Positive selection of somatically mutated clones identifies adaptive pathways in metabolic liver disease

Highlights

• Clones with Mboat7 loss-of-function mutations are selected against in NASH livers

• Clones with Gpam loss-of-function mutations are positively selected for in NASH

• In vivo screening was built to identify genes that alter cell fitness and steatosis

• Screening of transcription and epi-factors identified unexpected NASH target genes

Summary

Somatic mutations in nonmalignant tissues accumulate with age and injury, but whether these mutations are adaptive on the cellular or organismal levels is unclear. To interrogate genes in human metabolic disease, we performed lineage tracing in mice harboring somatic mosaicism subjected to nonalcoholic steatohepatitis (NASH). Proof-of-concept studies with mosaic loss of Mboat7, a membrane lipid acyltransferase, showed that increased steatosis accelerated clonal disappearance. Next, we induced pooled mosaicism in 63 known NASH genes, allowing us to trace mutant clones side by side. This in vivo tracing platform, which we coined MOSAICS, selected for mutations that ameliorate lipotoxicity, including mutant genes identified in human NASH. To prioritize new genes, additional screening of 472 candidates identified 23 somatic perturbations that promoted clonal expansion. In validation studies, liver-wide deletion of Tbx3, Bcl6, or Smyd2 resulted in protection against hepatic steatosis. Selection for clonal fitness in mouse and human livers identifies pathways that regulate metabolic disease.

Introduction

Somatic mutations are common in most individuals, and there is accumulating evidence that the mutation burden increases with age and chronic tissue damage.^1,2,3,4 While the identity and abundance of these mutations are becoming increasingly understood through deep sequencing, fundamental questions about the relevance of these mutations remain unanswered. The detection of mutant clone expansion, recurrent mutations, or convergent mutations using sequencing provides correlative evidence for increased clonal fitness. However, such fitness increases can be caused by adaptive or pathogenic mechanisms, and it is uncertain if these ever contribute to organ health or function. Even though most somatically mutated clones are not fated to become cancerous, it is possible that increased proliferation/survival could be selfish and have no beneficial effects on tissue function. Therefore, it is unclear how somatic mutations contribute to organismal aging or disease pathogenesis and whether or not somatic mutations can cause a reversal or adaptation to disease.

Recent evidence from human liver sequencing suggests that mutations could be adaptive. Our previous work indicates that some mutations in cirrhotic livers can result in the regenerative expansion of clones during injury³; however, it is unclear if these expansion events protect against clinically relevant causes of liver disease. Nonalcoholic steatohepatitis (NASH) is becoming the leading cause of liver disease worldwide.⁵ NASH is usually conceptualized at the organismal and tissue levels, and less thought has been given to genetic heterogeneity between clones in the liver. In NASH livers, Ng et al. identified convergent mutations in genes central to insulin signaling and lipogenesis.⁶ These mutations in metabolic enzymes that generate hepatic lipids suggest that some somatic alterations can confer increased fitness through a reversal of the driving etiology of disease.

To understand the impact of somatic mutations at the cellular, tissue, and organismal levels, we developed mouse models that replicate a high density of mutations in the context of common liver diseases. The somatic mutations observed in human liver tissues were also the most positively selected in mouse models of fatty liver but were not selected for in the absence of disease. Mechanistically, these mutations mitigated lipotoxic phenotypes, thereby increasing the survival of hepatocyte clones. These findings uncover the biological basis for positive selection of somatic mutations in NASH patient livers. We reasoned that identifying mutant cells with greater fitness than wild-type (WT) cells within diseased environments might nominate therapeutic targets. This encouraged us to explore genes beyond those that are known to be somatically mutated by performing additional in vivo CRISPR screens for genes that are dysregulated in chronic liver disease. These screens identified genes that, when inhibited, promote liver fitness through the suppression of lipotoxicity. We propose that evolutionary selection in somatically mosaic tissues is a high-throughput approach for the identification of adaptive metabolic disease pathways.