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Here we describe a novel class of pan-RAS inhibitor with highly potent and selective anticancer activity by killing cancer cells harboring mutations in RAS or with constitutively activated RAS resulting from mutations in upstream signaling components. A lead compound from this chemical family, ADT-007, binds RAS when in a nucleotide free transitional state to block loading of GTP, thereby interfering with RAS activation and disruption of binding to effectors such as RAF and PI3K to suppress MAPK and AKT signaling. ADT-007 potently inhibits the growth of cultured human and murine cancer cell lines with single-digit nM IC50 values irrespective of specific RAS isozyme or mutational codon. ADT-007 also inhibits tumor growthin vivothrough inhibition of RAS-MAPK signaling in syngeneic, immune competent and xenogeneic, immune deficient mouse models of colon and pancreatic cancer. In RAG 1-/-mice the activity of ADT-007 is partially inhibited indicating a role for the adaptive immune system in ADT-007-mediated tumor growth inhibition.Ex vivoanalyses of tumor infiltrating leukocytes, reveals that ADT-007 enhances T cell functions in the pancreatic and colorectal tumor immune microenvironment.SIGNIFICANCEADT-007 represents a 1st-in-class pan-RAS inhibitor with broad anticancer activity across all RAS driven cancers and unique chemical selectivity to allow normal cells to be spared from pan-RAS inhibition. ADT-007 also has the potential to modulate the adaptive immune response in colorectal cancer (CRC) and pancreatic ductal adenocarcinoma (PDA). These data support future clinical trials of an orally bioavailable prodrug of ADT-007 as a monotherapy for the treatment of patients with CRC or PDAC regardless of the underlying mutation or in combination with immunotherapy.

Supplemental Figure Legends

HPLC chromatogram of ADT 061

HPLC chromatogram of compound 4.

AST and ALT activity of male APC+/Min-FCCC mice administered varying doses of ADT 061 in the diet. Enzyme activity was measured in plasma samples. Results are presented as mean {plus minus} SEM.

Reaction steps for synthesis of ADT 061 (a) 3-(4-methoxyphenyl)-2-methylacrylic acid (compound 1): 4-Methoxybenzaldehyde (219 g, 1.61 mole), propionic anhydride (315g, 2.42 mole), and sodium propionate (155g, 1.61 mole) were stirred at 140 ºC until a clear solution was achieved (~48 h). The solution was cooled to room temperature and poured into 8L of ice water. The precipitation formed was collected by filtration, transferred into a 2L round-bottom flask, and refluxed in 1.5 L of ethanol for 3h. The flask was stored at -20 ºC overnight. Compound 1 was obtained as a colorless crystal (213g) after filtration. Compound 1 is also commercially available from AstraTech (# W18287,95% purity). (b) 3-(4-Methoxyphenyl)-2-methylpropanoic acid (compound 2): p-Methoxy-α-methylcinnamic acid (213g) and palladium on active charcoal (Pd-C, 10%, 2g) were suspended in 1.5 L of 95% ethanol and warmed to 60 ºC in a water bath. The warm suspension was immediately put on a catalytic hydrogenator, treated with hydrogen (40 psi). The reaction was completed within 45 min, as indicated by the complete dissolving of the solid starting material. The catalyst was removed by filtration, and the filtrate was concentrated to give compound 2 as a colorless oil (215g), which was used for the next reaction step without further purification and characterization. Compound 2 is also commercially available from PharmaBlocks (# PBTQ6955, 97% purity). (c) 6-Methoxy-2-methyl-2,3-dihydro-1H-inden-1-one (compound 3): Phosphoryl acid (98%, 500g) and polyphosphoryl acid (PPA, 450g) were pre-warmed separately to 70{degree sign}C in a water bath before being transferred to a 2L, three-necked flask equipped with a thermometer, a mechanical stirrer, and a dropping funnel. The flask was kept in an oil bath at 55-60 {degree sign}C for 1 h until smooth stirring was achieved. Compound 2 (95 g) was added dropwise over a period of 5 min. The temperature was carefully raised to 70-75{degree sign}C for 15 min. The reaction solution was immediately transferred to 6L of ice water and stirred until the PPA was completely dissolved. The mixture was extracted with ethyl ether (1L Ã- 3). The organic layer was dried with sodium sulfate and concentrated. The residue was purified by a silica gel column and eluted with hexane and acetone. Purity was monitored by TLC. Compound 3 was obtained as a clear, colorless oil (76g) that was used for the next reaction step without further purification and characterization. Compound 3 is also commercially available from AstraTech (# 96721, 95% purity). (d) 2-(5-Methoxy-2-methyl-1H-inden-3-yl) acetic acid (compound 4): A mixture of compound 3 (76g, 0.39 mole), cyanoacetic acid (36.6g, 0.43 mole), acetic acid (40 mL) and ammonium acetate (9 g) in 500 mL of toluene was refluxed with stirring for 48h. The liberated water was collected by a Dean-Stark trap. The reaction mixture was cooled and filtered, and the filtrate was concentrated. The residue was dissolved in 250 mL of ethanol. A solution of potassium hydroxide (90 g) in 360 mL of water was added to the solution. The mixture was refluxed overnight under argon. The organic solvents were removed under vacuum, and the remaining aqueous solution was diluted with 500 mL of water, extracted with ethyl ether, boiled with active charcoal for 1h, then filtered. The filtrate was acidified with 6N HCl and sonicated for 2h. The precipitate was collected by filtration, washed with water, refluxed in 180 mL of acetone for 2h, and stored at -20 ºC overnight. The precipitate was collected by filtration and dried under vacuum to give compound 4 as a colorless solid (42g). The mother liquor was concentrated, and a second crop (3.5g) was obtained after repeating the recrystallization procedure. The structure was confirmed by mass spectrometry (M+H: 219.04) and 1H-NMR (DMSO-d6) Î'(ppm): 7.240(d, 1H); 6.804(d, 1H); 6.658(m, 1H); 3.781 (s, 3H); 3.741(s, 2H); 3.250(s, 2H); 2.050(s, 3H). Purity was determined by HPLC (98.7%) (Suppl. Fig. 2a-c). (e) (Z)-2-(5-methoxy-2-methyl-1-(3,4,5-trimethoxybenzylidene)-1H-inden-3-yl)acetic acid (compound 5): Compound 4 (32.7g, 0.150 mole), 3, 4, 5-trimethoxybenzaldehyde (35.3g, 0.180 mole) and sodium methoxide (21g) in 300 mL of anhydrous methanol were refluxed in a 500 mL round-bottomed flask overnight. After cooling, the reaction mixture was diluted with 150 mL of acetone and sonicated for 30 min. The precipitate was collected by filtration and washed twice with acetone, then dissolved in 150 mL of water. The aqueous solution was acidified with 6N HCl and sonicated for 30 min. The precipitate was collected by filtration, washed with water, recrystallized from methanol, and dried under vacuum to afford compound 5 as a yellow crystal (51.7g). The structure was confirmed by mass spectrometry (M+H: 397.11) and 1H-NMR (DMSO-d6) Î'(ppm): 12.377 (s, 1H); 7.429 (d, 1H); 7.139 (s, 1H); 6.877(s, 2H); 6.669 (d, 1H); 6.546(dd, 1H); 3.780 (s, 3H); 3.780(s, 3H); 3.737(s, 6H); 3.545(s, 2H); 21.121(s, 3H). Purity was determined by HPLC (99.64%) (Suppl. Fig. 3a-c). (f) (Z)-2-(5-methoxy-2-methyl-1-(3,4,5-trimethoxybenzylidene)-1H-inden-3-yl)-N-(pyridin-3-yl) acetamide (ADT 061): To a 500 mL round-bottomed flask containing compound 5 (39.6g, 0.100 mole) and 300 mL of anhydrous dichloromethane, 1,1'-carbonyldiimidazole (CDI, 20.0g, 0.115 mole) was added in portions and stirred for 15 min at room temperature. 3-Amino-pyridine (10.6g, 0.115 mole) was added, followed by anhydrous pyridine (80 mL). The solution was stirred at 40{degree sign}C overnight, treated with 5g of sodium hydroxide in 20 mL of water for 10 min, then diluted with 300 mL of dichloromethane, and washed with water (250 mL x 3). The solution was concentrated, and the residue was purified with a silica gel column. Recrystallization from methanol and then from ethyl acetate afforded ADT 061 as a yellow crystal (32.7g). The structure of ADT 061 was confirmed by mass spectrometry (M+H: 473.2) and 1H-NMR (DMSO-d6) Î'(ppm): 10.453(sb, 1H); 8.765(d, 1H); 8.276(dd, 1H); 8.044(m, 1H); 7.431(d, 1H); 7.351(dd, 1H); 7.142(s,1H); 6.914(s, 1H); 6.880(s, 2H); 6.542(m, 1H); 3.781 (s, 6H); 3.731(s, 3H); 3.726(s, 3H); 3.696(s, 2H); 2.194(s, 3H). Purity was determined by HPLC (>99.7%) (Suppl. Fig. 4a-c).

