RAS-RAF binding was determined by pre-incubation of GST-RAF1-RBD beads with cell lysates or recombinant RAS in the presence of test compounds for 30 min. Cell cycle distribution was measured by DNA content and immunofluorescent detection of cell cycle proteins. Antitumor activity was determined in a subcutaneous mouse tumor model involving KRAS mutant colon tumors. Results: Low nanomolar concentrations of DC070-547 selectively inhibited growth of a diverse panel of tumor cell lines harboring activated RAS relative to tumor cell lines lacking activated RAS. Transfection of HT-29 cells lacking activated RAS with mutant RAS conferred sensitivity to DC070-547. The compounds blocked binding of RAF1-RBD to recombinant RAS, RAS from cell lysates, as well as RAS in intact cells. Sustained and potent growth inhibitory effects of DC070-547 were demonstrated by colony formation assays. Immunoblotting showed that DC070-547 inhibited EGF-induced signaling in HCT-116 colon tumor cells with activated RAS at concentrations that inhibit growth. DC070-547 also induced apoptosis as evident by Annexin V labeling and M-phase arrest in HCT116 cells as shown by DNA content and immunostaining of phospho-Histone H3B and Cdc25C, an important downstream mediator of RAS signaling. DC070-547 and three analogs from this series were evaluated for antitumor activity in an athymic mouse model using subcutaneously implanted KRAS mutant colon tumor cells. Treatments either completely suppressed tumor growth or caused tumor regression with no discernible toxicity. Conclusion: While RAS is widely considered to be non-druggable, a novel compound series was identified that potently and selectivity inhibit in vitro and in vivo the growth of tumor cells harboring activated RAS by inhibiting RAS-effector binding. Together, these findings support further preclinical development of this compound class for Phase I/II clinical evaluation for the treatment of RAS-driven cancers. Citation Format: Antonio Ward, Xi Chen, Jacob Valiyaveettil, Bing Zhu, Veronica Ramirez-Alcantara, Kevin J. Lee, Ashley Lindsey, Kristy Berry, Tyler E. Mattox, Kate McConnell, Michael R. Boyd, Gary A. Piazza, Adam B. Keeton. Characterization of a novel class of RAS inhibitory compounds with potent anti-tumor activity [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 5159. doi:10.1158/1538-7445.AM2017-5159

Abstract B23: DC070-547, a novel Ras inhibitor potently and selectivity inhibits colon tumor growth in vitro and in vivo

