Numerous Chk1 inhibitors have been described to date and demonstrate exciting anti-tumour efficacy as single agents, in combination with cytotoxic chemotherapy, and in combination with novel targeted therapies (such as PARP inhibitors). Despite these initially exciting pre-clinical results, no inhibitors have yet to progress beyond Phase 2 into Phase 3 registration trials with the majority terminating after Phase 1 6,15,16. The reasons behind this are numerous and complex including (but not limited to) lack of efficacy, dose limiting toxicities (especially in combination), poor pharmaceutical properties (oral bioavailability, half-life), target patient population selection and drug combination selection.
To further evaluate the clinical potential of our own Chk1 inhibitor, V158411 8, we utilised RNAseq analysis of V158411 treated cancer cells to evaluate the genome wide changes to inhibitor treatment. One of the most upregulated genes in response to V158411 treatment in the U2OS osteosarcoma cell line was BCL2A1 (BFL1). BFL1 is a member of the Bcl-2 family of antiapoptotic proteins that also includes the much wider studied Bcl-2, Mcl-1 and Bcl-XL members 17,18. BFL1 has been implicated in the development of lymphomas and leukaemia’s 19–21, and increased BCL2A1 is associated with resistance to the clinically approved Bcl-2 inhibitor Venetoclax 22,23. Upregulated BCL2A1 / BFL1 has been identified in melanoma 24,25 where it confers resistance to Braf inhibitors. Like the other members of the Bcl-2 family of proteins, BFL1 binds to pro-apoptotic Bcl-2 proteins (such as BAK and BAX) and pro-apoptotic BH3-only proteins (such as tBID, BIM, PUMA and NOXA) thereby preventing mitochondrial membrane permeability, cytochrome c release and caspase activation in response to apoptotic stimuli.
Chk1 inhibition by V158411 induces replication stress and DNA damage in a wide range of human cancer cell lines of differing tumour types 9,14,26. Out of the 24 cell lines studied, U2OS cells were the only cell line with low basal BFL1 protein levels that were increased by V158411. The increase in BCL2A1 mRNA expression in response to Chk1 inhibitor induced DNA damage appeared restricted to a subset of around 30-40% of the U2OS cells. Three additional osteosarcoma cell lines were evaluated (HOSTE85, SAOS-2 and SJSA-1) but did not induce BFL1 protein in response to V158411-induced DNA damage. Increases were also observed in the HL60 myeloid leukaemia, and PANC-1 pancreatic cancer cell line but these two cell lines had high basal BFL1 expression levels. The reason why U2OS, HL60 and PANC-1 cells were sensitive to BFL1 induction by V158411 was not immediately apparent. The cells all come from diverse tissue types (bone, blood and pancreas respectively), have no obvious common driver mutations, and vary widely in their sensitivity to growth inhibition by V158411 (0.82, 0.21 and 8.0 µM respectively). Further work is ongoing to understand these differences in BFL1 induction response between different cell lines.
The increase in BCL2A1 / BFL1 in U2OS cells was not limited to V158411 but was also induced by other Chk1 inhibitors, the Wee1 inhibitor AZD1775 and topoisomerase inhibitors. The topoisomerase inhibitors etoposide 10 and camptothecin 27 have both previously been demonstrated to increase BFL1. However, other inducers of DNA damage such as the cytotoxic chemotherapy drugs gemcitabine, hydroxyurea and cisplatin, and inhibitors of the DNA damage response proteins ATR, ATM and DNA-PKcs did not increase BFL1 expression. The fact that not all DNA damage resulted in BFL1 induction suggests that the cells are responding to either a specific kind of DNA damage and/or DNA damage induced at a specific point in the cell cycle.
In addition to increasing BCL2A1, V158411 increased BCL2L10 (Bcl-b) mRNA though to a much lesser extent (5.8-fold) than BCL2A1 (444-fold). Coupled to this was a decrease in BIM mRNA and protein levels. The mechanism by which BCL2A1 mRNA was increased in U2OS cells by V158411 so far appears unclear. Previous studies have demonstrated NF-κB as a significant transcriptional inducer of BCL2A1 11–13. NF-κB also upregulates Bcl-XL 12,28,29 with the two proteins often co-induced by NF-κB activating stimuli. Here we observed only an increase in BFL1 and not Bcl-XL and found no evidence for NF-κB activation in response to Chk1i, Wee1i or topoisomerasei treatment in U2OS cells. In melanoma cells, BCL2A1 is upregulated via MITF 24,25 but as U2OS are osteosarcoma cells, it would appear unlikely that MITF is responsible in this cell line.
