Project Summary
While impressive progress has been made in tumor immunology and clinical application of immunomodulatory
agents in solid tumors, we don’t fully understand how to effectively incorporate immunotherapeutic strategies in
Acute Myeloid Leukemia (AML) (Perna F et al., Cancer Treat Res 2022). Advances in genomic and epigenomic
characterization of AML have fostered better understanding of leukemogenesis. The average AML genome may
have only 13-16 coding mutations, of which around five are recurrent mutations in suspected driver genes.
Despite approval of novel targeted therapies, the clinical outcome of AML patients remains dismal, with a 5-year
overall survival of approximately 27%, prompting the search for additional and synergistic therapeutic rationales.
Developing immune-based therapies for AML has been challenged by the lack of surface targets. I previously
developed a target discovery strategy to identify Chimeric Antigen Receptor (CAR) targets in AML (Perna F. et
al., Cancer Cell 2017). As we could not identify single targets with favorable expression profiles, we showed that
combinatorial pairings hold promise for CAR T-cell therapy of AML.
Over the past two years, my lab investigated whether cancer-specific mechanisms such as alternative
mRNA splicing driven by founder mutations (i.e. genetic mutations affecting splicing factors) may shape the
cancer surface proteome, providing targets for promoting disease initiation and progression and use of
immunotherapy (Perna F. Molecular Therapy 2021 and Dong et al., Oncogene 2021). We have now developed
a novel pipeline that we called Spliced-ImmuneTargetFinder based on 5 steps to identify splice variants of cell
surface molecules: 1) RNA-seq analysis of normal cord blood CD34+ HSPCs virally expressing splicing factor
mutations such as SRSF2P95H mutation 2) custom transcriptome analysis for isoform quantification 3) isoform
switch analysis and prediction of functional consequences based on isoform sequences such as coding potential
and domain gain 4) cell surface molecule annotation based on an integrated dataset developed in our laboratory,
and 5) protein candidates validation in primary AML patient samples beyond SRSF2 mutant patients. Given that,
we identified six top candidate targets that are a) involved in at least one switch isoform b) upregulated in SRSF2
mutant cells compared to controls and c) derive from genes coding for cell surface proteins. We validated their
aberrant protein expression in several AML cell lines and primary AML patient samples by developing custom
antibodies specifically recognizing isoform unique domains derived from splicing events.
Thus, we propose to investigate the functional roles of these promising variants and the path for targeting
the extracellular isoform-specific domains with immune-therapeutic interventions. This method for
unconventional target antigens discovery is generalizable to other cancers as well and will provide useful
information to prepare for a phase-I clinical trial in AML patients. Identification of these attractive antigens may
also open new avenues for understanding the role of splice isoforms in leukemogenesis.