Introduction: Activating mutations in Ras oncogenes play a critical role in the development of colorectal cancer and are an indicator of poor prognosis. Constitutive activation of Ras proteins is also associated with resistance to chemotherapy and radiation in which treatment options are limited. Despite extensive efforts, no drugs have been successfully developed that target these aberrant gene products, in part because of the high affinity of Ras binding to GTP, which is essential for Ras activation. Using a phenotypic screening assay designed to select for Ras inhibitors and follow-up chemical optimization, a novel Ras inhibitor, DC070-547 was identified, which potently and selectively inhibits the growth of colon tumor cells with activated Ras in vitro and in vivo. Methods: Tumor cell growth inhibitory activity of DC070-547 was measured in a panel of human colon tumor cell lines using the CellTiter Glo assay following 72 h of treatment. Ras activation levels were measured by precipitating GTP-bound Ras from human colon tumor cell lysates with GST-Raf1-RBD/GSH Sepharose followed by western blotting using anti-Ras antibody. Isogenic cell lines were established by transfecting Ras wild-type HT-29 colon tumor cells with mutant H-Ras. Disruption of Ras-Raf binding was determined by pre-incubating cell lysates or intact cells in the presence of DC070-547 for 30 or 45 min followed by Ras pull-down with GST-Raf beads and western blotting using anti-Ras or an anti-GST antibody as a loading control. Cell cycle arrest and apoptosis were measured by DNA content and annexin V levels, respectively. Antitumor activity was determined in a mouse xenograft model subcutaneously implanted with mutant K-Ras HCT-116 colon tumor cells. Mice were treated with DC070-547 administered i.p. for 14 days at a dose of 2.5 mg/kg bid in a co-solvent formulation. Results: DC070-547 potently inhibits the growth of mutant K-Ras HCT-116 and other colon tumor cell lines having constitutively activated Ras with IC50 values as low as 2 nM and selectivity indices of approximately 100-fold for HT-29 and other colon tumor cells lacking activated Ras. Among a panel of six colon tumor cell lines, a strong correlation was measured between IC50 values for growth inhibition and the level of Ras activation, suggesting that activated Ras is the primary target. Isogenic cell line pairs involving transfecting Ras wild-type cells with mutant H-Ras confirmed that sensitivity to DC070-547 required activated Ras. DC070-547 also blocked Ras-Raf binding in cell lysates and intact cells at concentrations that inhibit the growth of tumor cells with activated Ras. The mechanism of growth inhibition by DC070-547 appears to involve mitotic arrest and apoptosis. Moreover, cells obtained from normal colon mucosa were essentially refractory to treatment with DC070-547. In a mouse xenograft model involving mutant K-Ras human HCT-116 colon tumors, DC070-547 was well tolerated and caused a sustained inhibition of tumor growth in which there was complete tumor regression in 3 of 7 mice. Conclusions: DC070-547 potently and selectively inhibits the growth of colon tumor cells with constitutively activated Ras by disrupting Ras-effector interactions and represents a first-in-class drug development candidate for the treatment of Ras-driven colorectal cancer. Citation Format: Veronica Ramirez-Alcantara, Adam B. Keeton, Bing Zhu, Kevin J. Lee, Joshua Canzoneri, Ashley S. Lindsey, Luciana Madeira da Silva Barnes, Kristy Berry, Jacob Valiyaveettil, Xi Chen, Michael R. Boyd, Gary Piazza. DC070-547, a novel Ras inhibitor potently and selectivity inhibits colon tumor growth in vitro and in vivo. [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer: From Initiation to Outcomes; 2016 Sep 17-20; Tampa, FL. Philadelphia (PA): AACR; Cancer Res 2017;77(3 Suppl):Abstract nr B23.

Abstract 5165: Novel RAS inhibitors DC070-547 and ADT-006 potently and selectively inhibit the growth of pancreatic tumor cells harboring constitutively activated RAS by blocking RAS-effector binding and signaling

Gain-in-function mutations in RAS genes cause constitutive activation of RAS and induction of downstream signaling pathways that drive tumor cell proliferation, survival, and metastasis. Such driver mutations occur in over 90% of pancreatic ductal adenocarcinomas (PDACs), making RAS an attractive cancer target. However, RAS is considered “undruggable”, given the lack of suitable surfaces on the protein for small molecule binding, as well as its high affinity for GTP binding. A phenotypic assay was developed to screen a library of indene derivatives for growth inhibitory activity in tumor cells harboring constitutively active RAS versus tumor cells with low levels of active RAS. A novel compound series was identified that potently and selectively inhibits the growth of tumor cells with constitutively active RAS, while having minimal effects on tumor cells lacking constitutively active RAS or cells derived from normal tissues. Chemical optimization resulted in a drug development candidate, DC070-547, and several back-up analogs (e.g. ADT-006), which showed strong antitumor activity at doses that do not cause any discernible toxicity in preclinical mouse models. Here we report DC070-547 and ADT-006 inhibited the growth of PDAC cells with IC50 values of approximately 2 nM and 20 nM, respectively, in MIA PaCa-2 cells harboring constitutively active RAS and greater than 350-fold selectivity over BxPC-3 cells, which lack constitutively active RAS. Both compounds also decreased the clonogenic potential of MIA PaCa-2 cells at concentrations that inhibit their growth. Within the same low nanomolar concentration range, treatment of recombinant human K-RAS, MIA PaCa-2 cell lysates, and intact MIA PaCa-2 cells blocked RAS-effector interactions as evident by performing active RAS pull-down assays using GST-RAF1-RBD/glutathione agarose. Treatment of intact MIA PaCa-2 cells also inhibited RAF/MAPK and PI3K/AKT phosphorylation within the same concentration range. These results demonstrate that DC070-547 and ADT-006 inhibit PDAC cell growth by blocking RAS-effector interactions, supporting further evaluation of this novel class of RAS inhibitors for the treatment of pancreatic cancer, as well as other RAS-driven cancers. Citation Format: Tyler E. Mattox, Kevin J. Lee, Xi Chen, Jacob Valiyaveettil, Luciana Madeira da Silva, Ashley S. Lindsey, Adam B. Keeton, Bing Zhu, Michael Boyd, Gary A. Piazza. Novel RAS inhibitors DC070-547 and ADT-006 potently and selectively inhibit the growth of pancreatic tumor cells harboring constitutively activated RAS by blocking RAS-effector binding and signaling [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 5165. doi:10.1158/1538-7445.AM2017-5165

