Small molecule inhibitors and a kinase-dead expressing mouse model demonstrate that the kinase activity of Chk1 is essential for mouse embryos and cancer cells

Abstract

Chk1 kinase is downstream of the ATR kinase in the sensing of improper replication. Previous cell culture studies have demonstrated that Chk1 is essential for replication and Chk1 inhibitors are efficacious against tumors with high-level replication stress such as Myc-induced lymphoma cells. Treatment with Chk1 inhibitors also combines well with certain chemotherapeutic drugs and effects associates with induction of DNA damage and reduction of Chk1 protein levels. Most studies of Chk1 function has relied on the use of inhibitors. Whether or not a mouse or cancer cells could survive if a kinase-dead form of Chk1 is expressed has not been investigated before. Here we generate a mouse model that expresses a kinase-dead (D130A) allele in the mouse germline. We find that this mouse is overtly normal and does not have problems with erythropoiesis with ageing as previously been shown for a mouse expressing one null allele. However, similar to a null allele, homozygous kinase-dead mice cannot be generated and timed pregnancies of heterozygous mice suggest lethality of homozygous blastocysts at around the time of implantation. By breeding the kinasedead Chk1 mouse with a conditional allele we are able to demonstrate that expression of only one kinase-dead allele, but no wildtype allele, of Chek1 is lethal for Mycinduced cancer cells. Finally, treatment of melanoma cells with tumor-infiltrating T cells or CAR-T cells is effective even if Chk1 is inhibited, suggesting that Chk1 inhibitors can be safely administered in patients where immunotherapy is an essential component of the arsenal against cancer.

CHK1-CDC25A-CDK1 regulate cell cycle progression in early mouse embryos to protect genome integrity

“…CHK1 inhibition causes premature mitosis in both mouse (Ju et al, 2020) and human (Palmerola et al, 2022) zygotes. CHK1 kinase activity itself is necessary for early embryos (Muralidharan et al, 2020). It is also consistent with more other studies in mice and (Fishler et al, 2010; Lam et al, 2004; Niida et al, 2005; Peddibhotla et al, 2009; Ruth et al, 2021), humans (Branigan et al, 2021; Durkin et al, 2006; Niida et al, 2005; Syljuåsen et al, 2005) and other species (Zachos et al, 2007, 2003, 2005).…”

“…CHK1 is activated after replication stress in somatic cells and early embryos (González Besteiro & Gottifredi 2015; Lam et al, 2004; Lebrec et al, 2022; Lemmens et al, 2018; Michelena et al, 2019; Moiseeva et al, 2019; Muralidharan et al, 2020; Palmerola et al, 2022; Syljuåsen et al, 2005; Zachos et al, 2005). Replication stress signalling is usually orchestrated by ATR kinase (Eykelenboom et al, 2013; Saldivar et al, 2018).…”

“…This finding is consistent with other studies (Branigan et al, 2021;Lam et al, 2004), where Chk1 depletion or inhibition causes premature mitosis in cells with only mildly, but not severely, under-replicated DNA. CHK1 is activated after replication stress in somatic cells and early embryos (González Besteiro & Gottifredi 2015;Lam et al, 2004;Lebrec et al, 2022;Lemmens et al, 2018;Michelena et al, 2019;Moiseeva et al, 2019;Muralidharan et al, 2020;Palmerola et al, 2022;Syljuåsen et al, 2005;Zachos et al, 2005). Replication stress signalling is usually orchestrated by ATR kinase (Eykelenboom et al, 2013;Saldivar et al, 2018).…”

“…CHK1 also regulates cell cycle progression during embryonic development in zebrafish (Dalle Nogare et al, 2009; Zhang et al, 2014), Drosophila (Deneke et al, 2016) and Xenopus (Petrus et al, 2004; Shimuta et al, 2002). Moreover, CHK1 inhibition or depletion in human (Palmerola et al, 2022) and mouse embryos (Ju et al, 2020; Liu et al, 2000; Muralidharan et al, 2020; Takai et al, 2000) exacerbates the incidence of aneuploidy and induces genome fragmentation.…”

“…CHK1 also regulates cell cycle progression during embryonic development in zebrafish (Dalle Nogare et al, 2009;Zhang et al, 2014), Drosophila (Deneke et al, 2016) and Xenopus (Petrus et al, 2004;Shimuta et al, 2002). Moreover, CHK1 inhibition or depletion in human (Palmerola et al, 2022) and mouse embryos (Ju et al, 2020;Liu et al, 2000;Muralidharan et al, 2020;Takai et al, 2000) exacerbates the incidence of aneuploidy and induces genome fragmentation. Although the mechanism underlying the role of CHK1 in cell cycle checkpoint signaling is well understood in somatic cells, little is known regarding the function of CHK1 signaling in regulating embryonic cell division and genome integrity in early mammalian embryos.…”