Mass spectroscopy of compound 5.

ADT 061 pharmacokinetics and tissue distribution. ADT 061 was detected in plasma at a concentration of 2.8 µM 0.5 hr after a single oral administration of a dose of 100 mg/kg in 0.5% CMC/0.25% T80 in water to female C57BL/6 mice and reached plasma Cmax of 4.9 µM 4 hr after administration, which is >10 times higher than human colon cancer cell growth inhibition IC50 values. ADT 061 reached a Cmax in colon mucosa within 2 hr after oral administration (62.6 nmol/g) and was still present at concentrations exceeding IC50 values 8 hr after administration (28.9 nmol/g). ADT 061 concentrations in lungs, ovaries, and uterus (1-2 nmol/g) were appreciably lower than colon mucosa levels, while the ADT 061 concentration in the brain was nearly undetectable 2 and 8 hr post treatment (0.1 and 0.2 nmol/g, respectively) (n=4, mean {plus minus} SD).

Eadie-Hofstee plot of data from Fig. 2C. Error bars = SEM

Previous studies have reported that phosphodiesterase 10A (PDE10) is overexpressed in colon epithelium during early stages of colon tumorigenesis and essential for colon cancer cell growth. Here we describe a novel non-COX inhibitory derivative of the anti-inflammatory drug, sulindac, with selective PDE10 inhibitory activity, ADT 061. ADT 061 potently inhibited the growth of colon cancer cells expressing high levels of PDE10, but not normal colonocytes that do not express PDE10. The concentration range by which ADT 061 inhibited colon cancer cell growth was identical to concentrations that inhibit recombinant PDE10. ADT 061 inhibited PDE10 by a competitive mechanism and did not affect the activity of other PDE isozymes at concentrations that inhibit colon cancer cell growth. Treatment of colon cancer cells with ADT 061 activated cGMP/PKG signaling, induced phosphorylation of oncogenic β-catenin, inhibited Wnt-induced nuclear translocation of β-catenin, and suppressed TCF/LEF transcription at concentrations that inhibit cancer cell growth. Oral administration of ADT 061 resulted in high concentrations in the colon mucosa and significantly suppressed the formation of colon adenomas in the Apc+/min-FCCC mouse model of colorectal cancer without discernable toxicity. These results support the development of ADT 061 for the treatment or prevention of adenomas in individuals at risk of developing colorectal cancer.Prevention Relevance:PDE10 is overexpressed in colon tumors whereby inhibition activates cGMP/PKG signaling and suppresses Wnt/β-catenin transcription to selectively induce apoptosis of colon cancer cells. ADT 061 is a novel PDE10 inhibitor that shows promising cancer chemopreventive activity and tolerance in a mouse model of colon cancer.

Previous studies have reported that phosphodiesterase 10A (PDE10) is overexpressed in colon epithelium during early stages of colon tumorigenesis and essential for colon cancer cell growth. Here we describe a novel non-COX inhibitory derivative of the anti-inflammatory drug, sulindac, with selective PDE10 inhibitory activity, ADT 061. ADT 061 potently inhibited the growth of colon cancer cells expressing high levels of PDE10, but not normal colonocytes that do not express PDE10. The concentration range by which ADT 061 inhibited colon cancer cell growth was identical to concentrations that inhibit recombinant PDE10. ADT 061 inhibited PDE10 by a competitive mechanism and did not affect the activity of other PDE isozymes at concentrations that inhibit colon cancer cell growth. Treatment of colon cancer cells with ADT 061 activated cGMP/PKG signaling, induced phosphorylation of oncogenic β-catenin, inhibited Wnt-induced nuclear translocation of β-catenin, and suppressed TCF/LEF transcription at concentrations that inhibit cancer cell growth. Oral administration of ADT 061 resulted in high concentrations in the colon mucosa and significantly suppressed the formation of colon adenomas in the Apc+/min-FCCC mouse model of colorectal cancer without discernable toxicity. These results support the development of ADT 061 for the treatment or prevention of adenomas in individuals at risk of developing colorectal cancer. Prevention Relevance: PDE10 is overexpressed in colon tumors whereby inhibition activates cGMP/PKG signaling and suppresses Wnt/β-catenin transcription to selectively induce apoptosis of colon cancer cells. ADT 061 is a novel PDE10 inhibitor that shows promising cancer chemopreventive activity and tolerance in a mouse model of colon cancer.

Pancreatic ductal adenocarcinoma (PDA) is the 4th leading cause of cancer death in the U.S. with only about a 10% five-year survival rate and an estimated 60,000 deaths/year by 2030. Poor survival is frequently due to advanced disease at the time of diagnosis, as well as the high prevalence of KRAS driver mutations. Currently, approved KRAS-targeted therapeutics are designed to covalently inactivate only KRAS-G12C mutants, whereas the most common mutations in PDA are KRAS-G12D, KRAS-G12V, and KRAS-G12R. Thus, the need for novel RAS-targeted therapeutics with efficacy in tumors with these more common mutations is of utmost urgency to reduce PDA disease burden. We have synthesized a novel chemical class of pan-RAS inhibitors with unique biological properties and attractive drug-like properties. As a prodrug, ADT-1004, generates an active metabolite, ADT-007, that potently inhibits the growth of human and murine PDA cell lines in vitro with single-digit nM IC50 irrespective of specific RAS mutational codon or isozyme by inhibiting activated RAS and signaling downstream of KRAS (ERK1/2 and AKT phosphorylation). ADT-1004 is well tolerated with no discernable toxicity at oral dosages of up to 175 mg/kg bid. Pharmacokinetic studies demonstrated that ADT-1004 produces sustained plasma concentrations of ADT-007 ~50-fold above IC50 values, with even higher concentrations in both subcutaneous and orthotopic pancreatic tumors. Once daily oral administration of ADT-1004 significantly inhibited the growth of orthotopically implanted PDA tumors with a corresponding reduction in RAS/MAPK signaling. ADT-1004 represents a 1st-in-class pan-RAS inhibitor with potential advantages over mutant-specific KRAS inhibitors for greater efficacy and ability to avert mechanisms of resistance. These data support future clinical trials of ADT-1004 as a monotherapy for the treatment of patients with PDA regardless of the underlying mutation. Citation Format: Yulia Y. Maxuitenko, Jeremy B. Foote, Adam B. Keeton, Xi Chen, Kristy Berry, Khalda Fadlalla, Jacob Valiyaveettil, Chung-Hui Huang, Austin M. Moore, Emily C. Graff, Ganji P. Nagaraju, Julienne L. Carstens, Bassel F. El-Rayes, Donald J. Buchsbaum, Gary A. Piazza. Antitumor activity of 1st-in-class pan-RAS inhibitor ADT-1004 in mouse models of pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB322.

