Project Summary/Abstract Project 2: Genomics of allogeneic transplantation. The goal of this project is to identify and characterize the genomic and transcriptional variants that provide long-term, relapse-free survival to AML patients after allogeneic hematopoietic cell transplantation (alloHCT). Relapse after alloHCT remains common and challenging to resolve. Donor T-cell responses to minor histocompatibility antigens (mHAs) presented on the surface of recipient cells are important drivers of the beneficial graft-versus-leukemia (GVL) effect and harmful graft-versus-host disease (GVHD) pathology following major histocompatibility complex (MHC)-matched alloHCT. mHAs are a diverse collection of peptides that are bound by the MHC and are derived from polymorphic proteins that differ between the donor and recipient. In this proposal, we will utilize state-of-the-art genome and transcriptome sequencing to study the role of mHAs in alloHCT as follows: Specific Aim 1: We will define human mHAs that provide strong GVL activity, but not GVHD. We will evaluate 60 AML patients who have maintained a morphologic complete remission for at least 24 months after MHC-matched sibling alloHCT. To segregate the “therapeutic” mHAs providing a GVL response, from the “pathogenic” targets of GVHD, we will evaluate equal numbers of AML patients who did (n=30) or did not (n=30) develop grade II-IV acute or chronic GVHD after transplantation. All mHA disparities that are expressed within each donor-recipient pair in the graft-versus-host direction (i.e., variant present in the recipient but not donor) will be defined by enhanced whole genome (eWGS) and transcriptome sequencing. Candidate mHAs will be predicted using in silico peptide binding prediction algorithms and validated using immunological assays, mass spectrometry, and correlative studies with recipient samples collected post-alloHCT. Specific Aim 2: We will define the genetic and epigenetic changes that contribute to AML relapse after allogeneic “selection” in mice and humans. Our hypothesis is that relapse after alloHCT is mediated via downregulation or loss of immunogenic mHAs and/or selection of rare cells that lacked expression of the immunogenic mHAs. In humans, we will evaluate patients who have relapsed after undergoing an MHC- matched sibling alloHCT. RNA-seq (bulk and single cell), coupled with eWGS will be performed on primary and post-alloHCT relapse AML sample pairs, as well as the donor’s blood mononuclear cells, to identify mHAs and evaluate the events that make subclones progress after an alloHCT. In mice we will use novel C57Bl/6 (B6)- derived Dnmt3a R878H/FLT3-ITD AML tumors and apply allogeneic immunological pressure using MHC- mismatched mice (BALB/c) or MHC-matched mice that differ from B6 mice at multiple mHAs (C3H.SW and 129Sv/J). We will characterize the allogeneic-resistant AML cells by WGS and RNA-seq to identify mechanisms that contribute to immunologic “escape”. Taken together, these s...