Elucidate the Role of RNA Editing in Acute T-cell Lymphoblastic Leukemia Initiating Cells

SUMMARY/ABSTRACT Relapsed pediatric T-cell acute lymphoblastic leukemia (T-ALL) is often refractory to conventional therapy and is associated with a dismal survival rate of less than 25%. Thus, the development of novel therapies for relapsed T-ALL represents an urgent unmet medical need in children. Relapsed/refractory T-ALL is often enriched with

leukemia initiating cells (LICs), which exhibit enhanced survival and self-renewal capacity. Our long-term goal is to identify new targets for development of less toxic and more effective therapies by eliminating this cell population. We discovered that adenosine deaminase acting on RNA 1 (ADAR1) activation is required LIC

maintenance by directing cell-type-specific gene programs to prevent interferon activation and aberrant double- stranded RNA (dsRNA) sensing. Approximately 70% of T-ALL patients exhibit high expression of ADAR1, and this is associated with a significantly worse clinical outcome. Strikingly, we found that inhibiting ADAR1 impairs

malignant T-ALL progenitor prorogation. These data suggest that ADAR1 has a fundamental role in LIC self- renewal and therapeutic resistance in T-ALL. The overall objective of this study is to dissect the mechanism through which ADAR1 and RNA editing promote LIC self-renewal in T-ALL patients. Our central hypothesis is

that activation of ADAR1 supports T-ALL LIC maintenance by suppressing aberrant dsRNA sensing in an isoform-specific manner. We will test this hypothesis in three independent aims: 1) Examine if ADAR1 promotes LIC self-renewal in relapsed T-ALL by increasing RNA editing, 2) elucidate the molecular mechanism by which

ADAR1 promotes LIC activity, and 3) investigate if NOTCH1 signaling induces ADAR1 activation in T-ALL LIC. We will directly evaluate the isoform-specific function of ADAR1 p150 and p110 isoforms in LICs within human thymic organoids and patient-derived xenograft mouse models of T-ALL. We will use single cell RNA-sequencing

to investigate the mechanisms by which p150 and p110 isoforms regulate cellular function and gene expression in T-ALL LICs. In Aim 2, we will perform concurrent knockdown of dsRNA sensors in combination with ADAR1 isoform knockout in LICs to determine if aberrant dsRNA sensing disrupts LIC activities in patient-derived

xenograft mouse models. Mechanistically, we will map dsRNA-containing genes suppressed by either RNA editing or dsRNA activity of ADAR1. In Aim 3, we will use g-secretase inhibitor (GSI) and DDL1-expressing thymic co-culture system to examine if NOTCH signaling contributes to ADAR1 activation, and if ectopic expression of

ADAR1 can bypass the need for NOTCH signaling and induces resistance to GSI inhibition. Lastly, we will investigate if ADAR1 is a direct transcriptional target of NOTCH1. These studies will improve our understanding of how dysregulation of ADAR1 contributes to LIC self-renewal and T-ALL progression. Furthermore, these

studies will expand our fundamental understanding of the mechanisms underlying LICs-driven malignancies and may be broadly applicable to other cancer stem cell-driven malignancies.