RAS is a critically important oncogenic protein that is mutated in approximately 1/3 of cancers resulting in aberrant activation of downstream signaling, which drives malignant transformation. Current molecular targeted therapeutics, and several in development, inhibit only specific mutant alleles (G12C, G12D). In addition, compounds which directly inhibit RAS via proteolytic degradation or inhibit RAS activation by SOS1 are in preclinical development and are referred to as Pan-KRAS inhibitors. Previously investigators have reported that the NSAID, sulindac, can selectively inhibit RAS mutant tumorigenesis by a cyclooxygenase (COX)-independent mechanism. Sulindac, and more potent analogs have also previously been reported to inhibit RAS-mediated transformation and directly bind RAS. Here we describe an ultra-potent non-COX inhibitory derivative of sulindac, ADT-007, which binds to and inhibits RAS nucleotide binding and RAS-effector association. ADT-007 binding to KRAS was evaluated by Micro-Tag cell target engagement and by Cellular Thermal Shift Assay (CETSA) which demonstrated a potency of target engagement (EC50) value in the subnanomolar range. Consistent with molecular docking studies, HSQC NMR spectroscopy using recombinant KRAS revealed that ADT-007 interacted with KRAS after Mg2+ chelation to obtain a nucleotide free (NF) state, resulting in chemical shift changes and signal attenuation of residues in the P-loop and nucleotide binding domain. Similarly, biochemical assays confirmed that ADT-007 prevented MANT-GTP binding to recombinant NF KRAS but did not compete with bound GTP. Functional assays also showed that KRAS binding to RAF-RBD(GST) was inhibited by ADT-007. The compound inhibited constitutive RAS activation (RAF-RBD pulldown) in serum starved MiaPaCa-2 pancreatic cancer cells harboring a KRAS-G12C mutation and demonstrated Pan-RAS inhibition in serum- or EGF-stimulated cells. Further, ADT-007 inhibited AKT phosphorylation and EGF-stimulated downstream ERK1/2 phosphorylation. Finally, ADT-007 demonstrated RAS-selective growth inhibition in isogenic pancreatic and colorectal cancer cell pairs (BxPC-3, HT29). Together, these experiments support further development of ADT-007 and related analogs for treatment of RAS-driven cancers. Citation Format: Adam B. Keeton, Xi Chen, Jacob Valiyaveettil, Chung-Hui Huang, Tyler E. Mattox, Khalda Fadlalla, Jeremy B. Foote, Donald J. Buchsbaum, Kristy L. Berry, Elmar Nurmemmedov, Ivan Babic, Vadim Gaponenko, Gregory Gorman, Lori Coward, Yulia Y. Maxuitenko, Forrest T. Smith, Gary A. Piazza. ADT-007 binds RAS and inhibits RAS signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1658.

Background: Pancreatic ductal adenocarcinoma (PDAC) has low (90% of human PDAC tumors, provides an opportunity to inhibit a critical pathway and significantly impact standard of care in patients diagnosed with PDAC. Furthermore, evidence indicates that inhibiting-oncogenic KRAS signaling modulates anti-tumor immune responses and demonstrates synergy with immune checkpoint blockade in pre-clinical models. The purpose of this study is to evaluate efficacy and impact on anti-tumor immunity of a novel inhibitor, ADT-007 that demonstrates pan-RAS inhibitory activity. Methods: Female C57BL/6J, C57BL/6J. Ifng/Thy1.1 KI;Cg-Tg-IL2tm1/eGFP/Weav (IFNg-Thy1.1/IL-2-GFP reporter mice), or immune deficient (T/B cells) Rag 1 −/− mice (n=5-10/group) were implanted SQ with 106 mouse derived PDAC cell lines (7160c2 and 2838c3) bearing KRASG12D mutation, and then randomized (day 7), into vehicle (5% DMSO/5% Kolliphor/90% H2O) or ADT-007 treatment, which was administered SQ (peritumoral), BID at 5 mg/kg for 2 - 3 weeks. Tumor growth in vitro (IC50), KRAS-MAPK/AKT signaling, in vivo tumor volumes (tv = L x W2/2), and tumor immune responses were assessed by CellTiter Glo, phosphoflow/western blotting, bi-weekly caliper measurements, and multi-parameter flow cytometry, respectively. Results: ADT-007 inhibited KRAS-MAPK/AKT signaling in human and mouse PDAC cell lines inducing G2-M phase arrest and apoptosis at low nanomolar concentrations. Peritumoral administration of ADT-007 inhibited tumor cell growth in vivo, modulating T cells in the tumor microenvironment (TME), specifically, an increase in tumor infiltrating, CD44+ CD62L− CD4+ and CD8+ T cells and increases in production of TNFα, IFN-γ, IL-2, and granzyme B upon stimulation with PMA/ionomycin. Conclusion: The small molecule inhibitor, ADT-007 blocks oncogenic KRAS signaling and modulates T cell activation and function in the TiME of immune competent, syngeneic mouse models of PDAC. More broadly, these findings indicate that a pan-RAS inhibitor is capable of modulating anti-tumor immune responses extending previous observations that targeted inhibition of oncogenic KRASG12C modulates tumor immunity in animal models of colon and lung cancer. Citation Format: Jeremy B. Foote, Tyler E. Mattox, Adam B. Keeton, Ganji N. Purnachandra, Yulia Maxuitenko, Xi Chen, Jacob Valiyaveettil, Donald J. Buchsbaum, Gary A. Piazza, Bassel F. El-Rayes. Oncogenic KRAS inhibition with ADT-007 primes T cell responses in pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4140.

Metastatic prostate cancer is the second leading cause of cancer deaths in US men. Most early-stage PCa are dependent on overexpression of the androgen receptor (AR) and are therefore sensitive to androgen deprivation therapies. However, eventual resistance to standard medical castration and secondary chemotherapies including next-generation AR-targeting agents like abiraterone or enzalutamide and taxanes (Docetaxel or cabazitaxel) is nearly universal. Further, neuroendocrine PCa or NEPC, a poorly differentiated aggressive variant of PCa that lacks AR expression, and the presence of cancer stem-like cells with self-renewal and differentiation (epithelial to mesenchymal trans-differentiation or EMT) capacities significantly contribute to the development of clinically most aggressive and lethal variants of PCa. Endogenous Ras/ERK/MAPK signaling is known to drive prostate tumor formation, cancer progression, cell migration, and the acquisition of mesenchymal phenotype (EMT) in metastatic prostate cancers. Ras family members are responsible for modulating the function of most of the receptors upregulated in advanced PCa, including AR, so that AR can be functional even in the absence of physiologic levels of androgen. Previous studies have reported that the NSAID, sulindac, inhibits Ras-induced transformation and directly binds Ras. In this study, we show that a non-COX inhibitory sulindac derivative, ADT-007, is a potent drug candidate against lethal PCa. First, we performed single-cell RNA sequencing as a biomarker-based drug screen and showed that chemo-resistant, drug-tolerant, stem-cell-like PCa cell clusters had high expression of Ras genes, indicating that the Ras inhibitor, ADT-007, may be effective in targeting these aggressive subclones. Next, using several cell-based assays on a large panel of lethal PCa cell lines representing metastatic castration-resistant PCa (mCRPC), NEPC as well as African American ancestry, we showed that ADT-007 is highly effective as a single-agent and in combination with taxanes. Combination index values were 10, indicating very high synergy. Finally, using pre-vs-post-treatment bulk RNAseq, we demonstrated that ADT-007 is indeed effective in downregulating KRas and NRas in a dose-dependent manner. In addition, ingenuity pathway analysis (IPA) predicted significant downregulation of cell cycle, DNA replication, CCND1, AR, and the lncRNA ELDR, and upregulation of autophagy and interferon pathway genes, and the microRNA miR-96. Finally, using PCa patient datasets, we showed that ADT-007 is potentially capable of reversing the expression of several genes associated with biochemical recurrence. Thus, our results show that ADT-007 is a novel drug candidate that circumvents subclonal aggressiveness, drug resistance, and stemness in lethal PCa through simultaneous inhibition of multiple oncogenic factors/pathways. Citation Format: Taraswi Mitra Ghosh, Adam B. Keeton, Xi Chen, Salsabil Ahmed, Razan S. Waliagha, Gary A. Piazza, Amit K. Mitra. Validation of a novel pan-Ras inhibitor, ADT-007, against lethal variants of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1638.

