PARP inhibition synergizes with melphalan but does not reverse resistance completely
High dose melphalan (MEL) and autologous stem cell transplant (ASCT) is the standard of care in the treatment of multiple myeloma (MM). Resistance to MEL has been linked to increased DNA repair. Here we sought to identify whether inhibition of PARP1 (PARPi) synergizes with MEL and can overcome resistance. We tested the synergistic cytotoxicity of PARPi with veliparib (VEL), olaparib (OLA) or niraparib (NIRA) combined with MEL in RPMI8226 and U266 MM cell lines as well as their MEL resistance counterparts, RPMI8226-LR5 (LR5) and U266-LR6 (LR6). The addition of VEL, OLA and NIRA to MEL reduced the IC50 in RPMI8226 cells from 27.8 to 23.1, 22.5 and 18.0µM respectively. Similarly, MEL IC50 decreased in U266 cells from 6.2 to 3.2, 3.3 and 3.0 µM. In LR5 and LR6 cells, PARPi did not reverse MEL resistance. We confirmed this in a NOD/SCID/gamma null xenograft mouse model with either MEL sensitive (RPMI8226) or resistant (LR5) MM. Treatment with a MEL-VEL combination prolonged survival in RPMI8226 mice compared to MEL alone (107 vs. 67.5 days, p=0.0009), but not in LR5 mice (41 vs. 39 days, p=0.09). Next we tested whether the double strand DNA repair mechanisms, homologous recombination (HR) and non-homologous end joining (NHEJ), cause MEL resistance in LR5 and LR6 cells. Using an HR assay, LR6 cells had 4.5 fold higher HR ability than parent U226 cells (p=0.05). However, LR5 cells had equivalent HR ability to parent RPMI8226 cells. We hypothesized that NHEJ may be a mediator of MEL resistance in LR5 cells. As DNA-PK is integral to NHEJ and may be a therapeutic target, we treated LR5 cells with the DNA-PK inhibitor NU7026 in combination with MEL. Although NU7026 alone at 2.5µM had no cytotoxicity, in combination it completely reversed resistance to MEL (MEL IC50 46.4 vs. 14.4µM). We examined the clinical implications of our findings in a dataset of 414 patients treated with tandem ASCT. High PARP1 expressers had a reduced survival compared to patients with low expression (42.7 months vs. median not reached, p=0.003). We hypothesized that combined expression of the HR gene BRCA1, the NHEJ gene PRKDC (DNA-PK), and PARP1 may predict survival and found that overexpression of 0 (n=101), 1-2 (n=287), or all 3 genes (n=26) had a negative impact on median survival (undefined vs. 57.8 vs. 14.8 months, p<0.0001). Here we demonstrated PARPi synergized with MEL, but resistance (which may be due to HR and NHEJ pathways) is not completely reversed by PARPi. In addition, we observed that a 3 gene analysis may be tested to identify patients resistant or sensitive to high dose MEL.