THERAPEUTIC POTENTIAL OF TARGETING THE FLNA-REGULATED WEE1 KINASE PATHWAY IN ADRENOCORTICAL CARCINOMAS.

Adrenocortical carcinomas (ACC) are highly aggressive tumors with very limited treatment options. Alterations in cell cycle-related genes have been identified as key ACC drivers, and they are largely present in the most aggressive tumor subtypes. Compared to benign adenomas (ACA), the cytoskeletal protein filamin A (FLNA) is poorly expressed in ACC. Of note, its presence, even at low levels, is associated to a less aggressive tumor behaviour, potentially due to its role in negatively regulating the insulin-like growth factor 1 receptor (IGF1R) signalling. Upregulation of Wee1 nuclear kinase, a key regulator of G2/M cell cycle checkpoint, was shown in mouse neural progenitor cells lacking FLNA, and it has been reported in several cancers. The first part of this PhD thesis is focused 1) on the analysis of Wee1 expression in ACC and its regulation by FLNA, 2) on the effects of the specific Wee1 inhibitor AZD1775, 3) and lastly on the impact of FLNA on its efficacy in three different ACC cell lines (H295R, MUC-1, and TVBF-7) and in patient-derived primary cultured ACC cells. Analysis of FLNA and Wee1 proteins revealed elevated Wee1 and reduced FLNA in ACC compared to normal adrenal gland (NAG). FLNA knockdown increased Wee1 protein in H295R, MUC-1, and in primary ACC cells. Higher p-CDK1 and cyclin B1 were shown in FLNA-silenced MUC-1, while decreased Wee1, p-CDK1 and cyclin B1 resulted after FLNA overexpression. Notably, Wee1 depletion was reverted by lactacystin treatment, and FLNA transfection increased Wee1 phosphorylation at Ser123 residue, suggesting FLNA role in targeting Wee1 for proteasomal degradation. AZD1775 dose-dependently reduced proliferation and viability in ACC cell lines and primary cultures, and it triggered MUC-1 cells death. Similar effects were induced by Wee1 silencing. FLNA depletion augmented AZD1775 efficacy in reducing proliferation and potentiating apoptosis in MUC-1 and primary cells. In conclusion, we demonstrated that FLNA regulates Wee1 expression by promoting its degradation, suggesting that low FLNA typical of ACC leads to increased Wee1 with consequent cancer cells growth. Therefore, it proposes Wee1 inhibition as a new potential therapeutic approach for ACC, particularly for those lacking FLNA. Polo-like kinase 1 (PLK1) and cyclin-dependent kinases (CDKs) 1/2/4 are among the most overexpressed cell cycle-related genes in ACC human samples. Previously, the efficacy of the polo-box domain (PBD)-targeting PLK1 inhibitor Poloxin has been demonstrated in ACC cell lines (H295R, MUC-1, CU-ACC2). The second part of this PhD thesis is centred on testing 1) the efficacy of Poloxin and of the kinase domain (KD)-targeting PLK1 inhibitor Plogosertib, and 2) the efficacy of the multi-CDKs inhibitors Dinaciclib, SNS-032, and AT7519, as well as that of the CDK1-cyclin B1 inhibitor Cucurbitacin E, on cell proliferation, viability, and apoptosis; lastly, 3) the combinatorial effect of Plogosertib and Dinaciclib on ACC cells proliferation. Experiments were carried out in four human ACC cell lines, including H295R, MUC-1, TVBF-7 and the recently acquired JIL-2266. PLK1 inhibitor Poloxin reduced cell proliferation and viability at very high doses, reaching a maximum effect at 100 µM, and increased apoptosis at 10 µM. At much lower doses, Plogosertib induced a dose-dependent reduction of cell proliferation and viability, and an increase of caspase 3/7 activity. The multi-CDKs inhibitor Dinaciclib reduced cell proliferation at low nanomolar concentrations, having a very strong effect in the MUC-1 and JIL-2266 cell lines. It also increased apoptosis, particularly of TVBF-7 cells. Cucurbitacin E reduced proliferation in all ACC cells, but its effects were less pronounced than that induced by Dinaciclib. Synergistic inhibition of cell proliferation by combined treatment with the PLK1 inhibitor Plogosertib and multi-CDKs inhibitor Dinaciclib was observed in H295R and TVBF-7 cells. In conclusion, we identified Plogosertib and Dinaciclib as the most effective PLK1 and multi-CDKs inhibitors on all cell lines, respectively, therefore representing interesting novel treatment options for ACC. Moreover, the combination of these drugs showed a significant synergistic effect, suggesting a potential benefit of using both of them to increase therapeutic efficacy and to reduce potential side effects.