Arturo Orlacchio

Obatoclax kills anaplastic thyroid cancer cells by inducing lysosome neutralization and necrosis

Poorly differentiated and anaplastic thyroid carcinomas are very aggressive, almost invariably lethal neoplasms for which no effective treatment exists. These tumors are intrinsically resistant to cell death, even when their driver oncogenic signaling pathways are inhibited. We have undertaken a detailed analysis, in mouse and human thyroid cancer cells, of the mechanism through which Obatoclax, a pan-inhibitor of the anti-apoptotic proteins of the BCL2 family, effectively reduces tumor growth in vitro and in vivo. We demonstrate that Obatoclax does not induce apoptosis, but rather necrosis of thyroid cancer cells, and that non-transformed thyroid cells are significantly less affected by this compound. Surprisingly, we show that Obatoclax rapidly localizes to the lysosomes and induces loss of acidification, block of lysosomal fusion with autophagic vacuoles, and subsequent lysosomal permeabilization. Notably, prior lysosome neutralization using different V-ATPase inhibitors partially protects cancer cells from the toxic effects of Obatoclax. Although inhibition of autophagy does not affect Obatoclax-induced cell death, selective down-regulation of ATG7, but not of ATG5, partially impairs Obatoclax effects, suggesting the existence of autophagy-independent functions for ATG7. Strikingly, Obatoclax killing activity depends only on its accumulation in the lysosomes, and not on its interaction with BCL2 family members. Finally, we show that also other lysosome-targeting compounds, Mefloquine and LLOMe, readily induce necrosis in thyroid cancer cells, and that Mefloquine significantly impairs tumor growth in vivo, highlighting a clear vulnerability of these aggressive, apoptosis-resistant tumors that can be therapeutically exploited.

MCM5 as a target of BET inhibitors in thyroid cancer cells

Anaplastic thyroid carcinoma (ATC) is an extremely aggressive thyroid cancer subtype, refractory to the current medical treatment. Among various epigenetic anticancer drugs, bromodomain and extra-terminal inhibitors (BETis) are considered to be an appealing novel class of compounds. BETi target the bromodomain and extra-terminal of BET proteins that act as regulators of gene transcription, interacting with histone acetyl groups. The goal of this study is to delineate which pathway underlies the biological effects derived from BET inhibition, in order to find new potential therapeutic targets in ATC. We investigated the effects of BET inhibition on two human anaplastic thyroid cancer-derived cell lines (FRO and SW1736). The treatment with two BETis, JQ1 and I-BET762, decreased cell viability, reduced cell cycle S-phase, and determined cell death. In order to find BETi effectors, FRO and SW1736 were subjected to a global transcriptome analysis after JQ1 treatment. A significant portion of deregulated genes belongs to cell cycle regulators. Among them, MCM5 was decreased at both mRNA and protein levels in both tested cell lines. Chromatin immunoprecipitation (ChIP) experiments indicate that MCM5 is directly bound by the BET protein BRD4. MCM5 silencing reduced cell proliferation, thus underlining its involvement in the block of proliferation induced by BETis. Furthermore, MCM5 immunohistochemical evaluation in human thyroid tumor tissues demonstrated its overexpression in several papillary thyroid carcinomas and in all ATCs. MCM5 was also overexpressed in a murine model of ATC, and JQ1 treatment reduced Mcm5 mRNA expression in two murine ATC cell lines. Thus, MCM5 could represent a new target in the therapeutic approach against ATC.

