Undergraduate Student University of Minnesota - Twin Cities Minneapolis, Minnesota, United States
Disclosure(s):
Bhavya Kanagala: No financial relationships to disclose
Introduction/Rationale: Chimeric Antigen Receptor (CAR) T cells are a revolutionary treatment option for aggressive relapsed and refractory blood cancers. Unfortunately, CAR T cell cure rates remain low (~50%), in part due to issues with T cell persistence and in vivo function. Treating CAR T cells with Compound 991, an AMPK agonist, has shown promise by increasing the in vivo efficacy of CAR T cells. However, the mechanism behind how AMPK activation improves in vivo CAR T cell performance is unknown.
Methods: To identify candidates downstream of AMPK in human T cells, we performed phosphoproteomic analysis on cell lysates following 991 treatment. Phosphorylation results were then confirmed by immunoprecipitation of candidate samples and immunoblotting using an anti-phosphoserine antibody. CRISPR technologies were used in parallel to eliminate the target protein of interest.
Results: Phosphoproteomic analysis of cell lysates from 991-treated T cells revealed increased phosphorylation of several proteins. Rho/Rac guanine nucleotide exchange factor 2 (ARHGEF2), known to be a direct target of AMPKs when phosphorylated on site (Ser151) stood out with an observed log2 fold change in phosphorylation of 8.081 following 991 treatment (p-value < 0.001). ARHGEF2 protein could be immunoprecipitated from 991-treated cells, with detection by phosphoserine antibodies ongoing. Further, we have identified a guide RNA which convincingly targets ARHGEF2 expression, with initial decreases of >34% at the protein level following CRISPR treatment.
Conclusion: Phosphoproteomic analysis following 991 treatment detects increased phosphorylation of ARHGEF2, a protein that serves a diverse array of functions in human cells. These data suggest that AMPK, working through ARHGEF2 phosphorylation, could be the mechanistic link to improved T cell metabolism following AMPK activation. We now have the tools to both define phosphorylation of ARHGEF2, as well as delete ARHGEF2 in T cells, to determine whether our suggested, novel mechanism is true.
