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.