BACKGROUND: Previous publications have reported that the antineoplastic activity of sulindac occurs by a cyclooxygenase independent mechanism, possibly involving direct inhibition of RAS. Suppression of β-catenin/TCF transcription has also been reported by inhibiting cyclic guanosine monophosphate (cGMP) phosphodiesterase (PDE) and the activation of cGMP-dependent protein kinase (PKG). Here we characterize a potent and selective non-COX inhibitory sulindac derivative, ADT-030, that inhibits cancer cell growth by blocking RAS and β-catenin signaling. METHODS AND RESULTS: ADT-030 inhibited the proliferation of lung cancer cells expressing the PDE10A isozyme, while normal airway cells lacking PDE10A displayed reduced sensitivity. ADT-030 inhibited recombinant PDE10 activity at concentrations that inhibit lung cancer cells proliferation. ADT-030 induced cell cycle arrest, apoptosis, and morphological characteristics of autophagy blockage. A cellular thermal stability assay showed that ADT-030 binds both RAS and PDE10 in lysates from cancer cells with nanomolar affinity. Consistent with its PDE10 inhibitory activity, ADT-030 activated PKG and phosphorylated β-catenin on amino acid residues known to induce proteasomal degradation, and reduced levels of the unphosphorylated (oncogenic stabilized) form of β-catenin. Consistent with its ability to directly bind RAS, ADT-030 also inhibited EGF-stimulated MAPK/AKT signaling. Oral administration of ADT-030 inhibited tumor growth in an orthotopic xenograft mouse model of lung cancer, providing a durable survival response with 25% tumor-free mice at dosages having no discernable toxicity. ADT-030 was also highly efficacious in two different chemical-induced mouse models of lung tumorigenesis suppressing the carcinogen-activation of RAS, as well as in multiple mouse subcutaneous tumor models, including highly aggressive models of metastasis. CONCLUSIONS: These results support further development of ADT-030, a novel inhibitor of RAS and β-catenin signaling for the treatment of lung cancer and other RAS driven cancers, including those with co-occurring mutations in the Wnt/β-catenin pathway. Supported by NIH grants R01 CA238514, R01 CA197147, R01 CA254197. Citation Format: Veronica Ramirez-Alcantara, Xi Chen, Michele A. Schuler, Dennis Otali, William Grizzle, Kristy Berry, Antonio Ward, Ivan Babic, Elmar Nurmemmedov, Gang Zhou, Chengguo Xing, Greg Gorman, Lori Coward, Yulia Maxuitenko, Adam B. Keeton, Gary A. Piazza. ADT-030, a novel dual-acting RAS and β-catenin inhibitor with robust antitumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1633.

Approximately 30% of human cancers harbor a gain‐in‐function mutation in the RAS gene, resulting in constitutive activation of the RAS protein to stimulate downstream signaling, including the RAS‐mitogen activated protein kinase pathway that drives cancer cells to proliferate and metastasize. RAS‐driven oncogenesis also promotes immune evasion by increasing the expression of programmed cell death ligand‐1, reducing the expression of major histocompatibility complex molecules that present antigens to T‐lymphocytes and altering the expression of cytokines that promote the differentiation and accumulation of immune suppressive cell types such as myeloid‐derived suppressor cells, regulatory T‐cells, and cancer‐associated fibroblasts. Together, these changes lead to an immune suppressive tumor microenvironment that impedes T‐cell activation and infiltration and promotes the outgrowth and metastasis of tumor cells. As a result, despite the growing success of checkpoint immunotherapy, many patients with RAS‐driven tumors experience resistance to therapy and poor clinical outcomes. Therefore, RAS inhibitors in development have the potential to weaken cancer cell immune evasion and enhance the antitumor immune response to improve survival of patients with RAS‐driven cancers. This review highlights the potential of RAS inhibitors to enhance or broaden the anticancer activity of currently available checkpoint immunotherapy.

Background Cancer remains the leading cause of human morbidity universally. Hence, we sought to assess the in vitro antiproliferative activity of new isatin-based conjugates (5a-s) against three human cancer cell lines. Methods The antiproliferative activities of compounds 5a-s were evaluated in vitro and their ADME (absorption, distribution, metabolism and excretion) was carried out using standard protocols. Subsequently, Western blot analysis was conducted to elucidate the potential antiproliferative mechanism of compounds 5a-s. Results The in vitro antiproliferative activities of compounds 5a-s against the tested cancer cell lines ranged from 20.3 to 95.9%. Compound 5m had an IC50 value of 1.17 µM; thus, its antiproliferative potency was approximately seven-fold greater than that of sunitinib (IC50 = 8.11 µM). In-depth pharmacological testing was conducted with compound 5m to gain insight into the potential antiproliferative mechanism of this class of compounds. Compound 5m caused an increase in the number of cells in the G1 phase, with a concomitant reduction of those in the G2/M and S phases. Additionally, compound 5m significantly and dose-dependently reduced the amount of phosphorylated retinoblastoma protein detected. Compound 5m enhanced expression of B cell translocation gene 1, cell cycle-associated proteins (cyclin B1, cyclin D1, and phosphorylated cyclin-dependent kinase 1), and a pro-apoptotic protein (Bcl-2-associated X protein gene), and activated caspase-3. ADME predictions exposed the oral liability of compounds 5a-s. Conclusion Herein, we revealed the antiproliferative activity and ADME predictions of the newly-synthesized compounds 5a-s and provided a detailed insight into the pharmacological profile of compound 5m. Thus, compounds 5a-s can potentially be exploited as new antiproliferative lead compounds for cancer chemotherapeutic.

Approximately 28 million people in the United States have precancerous colonic adenomas with many at high risk of developing colorectal cancer who could benefit from a pharmacological approach to prevent malignant progression. Clinical, epidemiological, and preclinical studies have reported or provided mechanistic evidence that nonsteroidal anti-inflammatory drugs (NSAIDs) can reduce adenoma formation and the risk of developing colorectal cancer. Unfortunately, the long-term use of NSAIDs is not recommended because of potentially fatal toxicities resulting from cyclooxygenase (COX) inhibition and the depletion of physiologically important prostaglandins. However, multiple investigators have concluded that the mechanism responsible for the antineoplastic activity of NSAIDs does not require COX inhibition, which suggests the feasibility of developing safer and more efficacious derivatives for the treatment or prevention of colorectal cancer by targeting such mechanisms. We have widely reported that the NSAID, sulindac, inhibits certain phosphodiesterase (PDE) isozymes to increase intracellular cyclic guanosine monophosphate (cGMP) levels and activate protein kinase G (PKG) at concentrations that inhibit cancer cell growth in vitro. PDE5 and PDE10 are potential targets given that both are overexpressed in colon adenomas and adenocarcinomas and essential for colon cancer cell proliferation. An extensive medicinal chemistry campaign was conducted to identify novel sulindac derivatives lacking COX inhibitory activity with increased potency and selectivity to inhibit cancer cell growth. From a large collection of over 1500 compounds sharing the indene scaffold of sulindac, a series of derivatives were identified that inhibit cancer cell growth with high potency and selectivity by a mechanism involving the activation of cGMP/PKG signaling. Development candidates have been identified that block the oncogenic effects from APC gene mutations responsible for most human colorectal cancers by suppressing β-catenin-dependent transcriptional activity. This review describes the scientific rationale for the development of sulindac derivatives lacking COX inhibitory activity with improved potency and selectivity to inhibit PDE5 and/or PDE10 for the treatment or prevention of colorectal cancer.

Mutations in the three RAS oncogenes are present in approximately 30% of all human cancers that drive tumor growth and metastasis by aberrant activation of RAS-mediated signaling. Despite the well-established role of RAS in tumorigenesis, past efforts to develop small molecule inhibitors have failed for various reasons leading many to consider RAS as "undruggable." Advances over the past decade with KRAS(G12C) mutation-specific inhibitors have culminated in the first FDA-approved RAS drug, sotorasib. However, the patient population that stands to benefit from KRAS(G12C) inhibitors is inherently limited to those patients harboring KRAS(G12C) mutations. Additionally, both intrinsic and acquired mechanisms of resistance have been reported that indicate allele-specificity may afford disadvantages. For example, the compensatory activation of uninhibited wild-type (WT) NRAS and HRAS isozymes can rescue cancer cells harboring KRAS(G12C) mutations from allele-specific inhibition or the occurrence of other mutations in KRAS. It is therefore prudent to consider alternative drug discovery strategies that may overcome these potential limitations. One such approach is pan-RAS inhibition, whereby all RAS isozymes co-expressed in the tumor cell population are targeted by a single inhibitor to block constitutively activated RAS regardless of the underlying mutation. This chapter provides a review of past and ongoing strategies to develop pan-RAS inhibitors in detail and seeks to outline the trajectory of this promising strategy of RAS inhibition.

