Activated RAS is a common driver of cancer that was considered undruggable for decades. Recent advances have enabled the development of RAS inhibitors, but the efficacy of these inhibitors remains limited by resistance. In this study, we developed a pan-RAS inhibitor, ADT-007, (Z)-2-(5-fluoro-1-(4-hydroxy-3,5-dimethoxybenzylidene)-2-methyl-1H-inden-3-yl)-N-(furan-2-ylmethyl)acetamide, that binds nucleotide-free RAS to block GTP activation of effector interactions and MAPK/AKT signaling, resulting in mitotic arrest and apoptosis. ADT-007 potently inhibited the growth of RAS-mutant cancer cells irrespective of the RAS mutation or isozyme. Wild-type RAS (RASWT) cancer cells with GTP-activated RAS from upstream mutations were equally sensitive. Conversely, RASWT cancer cells harboring downstream BRAF mutations and normal cells were essentially insensitive to ADT-007. Sensitivity of cancer cells to ADT-007 required activated RAS and dependence on RAS for proliferation, whereas insensitivity was attributed to metabolic deactivation by UDP-glucuronosyltransferases that were expressed in RASWT and normal cells but repressed in RAS-mutant cancer cells. ADT-007 displayed unique advantages over KRAS mutant-specific, pan-KRAS, and pan-RAS inhibitors that could impact in vivo antitumor efficacy by escaping compensatory mechanisms that lead to resistance. Local administration of ADT-007 showed robust antitumor activity in syngeneic immunocompetent and xenogeneic immune-deficient mouse models of colorectal and pancreatic cancers. The antitumor activity of ADT-007 was associated with the suppression of MAPK signaling and activation of innate and adaptive immunity in the tumor immune microenvironment. Oral administration of ADT-007 prodrug also inhibited tumor growth. Thus, ADT-007 has the potential to address the complex RAS mutational landscape of many human cancers and to improve treatment of RAS-driven tumors.Significance: ADT-007, a first-in-class pan-RAS inhibitor, has unique selectivity for cancer cells with mutant RAS or activated RAS protein and the capability to circumvent resistance to suppress tumor growth, supporting further development of ADT-007 analogs.

BACKGROUND:Oncogenic KRAS mutations occur in nearly, 90% of patients with pancreatic ductal adenocarcinoma (PDAC). Targeting KRAS has been complicated by mutational heterogeneity and rapid resistance. We developed a novel pan-RAS inhibitor, ADT-1004 (an oral prodrug of ADT-007) and evaluated antitumor activity in murine and human PDAC models. METHODOLOGY:Murine PDAC cells with KRASG12D mutation (KPC-luc or 2838c3-luc) were orthotopically implanted into the pancreas of C57BL/6J mice, and four PDX PDAC tumors with KRAS mutations were implanted subcutaneously in NSG mice. To assess potential to overcome RAS inhibitor resistance, parental and resistant MIA PaCa-2 PDAC cells (KRASG12C mutation) were implanted subcutaneously. Subcutaneously implanted RASWT BxPC-3 cells were used to assess the selectivity of ADT-1004. RESULTS:ADT-1004 potently blocked tumor growth and RAS activation in mouse PDAC models without discernable toxicity with target engagement and reduced activated RAS and ERK phosphorylation. In addition, ADT-1004 suppressed tumor growth in PDX PDAC models with KRASG12D, KRASG12V, KRASG12C, or KRASG13Q mutations and increased CD4+ and CD8+ T cells in the TME consistent with exhaustion and increased MHCII+ M1 macrophage and dendritic cells. ADT-1004 demonstrated superior efficacy over sotorasib and adagrasib in tumor models resistant to these KRASG12C inhibitors and MRTX1133 resistant KRASG12D mutant cells. As evidence of selectivity for tumors with mutant KRAS, ADT-1004 did not impact the growth of tumors from RASWT PDAC cells. CONCLUSION/SIGNIFICANCE:ADT-1004 has strong antitumor activity in aggressive and clinically relevant PDAC models with unique selectivity to block RAS-mediated signaling in RAS mutant cells. As a pan-RAS inhibitor, ADT-1004 has broad activity and potential efficacy advantages over allele-specific KRAS inhibitors. These findings support clinical trials of ADT-1004 for KRAS mutant PDAC.

