Pre-clinical investigation of inhibition of the DNA damage response as a targetted therapy in myeloproliferative neoplasms shows synergism of ATR inhibitors with standard-of-care treatment.

Abstract

Introduction Myeloproliferative neoplasms (MPNs) are a group of haematological malignancies arising from haematopoietic stem cells with acquired driver mutations in JAK2, MPL and CALR. Increased replication stress is seen in the presence of JAK2V617F. Genes involved in the DNA double-strand break (DSB) repair pathways – BRCA-dependent homologous recombination repair (HRR) and DNA-dependent protein kinase-mediated non-homologous end-joining (D-NHEJ) are upregulated in MPN cells expressing mutated JAK2. Aims Using JAK2V617F and CALR (del 52) mutant cell lines: Determine the effect of single-agent DNA damage repair (DDR) inhibitors on cell viability and apoptosis. Evaluate the efficacy of DDR inhibitors in combination with hydroxyurea or ruxolitinib. Materials and Methods Cell lines expressing JAK2 (V617F)- HEL and CALR (del52)- MARIMO were treated with a drug panel comprising hydroxyurea, ruxolitinib, methotrexate, AZD6738 (ATRi), NU7441 (DNA-PKi), Olaparib (PARPi) and VE-821 (ATRi). AlamarBlue assay for cell proliferation and annexin V/ propidium iodide staining for flow cytometry were used to evaluate the toxicity. Results In JAK2 and CALR mutated cell lines, ATR inhibition by AZD6738 or VE-821, DNA-PKs inhibition by NU7441 and hydroxyurea each reduced viability, whereas PARP inhibition by olaparib had a minimal effect. The combination of ATR inhibition and hydroxyurea demonstrated high synergism in both apoptosis induction and proliferation arrest. Ruxolitinib alone had a modest effect in the presence of JAK2V617F and a minimal effect in CALR (del 52) mutated cells. Synergistic toxicity was observed for ruxolitinib and AZD6738/ VE-821 combination in JAK2 mutated cell line. Conclusions DDR inhibition reduces viability in cells expressing the driver mutations seen in MPNs. Most notably, ATR kinase inhibitors have a synergistic effect with the current standard-of-care treatment hydroxyurea. This study provides preliminary evidence that ATR inhibitors combined with standard therapies may be exploited in MPNs harbouring JAK2 and CALR mutations.