Abstract PO-044: ATG4B loss reduces pancreatic cancer cell viability and enhances the utilization of survival-promoting GABARAP-L2

Autophagy is a lysosomal-dependent intracellular recycling pathway that was found to be essential for the survival of some pancreatic ductal adenocarcinoma (PDAC) cells in vitro and in vivo. Hence, there is interest in identifying potential targets within the autophagy pathway for PDAC therapy. The ATG4 cysteine protease family (ATG4A, ATG4B, ATG4C and ATG4D) and their substrates – LC3B, GABARAP, GABARAP-L1 and GABARAP-L2 – are important in the formation of autophagosomes, which are vesicles that encapsulate and transport cargo to lysosomes for degradation. ATG4B, in particular, has garnered attention because of its ability to efficiently recognize and cleave all of the substrates, and several cancer types have shown sensitivity to ATG4B inhibition. To determine the effects of targeting ATG4B in PDAC cells, stable knockdown (KD) of ATG4B was generated using PDAC cell lines, MiaPaCa2 and Panc1. ATG4B KD significantly reduced cell viability under fed conditions (10% serum) compared to the control parental lines. Since autophagy is a stress response mechanism, cell viability was tested under low serum (0.8%) conditions, which showed that the ATG4B KD lines were significantly more sensitive to low serum compared to parental lines. To determine the association between ATG4B and PDAC clinical parameters, 252 PDAC tumors were analyzed for ATG4B expression levels. Unexpectedly, tumors categorized as ATG4B-negative were associated with poor differentiation and worse disease-specific survival of PDAC patients (pLogRank = 0.0006). ATG4B knockout lines (KO) were derived from MiaPaCa2 and Panc1 to study the effects of complete loss of ATG4B KO in PDAC. Under fed conditions, the ATG4BKO lines displayed significantly reduced viability compared to the parental lines. In low serum conditions, however, the ATG4B KO lines displayed similar sensitivity as the parental lines, suggesting the induction of compensatory mechanisms. To determine if the observed differences in viability were associated with changes in ATG4 substrates, the expression and lipidation of LC3B, GABARAP, GABARAP-L1 and GABARAP-L2 were analyzed. While there were no consistent differences for LC3B, GABARAP and GABARAP-L1, both the ATG4B KD and KO lines showed an increase in GABARAP-L2 lipidation under fed and low serum conditions. GABARAP-L2 knockdown resulted in apoptosis of MiaPaCa2 cells regardless of ATG4B status. This finding indicates that GABARAP-L2 is required for the survival of MiaPaca2 cells, and further analyses of GABARAP-L2 in PDAC and normal pancreatic cell lines are in progress. Citation Format: Paalini Sathiyaseelan, Nancy E. Go, Steve E. Kalloger, Daniel J. Renouf, David F. Schaeffer, Sharon M. Gorski. ATG4B loss reduces pancreatic cancer cell viability and enhances the utilization of survival-promoting GABARAP-L2 [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-044.