PROJECT SUMMARY Germline predisposition to hematopoietic malignancies (HMs) is more common than previously appreciated, but individuals are spread throughout the country, with few local experts. The NIH provides focused clinics for patients with germline GATA2 mutations under Drs. Holland and Hickstein and for those with germline RUNX1 mutations under Dr. Liu. The proposed U01 consortium will engage four extramural investigators, Drs. Lucy Godley (The University of Chicago), Anupriya Agarwal (Oregon Health & Science University), Emery Bresnick (University of Wisconsin-Madison), and Nancy Speck (University of Pennsylvania) to perform complementary studies using primary samples from patients seen at the NIH Clinical Center. Increased inflammation is characteristic of the infections suffered by immunodeficient individuals with germline GATA2 mutations, and preliminary data from the extramural investigators suggest that germline RUNX1-mutant cells produce increased levels of inflammatory molecules. The U01 investigators hypothesize that inflammation derived from intrinsic and extrinsic sources drives bone marrow failure (BMF) in GATA2-mutant patients, plus clonal hematopoiesis (CH) that frequently evolves into HMs in both syndromes. The U01 consortium will test this hypothesis using three Aims: Aim 1- Identify which germline GATA2 and RUNX1 VUSs are deleterious by analyzing their influences on BM hematopoietic stem and progenitor cells (HSPCs) and mesenchymal stromal cells (MSCs). VUSs identified in patients presenting to the NIH Clinical Center will be characterized functionally using growth and differentiation assays of patient-derived HSPCs, non-hematopoietic MSCs, and an MSC cell line benchmarking them against wild-type and known deleterious or benign variants. Aim 2- Establish how inflammatory mechanisms interface with germline GATA2 and RUNX1 mutations to alter HSPC and MSC growth and differentiation. We will identify the inflammatory cytokines/chemokines overproduced by RUNX1- and GATA2-mutant BM cells in response to TLR4 ligands and determine their impact as well as that of LPS on colony formation, serial-replating, and differentiation of RUNX1- and GATA2-mutant HSPCs and MSCs. We will establish a pre-clinical model of inflammation-induced BMF in Gata2-deficient mice and determine the contribution of elevated TLR signaling to their hematopoietic defects. Aim 3- Determine how acquired mutations and inflammation promote the expansion of germline-mutant HSCs. CH occurs more frequently and at an earlier age in patients with germline GATA2- and RUNX1-mutations compared to the general population. We will use xenograft and syngeneic mouse models to determine how inflammation drives bone marrow failure in Gata2-deficient mice and facilitates the growth/survival of germline GATA2- or RUNX1-mutant HSPCs with acquired somatic mutations, providing a clonal advantage that ultimately evolves into leukemia. The results of these studies will be used to desig...