Small molecule stabilization of the KSR inactive state antagonizes oncogenic Ras signalling.

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 therapies. Sustained signalling through this pathway is caused most often by mutations in K-Ras, which biochemically favours the stabilization of active RAF signalling complexes. Kinase suppressor of Ras (KSR) is a MAPK scaffold that is subject to allosteric regulation through dimerization with RAF. 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.

Reengineered tricyclic anti-cancer agents.

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.

Direct activation of PP2A for the treatment of tyrosine kinase inhibitor-resistant lung adenocarcinoma.

Although tyrosine kinase inhibitors (TKIs) have demonstrated significant efficacy in advanced lung adenocarcinoma (LUAD) patients with pathogenic alterations in EGFR, most patients develop acquired resistance to these agents via mechanisms enabling the sustained activation of the PI3K and MAPK oncogenic pathways downstream of EGFR. The tumor suppressor protein phosphatase 2A (PP2A) acts as a negative regulator of these pathways. We hypothesize that activation of PP2A simultaneously inhibits the PI3K and MAPK pathways and represents a promising therapeutic strategy for the treatment of TKI-resistant LUAD. After establishing the efficacy of small molecule activators of PP2A (SMAPs) in a transgenic EGFRL858R model and TKI-sensitive cell lines, we evaluated their therapeutic potential in vitro and in vivo in TKI-resistant models. PP2A activation resulted in apoptosis, significant tumor growth inhibition, and downregulation of PI3K and MAPK pathways. Combination of SMAPs and TKI afatinib resulted in an enhanced effect on the downregulation of the PI3K pathway via degradation of the PP2A endogenous inhibitor CIP2A. An improved effect on tumor growth inhibition was observed in a TKI-resistant xenograft mouse model treated with a combination of both agents. These collective data support the development of PP2A activators for the treatment of TKI-resistant LUAD.

Activation of tumor suppressor protein PP2A inhibits KRAS-driven tumor growth.

Targeted cancer therapies, which act on specific cancer-associated molecular targets, are predominantly inhibitors of oncogenic kinases. While these drugs have achieved some clinical success, the inactivation of kinase signaling via stimulation of endogenous phosphatases has received minimal attention as an alternative targeted approach. Here, we have demonstrated that activation of the tumor suppressor protein phosphatase 2A (PP2A), a negative regulator of multiple oncogenic signaling proteins, is a promising therapeutic approach for the treatment of cancers. Our group previously developed a series of orally bioavailable small molecule activators of PP2A, termed SMAPs. We now report that SMAP treatment inhibited the growth of KRAS-mutant lung cancers in mouse xenografts and transgenic models. Mechanistically, we found that SMAPs act by binding to the PP2A Aα scaffold subunit to drive conformational changes in PP2A. These results show that PP2A can be activated in cancer cells to inhibit proliferation. Our strategy of reactivating endogenous PP2A may be applicable to the treatment of other diseases and represents an advancement toward the development of small molecule activators of tumor suppressor proteins.

Targeting the FOXO1/KLF6 axis regulates EGFR signaling and treatment response.

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.

Gene-protein correlation in single cells.

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.