Abstract 4145: Concurrent treatment with Pim kinase inhibitor decreases alternative non-homologous end-joining repair of DNA damage induced by topoisomerase 2 inhibitors in cells with FLT3-ITD
Authors: Doshi Kshama A. , Nagaria Pratik K. , Tron Adriana E. , Rassool Feyruz V. , Baer Maria R. 
Published: July 2017

Purpose: Internal tandem duplication of fms-like tyrosine kinase 3 (FLT3-ITD) is a common (30%) molecular abnormality in acute myeloid leukemia (AML), associated with a high relapse rate and short disease-free survival. The oncogenic kinase Pim-1 is upregulated downstream of FLT3-ITD and contributes to its proliferative and anti-apoptotic effects. We recently showed that Pim kinase inhibition sensitizes AML cells with FLT3-ITD to apoptosis induction by topoisomerase 2 (TOP-2) inhibitors by increasing DNA double-strand breaks (DSBs) and oxidative stress. Here, we studied whether altered DSB repair activity contributes to the increase in DNA damage.

Methods: Ba/F3 cells expressing FLT3-ITD (Ba/F3-ITD) or wild-type FLT3 (Ba/F3-WT) were studied. The pan-Pim kinase inhibitor AZD1208 was provided by AstraZeneca. TOP-2 inhibitors included daunorubicin (DNR), mitoxantrone (MXR) and etoposide (VP-16). Nuclear expression of repair proteins was measured by immunoblotting. Functional changes in DSB repair activities were measured in cell lines with stable chromosomal integration of the green fluorescent protein (GFP)-based DSB repair reporters DR-GFP, EJ5-GFP and EJ2-GFP, measuring homologous recombination (HR), classical non-homologous end-joining (C-NHEJ) and alternative (Alt-) NHEJ, respectively.

Results: Nuclear expression of the HR component Rad51 decreased progressively (to 40%) in Ba/F3-ITD cells after 12 hours of co-treatment with AZD1208 and DNR, compared to DNR alone. Progressive decrease (to 60%) in nuclear expression of the Alt-NHEJ component DNA ligase 3 and the C-NHEJ component Ku-70 was also seen with combination treatment. Functionally, co-treatment with AZD1208 and DNR led to a 50% decrease in Alt-NHEJ repair activity, compared to DNR alone, but no significant change in C-NHEJ or HR repair activity. Ba/F3-WT cells did not show a significant change in Alt-NHEJ, HR or C-NHEJ activity with combination treatment. Decreased Alt-NHEJ activity was also seen in Ba/F3-ITD, but not Ba/F3-WT, cells co-treated with AZD1208 and MXR or VP-16.

Conclusions: While nuclear expression of multiple DNA DSB repair proteins decreased, only Alt-NHEJ repair activity decreased in FLT3-ITD cells co-treated with Pim kinase and TOP-2 inhibitors. Alt-NHEJ, a highly error-prone DNA DSB repair pathway, is upregulated in cells with FLT3-ITD and contributes to genomic instability in these cells. Decreasing Alt-NHEJ repair activity in cells treated with TOP-2 inhibitors has the potential to decrease the genomic instability that may contribute to disease progression in AML patients with FLT3-ITD.

Citation Format: Kshama A. Doshi, Pratik K. Nagaria, Adriana E. Tron, Feyruz V. Rassool, Maria R. Baer. Concurrent treatment with Pim kinase inhibitor decreases alternative non-homologous end-joining repair of DNA damage induced by topoisomerase 2 inhibitors in cells with FLT3-ITD [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4145. doi:10.1158/1538-7445.AM2017-4145