Project Summary Replication stress (RS) is defined as the slowing or stalling of the replication fork progression during DNA synthesis and is widely recognized as a significant cause of genomic instability and a critical feature of cancer cells. The response to RS is facilitated by ATR kinase which induces a cascade of molecular events to facilitate cell cycle arrest, stabilization of the replication forks and DNA repair. Inhibition of ATR may be an effective strategy against tumors with high degree of RS. However, thus far, clinical evaluation of ATR inhibitors (ATRi) has demonstrated limited efficacy as monotherapy outside the setting of BRCA deficient and ATM deficient tumors. Using two high-throughput approaches, i.e., PRISM screening of ~800 genomically characterized human cancer cell-line models and genome-wide CRISPR-based functional screen of ATRi elimusertib, we found that inhibition of the PI3K pathway may synergize with ATRi. Preliminarily, we have also demonstrated synergism between PI3K inhibitor copanlisib and ATRi elimusertib in vitro and in vivo. Clinically, this synergism is particularly relevant to uterine cancer (UC) because of the prevalence of genomic alterations associated with high RS in uterine serous tumors (e.g. CCNE1 amplification, MYC amplification, FBXW7 mutations) and because UC (predominantly those with endometrioid or clear cell histology) frequently harbor ARID1A mutations which have been shown to be associated with cell cycle progression defects in both S and G2/M phases of the cell cycle leading to increased RS and sensitivity to ATR inhibition. Additionally, although all histologic subtypes of UC are characterized by PI3K pathway alterations, response to PI3K inhibition as monotherapy has been disappointingly low and no PI3K inhibitor is currently approved in UC. For all these reasons, we hypothesize that combined ATR and PI3K pathway inhibition may be an effective therapeutic strategy to capitalize on concomitant PI3K pathway alterations and high degree of RS in uterine serous and ARID1A-mutated uterine tumors. In Aim 1, we will perform in vitro studies of targeted inhibition of ATR alone and in combination with PI3K inhibition across a collection of established patient-derived organoids with various genomic backgrounds to assess for synergism and elucidate its underlying mechanisms. In Aim 2, we will evaluate in vivo synergism between ATRi elimusertib and PI3Ki copanlisib in PDX and GEMM models of UC and will explore whether this correlates with IHC assays of replication stress. Finally, in Aim 3, we will conduct a Phase 1b dose escalation trial of PI3K inhibitor copanlisib and ATR inhibitor elimusertib in UC with two dose expansion cohorts in uterine serous (cohort A) and ARID1A mutated uterine tumors (cohort B). It is our expectation that the research proposed herein, if successful, will provide patients with recurrent UC with a new therapeutic option that is applicable across different histologic subtypes ...