Mechanisms of physioxia-dependent cancer cell adaptation to targeted drugs along a resistance continuum

Background and Innovation: When preclinical models fail to replicate human biology, scientific progress stalls, clinical trials falter, and patients continue to suffer. While many factors contribute to these failures, lack of attention to pre-analytic variability in drug discovery and assays of drug resistance mechanisms are some of the contributing factors. We recently reported that even short-term exposure to ambient air is sufficient to trigger signaling changes in tumor and non-malignant biospecimens. Those changes in turn alter their biology and responsiveness to targeted therapies. Oxygen (O2) tension in various organs ranges from 3-9%, whereas O2 in ambient air is 21%. Using transgenic models of breast cancer and ascitic fluid cells from patients with ovarian cancer, we demonstrated differential expression of various signaling molecules including YAP1, NRF2/KEAP1 and WNT/b-Catenin when biospecimens are collected and processed under physioxia (3-5% O2) compared to the same biospecimen collected and processed under ambient air. Key signaling molecules affected include pEGFR(Y1068), pPDGFRb(Y751), pAKT(S473), pERK(T202/Y204), DNMT3A, TET2, HDAC10, and BRD4. EGFR signaling was more active under ambient air, whereas PDGFRb signaling was active under physioxia. These differences translated into differences in sensitivity to EGFR and PDGFR inhibitors in both O2 tensions and allowed identification of new effective combination therapy. We uncovered that PTPRK, a tumor suppressor that dephosphorylates EGFR, is expressed at a higher level under physioxia, whereas DUSP5, a dual specificity phosphatase that regulates nuclear ERK activity, is expressed at a higher level under ambient air. We also observed that O2 tension in experimental conditions profoundly affects the activity levels of key signaling molecules such as NRF2 involved in cellular adaptation to targeted therapy-induced resistance continuum. Based on these results, we hypothesize that experimental conditions that mimic intra-tumoral O2 levels would allow discovery of the most effective single or combination targeted therapies and elucidation of physiologically/pathologically relevant drug resistance mechanisms. Mechanistically, O2 tension-dependent variability in expression of two phosphatases could play a central role. Experiments described in three aims will test these hypotheses. 1) To investigate how physioxia-dependent PTPRK expression impacts sensitivity- resistance continuum to EGFR and TGFb targeted therapies; 2) To investigate how O2 tension-dependent variability in DUSP5 expression impacts sensitivity-resistance continuum to targeted therapies, particularly MEK/ERK targeted therapies; and 3) To determine how physioxia impacts resistance continuum of cancer cells to epigenome targeted therapies due to the effects of O2 tension on expression of DNMT3A, BRD4, and TET2. Significance and Impact on Veterans Healthcare: 90% of new candidate drugs encounter a “Valley of Death” at clinical trial stages, despite many strategies to improve drug optimization and target validation. These failures not only increase cost of healthcare but also add unnecessary toxicity. By ensuring that biomarker discovery, targeted drug testing and elucidation of drug resistance mechanisms are done under physiologically relevant experimental conditions, particularly O2 levels, this proposal will have broader implications in enhancing success of clinical trials across many cancer types. Cancer affects ~43,000 Veterans per year and ~450,000 Veterans receive cancer care at VA institutions every year. Efforts to improve response to treatment would benefit a significant number of Veterans and this proposal is geared towards developing a methodology to screen for effective single and combination therapies. Path to Translation (T0-1B and T0-2B): This work will force the development of new technologies for tissue collection for diagnostic purposes to allow proper assessment of tumors and select app Project Number: 1I01BX007249-01 | Fiscal Year: 2026 | NIH Institute/Center: Veterans Affairs (VA) | Principal Investigator: Harikrishna Nakshatri | Institution: RLR VA MEDICAL CENTER, INDIANAPOLIS, IN | Activity Code: I01 | Study Section: Special Emphasis Panel[ZRD1 ONCA-S (01)] View on NIH RePORTER: https://reporter.nih.gov/project-details/11189074