Abstract Background: The Integrated Stress Response (ISR) is a major adaptive stress response pathway in cancer cell maintenance. The ISR kinase family member PERK controls one of the three arms of the Unfolded Protein Response (UPR). The UPR is considered an Achilles’ heel in B-cell cancers. Multiple myeloma (MM) and B-cell lymphomas are dependent on a well-balanced UPR pathway to cope with the high demand for protein folding and their secretory nature. Given the double-edge sword nature of the UPR, the activation of PERK and downstream pathway can have cytoprotective or cytotoxic effects. In B-cell cancers the UPR is at close to maximum cytoprotective capacity, such that further pharmacological stimulation of PERK can potentially be leveraged to cause a cancer cell cytotoxic response and induce antitumoral effects. Methods: Modulation of ISR kinases was characterized using enzymatic assays. Kinome selectivity profiling was determined using enzymatic and cellular assays. Cellular assays of PERK activation assessed ATF4 by ELISA. Cellular assays of GCN2 modulation assessed phospho-GCN2 and ATF4 by Western blot or ELISA (under basal or low amino acid conditions). DP-9024-induced upregulation of components of the ISR/UPR pathway (ATF4, CHOP) or the apoptosis pathway (PARP and Caspase 3/7) was measured by Western blot or ELISA assays. Compound-mediated PERK activation was investigated mechanistically using a cellular nanoBRET dimerization assay. In vivo upregulation of tumoral ATF4 was determined in a MM PK/PD xenograft model. In vivo inhibition of tumor growth was determined in MM and B-cell lymphoma xenografts. Results: DP-9024 was designed as a selective and potent modulator of PERK and GCN2. DP-9024 was found to upregulate the ISR/UPR pathway (ATF4, CHOP). The mechanism by which DP-9024 treatment induced the UPR pathway was found to be through the dimerization and activation of PERK. Upregulation of the UPR pathway downstream of PERK led to induction of apoptosis (PARP and Caspase 3/7) in MM and B-cell lymphoma lines in vitro. DP-9024 mediated activation of the UPR pathway in cell lines with high basal level of endoplasmic reticulum (ER) stress led to growth arrest in combination with FDA approved therapies. Oral dosing of DP-9024 in MM xenograft models induced ATF4, and combination efficacy was observed in MM and B-cell lymphoma xenografts in combination with FDA approved agents in vivo. Conclusions: The ISR/UPR is a targetable vulnerability in cancers with high basal levels of ER stress. DP-9024 increases UPR signaling via activating PERK dimerization. This novel mechanism leads to antitumoral effects in B-cell cancers in vitro and in vivo likely through the induction of unresolved ER stress, which may potentially provide an alternative mechanism to current UPR targeting therapies. Citation Format: Gada Al-Ani, Qi Groer, Aaron J. Rudeen, Kristin M. Elliott, Patrick C. Kearney, Jeffery D. Zwicker, Yu Mi Ahn, Stacie L. Bulfer, Cale L. Heiniger, Molly M. Hood, Salim Javed, Joshua W. Large, Max D. Petty, Kristen L. Stoltz, Bertrand Le Bourdonnec, Bryan D. Smith, Daniel L. Flynn. DP-9024, an investigational small molecule modulator of the Integrated Stress Response kinase PERK, causes B-cell cancer growth inhibition as single agent and in combination with standard-of-care agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1640.