Stepwise-edited, human melanoma models reveal mutations’ effect on tumor and microenvironment

Abstract

Establishing causal relationships between genetic alterations of human cancers and specific phenotypes of malignancy remains a challenge. We sequentially introduced mutations into healthy human melanocytes in up to five genes spanning six commonly disrupted melanoma pathways, forming nine genetically distinct cellular models of melanoma. We connected mutant melanocyte genotypes to malignant cell expression programs in vitro and in vivo, replicative immortality, malignancy, rapid tumor growth, pigmentation, metastasis, and histopathology. Mutations in malignant cells also affected tumor microenvironment composition and cell states. Our melanoma models shared genotype-associated expression programs with patient melanomas, and a deep learning model showed that these models partially recapitulated genotype-associated histopathological features as well. Thus, a progressive series of genome-edited human cancer models can causally connect genotypes carrying multiple mutations to phenotype.

Functional analysis of recurrent non-coding variants in human melanoma

“…Present in ∼80% of cutaneous melanomas, the TERT promoter mutation creates a novel ETS motif that leads to binding of GABPA and derepression of TERT 4 . The full extent of TERT’s influence on tumorigenesis, particularly via this regulatory variant, is still emerging, including its canonical role on telomere maintenance 3, 5 . Beyond TERT promoter variants, few other CRVs have been identified and characterized in melanoma 1, 2, 6–10 .…”

Type 1 interferon remodels normal and neoplastic hematopoiesis in human

“…As IFNa has been shown to induce HSC and progenitors into cell cycle entry 43 , we posited that differential induction of progenitor subtypes into proliferation may underlie the shifts in differentiation. Therefore, we examined the gene signatures for proliferation in the context of cell identity shown to be an accurate assessment of proliferation state 44 . Consistent with previous reports in mice 43 , the proportions of HSCs expressing G2/M/S-phase genes were enhanced upon IFNa treatment ( Fig.…”

Modeling clonal evolution and oncogenic dependency in vivo in the context of hematopoietic transformation

“…This led to insights into leukemic transformation and will inform studies of how mutational order, cell type of mutation acquisition, and mutational clonal representation impact leukemic transformation and the response to therapy. Most importantly, these studies provide a template by which multiple mutations can be deterministically and sequentially induced in vivo, without the selection bottlenecks induced by transplantation, 32 simultaneous CRISPR editing of different mutant alleles, 17,18 or ectopic mutant gene expression 32 33,34 . Each of these approaches will have an important role in modeling cancer evolution and clonal complexity, and there may be advantages to combining endogenous targeting and multiplex CRISPR editing to better model mutational complexity in vivo.…”

“…Experimental systems have largely relied upon ectopic overexpression models, Cre-Lox technology 14 , and the increasing use of CRISPR/CAS9 genome editing in different species [15][16][17][18] .…”