Abstract Due to their higher proliferation rate, most cancers experience impaired ATP generation, hypoxia, hypoglycemia, and increased genetic mutations that may perturb cellular and protein homeostasis. Persistent stress conditions induce the unfolded protein response (UPR) as well as increased expression of molecular chaperones, such as Hsp70, to modulate cell fate. Our recent studies demonstrate how perturbing cytosolic Hsp70 activity with a small molecule modulator, MAL3-101, induces rhabdomyosarcoma cell (RMS13) death, patient-derived cells of one of the most common soft-tissue cancers in children. MAL3-101 addition activates PERK- and CHOP-dependent RMS13 cell death in a dose dependent manner (Sabnis et al., PNAS, 2016). To identify the mechanism that links UPR-triggered cell death and cytosolic Hsp70 inhibition, an isogenic MAL3-101-resistant cell line was generated (MAR4C). Even though both RMS13 and MAR4C cells were equally sensitive to a diverse panel of therapeutic compounds, we now find that they have different efficiencies of ER-associated degradation (ERAD), a quality control pathway that degrades misfolded proteins in the secretory pathway and that lessens proteotioxic stress. Specifically, MAR4C cells remove both soluble and membrane associated ERAD substrates faster than RMS13 cells and possess increased levels of ubiquitinated proteins upon proteasomal inhibition. These data suggest that higher ERAD efficiency is required for MAR4C cell survival upon Hsp70 inhibition and highlight an unexpected link between cytosolic Hsp70 inhibition and the ERAD pathway in cancer cells. Moreover, RNAseq analysis suggests that select redox-related ER quality control chaperones are differentially expressed between MAR4C and RMS13 cells, and lower levels of the BiP and PDI chaperones and Peroxiredoxin 4 are found in MAR4C cells. Because new therapeutic agents as well as reliable biomarkers are critically needed in rhabdomyosarcoma and other cancers, this study sheds light on one mechanism of cancer pathogenesis and suggests that different cancer subtypes modulate ER quality protein quality uniquely. Citation Format: Sara Sannino, Christopher J. Guerriero, Jeffrey L. Brodsky. Role of ER-associated degradation in rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4503. doi:10.1158/1538-7445.AM2017-4503