Abstract A132: Deciphering the molecular regulators of tumor resistance to VEGF-receptor tyrosine kinase inhibitors via in vivo genome-wide RNAi screen
Authors: Fahim Golestaneh Azadeh , Sun Maoyun , Schwager Christian , Tang Zili , Ma Lili , Hahnfeldt Philip , Moustafa Mahmoud , Weichert Wilko , Debus Juergen , Hlatky Lynn , Abdollahi Amir 
Published: August 2018

Inhibiting angiogenesis, which is a shared characteristic of all solid tumors, is a promising approach for treating cancer. However, emerging resistance after initial response is still a drawback. We sought to decipher the molecular landscape of tumor resistance to continuous treatment with VEGF-receptor tyrosine kinase inhibitor (RTKi) sunitinib (SU11248, SU). To address that, we performed serial transplantation of a syngeneic LLC tumor model in C57bl6 mice using a genome-wide lentiviral shRNA library targeting about 39,000 transcripts (150K complexity). The abundance of each single knockdown (kd) was longitudinally traced via molecular barcodes towards development of resistant tumors. After three in vivo panning rounds, distinct shRNA constructs were found to be significantly enriched in tumors passaged under SU selection pressure (p3-SU) compared to those passaged without treatment (p3-ctrl). We focused on validation of three candidate shRNA targeting Kdm5c, Csf1, and Cd44. Knockdown of each of these three genes confirmed their role in emerging resistance to sunitinib in vivo in syngeneic model. Furthermore, knockdown of Kdm5c promoted resistance in 786-0 human renal cell carcinoma xenograft model, in which sunitinib is used as the first-line therapy. Transcriptome analysis of shKdm5c, shCsf1, and shCd44 harboring vs. shCtrl cells suggested that the development of resistance is partly due to modulation of cellular pathways as well as alteration of interaction between cells and their microenvironment. In addition, shKdm5c tumors demonstrated resistance towards pazopanib, an VEGF-RTKi with different inhibitory spectrum compared to sunitinib, which suggests a general mechanism of resistance to inhibitors of VEGF signaling after Kdm5c knockdown. Furthermore, cells with shKdm5c showed an increase in survival under hypoxia induced by VEGF-RTKi. Expression profiling of these cells in hypoxia illustrated a network of genes under Kdm5c control, including three components of PRC2 epigenetic modulator, Ezh2, Eed, and Suz12. We found that knockdown of Kdm5c interrupts the imposed break on these genes under hypoxia. In conclusion, we have shown that loss of Kdm5c, CSf1, and Cd44 renders tumor cells resistant to VEGF-RTKi partly through compensatory upregulation of angiogenic factors. In addition, loss of Kdm5c leads to aberrant activation of a gene-regulatory circuitry controlling cellular response to hypoxia.

Citation Format: Azadeh Fahim Golestaneh, Maoyun Sun, Christian Schwager, Zili Tang, Lili Ma, Philip Hahnfeldt, Mahmoud Moustafa, Wilko Weichert, Juergen Debus, Lynn Hlatky, Amir Abdollahi. Deciphering the molecular regulators of tumor resistance to VEGF-receptor tyrosine kinase inhibitors via in vivo genome-wide RNAi screen [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A132.