Abstract 1140: Characterization of a novel PDE10 inhibitor in lung tumor cells and an orthotopic mouse model of lung cancer

BACKGROUND: Screening a focused library of indene derivatives for PDE10 inhibitory activity identified novel leads with potent and selective tumor cell growth inhibitory activity. ADT-030 emerged from lead optimization chemistry with excellent drug-like properties and oral bioavailability. Here we characterize the anti-tumor activity of ADT-030 in human lung tumor cells and an orthotopic mouse model of lung cancer. METHODS: Growth inhibitory activity of ADT-030 was measured in a panel of human lung tumor cell lines by ATP quantification following 72 h of treatment. The effect of ADT-030 on intracellular cGMP/cAMP was measured in whole cell lysates using a competitive ELISA assay. PDE inhibitory activity of ADT-030 was evaluated in lysates of human lung tumor cells and by recombinant PDE isozymes using the IMAP fluorescence polarization PDE assay. Activation of PKG signaling and suppression of β-catenin levels in response to ADT-030 treatment was evaluated by Western blot using whole cell lysates of human lung tumor cells. ADT-030 was orally administrated to C57BL/6 mice and free levels quantified in plasma and tissues by LC-MS. Anti-tumor activity of ADT-030 was evaluated in athymic nude-Foxn1nu mice after inoculating the left lung with 1x106 A549 lung tumor cells and treating once daily by oral administration at dosages ranging from 25 - 125 mg/kg. Tumor growth was monitored by in situ bioluminescence using IVIS as well as necropsy and pathological grading after 4 weeks of treatment. RESULTS: ADT-030 inhibited the growth of human lung tumor cell lines with IC50 values in the low micromolar range by inducing apoptosis, while appreciably higher concentrations were required to affect the growth of normal human airway epithelial cells. ADT-030 treatment of human lung tumor cells increased both intracellular cGMP and cAMP levels, activated PKG and suppressed β-catenin within the same concentration range as required for tumor cell growth inhibition. Pharmacokinetic studies in mice demonstrated a half-life suitable for once a day dosing. Tissue distribution studies revealed appreciably higher concentrations of ADT-030 in lungs relative to plasma and other tissues, with the highest accumulation measured in the parenchyma. ADT-030 was well tolerated in mice implanted with A549 tumor cells and displayed strong anti-tumor activity as evident by reduced luminescence, tumor grading, and double-blinded pathological evaluation. CONCLUSIONS: ADT-030 represents a prospective drug development candidate with favorable drug-like properties that concentrates in lung after oral administration exhibiting a strong anti-tumor activity in a pre-clinical mouse model. The mechanism of lung tumor cell growth inhibition involves PDE10 inhibition, elevation of cGMP, activation of PKG, and attenuation of β-catenin. Citation Format: Veronica Ramirez-Alcantara, Bing Zhu, Xi Chen, Rajkumar Savai, Prema Subbarayal, Michele A. Schuler, Kevin J. Lee, Ashley S. Lindsey, Kristy L. Berry, Dennis Otali, Joshua Canzoneri, Jacob Valiyaveettil, Adam Keeton, Lori Coward, Gregory Gorman, William Grizzle, Michael Boyd, Gary A. Piazza. Characterization of a novel PDE10 inhibitor in lung tumor cells and an orthotopic mouse model of lung cancer [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 1140. doi:10.1158/1538-7445.AM2017-1140

Abstract 4972: Novel Ras inhibitor DC070-547 potently and selectively blocks Ras-RBD binding, EGFR binding to Ras signaling complex, EGFR activation of Ras signaling, and growth of Ras-driven lung tumor cells