e15084 Background: Pancreatic ductal adenocarcinoma (PDAC) remains a formidable challenge due to its late diagnosis and limited treatment options. KRAS mutations and activation of Wnt/β-catenin pathway components that drive aberrant signaling cascades resulting in uncontrolled cellular proliferation and metastasis occur in a majority of PDAC patients. The development of mutant specific KRAS inhibitors has emerged as a promising approach to counter the oncogenic impact of KRAS mutations, although have limited use for PDAC given the complex mutational landscape, while inhibitors of Wnt/β-catenin signaling remain elusive. Novel target-directed drugs that effectively tackle the complex mutational status in KRAS mutant PDAC are urgently needed. A novel pan-RAS/β-catenin inhibitor, ADT-030, was evaluated in mouse tumor models of PDAC harboring KRASG12C or KRASG12D mutations. Methods: PDAC cell lines 2838c3 (KRASG12D), MIA PaCa-2 (KRASG12C), and BxPC-3 (RASWT) were utilized to evaluate in vitro growth inhibitory potency and selectivity of ADT-030. Assays for colony formation, apoptosis, and cell cycle, along with western blotting for RAS signaling were also conducted. A murine model involving 2838c3-Luc KRASG12D PDAC cells injected into the pancreas of C57BL/6J mice was used to evaluate in vivo antitumor activity of ADT-030. ADT-030 was administered orally once daily at dosages of 50, 100, and 150mg/kg body weight, 5x/week for 4 weeks. Tumor growth was monitored by bioluminescence imaging using an IVIS Xenogen system. Body weights were measured twice weekly. Pancreatic tumors were collected and weighed at the end of treatment. Results: ADT-030 produced potent inhibition of proliferation of human and murine PDAC cells harboring KRASG12D and KRASG12C mutations. Annexin V assay revealed the capacity of ADT-030 to trigger apoptosis, specifically in KRAS mutant cells with minimal impact on BxPC3 RASWT cells. Cell cycle analysis using Propidium Iodide staining revealed the ability of ADT-030 to arrest cells in the G2/M phase of the cell cycle. Western blot for detecting the key RAS signaling molecule P-ERK1/2 showed decreased levels following ADT-030 treatment. Other experiments revealed that ADT-030 and related analogs simultaneously suppress both MAPK/AKT signaling and β-catenin transcriptional activity (doi: 10.1186/s13048-022-01050-9). In an orthotopic xenograft model in which 2838c3-Luc cells were implanted in the pancreas, oral administration of 50, 100, and 150mg/kg of ADT-030 was well tolerated and inhibited tumor growth by 64.2%, 72.8%, and 80%, respectively, after 4 weeks of treatment. Conclusions: These compelling results position ADT-030 as a novel dual pan-RAS/β-catenin inhibitor for combating the challenges posed by complex mutations in PDAC and other RAS driven cancers and support its clinical evaluation as a valuable therapeutic alternative to mutant-specific KRAS inhibitors.

