Abstract
Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal cancer with high incidences of p53 mutations. AZD1775 (adavosertib, previously MK-1775) is a small molecule WEE1 inhibitor that abrogates the G2M checkpoint and can potentially synergize with DNA damaging therapies commonly used in PDAC treatment. The purpose of this study was to identify combination partners for AZD1775, including standard chemotherapy or targeted agents, in PDAC preclinical models. Low powered preliminary screens demonstrated that two of the four PDX models responded better to the combinations of AZD1775 with irinotecan or capecitabine than to either single agent. Following the screens, two full powered PDAC PDX models of differing p53 status were tested with the combinations of AZD1775 and irinotecan or capecitabine. The combinations of AZD1775 and SN38 or 5-FU were also tested on PDAC cell lines. Cellular proliferation was measured using an IncuCyte Live Cell Imager and apoptosis was measured using a Caspase-Glo 3/7 assay. Flow cytometry was conducted to measure alterations in cell cycle distribution. Western blot analysis was used to determine the effects of the drug combinations on downstream effectors. In PDX models with mutated p53 status, there was significant tumor growth inhibition from the combination of AZD1775 with irinotecan or capecitabine (P ≤ 0.03), while PDX models with wild type p53 did not show anti-tumor synergy from the same combinations (P ≥ 0.08). The combination of AZD1775 with SN38 or 5-FU significantly decreased proliferation in all PDAC cell lines, and enhanced apoptosis in multiple cell lines. Cell cycle distribution was disrupted from the combination of AZD1775 with SN38 or 5-FU which was recorded as G2M arrest and decreased G1 phase. AZD1775 inhibited phospho-CDC2 and increased the expression of γH2AX that was either maintained or enhanced after combination with SN38 or 5-FU. The combination of AZD1775 with irinotecan/SN38 or capecitabine/5-FU showed anti-tumor effects in vivo and in vitro in PDAC models. These results support further investigation for these combination strategies to enhance outcomes for PDAC patients.
Highlights
Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer death in men and women, with approximately 35% of patients presenting with locally advanced disease at diagnosis [1]
In at least two of the four PDAC patient-derived xenograft (PDX) models tested, the combination of AZD1775 with irinotecan or navitoclax increased the tumor growth inhibition (TGI) values, these drug combinations were selected for further validation in full powered p53 mutant (MT) and wild type (WT) PDX models (Figure 1)
WEE1 is a key regulator of cell cycle progression at the G2M checkpoint and controls entry into mitosis
Summary
Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer death in men and women, with approximately 35% of patients presenting with locally advanced disease at diagnosis [1]. Activation of the DNA damage response (DDR) pathway in cancer cells with chemotherapy can lead to treatment resistance [5]. Targeting the cell cycle checkpoint has potential to decrease the activation of the DDR and to sensitize PDAC cells to chemotherapy or targeted agents for improved patient outcomes. PDAC tumors frequently have mutated p53 which commonly results in a defective G1 checkpoint, forcing cancer cells to rely primarily on the G2M checkpoint to repair DNA damage before mitosis [6,7,8]. Inhibition of WEE1 prevents the arrest of damaged DNA, which enhances CDC2 activity and drives cells in S phase to prematurely enter mitosis before repair [10]. Phase 1 clinical data has shown AZD1775 is clinically viable and can safely be combined with chemotherapies in advanced solid tumors [12]
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