David Kastrinsky

All roads lead to PP2A: exploiting the therapeutic potential of this phosphatase

Protein phosphatase 2A (PP2A) is a serine/threonine phosphatase involved in the regulation of many cellular processes. A confirmed tumor suppressor protein, PP2A is genetically altered or functionally inactivated in many cancers highlighting a need for its therapeutic reactivation. In this review we discuss recent literature on PP2A: the elucidation of its structure and the functions of its subunits, and the identification of molecular lesions and post‐translational modifications leading to its dysregulation in cancer. A final section will discuss the proteins and small molecules that modulate PP2A and how these might be used to target dysregulated forms of PP2A to treat cancers and other diseases.

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.

Identifying a small molecule blocking antigen presentation in autoimmune thyroiditis

We previously showed that an HLA-DR variant containing arginine at position 74 of the DRβ1 chain (DRβ1-Arg74) is the specific HLA class II variant conferring risk for autoimmune thyroid diseases (AITD). We also identified 5 thyroglobulin (Tg) peptides that bound to DRβ1-Arg74. We hypothesized that blocking the binding of these peptides to DRβ1-Arg74 could block the continuous T-cell activation in thyroiditis needed to maintain the autoimmune response to the thyroid. The aim of the current study was to identify small molecules that can block T-cell activation by Tg peptides presented within DRβ1-Arg74 pockets. We screened a large and diverse library of compounds and identified one compound, cepharanthine that was able to block peptide binding to DRβ1-Arg74. We then showed that Tg.2098 is the dominant peptide when inducing experimental autoimmune thyroiditis (EAT) in NOD mice expressing human DRβ1-Arg74. Furthermore, cepharanthine blocked T-cell activation by thyroglobulin peptides, in particular Tg.2098 in mice that were induced with EAT. For the first time we identified a small molecule that can block Tg peptide binding and presentation to T-cells in autoimmune thyroiditis. If confirmed cepharanthine could potentially have a role in treating human AITD.

Small-Molecule Activators of Protein Phosphatase 2A for the Treatment of Castration-Resistant Prostate Cancer

Primary prostate cancer is generally treatable by androgen deprivation therapy, however, later recurrences of castrate-resistant prostate cancer (CRPC) that are more difficult to treat nearly always occur due to aberrant reactivation of the androgen receptor (AR). In this study, we report that CRPC cells are particularly sensitive to the growth-inhibitory effects of reengineered tricyclic sulfonamides, a class of molecules that activate the protein phosphatase PP2A, which inhibits multiple oncogenic signaling pathways. Treatment of CRPC cells with small-molecule activators of PP2A (SMAP) in vitro decreased cellular viability and clonogenicity and induced apoptosis. SMAP treatment also induced an array of significant changes in the phosphoproteome, including most notably dephosphorylation of full-length and truncated isoforms of the AR and downregulation of its regulatory kinases in a dose-dependent and time-dependent manner. In murine xenograft models of human CRPC, the potent compound SMAP-2 exhibited efficacy comparable with enzalutamide in inhibiting tumor formation. Overall, our results provide a preclinical proof of concept for the efficacy of SMAP in AR degradation and CRPC treatment.Significance: A novel class of small-molecule activators of the tumor suppressor PP2A, a serine/threonine phosphatase that inhibits many oncogenic signaling pathways, is shown to deregulate the phosphoproteome and to destabilize the androgen receptor in advanced prostate cancer. Cancer Res; 78(8); 2065-80. ©2018 AACR.

