Functional analysis of the leukemic evolution in children with Down Syndrome utilizing CRISPR-Cas genome editing

• Applicant: Professor Dr. Dirk Heckl
• Subject Area: Pediatric and Adolescent Medicine ; Hematology, Oncology
• Term: from 2015 to 2019
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 276311671

Acute myeloid leukemias are a heterogeneous group of clonal diseases originating from the hematopoietic stem- and progenitor cells (HSPCs), which transform through stepwise acquisition of cooperating genetic lesions. Due to the lack of appropriate technologies to modify the genomes of human cells at the degree of complexity found in leukemia, this evolutionary process of stepwise transformation has been impractical to model and interrogate in the human system. Even in mice, where these modifications are feasible, combinatorial genetics of more than two modifications are time consuming and expensive. We have recently developed a lentivirus-based CRISPR-Cas (clustered regularly interspaced short palindromic repeats (CRISPR) - CRISPR-associated) system, which allowed us to simultaneously modify up to five genes at their endogenous loci in a single HSPC. Here, we will leverage this novel genome modification tool to delineate the leukemic evolution of myeloid leukemia in children with Down syndrome (ML-DS). 5-30% of infants with DS are diagnosed with transient abnormal myelopoiesis (TAM), which has typical characteristics of leukemia. TAM regresses in most of the infants without anti-leukemic treatment, however, in 20-30% of these children an AML develops within the first four years of life. Both TAM and ML-DS almost universally (95%) harbor mutations in the hematopoietic transcription factor GATA1, which lead to the exclusive expression of a truncated protein (GATA1s), and have been identified as the causative lesion inducing TAM in infants with DS. Nevertheless, GATA1s mutations are insufficient to cause ML-DS and cooperating lesions remain unknown.To identify the genetic aberrations during the transition from TAM to ML-DS, we have joined an international consortium and sequenced exomes of 22 paired TAM/ML-DS patients, followed by targeted sequencing of candidate mutations in 400 ML-DS samples. Besides high prevalence of mutations in tyrosine kinases and cytokine receptor signaling-associated genes, a particularly high percentage of patients presented with mutations in cohesin-complex-associated genes or histone modifiers, which contrasts the mutational spectrum seen in pediatric non-DS AML. Utilizing our lentiviral CRISPR-Cas genome editing tool and classical lentiviral cDNA expression -both in Gata1s mice and primary TAM patient samples- we will interrogate the functional and molecular consequences of cohesin-complex- and histone modifier mutations in the transformation of TAM to ML-DS. We will thereby unravel the molecular processes that guide leukemic transformation in the context of GATA1s and trisomy 21. In a CRISPR-Cas based loss of function screening we will leverage this knowledge about ML-DS transformation to identify novel therapeutic targets that will allow to eradicate ML-DS with targeted and reduced toxicity therapy, or open even new venues to eliminate preleukemic TAM clones and thereby transformation of TAM to ML-DS.

• DFG Programme: Research Grants