PROJECT SUMMARY
ASXL1 is one of the most mutated genes in hematological disorders. ASXL1 mutations occur in all types of
myeloid malignancies, including 45% of chronic myelomonocytic leukemia cases where they are associated with
poor prognosis; and ASXL1 is the third-most mutated gene in clonal hematopoiesis, a condition defined by
aberrant expansion of hematopoietic stem cells (HSCs), which increases the risk of cardiovascular disease and
other hematological disorders. Despite the prevalence and significance of ASXL1 mutations, it is poorly
understood how they are mechanistically linked with these hematological disorders as very little is known about
the protein itself.
ASXL1 is a scaffolding component of the Polycomb-Repressive Deubiquitinase (PR-DUB) complex, which
antagonizes Polycomb-mediated repression to regulate critical gene expression programs. Many ASXL1
mutations are frameshift or nonsense mutations in the final exon that result in truncated protein products. While
early work focused on the effects of knocking out ASXL1, recent work has shown that truncated ASXL1 is actually
present at higher levels than the wild-type protein. This stabilizes and hyperactivates PR-DUB, ultimately leading
to epigenetic dysregulation. However, the molecular basis of how ASXL1 truncation increases its abundance
and whether this drives its disease function remain unclear.
In my preliminary studies, I discovered a degron in the last exon of ASXL1 that is necessary and sufficient
for its degradation by the ubiquitin proteasome system. This proposal will test my hypothesis that the highly
recurrent ASXL1-truncating mutations remove this degron to aberrantly stabilize the protein, leading to
formation of hyperactive PR-DUB complexes, de-repression of Polycomb-target genes, and HSC
expansion. To test this hypothesis, I will use a fluorescence-based cellular reporter of protein stability to further
characterize this degron and perform a CRISPR screen to identify the E3 ubiquitin ligase that recognizes the
degron. Then I will establish the relevance of this mechanism to the clonal expansion phenotype in primary HSCs
by CRISPR mutagenesis paired with single-cell RNA sequencing (i.e., Perturb-seq). In the long term, this
knowledge will advance our fundamental understanding of ASXL1’s role in primary hematopoiesis, how clinical
mutations disrupt that process, and propel the development of new therapeutic strategies for a wide array of
hematological disorders.