Background. PDK1, is a master activator of multiple prosurvival and oncogenic protein kinases such as AKT, PKC, RSK and SGK families in oncology[1]. On the other hand, overexpression and aberrant activation of Aurora-A kinase (AurA) have been functionally linked to oncogenic transformation mainly through development of centrosome amplification and chromosomal instability[2]. In this study, the molecular mechanisms at the simultaneous PDK1/AurA inhibition using OXID-pyridonyl[3] compounds were explored in panel of tumor cell lines carrying different oncogenic mutations (PI3KCA, K-RAS, PTEN, RB-1, EGFR and TP53).

Materials and Methods. Established human cancer cell lines from breast, glioblastoma, renal prostate, colon, lung and Ewing sarcoma were studied. Anti-proliferative effects of OXID-pyridonyl compounds (SA16, IB35, VI8, VI18) were assessed by soft agar clonogenic formation and MTT assays after 72h-exposure with increasing concentrations (from 0.014 to 30 µM). Protein levels were analyzed by Western Blot using commercial antibodies. OSU-03012 (PDK1 inhibitor) and MK-5108 (AurA inhibitor) were used as reference compounds to investigate the functional crosstalk between the two pathways in sensitive cancer cell lines.

Results. We assessed anti-proliferative activity of single-agents SA16, IB35, VI8 and VI18 in a panel of 15 cancer cell lines and observed that three Ewing sarcoma and one colon carcinoma cell lines were sensitive (6647,TC71, SKNMC and HCT116), with IC50s values between 2.6 and 25 µM after 72h-exposure, whereas all the studied cell lines were resistant to IB35 and VI18. These results were confirmed by soft agar clonogenic formation studies. AurA, PDK-1, p-PDK1 ser241, AKT, p-AKT ser473 protein levels were characterized but no differences in their basal levels were observed between OXID-pyridonyl compounds -sensitive or -resistant cell lines. SA16 effect at the protein level was evaluated after 5 to 120 min in sensitive cell lines. In SKNMC, TC71 and HCT116 cells, we didn't observe a modulation of AurA or p-PDK1 ser241 while a decrease of p-AKT was observed in SKNMC and HCT116 cells. In addition, a decrease of the p-p44/42 MAPK was observed after 30 min-exposure in SKNMC cells. On the other hand, p-RB1, p-Cdc2, P21 and C-MYC were not regulated at the protein level with SA16 treatment up to 120 min-exposure. In resistant cell lines, U87MG and PC3, both p-PDK1 ser241 and AurA levels were not modulated up to 2h of treatment with SA16.

Conclusion. Here we report the characterization of two potent first-in-class dual PDK1-AurA inhibitors, SA16 and VI8. Our findings suggest that innovative PDK1/AurA dual-target molecules which could represent an attractive lead scaffold for the design and synthesis of a new multitarget strategy for Ewing sarcoma.

[1] Nagashima K et al. J Biol Chem. 2011. [2] Yan M et al. Med Res Rev. 2016. [3] Sestito, S et al. Proceedings 2019.

Citation Format: Maria Eugenia Riveiro, Simona Rapposelli, Samuel Huguet, Ramiro Vazquez, Mohamed Bekradda, Guido Puricelli, Simona Sestito, Olivier Madar, Keyvan Rezai. Dual targeting of PDK1 and Aurora A using first-in class OXID-pyridonyl compounds in preclinical models of Ewing sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1294.