SPATIAL EPIGENOMIC PROFILING OF IMMUNE CELL SIGNATURES AT SUBCELLULAR RESOLUTION IN HEALTH AND DISEASE More than ten percent of childhood cancers are still incurable and need novel therapies. Epigenetic treatments deserve special attention with their specificity and reduced toxicity. Here I plan to explore epigenetic profiles of immune and cancer cells in normal development and blood cancer patients under the mentorship of Garry Nolan for single cell proteomics technology development, in collaboration with Howard Chang for implementation of epigenomic methods such as chromosome accessibility assays, and with Kara Davis for epigenetics studies of treatment resistant B cell subtypes in acute lymphoblastic leukemia (ALL). Epigenetic measurements have been limited to bulk level sequencing and ligation assays or limited number of imaging markers. To address these limitations, I will use an emerging three dimensional (3D) proteomic imaging technology in individual cells, termed as 3D Multiplexed ion beam imaging (MIBI) or 3D MIBI. Epigenetics research by 3D MIBI benefits from high degree multiplexing (up to 100 markers) and super resolution imaging capability (20 nm x-y; 5 nm z resolution), providing exciting opportunities to study genomic sites, methylated DNA, protein factors, and chromosome accessibility, all within the same experiments in single immune and aberrant (leukemic) cells. To systematically determine epigenetic states, I plan to utilize clonal B cell lines to decipher variability of epigenetic components including chromatin states, protein factors and modifiers by a fifty-marker 3D MIBI panel (Aim 1). These experiments will show distribution of epigenetic factors (linear or log-scale) in their expression levels and spatial variations (global or local) in the chromatin states. I will then perform experiments with primary B cells isolated from six different bone marrow aspirates of normal human subjects (Aim 2). I will correlate epigenetic signatures of each B cell subtype to corresponding development state (progenitor, pre, post, or mature). I will then perform an ex vivo co-culture of primary B cells on OP9 stromal cells over 1-6 weeks of culturing, which will be followed by fixation and profiling by 3D MIBI. These perturbation experiments will show how signaling events from neighboring cells drive necessary epigenetic conditions that are required for reaching a B cell subset. Finally, I will turn to primary B cells that are isolated from twenty newly diagnosed ALL patients (Aim 3). I will dissect differentiation and spatial epigenomic remodeling of responder B cell subsets and treatment resistant B cell subtypes from bone marrow aspirates using the OP9 co-culture. These will show how treatment resistance arises from a single epigenetic state or multiple distinct epigenetic signatures. I will then screen Histone deacetylase inhibitors (HDACi) on the same co-culture of B cell subtypes from ALL and stromal cells. By varying concentration and duration of inhibition conditions, I will dissect the role of epigenetic drugs in spatial chromatin remodeling toward development of epigenetic therapies in ALL. Together, these experiments will shed light on the role of epigenetic programming for cancer treatment applications from immune cell signatures in normal subjects and blood cancer patients.

Public Health Relevance

Epigenetic alterations in immune and cancer cells regulate normal development and aberrant formation in humans. We reveal spatial heterogeneity and dynamic changes in epigenetic states of B cell subtypes from healthy and diseased subjects using a novel multiplex imaging mass cytometry. Deciphering the role of epigenetic perturbations in treatment resistant B cells will guide development of efficient and safe epigenetic therapies for pediatric blood cancers.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Mentored Quantitative Research Career Development Award (K25)
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Allergy, Immunology, and Transplantation Research Committee (AITC)
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Gondre-Lewis, Timothy A
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Stanford University
Schools of Medicine
United States
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