Potential Pre -Leukemic Mutations in PPM1D Confer Chemotherapy Resistance to Aged HSC Clones

Abstract

Michael D. Milsom Division of Experimental Hematology, German Cancer Research Center (DKFZ), Heidelberg, Germany. Heidelberg Institute for Stem Cell Technology and Experimental Medicine (HI-STEM), Heidelberg, Germany. The authors have indicated they have no potential conflicts of interest to disclose. Copyright © 2018 the Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the European Hematology Association. This is an open access article distributed under the Creative Commons Attribution License 4.0 (CCBY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. HemaSphere (2019) 3:1(e171) Citation: Milsom MD. Potential PreLeukemic Mutations in PPM1D Confer Chemotherapy Resistance to Aged HSC Clones. HemaSphere, 2019;3:1. http://dx.doi.org/10.1097/ HS9.0000000000000171 hematopoiesis (CH), which arises in apparently healthy humans during the process of aging, and is frequently referred to in the literature as clonal hematopoiesis of Tindeterminate potential (CHIP) or age-related clonal hematopoiesis (ARCH). The growing activity in this research field has predominantly been driven by the observation that the incidence of CH correlates with risk of developing diseases such as atherosclerosis, stroke and hematologic malignancies, suggesting that a causal relationship may exist. However, for the vast majority of the somatic mutations that are frequently found in dominant age-associated clones, it is unclear why the mutant clone enjoys a selective growth advantage over its normal counterparts. Two recent studies, from the groups of Benjamin Ebert and Margaret Goodell, independently studied the biological relevance of mutations within exon 6 of the gene encoding protein phosphatase Mg2+/Mn2+ 1D (PPM1D), since analogous mutations are frequently found in CH. PPM1D, which is also known aswild-type p53-induced phosphatase 1 (WIP1), had been previously identified as a potential oncogene in several solid tumor entities, including breast and ovarian cancer and neuroblastoma. The putative mechanism of action of PPM1D relates to its capacity to dephosphorylate regulatory residues of key proteins involved in mediating the DNA damage response (DDR), including p53. Thus, aberrant activation of PPM1D would result in downmodulationof theDDR,potentially rendering cells resistant to various formsofDNAdamage. In line with this hypothesis, both groups found that mutations in PPM1D were enriched in CH where the individual had prior exposure to chemotherapy, for example in the treatment of solid cancer or lymphoid malignancies. These mutations were also found in samples from patients with therapyrelatedMDS and secondaryAML, suggesting thatPPM1Dmutant clones can act as the cell of origin for hematologic malignancies arising as a consequence of chemotherapeutic challenge. Using gene editing to recapitulate truncating mutations in exon 6 of PPM1D in AML cell lines, both groups could observe that basal levels of mutant PPM1Dwere elevated compared to the wildtype protein. Further experiments strongly supported the conclusion that a proteasome degradation signal was present at this site in the protein and that this was lost in mutant cells, resulting in increased stability of the protein. In line with this concept, an unbiased CRISPR/Cas9 screen for functional domains using tiled guides spanning the PPM1D gene, revealed that mutations in amino acids 400–585, corresponding to exon 6, conferred resistance to cytarabine treatment in an AML cell line. Cell lines harboring mutant PPM1D demonstrated increased viability and a selective growth advantage compared to their isogenic counterparts upon treatment with clinically relevant chemotherapeutic agents such as cytarabine, cisplatin, doxorubicin and etoposide. This corresponded to an attenuated DDR, as classical DNA repair intermediates such as phosphorylated gH2AX and TP53, were suppressed in mutant cells upon chemotherapeutic challenge. Phosphoproteome analysis revealed that truncated PPM1D resulted in differential phosphorylation of multiple DDR pathway members in response to DNA damage, including TP53, CHECK1, CHECK2 and MDM4. Both groups could show that this compromised DDR resulted in decreased levels of apoptosis in response to chemotherapeutic challenge, while the Ebert group could additionally characterize that the cell cycle arrest was curtailed in mutant cells following cytarabine treatment. Importantly, overexpression of wild-type PPM1D could also confer increased chemo-resistance to AML cell lines, strongly suggesting that the attenuated DDR in cells with mutant PPM1D is predominantly driven by increased levels of the enzyme, resulting from enhanced protein stability, as opposed to any alternative biological effect resulting from the