Modifier Genetics in Zebrafish Identify Chk1 and an Associated Survival Pathway as Targets for Pharmacotherapy of MDS/AML with P53 Mutations.

P53 mutations are frequently detected in high-risk MDS and AML cases with complex aberrant karyotypes (~20% of de novo MDS and AML cases). Pro-apoptotic signaling is a predominant mechanism through which wild-type p53 suppresses malignant transformation, and defects in this process account for the chemo/radio-resistance of many human tumors. The ability to sensitize p53 mutant myeloid cells to undergo apoptosis after drug or radiation therapy could accelerate the development of novel therapies for MDS, AML and other malignancies with p53 mutations. We have engineered a p53 mutant zebrafish line in which the allele, p53^e7 (p53^M214K), mimics a mutation found in multiple primary human tumors. The line is homozygous viable and fertile with homozygotes developing malignant peripheral nerve sheath tumors with 30% penetrance by 1.5 years of age. We show that p53^e7/e7 embryos exhibit fully penetrant radioresistance in both the nervous system and myeloid lineage, as visualized in live embryos stained with acridine orange or carrying a pu.1-EGFP transgene. In a pilot morpholino (MO) screen that targeted 8 checkpoint kinases, we found that chk1 knockdown restores radiosensitivity in neurons and myeloid progenitors of p53^e7/e7 mutants, p53 protein-null morphants and p53^e6 (p53^N168K) homozygotes. Remarkably, chk1 knockdown is compatible with normal zebrafish development and viability in the absence of IR, regardless of p53 status. The radiosensitizing effects of chk1 loss are phenocopied by a recently developed, specific inhibitor of human CHK1, but not by inhibitors of ATM and CHK2. The CHK1 inhibitor shows no apparent toxicity in non-irradiated p53^+/+ specimens. Radiosensitization of p53 mutants through Chk1-loss correlates with G²/M checkpoint malfunction and TUNEL-positive DNA fragmentation occurring throughout the cell cycle. Cell death in irradiated p53^e7/e7;chk1MO embryos preferentially occurs during mitosis, as evidenced by an increased number of TUNEL/pH3 doubly labeled cells compared to irradiated wild-type embryos. Despite prominent TUNEL staining, p53^e7/e7;chk1MO embryos analyzed by whole-mount immunochemistry are devoid of IR-induced activated cellular Caspase-3. Therefore, Chk1-loss in p53 mutants does not restore the usual Casp-3-mediated apoptotic response to IR, a notion supported by the inability of forced Bcl-xL expression to completely block cell-death recovery in p53^e7/e7;chk1MO embryos. Rather, Chk1-loss unbuffers a p53-independent pathway that is normally suppressed by Chk1 after DNA damage. Through epistasis analyses, we show that this alternative cell death pathway is activated by ATR and requires Casp-2, but not Casp-8 or -9 activity. Altogether, our data indicate that Chk1 acts as a survival factor in p53 mutants after DNA damage, by blocking an ATR/Casp-2 cell-death program that would otherwise lead to widespread cellular death. As such, our results provide in vivo evidence supporting the use of CHK1 inhibitors in the treatment of MDS and AML cases with p53 mutations, as well as other types of human cancers associated with P53 deficiency.