Bing Zhu

Sulindac selectively inhibits colon tumor cell growth by activating the cGMP/PKG pathway to suppress Wnt/β-catenin signaling

Nonsteroidal anti-inflammatory drugs (NSAID) display promising antineoplastic activity for colorectal and other cancers, but toxicity from COX inhibition limits their long-term use for chemoprevention. Previous studies have concluded that the basis for their tumor cell growth inhibitory activity does not require COX inhibition, although the underlying mechanism is poorly understood. Here, we report that the NSAID sulindac sulfide inhibits cyclic guanosine 3′,5′-monophosphate phosphodiesterase (cGMP PDE) activity to increase intracellular cGMP levels and activate cGMP-dependent protein kinase (PKG) at concentrations that inhibit proliferation and induce apoptosis of colon tumor cells. Sulindac sulfide did not activate the cGMP/PKG pathway, nor affect proliferation or apoptosis in normal colonocytes. Knockdown of the cGMP-specific PDE5 isozyme by siRNA and PDE5-specific inhibitors tadalafil and sildenafil also selectively inhibited the growth of colon tumor cells that expressed high levels of PDE5 compared with colonocytes. The mechanism by which sulindac sulfide and the cGMP/PKG pathway inhibits colon tumor cell growth involves the transcriptional suppression of β-catenin to inhibit Wnt/β-catenin T-cell factor transcriptional activity, leading to downregulation of cyclin D1 and survivin. These observations suggest that safer and more efficacious sulindac derivatives can be developed for colorectal cancer chemoprevention by targeting PDE5 and possibly other cGMP-degrading isozymes. Mol Cancer Ther; 12(9); 1848–59. ©2013 AACR.

Phosphodiesterase 10A: a novel target for selective inhibition of colon tumor cell growth and β-catenin-dependent TCF transcriptional activity

The cyclic nucleotide phosphodiesterase 10A (PDE10) has been mostly studied as a therapeutic target for certain psychiatric and neurological conditions, although a potential role in tumorigenesis has not been reported. Here we show that PDE10 is elevated in human colon tumor cell lines compared with normal colonocytes, as well as in colon tumors from human clinical specimens and intestinal tumors from ApcMin/+ mice compared with normal intestinal mucosa, respectively. An isozyme and tumor-selective role of PDE10 were evident by the ability of small-molecule inhibitors and small interfering RNA knockdown to suppress colon tumor cell growth with reduced sensitivity of normal colonocytes. Stable knockdown of PDE10 by short hairpin RNA also inhibits colony formation and increases doubling time of colon tumor cells. PDE10 inhibition selectively activates cGMP/cGMP-dependent protein kinase signaling to suppress β-catenin levels and T-cell factor (TCF) transcriptional activity in colon tumor cells. Conversely, ectopic expression of PDE10 in normal and precancerous colonocytes increases proliferation and activates TCF transcriptional activity. These observations suggest a novel role of PDE10 in colon tumorigenesis and that inhibitors may be useful for the treatment or prevention of colorectal cancer.

New NSAID targets and derivatives for colorectal cancer chemoprevention.

Clinical and preclinical studies provide strong evidence that nonsteroidal anti-inflammatory drugs (NSAIDs) can prevent numerous types of cancers, especially colorectal cancer. Unfortunately, the depletion of physiologically important prostaglandins due to cyclooxygenase (COX) inhibition results in potentially fatal toxicities that preclude the long-term use of NSAIDs for cancer chemoprevention. While studies have shown an involvement of COX-2 in colorectal tumorigenesis, other studies suggest that a COX-independent target may be at least partially responsible for the antineoplastic activity of NSAIDs. For example, certain NSAID derivatives have been identified that do not inhibit COX-2 but have demonstrated efficacy to suppress carcinogenesis with potential for reduced toxicity. A number of alternative targets have also been reported to account for the tumor cell growth inhibitory activity of NSAIDs, including the inhibition of cyclic guanosine monophosphate phosphodiesterases (cGMP PDEs), generation of reactive oxygen species (ROS), the suppression of the apoptosis inhibitor protein, survivin, and others. Here, we review several promising mechanisms that are being targeted to develop safer and more efficacious NSAID derivatives for colon cancer chemoprevention.

Suppression of β-catenin/TCF transcriptional activity and colon tumor cell growth by dual inhibition of PDE5 and 10

Previous studies suggest the anti-inflammatory drug, sulindac inhibits tumorigenesis by a COX independent mechanism involving cGMP PDE inhibition. Here we report that the cGMP PDE isozymes, PDE5 and 10, are elevated in colon tumor cells compared with normal colonocytes, and that inhibitors and siRNAs can selectively suppress colon tumor cell growth. Combined treatment with inhibitors or dual knockdown suppresses tumor cell growth to a greater extent than inhibition from either isozyme alone. A novel sulindac derivative, ADT-094 was designed to lack COX-1/-2 inhibitory activity but have improved potency to inhibit PDE5 and 10. ADT-094 displayed >500 fold higher potency to inhibit colon tumor cell growth compared with sulindac by activating cGMP/PKG signaling to suppress proliferation and induce apoptosis. Combined inhibition of PDE5 and 10 by treatment with ADT-094, PDE isozyme-selective inhibitors, or by siRNA knockdown also suppresses β-catenin, TCF transcriptional activity, and the levels of downstream targets, cyclin D1 and survivin. These results suggest that dual inhibition of PDE5 and 10 represents novel strategy for developing potent and selective anticancer drugs.

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 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