The checkpoint kinases ATR and WEE1 protect cells from replication stress (RS), a hallmark of cancer that has potential to be exploited for cancer treatment. ATR and WEE1 inhibitors are in early clinical trials. Here we report selective anti-tumour activity of inhibitors to ATR (AZD6738) and WEE1 (AZD1775) in a subset of diffuse large B-cell lymphoma (DLBCL) cell lines. Using the cell-of-origin (COO) gene expression scoring system, we found enhanced sensitivity to AZD6738 in the activated B-cell like (ABC, n=6) and unclassified cell lines (n=6) with a combined median GI50 of 0.372 µM, compared to germinal centre B-cell like (GCB, n=7) cell lines, with median GI50 of 1.554 µM. Grouping the cells into AZD6738 sensitive (GI50 ≤ 0.5 µM) or insensitive (≥ 1 µM), we show that the ABC/unclassified COO score, together with high c-MYC protein or CDKN2A/B deletion identified 7/8 sensitive cell lines and 0/7 insensitive cell lines. Furthermore, markers of RS correlated with cell line sensitivity. Treatment of cells with 0.5 µM AZD6738 increased the quantity of 53BP1 nuclear bodies by 3-6 fold across the sensitive cell lines compared to only 2 fold across the insensitive cell lines. DNA fibre analysis also showed that sensitive cells lines had slower rates of replication fork progression (0.80 - 1.12 kbmin-1) compared to insensitive cell lines (1.22 - 1.41 kbmin-1). Across the same DLBCL cell line panel sensitivity to the WEE1 inhibitor AZD1775 was positively correlated with AZD6738 sensitivity, although at lower concentrations. However, using OCI-LY19 and DB cells to reflect high and low replication stress respectively, we show that AZD6738 and AZD1775 differ mechanistically in the extent of DNA damage and apoptosis induced within cell cycle phases. Treatment with 1 µM AZD1775 induced S-phase arrest and increased γH2AX+ cells in S/G2 from 4% to 20% in both cell lines at 8 h, indicating replication-associated DNA damage. However, earlier onset of cell death was observed in the OCI-LY19 cells, with 30% γH2AX+ cells undergoing apoptosis at 24 h compared to 25% γH2AX+ DB cells at 72 h. In contrast, AZD6738 only caused G1 arrest in OCI-LY19 daughter cells, with 20% apoptosis at 72 h. These findings are reflected in the anti-tumour activity of the inhibitors in xenograft DLBCL models in vivo. Once-daily, oral dosing of 50 mg/kg/day AZD6738 or 120 mg/kg/day AZD1775 in OCI-LY19 xenograft model showed efficacy with mean % tumour growth inhibition (%TGI) at day 8 of 104.1% (n=10) and 114.3% (n=10) respectively. In addition, the combination of AZD1775 (60 mg/kg/day; monotherapy %TGI=88.7, n=10) and AZD6738 (25 mg/kg/day; monotherapy %TGI=89.8%, n=10) dosed using a 5 day on, 9 day off dosing schedule was tolerated in mice and resulted in tumour regressions (combination %TGI=108.4%, n=8). These data suggest potential for both single agent and combination opportunities for ATR and WEE1 inhibitors in the DLBCL ABC subtype.
Citation Format: Lucy A. Young, Lenka Oplustil O'Connor, Zena Wilson, Corinne Reimer, Alan Lau, Jiri Bartek, Mark J. O'Connor. Differential activity of ATR and WEE1 inhibitors in DLBCL subtypes is linked to replication stress and differences in mode of action [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2826.