A functional sgRNA-CRISPR screening method for generating murine RET and NTRK1 rearranged oncogenes.

CRISPR/Cas9 gene editing represents a powerful tool for investigating fusion oncogenes in cancer biology. Successful experiments require that sgRNAs correctly associate with their target sequence and initiate double stranded breaks which are subsequently repaired by endogenous DNA repair systems yielding fusion chromosomes. Simple tests to ensure sgRNAs are functional are not generally available and often require single cell cloning to identify successful CRISPR-editing events. Here, we describe a novel method relying on acquisition of IL3-independence in Ba/F3 cells to identify sgRNA pairs that generate oncogenic gene rearrangements of the Ret and Ntrk1 tyrosine kinases. The rearrangements were confirmed with PCR, RT-PCR and sequencing and Ba/F3 cells harboring Ret or Ntrk1 rearrangements acquired sensitivity to RET and TRK inhibitors, respectively. Adenoviruses encoding Cas9 and sgRNA pairs inducing the Kif5b-Ret and Trim24-Ret rearrangements were intratracheally instilled into mice and yielded lung adenocarcinomas. A cell line (TR.1) established from a Trim24-Ret positive tumor exhibited high in vitro sensitivity to the RET inhibitors LOXO-292 and BLU-667 and orthotopic TR.1 cell-derived tumors underwent marked shrinkage upon LOXO-292 treatment. Thus, the method offers an efficient means to validate sgRNAs that successfully target their intended loci for the generation of novel, syngeneic murine oncogene-driven tumor models.

Upregulation of complement proteins in lung cancer cells mediates tumor progression.

Introduction: In vivo, cancer cells respond to signals from the tumor microenvironment resulting in changes in expression of proteins that promote tumor progression and suppress anti-tumor immunity. This study employed an orthotopic immunocompetent model of lung cancer to define pathways that are altered in cancer cells recovered from tumors compared to cells grown in culture. Methods: Studies used four murine cell lines implanted into the lungs of syngeneic mice. Cancer cells were recovered using FACS, and transcriptional changes compared to cells grown in culture were determined by RNA-seq. Results: Changes in interferon response, antigen presentation and cytokine signaling were observed in all tumors. In addition, we observed induction of the complement pathway. We previously demonstrated that activation of complement is critical for tumor progression in this model. Complement can play both a pro-tumorigenic role through production of anaphylatoxins, and an anti-tumorigenic role by promoting complement-mediated cell killing of cancer cells. While complement proteins are produced by the liver, expression of complement proteins by cancer cells has been described. Silencing cancer cell-specific C3 inhibited tumor growth In vivo. We hypothesized that induction of complement regulatory proteins was critical for blocking the anti-tumor effects of complement activation. Silencing complement regulatory proteins also inhibited tumor growth, with different regulatory proteins acting in a cell-specific manner. Discussion: Based on these data we propose that localized induction of complement in cancer cells is a common feature of lung tumors that promotes tumor progression, with induction of complement regulatory proteins protecting cells from complement mediated-cell killing.