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.

大剂量美法仑（MEL）和自体干细胞移植（ASCT）是治疗多发性骨髓瘤（MM）的护理标准。对MEL的抗性与DNA修复的增加有关。在这里，我们试图确定聚（ADP-核糖）聚合酶（PARP）的抑制作用是否与MEL协同作用并可以克服耐药性。我们测试了RPMI8226和U266 MM细胞系中的MEL与MEL结合使用的三种PARP（PARPi）抑制剂（veliparib（VEL），olaparib（OLA）和niraparib（NIRA））与MEL的协同细胞毒性， RPMI8226-LR5（LR5）和U266-LR6（LR6）。在MEL中添加VEL，OLA和NIRA可将RPMI8226细胞中的半数最大抑制浓度（IC ₅₀）分别从27.8 µM降至23.1 µM，22.5 µM和18.0 µM。同样，IC ₅₀U266电池中的MEL含量分别从6.2 µM降低到3.2 µM，3.3 µM和3.0 µM。在LR5和LR6细胞中，PARPi不能逆转MEL抵抗力。我们在具有MEL敏感（RPMI8226）或MEL抵抗（LR5）MM的NOD / SCID /γ无效异种移植小鼠模型中证实了这一点。与单独的MEL相比，在RPMI8226小鼠中用MEL-VEL组合治疗可延长生存期（107天vs. 67.5天；P  = .0009），但在LR5小鼠中则不是（41 vs 39天；P  = .09）。接下来，我们测试了2个双链DNA修复机制（同源重组（HR）和非同源末端连接（NHEJ））是否引起LR5和LR6细胞的MEL抗性。在HR分析中，LR6细胞的HR能力比亲本U226细胞高4.5倍（P = .05）; 然而，LR5细胞具有与亲本RPMI8226细胞相同的HR能力。我们假设NHEJ可能是LR5细胞中MEL抗性的介体。鉴于DNA-PK是NHEJ不可或缺的并且可能是治疗靶点，我们用DNA-PK抑制剂NU7026与MEL结合治疗了LR5细胞。尽管单独使用浓度为2.5 µM的NU7026没有细胞毒性，但结合使用它可以完全逆转对MEL的耐药性（MEL IC _50为46.4 µM，而14.4 µM）。我们在414名接受串联ASCT治疗的患者的数据集中检查了我们的发现的临床意义。与低表达患者相比，高表达PARP1的患者生存率较低（中位数为42.7个月，而中位数未达到；P  = 0.003）。我们假设HR基因的联合表达BRCA1，NHEJ基因PRKDC（DNA-PK）和PARP1可以预测存活率，并发现0（n = 101），1或2（n = 287）或所有3（n = 26）基因的过表达都为阴性对中位生存期的影响（未定义vs. 57.8个月vs. 14.8个月；P <.0001）。在这里，我们证明PARPi与MEL协同作用，但是PARPi不能完全逆转耐药性（这可能是由于HR和NHEJ途径引起的）。此外，我们观察到可以测试3基因分析以鉴定对大剂量MEL耐药或敏感的患者。