A novel series of compounds that potently and selectively inhibits the growth of tumor cells harboring constitutively activated Ras relative to cells lacking activated Ras were identified by screening a proprietary library of indene derivatives in a phenotypic, cell-based assay. Lead-optimization produced a drug development candidate, DC070-547, which showed strong antitumor activity at doses not causing any discernible toxicity in preclinical mouse models. Here we characterize the underlying mechanism of growth inhibition in lung tumor cells. A panel of non-small cell lung cancer lines with constitutively activated Ras were highly sensitive to DC070-547 with IC50 values as low as 2 nM, while normal airway epithelial cells were essentially insensitive. Transfection of wild-type ras H322 bronchioalveolar tumor cells with mutant ras (G12V) confirmed that activated Ras is required for the selective growth inhibitory activity of DC070-547. Ras-RBD binding assays showed that DC070-547 disrupts Ras-RBD binding at low nanomolar concentrations that parallel those required to inhibit the growth of lung tumor cells with activated Ras. Similar concentrations of DC070-547 were found to inhibit the binding of phosphorylated EGFR (Y1068) to Ras immunoprecipitates in mutant ras transfected H322 cells, but not in control H322 cells. DC070-547 also inhibited the binding of SOS, Grb2, Gab1, S338 phosphorylated c-Raf (pc-Raf), S473 phosphorylated Akt-1 (pAkt-1) and T202/Y204 phosphorylated Erk1/2 (pERK1/2) to Ras or EGFR immunoprecipitates. To determine if DC070-547 can inhibit EGF-stimulated Ras signaling, serum-starved mutant ras transfected H322 cells were treated with EGF and probed for effects on Ras signaling components. DC070-547 caused a concentration-dependent inhibition of EGF-induced Y1068-EGFR as measured in Ras immunoprecipitates, and also reduced pc-Raf, pAkt-1, pErk1/2 and pGab1 (Y627) levels in Ras or EGFR immunoprecipitates. In addition, DC070-547 caused a concentration-dependent decrease in Erk1/2 and Akt-1-mediated phosphorylation of Bad proteins (S112, S136 and S155) to induce apoptosis. These results show that DC070-547 prevents Ras-RBD binding to block EGF-induced Raf/MAPK and Akt signaling to potently and selectively inhibit the growth of lung tumor cells harboring constitutively activated Ras. Support provided by NCI grants 1R01CA197147 and 1R21CA182941. Citation Format: Bing Zhu, Xi Chen, Jacob Valiyaveettil, Joshua Canzoneri, Kevin Lee, Kate Saville, Kristy Berry, Luciana Barnes, Tyler Maddox, Ashley Lindsey, Antonio Ward, Veronica Ramirez-Alcantara, Adam Keeton, Michael Boyd, Gary Piazza. Novel Ras inhibitor DC070-547 potently and selectively blocks Ras-RBD binding, EGFR binding to Ras signaling complex, EGFR activation of Ras signaling, and growth of Ras-driven lung tumor cells [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 4972. doi:10.1158/1538-7445.AM2017-4972

