Microsatellite Instability Induced Mutation in DNA Repair Genes, CTiP and Mre11 Confer Hypersensitivity to PARP Inhibitors in Myeloid Malignancies

Abstract 276

In both myelodysplatic syndrome (MDS) and acute myeloid leukaemia (AML), relapse from standard chemotherapeutic intervention is common with only 20–30% enjoying long-term disease-free survival. Allogeneic stem cell transplantation still remains the only curative treatment in MDS/AML, but only in 15–20% of 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. Given the high number of patients refractory to conventional therapy and the relatively high rate of therapy relapse, efforts have been made in the search for alternative treatment strategies. Inhibition of Poly ADP ribose polymerase (PARP) activity can selectively target cancer cells through exploitation of inherent DNA repair defects. Significant single agent anti-tumour responses coupled with a wide therapeutic index have been influential in moving PARP inhibitors (PARPi) to the clinical arena. MDS/AML is characterized by chromosomal instability (CI) manifesting as deletions, translocations and chromosome losses. Single nucleotide polymorphism arrays (SNPA) karyotyping show that loss of heterozygosity (LOH) and uniparental disomy (UPD) are common in MDS/AML and thus it has been suggested that the underlying cause of this CI is a defect in double strand DNA repair. We have previously shown that 15% of AML and MDS primary patient cells and cell lines are sensitive to PARPi through exploitation of homologous recombination DNA repair defects. To explicate still further the mechanisms that support PARPi sensitivity in MDS/AML we tested for microsatellite instability (MSI) in primary high risk MDS and primary AML for the presence of frameshift mutations in specific DNA repair genes. 13 of 63 (21%) high risk MDS patients possessed MSI (9 MSI-low and 4 MSI-high). Significantly, all 13 MSI positive patients possessed chromosomal abnormalities, both gross and cryptic UPD determined by SNPA, whilst 12 patients (19%) found to be have normal cytogenetics and lacking UPD failed to demonstrate MSI. Moreover, 3 patients with MSI-high and 1 patient with MSI-low possessed a mono-allelic 1bp deletion in the CTiP exon coding microsatellite. 1bp deletion within the coding exon of CTiP would result in an abbreviated gene CTiP transcript in these patients. From a panel of 18 primary AML samples, 5 primary AML demonstrated sensitivity to the PARPi, BMN673 (100nM). Immunocytochemical staining also showed that PARPi sensitive AML cells demonstrated severely reduced rad51 and increased phospho-γH2AX foci formation compared to PARPi insensitive AML cells. This confirmed that BMN673 targeted HR deficiencies in AML PARPi sensitive cells. Strikingly, 2 of the 5 PARPi responders exhibited MSI, with 1 patient displaying a bi-allelic 1bp deletion in MRe11 and 1 patient exhibiting a mono-allelic 1bp deletion in CTiP. MSI was not observed in the 13 PARPi insensitive AML patients. Western blotting analysis identified the loss of mismatch repair proteins MLH-1 and MSH-2 respectively, in the 2 MSI positive primary AML underlying the MSI observed in the AML patients cells. Moreover, Western blotting analysis also revealed aberrant expression of Mre11 and CTiP in these patients. Finally, to confirm the relative contribution of mutant MRe11 and CTiP to PARPi sensitivity, an expression construct of MRe11 missing exons 5 to 7 (δ_5–7MRe11) was transfected into the MSI negative and PARPi insensitive cell line, U937. Cytotoxicity assays and immunocytochemical staining revealed that U937 + δ_5–7MRe11 demonstrated significant sensitivity to PARPi with concomitant HR DNA repair defects compared to U937 + vector control. Similarly, Si-RNA knockdown of CTiP in U937 also conferred hypersensitivity to PARPi as a result of an abrogation of functional HR DNA repair. In conclusion, we make the unique observation that MSI dependent mutations in genes that are essential for DNA repair signalling confer PARPi sensitivity in myeloid malignancy. Identification of a cohort of MDS/AML patients with MSI would signify a major development in the identification of candidates for PARPi therapy.