Abstract 1935: The NEDD8 and EGFR pathways are independent therapeutic targets in colorectal cancer
Authors: Invrea Federica , Carugo Alessandro , Petti Consalvo , Bristow Cristopher , Peoples Michael , Cancelliere Carlotta , Corrado Alessia , Bardelli Alberto , Isella Claudio , Draetta Giulio F. , Medico Enzo 
Published: September 2020

Abstract
Colorectal cancer (CRC) develops and progresses through an accumulation of genetic and epigenetic alterations in multiple molecular pathways. Currently, targeted therapies combined with standard chemotherapy are the first-line therapy for metastatic CRC; nevertheless, the effectiveness of these drugs is limited by primary and acquired resistance. Since pathways are highly interconnected, the development of rational combinations of targeted therapies can be exploited to circumvent resistance mechanisms. As a possible alternative pathway for CRC treatment, an attractive option is the blockade of the NEDD8-ubiquitin like protein conjugation pathway. We previously found that the NEDD8-inhibitor pevonedistat has significant in vitro and in vivo activity on a subset of CRCs. However, the in vivo response is limited to tumor stabilization rather than regression, highlighting the need for therapeutic combinations. When the optimal drug combination cannot be readily predicted, functional genetic screens represent a powerful tool for unbiased exploration. We therefore carried out a synthetic lethality screening with an shRNA library covering 200 genes associated with FDA-approved drugs. The screening was aimed at identifying shRNAs that would be depleted only in the presence of pevonedistat: such constructs were expected to target genes that could harbor synergistic effects with NEDD8 inhibition. After shRNA library transduction, CRC cell lines were grown in the absence or presence of low-dose pevonedistat; Next Generation Sequencing and bioinformatics analysis allowed comparing the transduced library repertoire with or without pevonedistat, and identifying candidate synthetic lethal genes. The screening results revealed a strong, pevonedistat-dependent depletion of constructs targeting different tyrosine kinases (i.e. EGFR, BRAF, FYN and FLT4) in specific CRC cell lines (CAR1, WIDR, LIM2099 and DIFI, respectively). Firstly, we focused on the remarkable drop-out of EGFR-targeting shRNAs in CAR1 cells, which represent a subgroup of aggressive tumors refractory to cetuximab treatment, despite the lack of currently known markers of resistance. We discovered an independent and additive effect of anti-EGFR drugs (cetuximab and lapatinib) combined with pevonedistat, which was then validated in vitro on further cell lines (HCA7 and HROC69) and in vivo on HCA7-transplanted mice. Furthermore, we considered the depletion of constructs targeting BRAF, a member of EGFR pathway, observed in WIDR BRAF-mutant cells: we found an additive effect of BRAF inhibitor vemurafenib and pevonedistat in reducing in vitro cell growth. Altogether our results suggest that the concomitant pharmacological inhibition of NEDD8 and EGFR-pathway could be a novel effective approach to treat clinically aggressive CRCs, worthy of further characterization.
Citation Format: Federica Invrea, Alessandro Carugo, Consalvo Petti, Cristopher Bristow, Michael Peoples, Carlotta Cancelliere, Alessia Corrado, Alberto Bardelli, Claudio Isella, Giulio F. Draetta, Enzo Medico. The NEDD8 and EGFR pathways are independent therapeutic targets in colorectal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1935.