90 Background: Pancreatic ductal adenocarcinoma (PDAC) is a challenging cancer with a 5-year survival rate of under 12%. More than 90% of PDAC cases involve KRAS mutations. KRASG12C inhibitors recently developed have limited use because only 3% of PDAC express this mutation, highlighting the urgent need for pan-RAS inhibitors to address the complex mutation landscape in PDAC while avoiding resistance mechanisms. We synthesized and evaluated a novel pan-RAS inhibitor, ADT-1004, in mouse tumor models transplanted with mouse PDAC cell lines or patient-derived xenografts. ADT-1004 is an orally bioavailable prodrug of ADT-007, a highly potent and selective pan-RAS inhibitor (doi.org/10.1101/2023.05.17.541233). Methods: Human and mouse PDAC cell lines were utilized to evaluate in vitro growth inhibitory potency and selectivity of ADT-007. For in vivo experiments, KPC-Luc (1 × 105) and 2838C3-Luc (1.5 × 105) PDAC cells harboring the KRASG12D mutation were injected into the pancreas of C57BL/6J mice. After one week, the mice were randomly divided into treatment groups (n = 7 per group) and received oral ADT-1004 at a dose of 40mg/kg body weight five times a week for 4 weeks. Tumor burden was assessed weekly by monitoring bioluminescence signals using the IVIS Xenogen imaging system. Four patient-derived xenografts from PDAC patients with KRASG12C, KRASG12D, KRASG12V, KRASG13Q mutations were subcutaneously implanted in NSG mice by a small incision in the right flank. A week later, when the tumors reached a size of 100mm3 mice were randomized (n=7 per group) and treated orally with 40mg/kg of ADT-1004 five times a week for 6 weeks. Body weight and tumor size were measured twice weekly. Results: ADT-007 potently and selectively inhibited the growth of human and mouse PDAC cell lines. For example, the IC50 value pf ADT-007 for KRASG12C MIA PaCA-2 PDAC cells was as low as 2 nM compared to 2500 nM for RASWT BxPC3 PDAC cells. Growth inhibition was dependent on activated RAS and associated with reduced GTP-RAS levels and MAPK/AKT signaling. ADT-1004 was well tolerated in mice at a dose up to 175 mg/kg bid orally with sustained plasma levels of ADT-007 far exceeding growth IC50 values. When administered orally at 40 mg/kg body weight, ADT-1004 displayed robust antitumor activity in mouse tumor models using patient or mouse derived PDAC cell lines with G12D, G12V, G12C, or G13Q KRAS mutations, causing significant inhibitory effects on ERK phosphorylation. Conclusions: ADT-1004 inhibited tumor growth of KRAS mutant PDA tumors by targeting activated RAS to disrupt downstream MAPK/AKT signaling. The results highlight the promise of ADT-1004 as a novel pan-RAS inhibitor and open new strategies for addressing the complex genetic landscape of PDAC and other RAS driven cancers.

Here, we describe a novel pan-RAS inhibitor, ADT-007, that potently inhibited the growth of RAS mutant cancer cells irrespective of the RAS mutation or isozyme. RAS WT cancer cells with GTP-activated RAS from upstream mutations were equally sensitive. Conversely, RAS WT cancer cells harboring downstream BRAF mutations and normal cells were essentially insensitive to ADT-007. Sensitivity of cancer cells to ADT-007 required activated RAS and dependence on RAS for proliferation, while insensitivity was attributed to metabolic deactivation by UDP-glucuronosyltransferases expressed in RAS WT and normal cells but repressed in RAS mutant cancer cells. ADT-007 binds nucleotide-free RAS to block GTP activation of effector interactions and MAPK/AKT signaling, resulting in mitotic arrest and apoptosis. ADT-007 displayed unique advantages over mutant-specific KRAS and pan-KRAS inhibitors, as well as other pan-RAS inhibitors that could impact in vivo antitumor efficacy by escaping compensatory mechanisms leading to resistance. Local administration of ADT-007 showed robust antitumor activity in syngeneic immune-competent and xenogeneic immune-deficient mouse models of colorectal and pancreatic cancer. The antitumor activity of ADT-007 was associated with the suppression of MAPK signaling and activation of innate and adaptive immunity in the tumor immune microenvironment. Oral administration of ADT-007 prodrug also inhibited tumor growth, supporting further development of this novel class of pan-RAS inhibitors for RAS-driven cancers. SIGNIFICANCE:ADT-007 has unique pharmacological properties with distinct advantages over other RAS inhibitors by circumventing resistance and activating antitumor immunity. ADT-007 prodrugs and analogs with oral bioavailability warrant further development for RAS-driven cancers.

ABSTRACT 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 growth in vivo through 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 vivo analyses of tumor infiltrating leukocytes, reveals that ADT-007 enhances T cell functions in the pancreatic and colorectal tumor immune microenvironment. SIGNIFICANCE ADT-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.

# Abstract 45. Determining clinically important improvement following surgery for degenerative conditions of the spine: analysis of the Canadian Spine Outcomes and Research Network (CSORN) Registry {#article-title-2} There is significant variability in clinically important improvement (CII)

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.

