Barnaba Pilot Project Summary

Cancer cells exhibit an elevated metabolic demand due to their continuous proliferation. Consequently, cancer cells boost their catabolic potential through the upregulation of metabolic pathways such as autophagy. Autophagy upregulation serves a dual purpose: it recycles cytosolic material to sustain metabolism and facilitates cell response to withstand stress imposed by chemotherapy. AMPK is the central regulator of autophagy under conditions of energy stress. Recently, research performed by the PI has revealed that AMPK has a suppressive role on autophagy, challenging the common notion that energy stress stimulates autophagy. Since AMPK is currently a target in several cancer clinical trials, this redefinition of AMPK's role in autophagy will have a significant impact in cancer therapy. Preliminary data suggest that AMPK inhibits autophagy by blocking WIPI-mediated tethering of ATG9 vesicle, the seed of autophagosomes. To date, however, the specific mechanism(s) though which AMPK activates catabolic pathways remains unclear.

Here we propose to determine the role of AMPK in autophagy suppression through two key objectives. In Aim 1, our focus is on discerning metabolic reprogramming in a cancer cell model by analyzing changes in the transcriptome under conditions that activate AMPK signaling, using RNA-seq analysis. The transcriptome analysis will enable us to identify the specific genes influenced during AMPK signaling and to understand how autophagy is suppressed. Changes in the metabolic reprogramming will be correlated with autophagy flux using our recently developed single-cell live-cell imaging pipeline. In Aim 2, our goal is to unravel the mechanism behind AMPK-driven autophagy suppression, focusing on ATG9 vesicle tethering. To understand how AMPK blocks vesicle tethering, we will generate cancer cell lines depleted of WIPI1-4 isoforms using genetic knockout and evaluate both cancer metabolic adaptation and autophagy flux. By uncovering how AMPK suppresses autophagy, we will advance cancer therapy and paving the way for innovative treatments, offering profound implications for drug discovery. Results from this Pilot Study will also provide critical preliminary data to support a major grant application to the NIH NIGMS (R35 MIRA Early-Stage Investigator). These funding applications will seek to uncover the intricacies of metabolic reprogramming in cancer cells and their link to chemoresistance, ultimately leading to novel therapeutic strategies for more effective cancer treatment.