Abstract 5166: Sensitivity of melanoma cells to a novel class of Ras inhibitors

A high percentage of human cancers arise from mutations in the ras gene that encodes an abnormal Ras protein locked in a constitutively active GTP-bound state that promotes tumor cell proliferation, survival, and metastasis. These gain-in-function mutations in the ras gene or constitutive activation of tyrosine kinase receptors upstream of the Ras protein drive tumor cell growth by activating Raf/MAPK and PI3K/AKT signaling pathways. Ras has been an elusive drug target for which no inhibitors are available to treat Ras-driven cancers. Screening a library of indene derivatives in a differential phenotypic assay identified a novel compound class displaying high potency and selectivity to inhibit the growth of tumor cells harboring mutant Ras relative to tumor cells with wild type (WT) Ras. Lead optimization resulted in a drug development candidate (DC070-547) and several back-up analogs (e.g. ADT-006) with IC50 values in the low nanomolar range and selectivity indices of 100 fold or greater to inhibit the growth of tumor cells with constitutively activated Ras relative to tumor cells with low levels of activated Ras. Sensitivity among a large panel of tumor cell lines to this compound class strongly correlated with levels of activated Ras, but did not appear to be limited to a specific ras gene mutation or Ras protein isoform. Here, we report that DC070-547 and ADT-006 potently and selectively inhibit the growth of the human melanoma cell line, SK-MEL-2, harboring a mutation in the ras gene that encodes the constitutively active N-Ras protein with IC50 values of 7 and 25 nM, respectively. In addition, both compounds potently inhibited the growth of the murine melanoma cell line, B16-F10 with WT ras, but harboring a mutation in a tyrosine kinase receptor upstream of Ras, specifically PDGFRα, which results in high levels of active, GTP-bound Ras as confirmed by Ras-RBD pull-down assays. To determine if treatment can disrupt Ras signaling, B16-F10 cells were incubated with the compounds before being subjected to Western blotting for phosphorylated signaling molecules downstream of Ras. Treatment reduced levels of phosphorylated c-Raf and MEK at concentrations that inhibit tumor cell growth. These findings support further investigation of this novel class of Ras inhibitors for the treatment of Ras-driven melanoma. Citation Format: Kate M. Saville, Kevin Lee, Tyler E. Mattox, Xi Chen, Jacob Valiyaveettil, Kristy Berry, Veronica Ramirez-Alcantara, Bing Zhu, Adam Keeton, Michael Boyd, Gary Piazza, Ashley S. Lindsey. Sensitivity of melanoma cells to a novel class of Ras inhibitors [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 5166. doi:10.1158/1538-7445.AM2017-5166

Abstract 1238: A novel Ras inhibitor potently and selectively suppresses lung tumor cell growth by blocking Ras-Raf binding

Mutations in ras genes that result in constitutive activation of Ras proteins are key drivers of oncogenesis, but no effective drugs have been developed that target these aberrant gene products. Screening using a phenotypic assay designed to select for Ras inhibitors and iterative chemical synthesis, identified a preclinical drug development candidate with attractive drug-like properties designated as DC070-547. Here we evaluated the sensitivity of non-small cell lung cancer (NSCLC) cell lines to DC070-547. The compound potently suppresses the growth of human A549, H460, HOP62, H1299, H1975 lung tumor cells having high levels of activated Ras with low nanomolar IC50 values. By contrast, normal human airway epithelial cells (NHAECs) and H322 lung tumor cells with low levels of activated Ras displayed IC50 values in the mid micromolar range. The Ras selectivity of DC070-547 was confirmed by ectopic expression of mutant H-Ras-G12V (H-Rasm) in H322 cells using a retroviral construct, in which the IC50 value of DC070-547 for growth inhibition was reduced approximately 2,500 fold compared with vector control cells. To study the mechanism of action, Ras activation status was measured by Ras-Raf pull-down assays using GTP-bound Ras from cell lysates with GST-Raf1-RBD/GSH sepharose and followed by western blotting with Ras antibodies. Direct binding to Ras was apparent by the ability of DC070-547 to inhibit Ras-Raf binding at concentrations that inhibit Ras-dependent tumor cell growth. Pull-down assays also showed a dose-dependent inhibition of Ras-GTP levels in intact H-Rasm-H322 cells treated with DC070-547 at the same concentration range. Western blot analysis of Ras-immunoprecipitated proteins revealed that DC070-547 also attenuated high levels of EGFR phosphorylation at Y1068 in H-Rasm-H322 cells but not in control cells. These results demonstrate novel Ras inhibitory activities of DC070-547 in NSCLC cell lines. DC070-547 and its pipeline analogs are being evaluated for antitumor efficacy in preclinical mouse models of lung cancers. Citation Format: Bing Zhu, Xi Chen, Veronica Ramirez-Alcantara, Kevin Lee, Jacob Valiyaveettil, Joshua Canzoneri, Sara Sigler, Kristy Berry, Ashley Lindsey, Adam Keeton, Michael R. Boyd, Gary A. Piazza. A novel Ras inhibitor potently and selectively suppresses lung tumor cell growth by blocking Ras-Raf binding. [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 1238.