We previously reported that phosphodiesterase 10A (PDE10) is overexpressed during early stages of lung cancer and is essential for lung tumor cell growth. Here we characterize a novel PDE10 inhibitor, MCI-048, that was identified by screening a library of indene compounds. MCI-048 potently inhibited the growth and induced apoptosis of multiple human lung cancer cell lines expressing PDE10, while normal human airway epithelial cells lacking PDE10 expression were appreciably less sensitive. The mechanism of action of MCI-048 involves PDE10 inhibition, cGMP elevation, PKG activation, and phosphorylation of β-catenin at key residues that induce ubiquitination and proteosomal degradation of the oncogenic pool of β-catenin in cytoplasm to suppress the translocation of active β-catenin to the nucleus and Lef/Tcf-mediated transcription of genes encoding for proteins such as c-myc, cyclin D, and survivin, which are essential for tumor cell proliferation and survival. Pharmacokinetic and tissue distribution studies revealed a unique feature of MCI-048 to accumulate at high concentrations in lungs relative to plasma and other tissues. To assess the potential of MCI-048 for the treatment of lung cancer and blocking metastasis, we tested the drug in two mouse models of lung cancer involving either orthotopic implantation of KRAS mutant A549 lung tumor cells or the chemical carcinogen, urethane. Oral administration of MCI-048 significantly inhibited tumor growth and extended survival in the orthotopic model using two different protocols to either treat the primary tumor or metastasis. Similarly, MCI-048 significantly reduced tumor burden as measured by both surface counting and histopathological analysis in the urethane-induced model of lung tumorigenesis. Biochemical analysis showed that MCI-048 reduced levels of urethane-induced active Ras-GTP and PDE10 levels, as well as other oncogenic markers, including pEGFR and c-Myc. Both mouse models revealed that MCI-048 was well tolerated with no discernable toxicity, thus supporting preclinical development for the treatment of lung cancer. Funding provided by NCI grants R21CA182941, R01CA131378, R01CA148817, R01CA197147, and R01CA155638. Citation Format: Bing Zhu, Veronica Ramirez-Alcantara, Antonio Ward, Kristy Berry, Adam B. Keeton, Michael R. Boyd, Yulia Maxuitenko, Xi Chen, Gary A. Piazza. A novel PDE10/beta-catenin inhibitor, MCI-048, suppresses lung tumorigenesis to block metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2731.

Oncogenic mutations in RAS genes result in an intracellular elevation of active RAS and increased cellular propagation through downstream signaling pathways responsible for tumor cell growth and survival. Mutations in KRAS drive over 90% of pancreatic ductal adenocarcinomas (PDACs), presenting promising therapeutic potential for a RAS inhibitor. However, many have deemed RAS “undruggable” as a result of its relatively smooth structure and its high affinity for guanine nucleotide substrates. We have identified a novel series of indene derivatives that potently and selectively inhibits the growth of tumor cells with high levels of active RAS, while having minimal effects on tumor cells lacking constitutively active RAS, or cells derived from normal tissues. Here we report that our lead compound, MCI-062, potently and selectively inhibits the growth of KRAS mutant MIA PaCa-2 PDAC cells harboring oncogenic KRAS with an IC50 value of approximately 5 nM and greater than 300-fold selectivity over BxPC-3 PDAC cells that lack constitutively active RAS. MCI-062 also completely inhibits colony formation of a panel of PDAC cell lines with various KRAS mutations at low nanomolar concentrations. MCI-062 treatment of MIA PaCa-2 cells in basal and EGF-stimulated conditions depletes RAS-GTP levels in a dose-dependent manner. Consequently, MCI-062 treatment leads to inhibition of downstream MAPK and AKT signaling, cell cycle arrest, and induction of apoptosis in MIA PaCa-2 cells. Further investigation of the mechanism of action in cell-free systems demonstrated that this class of compounds inhibits GTP loading of RAS. MCI-062 inhibits binding of MANT-GTP to recombinant K-RAS in a dose-dependent manner. In a functional assay, MCI-062 reduces RAS-RAF-RBD binding when recombinant K-RAS is treated in a nucleotide-free state, but not when K-RAS is treated in a nucleotide-bound state. Testing in 3D spheroid models involving MIA PaCA-2 tumor cells revealed that MCI-062 potently inhibits growth of non-adherent cells. MCI-062 also has anti-tumor activity in a KRAS mutant CT26 mouse tumor xenograft model with no discernable toxicity. Western blotting of tumor lysates from mice treated with MCI-062 showed a reduction of active, GTP-bound RAS when compared to tumor lysates from control mice. These results provide in vitro and in vivo evidence that MCI-062 inhibits RAS-driven tumor cell growth by blocking GTP loading of RAS, supporting further evaluation of this novel class of RAS inhibitors for the treatment of pancreatic cancer, as well as other RAS-driven cancers. Funding provided by NCI grants R01CA131378, R01CA148817, R01CA197147, and R01CA155638. Citation Format: Tyler E. Mattox, Xi Chen, Jacob Valiyaveettil, Yulia Maxuitenko, Bing Zhu, Antonio Ward, Veronica Ramirez-Alcantara, Kristy Berry, Michael Boyd, Adam Keeton, Gary A. Piazza. Novel RAS inhibitor, MCI-062, potently and selectively inhibits the growth of KRAS mutant pancreatic tumor cells by blocking GTP loading of RAS [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 345.

