Research Focus 1: Identify and delineate the mechanisms of haploinsufficient tumor suppressor genes 

Somatic genetic alterations, including mutations, translocations, and chromosome and copy number changes, are the driving force of cancer. Chromosome and copy number abnormalities account for approximately 50~60% AML,  50~80% MDS, and 25~50% MPN cases. While substantial efforts have been made in recent years to decipher the role of recurrent genetic mutations and chromosome translocations in leukemia, the pathogenesis and therapeutic vulnerabilities of chromosome and copy number aberrations remain largely uncharacterized due to the cooperation between multiple haploinsufficient genes within the deleted segments, the technical challenges of modeling these events in vitro and targeting loss of function aberrations. To address this shortcoming, our lab has recently developed and optimized a workflow that couples CRISPR/Cas screening with a sensitive cellular transformation assay to identify loss-of-function genes that yield growth factor independence and transformation of pre-leukemic cells. We have also combined CRISPR nickase with Homology DNA repair (HDR) to generate heterozygous deletions of specific genes or large chromosome segments to model clinically relevant copy number and chromosome deletions. Using these advanced models, we aim to identify novel tumor suppressor gene (TSGs) associated with chromosome and copy number deletions. The discovery and characterization of novel TSGs associated with chromosome loss and deletion will fundamentally advance our understanding of how dose perturbations of TSGs predispose to leukemia development. Ultimately, I aim to leverage these mechanistic insights to identify therapies to target the underlying dysregulated pathways of transformative cytogenetic events and ultimately improve patient outcomes.

Research Focus 2: Target haploinsufficient essential genes to treat myeloid malignancies

With newly approved drugs and drug combinations adding to clinical practice, the therapeutic landscape for leukemia is changing. Despite these advances, challenges remain. Given the aggressive clinical course of leukemia, identification of biomarkers to stratify patients onto the most effective therapies and developing agents with enhanced efficacy on leukemia cells and reduced toxicity for normal tissues is an urgent need. The cellular requirement for a normal dosage of essential genes creates an opportunity to target vulnerabilities due to a reduction in the level of the protein product of a haploinsufficient gene, termed allelic haploinsufficient gene or Copy-number alterations Yielding Cancer Liabilities Owing to Partial losS (CYCLOPS) gene. Such CYCLOPS genes represent ideal biomarkers for patient stratification and synthetically lethal targets. We hypothesize there are CYCLOPS genes in the common chromosome and copy number deletion regions that could be therapeutically targeted. We will curate a list of CYCLOPS genes by analyzing gene expression, copy number, and gene dependency data from leukemia cell lines and validate clinically relevant CYCLOPS genes using shRNA and CRISPR/cas9 HDR method for future drug development.

Research Focus 3: Characterize the function and therapeutics targeting leukemia associated WT1 transcripts and mutations

The Wilms’ Tumor 1 (WT1) gene is upregulated in ~90% leukemia patient samples and the majority of AML cell lines. Elevated WT1 expression is used as a biomarker for minimal residual disease and early disease relapse during and after chemotherapy. WT1 mutations are present in approximately 6–15% of de novo AML cases and are associated with chemotherapy resistance and poor prognosis. The oncogenic potential, the drug sensitivity, and the structural basis and mechanisms of various WT1 transcripts and mutations remain uncharacterized. We will determine the distribution of different WT1 transcripts across different leukemia subtypes using RNAseq. We will use ectopic WT1 transcript viral vector, shRNA, and CRISPR/cas9 HDR methods to overexpress or knockdown/knockout WT1 and perform in vitro and in vivo functional characterizations to uncover the roles of WT1 transcripts and mutations in hematopoiesis and leukenogenesis. Collectively, these studies will advance our understanding of which and how WT1 transcripts and mutations contributes to leukemia development and chemoresistance and how could design drug combinations to target these events.