Epigenetic modifications regulate chromatin structure and function, playing important roles in altering DNA transcription levels and subsequently cell fate decisions. Various next-generation sequencing (NGS) methods have been developed to detect these epigenetic changes in the genome, such as chromatin immunoprecipitation followed by sequencing (ChIP-seq). Even though ChIP-seq is extensively used to analyze DNA and histone modification levels, this method is limited to one histone marker at a time and requires significant amount of input cells, which masks the profiles of cell-to-cell variation and the complex interaction between the epigenome and gene expression. To overcome these limitations in current next-generation sequencing methods, we have been developing a multiplexed assay that could detect multiple epigenetic modifications in single cells. So far, I have developed a NGS data analysis pipeline to identify potential gene candidates that are highly differentially modified by histone markers. In the future, we hope to use these gene candidates as templates to design DNA-fluorescent in situ hybridization (DNA-FISH) probes and perform Expansion Microscopy and DNA-FISH with these probes to link histone modifications to specific gene loci at high resolution. After the assay is fully developed and validated, we plan to utilize the assay to take the epigenetic profiles of hematopoietic stem cells and study cell fate decisions in hematopoiesis.

Rhabdomyosarcoma (RMS) is a rare and devastating pediatric soft tissue sarcoma, predominantly diagnosed in children and adolescents. Metastases and disease relapse rates continue to remain poor with a 5-year survival rate of less than 30%. Current therapeutic methods continue to remain inefficient in causing complete remission. Cancer stem cells (CSCs), a subpopulation of cells within tumors, are able to resist standard therapeutic treatments leading to disease relapse and metastases. Studies using human cells and a zebrafish model of RMS has shown that a population of CSCs exists within RMS. Thus, I am interested in characterizing potential genes that serve as a marker for CSCs in RMS. I am currently pursuing two candidate genes called PAX7 and CD82. Both PAX7 and CD82 have been demonstrated to play an essential role in regulating the function of skeletal muscle stems cells. The molecular signature of the CSCs in RMS is similar to that of skeletal muscle stem cells. Our preliminary data in the Chen lab also demonstrated increased expression of CD82 in a sphere assay, a surrogate in vitro assay to assess stem-like features in tumor cells. Based on these findings, my central hypothesis is that PAX7 and CD82 can potentially serve as specific markers of the CSCs in RMS. To test the hypothesis, I tagged PAX7 and CD82 with the aid of the CRISPR/Cas9 genome editing technology in order to isolate populations of RMS cells that either express PAX7 or CD82. I will perform cell-based assays in order to assess whether the stem-like qualities are enriched in isolated PAX7 and CD82-labeled RMS cell population. The identification of the CSCs in RMS will provide insight for a novel solution in overcoming drug-resistant RMS, tumor recurrence, and metastasis, through CSC-targeted drug therapy.

T cells of the immune system protect humans from most threats because they can recognize and eliminate foreign targets, as well as protect from reinfections. However, the persistence of an infection in the body leads to chronic stimulation of T cells, causing them to lose their effector function and enter a state known as “exhaustion”. Exhausted T cells are defined by increased expression of inhibitory receptors, loss of immune cell regulation, and most importantly, loss of cytotoxicity and effector function. Studies have shown that the transcription factors, T-bet and Eomes, play crucial roles in regulating T cell differentiation, with T-bet being highly associated with effector T cell differentiation. T-bet expression is dampened in exhausted T cells, and therefore, I hypothesize that controlled induction of T-bet expression can reverse the exhausted phenotype in antigen-experienced T cells. I constructed a T-bet overexpression vector containing T-bet cDNA fused to a fluorescent protein and destabilizing domain. The destabilizing domain, or degron, facilitates degradation of the constitutively expressed T-bet transgene in the absence of the ligand, Shield-1, which when added at varying concentrations allows for a range of protein stability. This method of overexpression confers faster control kinetics compared to commonly used transcriptional approaches, such as inducible promoters. I have characterized the range of the T-bet transgene in Jurkat cells by titrating Shield-1 to provide a working range of 5-60% overexpression of T-bet compared to endogenous levels. Validating these parameters in primary T cells will allow me to apply this T-bet overexpression vector in a mouse model of T cell exhaustion. This tool has significant implications for improving immunotherapy strategies, such as TIL and CAR-T therapies, where exhaustion of the therapeutic T cells has led to reduced efficacy of the treatment.

The evolutionarily conserved Hippo cell signaling pathway is important in tissue and organ development through its regulation of cell death, proliferation, and differentiation; as such, the Hippo pathway is often dysregulated in cancer. The phosphotransferases serine/threonine-protein kinase 3 and 4 (STK3 and STK4), are core components of the Hippo pathway. When active, these kinases negatively regulate the transcription factors yes-associated protein 1 (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ). The activities of STK3 and 4 are regulated by phosphorylation on multiple serine, threonine and tyrosine residues. Recently, we discovered that the central cellular signaling regulator cAMP-dependent protein kinase (PKA) can phosphorylate STK3 on serine 15 (S15). Since it has been shown before that PKA can suppress the transcriptional activity of YAP/TAZ, we hypothesized that this phosphorylation event activates STK3. To investigate the functional consequences of STK3-S15 phosphorylation, I used molecular cloning to construct active and kinase-dead (KD) FLAG-tagged STK3 mutants in which S15 was replaced by alanine (S15A, non-phosphorylatable) or aspartate (S15D, phosphomimic), respectively. I transfected plasmid DNA encoding these mutants into human embryonic kidney cells (HEK293T) to investigate if overexpression of each STK3 variant differentially affects activation of the Hippo pathway. Western blot analyses of transfected cells showed that active STK3 S15A and S15D but not the kinase-dead versions were able to phosphorylate known STK3 substrates. Further, I used anti-FLAG antibodies to enrich FLAG-tagged STK3 variants and co-precipitating protein complexes for mass spectrometric (MS) analysis to examine the effect of STK3-S15 phosphorylation on protein-protein interactions. MS analysis revealed that the STK3 S15D mutant preferentially bound to STK4, suggesting pathway activation. Insights into the mechanism of Hippo pathway regulation by PKA will have important implications for cancer research since targeted therapeutics modulating the cAMP - PKA signaling axis may be directly applicable in tumors with aberrant YAP/TAZ activity.