SGK1 is a critical component of an AKT-independent pathway essential for PI3K-mediated tumor development and maintenance

Activation of the PI3K-AKT signaling cascade is a common critical event during malignant transformation. In this study, we used thyroid gland epithelial cells and a series of genetically engineered mouse strains as model systems to demonstrate that, although necessary, AKT activation is not sufficient for PI3K-driven transformation. Instead, transformation requires the activity of the PDK1-regulated AGC family of protein kinases. In particular, SGK1 was found to be essential for proliferation and survival of thyroid cancer cells harboring PI3K-activating mutations. Notably, cotargeting SGK1 and AKT resulted in significantly higher growth suppression than inhibiting either PI3K or AKT alone. Overall, these findings underscore the clinical relevance of AKT-independent pathways in tumors driven by genetic lesions targeting the PI3K cascade. Cancer Res; 77(24); 6914-26. ©2017 AACR.

PI3K blockage synergizes with PLK1 inhibition preventing endoreduplication and enhancing apoptosis in anaplastic thyroid cancer

Anaplastic thyroid cancer (ATC) is among the most lethal malignancies. The mitotic kinase PLK1 is overexpressed in the majority of ATCs and PLK1 inhibitors have shown preclinical efficacy. However, they also cause mitotic slippage and endoreduplication, leading to the generation of tetraploid, genetically unstable cell populations. We hypothesized that PI3K activity may facilitate mitotic slippage upon PLK1 inhibition, and thus tested the effect of combining PLK1 and PI3K inhibitors in ATC models, in vitro and in vivo. Treatment with BI6727 and BKM120 resulted in a significant synergistic effect in ATC cells, independent of the levels of AKT activity. Combination of the two drugs enhanced growth suppression at doses for which the single drugs showed no effect, and led to a massive reduction of the tetraploid cells population. Furthermore, combined treatment in PI3Khigh cell lines showed a significant induction of apoptosis. Finally, combined inhibition of PI3K and PLK1 was extremely effective in vivo, in an immunocompetent allograft model of ATC. Our results demonstrate a clear therapeutic potential of combining PLK1 and PI3K inhibitors in anaplastic thyroid tumors.

Abstract 4376: Beyond AKT: Critical pathways for PI3K-dependent transformation

Proceedings: AACR 107th Annual Meeting 2016; April 16-20, 2016; New Orleans, LA Genetic alterations activating the PI3K/AKT pathway have been identified in most human cancers, including those originating in the thyroid gland, particularly follicular thyroid cancer (FTC) and anaplastic thyroid cancer (ATC). However, although PI3K and AKT represent relevant targets for therapeutic purposes, the number of essential roles played by this pathway in normal cells poses the problem of reaching therapeutic efficacy without harming healthy tissues. Ideally, the toxicity associated with wide-range inhibitors could be reduced by targeting only molecules directly involved in the transformation process. The PDK1 kinase is a master regulator of PI3K downstream effectors belonging to the AGC kinases family, such as AKT and S6K1. Additional AGC kinases are controlled by PDK1 independently of PI3K activity. If PDK1-controlled AGC kinases contribute to PI3K-dependent transformation, they would clearly represent novel important therapeutic targets. PDK1 possesses a substrate-docking site, the “PIF pocket”, required for the phosphorylation of most AGC kinases (including S6K, PKC, SGK, and RSK) but not of AKT. The L155E mutation in PDK1disrupts the PIF pocket, without affecting AKT activation. This differential mechanism of substrate selection allows us to clearly define the relative contribution of AKT and other PDK1 targets to the neoplastic transformation process. Using in vivo as well as ex vivo and in vitro genetic and pharmacological approaches, we have shown that Akt activation is not sufficient to transform thyroid epithelial cells. Mice in which the PI3K pathway is constitutively activated in the thyroid epithelium through loss of Pten show thyroid hyperplasia at birth that progresses to invasive and metastatic follicular carcinoma. While Pdk1 deletion, as expected, completely rescue the phenotype observed in Pten−/- thyroids, we found that the simple impairment of the PIF-pocket is able to completely abrogate neoplastic transformation, despite AKT and mTOR being constitutively activated. We show that members of two families of AGC kinases, SGK (1and 3) and RSK (1-3) are essential components of this PDK1-dependent pathway required for PI3K-dependent transformation. Genetic and pharmacological SGK and RSK inhibition strongly reduces cell proliferation in Pten−/− as well as Pik3ca mutant cell lines, both in 2d and in 3D systems, with SGK1 and RSK3 being the most critical isoforms. Furthermore, genetic inhibition of SGK and RSK drastically impairs tumor growth in syngeneic allograft and metastasis models. Taken together, our data identify two new signaling cascades that are essential for neoplastic transformation of thyroid epithelial cells, and thus novel strong candidates for drug development. Citation Format: Arturo Orlacchio, Antonio Di Cristofano. Beyond AKT: Critical pathways for PI3K-dependent transformation. [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 4376.

