Aleena Alex, COL ’24, Philadelphia, PA
This summer I got the opportunity to conduct research with the Paralkar Lab. The Paralkar Lab studies how transcription factors and other related proteins affect normal hematopoiesis or blood cell development. The lab focuses on understanding the molecular mechanisms through which dysregulation of these proteins might lead to Acute Myeloid Leukemia (AML). The overall goal of the lab is to suppress myeloid leukemia by investigating normal hematopoiesis and myeloid differentiation.
Acute Myeloid Leukemia is characterized by the clonal expansion of undifferentiated myeloid blast cells, which hinders normal hematopoiesis. Researchers have been able to differentiate classes of AML based on the mutation profiles of AML genes. Plant Homeodomain Finger 6 (PHF6) gene is a newly identified X-linked chromatin-binding regulator protein. The presence of recurrent mutations in PHF6 suggests that dysregulation of PHF6 function could contribute to AML development. Moreover, patient missense mutations are frequently seen in the ePHD2 domain of PHF6. However, much is not known about its role within the chromatin and the mechanism through which a loss of function mutation of PHF6 might contribute to AML or a myeloid phenotype.
My project thus focused on investigating PHF6 missense mutations in the ePHD2 domain to further understand the role of the ePHD2 domain within the protein and analyze the impact of these mutations on the protein. I utilized computational analysis as well as in vivo techniques to investigate these mutations. During the first half of this summer, I completed the in silico analysis, obtaining predictions regarding the stability of the ePHD2 domain mutants structures, and potential predictions relating to the pathogenicity of the mutant protein.
Currently, I am working on generating and validating CRISPR knockouts for some of the commonly seen missense mutations of the PHF6-ePHD2 domain in the THP1 cell line. This process includes designing and validating the guide RNAs, HDR templates, and primers for the particular mutations. After transfection and clonal selection, these clones would be validated and narrowed down using PCR, Sanger sequencing, and Western Blots. Through this project, I was able to attain proficiency in performing PCRs and Western blots. I am currently learning how to effectively and efficiently electroporate the guide RNA and template into my cells. This opportunity also allowed me to step outside of my comfort zone when it came to analyzing the gels and westerns, troubleshooting, tweaking the experimental procedure, etc.
Furthermore, attending lab meetings and journal clubs allowed me to improve my presentation and communication skills along with providing me with a diverse background and knowledge about the current workings within the lab. It has also helped me achieve a diverse perspective regarding myeloid leukemia and understand AML more thoroughly.
This would not have been possible without the constant support and guidance from my mentors, who prioritized my growth and development throughout this project. Their detailed and immediate feedback helped to improve my skills and work independently in parts of this project. Thanks to Dr. Vikram Paralkar and Aishwarya Pawar, whose expertise and profound mentorship helped me in achieving my research goals this summer.