The Telomerase Inhibitor RHPS4 Induces Telomere Shortening, DNA-Damage and Cell Death in AML Cells and Chemosensitises Cells to Daunorubicin.

Abstract 2760

Poster Board II-736

The quadruplex ligand RHPS4 is the lead compound in a drug discovery program at the University of Nottingham. It has been shown to bind to telomeres and inhibit telomerase, and subsequently induces growth arrest in progenitor cells from cancer cell lines whilst sparing normal haematopoietic progenitor cells. We explored its in vitro effects in AML cells, which are reported generally to have considerably shorter telomeres than normal CD34+ cells. AML cell lines were grown for 21 days in suspension culture. Primary samples were cultured for 14 days in semi-solid medium. Telomere length was measured by Southern blotting. γH2A.X was used to identify a DNA damage response, and cell viability was measured flow cytometrically with 7-amino actinomycin D. As reported in other tumour cell types, sensitivity to RHPS4 was found to be greatest in those AML cells with the shortest telomeres. In the OCI-AML3 cell line 0.3 μM RHPS4 inhibited cell growth by 50% in a 21 day clonogenic assay, accompanied by shortening of telomeres from 2.6 Kb to <1 Kb. Molm 13 cells (initial telomere length 3.2kB) also underwent telomere shortening in the presence of 0.3 μM RHPS4 (2.8Kb), whereas TF1a and U937 (both with initial telomere lengths approximately 6.5 kB) were insensitive at that concentration. After 6 days at 0.3 μM, RHPS4 was cytostatic, but at higher concentrations (1 μM) the drug was found to induce a substantial DNA damage response and loss of viability to OCI-AML3 cells. Moreover 0.3 μM RHPS4 enhanced the γH2A.X expression and cell death induced by the chemotherapy drug daunorubicin in these cells. Using 14 day clonogenic assays in primary AML samples (n=6), we found that the IC₅₀ for RHPS4 alone was 0.7 μM. However, in the presence of 0.3 μM RHPS4, the median IC₅₀ to daunorubicin was reduced from 19 nM to 5.5 nM. In conclusion we have determined that RHPS4 has telomere-shortening, cytostatic, cytotoxic and chemosensitising properties in AML cells.