In prostate cancer, BCL2A1 was induced by both PMA and etoposide via a PKCδ-dependent mechanism 10. V158411 induced BCL2A1 via a different mechanism as the PKC inhibitor sotrastaurin inhibited BFL1 induction by PMA but not that by V158411. AKT and ERK phosphorylation was increased in V158411 treated U2OS cells and the induction of BFL1 by V158411 appeared dependent on signalling through the MEK/PI3K/AKT/mTOR dependent pathways. Inhibitors targeting these pathways effectively blocked BFL1 induction not only by Chk1 inhibitors but also by topoisomerase inhibitors as well. The MEK inhibitor Trametinib appeared the most active inhibitor of BFL1 induction by Chk1 or topoisomerase inhibitors. There is limited literature to date linking BCL2A1 with either the MEK or PI3K/AKT pathways. BCL2A1 has been demonstrated to be regulated by PI3K/AKT to control neutrophil survival and homeostasis 30 and eosinophil adhesion to IMR-32 cells increased BCL2A1 in a PI3K-MEK/ERK-NF-κB dependent mechanism to protect against neuronal induced apoptosis 31. Clear synergy between Chk1 inhibitors and mTOR inhibitors 32–34 and MEK inhibitors 35–37 has been demonstrated with potentially the control of anti-apoptotic protein expression contributing to this.
A very recent study has demonstrated that BFL1 (BCL2A1) and Bcl-b (BCL2L10) are stabilised by Ubiquilin4 (UBQLN4). UBQLN4 is a substrate of ATM and is phosphorylated (and thereby activated) in response to ATM-activating DNA damage including that induced by the topoisomerase inhibitor camptothecin 38. BCL2A1 and BCL2L10 stabilisation by UBQLN4 reduced apoptosis in response to DNA damage in a mesothelioma cell model. Chk1 and Wee1 inhibitors activate ATM 9,39,40. In this study, the induction of BFL1 protein by Chk1i, Wee1i and topoisomerasei-induced DNA damage appears to be via an increase in BCL2A1 mRNA with BCL2A1 mRNA increased over 400-fold following V158411 treatment. BCL2L10 was also increased by V158411 but to a much lesser extent (around 5.8-fold). Likewise, inhibition of ATM with KU-60019 did not block increased BFL1 expression following V158411 treatment. However, increased protein stability through UBQLN4-induced stabilisation may contribute to increased BFL1 protein levels through reduced protein turnover.
Whilst BFL1 was induced following V158411-induced DNA damage in U2OS cells, it did not appear to block apoptosis in these cells with high levels of BFL1 protein observed in apoptotic cells. Likewise cells with high basal levels of BFL1 (namely A2058 and HL60 14) appeared to readily undergo caspase dependent apoptosis in response to Chk1 inhibition. Blocking the V158411 induced induction of BFL1 with Trametinib did not increase the fraction of U2OS cells undergoing caspase-dependent apoptosis. There was still a subset of around 30-40% of the cells that were resistant to V158411 induced apoptosis. This is in direct contrast to studies with inhibitors of other Bcl-2 family proteins. The Bcl-2 inhibitor venetoclax (ABT-199) increased the fraction of apoptotic AML cells when combined with a Chk1 inhibitor 41 whilst the Bcl-2/Bcl-XL inhibitor navitoclax sensitised pancreatic cancer cells to apoptosis induced by the Chk1 inhibitor prexasertib 42.
In conclusion, DNA damage induced by Chk1, Wee1 or topoisomerase inhibitors increased BFL1 expression and downregulated BIM via an PI3K/AKT/MEK dependent (and NF-κB independent) pathway in a subset of U2OS cells. This increase in BFL1 appeared to be an attempt by the cells to block DNA damage induced apoptosis. This attempt to inhibit apoptosis was ultimately futile as the cells still underwent apoptosis. Further work is ongoing to further understand the mechanism of BFL1 induction in this cell model.