Vitamin-Containing Antioxidant Formulation Reduces Carcinogen-Induced DNA Damage through ATR/Chk1 Signaling in Bronchial Epithelial Cells In Vitro

Lung cancer has the highest mortality rate worldwide and is often diagnosed at late stages, requiring genotoxic chemotherapy with significant side effects. Cancer prevention has become a major focus, including the use of dietary and supplemental antioxidants. Thus, we investigated the ability of an antioxidant formulation (AOX1) to reduce DNA damage in human bronchial epithelial cells (BEAS-2B) with and without the combination of apple peel flavonoid fraction (AF4), or its major constituent quercetin (Q), or Q-3-O-d-glucoside (Q3G) in vitro. To model smoke-related genotoxicity, we used cigarette-smoke hydrocarbon 4-[(acetoxymethyl)nitrosamino]-1-(3-pyridyl)-1-butanone (NNKOAc) as well as methotrexate (MTX) to induce DNA damage in BEAS-2B cells. DNA fragmentation, γ-H2AX immunofluorescence, and comet assays were used as indicators of DNA damage. Pre-exposure to AOX1 alone or in combination with AF4, Q, or Q3G before challenging with NNKOAc and MTX significantly reduced intracellular reactive oxygen species (ROS) levels and DNA damage in BEAS-2B cells. Although NNKOAc-induced DNA damage activated ATM-Rad3-related (ATR) and Chk1 kinase in BEAS-2B cells, pre-exposure of the cells with tested antioxidants prior to carcinogen challenge significantly reduced their activation and levels of γ-H2AX (p ≤ 0.05). Therefore, AOX1 alone or combined with flavonoids holds promise as a chemoprotectant by reducing ROS and DNA damage to attenuate activation of ATR kinase following carcinogen exposure.

CBIO-16. SUPPRESSION OF HISTONE H3.3 MITOTIC PHOSPHORYLATION DRIVES DEVELOPMENT OF H3K27M-MUTANT DIFFUSE MIDLINE GLIOMAS

Pediatric midline gliomas – including DIPG – are lethal brain tumors in children, with poor prognosis and limited treatment options that provide only short-term benefits. The majority have a lysine-to-methionine substitution at residue 27 (H3K27M) in genes expressing histone H3 – predominantly in the H3.3 variant. This causes a global reduction in H3 Lys27 tri-methylation (H3K27Me3), comprehensive epigenetic reprogramming, and is a key driver in gliomagenesis. We show that the H3.3K27M mutation also induces chromosome segregation defects, which in high-grade tumors, results in extensive copy number alterations (CNAs). Ser31 is one of five amino acid substitutions differentiating H3.3 from canonical H3.1. Mitotic phosphorylation of H3.3 Ser31 by Chk1 kinase is restricted to pericentromeric heterochromatin, where it plays a role in chromosome segregation. We show that the K27M mutation affects neighboring Ser31 phosphorylation and pericentromeric heterochromatin organization. We demonstrate that (i) H3.3 K27M protein is defective for Ser31 phosphorylation by Chk1 kinase in vitro; (ii) DIPG cell lines have significantly decreased mitotic Ser31 phosphorylation, and are chromosomally unstable; and (iii) CRISPR-reversion of H3.3K27M to Lys27 restores phospho-Ser31 (and Lys27 tri-methylation) and significantly decreases chromosome instability. Expression of H3.3K27M or non-phosphorylatable H3.3S31A mutants in WT cells results in chromosome missegregation; this is suppressed by co-expression of phospho-mimetic H3.3K27M/S31E. In normal cells, chromosome missegregation stimulates p53-dependent cell cycle arrest in G1 to prevent the proliferation of aneuploid daughters. However, cells expressing H3.3 K27M or S31A failed to arrest following missegregation - despite having WT p53. Finally, in a novel mouse model of glioma, mean survival of mice with tumors induced with H3.3K27M and H3.3S31A was 81 and 68 days: 100% of H3.3S31A mice developed high-grade tumors. H3.3 WT controls developed only low-grade tumors and all survived 100 days. H3.3S31A is WT for Lys27 tri-methylation and thus, loss of Ser31 phosphorylation alone is oncogenic.

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

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. Indeed, 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 associate with the induction of DNA damage and reduction of Chk1 protein levels. Most studies of Chk1 function have 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 germ line. We find that this mouse is overtly normal and does not have problems with erythropoiesis with aging 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 kinase-dead Chk1 mouse with a conditional allele, we are able to demonstrate that expression of only one kinase-dead allele, but no wild-type allele, of Chek1 is lethal for Myc-induced 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.

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

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 kinase-dead 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 Myc-induced 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.