The nonsteroidal anti-inflammatory drug (NSAID) sulindac demonstrates attractive anticancer activity, but the toxicity resulting from cyclooxygenase (COX) inhibition and the suppression of physiologically important prostaglandins precludes its long-term, high dose use in the clinic for cancer prevention or treatment. While inflammation is a known tumorigenic driver, evidence suggests that sulindac’s antineoplastic activity is partially or fully independent of its COX inhibitory activity. One COX-independent target proposed for sulindac is cyclic guanosine monophosphate phosphodiesterase (cGMP PDE) isozymes. Sulindac metabolites, i.e., sulfide and sulfone, inhibit cGMP PDE enzymatic activity at concentrations comparable with those associated with cancer cell growth inhibitory activity. Additionally, the cGMP PDE isozymes PDE5 and PDE10 are overexpressed during the early stages of carcinogenesis and appear essential for cancer cell proliferation and survival based on gene silencing experiments. Here, we describe a novel amide derivative of sulindac, sulindac sulfide amide (SSA), which was rationally designed to eliminate COX-inhibitory activity while enhancing cGMP PDE inhibitory activity. SSA was 68-fold and 10-fold less potent than sulindac sulfide (SS) in inhibiting COX-1 and COX-2, respectively, but 10-fold more potent in inhibiting growth and inducing apoptosis in breast cancer cells. The pro-apoptotic activity of SSA was associated with inhibition of cGMP PDE activity, elevation of intracellular cGMP levels, and activation of cGMP-dependent protein kinase (PKG) signaling, as well as the inhibition of β-catenin/Tcf transcriptional activity. SSA displayed promising in vivo anticancer activity, resulting in a 57% reduction in the incidence and a 62% reduction in the multiplicity of tumors in the N-methyl-N-nitrosourea (MNU)-induced model of breast carcinogenesis. These findings provide strong evidence for cGMP/PKG signaling as a target for breast cancer prevention or treatment and the COX-independent anticancer properties of sulindac. Furthermore, this study validates the approach of optimizing off-target effects by reducing the COX-inhibitory activity of sulindac for future targeted drug discovery efforts to enhance both safety and efficacy.

A leading theory for ovarian carcinogenesis proposes that inflammation associated with incessant ovulation is a driver of oncogenesis. Consistent with this theory, nonsteroidal anti-inflammatory drugs (NSAIDs) exert promising chemopreventive activity for ovarian cancer. Unfortunately, toxicity is associated with long-term use of NSAIDs due to their cyclooxygenase (COX) inhibitory activity. Previous studies suggest the antineoplastic activity of NSAIDs is COX independent, and rather may be exerted through phosphodiesterase (PDE) inhibition. PDEs represent a unique chemopreventive target for ovarian cancer given that ovulation is regulated by cyclic nucleotide signaling. Here we evaluate PDE10A as a novel therapeutic target for ovarian cancer. Analysis of The Cancer Genome Atlas (TCGA) ovarian tumors revealed PDE10A overexpression was associated with significantly worse overall survival for patients. PDE10A expression also positively correlated with the upregulation of oncogenic and inflammatory signaling pathways. Using small molecule inhibitors, Pf-2545920 and a novel NSAID-derived PDE10A inhibitor, MCI-030, we show that PDE10A inhibition leads to decreased ovarian cancer cell growth and induces cell cycle arrest and apoptosis. We demonstrate these pro-apoptotic properties occur through PKA and PKG signaling by using specific inhibitors to block their activity. PDE10A genetic knockout in ovarian cancer cells through CRISP/Cas9 editing lead to decreased cell proliferation, colony formation, migration and invasion, andin vivotumor growth. We also demonstrate that PDE10A inhibition leads to decreased Wnt-induced β-catenin nuclear translocation, as well as decreased EGF-mediated activation of RAS/MAPK and AKT pathways in ovarian cancer cells. These findings implicate PDE10A as novel target for ovarian cancer chemoprevention and treatment.

The RAS oncogene is both the most frequently mutated oncogene in human cancer and the first confirmed human oncogene to be discovered in 1982. After decades of research, in 2013, the Shokat lab achieved a seminal breakthrough by showing that the activated KRAS isozyme caused by the G12C mutation in the KRAS gene can be directly inhibited via a newly unearthed switch II pocket. Building upon this groundbreaking discovery, sotorasib (AMG510) obtained approval by the United States Food and Drug Administration in 2021 to become the first therapy to directly target the KRAS oncoprotein in any KRAS-mutant cancers, particularly those harboring the KRASG12C mutation. Adagrasib (MRTX849) and other direct KRASG12C inhibitors are currently being investigated in multiple clinical trials. In this review, we delve into the path leading to the development of this novel KRAS inhibitor, starting with the discovery, structure, and function of the RAS family of oncoproteins. We then examine the clinical relevance of KRAS, especially the KRASG12C mutation in human cancer, by providing an in-depth analysis of its cancer epidemiology. Finally, we review the preclinical evidence that supported the initial development of the direct KRASG12C inhibitors and summarize the ongoing clinical trials of all direct KRASG12C inhibitors.

Background Pancreatic Ductal Adenocarcinoma (PDAC) is an aggressive and lethal disease, lacking effective therapeutic approaches. Available therapies only marginally prolong patient survival and are frequently coupled with severe adverse events. It is therefore pivotal to investigate novel and safe pharmacological approaches. We have recently identified the ABC transporter, ABCC3, whose expression is dependent on mutation of TP53, as a novel target in PDAC. ABCC3-mediated regulation of PDAC cell proliferation and tumour growth in vivo was demonstrated and was shown to be conferred by upregulation of STAT3 signalling and regulation of apoptosis. Methods To verify the potential of ABCC3 as a pharmacological target, a small molecule inhibitor of ABCC3, referred to here as MCI-715, was designed. In vitro assays were performed to assess the effects of ABCC3 inhibition on anchorage-dependent and anchorage-independent PDAC cell growth. The impact of ABCC3 inhibition on specific signalling pathways was verified by Western blotting. The potential of targeting ABCC3 with MCI-715 to counteract PDAC progression was additionally tested in several animal models of PDAC, including xenograft mouse models and transgenic mouse model of PDAC. Results Using both mouse models and human cell lines of PDAC, we show that the pharmacological inhibition of ABCC3 significantly decreased PDAC cell proliferation and clonal expansion in vitro and in vivo, remarkably slowing tumour growth in mice xenografts and patient-derived xenografts and increasing the survival rate in a transgenic mouse model. Furthermore, we show that stromal cells in pancreatic tumours, which actively participate in PDAC progression, are enriched for ABCC3, and that its inhibition may contribute to stroma reprogramming. Conclusions Our results indicate that ABCC3 inhibition with MCI-715 demonstrated strong antitumor activity and is well tolerated, which leads us to conclude that ABCC3 inhibition is a novel and promising therapeutic strategy for a considerable cohort of patients with pancreatic cancer. Electronic supplementary material The online version of this article (10.1186/s13046-019-1308-7) contains supplementary material, which is available to authorized users.

Background Cancer is one of the most dreaded human diseases, that has become an ever-increasing health problem and is a prime cause of death globally. The potential antiproliferative activity of certain indole-isatin molecular hybrids 5a-w was evaluated in vitro against three human cancer cell lines. Methods Standard protocols were adopted to examine the antiproliferative potential and mechanisms of compounds 5a-w. Western blot analysis was carried out on compound 5o. Results Compounds 5a-w demonstrated in vitro antiproliferative activity in the range of 22.6-97.8%, with compounds 5o and 5w being the most active antiproliferative compounds with IC50 values of 1.69 and 1.91 µM, which is fivefold and fourfold more potent than sunitinib (IC50=8.11 µM), respectively. Compound 5o was selected for in-depth pharmacological testing to understand its possible mechanism of antiproliferative activity. It caused a lengthening of the G1 phase and a reduction in the S and G2/M phases of the cell cycle and had an IC50 value of 10.4 μM with the resistant NCI-H69AR cancer cell line. Moreover, compound 5o significantly decreased the amount of phosphorylated Rb protein in a dose-dependent fashion, which was confirmed via Western blot analysis. Conclusion The current investigation highlighted the potential antiproliferative activity of compounds 5a-w as well as the antiproliferative profile of compound 5o. These compounds can be harnessed as new lead antiproliferatives in the preclinical studies of cancer chemotherapy.

Previous studies have reported that phosphodiesterase 10A (PDE10) is overexpressed in colon epithelium during early stages of colon tumorigenesis and essential for colon cancer cell growth. Here we describe a novel non-cyclooxygenase inhibitory derivative of the anti-inflammatory drug, sulindac, with selective PDE10 inhibitory activity, ADT 061. ADT 061 potently inhibited the growth of colon cancer cells expressing high levels of PDE10, but not normal colonocytes that do not express PDE10. The concentration range by which ADT 061 inhibited colon cancer cell growth was identical to concentrations that inhibit recombinant PDE10. ADT 061 inhibited PDE10 by a competitive mechanism and did not affect the activity of other PDE isozymes at concentrations that inhibit colon cancer cell growth. Treatment of colon cancer cells with ADT 061 activated cGMP/PKG signaling, induced phosphorylation of oncogenic β-catenin, inhibited Wnt-induced nuclear translocation of β-catenin, and suppressed TCF/LEF transcription at concentrations that inhibit cancer cell growth. Oral administration of ADT 061 resulted in high concentrations in the colon mucosa and significantly suppressed the formation of colon adenomas in the Apc(+/min-FCCC) mouse model of colorectal cancer (CRC) without discernable toxicity. These results support the development of ADT 061 for the treatment or prevention of adenomas in individuals at risk of developing CRC.

