Inhibition of CHK1 and WEE1 as a new therapeutic approach in diffuse large B cell lymphomas with MYC deregulation

Diffuse large B cell lymphoma (DLBCL) is the most common type of aggressive lymphoma. Despite the remarkable progress made with the addition of rituximab to standard chemotherapy (CHOP: doxorubicin, cyclophosphamide, vincristine, prednisone), ~40% of patients present refractory disease or disease that will relapse after initial response. Unfortunately, the majority of patients with relapsed DLBCL will succumb to the disease. Aberrations of the oncogene MYC are common in DLBCL and define patients with a more aggressive phenotype and a poor outcome with an overall survival below 30% after 2 years (Barrans et al, 2010). Moreover, up to 40% of DLBCLs display high MYC protein expression, irrespective of the presence of translocations but still associated with inferior outcome, suggesting that mechanisms other than gene alterations are responsible for high protein expression (Testoni et al, 2015). Thus MYC is a crucial target for DLBCL therapy but the inability to effectively target this transcription factor limits the possibility to find new therapeutic strategies. Synthetic lethal approaches have been expanded to target essential signalling pathways downstream of MYC. The hypothesis that CHK1 protein kinase (also known as CHEK1), by allowing the repair of the insults caused by MYC (endogenous DNA damage from replicative stress) may be a crucial target to inhibit in MYCdriven malignancies has been recently demonstrated (Hoglund et al, 2011; Ravi et al, 2016). CHK1 inhibitors are strongly synergistic with WEE1 inhibitors in many experimental models (Carrassa et al, 2012) and both kinase inhibitors are under early clinical investigation (Daud et al, 2015; Scagliotti et al, 2016). We had recently reported that DLBCL cell lines are more sensitive to the single inhibition of CHK1 and WEE1 inhibitors than epithelial cancer cell lines, albeit at a lesser extent than mantle cell lymphoma (MCL) cell lines (Chila et al, 2015). Here, we investigated the effects of CHK1 and WEE1 inhibition in a panel of DLBCL cell lines (Fig 1A) with different MYC status (Table S1). We did not observe any specific correlation between MYC status and the extent of sensitivity to the CHK1 inhibitor PF-00477736 and the WEE1 inhibitor AZD-1775 (Fig S1). MYC, CHK1 and WEE1 protein levels and the degree of endogenous constitutive DNA damage (cH2AX and pS317CHK1) detected in DLBCL cell lines did not correlate with the sensitivity to PF00477736 or to AZD-1775. Similar results were seen at the mRNA level for MYC, CHEK1 and WEE1. The isobologram (Fig 1B) summarizes the combination index (CI) value of each cell line at the 50% inhibitory concentration IC50 dose when the two drugs were combined and clearly shows a synergistic effect in all the DLBCL cell lines regardless of molecular subtype and of MYC status, suggesting that this drug combination has a strong and broad effect in DLBCLs. A similar synergistic effect was observed by combining the WEE1 inhibitor AZD-1775 with another CHK1 inhibitor (AZD-7762) (data not shown). The strong synergistic effect was associated with cell death by apoptosis as suggested by the detection of increased caspase-3 activity observed after combined treatment (Fig S2). The CI values at IC50 in the different cell lines and their WEE1 protein or mRNA levels negatively correlated (P = 0 0015 and P = 0 0029 respectively) (Fig 1C), while the protein or mRNA levels of CHK1, MYC and constitutive DNA damage did not correlate with the extent of the synergistic effect. A significant correlation with WEE1 was confirmed also when five MCL cell lines were included in the analysis (data not shown). These data suggest that high WEE1 expression levels may be considered a molecular biomarker for the detection of lymphoma patients that could better benefit from this combined treatment. We took advantage of a large series of publicly available gene expression profiles for CHOP and for rituximab plus CHOP treated DLBCL cases (GSE10846) (Lenz et al, 2008) to further investigate the role of CHK1 and WEE1 in DLBCL. WEE1 was expressed at higher levels in germinal centre B (GCB) than activated B cell-like (ABC) DLBCL, while the opposite was true for CHEK1, albeit at lower extent (Fig S3), suggesting that GCB-DLBCL could potentially be more sensitive to the combined therapy with CHK1 and WEE1 inhibitors. The combined treatment induced an increased cH2AX in all the cell lines (Fig 2A). Interestingly, a decreased MYC protein level was observed after combined treatment in all except one cell lines (OCI-Ly10) bearing a wildtype MYC (Fig 2A). In this cell line, despite an observed increased cH2AX, no decreased pCDK2 (Fig 2B) and less activity of the combination in terms of CI (0 77) was observed. A similar decrease in MYC protein levels after this drug combination was observed in MCL cell lines (Fig S4), while CHK1 protein expression remained unchanged (Chila et al, 2015 and data not shown). The striking decrease in MYC protein levels was not associated with a significant decrease in MYC mRNA (Fig 2C). Studies on MYC protein stability in OCI-Ly7 cells treated with the two drugs and then with cycloheximide (CHX), showed that MYC protein Correspondence