Dysregulated Nucleotide Excision Repair (NER) Is a New Target in Multiple Myeloma

Nucleotide excision repair (NER) is involved in the removal of bulky adducts and DNA crosslinks induced by various genotoxins including alkylating agents. In cancer, somatic mutations, genes’ up-regulation, and epigenetic silencing of NER may lead to abnormal DNA damage responses. Little is known about the role of NER in biology of multiple myeloma (MM), a heterogeneous disease characterized by genomic instability which is often treated by alkylating agents.

In our genome sequencing data we did not observe recurrent mutation in NER although non silent mutations affected 13 genes involved in NER in 20 out of 272 distinct patients. Using a functional assay based on the purified DNA-Damage Binding protein 2 (DDB2) proteo-probe complex that allows monitoring of photoproducts removal after UV irradiation, we observed NER variability in MM cell lines (MMCL) allowing to separate the cell lines in 2 groups according to their ability to repair : namely rapid (repair>90% after 2 hours) versus slow (repair < 85% after 2 hours).

The separation in two NER phenotypes did not correlate with P53 loss of function or any cytogenetic event with the exception of t(4;14) translocation which was associated with the group showing a repair rate > 90%. Interestingly, the LR5 melphalan resistant cell line harbored a rapid repair phenotype as compared to the parental RPMI-8226 cell line which exhibited a slow repair.

We evaluated melphalan sensitivity in relationship to NER activity in rapid and slow MMCL and observed that MMCL with slow NER were more sensitive to melphalan. We next evaluated whether inhibiting NER modified drug sensitivity. We utilized spironolactone which is known to inhibit NER. With the DDB2 proteo-probe assay, we confirmed the ability of spironolactone to inhibit NER in MMCL by degrading the Xeroderma Pigmentosum group B (XPB) protein. Combination of melphalan with NER inhibition in the 20 MMCL induced a significant increase in melphalan sensitivity in MMCL (increase from 15 to 79%).

We have also confirmed similar NER activity in CD138 positive patient plasma cells. We then developed a gene expression signature using rapid and slow NER cell lines. In a multivariate analysis, the rapid NER signature was significantly associated with low survival in the IFM 2005-01 trial dataset that included 170 patients treated with high-dose melphalan.

In conclusion, we have observed that NER affects MM cells and plays a significant role in drug sensitivity; we have also shown that NER can be targeted to increase melphalan sensitivity and potentially improve patient outcome.