The Anti-Proliferative Effect of PI3K/mTOR and ERK Inhibition in Monolayer and Three-Dimensional Ovarian Cancer Cell Models

Most ovarian cancer patients are diagnosed with advanced stage disease, which becomes unresponsive to chemotherapeutic treatments. The PI3K/AKT/mTOR and the RAS/RAF/MEK/ERK kinase signaling pathways are attractive targets for potential therapeutic inhibitors, due to the high frequency of mutations to PTEN, PIK3CA, KRAS and BRAF in several ovarian cancer subtypes. However, monotherapies targeting one of these pathways have shown modest effects in clinical trials. This limited efficacy of the agents could be due to upregulation and increased signaling via the adjacent alternative pathway. In this study, the efficacy of combined PI3K/mTOR (BEZ235) and ERK inhibition (SCH772984) was investigated in four human ovarian cancer cell lines, grown as monolayer and three-dimensional cell aggregates. The inhibitor combination reduced cellular proliferation in a synergistic manner in OV-90 and OVCAR8 monolayers and in OV-90, OVCAR5 and SKOV3 aggregates. Sensitivity to the inhibitors was reduced in three-dimensional cell aggregates in comparison to monolayers. OV-90 cells cultured in large spheroids were sensitive to the inhibitors and displayed a robust synergistic antiproliferative response to the inhibitor combination. In contrast, OVCAR8 spheroids were resistant to the inhibitors. These findings suggest that combined PI3K/mTOR and ERK inhibition could be a useful strategy for overcoming treatment resistance in ovarian cancer and warrants further preclinical investigation. Additionally, in some cell lines the use of different three-dimensional models can influence cell line sensitivity to PI3K/mTOR and RAS/RAF/MEK/ERK pathway inhibitors.

Combined SHP2 and ERK inhibition for the treatment of KRAS-driven Pancreatic Ductal Adenocarcinoma

Mutant KRAS is present in over 90% of pancreatic as well as 30-40% of lung and colorectal cancers and is one of the most common oncogenic drivers. Despite decades of research and the recent emergence of isoform-specific KRASG12C-inhibitors, most mutant KRAS isoforms, including the ones frequently associated with pancreatic ductal adenocarcinoma (PDAC), cannot be targeted directly. Moreover, targeting single RAS downstream effectors induces adaptive mechanisms leading to tumor recurrence or resistance. We report here on the combined inhibition of SHP2, a non-receptor tyrosine phosphatase upstream of KRAS, and ERK, a serine/threonine kinase and a key molecule downstream of KRAS in PDAC. This combination shows synergistic anticancer activity in vitro, superior disruption of the MAPK pathway, and significantly increased apoptosis induction compared to single-agent treatments. In vivo, we demonstrate good tolerability and efficacy of the combination. Concurrent inhibition of SHP2 and ERK induces significant tumor regression in multiple PDAC mouse models. Finally, we show evidence that 18F-FDG PET scans can be used to detect and predict early drug responses in animal models. Based on these compelling results, we will investigate this drug combination in a clinical trial (SHERPA, SHP2 and ERK inhibition in pancreatic cancer, NCT04916236), enrolling patients with KRAS-mutant PDAC.

Statins and Bempedoic Acid: Different Actions of Cholesterol Inhibitors on Macrophage Activation

Statins represent the most prescribed class of drugs for the treatment of hypercholesterolemia. Effects that go beyond lipid-lowering actions have been suggested to contribute to their beneficial pharmacological properties. Whether and how statins act on macrophages has been a matter of debate. In the present study, we aimed at characterizing the impact of statins on macrophage polarization and comparing these to the effects of bempedoic acid, a recently registered drug for the treatment of hypercholesterolemia, which has been suggested to have a similar beneficial profile but fewer side effects. Treatment of primary murine macrophages with two different statins, i.e., simvastatin and cerivastatin, impaired phagocytotic activity and, concurrently, enhanced pro-inflammatory responses upon short-term lipopolysaccharide challenge, as characterized by an induction of tumor necrosis factor (TNF), interleukin (IL) 1β, and IL6. In contrast, no differences were observed under long-term inflammatory (M1) or anti-inflammatory (M2) conditions, and neither inducible NO synthase (iNOS) expression nor nitric oxide production was altered. Statin treatment led to extracellular-signal regulated kinase (ERK) activation, and the pro-inflammatory statin effects were abolished by ERK inhibition. Bempedoic acid only had a negligible impact on macrophage responses when compared with statins. Taken together, our data point toward an immunomodulatory effect of statins on macrophage polarization, which is absent upon bempedoic acid treatment.

