Microsatellite Instability (MSI) In High Risk Myelodysplastic Syndrome (MDS) and Acute Myeloid Leukaemia (AML) Cells Promotes Frameshift Mutations In DNA Repair Genes: Indications for PARP Inhibitor Therapy. (2010)

Abstract Abstract 1194 Despite major advances in the biology and pathogenesis of myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML) identification of the most effective and safest form of treatment continue to present a formidable challenge particularly in older patients. Older patients (>70 years) that constitute the majority of MDS/AML patients are often resistant to chemotherapy, achieve short lived remission and are not candidates for stem cell transplantation. Therefore the emphasis is to prolong survival or improve the quality of life. Currently, a number of therapeutic strategies are being evaluated and these include treatment with DNA methyltransferase or histone deacetylase inhibitors. Seminal work in breast cancer have shown that inhibitors of poly ADP ribose polymerase (PARP) activity can selectively target tumour cells through exploitation of inherent DNA repair defects. MDS/AML are characterized by genomic instability (GI) and single nucleotide polymorphism arrays (SNPA) karyotyping show that loss of heterozygosity (LOH) and uniparental disomy (UPD) are common in MDS/AML and it has been suggested that the underlying cause of this GI is a defect in double strand DNA repair. We have demonstrated that non homologous end joining, a major pathway for the repair of double strand DNA breaks is overactive and associated with extensive joining errors in primary AML cells. Hence, potentially MDS/AML patients are candidates for PI therapy. We have also shown more recently, that 15% of MDS/AML primary patient cells and cell lines are sensitive to PARP inhibitors (PI) through exploitation of homologous recombination DNA repair defects. To further elucidate the mechanisms that underlie PI sensitivity in MDS/AML we tested for microsatellite instability (MSI) in MDS/AML cell lines and high risk MDS patients and the presence of frameshift mutations in specific DNA repair genes that confer PI sensitivity. MSI is a change in length of a microsatellite allele caused by insertion or deletion of nucleotides that are misincorporated during DNA replication and not removed by the mismatch repair pathway. Using fluorescent PCR analysis, PI sensitive cell lines, P39, KG-1 and Molm-13 showed MSI-high (instability at ≥ 2 loci) at 5 mononucleotide microsatellites, in contrast to 12 PI insensitive cell lines that showed no MSI at these loci. We also show using fluorescent PCR and DNA sequencing that these MSI positive cell lines demonstrate MSI (monoallelic 1–2 base pair [bp] deletion) in the coding region microsatellites of DNA repair genes, Ataxia telancgiectasia mutated gene (ATM), CTiP, and MRE11. Monoallelic 1–2 bp base pair deletions at these loci produced frameshift mutations that induced aberrant gene splicing transcripts in ATM and MRE11 and a markedly truncated CTiP gene transcript. No MSI was detected in DNA repair genes CHK1, RAD50, PTEN, BLM and ATR in these cell lines and no mutations were observed at any DNA repair gene microsatellite in the 13 PI insensitive cell lines. We then determined MSI in high risk MDS patients with or without monosomy 7 (-7/del7q). 13 of 63 (21%) high risk MDS patients showed MSI (9 MSI-low and 4 MSI-high). Of the 13 MSI positive patients, 7 (4 MSI-high, 3 MSI-low) had monosomy 7 and other complex chromosomal abnormalities (Group 1, 54%), 2 (MSI-low) patients had isolated monosomy 7 (Group 2, 15%) whilst 4 patients (MSI-low) had normal cytogenetics (Group 3, 30%). Constitutional DNA from these patients did not show MSI at these loci. Significantly, however, Group 3 with MSI and normal cytogenetics all had widespread UPD and cryptic chromosome changes determined by SNPA. Strikingly, thus all 13 patients with MSI possessed chromosomal abnormalities, both gross and cryptic. Furthermore, 12 patients (19%) found to be cytogenetically normal and lacking UPD and genomic aberrations by SNPA did not show MSI. We have also identified that 3 patients with MSI-high (Group 1) and 1 patient with MSI-low (Group 3) had a monoallelic 1 bp deletion in the CTiP exon coding microsatellite. 1 bp deletion within the coding exon of CTiP resulted in an abbreviated CTiP gene transcript. In conclusion, we have made the important correlation between MSI and subsequent frameshift mutations in specific DNA repair genes with the gross and cryptic chromosomal changes observed in MDS/AML. Identification of a cohort of MDS/AML patients with MSI would herald a significant advancement for the selection of candidates for PI therapy. Disclosures: No relevant conflicts of interest to declare.