Targeting AGC Kinases in Anaplastic Thyroid Cancer: Kinome Screening and PI3K Pathway Inhibitor Validation

In this project, I led a target discovery initiative in anaplastic thyroid cancer (ATC), focusing on the identification of AGC family kinases as key oncogenic drivers. Through integrated genetic and pharmacological perturbation approaches, I demonstrated the functional relevance of specific kinases within the PI3K signaling axis. I collaborated with industry partners to screen and validate small molecule inhibitors targeting novel nodes of the pathway, supporting early-stage therapeutic development. To enable robust functional assays, I established and optimized primary thyrocyte-derived cell culture systems that preserved lineage-specific features. My work contributed to the generation of over 30 cellular models and supported more than 100 in vivo and in vitro drug studies across thyroid cancer research initiatives. These efforts advanced our mechanistic understanding of ATC and provided new leads for therapeutic intervention.

SGK1 activation is essential for PI3K-dependent tumor development

Arturo Orlacchio, Antonio Di Cristofano

AACR Annual Meeting 2017

Driver pathway blockage synergizes with PLK1 inhibition in anaplastic thyroid cancer

Daniela De Martino, Emrullah Yilmaz, Arturo Orlacchio, Antonio Di Cristofano

AACR Annual Meeting 2017

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

Emrullah Yilmaz, Arturo Orlacchio, Antonio Di Cristofano

AACR Annual Meeting 2016

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

Arturo Orlacchio, Antonio Di Cristofano

AACR Annual Meeting 2015

MYC amplification and overexpression in metastatic anaplastic thyroid cancer dictates response to therapy

Emrullah Yilmaz, Marika A. Russo, Arturo Orlacchio, Toni Forde, Antonio Di Cristofano

AACR Annual Meeting 2015

SGK1 Is a Critical Component of an AKT-Independent Pathway Essential for PI3K-Mediated Tumor Development and Maintenance

Arturo Orlacchio, Michela Ranieri, Martina Brave, Valeria Antico Arciuch, Toni Forde, Daniela De Martino, Karen E Anderson, Phillip Hawkins, Antonio Di Cristofano

Abstract

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 Research 2017

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

Daniela De Martino, Emrullah Yilmaz, Arturo Orlacchio, Michela Ranieri, Ke Zhao, Antonio Di Cristofano

Abstract

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.

Cancer Letters 2018

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

Devora Champa, Arturo Orlacchio, Bindi Patel, Michela Ranieri, Anton A Shemetov, Vladislav V Verkhusha, Ana Maria Cuervo, Antonio Di Cristofano

Abstract

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.

Oncotarget 2018

MCM5 as a target of BET inhibitors in thyroid cancer cells

Catia Mio, Elisa Lavarone, Ketty Conzatti, Federica Baldan, Barbara Toffoletto, Cinzia Puppin, Sebastiano Filetti, Cosimo Durante, Diego Russo, Arturo Orlacchio, Antonio Di Cristofano, Carla Di Loreto, Giuseppe Damante

Abstract

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.

Endocrine Related Cancer 2016

Targeting AGC Kinases in Anaplastic Thyroid Cancer: Kinome Screening and PI3K Pathway Inhibitor Validation

SGK1 activation is essential for PI3K-dependent tumor development

Driver pathway blockage synergizes with PLK1 inhibition in anaplastic thyroid cancer

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

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

MYC amplification and overexpression in metastatic anaplastic thyroid cancer dictates response to therapy

SGK1 Is a Critical Component of an AKT-Independent Pathway Essential for PI3K-Mediated Tumor Development and Maintenance

Abstract

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

Abstract

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

Abstract

MCM5 as a target of BET inhibitors in thyroid cancer cells

Abstract

Beyond AKT: Critical pathways for PI3K-dependent transformation

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