Approximately 45% of colorectal cancers harbor mutations in the KRAS gene, resulting in constitutive activation of RAS signaling through effector proteins, primarily RAF and PI3K. Members of the RAS family are GTPases that function as a molecular switch, cycling between inactive (GDP-bound) and active (GTP-bound) states in cells to regulate proliferation and survival. Constitutive signaling from mutant RAS drives tumorigenesis, in part, by deregulation of the cell cycle, resulting in increased proliferation (mitosis) and decreased apoptosis of tumor cells. From an extensive medicinal chemistry/screening campaign, we identified a novel compound class that potently and selectively inhibits the growth of tumor cell lines harboring constitutively activated RAS by a mechanism involving the disruption of RAS nucleotide loading, blockage of effector activation, and consequent inhibition of signaling. A prototype of the class, MCI-062, inhibits the growth of KRAS-driven (HCT-116) colon tumor cells with low nanomolar IC50 values, while RAS-independent (HT-29) colon tumor cells harboring the BRAF V600E mutation, are essentially insensitive. A strong correlation was measured among a larger panel of tumor cell lines between potency to inhibit tumor cell growth and levels of activated RAS. The growth inhibitory effects of MCI-062 were sustained and irreversible as demonstrated by colony formation and apoptosis assays. MCI-062 treatment of HCT-116 colon tumor cells reduced levels of activated RAS and RAS-mediated signaling as measured by GST-RBD pulldown assays and phospho-specific immunoblotting. Within the same concentration range, MCI-062 induced mitotic arrest as measured by cell cycle analysis of DNA content and phospho-histone H3B immunofluorescence. Further analysis revealed that MCI-062 interfered with localization of the mitosis-inducing protein, PLK1 to kinetochores and decreased nuclear localization of its substrate, Cdc25C, a downstream target of RAS-RAF signaling involved in both mitotic entry and exit checkpoints. In vivo testing of MCI-062 in a syngeneic mouse model of KRAS-driven colon cancer (CT-26) demonstrated that MCI-062 engages its molecular target, depleting GTP-RAS and suppressing activation of the MAPK signal transduction pathway, and inhibiting tumor growth. MCI-062 also suppresses PD-L1 expression and activates anti-tumor immunity, which may contribute to its antitumor activity and suggests potential benefits of combining with immunotherapy. From these studies, we have identified a novel class of RAS inhibitors that potently and selectively inhibits RAS-driven tumor growth by disrupting downstream signaling, leading to cell cycle arrest and apoptosis. These findings support further development of MCI-062 for treatment of RAS-driven colorectal and other cancers. Funding provided by NCI grants R01CA131378, R01CA148817, R01CA197147, and R01CA155638. Citation Format: Adam B. Keeton, Antonio Ward, Xi Chen, Jacob Valiyaveettil, Bing Zhu, Veronica Ramirez-Alcantara, Yulia Maxuitenko, Kristy Berry, Tyler E. Mattox, Michael R. Boyd, Gary A. Piazza. A novel RAS inhibitor, MCI-062, inhibits colon tumor growth in vivo and activates antitumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2707.

