Pan-Aurora Kinases and TRAIL Combination Therapy Has Potent Antitumor Activity In Vivo Against Drug-Resistant Human Multiple Myeloma Xenograft Model

Abstract 1846

Although novel drugs such as Bortezomib and Thalidomide have extended the overall survival of multiple myeloma patients they often do not achieve lasting cures, providing an impetus to search for novel therapeutic modalities. Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL, also known as Apo2L) is a member of the TNF family of death receptor ligands and has significant potential for use in cancer therapy because of its potent ability to selectively kill cancer cells while sparing normal cells. Preclinical studies have demonstrated that combinations with cytotoxic chemotherapy or other targeted agents may enhance the antitumor activity of TRAIL through cross-talk between the intrinsic and extrinsic pathways. We here demonstrate that targeting Aurora A and B kinases with selective pan-Aurora kinases inhibitors (AKIs) (VX-680, Vertex/Merck; or PHA-680632, Nerviano Medical Sciences, Pfizer) strikingly enhances TRAIL-induced cytotoxicity in TRAIL-sensitive and -resistant human myeloma cell lines (HMCLs) via potentiation of both the death receptor-mediated and mitochondrial apoptotic pathways.

In particular, we found that co-treatment with pan-AKIs elevated (DR4+DR5)/(DcR1+DcR2) ratio, caspase-8 activation, Bid fragmentation, mitochondrial depolarization and caspase-9 activation of TRAIL-treated HMCLs. Furthermore we found that the combination of pan-AKIs with TRAIL enhanced the levels of the pro-apoptotic protein Bak and reduced the basal and/or TRAIL-induced expression of the anti-apoptotic Bfl-1/A1 and Mcl-1 at the mitochondrial level; notably, loss of either Mcl-1 or Bfl-1/A1 expression greatly increased TRAIL sensitivity in both sensitive and resistant HMCLs analyzed.

To assess the in vivo efficacy of combining TRAIL and Pan-AKIs, we tested these compounds using mouse tumor human plasmacytoma xenograft model in which the drug-resistant RPMI 8226/R5 cells (1.0×10⁷ cells per mouse) were injected subcutaneously into NOD-SCID mice.

When the tumors reached approximately 250 mm³ mice bearing RPMI 8226/R5 tumors were randomized (n=12/group) to receive vehicle or MK-0457 or PHA-680632 at 50mg/kg or TRAIL (300μg/per mouse) or MK-0457/TRAIL or PHA-680632/TRAIL. Both Pan-AKIs and TRAIL were administered by intraperitoneal injection. Mice were treated with daily doses of Pan-Aurora inhibitors for 9 days, and two doses of TRAIL. Treatment of RPMI 8226/R5 MM-tumor-bearing mice with MK-0457 or PHA-680632 significantly reduced MM-tumor growth as compared to control (P <.01, Tukey-Kramer test), TRAIL had minimal effect on the growth of tumors, which increased as in control mice. Importantly, when Pan-Aurora inhibitors were combined with TRAIL, there was a significant (P<.001 Dunnet test) reduction in tumor growth relative to either treatment alone.

Furthermore, the combination pan-AKIs/TRAIL significantly (P<.001; Kaplan-Meier method and compared using the log-rank test, followed by a Bonferroni correction for multiple comparisons) prolonged survival compared with treatment with either drug alone and was well tolerated in vivo. We next investigated the in vivo effects of the drug combination on proliferation and apoptosis; whole tumor-cell tissues and tumor lysates from mice treated for six days (n=3/group) were subjected to immunohistochemical staining and immunoblotting to assess in vivo phosphorylation histone H3 on Ser10 (phospho-H3), a direct downstream target of the Aurora Kinases, cleaved caspase-3 and PARP fragmentation. Tumor tissues from pan-AKIs treatments resulted in profound phospho-Histone H3 inhibition compared with tumor tissues from vehicle control or TRAIL-treated animals thereby confirming the tumors growth retardation observed in pan-AKIs-treated mice. Either pan-AKIs or TRAIL alone slightly increased caspase-3 cleavage/activation and PARP fragmentation compared with tumors from control cohorts. However, the combination pan-AKIs/TRAIL dramatically increased caspase-3 cleavage/activation, PARP degradation in tumors. In conclusion, our preclinical in vitro and in vivo studies provide the framework for testing pan-AKIs and TRAIL combination therapy in clinical trials aimed to improve patient outcome in MM.