Generation of Models of Human Hematologic Malignancies Using CRISPR Genome Engineering

Hematopoietic malignancies are genetically complex diseases in which the serial acquisition of somatic mutations results in clonal diversity with distinct responses to therapy. While tremendous progress has been made in defining the genetic basis of hematologic malignancies through large-scale sequencing studies, models are now needed that reflect the specific combinations of mutations identified and the clonal complexity of human disease. Such models would be powerful tools to probe the biology of malignant transformation and to identify genetic subtypes that are sensitive or resistant to therapeutic agents.

We used CRISPR/Cas9 genome engineering of primary human hematopoietic stem and progenitor cells (HSPCs), the cells of origin for myeloid malignancies, followed by transplantation into immunodeficient mice, to generate models of clonal hematopoiesis and malignancy. We targeted nine recurrently mutated genes in MDS/AML with predicted loss-of-function (LOF) mutations: TET2, ASXL1, DNMT3A, RUNX1, TP53, NF1, EZH2, STAG2 and SMC3, in both umbilical cord and adult CD34+ cells. We developed a next-generation sequencing and computational strategy to identify and track the allelic fractions of specific insertions or deletions introduced by CRISPR/Cas9. We demonstrated feasibility and efficiency of multiplex targeting at a single cell level, with 42% of clones showing LOF mutations in at least one gene and 30% of clones demonstrating targeting in 2-6 genes.

In vivo, we first modeled clonal hematopoiesis of indeterminate potential (CHIP) and noted clonal expansion of TET2 and DNMT3A LOF clones over the course of 5 months. Since overt myeloid malignancies are generally associated with the acquisition of somatic mutations in multiple driver genes in a single clone, we performed multiplex genome editing in vivo using a combination of CRISPR/Cas9 and overexpression of gain of function oncogenes, such as FLT3-ITD and NPM1. Human cells bearing mutations in combinations of genes observed in myeloid malignancies generated neoplastic clones capable of long-term, multi-lineage reconstitution and serial transplantation. The genetic lesions introduced into human HSPCs generated diverse morphologic phenotypes, such as a clonal expansion of immature human myeloid forms in mice targeted with SMC3 and FLT3-ITD, a combination frequently seen in patients. In addition, multiplex targeting also allowed us to monitor in vivo clonal dynamics of human cells over time and model selective dominance of an individual genetic clone. Finally, employing these models to investigate therapeutic efficacy, we recapitulated differential sensitivity of TET2 and ASXL1 clones to treatment with the hypomethylating agent azacitidine observed in patients. Of note, we found that STAG2 and SMC3 mutated hematopoietic cells were also sensitive to treatment with hypomethylating agents.

Our approach of modeling mutations in human cells in vivo is highly customizable, recapitulates the genetic complexity of human myeloid diseases, and enables the study of clonal dynamics over time. These findings demonstrate the potential for generating genetically defined models of hematologic malignancies that reflect human disease and are suitable for examining the biological consequences of somatic mutations and the testing of therapeutic agents.