HER2 + breast cancers evade anti-HER2 therapy via a switch in driver pathway

Inhibition of HER2 in HER2-amplified breast cancer has been remarkably successful clinically, as demonstrated by the efficacy of HER-kinase inhibitors and HER2-antibody treatments. Whilst resistance to HER2 inhibition is common in the metastatic setting, the specific programs downstream of HER2 driving resistance are not established. Through genomic profiling of 733 HER2-amplified breast cancers, we identify enrichment of somatic alterations that promote MEK/ERK signaling in metastatic tumors with shortened progression-free survival on anti-HER2 therapy. These mutations, including NF1 loss and ERBB2 activating mutations, are sufficient to mediate resistance to FDA-approved HER2 kinase inhibitors including tucatinib and neratinib. Moreover, resistant tumors lose AKT dependence while undergoing a dramatic sensitization to MEK/ERK inhibition. Mechanistically, this driver pathway switch is a result of MEK-dependent activation of CDK2 kinase. These results establish genetic activation of MAPK as a recurrent mechanism of anti-HER2 therapy resistance that may be effectively combated with MEK/ERK inhibitors.

ERK1/2 Inhibition Overcomes Resistance in Acute Myeloid Leukemia (AML) and Alters Mitochondrial Dynamics

Background: Presence at diagnosis or acquisition of activating RAS pathway mutations is a pervasive mechanism of resistance to therapy in AML. Efforts to directly target mutant RAS have been unsuccessful and the efficacy of BRAF and MEK inhibitors has been limited due to compensatory reactivation of MAPK signaling. ERK1/2 (ERK) is a key downstream component in the MAPK pathway and therefore represents an attractive target for inhibiting MAPK signaling. Compound 27 (1) is a dual-mechanism inhibitor of ERK that inhibits both the catalytic activity of ERK and its phosphorylation by MEK. It is a close analog of ASTX029, a dual-mechanism ERK inhibitor currently under clinical investigation in solid tumors (NCT03520075). Objectives: We analysed the preclinical activity of Compound 27 in AML, investigated its mechanism of action and ability to overcome resistance. Results: Using a panel of 9 AML cell lines, the IC50 value for single agent Compound 27 was in the low to intermediate nanomolar range (1.89-388 nM). Decreased ERK phosphorylation was confirmed by Western blot analysis. To better characterize the biological effects of Compound 27, we performed mass cytometry (CyTOF) analysis of NRAS-mutated OCI-AML3 cells. This experiment showed approximately 75% downregulation of CyclinB1 and cMyc in 250 nM drug-treated cells versus untreated cells (Figure 1a). The expression of anti-apoptotic proteins, including MCL1, BclXL and Bcl2, were also decreased. Western blot analysis confirmed increased cleaved PARP, and reduced cMyc and cell cycle-related proteins CyclinB1, CyclinD1 and CDK4 with Compound 27 treatment. In isogenic cells, p53 knock-down had no effect on the efficacy of Compound 27. We next investigated the efficacy of simultaneous inhibition of ERK and Bcl-2 in AML cells. Compound 27 sensitized OCI-AML3 cells, which are intrinsically resistant to ABT-199 (a Bcl-2 inhibitor), to treatment with ABT-199 and shifted the cytostatic effect of the single agents to a cytotoxic effect with a combination index (CI) of 0.008 (cell death 91% for combination versus 20% with ABT-199 alone). This suggests strong synergistic effects of combination treatment (Figure 1b). In