Neil Dhawan

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.

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 A&agr; 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.

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.

Abstract 1885: Targeting the FOXO1/KLF6 transcriptional network to modulate response to anti-EGFR based therapy

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

Abstract A218: Simultaneous inhibition of both the PI3K-AKT and MAPK-ERK pathways using a single small molecule based approach for the treatment of advanced cancer.

Background: Activation of the PI3K-AKT and MAPK-ERK signaling pathways drives a significant percentage of human cancer and serve as the target for multiple drug development efforts and clinical trials. Due to defined molecular crosstalk, dual inhibition of both pathways is necessary for optimal therapeutic efficacy, and therefore, combinations of PI3K-AKT and MAPK-ERK specific drug therapies are being evaluated. In this study, we have identified compounds that are capable of simultaneously inhibiting both the PI3K-AKT and MAPK-ERK pathway to induce apoptosis both in vitro and in mouse models of the disease. Moreover, we have performed additional derivatization of these small molecules to limit their toxicity and significantly improve their therapeutic window in cell culture and in vivo. Methods: Our new series of molecules are derived from the phenothiazine, exemplified by trifluoperazine(TFP), and dibenzazepine structural backbones, exemplified by clomipramine(CIP). While the antiproliferative properties of neuroleptic tricyclics have been identified, previous clinical trials failed due to dose limiting CNS toxicities related to their potent antidopaminergic properties. We rendered the pendant amine non-basic to attempt to abolish the antidopaminergic effects of this class of drugs. We screened 100 of these novel compounds in the PTEN-null, EGFR-activated H1650 cell line. Subsequently, we determined the effect of two candidate molecules on the PI3K-AKT and MAPK-ERK pathways and their ability to induce apoptosis in vitro and in vivo. Results: Through multiple rounds of SAR (structure activity relationship) analysis, we sequentially derivatized the parent compounds and identified two potent small molecule candidates that efficiently decouple the dose limiting CNS toxicity from the anti-proliferative and anti-tumorigenic properties of this class of FDA approved drugs. Treatment of a panel of lung adenocarcinoma cancer cell lines with these compounds, DBK-368 and DBK-382, led to a decrease in cell viability through the induction of spontaneous apoptosis. These compounds specifically induce caspase-dependent apoptosis as indicated by ZVAD-mediated inhibition of Annexin V staining. Upon mechanistic analysis, DBK-368 and DBK-382 display the ability to directly inhibit AKT and ERK downstream of PI3K and MEK, efficiently and potently decoupling the crosstalk between these two signaling pathways. Furthermore, in a transgenic inducible EGFR-activated mouse model of lung adenocarcinoma, we demonstrated that the novel derivative compounds inhibit AKT and ERK signaling and induce apoptosis in vivo. Lastly, in vivo toxicology studies demonstrated that while TFP exhibited dose limiting CNS toxicities at 15 mg/kg, DBK-368 and DBK-382 displayed no significant effects up to 60 mg/kg. Conclusions: We have identified a series of novel small molecules through a reverse engineering effort of the tricyclic class of FDA approved drugs. Specifically, DBK-368 and DBK-382 appear to be promising monotherapy for advanced cancer as they exhibit dual functionality in the inhibiting both the PI3K-AKT and MAPK-ERK pathways simultaneously, both in vitro and in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A218.

Abstract 2899: Restoring sensitivity to anti-EGFR-based therapy in lung adenocarcinoma by downstream modulation of the FOXO1/KLF6 transcriptional network

Lung cancer is currently the leading cause of cancer-related death. This high mortality rate has prompted numerous exhaustive efforts to identify novel therapeutic targets and treatment modalities for this deadly disease. The characterization of specific signaling cascades involved in the development and progression of lung cancer has been instrumental in developing novel therapeutic strategies. This paradigm is best exemplified by recent studies demonstrating that the epidermal growth factor receptor (EGFR) is a critical diagnostic and therapeutic target in metastatic lung adenocarcinoma. The clinical utility of anti-EGFR-based strategies is, however, ultimately limited by primary or acquired drug resistance, that can develop through several distinct molecular mechanisms. Thus, a more complete molecular characterization of downstream mediators of both cancer progression and treatment resistance will allow for the development of rationally designed combination based therapies to overcome the specific molecular lesions driving the reistant phenotype. 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. Here we identify a transcriptional network involving the KLF6 and FOXO1 tumor suppressor genes that regulates response to anti-EGFR-based therapies in both cell culture and in vivo models of the disease. Specifically, inhibition of AKT signaling promotes FOXO1 stabilization resulting in transactivation of the KLF6 tumor suppressor gene and induction of apoptosis in lung adenocarcinoma cell lines. Furthermore, the use of the FDA-approved drug Trifluoperazine Hydrochloride (TFP), which has been shown to inhibit FOXO1 nuclear export, restores sensitivity to erlotinib-resistant cell lines through modulation of the KLF6/FOXO1 signaling cascade in both cell culture and xenograft lung adenocarcinoma models. Further validation of the requirement for KLF6 activation in erlotinib response was demonstrated by loss-of-function experiments, in which targeted reduction of KLF6 using sequence specific siRNAs abrogated the utility of erlotinib on lung tumors in vivo. In addition, a significant correlation between activated oncogenic EGFR signaling and downregulation of the FOXO1 and KLF6 tumor suppressor gene network in both primary human lung adenocarcinoma patient samples and a transgenic mouse model of the disease was observed. Combined, these studies define a novel transcriptional network regulating oncogenic EGFR signaling and identify a class of FDA-approved drugs 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 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2899. doi:10.1158/1538-7445.AM2011-2899