A series of 2-arylthiazolidine-4-carboxylic acid amide derivatives were synthesized and their cytotoxic activity against three cancer cell lines (PC-3, SKOV3 and MDA-MB231) was evaluated. Various structural modifications were tried including modifications of the length of the amide chain and modifications of the 2-aryl part using disubstituted phenyl and thiophene derivatives. The structure activity relationship was evaluated based on the in vitro biological evaluation against the above mentioned three cancer cell lines. The most selective compounds towards cancer cells were further evaluated against DLD-1, NCI-H520, Du145, MCF-7 and NCI-N87 cancer cells. The dodecyl amide having the 4-bromothienyl as the 2-aryl, compound 2e, exhibited the highest selectivity for cancer cells vs non-tumor cells. Mechanistic studies of the anticancer effect of compound 2e in prostate cancer PC-3 and colorectal cancer DLD-1 cells revealed that 2e could prevent the cell cycle in the G0/G1 phase by up-regulating the expression of p21 and reducing the expression of CDK2 and cyclin E. It increased the pro-apoptotic protein Bax and cleaved caspase 3, and down-regulated the expression of anti-apoptotic protein Bcl-2 to induce apoptosis. In addition, 2e also downregulated AKT, N-cadherin, and vimentin proteins expression giving indication that 2e inhibit the PI3K/AKT pathway to regulate cell cycle arrest and induce apoptosis, and can regulate the expression of epithelial-mesenchymal transition-related proteins.

A ligand-based approach involving systematic modifications of a trisubstituted pyrazoline scaffold derived from the COX2 inhibitor, celecoxib, was used to develop novel PDE5 inhibitors. Novel pyrazolines were identified with potent PDE5 inhibitory activity lacking COX2 inhibitory activity. Compound d12 was the most potent with an IC50 of 1 nM, which was three times more potent than sildenafil and more selective with a selectivity index of >10,000-fold against all other PDE isozymes. Sildenafil inhibited the full-length and catalytic fragment of PDE5, while compound d12 only inhibited the full-length enzyme, suggesting a mechanism of enzyme inhibition distinct from sildenafil. The PDE5 inhibitory activity of compound d12 was confirmed in cells using a cGMP biosensor assay. Oral administration of compound d12 achieved plasma levels >1000-fold higher than IC50 values and showed no discernable toxicity after repeated dosing. These results reveal a novel strategy to inhibit PDE5 with unprecedented potency and isozyme selectivity.

Expression of ATP-binding cassette (ABC) transporters has long been implicated in cancer chemotherapy resistance. Increased expression of the ABCC subfamily transporters has been reported in prostate cancer, especially in androgen-resistant cases. ABCC transporters are known to efflux drugs but, recently, we have demonstrated that they can also have a more direct role in cancer progression. The pharmacological potential of targeting ABCC1, however, remained to be assessed. In this study, we investigated whether the blockade of ABCC1 affects prostate cancer cell proliferation using both in vitro and in vivo models. Our data demonstrate that pharmacological inhibition of ABCC1 reduced prostate cancer cell growth in vitro and potentiated the effects of Docetaxel in vitro and in mouse models of prostate cancer in vivo. Collectively, these data identify ABCC1 as a novel and promising target in prostate cancer therapy.

A series of thirty compounds of quinazolinone-Schiff's base hybrids were rationally designed, synthesized, and evaluated for their in vitro Phosphodiesterase 4B inhibition, anti-lung and anti-colon cancer activities. Compounds 9, 16, 23, 29, 30, 31, 32 and 33, each possessing a tri-hydroxy moiety, showed the highest inhibitory activity (56.05-89.07%) at 10 µM concentration if compared to rolipram. Compounds 16 and 23 showed good anti-lung cancer activity with IC50 1.55 and 1.30 µM, respectively. Moreover, compound 16 showed high anti-colon cancer activity with IC50 0.6 µM. Compound 33 showed good affinity and molecular binding modes towards the key amino acid Gln 443 and Phe 446 for inhibition of the target enzyme. Finally, active compounds showed good ADME calculations.

Although numerous reports conclude that nonsteroidal anti-inflammatory drugs (NSAIDs) have anticancer activity, this common drug class is not recommended for long-term use because of potentially fatal toxicities from cyclooxygenase (COX) inhibition. Studies suggest the mechanism responsible for the anticancer activity of the NSAID sulindac is unrelated to COX inhibition but instead involves an off-target, phosphodiesterase (PDE). Thus, it might be feasible develop safer and more efficacious drugs for cancer indications by targeting PDE5 and PDE10, which are overexpressed in various tumors and essential for cancer cell growth. In this review, we describe the rationale for using the sulindac scaffold to design-out COX inhibitory activity, while improving potency and selectivity to inhibit PDE5 and PDE10 that activate cGMP/PKG signaling to suppress Wnt/β-catenin transcription, cancer cell growth, and tumor immunity.

In connection with our research program on the development of novel indolin-2-one-based anticancer candidates, herein we report the design and synthesis of different series of hydrazonoindolin-2-ones 3a-e, 5a-e, 7a-c, and 10a-l. The synthesised derivatives were in vitro evaluated for their anti-proliferative activity towards lung A-549, colon HT-29, and breast ZR-75 human cancer cell lines. Compounds 5b, 5c, 7b, and 10e emerged as the most potent derivatives with average IC50 values of 4.37, 2.53, 2.14, and 4.66 µM, respectively, which are superior to Sunitinib (average IC50 = 8.11 µM). Furthermore, compounds 7b and 10e were evaluated for their effects on cell cycle progression and levels of phosphorylated retinoblastoma (Rb) protein in the A-549 cancer cell line. Moreover, 7b and 10e inhibited the cell growth of the multidrug-resistant lung cancer NCI-H69AR cell line with IC50 = 16 µM. In addition, the cytotoxic activities of 7b and 10e were assessed towards three non-tumorigenic cell lines (Intestine IEC-6, Breast MCF-10A, and Fibroblast Swiss-3t3) where both compounds displayed mean tumor selectivity index (1.6 and 1.8) higher than that of Sunitinib (1.4)., Graphical Abstract

Objective: A leading theory for ovarian carcinogenesis proposes that the inflammation associated with incessant ovulation is a driver of oncogenesis. Consistent with this theory, epidemiological studies have shown that nonsteroidal anti-inflammatory drugs (NSAIDs) decrease ovarian cancer risk. Previous studies suggest, however, that the antineoplastic activity of NSAIDs does not require the traditional cyclooxygenase (COX) enzyme inhibition, and rather may be exerted through phosphodiesterase (PDE) inhibition. PDEs represent a potentially unique chemopreventive target for ovarian cancer given that ovulation is regulated by cyclic nucleotide signaling. This study evaluates the effects of phosphodiesterase 10A (PDE10A) inhibition as a novel chemopreventive and therapeutic approach for ovarian cancer. Methods: We investigated the effects of PDE10A small molecule inhibitors, Pf-2545920 and MCI-030 (a novel non-COX inhibitory NSAID-derivative), and PDE10A knockout by CRISPR/Cas9 gene editing in various ovarian cancer cell lines using in vitro assays that measured cell proliferation, cell viability, cell cycle arrest, apoptosis, cell migration and invasion. Downstream signaling pathways affected by PDE10A inhibition and gene knockout (KO) were assessed by western-blotting, confocal microscopy, and RNA sequencing. Results: Analysis of The Cancer Genome Atlas (TCGA) ovarian cancer database and an institutional cohort of ovarian cancer patients revealed that high PDE10A mRNA expression was associated with significantly worse overall survival. PDE10A expression was also positively correlated with oncogenic and inflammatory signaling pathways in the TCGA dataset. PDE10A inhibition with Pf-2545920 or MCI-030 decreased ovarian cancer cell proliferation, while inducing cell cycle arrest and apoptosis. Using inhibitors to block PKA (H89) and PKG (KT5823) kinase activity, we demonstrated that the pro-apoptotic effects of PDE10A inhibition were mediated by activation of cGMP/PKG and cAMP/PKA signaling. SKOV3 and OV-90 PDE10A KO cells showed decreased colony formation, cell proliferation, and migration and invasion properties compared to their wild-type (WT) counterparts. Moreover, PDE10A inhibition decreased Wnt-induced β-catenin nuclear translocation as well as decreased EGF-mediated activation of RAS/MAPK and AKT pathways in ovarian cancer cells. RNA sequencing of SKOV3 PDE10A KO clones revealed that pathways associated with cancer, Wnt and TGF-β signaling were downregulated compared to WT control cells. Conclusions: Our data demonstrate that PDE10A has pro-tumorigenic effects in ovarian cancer cells and its high expression in ovarian cancer patients is associated with poor prognosis. Altogether, our results warrant future studies of PDE10A as a novel target for ovarian cancer chemoprevention and/or treatment. Citation Format: Rebecca M. Barber, Elaine Gavin, Alla Musiyenko, Wito Richter, Kevin J. Lee, Annelise Wilhite, Joel F. Andrews, Steve McClellan, Ileana Aragon, Antonio Ward, Xi Chen, Adam Keeton, Kristy Berry, Gary A. Piazza, Jennifer M. Scalici, Luciana Madeira da Silva. PDE10A as a novel target to suppress Wnt/β-catenin signaling and other oncogenic pathways in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1213.

