A Novel DNA Repair Chip Assay Allows Rapid Identification of DNA Repair Abnormalities Induced By BCR-ABL
Authors: Turhan Ali G , Sarrazy Fanny , Bonnet Marie Laure , Bertrand Angelina , Meunier Marie Claude , Aggoune Djamel , Chomel Jean-Claude , Feraud Olivier , Caillat Sylvain , Bennaceur-Griscelli Annelise , Sauvaigo Sylvie 
Published: October 2019

BCR-ABL expression in leukemic cells is associated with several types of DNA repair abnormalities, involving homologous recombination (HR), non-homologous end joining (NHEJ), nucleotide excision repair (NER), mismatch repair (MMR) and base-excision repair (BER) pathways. These defects, combined with the genetic instability inherent to chronic myeloid leukemia (CML), are thought lead to ABL-kinase domain mutations which remain poorly repaired and selected by tyrosine kinase inhibitor (TKI) therapies, leading to drug resistance. Currently, there is no functional, practical, single step assay to determine if BCR-ABL expression in a given leukemic cell is predominantly associated with a specific DNA repair pathway. For this purpose, we have used a novel DNA repair test based on biopchips evaluating the functional capacity of cells to achieve DNA repair through the use of a functional excision/synthesis repair assay that establish DNA Repair Enzyme Signatures. This assay allows monitoring the repair of a set of DNA lesions handled by BER (8oxoG, alkylated bases, glycols and Abasic sites) and NER (photoproducts, psoralen and cisplatin adducts). For modelling CML, we have used the UT7 cell line expressing BCR-ABL and T315I-mutated BCR-ABL. Parental (P)-UT7 cells were used as controls. We have tested the effects of BCR-ABL tyrosine kinase activity on the DNA repair assays using two strategies: The first was the evaluation of the effects of the nuclear extracts of UT7 cells expressing either native (N) BCR-ABL or T315I-mutated BCR-ABL before and after incubation of the cells in the presence of Imatinib (IM) or Nilotinib whereas the second tested the effects of BCR-ABL in an inducible system without the use of TKI. As expected, UT7 cells with N-BCR-ABL were sensitive to the inhibitory effects of these drugs whereas UT7 cells with T315I-mutated BCR-ABL were totally resistant. Using this system, we have found that the inhibition of BCR-ABL by IM stimulated the DNA repair capacity of the cells and specially BER (8oxoG, alkylated bases and glycols) and NER (photoproducts and psoralen adducts) after 4 or 8 hours of treatment, as compared to baseline repair capacities ( p< 0.05). No effect was detected for the repair of Abasic sites and Cisplatin adducts. When the cells were treated with Nilotinib in the same conditions, we have observed also the stimulation of DNA repair capacities for all lesions except for the Abasic sites ( p< 0.125). The fact of treating P-UT-7 cells or UT7-T315I cells with IM or Nilotinib had no effect and led conversely, to the inhibition of DNA repair activity via Abasic and Cisplat adducts pathways (p< 0.05) or Pso (p < 0.125). To confirm directly the effect of the BCR-ABL activity in the repair modifications observed, we have used UT7 cell lines expressing N-BCR-ABL or T315I-mutated BCR-ABL under the control of a Doxycycline-inducible promoter. In this TET-OFF system, BCR-ABL expression is inhibited upon Doxycycline addition to the culture and BCR-ABL protein becomes undetectable by Western blots at day 8. In this system, the analysis of DNA repair capacity demonstrated a statistically significant stimulation of repair of Alkylated bases, Glycols, Psoralen adducts and photoproducts ( p < 0.05) upon inhibition of N-BCR-ABL or that of T315I-mutated BCR-ABL. Interestingly, the fact of reducing BCR-ABL T315I in the DOX-inducible system stimulated DNA repair capacity whereas in the non-inducible UT7 cells expressing T315I, IM did not have a stimulatory effect, whereas Nilotinib induced a slight stimulation of both NER and BER pathways after 8 hours of treatment. Overall, these results demonstrate that the novel microarrays that we describe can be efficiently detect the DNA repair ability of the leukemic cells and the reversal of these abnormalities on TKI therapies. The preliminay results suggest clearly a differential effect of IM as compared to Nilotinib in terms of the stimulation of DNA repair capacity. This new methodology can now be used to quantify the abnormality of the DNA repair pathways in CML cells at diagnosis, allowing the monitoring of the tyrosine kinase inhibitor therapies and potentially predicting the occurrence of ABL-kinase mutations according to the preferential involvement of NER or BER pathways.

Disclosures

Turhan: Bristol Myers Squibb, Novartis: Honoraria, Research Funding. Sauvaigo:LXRepair: Employment, Equity Ownership.