Overcoming cytidine deaminase (CDA) mediated resistance via EO-4426 dual DNA-replication targeting: Implications for CDA-high solid tumors and mesenchymal GBM

Background: EO-4426 (tezacitabine) is a brain-penetrant, deamination-resistant cytidine analog that uniquely inhibits both DNA polymerase-alpha (Polalpha) and ribonucleotide reductase (RNR), leading to dNTP-pool depletion, replication-fork collapse, and DNA-damage accumulation in rapidly proliferating tumor cells. Unlike gemcitabine and hydroxyurea, EO-4426 resists cytidine deaminase (CDA)-mediated inactivation. Tumors with high CDA expression or APOBEC3G-linked cytidine-metabolism activity-including mesenchymal GBM, TNBC, NSCLC, and ovarian cancer-are predicted to exhibit heightened dependence on Polalpha/RNR function and may therefore be particularly susceptible to dual-targeting strategies. Methods: Ongoing invitro studies are assessing EO-4426 across engineered and naturally CDA-high cancer cell models to define mechanistic pharmacology and biomarker-enrichment hypotheses. Experimental approaches include: (i) LCMS quantification of EO-4426-induced dNTP-pool depletion; (ii) evaluation of replication-stress and DNA-damage biomarkers (gammaH2AX, pRPA32, pCHK1) by immunoblot and high-content imaging; (iii) characterization of APOBEC3G→CDA pathway activity and its impact on drug sensitivity; (iv) flow cytometric analysis of S-phase arrest and replication-fork perturbation; and (v) combinatorial studies with ionizing radiation and DDR-targeting agents, including ATR inhibition. Preliminary Findings: Prior nonclinical studies demonstrate that EO4426 retains potent activity in CDA-high tumor environments and is not susceptible to rapid deamination, distinguishing it from gemcitabine, which is efficiently converted to inactive dFdU. Published work also reports enhanced antitumor activity in combination with DNA-directed agents and fluoropyrimidines, consistent with strong replication-stress induction and dNTP-depletion biology. Clinically, EO4426 has been evaluated in multiple third-party sponsored Phase I/II trials, showing predictable myelosuppression as the principal toxicity and evidence of antitumor activity, including objective responses and durable disease stabilization, in heavily pretreated solid-tumor populations. Conclusions: New data are being developed to support further investigation of EO-4426 in cytidine metabolism-driven tumor contexts and biomarker guided clinical development strategies for CDA-high solid tumors and mesenchymal/APOBEC3G-enriched GBM. Comprehensive in vitro mechanistic data, biomarker correlations, and combination treatment findings will be presented at the Meeting.