Abstract 2713: Combination of PLK1 and PI3K inhibitors shows strong synergy in anaplastic thyroid cancer

Anaplastic thyroid cancer (ATC) is the most aggressive thyroid cancer with median of 6 months of survival from diagnosis. Molecular alterations are well defined recently. p53 mutations, and activation of PI3K, RAS and BRAF are among the most common alterations. PLK1 overexpression has also been identified in ATC. We have previously shown that PLK1 inhibitors are effective in ATC cell lines. However PLK1 inhibition in cell lines with PI3K activation resulted in escaping growth arrest and subsequent mitotic catastrophe through mitotic slippage. Since the PI3K activation is common in ATC, there is risk of generating polyploid, genetically unstable cell populations by targeting these tumors with a PLK1 inhibitor. So we tested the effect of combining PLK1 and PI3K inhibitors in ATC cell lines. We combined PLK1 inhibitor BI6727 and PI3K inhibitor BKM120 and measured the cell viability using Alamar Blue. Combination of these two drugs resulted in significant synergy in mouse derived PTEN deleted ATC cell lines. We observed the same synergy in the human ATC cell lines with PIK3CA mutations. BI6727 showed significant G2/M arrest in the cell lines. The combination two drugs enhanced the G2/M arrest. In a low dose that the single drugs showed no inhibition in cell cycle, combination resulted in G2/M arrest. We treated the cells with a dual PI3K and PLK1 inhibitor Rigosertib. Although we have seen some sensitivity of the cells with PIK3CA mutation, the cell lines with PTEN were strongly resistant to Rigosertib. Our results show that combination of PLK1 and PI3K inhibitors is an effective treatment for ATC cells with PI3K activation. This combination results in cell cycle arrest suggesting the role of using combination therapies in this aggressive cancer. Citation Format: Emrullah Yilmaz, Arturo Orlacchio, Antonio Di Cristofano. Combination of PLK1 and PI3K inhibitors shows strong synergy in anaplastic thyroid 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 2713.

Abstract 2158: PDK1-dependent activation of RSK is an absolute requirement for PI3K oncogenic activity in the thyroid gland

