WEE1 Inhibition in Combination With Targeted Agents and Standard Chemotherapy in Preclinical Models of Pancreatic Ductal Adenocarcinoma

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.

The expression of m6A regulators correlated with the immune microenvironment plays an important role in the prognosis of pancreatic ductal adenocarcinoma

“…To illustrate the biological function in the two subtypes of PDAC patients, we carried out the GSVA enrichment analysis, and the results found that, compared with the cluster A subtype, immune-related pathways, such as the T cell receptor signaling pathway and the B cell receptor signaling pathway, were significantly inhibited in the cluster B subtype, while other pathways related to PDAC progress, such as the cell cycle and the p53 signaling pathway, were significantly increased. It is well known that the cell cycle and the p53 signaling pathway play important roles in the development and progression of PDAC (25)(26)(27). The results of this study further suggest that m 6 A methylation regulators may play an important role in the pathologic progression of PDAC by regulating related signaling pathways.…”

Determinants of Homologous Recombination Deficiency in Pancreatic Cancer

“…Wee1, a kinase which phosphorylates and represses Cdk1 activity, has dual roles in regulating entry into mitosis and maintaining HR proficiency [ 184 ]. In preclinical work, Wee1 inhibition sensitizes HR-proficient pancreatic cancer to chemotherapy and radiation [ 185 , 186 , 187 ]. Furthermore, Wee1 inhibition reduced Cdk1 phosphorylation in patients and shows significant potential as a radio-sensitizing strategy in combination with gemcitabine for the treatment of patients with locally advanced pancreatic cancer [ 188 ].…”

Assessing the performance of different outcomes for tumor growth studies with animal models

“…The data used to illustrate and implement these approaches examined the effect of the combination of AZD1775 provided by AstraZeneca or purchased from MolPool (Hong Kong) with Navitoclax purchased from Active Biochem (“drug AB”) versus a placebo treatment (“vehicle”). A subset of data from a past experiment on 1 PDX line for triple‐negative breast cancer (TNBC012) is used to illustrate the different methods where 2 tumors were planted into the hind flanks of each mouse as described previously 2,15,16 . These tumors were repeatedly measured at unequally spaced days.…”