Abstract 4216: Targeting NANOG: genes, proteins and response to viral RNAi in preclinical models

Purpose: Since the NANOG family controls stemness and neoplastic progression in human colorectal carcinoma (CRC), our postulate is that targeting the dominant NANOG and NANOGP8 members with viral vector delivered shRNA will inhibit CRC progression in preclinical models and that replicating viral vectors are more active than non-replicating vectors. Methods: Cells were human CRC lines (Clone A, CX-1 and LS174T) and mouse fibroblasts (3T3). shRNA specific to NANOG (shNG-1), NANOGP8 (shNp8-1) or a control (shNEG) were created in lentivirus (LV) and chimeric adenovirus 5/3 (Ad5/3). CRC and 3T3 were cultured in monolayer with serum-containing medium or in suspension in ultra low attachment plates without serum. Nanostring 770 gene cancer panel was analyzed in 72 hr cultures with NMF clustering and GSEA analysis. Annexin V expression and caspase-3, 8 or 9 activation in suspension culture assessed Apoptosis. LV or Ad5/3 shRNAs were added to spheroids in 3-D suspension cultures to test therapy in vitro. Intratumoral injection of LV or Ad5/3 shRNA into 5-10 mm sub cutis nodules in groups of 5-10 NOD/SCID mice measured activity in vivo. Results: LV shNp8-1 in both Clone A and CX-1 caused significant inhibition of TGF-beta, EGFR, NRF2, E2F3 and the Cyclin D1 pathways by NMF and GSEA analysis in monolayer culture. LV shNp8-1 inhibited growth of LS174T, Clone A and CX-1 by 50% in 3 days in 3-D suspension cultures without further decrease. Apoptosis was due to Caspase 9 activation. In contrast, Ad5/3-shNp8-1 decreased spheroid growth by up to 94% at 9 -14 days while Ad5/3 decreases spheroid growth by 80%. Ad5/3-shNp8-1 but nit Ad5/3 was active in co-cultures with 3T3 to a 2:1 CRC:3T3 ratio. Transduction level was ∼50% in LV treated cells but ∼90% in Ad5/3 treated cells. Intratumoral injection of LVshNG-1 or LVshNp8-1, decreased tumor size by 50% compared to LVshNEG or untreated CX-1 tumors for 11 days after which tumor growth rebounded to match controls. Ad5/3-shNp8-1 injection into 5-9 mm nodules inhibited CX-1 and LS14T growth by ∼50% until euthanized. Conclusions: Targeting NANOGP8 more than NANOG inhibits proliferation and activates apoptosis through multiple pathways. Nonreplicating LV delivered shRNA inhibited tumor growth in in vitro and in vivo preclinical models but replicating oncolytic Ad5/3 infects more cells and promotes shRNA efficacy. Replicating vectors targeting NANOG are important for further development. Citation Format: J. Milburn Jessup, Abid R. Mat