Abstract C132: Therapeutic reactivation of PP2A for prostate cancer treatment

Several new therapies have recently been approved for patients with castration-resistant prostate cancer (CRPC), however, none are curative and tumors ultimately develop resistance. Advances in the treatment of CRPC require novel approaches and therapies such as those outlined in this study. Most drug development efforts have focused on targeting single oncogenic proteins, an approach limited by the complexity of signaling networks and associated cross talk. Targeting phosphatases, the key negative regulators of signaling proteins, on the other hand, may overcome some of these limitations, particularly if these negative regulators themselves are altered.Through reverse engineering of tricyclic neuroleptic drugs, we have developed a series of small molecule activators of the serine/threonine phosphatase 2A (PP2A), a key negative regulator of numerous oncogenic signaling pathways. PP2A acts as a tumor suppressor and dephosphorylates several critical nodes in prostate cancer pathogenesis including the androgen receptor (AR). Decreased PP2A expression and/or activity have been correlated with castration-resistance in cell culture and human prostate cancer studies. These small molecule activators of PP2A (SMAPs), as represented by TRC-794, TRC-1154, and DT-061, directly bind and activate PP2A and have favorable pharmaceutical properties. In this study we sought to determine the activity of SMAPs in clinically relevant preclinical models of prostate cancer. Treatment of prostate cancer cell lines with SMAPs resulted in decreased cell viability and colony formation, cell cycle arrest, and an increase in apoptosis. Global Phosphoproteomic analysis of TRC-794 treated prostate cancer cells revealed that the AR and MYC were significantly perturbed in drug treated cells compared to controls which was subsequently confirmed by western blotting. Western blot analysis of prostate cancer cells demonstrated dose-dependent degradation of the AR resulting in PSA reduction and changes in canonical AR target gene expression. In order to investigate whether PP2A was mediating SMAP induced AR degradation, LNCAP cells were stably transduced with the SV40 small t antigen (ST), a potent oncoprotein that perturbs PP2A function. SMAPs were unable to degrade AR in LNCAP cells transduced with ST, suggesting that PP2A mediates SMAP induced AR degradation. SMAPs were evaluated in vivo in xenograft models representing prostate cancers that are sensitive to conventional therapy and resistant to enzalutamide, the current gold standard, due to overexpression of the AR or expression of androgen receptor splice variants (AR-SV). Single agent treatment with DT-1154 or DT-061 in vivo resulted in either significant tumor growth inhibition or tumor regression and induction of tumor cell apoptosis comparable to enzalutamide. Western blot analysis of the tumors demonstrated that the effects on tumor volume correlated strongly with target engagement as evidenced by significant decreases in PSA and AR expression in vivo. Additionally, these compounds demonstrated favorable pharmacokinetics and showed no overt toxicity. Combined these data highlight the potential for PP2A activation for both the treatment of CRPC and potentially for diverse PP2A inactivated tumor types and diseases. Citation Format: Kim McClinch, Rita Avelar, David Callejas, David Kastrinsky, Michael Ohlmeyer, Stephen Plymate, Matthew Galsky, Goutham Narla. Therapeutic reactivation of PP2A for prostate cancer treatment. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C132.

Abstract 3865: Therapeutic activation of protein phosphatase 2A for the treatment of lung cancer

PP2A is a phosphatase tumor suppressor that is dysregulated and deactivated in lung cancer. It is one of the most abundant cellular proteins and regulates the activity of numerous kinases Where achievable, restoration of PP2A function inhibits cancer progression, and notably, by the inhibition of the downstream effectors of the oncogenic kinases that initiate and drive cancer progression. In this study, we determined PP2A inactivation in human lung cancer with specific molecular genotypes and we ascertained the biological and functional consequences of PP2A reactivation. In assessing a lung cancer TMA, we identified that PP2A inactivation was correlated with poor survival and was significantly higher in patients with Kras mutations. In order to understand the therapeutic potential of restoration of PP2A activity in KRAS mutant lung cancer, our lab developed a series of small molecule activators of PP2A (SMAPs) through reverse engineering of tricyclic neuroleptic drugs. SMAP treatment of lung cancer cell lines resulted in an induction of apoptosis and decreased cell viability. Structural and biophysical studies have identified the site of drug binding and mechanism for PP2A activation by this small molecule series. Additionally, cell lines harboring drug-binding mutations were resistant to SMAP therapy as compared to wild type PP2A and EGFP control. Global phosphoproteomic analysis of SMAP treated KRAS lung cancer cell lines revealed ERK signaling as a commonly perturbed pathway in drug treated cell lines. Given the marked dephosphorylation of ERK upon treatment of cell lines with SMAPs, we overexpressed a constitutively active form of MEK (MEKDD) to blunt SMAP mediated ERK dephosphorylation to determine the relevance of ERK inactivation for the biological effects of SMAPs on cellular apoptosis. Overexpression of MEKDD resulted in a blunted apoptotic response to SMAP treatment. Single agent SMAP treatment of KRAS GEMM and xenograft mouse models of lung cancer resulted in tumor stasis, induction of tumor cell apoptosis and cell cycle arrest to comparable levels seen with a combination of AKT and MEK inhibitors. Western blotting and immunohistochemical analysis of the tumors demonstrated that SMAP treatment resulted in of ERK, AKT, and PP2A-Y307 dephosphorylation in vivo. Additionally, these compounds demonstrate favorable pharmacokinetics and show no overt toxicity. Furthermore, combination of SMAPs with kinase inhibitors further decreased tumor growth in vivo. Taken together, these findings point to therapeutic activation of PP2A as a novel strategy for the treatment of KRAS-mutant NSCLC. Citation Format: Jaya Sangodkar, Rita Tohme, Janna Kiselar, Sudeh Izadmehr, Divya Hoon, Sahar Mazhar, Abbey Perl, Danica Wiredja, Daniela Schlatzer, Shen Yao, David Kastrinsky, Neelesh Sharma, David Brautigan, Mark Chance, Alain Borczuk, Michael Ohlmeyer, Yiannis Ioannou, Goutham Narla. Therapeutic activation of protein phosphatase 2A for the treatment of lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3865.

