D lately that the abnormal DSB repair in BCR-ABL1-positive CML was due to reduced activity of DNA PK-dependent NHEJ and improved activity of ALT NHEJ (29). Furthermore, “knockdown” of DNA ligase III, a participant in ALT NHEJ, resulted in increased accumulation of unrepaired DSBs and decreased survival, suggesting that ALT NHEJ pathway components, such as PARP1 and DNA ligase III (295) might be novel therapeutic targets in cancer cells which might be additional dependent on ALT NHEJ for DSB repair. The recent development of PARP inhibitors, which selectively target the DSB repair defect in hereditary breast cancers (36, 37), has stimulated interest inside the use of DNA repair inhibitors as cancer therapeutics. Because DNA ligation would be the final step of nearly all DNA repair pathways, we applied a structure-based drug design method to identify little molecule inhibitors with PRMT4 Inhibitor MedChemExpress different specificities for the three human DNA ligases (38, 39). As expected, a subset of those inhibitors potentiated the cytotoxicity of DNA-damaging agents, but, interestingly, this effect was a lot more pronounced in cancer cells (38, 39). Considering that BCR-ABL1positive CML cells have abnormal DSB repair (29), we’ve examined the impact of PARP1 inhibitors on TKI-sensitive and -resistant CML cells in the presence or absence of a DNA ligase inhibitor. Our outcomes give proof that targeting ALT NHEJ using a combination of DNA ligase and PARP inhibitors is a potentially novel therapeutic approach for CML sufferers who fail TKI therapy.NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptOncogene. Author manuscript; accessible in PMC 2013 August 26.Tobin et al.PageResultsGeneration and characterization of IMR BCR-ABL1-positive cell linesNIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptIMR derivatives in the CML IM sensitive (IMS) cell line K562, along with the hematopoietic cell lines, Mo7e-P210 and Baf3-P210 that had been engineered to stably express BCR-ABL1 (Figure S1A and Table S1), have been chosen by growth in IM-containing media. The IMR cell lines, Mo7e-P210 IMR2 and Baf3-P210 IMR, had acquired mutations within BCRABL1 resulting in D276G and T315I amino acid modifications, respectively. Notably, these amino acid changes happen to be observed in IMR CML patients (Table S1, 6, 9). Whilst BCRABL1 was neither overexpressed nor mutated within the K562 IMR and Mo7e-P210 IMR1 cell lines, the Mo7e-P210 IMR1 cells had increased RAS activation and phosphorylation of AKT PKCĪ² Modulator Species compared to Mo7e-P210 (Figure S1D ), suggesting that activation of parallel signaling pathways could contribute to the IMR of these cells(40). Importantly, our IMR cell lines recapitulate different mechanisms of resistance to TKIs which have been described in IMR CML patients (six, 7, 9). Altered expression of DNA repair proteins in IMS and IMR BCR-ABL1-positive cell lines Due to the fact we had shown previously that the steady-state levels in the ALT NHEJ protein, DNA ligase III were greater in K562 leukemia cells compared with B cell lines established from regular individuals (29), we examined the steady state protein levels of important DNAPKdependent and ALT NHEJ proteins in other cell lines expressing BCR-ABL1. Along with DNA ligase III, the steady-state levels of an additional ALT NHEJ protein, PARP1 (295), was also elevated in K562 in comparison to NC10 cells (p0.05, Figure 1A ). The NC10 cells are not genetically related to K562 cells so the alterations within the steady state levels of DNA ligase III and PARP1 may be due to intrinsic.
