Mechanisms of Bone Marrow Failure and Leukemia Progression in Primary Human Fanconi Anemia Stem Cells in Novel FA PDX Model

ABSTRACT

The goal of this research proposal is to provide better treatments for Fanconi Anemia (FA), an inherited bone marrow failure disorder that affects approximately 1 in 100,000 children. The combination of hematopoietic stress and inherent genomic instability leads to cancer and accumulation of genetic defects is likely the cause of AML progression. We proposed to study primary human cell defective in the FA pathway to delineate pathways of leukemia progression and eventually prevent progression to bone marrow failure or progression to leukemia. Our two aims are to 1)identify molecular vulnerabilities and genetic changes promoting oncogenesis in FA deficient CD34+ cells in vitro and to 2) determine molecular changes at the root of disease progression in primary human FA bone marrow and test potential therapeutic approaches in vivo in MISTRG-kitMUT mice. To achieve this goal we have to i) obtain primary FA patient cells and ii) generate human FANC gene KO CD34+ cells. Note that the COVID pandemic has significantly impaired our progress since 3/15/2020. We have focused our efforts on generating FA defective cells via two mechanisms a) shRNA mediated knockdown and b) via CRISPR/Cas9 mediated deletion. We have encountered 2 difficulties which we are still addressing inefficiency of deleting FA genes and selection against deleted cells;silencing of rescue lentiviral vectors in primary hematopoietic cells. With COVID all work had to halt and mouse work was minimal we are expanding colonies and actively transplanting primary FA samples with goal to further optimize engineering of FA samples and transplantation in MISTRG mice.