OCI-AML2 cells with acquired resistance to ABT-199, the combination increased apoptosis to 80% as compared to 20% with ABT-199 alone. Compound 27 sensitized bulk CD45+ as well as CD34+CD38-leukemia progenitor cells to ABT-199. Compound 27 also sensitized FLT3-ITD mutant human AML cell lines MOLM13, MOLM14, MV-4-11 and murine Ba/F3-ITD cells to the FLT3 inhibitor AC220 (CI in MOLM13=0.3). Synergy of Compound 27 and 5-azacitidine was also observed (p=0.009). Leukemia microenvironment-mediated resistance to therapy is partly mediated by MAPK activation. We co-cultured OCI-AML3 and MOLM13 cells with normal bone marrow-derived mesenchymal stromal cells (NMSCs) to mimic the bone marrow microenvironment and analysed the effect of Compound 27 in combination with either ABT-199 or AC220. Combination drug treatment were more effective in terms of cytoreduction and apoptosis induction in coculture. However, neither combination was able to completely overcome stroma-mediated resistance (Figure 1b). Analysis of other stroma-relevant molecules in coculture showed that CXCR4 was increased while CD44 was decreased in response to ERK inhibition. Effective reactive oxygen species (ROS) mitigation and hyper-active mitochondrial fission is important for maintaining "stemness" of AML cells (2). ERK phosphorylates DRP1, which is necessary for mitochondrial fission. Treatment of OCI-AML3 cells with Compound 27 led to increased mitochondrial ROS, decreased levels of pDRP1(Ser616) and increased mitochondrial length, suggesting impaired fission and reduced "stemness" of AML cells (Figure 1c). Conclusion: ERK inhibition by Compound 27 synergizes with 5-azacitidine, ABT-199 and AC220 and can overcome primary or acquired resistance. The impact on mitochondrial dynamics suggests a potential impact on leukemia stem cells. Additional mechanistic confirmatory work is in progress. References: 1. Heightman TD, Berdini V, Braithwaite H, et al. Fragment-based discovery of a potent, orally bioavailable inhibitor that modulates the phosphorylation and catalytic activity of ERK1/2. J Med Chem. 2018;61(11):4978-4992. 2. Schimmer AD. Mitochondrial Shapeshifting Impacts AML Stemness and Differentiation. Cell Stem Cell. 2018;23(1):3-4. Figure 1 Figure 1. Disclosures Hindley: Astex Pharmaceuticals: Current Employment. Dao: Astex Pharmaceuticals, Inc.: Current Employment. Sims: Astex Pharmaceuticals: Current Employment. Andreeff: Medicxi: Consultancy; Syndax: Consultancy; Aptose: Consultancy; ONO Pharmaceuticals: Research Funding; AstraZeneca: Research Funding; Amgen: Research Funding; Reata, Aptose, Eutropics, SentiBio; Chimerix, Oncolyze: Current holder of individual stocks in a privately-held company; Breast Cancer Research Foundation: Research Funding; Karyopharm: Research Funding; Glycomimetics: Consultancy; Senti-Bio: Consultancy; Oxford Biomedica UK: Research Funding; Daiichi-Sankyo: Consultancy, Research Funding; Novartis, Cancer UK; Leukemia & Lymphoma Society (LLS), German Research Council; NCI-RDCRN (Rare Disease Clin Network), CLL Foundation; Novartis: Membership on an entity's Board of Directors or advisory committees. Borthakur: University of Texas MD Anderson Cancer Center: Current Employment; Takeda: Membership on an entity's Board of Directors or advisory committees; Astex: Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Ryvu: Research Funding; ArgenX: Membership on an entity's Board of Directors or advisory committees; Protagonist: Consultancy; GSK: Consultancy.

