Metabolomics of cGVHD

Abstract Our goal is to develop new therapies for chronic GVHD (cGVHD), the leading cause of late morbidity and mortality after allotransplant. We made the important observation that T cell:B cell cooperativity and class-switched Ig tissue deposition caused multi-organ system cGVHD with bronchiolitis obliterans (BO), a non-infectious airway obstructive and epithelial remodeling disorder that portends an abysmal 5 year survival for patients. From proof-of- concept in cGVHD models, we provided key data leading to clinical trials of 6 new therapies (2 now FDA approved) for patients failing first-line steroids. CGVHD can be induced by germinal center (GC) T- and B- cell cooperativity, to produce anti-host Abs and fibrosis. While we observed GC Tfollicular helper cells (Tfh) to have increased glycolysis in the early cGVHD/BO phase, glycolysis decreased over time, consistent with exhaustion. cGVHD pathogenic Tfh and Tfollicular regulatory cells (Tfr) that restrain GCs must adapt and thrive in GCs with high reactive oxygen species and limited energy sources. Our central hypothesis is that cGVHD imposes unique metabolic demands on GC cells for cGVHD pathogenesis and distinct demands on damaged lung epithelial progenitors, impeding repair and regeneration. Choosing the best single or combined drug therapies to treat established cGVHD/BO optimally requires targeting pathogenic (Tfhs, GC B cells) and sparing Tfrs and lung stem/progenitor cells. We will test the hypothesis that rapidly proliferating Tfh that support GCs depend on multiple energy sources (glutaminolysis, glycolysis, fatty acid synthesis (FAS), while aberrant GC B cells rely on glycolysis and FAS. Relatedly, we will test the hypothesis that knowing metabolic pathways required by GC subsets (aim 1) and regenerating lung stem/ progenitor cells (aim 2) will lead to new druggable targets. Aim 1A proposes to: (1) Interrogate GC subsets for the precise pathways used for energy; (2). Test if inducing single metabolism gene deletion in a lineage-restricted GC subset in cGVHD/BO mice will improve pulmonary function and immune parameters; and (3). Test selected metabolism drug candidates in vivo to reverse ongoing cGVHD/BO. In aim 1B, we hypothesize that a focused CRISPR guide RNA metabolism library of ~40-50 gene targets from aim 1A drug results, U-[13C]-substrate (glucose, glutamine palmitate) labeling and RNA-seq data, transduced into Cas9 transgenic donor cells infused on day 0 will identify undiscovered metabolism genes critical for cGVHD/BO pulmonary dysfunction. Drugs to treat immune dysfunction may be offset by detrimental effects on lung injury repair/regeneration. In aim 2A, we will use scRNA-seq to define altered cell states and ligand-receptor interactions in distal and proximal airway epithelial and mesenchymal cells. Lung bronchiolar, alveolar and tracheosphere organoid cultures will be used to identify cells adversely affected by cGVHD/BO. In aim 2B, we hypothesize that aim 1 drugs effective in targeting GCs and aim 2 drugs in supporting lung cell regeneration will guide drug selection to inhibit cGVHD/ BO pathogenesis and repair lung injury with a high predictive value for superior cGVHD/BO outcomes. These novel approaches provide unprecedented mechanistic data to elucidate pathogenesis and lead to new therapies.