// Bing Zhu 1 , Ashley Lindsey 1 , Nan Li 2 , Kevin Lee 1 , Veronica Ramirez-Alcantara 1 , Joshua C. Canzoneri 1 , Alexandra Fajardo 1 , Luciana Madeira da Silva 1 , Meagan Thomas 1 , John T. Piazza 1 , Larry Yet 3 , Brian T. Eberhardt 3 , Evrim Gurpinar 4 , Dennis Otali 5 , William Grizzle 5 , Jacob Valiyaveettil 1 , Xi Chen 1 , Adam B. Keeton 1 and Gary A. Piazza 1 1 Drug Discovery Research Center, Mitchell Cancer Institute, University of South Alabama, Mobile, Alabama, USA 2 Department of Biochemistry and Molecular Biology, The University of Alabama at Birmingham, Birmingham, Alabama, USA 3 Department of Chemistry, University of South Alabama, Mobile, Alabama, USA 4 Department of Pharmacology, The University of Alabama at Birmingham, Birmingham, Alabama, USA 5 Department of Pathology, The University of Alabama at Birmingham, Birmingham, Alabama, USA Correspondence to: Gary A. Piazza, email: // Bing Zhu, email: // Keywords : lung cancer, PDE10, PKG, cGMP, PQ10 Received : June 30, 2017 Accepted : August 04, 2017 Published : August 27, 2017 Abstract 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.

Hatem A Abdel-Aziz,1 Wagdy M Eldehna,2 Adam B Keeton,3 Gary A Piazza,3 Adnan A Kadi,4 Mohamed W Attwa,4 Ali S Abdelhameed,4 Mohamed I Attia4,5 1Department of Applied Organic Chemistry, National Research Centre, Giza, 2Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Kafrelsheikh University, Kafrelsheikh, Egypt; 3Department of Oncologic Sciences and Pharmacology, Drug Discovery Research Center, Mitchell Cancer Institute, University of South Alabama, Mobile, AL, USA; 4Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia; 5Medicinal and Pharmaceutical Chemistry Department, Pharmaceutical and Drug Industries Research Division, National Research Centre, Giza, Egypt Abstract: In continuation of our endeavor with respect to the development of potent and effective isatin-based anticancer agents, we adopted the molecular hybridization approach to design and synthesize four different sets of isatin-quinazoline (6a–f and 7a–e)/phthalazine (8a–f)/quinoxaline (9a–f) hybrids. The antiproliferative activity of the target hybrids was assessed towards HT-29 (colon), ZR-75 (breast) and A-549 (lung) human cancer cell lines. Hybrids 8b–d emerged as the most active antiproliferative congener in this study. Compound 8c induced apoptosis via increasing caspase 3/7 activity by about 5-fold in the A-549 human cancer cell line. In addition, it exhibited an increase in the G1 phase and a decrease in the S and G2/M phases in the cell cycle effect assay. Furthermore, it displayed an inhibitory concentration 50% value of 9.5 µM against multidrug-resistant NCI-H69AR lung cancer cell line. The hybrid 8c was also subjected to in vitro metabolic investigations through its incubation with rat liver microsomes and analysis of the resulting metabolites with the aid of liquid chromatography-mass spectrometry. Keywords: isatins, hybridization approach, antiproliferative, apoptosis

About a third of all human cancers harbor mutations in one of the K-, N-, or HRAS genes that encode an abnormal RAS protein locked in a constitutively activated state to drive malignant transformation and tumor growth. Despite more than three decades of intensive research aimed at the discovery of RAS-directed therapeutics, there are no FDA-approved drugs that are broadly effective against RAS-driven cancers. Although RAS proteins are often said to be “undruggable,” there is mounting evidence suggesting it may be feasible to develop direct inhibitors of RAS proteins. Here, we review this evidence with a focus on compounds capable of inhibiting the interaction of RAS proteins with their effectors that transduce the signals of RAS and that drive and sustain malignant transformation and tumor growth. These reports of direct-acting RAS inhibitors provide valuable insight for further discovery and development of clinical candidates for RAS-driven cancers involving mutations in RAS genes or otherwise activated RAS proteins. Cancer Res; 77(2); 221–6. ©2017 AACR.

Background Cancer remains the leading cause of human morbidity universally. Hence, we sought to assess the in vitro antiproliferative activity of new isatin-based conjugates (5a–s) against three human cancer cell lines. Methods The antiproliferative activities of compounds 5a–s were evaluated in vitro and their ADME (absorption, distribution, metabolism and excretion) was carried out using standard protocols. Subsequently, Western blot analysis was conducted to elucidate the potential antiproliferative mechanism of compounds 5a–s. Results The in vitro antiproliferative activities of compounds 5a–s against the tested cancer cell lines ranged from 20.3 to 95.9%. Compound 5m had an IC50 value of 1.17 µM; thus, its antiproliferative potency was approximately seven-fold greater than that of sunitinib (IC50 = 8.11 µM). In-depth pharmacological testing was conducted with compound 5m to gain insight into the potential antiproliferative mechanism of this class of compounds. Compound 5m caused an increase in the number of cells in the G1 phase, with a concomitant reduction of those in the G2/M and S phases. Additionally, compound 5m significantly and dose-dependently reduced the amount of phosphorylated retinoblastoma protein detected. Compound 5m enhanced expression of B cell translocation gene 1, cell cycle-associated proteins (cyclin B1, cyclin D1, and phosphorylated cyclin-dependent kinase 1), and a pro-apoptotic protein (Bcl-2-associated X protein gene), and activated caspase-3. ADME predictions exposed the oral liability of compounds 5a-s. Conclusion Herein, we revealed the antiproliferative activity and ADME predictions of the newly-synthesized compounds 5a–s and provided a detailed insight into the pharmacological profile of compound 5m. Thus, compounds 5a–s can potentially be exploited as new antiproliferative lead compounds for cancer chemotherapeutic.

Background Cancer is one of the most dreaded human diseases, that has become an ever-increasing health problem and is a prime cause of death globally. The potential antiproliferative activity of certain indole–isatin molecular hybrids 5a-w was evaluated in vitro against three human cancer cell lines. Methods Standard protocols were adopted to examine the antiproliferative potential and mechanisms of compounds 5a-w. Western blot analysis was carried out on compound 5o. Results Compounds 5a-w demonstrated in vitro antiproliferative activity in the range of 22.6–97.8%, with compounds 5o and 5w being the most active antiproliferative compounds with IC50 values of 1.69 and 1.91 µM, which is fivefold and fourfold more potent than sunitinib (IC50=8.11 µM), respectively. Compound 5o was selected for in-depth pharmacological testing to understand its possible mechanism of antiproliferative activity. It caused a lengthening of the G1 phase and a reduction in the S and G2/M phases of the cell cycle and had an IC50 value of 10.4 μM with the resistant NCI-H69AR cancer cell line. Moreover, compound 5o significantly decreased the amount of phosphorylated Rb protein in a dose-dependent fashion, which was confirmed via Western blot analysis. Conclusion The current investigation highlighted the potential antiproliferative activity of compounds 5a-w as well as the antiproliferative profile of compound 5o. These compounds can be harnessed as new lead antiproliferatives in the preclinical studies of cancer chemotherapy.