Aberrant activation of the phosphatidylinositol-3 kinase (PI3K)/Akt pathway plays a key role in thyroid tumorigenesis, particularly in follicular thyroid cancer (FTC) and anaplastic thyroid cancer (ATC). Accordingly, conditional deletion of Pten in the mouse thyroid epithelium induces constitutive activation of the PI3K/AKT pathway, and causes thyroid hyperplasia at birth that progresses to invasive and metastatic follicular carcinoma. This phenotype is dramatically accelerated by simultaneous deletion of Trp53, leading to the development of ATC, or activation of Kras, leading to poorly differentiated carcinomas. The PDK1 kinase plays a central role in the PI3K signaling cascade, acting as a master regulator of PI3K downstream effectors belonging to the AGC kinases family, such as AKT and S6K1. Additional AGC kinases are controlled by PDK1 independent of PI3K activity. PDK1 possesses a substrate docking site, the “PIF pocket”, required for the phosphorylation of most AGC kinases (including S6K, PKC, SGK, and RSK) but not of AKT. The L155E PDK1 mutant disrupts the PIF pocket and impairs AGC kinase activation, without any effect on AKT activation. Thanks to this differential mechanism of substrate activation, we are defining the relative contribution of AKT and other PDK1 targets to the neoplastic transformation process in Pten-/- thyrocytes. Surprisingly, in vivo disruption of the PIF-pocket completely abrogates neoplastic transformation of Pten-/- thyroids, even in the face of AKT constitutive activation. This finding supports the hypothesis that the linear PI3K-AKT pathway is not sufficient to drive thyroid transformation and that concomitant activation of additional PDK1-dependent pathways is absolutely required. We now show that RSK family members (RSK1-3) are essential components of this PDK1-dependent accessory pathway. Remarkably, RSK3 levels in the thyroid are increased in the absence of Pten, and RSK activity in thyroid cells is abrogated when PDK1 PIF-pocket is mutated. Genetic and pharmacological inhibition of RSK drastically impairs [Pten,Trp53]-/- ATC cell proliferation both in vitro and in vivo, in syngeneic allograft models. Taken together, our data identify a new signaling cascade that is essential for neoplastic transformation of thyroid epithelial cells, and thus a new critical candidate for drug development. Citation Format: Arturo Orlacchio, Antonio Di Cristofano. PDK1-dependent activation of RSK is an absolute requirement for PI3K oncogenic activity in the thyroid gland. [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 2158. doi:10.1158/1538-7445.AM2015-2158

Abstract 4776: PDK1-dependent activation of AGC kinases is an absolute requirement for AKT oncogenic activity

The PI3K signaling cascade is frequently activated in human cancer, and AKT is commonly considered its major transforming conduit. Thus, PI3K and AKT represent relevant targets for therapeutic purposes. However, the many essential of roles played by this pathway in normal cells poses the problem of reaching therapeutic efficacy without harming healthy tissues. In principle, it would be ideal to target only molecules directly involved in the transformation process, thus reducing the toxicity associated with wide-range inhibitors. PDK1 plays a key role in the PI3K cascade, directly activating AKT. In addition, PDK1 possesses a substrate docking site named “PIF pocket” that is required to phosphorylate and activate members of the AGC kinase family, including S6K, PKC, SGK, and RSK. Mice in which the PI3K pathway is constitutively activated in the thyroid epithelium through loss of Pten are born with thyroid hyperplasia that progresses to invasive and metastatic follicular carcinoma, a process that is accelerated by the simultaneous loss of p27 or p53, and by Ras or Braf activation. Using in vivo as well as ex vivo and in vitro genetic and pharmacological approaches, we now show that although Akt activation is necessary, it is not sufficient to transform thyroid epithelial cells. Concomitant activation of Pdk1-dependent pathways is absolutely required to develop neoplastic lesions in vivo, and to induce cell proliferation ex vivo. Pten mutants develop adenomas and carcinomas by one year of age. Strikingly, complete rescue of this phenotype is observed in compound mutants in which, in addition to thyroid-specific Pten loss, the PIF-pocket of Pdk1 has been genetically disrupted, maintaining constitutive activation of Akt and mTor, but not of PIF-pocket dependent AGC kinases. We also show that the transcription factor STAT3 represents a key mediator of this essential Pdk1-dependent pathway: STAT3 phosphorylation is increased in Pten mutants, and abolished in compound mutants. Furthermore, genetic and pharmacological inhibition of STAT3 drastically impairs cell proliferation in vitro and rescues the hyperplastic phenotype of Pten mutant mice in vivo. Thus, we have identified an essential and druggable signaling cascade that critically cooperates with AKT activation to transform thyroid epithelial cells. Citation Format: Arturo Orlacchio, Valeria Antico Arciuch, Antonio Di Cristofano. PDK1-dependent activation of AGC kinases is an absolute requirement for AKT oncogenic activity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4776. doi:10.1158/1538-7445.AM2014-4776