Abstract B124: Drugging the undruggable: development of small molecule activators of protein phosphatase 2A for cancer treatment

KRAS is the most common recurrent oncogenomic mutations driving the growth of NSCLC. Patients with KRAS mutations respond poorly to current therapies. Thus, novel therapies, are critically needed, to improve the lives of patients suffering from KRAS driven lung cancers. While oncogenic kinases have proven to be successful targets for cancer treatment, the therapeutic targeting of phosphatases, the key negative regulators of these same pathways, has remained largely unexplored. Through reverse engineering of tricyclic neuroleptic drugs, we developed a first-in-class series of small molecule activators of PP2A activators (SMAPs) molecules, as represented that have favorable pharmaceutic properties directly bind and activate the serine/threonine phosphatase 2A (PP2A). A critical role for PP2A as a tumor suppressor has previously been established, and PP2A inactivation is common feature in human lung cancers. Furthermore, protein phosphatase 2A (PP2A) accounts for the majority of cellular serine/threonine phosphatase activity, and its dominant and best-defined targets are oncogenic protein kinases including ERK and AKT. In this study, we sought to determine both the association of PP2A inactivation in lung cancer with specific molecular genotypes and the biological and functional consequences of PP2A reactivation in lung cancer. To understand the effects of SMAPs on cell viability and survival, we used MTT and colony formation assays in lung cancer cell lines. Apoptosis was evaluated through annexin V staining and cell cycle profile analysis. Additionally, global phosphoproteomic profiling was performed. Effects of SMAPs in vivo were assessed using A549, HH41 and H358 xenograft and Kras LA2 transgenic mouse models. Treatment of lung cancer cell lines with TRC resulted in decreased cell viability, decreased colony formation, and an increase in apoptosis. Global phosphoproteomic analysis of SMAP treated cell lines revealed ERK signaling as a commonly perturbed pathway which was confirmed by western blotting. Single agent SMAP treatment of KRAS GEMM and xenograft mouse models of lung cancer resulted in tumor stasis, induction of tumor cell apoptosis and cell cycle arrest to comparable levels seen with a combination of AKT and MEK inhibitors. Furthermore, combination based therapy with kinase inhibitors and our novel phosphatase activators resulting in marked synergy and tumor regressions in vivo. Importantly, these compounds demonstrate favorable pharmacokinetics and show no overt toxicity both alone or in combination. Taken together, these findings point to therapeutic activation of PP2A as a novel strategy for the treatment of advanced KRAS-mutant NSCLC. While research and clinical effort has largely focused on development of inhibitors of oncogenic kinases, the identification of small molecule activators of tumor suppressor proteins has remained elusive. Activation of such proteins could offer the opportunity to identify novel synergistic strategies for the treatment of a number of cancer types. Nevertheless, translation of a PP2A activation strategy into clinical medicine has required pharmaceutically tractable agents for development. Our studies represent a first step into that new territory and highlight the potential for the development of small molecule activators of other protein phosphatases and tumor suppressor proteins Citation Format: Jaya Sangodkar, Daniel McQuaid, Janna Kisselar, David Brautigan, Mark Chance, Michael Ohlmeyer, David Kastrinsky, Yiannis Ioannou, Goutham Narla. Drugging the undruggable: development of small molecule activators of protein phosphatase 2A for cancer treatment. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B124.