Over 90% of colorectal cancers harbor mutations in β-catenin or pathway components (e.g. APC) that stabilize β-catenin, causing nuclear translocation and constitutive Tcf-mediated transcription of genes encoding proteins essential for the proliferation and survival of tumor cells. We recently reported that the cyclic nucleotide degrading phosphodiesterase (PDE) isozyme PDE10 is overexpressed in colorectal cancers relative to normal tissue. Its expression and enzymatic activity are essential for colon tumor cell growth, as evidenced by knockdown of PDE10 expression using siRNA or inhibition of enzyme activity using known inhibitors such as PF-2545920. PDE10 inhibition in tumor cells expressing high levels of PDE10 causes increased intracellular cGMP levels to activate PKG and phosphorylate β-catenin, which induces ubiquitination and proteasomal degradation to suppress nuclear translocation and Tcf transcriptional activity. Conversely, ectopic expression of PDE10 in normal colonocytes or precancerous adenoma cells causes increased levels of β-catenin and the expression of proteins (e.g. cyclin D and survivin) essential for the proliferation and survival of tumor cells. To identify novel antitumor PDE10 inhibitors, we screened a chemically diverse library of indenes for PDE10 and tumor cell growth inhibitory activity. Following extensive chemical optimization, MCI-030 emerged as a potent and selective inhibitor of tumor cell growth. Similar to PF-2545920, but with appreciably greater potency and tumor cell selectivity, MCI-030 inhibited colon tumor cell growth by activating cGMP/PKG signaling to phosphorylate and induce β-catenin degradation. MCI-030 also inhibited colon tumor cell spheroid formation and reduced spheroid size and growth at concentrations that inhibit PDE10. Oral administration of MCI-030 significantly inhibited colon tumor formation in the Apc+/min-FCCC mouse model without discernable toxicity. Importantly, unlike PDE10 inhibitors developed to cross the blood-brain barrier for the treatment of CNS disorders, MCI-030 lacks the sedation side effects. Together, these findings support preclinical development of MCI-030 for the treatment of colorectal cancer as a novel PDE10 inhibitor capable of selectively inhibiting the growth of tumors harboring β-catenin or APC mutations. Funding provided by NCI grants R01CA131378, R01CA148817, R01CA197147, and R01CA155638. Citation Format: Antonio B. Ward, Xi Chen, Jacob Valiyaveettil, Kevin Lee, Wen-Chi L. Chang, Yulia Maxuitenko, Veronica Ramirez-Alcantara, Kristy Berry, Luciana Madeira da Silva, Bing Zhu, Tyler Mattox, Michael R. Boyd, Adam B. Keeton, Margie L. Clapper, Harry S. Cooper, Gary A. Piazza. A novel PDE10/β-catenin pathway inhibitor, MCI-030, for the treatment of colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3864.

