Kelvin, a freshman studying Neuroscience and Computer Science, performed an In Vivo CRISPR screen hit validation on heme synthesis genes.
Cancer immunotherapy is a promising form of cancer treatment that activates the immune system to target cancer cells. The Broad exudes brilliance every corner you turn. It is such an awe-inspiring experience to be in an institution filled with scientists who value collaboration, mentorship, and conducting innovative research. My time at the Broad has sharpened my ability to critically think, effectively communicate, creatively solve problems, and adapt–all skills I will hold dearly and use as tools to bear my riches to the world.However, only a small subset of cancer patients respond to immunotherapy. Patients who do respond to immunotherapy often relapse, but there are potential genetic targets within tumor cells we could utilize to improve the efficacy of immunotherapy. An in-vivo CRISPR screen that we performed using humanized mice revealed genes in the heme pathway as some of the targets we can utilize to improve immunotherapy. Heme is an iron-containing compound that forms the non-protein section of hemoglobin, so it plays a crucial role in transporting oxygen to cells. There are eight genes responsible for the synthesis of heme, but FECH, HMBS, and UROS were the genes that showed up in our CRISPR screen We hypothesized that a deficiency in heme will increase tumor cells’ sensitivity to cytokines as well as cause ferroptosis, an iron-induced cell death. To validate our initial hits, we took cas12 expressing A375 cells and infected them with viruses containing our specific genetic knockouts (KOs). We then put the cells through antibiotic selection and ran a growth experiment with cytokines, TNFa and IFNy, and with an iron chelator, DFO, serving as a positive control. After our experiment, we performed a CellTiter Glo 2.0 assay, a luminescence-based method to detect viable cells in culture by quantifying ATP. The assay revealed that DFO had no effect on cell growth until a concentration of 5.0 mM at which it proved to be toxic to the cells. However, heme gene KOs produced less viable cells, which was further exacerbated by the addition of cytokines, implicating heme genes as a valid target for improving immunotherapy.
Project: In Vivo CRISPR screening reveals that knockout of heme synthesis genes may inhibit melanoma growth in a humanized mouse model
Mentors: Aiping Jiang & Rachel Fetterman