No abstract

Deregulation of the Ras–mitogen activated protein kinase (MAPK) pathway is an early event in many different cancers and a key driver of resistance to targeted therapies1. Sustained signalling through this pathway is caused most often by mutations in K-Ras, which biochemically favours the stabilization of active RAF signalling complexes2. Kinase suppressor of Ras (KSR) is a MAPK scaffold3–5 that is subject to allosteric regulation through dimerization with RAF6,7. Direct targeting of KSR could have important therapeutic implications for cancer; however, testing this hypothesis has been difficult owing to a lack of small-molecule antagonists of KSR function. Guided by KSR mutations that selectively suppress oncogenic, but not wild-type, Ras signalling, we developed a class of compounds that stabilize a previously unrecognized inactive state of KSR. These compounds, exemplified by APS-2-79, modulate KSR-dependent MAPK signalling by antagonizing RAF heterodimerization as well as the conformational changes required for phosphorylation and activation of KSR-bound MEK (mitogen-activated protein kinase kinase). Furthermore, APS-2-79 increased the potency of several MEK inhibitors specifically within Ras-mutant cell lines by antagonizing release of negative feedback signalling, demonstrating the potential of targeting KSR to improve the efficacy of current MAPK inhibitors. These results reveal conformational switching in KSR as a druggable regulator of oncogenic Ras, and further suggest co-targeting of enzymatic and scaffolding activities within Ras–MAPK signalling complexes as a therapeutic strategy for overcoming Ras-driven cancers.

The phenothiazine and dibenzazepine tricyclics are potent neurotropic drugs with a documented but underutilized anti-cancer side effect. Reengineering these agents (TFP, CPZ, CIP) by replacing the basic amine with a neutral polar functional group (e.g., RTC-1, RTC-2) abrogated their CNS effects as demonstrated by in vitro pharmacological assays and in vivo behavioral models. Further optimization generated several phenothiazines and dibenzazepines with improved anti-cancer potency, exemplified by RTC-5. This new lead demonstrated efficacy against a xenograft model of an EGFR driven cancer without the neurotropic effects exhibited by the parent molecules. Its effects were attributed to concomitant negative regulation of PI3K-AKT and RAS-ERK signaling.

EGFR activation is both a key molecular driver of disease progression and the target of a broad class of molecular agents designed to treat advanced cancer. Nevertheless, resistance develops through several mechanisms, including activation of AKT signaling. Though much is known about the specific molecular lesions conferring resistance to anti-EGFR-based therapies, additional molecular characterization of the downstream mediators of EGFR signaling may lead to the development of new classes of targeted molecular therapies to treat resistant disease. We identified a transcriptional network involving the tumor suppressors Krüppel-like factor 6 (KLF6) and forkhead box O1 (FOXO1) that negatively regulates activated EGFR signaling in both cell culture and in vivo models. Furthermore, the use of the FDA-approved drug trifluoperazine hydrochloride (TFP), which has been shown to inhibit FOXO1 nuclear export, restored sensitivity to AKT-driven erlotinib resistance through modulation of the KLF6/FOXO1 signaling cascade in both cell culture and xenograft models of lung adenocarcinoma. Combined, these findings define a novel transcriptional network regulating oncogenic EGFR signaling and identify a class of FDA-approved drugs as capable of restoring chemosensitivity to anti-EGFR-based therapy for the treatment of metastatic lung adenocarcinoma.

The Icahn School of Medicine at Mount Sinai, on behalf of GN, MO, and NSD, has filed patents covering composition of matter on the small molecules disclosed herein for the treatment of human cancer and other diseases (international application numbers: PCT/US15/19770, PCT/US15/19764; and US Patent: US 9,540,358 B2). RAPPTA Therapeutics LLC has licensed this intellectual property for the clinical and commercial development of this series of small molecule PP2A activators. GN, MO, and MDG have an ownership interest in RAPPTA Therapeutics LLC.

We present a novel methodology combining traditional fluorescent in situ hybridization with an in situ protein detection technology called proximity ligation assay. This method has potential to perform a detailed analysis of the relationship between gene status and corresponding protein expression in cells and tissues. We demonstrate that the fluorescent in situ gene protein assay methodology is capable of resolving gene and protein patterns simultaneously on a cell-by-cell basis.

No abstract

Epidermal growth factor receptor (EGFR) activation is both a key molecular driver of disease progression and the target of a broad class of molecular agents designed to treat advanced cancer. Nevertheless, resistance develops through several mechanisms including constitutive activation of AKT signaling. Additional molecular characterization of the downstream mediators of EGFR signaling may lead to the development of new classes of targeted molecular therapies to treat resistant disease. Here we identify a transcriptional network involving the KLF6 and FOXO1 tumor suppressor genes that negatively regulate activated EGFR signaling and that can be reactivated using the combination of two FDA approved agents in both cell culture and in vivo models of the disease. In both murine models and patient derived lung adenocarcinoma samples, EGFR activation is associated with FOXO1 mislocalization and decreased KLF6 expression. Furthermore, in a Kras driven mouse model, KLF6 expression is not significantly changed whereas AKT activation seen in the Pten/Mmac1+/− heterozygous mouse model results in FOXO1 mislocalization and decreased KLF6 expression. Consistent with these findings, inhibition of AKT signaling promotes increase in nuclear FOXO1 resulting in transactivation of the KLF6 tumor suppressor gene in lung adenocarcinoma cell lines. Correspondingly, the EGFRL858R mouse model demonstrates spontaneous tumor regression when treated with the anti-EGFR based therapy, erlotinib, an FDA-approved small-molecule inhibitor of EGFR signaling. We analyzed L858R mouse tumors samples treated with erlotinib and found increased KLF6 expression following EGFR inhibition. Conversely, targeted reduction of KLF6 resulted in decreased erlotinib response in both cell culture and in vivo models of disease suggesting a direct link between KLF6 upregulation and the induction of apoptosis by anti-EGFR based therapy. Therefore, we hypothesized that acquired resistance to anti-EGFR based therapies could be overcome by restoring downstream function of the FOXO1/KLF6 transcriptional network. Here we demonstrate that an FDA-approved drug, trifluoperazine hydrochloride (TFP), which has been shown to inhibit FOXO1 nuclear export, restores sensitivity to AKT-driven erlotinib-resistance through modulation of the KLF6/FOXO1 signaling cascade in both cell culture and xenograft models. Furthermore, silencing of FOXO1 blunts apoptosis mediated through combination erlotinib and TFP treatment suggesting that this transcriptional network is important for negatively regulating AKT signaling. Combined, these studies define a novel transcriptional network regulating oncogenic EGFR signaling and identify a class of FDA-approved drugs with the potential for rapid clinical translation to restore chemosensitivity to anti-EGFR-based therapy for the treatment of metastatic lung adenocarcinoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1885. doi:1538-7445.AM2012-1885