The main goal of the PPTP is to prioritize drugs being developed predominantly for adult cancer treatment for expedited clinical trials in children with relapsed/refractory cancers. MLN8237, which has 200-fold specificity for Aurora kinase A inhibition versus Aurora kinase B [16
], showed high-level activity at its MTD in its initial PPTP evaluation; therefore, it was critical to validate and extend these previous results. This was done by evaluating MLN8237 against an extensive number of Ewing sarcoma and neuroblastoma cancer lines in vitro, and by assessing its activity in vivo against neuroblastoma and ALL xenografts across a range of doses with pharmacokinetic and pharmacodynamic correlation.
Aurora kinase inhibitors have to date shown only modest clinical activity against solid tumors in adults, although more pronounced activity has been reported in leukemia patients [14
]. There are limited data available to support Aurora kinase A as a relevant molecular target in pediatric cancers besides the report by Shang et al. [38
] and the PPTP’s previous report of MLN8237 Stage 1 testing [23
]. In this latter publication, high levels of activity were obtained against several solid tumor models and against ALL xenografts of both T and B lineage. The most intriguing set of results was that MLN8237 performed more impressively than other investigational drugs, and even established drugs, against the neuroblastoma panel as a single agent at its MTD.
The Aurora kinases play critical roles in cell division, and alteration of their expression and function has been associated with oncogenesis. Knockdown of Aurora kinase A using RNA interference results in mitotic spindle defects, mitotic delay, and apoptosis in human cells [39
], while overexpression leads to transformation of normal cells [40
]. Also, Aurora kinase A is amplified or overexpressed in some adult cancers [41
], which supports its potential exploitation as a cancer therapeutic target [14
]. Similarly, the overexpression of Aurora kinase A has been postulated as predictive of susceptibility to inhibition of the specific kinase activity. Thus, Ewing sarcomas, with genetic alterations that enhance Aurora kinase A expression [37
], should have higher sensitivity than the lower expressing neuroblastoma or ALL panels. The results presented in this study confirm our previous results of high-level activity for MLN8237 against neuroblastoma and ALL xenografts, which express markedly lower Aurora kinase A levels compared to other PPTP xenografts [23
], thereby calling into question the premise that overexpression of Aurora kinase A is associated with more effective cell kill upon kinase inhibition. Although the Ewing sarcoma xenografts had slightly increased expression of AURKA compared to the median for all xenografts, our study did not confirm enhances in sensitivity to MLN8237 in vitro or in vivo. Indeed, the gene copy number analysis for AURKA appears to support an inverse relationship between Aurora kinase A expression and sensitivity. Increased copy number was present in half of the rhabdomyosarcomas and in 14 of the solid tumors. Loss of copy number was detected in 7 solid tumors and ALL-17. Further, the correlation between gene expression variation and copy number variation was strong, placing this locus in the top 1.6% of all genes tested. Although there is no absolute relationship between copy number variation and tumor sensitivity, of the 14 solid tumors with increased copy number, there were only two that showed sensitivity to MLN8237 (1 PR, 1 SD). In contrast, of the eight models demonstrating decreased copy number, there were five sensitive models (3 MCR, 1 CR, and 1 SD).
The in vitro activity of MLN8237 against the Ewing sarcoma and neuroblastoma extended panels (n
= 11 and n
= 17, respectively) is consistent with the PPTP’s Stage 1 results for MLN8237, which showed median relative and absolute IC50
values against all of the cell lines in the PPTP in vitro panel of 49 and 61 nM, respectively [23
]. The larger number of Ewing and neuroblastoma cell lines described in this report compared to those studied in Stage 1 testing allowed detection of significantly lower IC50
values for the neuroblastoma cell lines compared to the Ewing sarcoma cell lines. Further, one Ewing sarcoma cell line (CHLA-56) was resistant to MLN8237 (IC50
> 10 μM). The identification of this highly resistant cell line places it as a valuable tool for identifying resistance mechanisms and warrants further investigation. Recently, a functional Aurora kinase A mutation (T217D) that renders the kinase impervious to MLN8054 and MLN8237 inhibition has been reported [44
] and points to a mechanism of resistance independent from levels of expression.
The efficacy of MLN8237 treatment in vivo at its MTD was confirmed against the xenograft panel included in this report. Out of 10 xenografts also evaluated in the previous report, only one (OS-1) was scored more than one response category apart from its previous score [23
]. We have confirmed the high level of activity of MLN8237 against xenograft models of neuroblastoma and ALL, when administered as a single agent at its MTD. This further demonstrates the potential relevance of Aurora kinase A inhibition for neuroblastoma cancer treatment. However, the efficacy of MLN8237 (as indicated by the Median Group Response) was reduced or lost for most of the solid tumor models with dose reduction (Fig. ). Thus, at 0.5MTD, only two xenografts exhibited an objective response, and at 0.25MTD, only one xenograft was classified as PR. In contrast, the dose–response relationship for the ALL xenografts was not as steep, with all three models exhibiting objective responses at 0.5MTD and only one not reaching an objective response upon further reduction to 0.25MTD.
Data for the pharmacokinetics of MLN8237 in patients have recently been presented [45
]. In patients receiving 50 mg BID, the Cmax
and AUC0–24 h
were 1.3 and 40 μM h, respectively. At the recommended phase 2 dose of 50 mg BID for 7 days, average trough concentrations exceeded 1 μM, the efficacious concentration estimated in previous preclinical work. In mice receiving MLN8237 at 10 mg/kg, the Cmax
and AUC0–24 h
were 16 and 39μM h, respectively, with the 12 h level being 1.2 μM. Thus, results presented here suggest that drug exposures achievable in patients may induce responses in only the most sensitive of tumors and that dose intensity and scheduling may be critical as a minority of the solid tumor models exhibited objective responses at this level of drug exposure.
When comparing the plasma exposure of MLN8237 to the pharmacodynamic response, the peak of pharmacodynamic activity (~8–12 h) was delayed relative to the peak plasma exposure (0.5 h). This is consistent with previous observations using the Aurora kinase A inhibitor MLN8054 in a colon tumor xenograft [16
] and is likely due to the time it takes for a sufficient number of cells to transit the cell cycle and accumulate in mitosis subsequent to Aurora kinase A inhibition as well as to the time during which MLN8237 drug levels are above a threshold level required for Aurora kinase A inhibition. The comparable mitotic indices estimated using MPM2 and pHistH3 as mitotic markers are consistent with specific inhibition of Aurora kinase A by MLN8237 in vivo, as histone H3 is phosphorylated by Aurora kinase B [46
A likely critical step in the development of MLN8237 for use in the treatment of pediatric cancers is the development of effective drug combinations. The limited activity observed at reduced doses of MLN8237 as a single agent against most solid tumor xenografts may be overcome if synergistic interactions with other drugs can be identified. Combinations of MLN8237 with established drugs against in vivo models of pediatric solid tumors and ALL are under evaluation by the PPTP.
The cumulative evidence of anti-tumor activity observed in preclinical testing together with the results presented here provides strong rationale for expeditious evaluation of MLN8237 in the childhood cancer setting. A pediatric phase 1/2 trial was opened in the Children’s Oncology Group Phase 1 Consortium during 2008. As results from that clinical trial emerge, it will be crucial to correlate the observed anti-tumor activities with pharmacokinetic measurements to assess whether drug levels are in the range associated with substantial preclinical activity.