Twelve derivatives of the general formula 3-substituted-6-chloroindoles were synthesized and tested for their growth inhibitory effects versus p53(+/+) colorectal cancer HCT116 and its p53 knockout isogenic cells; colorectal cancer cell p53(-/-) SW480; the lung cancer cell line p53(-/-) H1299; mouse embryonic fibroblasts (MEF) p53(+/+) and its p53 knockout isogenic cells. The compounds were also evaluated for their ability to induce p53 nuclear translocation and binding to murine double minute 2 (MDM2) and murine double minute 4 (MDM4). Of these, compound 5a was the most active in inhibiting the growth of cells, with selectivity towards the p53(+/+) cell lines, and it showed stronger binding to MDM4 rather than MDM2. The activity profile of compound 5a is strongly similar to that of Nutlin-3.

In the present study, anti-proliferative activities of cranberry derived flavonoids and some of their in vivo metabolites were evaluated using a panel of human bladder tumor cell lines (RT4, SCABER, and SW-780) and non-tumorigenic immortalized human uroepithelial cells (SV-HUC). Among the compounds tested, quercetin 3-O-glucoside, isorhamnetin (3′-O-methylquercetin), myricetin and quercetin showed strong concentration-dependent cell growth inhibitory activities in bladder cancer cells with IC50 values in a range of 8–92 μM. Furthermore, isorhamnetin and myricetin had very low inhibitory activity against SV-HUC even at very high concentrations (>200 μM) compared to bladder cancer cells, indicating that their cytotoxicity is selective for cancer cells. To determine whether the differential cell growth inhibitory effects of isomeric flavonoids quercetin 3-O-glucoside (active) and hyperoside (quercetin 3-O-galactoside) (inactive) are related to their metabolism by the cancer cells, SW-780 cells were incubated with these compounds and their metabolism was examined by LC-MS/MS. Compared to quercetin 3-O-glucoside, hyperoside undergoes relatively less metabolic biotransformation (methylation, glucuronidation and quinone formation). These data suggest that isorhamnetin and quercetin 3-O-glucoside may be the active forms of quercetin in prevention of bladder cancer in vivo and emphasize the importance of metabolism for the prevention of bladder cancer by diets rich in cranberries.

// Kevin Lee 1 , Ashley S. Lindsey 1 , Nan Li 2 , Bernard Gary 1 , Joel Andrews 3 , Adam B. Keeton 1 , Gary A. Piazza 1 1 Drug Discovery Research Center, Mitchell Cancer Institute, University of South Alabama, Mobile, Alabama, USA 2 Department of Biochemistry and Molecular Genetics, The University of Alabama at Birmingham, Birmingham, Alabama, USA 3 Mitchell Cancer Institute, University of South Alabama, Mobile, Alabama, USA Correspondence to: Kevin Lee, e-mail: kjlee@health.southalabama.edu Keywords: β-catenin, PKG, PKA, PDE10A, colon cancer Received: September 09, 2015 Accepted: December 07, 2015 Published: December 21, 2015 ABSTRACT Phosphodiesterase 10A (PDE10) is a cGMP and cAMP degrading PDE isozyme that is highly expressed in the brain striatum where it appears to play an important role in cognition and psychomotor activity. PDE10 inhibitors are being developed for the treatment of schizophrenia and Huntington’s disease and are generally well tolerated, possibly because of low expression levels in most peripheral tissues. We recently reported high levels of PDE10 in colon tumors and that genetic silencing of PDE10 by siRNA or inhibition with small molecule inhibitors can suppress colon tumor cell growth with a high degree of selectivity over normal colonocytes (Li et al., Oncogene 2015). These observations suggest PDE10 may have an unrecognized role in tumorigenesis. Here we report that the concentration range by which the highly specific PDE10 inhibitor, Pf-2545920 (MP-10), inhibits colon tumor cell growth parallels the concentration range required to increase cGMP and cAMP levels, and activates PKG and PKA, respectively. Moreover, PDE10 knockdown by shRNA reduces the sensitivity of colon tumor cells to the growth inhibitory activity of Pf-2545920. Pf-2545920 also inhibits the translocation of β-catenin to the nucleus, thereby reducing β-catenin mediated transcription of survivin, resulting in caspase activation and apoptosis. PDE10 mRNA was also found to be elevated in colon tumors compared with normal tissues. These findings suggest that PDE10 can be targeted for cancer therapy or prevention whereby inhibition results in cGMP elevation and PKG activation to reduce β-catenin-mediated transcription of survival proteins leading to the selective apoptosis of cancer cells.

Herein we present the synthesis and characterization of a novel chemical series of tadalafil analogues that display different pharmacological profiles. Compounds that have the 6R, 12aR configuration and terminal carboxylic acid group at the side chain arising from the piperazinedione nitrogen were potent PDE5 inhibitors, with compound 11 having almost equal potency to tadalafil and superior selectivity over PDE11, the most common off–target for tadalafil. Modifying the stereochemistry into 6S, 12aS configuration and adopting the hydroxamic acid moiety as a terminal group gave rise to compounds that only inhibited HDAC. Dual PDE5/HDAC inhibition could be achieved with compounds having 6R, 12aR configuration and hydroxamic acid moiety as a terminal group. The anticancer activity of the synthesized compounds was evaluated against a diverse number of cell lines of different origin. The compounds elicited anticancer activity against cell lines belonging to lymphoproliferative cancer as well as solid tumors. Despite the previous reports suggesting anticancer activity of PDE5 inhibitors, the growth inhibitory activity of the compounds seemed to be solely dependent on HDAC inhibition. Compound 26 (pan HDAC IC50 = 14 nM, PDE5 IC50 = 46 nM) displayed the most potent anticancer activity in the present series and was shown to induce apoptosis in Molt-4 cells. HDAC isoform selectivity testing for compound 26 showed that it is more selective for HDAC6 and 8 over HDAC1 by more than 20-fold.

Oncogenic mutations in RAS genes result in the elevation of cellular active RAS protein levels and increased signal propagation through downstream pathways that drive tumor cell proliferation and survival. These gain-of-function mutations drive over 30% of all human cancers, presenting promising therapeutic potential for RAS inhibitors. However, many have deemed RAS “undruggable” after nearly 40 years of failed drug discovery campaigns aimed at identifying a RAS inhibitor with clinical activity. Here we review RAS nucleotide cycling and the opportunities that RAS biochemistry presents for developing novel RAS inhibitory compounds. Additionally, compounds that have been identified to inhibit RAS by exploiting various aspects of RAS biology and biochemistry will be covered. Our current understanding of the biochemical properties of RAS, along with reports of direct-binding inhibitors, both provide insight on viable strategies for the discovery of novel clinical candidates with RAS inhibitory activity.

The synthesis and molecular characterization of new isatin-based hydrazonoindolin-2-ones 4a-o and 7a-e are reported. The in vitro anti-proliferative potential of the synthesized compounds 4a-o and 7a-e was examined against HT-29 (colon), ZR-75 (breast) and A549 (lung) human cancer cell lines. Compounds 7b, 7d and 7e were the most active congeners against the tested human cancer cell lines with average IC50 values of 4.77, 3.39 and 2.37 μM, respectively, as compared with the reference isatin-based drug, sunitinib, which exhibited an average IC50 value of 8.11 μM. Compound 7e was selected for further pharmacological evaluation in order to gain insight into its possible mechanism of action. It increased caspase 3/7 activity by 2.4- and 1.85-fold between 4 and 8 h of treatment, respectively, at 10 μM and it caused a decrease in the percentage of cells in the G1 phase of the cell cycle with a corresponding increase in the S-phase. In addition, compound 7e increased phosphorylated tyrosine (p-Tyr) levels nearly two-fold with an apparent IC50 value of 3.8 μM. The 7e-loaded PLGA microspheres were prepared using a modified emulsion-solvent diffusion method. The average encapsulation efficiency of the 7e-loaded PLGA microspheres was 85% ± 1.3. While, the in vitro release profile of the 7e-loaded microspheres was characterized by slow and continuous release of compound 7e during 21 days and the release curve was fitted to zero order kinetics. Incorporation of 7e into PLGA microspheres improved its in vitro anti-proliferative activity toward the human cancer cell line A549 after 120 h incubation period with an IC50 value less than 0.8 μM.