An Inverse Switch in DNA Base Excision and Strand Break Repair Contributes to Melphalan Resistance in Multiple Myeloma Cells

Abstract

Alterations in checkpoint and DNA repair pathways may provide adaptive mechanisms contributing to acquired drug resistance. Here, we investigated the levels of proteins mediating DNA damage signaling and -repair in RPMI8226 multiple myeloma cells and its Melphalan-resistant derivative 8226-LR5. We observed markedly reduced steady-state levels of DNA glycosylases UNG2, NEIL1 and MPG in the resistant cells and cross-resistance to agents inducing their respective DNA base lesions. Conversely, repair of alkali-labile sites was apparently enhanced in the resistant cells, as substantiated by alkaline comet assay, autoribosylation of PARP-1, and increased sensitivity to PARP-1 inhibition by 4-AN or KU58684. Reduced base-excision and enhanced single-strand break repair would both contribute to the observed reduction in genomic alkali-labile sites, which could jeopardize productive processing of the more cytotoxic Melphalan-induced interstrand DNA crosslinks (ICLs). Furthermore, we found a marked upregulation of proteins in the non-homologous end-joining (NHEJ) pathway of double-strand break (DSB) repair, likely contributing to the observed increase in DSB repair kinetics in the resistant cells. Finally, we observed apparent upregulation of ATR-signaling and downregulation of ATM-signaling in the resistant cells. This was accompanied by markedly increased sensitivity towards Melphalan in the presence of ATR-, DNA-PK, or CHK1/2 inhibitors whereas no sensitizing effect was observed subsequent to ATM inhibition, suggesting that replication blocking lesions are primary triggers of the DNA damage response in the Melphalan resistant cells. In conclusion, Melphalan resistance is apparently contributed by modulation of the DNA damage response at multiple levels, including downregulation of specific repair pathways to avoid repair intermediates that could impair efficient processing of cytotoxic ICLs and ICL-induced DSBs. This study has revealed several novel candidate biomarkers for Melphalan sensitivity that will be included in targeted quantitation studies in larger patient cohorts to validate their value in prognosis as well as targets for replacement- or adjuvant therapies.

DNA repair of myeloma plasma cells correlates with clinical outcome: the effect of the nonhomologous end-joining inhibitor SCR7

“…We found that gH2AX foci reached maximal levels within 8 hours and decreased thereafter. In accordance with previous data showing that melphalan-induced gH2AX foci persist for a shorter time in melphalanresistant LR5 cells, 28 we found that RPMI 8226 cells showed BLOOD significantly lower gH2AX foci removal capacity and higher gH2AX foci burden than LR5 cells (Figure 2F-I; supplemental Table 1). All DSB/R inhibitors increased the accumulation of gH2AX in both cell lines (Figure 2G-I).…”

“…All DSB/R inhibitors increased the accumulation of gH2AX in both cell lines (Figure 2G-I). It is worth mentioning that, in line with the high levels of ligase IV observed in these cell lines, 28 we found that SCR7 showed the strongest effect. In harmony with gH2AX foci formation, Rad51 foci reached maximal levels within 8 hours and decreased thereafter, whereas LR5 cells showed lower levels of Rad51 foci compared with RPMI 8226 cells (Figure 2J-M; supplemental Table 1).…”

“…Although we did not address the mechanistic basis for the increased DSBs' repair efficiency of nonresponders' BMPCs, it is possible that it could be related to the upregulation of several factors involved in DSBs repair, such as ligase IV, XRCC4, RPA2, Artemis, and PARP-1 found in the melphalan-resistant LR5 cells in previous studies. 28 Moreover, individual DSB/R efficiencies were inversely correlated with apoptosis rates in the same cells, suggesting that the DSB/R capacity of BMPCs plays a crucial role in the successful outcome of chemotherapy. In addition, Sousa et al 28 have shown that LR5 cells were significantly more sensitive to PARP-1 inhibition than RPMI 8226 cells, and that the DNA-PK inhibitor markedly increased the melphalan sensitivity of the resistant LR5 cell line.…”

“…28 Moreover, individual DSB/R efficiencies were inversely correlated with apoptosis rates in the same cells, suggesting that the DSB/R capacity of BMPCs plays a crucial role in the successful outcome of chemotherapy. In addition, Sousa et al 28 have shown that LR5 cells were significantly more sensitive to PARP-1 inhibition than RPMI 8226 cells, and that the DNA-PK inhibitor markedly increased the melphalan sensitivity of the resistant LR5 cell line. Herein, we found that DSB/R inhibitors significantly increased the accumulation of DSBs and the melphalan sensitivity of BMPCs in all patients with MM.…”

“…Many inhibitors targeting DNA repair pathways (PARP1, DNA-PK, ATM, ATR, MGMT, APE) or cell-cycle checkpoints (CHK1, CHK2) have now been developed and might be useful to induce tumor cell apoptosis in combination with DNA damage-inducing drugs. [25][26][27][28][29][30] Herein, to further investigate the mechanistic basis for the link between DNA repair efficiency and response to antimyeloma therapy, we studied major DNA repair mechanisms in MM cell lines and malignant bone marrow plasma cells (BMPCs) from patients with MM before antimyeloma therapy. We found that BMPCs from responders to melphalan therapy are characterized by slower rates of NER and DSBs repair (DSB/R) compared with nonresponders.…”