Fully accessible fitness landscape of oncogene-negative lung adenocarcinoma

Abstract

Cancer genomes are almost invariably complex with genomic alterations cooperating during each step of carcinogenesis. In cancers that lack a single dominant oncogene mutation, cooperation between the inactivation of multiple tumor suppressor genes can drive tumor initiation and growth. Here, we shed light on how the sequential acquisition of genomic alterations generates oncogene-negative lung tumors. We couple tumor barcoding with combinatorial and multiplexed somatic genome editing to characterize the fitness landscapes of three tumor suppressor genes NF1, RASA1, and PTEN, the inactivation of which jointly drives oncogene-negative lung adenocarcinoma initiation and growth. The fitness landscape was surprisingly accessible, with each additional mutation leading to growth advantage. Furthermore, the fitness landscapes remained fully accessible across backgrounds with the inactivation of additional tumor suppressor genes. These results suggest that while predicting cancer evolution will be challenging, acquiring the multiple alterations that drive the growth of oncogene-negative tumors can be facilitated by the lack of constraints on mutational order.

Efficient and multiplexed somatic genome editing with Cas12a mice

“…We also assessed the number of clonal barcoded tumors in each mouse and found that H11 LSL-Cas12a mice had an average of 2-fold more tumors than Kras LSL-G12D mice when corrected for viral titer ( Figure 3c ). These results are consistent with the high tumorigenic potential of lung epithelial cells with combined inactivation of Nf1 , Rasa1 , and Pten 19,23 , as well as efficient multiplexed genome editing by Cas12a. Tumor sizes in H11 LSL-Cas12a mice were dramatically larger than in KT mice, with the log-normal mean tumor size around 4-fold greater in H11 LSL-Cas12a mice ( Figure 3d ), in line with an increased tumor growth rate for cr Nf1/Rasa1/Pten tumors compared to those driven by oncogenic KRAS.…”

Combinatorialin vivogenome editing identifies widespread epistasis during lung tumorigenesis

“…As a result, they may have relatively little room to increase their proliferation even further compared to tumors without such a strong oncogenic driver. Concordantly, we previously showed that the combined inactivation of Nf1, Rasa1 and Pten was highly synergistic in oncogene-negative lung adenocarcinoma 39,40 .…”

“…Conversely, while barcoded dual-sgRNA vectors have led to key insights into the tumorigenic potential of complex tumor genotypes in vivo, they have required laborious individual cloning [39][40][41][42] and/or have suffered from lentiviral template switching that decouples the barcode from the sgRNA 43 . To generate barcoded lentiviral-sgRNA vector pools for tumor barcoding and highthroughput barcode sequencing (Tuba-seq), we recently integrated a diverse barcode within the 21 nucleotides of the U6 promoter directly adjacent to a single sgRNA (U6 barcode Labeling with per-Tumor Resolution Analysis; Tuba-seq Ultra ) 44 .…”