Abstract 4749: Acrivon predictive precision proteomics (AP3) uncovers mechanism of resistance to ACR-368, a clinical-stage CHK1/2 inhibitor, and identifies rational combination treatment
Authors: Helén Nilsson, Lei Shi, Magnus E. Jakobsson, Joelle Baddour-Sousounis, Shahrzad Rafiei, Uthira Muralitharan, Zachary Best, Valentina Siino, Francisco J. Santana, Ignacio Arribas Diez, Kailash Singh, Portia Lombardo, William Dahlberg, Subodh Kumar, Ahmed Youssef, Reina Improgo, Corey Xu, Joon Jung, Jung-Min Lee, Ayesha Murshid, Michail Shipitsin, Jesper V. Olsen, Kristina Masson, David A. Proia, Caroline Wigerup, Peter Blume-Jensen
Journal: Cancer Research (2024)

Abstract ACR-368 (prexasertib) is a clinically advanced CHK1/2 inhibitor which has demonstrated durable activity across a proportion of patients with advanced solid tumors. Genomic biomarkers have proven unsuccessful in predicting response to ACR-368, limiting its clinical success. Using AP3, we previously developed a response-predictive proteomics-based test for ACR-368 (ACR-368 OncoSignature) for the identification of patients sensitive to ACR-368 monotherapy treatment as demonstrated in blinded preclinical studies. A Phase 2 clinical trial is ongoing where patients are treated with ACR-368 monotherapy based on OncoSignature-predicted sensitivity (NCT05548296). Here, we demonstrate the utility of AP3 for the identification of a key druggable resistance mechanism to ACR-368 and how to overcome that with low dose gemcitabine (gem), providing OncoSignature negative patients with a new potential therapeutic option. Five ovarian cancer cell lines were rendered durably resistant to ACR-368 by culturing in the presence of clinically relevant concentrations of ACR-368. Matched parental and ACR-368 resistant cell line pairs were profiled using AP3 mass spectrometry. Comprehensive pathway reconstitution and kinase activity analyses were performed to identify drug resistance mechanisms in an unbiased manner. Downregulation of DNA damage repair pathway activity was causally linked to the ACR-368-resistant phenotype, suggesting agents that restore replication stress around the CHK1/2 signaling axis may re-sensitize to ACR-368. To test this, a panel of ovarian cancer cell lines with intrinsic or drug-induced resistance to ACR-368 were screened for cell growth inhibition by ACR-368 combined with gem. Gem synergized with ACR-368 in 12/13 cell lines at low doses (1-40 nM). Moreover, Western blot analysis demonstrated protein markers of replication stress were induced by low dose gem (3-30 nM), suggesting a correlation between gem-induced replication stress and synergy with ACR-368. Comet assays showed that DMSO, gem (3 nM), or ACR-368 (100 nM) had minimal impact (4.4%, 4.5%, and 11.4%, respectively) on % comet tail DNA in ACR-368 resistant cells, while the gem combination led to 35% comet tail DNA (p&amp;lt;0.001). Finally, in a human tumor xenograft mouse model, low dose gem demonstrated a dose-dependent increase in replication stress markers (Cyclin E, pCHK1 S345) from 0.3-3 mg/kg, which allometrically scales to 1-10 mg/m2 in humans. These data supported a dose escalation Phase 1b/2 clinical study of low dose gem with ACR-368 to evaluate the efficacy and safety of the combination in ACR-368 OncoSignature negative patients (NCT05548296). This shows the potential of AP3 for unbiased elucidation of actionable drug resistance mechanisms and rapid clinical implementation in our trials, which have recently confirmed clinical activity. Citation Format: Helén Nilsson, Lei Shi, Magnus E. Jakobsson, Joelle Baddour-Sousounis, Shahrzad Rafiei, Uthira Muralitharan, Zachary Best, Valentina Siino, Francisco J. Santana, Ignacio Arribas Diez, Kailash Singh, Portia Lombardo, William Dahlberg, Subodh Kumar, Ahmed Youssef, Reina Improgo, Corey Xu, Joon Jung, Jung-Min Lee, Ayesha Murshid, Michail Shipitsin, Jesper V. Olsen, Kristina Masson, David A. Proia, Caroline Wigerup, Peter Blume-Jensen. Acrivon predictive precision proteomics (AP3) uncovers mechanism of resistance to ACR-368, a clinical-stage CHK1/2 inhibitor, and identifies rational combination treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4749.