Abstract Gain-of-function mutations in RAS genes occur at high frequency in several types of cancer, including pancreatic, colorectal, and lung adenocarcinomas. Such driver mutations result in constitutive activation of RAS and its downstream signaling pathways, promoting tumor cell proliferation, survival, and metastasis. The development of drugs directly targeting RAS has been hindered due to the lack of suitable surfaces on the protein for small-molecule binding, as well as its high affinity for GTP binding. We recently identified a novel series of indene derivatives that showed highly selective growth inhibitory activity in tumor cells harboring constitutively active RAS versus tumor cells with low levels of active RAS. Chemical optimization resulted in series of compounds that potently and selectively inhibit RAS-dependent tumor cell growth by blocking RAS-effector interactions. High-grade serous ovarian carcinoma (HGSOC) is invariably characterized by the key driver mutation in TP53, but other mutational drivers, such as mutations or altered methylation of BRCA1 and BRCA2, cyclin E1, PIK3CA and AKT1/2 amplifications, and loss of NF1, RB1 and PTEN, are also commonly found. Conversely, RAS mutations are usually associated with low-grade serous ovarian carcinoma (LGSOC) and mucinous ovarian tumors. Here we show that 5 (OVCAR5, OVCAR8, ES2, KURAMOCHI, and IGROV1) out of 12 ovarian cancer cell lines tested have high levels of constitutive RAS activation as measured by the active RAS pull-down assay, comparable or higher than those of MIA PaCa-2 pancreatic cancer cells, which harbors the activating mutation G12C on KRAS. The 7 remainder cell lines tested (A2780, SKOV3ip, CAOV3, OVCAR4, OVCAR3, OVSAHO, and OV90) had levels of active RAS comparable or lower than BxPC-3 pancreatic cells, which lacks constitutively active RAS. Interestingly, most ovarian cancer cell lines were highly sensitive to ADT-006 (IC50 ~20 nM) in vitro, with the exception of OV-90 cells, which showed IC50 ~300 nM and the lowest level of active RAS measured by the pull-down assay. Treatment of intact SKOV3ip and OVCAR8 cells with ADT-006 inhibited RAF/MAPK and PI3K/AKT phosphorylation within the same concentration range as the growth inhibitory activity of this compound. Xenograft studies performed with SKOV3ip cells implanted intraperitoneally in athymic nude mice and treated twice daily with ADT-006 i.p. at the dose of 10 mg/kg for two weeks showed significant antitumor activity with no discernible toxicity. Our results demonstrate that ADT-006 inhibits HGSOC cell growth by blocking RAS-effector interactions, and supports further evaluation of our novel RAS inhibitors for the treatment of ovarian cancer. Citation Format: Luciana Madeira da Silva, Tyler E. Mattox, Adam B. Keeton, Bing Zhu, Kristy L. Berry, Alla Musiyenko, Elaine Gavin, Kevin Lee, Veronica Ramirez-Alcantara, Yulia Y. Maxuitenko, Xi Chen, Jacob Valiyaveettil, Michael R. Boyd, Jennifer Scalici, Rodney Rocconi, Gary A. Piazza. Targeting constitutively active RAS signaling in high-grade serous ovarian carcinoma (HGSOC) with ADT-006, a novel small molecule that blocks RAS-effector interactions [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr B54.

Abstract 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 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 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 A high percentage of human cancers arise from mutations in the ras gene that encodes an abnormal Ras protein locked in a constitutively active GTP-bound state that promotes tumor cell proliferation, survival, and metastasis. These gain-in-function mutations in the ras gene or constitutive activation of tyrosine kinase receptors upstream of the Ras protein drive tumor cell growth by activating Raf/MAPK and PI3K/AKT signaling pathways. Ras has been an elusive drug target for which no inhibitors are available to treat Ras-driven cancers. Screening a library of indene derivatives in a differential phenotypic assay identified a novel compound class displaying high potency and selectivity to inhibit the growth of tumor cells harboring mutant Ras relative to tumor cells with wild type (WT) Ras. Lead optimization resulted in a drug development candidate (DC070-547) and several back-up analogs (e.g. ADT-006) with IC50 values in the low nanomolar range and selectivity indices of 100 fold or greater to inhibit the growth of tumor cells with constitutively activated Ras relative to tumor cells with low levels of activated Ras. Sensitivity among a large panel of tumor cell lines to this compound class strongly correlated with levels of activated Ras, but did not appear to be limited to a specific ras gene mutation or Ras protein isoform. Here, we report that DC070-547 and ADT-006 potently and selectively inhibit the growth of the human melanoma cell line, SK-MEL-2, harboring a mutation in the ras gene that encodes the constitutively active N-Ras protein with IC50 values of 7 and 25 nM, respectively. In addition, both compounds potently inhibited the growth of the murine melanoma cell line, B16-F10 with WT ras, but harboring a mutation in a tyrosine kinase receptor upstream of Ras, specifically PDGFRα, which results in high levels of active, GTP-bound Ras as confirmed by Ras-RBD pull-down assays. To determine if treatment can disrupt Ras signaling, B16-F10 cells were incubated with the compounds before being subjected to Western blotting for phosphorylated signaling molecules downstream of Ras. Treatment reduced levels of phosphorylated c-Raf and MEK at concentrations that inhibit tumor cell growth. These findings support further investigation of this novel class of Ras inhibitors for the treatment of Ras-driven melanoma. Citation Format: Kate M. Saville, Kevin Lee, Tyler E. Mattox, Xi Chen, Jacob Valiyaveettil, Kristy Berry, Veronica Ramírez-Alcántara, Bing Zhu, Adam Keeton, Michael Boyd, Gary Piazza, Ashley S. Lindsey. Sensitivity of melanoma cells to a novel class of Ras inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5166. doi:10.1158/1538-7445.AM2017-5166

Abstract 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