PARP Inhibition Synergizes with Melphalan but Does not Reverse Resistance Completely

Abstract

High-dose melphalan (MEL) and autologous stem cell transplantation (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 poly(ADP-ribose) polymerase (PARP) synergizes with MEL and can overcome resistance. We tested the synergistic cytotoxicity of 3 inhibitors of PARP (PARPi)-veliparib (VEL), olaparib (OLA), and niraparib (NIRA)-combined with MEL in RPMI8226 and U266 MM cell lines, as well as in their MEL resistance counterparts, RPMI8226-LR5 (LR5) and U266-LR6 (LR6). The addition of VEL, OLA, and NIRA to MEL reduced the half maximal inhibitory concentration (IC₅₀) in RPMI8226 cells from 27.8 µM to 23.1 µM, 22.5 µM, and 18.0 µM, respectively. Similarly, the IC₅₀ of MEL in U266 cells was decreased from 6.2 µM to 3.2 µM, 3.3 µM, and 3.0 µM, respectively. 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 MEL-resistant (LR5) MM. Treatment with a MEL-VEL combination prolonged survival compared with MEL alone in RPMI8226 mice (107 days versus 67.5 days; P = .0009), but not in LR5 mice (41 versus 39 days; P = .09). We next tested whether 2 double-stranded DNA repair mechanisms, homologous recombination (HR) and nonhomologous end-joining (NHEJ), cause MEL resistance in LR5 and LR6 cells. In an HR assay, LR6 cells had a 4.5-fold greater HR capability than parent U226 cells (P = .05); however, LR5 cells had an equivalent HR ability as parent RPMI8226 cells. We hypothesized that NHEJ may be a mediator of MEL resistance in LR5 cells. Given that 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 2.5 µM had no cytotoxicity, in combination it completely reversed resistance to MEL (MEL IC₅₀, 46.4 µM versus 14.4 µM). We examined the clinical implications of our findings in a dataset of 414 patients treated with tandem ASCT. High PARP1 expressers had lower survival compared with patients with low expression (median 42.7 months versus median not reached; P = .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 or 2 (n = 287), or all 3 (n = 26) genes had a negative impact on median survival (undefined versus 57.8 months versus 14.8 months; P < .0001). Here we demonstrate that PARPi synergized with MEL, but that 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.

Keywords: Alkylator; DNA repair; Melphalan; Myeloma; PARP1.