Abstract 5329: Development of small molecule activators of protein phosphatase 2A for the treatment of lung cancer

KRAS is the most common recurrent oncogenomic mutations driving the growth of NSCLC and accounting for ∼25% of patients with advanced NSCLC. Patients with KRAS mutations respond poorly to current therapies. Thus, novel therapies, are critically needed, to improve the lives of patients suffering from KRAS driven lung cancers. While oncogenic kinases have proven to be successful targets for cancer treatment, the therapeutic targeting of phosphatases, the key negative regulators of these same pathways, has remained largely unexplored. Through reverse engineering of tricyclic neuroleptic drugs, we developed a first-in-class series of small molecule activators of PP2A activators (SMAPs) molecules, as represented by TRC-794 and TRC-1154, that have favorable pharmaceutical properties directly bind and activate the serine/threonine phosphatase 2A (PP2A). PP2A accounts for the majority of cellular serine/threonine phosphatase activity, and its dominant and best-defined targets are oncogenic protein kinases including ERK and AKT. In this study, we sought to determine both the association of PP2A inactivation in lung cancer with specific molecular genotypes and the biological and functional consequences of PP2A reactivation in lung cancer. We determined the PP2A activation status by immunohistochemistry for the Y307 PP2A residue, a well documented inactivating site on the phosphatase, in a large cohort of primary lung tumors and identified that KRAS G12C mutant tumors displayed coordinate overexpression of both pERK and PP2A Y307. Global phosphoproteomic analysis of TRC-794 treated KRAS lung cancer cell lines revealed ERK signaling as the only commonly perturbed pathway in drug treated cell lines which was confirmed by western blotting. Treatment of lung cancer cell lines with TRC resulted in decreased cell viability, decreased colony formation, and an increase in apoptosis. Given the marked dephosphorylation of ERK upon treatment of cell lines with TRC-1154, we overexpressed a constitutively active form of MEK (MEKDD) to blunt SMAP mediated ERK dephosphorylation to determine the relevance of ERK inactivation to the biological effects of SMAPs on cellular apoptosis. Overexpression of MEKDD resulted in blunting the apoptotic response to TRC-1154 treatment. Single agent TRC-794 or TRC-1154 treatment of KRAS GEMM and xenograft mouse models of lung cancer resulted in tumor stasis, induction of tumor cell apoptosis and cell cycle arrest to comparable levels seen with a combination of AKT and MEK inhibitors. Western blotting and immunohistochemical analysis of the tumors demonstrated that SMAP treatment resulted in of ERK, AKT, and PP2A-Y307 dephosphorylation in vivo. Additionally, these compounds demonstrate favorable pharmacokinetics and show no overt toxicity. Taken together, these findings point to therapeutic activation of PP2A as a novel strategy for the treatment of advanced KRAS-mutant NSCLC. Citation Format: Jaya Sangodkar, Sudeh Izadmehr, Sahar Mahzar, Divya Hoon, Shen Yao, David Kastrinsky, Daniela Schlatzer, Neelesh Sharma, Alain C. Borczuk, Michael Ohlmeyer, Yiannis Ioannou, Goutham Narla. Development of small molecule activators of protein phosphatase 2A for the treatment of lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5329. doi:10.1158/1538-7445.AM2015-5329

Abstract A38: Therapeutic targeting of oncogenic KRAS signaling using a novel small molecule agonist of the PP2A tumor suppressor gene