Spatial Regulation of Reactive Oxygen Species via G6PD in Brown Adipocytes Supports Thermogenic Function

Reactive oxygen species (ROS) are associated with various roles of brown adipocytes. Glucose-6-phosphate dehydrogenase (G6PD) controls cellular redox potentials by producing NADPH. Although G6PD upregulates cellular ROS levels in white adipocytes, the roles of G6PD in brown adipocytes remain elusive. Here, we found that G6PD defect in brown adipocytes impaired thermogenic function through excessive cytosolic ROS accumulation. Upon cold exposure, G6PD-deficient mutant (G6PD^mut) mice exhibited cold intolerance and downregulated thermogenic gene expression in brown adipose tissue (BAT). In addition, G6PD-deficient brown adipocytes had increased cytosolic ROS levels, leading to ERK activation. In BAT of G6PD^mut mice, administration of antioxidant restored the thermogenic activity by potentiating thermogenic gene expression and relieving ERK activation. Consistently, body temperature and thermogenic execution were rescued by ERK inhibition in cold-exposed G6PD^mut mice. Taken together, these data suggest that G6PD in brown adipocytes would protect against cytosolic oxidative stress, leading to cold-induced thermogenesis.

Spatial Regulation of Reactive Oxygen Species via G6PD in Brown Adipocytes Supports Thermogenic Function

Reactive oxygen species (ROS) are associated with various roles of brown adipocytes. Glucose-6-phosphate dehydrogenase (G6PD) controls cellular redox potentials by producing NADPH. Although G6PD upregulates cellular ROS levels in white adipocytes, the roles of G6PD in brown adipocytes remain elusive. Here, we found that G6PD defect in brown adipocytes impaired thermogenic function through excessive cytosolic ROS accumulation. Upon cold exposure, G6PD-deficient mutant (G6PD^mut) mice exhibited cold intolerance and downregulated thermogenic gene expression in brown adipose tissue (BAT). In addition, G6PD-deficient brown adipocytes had increased cytosolic ROS levels, leading to ERK activation. In BAT of G6PD^mut mice, administration of antioxidant restored the thermogenic activity by potentiating thermogenic gene expression and relieving ERK activation. Consistently, body temperature and thermogenic execution were rescued by ERK inhibition in cold-exposed G6PD^mut mice. Taken together, these data suggest that G6PD in brown adipocytes would protect against cytosolic oxidative stress, leading to cold-induced thermogenesis.

Inhibition of PI3K/Akt and ERK signaling decreases visfatin-induced invasion in liver cancer cells

Objectives Visfatin is found in adipose tissue and is referred to as nicotinamide phosphoribosyltransferase (Nampt). Visfatin has anti-apoptotic, proliferative, and metastatic properties and may mediate its effects via ERK and PI3K/Akt signaling. Studies have yet to determine whether inhibition of kinase signaling will suppress visfatin-induced liver cancer. The purpose of this study was to determine which signaling pathways visfatin may promote liver cancer progression. Methods HepG2 and SNU-449 liver cancer cells were exposed to visfatin with or without ERK or PI3K/Akt inhibitor, or both inhibitors combined. These processes that were assessed: proliferation, reactive oxygen species (ROS), lipogenesis, invasion, and matrix metalloproteinase (MMP). Results Inhibition of PI3K/Akt and combination of inhibitors suppressed visfatin-induced viability. ERK inhibition in HepG2 cells decreased visfatin-induced proliferation. ERK inhibitor alone or in combination with PI3K inhibitors effectively suppressed MMP-9 secretion and invasion in liver cancer cells. PI3K and ERK inhibition and PI3K inhibition alone blocked visfatin’s ROS production in SNU-449 cells. These results corresponded with a decrease in phosphorylated Akt and ERK, β-catenin, and fatty acid synthase. Conclusions Akt and ERK inhibition differentially regulated physiological changes in liver cancer cells. Inhibition of Akt and ERK signaling pathways suppressed visfatin-induced invasion, viability, MMP-9 activation, and ROS production.