Metastatic non-small cell lung cancer (NSCLC) is the most common cause of cancer death. Cytotoxic chemotherapy has historically been the mainstay of therapy but is associated with only modest improvements in patient survival. Over the past decade, a better understanding of the pathogenesis of NSCLC, coupled with high throughput genomic technologies applied to patient tumor samples, has led to a molecular classification of NSCLC and a new generation of “precision” therapies. However, the most common recurrent oncogenomic mutation driving the growth of NSCLC, mutant KRAS, accounting for ∼25% of patients with advanced NSCLC, remains without an effective targeted therapy. Mutations in KRAS lead to downstream signaling through ERK, as well as cross talk with the PI3K-Akt pathway, the latter of which is amplified in the presence of inhibition of ERK pathway signaling alone. These findings likely explain, at least in part, why targeting ERK pathway signaling alone in NSCLC has been largely unsuccessful in the clinic, and suggest that coordinate inhibition of both ERK and Akt is necessary for optimal therapy. Approaches to inhibit both of these pathways simultaneously with co-administration of two small molecular kinase inhibitors has shown some promise, but has been limited by both “off-target” treatment-limiting side effects and suboptimal coordinate inhibition of both Akt and ERK signaling. Thus, novel therapies, are critically needed, to improve the lives of patients suffering from KRAS driven lung cancers and while oncogenic kinases have proven to be successful targets for cancer treatment, the therapeutic targeting of phosphatases, the key negative regulators of these same pathways, has remained largely unexplored. Starting with the observation that tricyclic neuroleptic drugs exert anticancer effects in xenograft models, we employed combinatorial chemistry to reverse engineer these drugs into a series of novel compounds that retain the anti-proliferative effects but are devoid of the dose-limiting effects on the central nervous system. We have demonstrated these agents exert potent anti-proliferative effects in both cell culture and in vivo lung cancer models and these effects are functionally linked with simultaneous inhibition of both PI3K-Akt and MAPK signaling. Importantly, these agents that have favorable pharmaceutic properties directly bind and activate the serine/threonine phosphatase 2A (PP2A) and we call these novel first-in-class agents Small Molecule Activators of PP2A (SMAPs). A critical role for PP2A as a tumor suppressor has previously been established, and inhibition and loss-of-function changes in PP2A occur in human lung cancers. Furthermore, protein phosphatase 2A (PP2A) accounts for the majority of cellular serine/threonine phosphatase activity, and its dominant and best defined targets are protein kinases and oncogenic proteins including ERK and AKT. Here we demonstrate for the first time the development and validation of a first-in-class orally bioavailable pharmacological agent that can directly bind and activate PP2A driving coordinate inhibition of both the MAPK and AKT effector pathways in cell culture and both xenograft and genetically engineered mouse models (GEMM) of human lung cancer. Global phosphoproteomic analysis of SMAP treated KRAS lung cancer cell lines reveals ERK signaling as the only commonly perturbed pathway in drug treated cell lines. Single agent SMAP treatment of KRAS GEMM and xenograft mouse models of lung cancer resulted in tumor stasis, induction of tumor cell apoptosis and cell cycle arrest to comparable levels seen with a combination of AKT and MEK inhibitors. Additionally, the compounds demonstrate favorable pharmacokinetics and show no overt toxicity. Taken together, these findings point to therapeutic activation of PP2A as a novel strategy for the treatment of advanced KRAS-mutant NSCLC. While research and clinical effort has largely focused on development of inhibitors of oncogenic kinases, the identification of small molecule activators of tumor suppressor proteins has remained elusive. Activation of such proteins could offer the opportunity to identify novel synergistic strategies for the treatment of a number of cancer types. Nevertheless, translation of a PP2A activation strategy into clinical medicine has required pharmaceutically tractable agents for development. Our studies represent a first step into that new territory and highlight the potential for the development of small molecule activators of other protein phosphatases and tumor suppressor proteins. Citation Format: Jaya Sangodkar, Sahar Mazhar, Danica Wiredja, Giridharan Gokulrangan, Daniela Schlatzer, David Kastrinsky, Analisa Difeo, Shen Yao, Sudeh Izadmehr, Neelesh Sharma, Yiannis Ioannou, Michael Ohlmeyer, Goutham Narla. Therapeutic targeting of oncogenic KRAS signaling using a novel small molecule agonist of the PP2A tumor suppressor gene. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr A38. doi: 10.1158/1557-3125.RASONC14-A38