Evaluation of the novel mitotic modulator ON 01910.Na in pancreatic cancer and preclinical development of an ex vivo predictive assay

Abstract

The pupose of this study was to evaluate the activity of ON 01910.Na, a mitotic inhibitor, in in vitro and in vivo models of pancreatic cancer and to discover biomarkers predictive of efficacy. Successive in vitro and in vivo models were used; these included cell line-derived and patient-derived tumors from our PancXenoBank, a live collection of freshly generated pancreatic cancer xenografts. ON 01910.Na showed equivalent activity to gemcitabine against pancreatic cancer cell lines in vitro. The activity of the agent correlated with suppression of phospho-CDC25C and cyclin B1. These markers were optimized for a fine-needle aspirate ex vivo rapid assay. Cyclin B1 mRNA evaluation yielded the most optimal combination of accuracy and reproducibility. Next, nine patient-derived tumors from the PancXenoBank were profiled using the assay developed in cell lines and treated with ON01910.Na for 28 days. Two cases were cataloged as potential responders and seven as resistants. There was a correlation between the ex vivo assay and sensitivity to the tested agent, as the two cases prospectively identified as sensitive met prespecified criteria for response. Of the seven tumors of predictive resistant, only one was found to be sensitive to ON 01910.Na. In addition, there was a good correlation between cyclin B1 downregulation ex vivo and changes in cyclin B1 protein post-treatment. The novel mitotic inhibitor, ON 01910.Na, showed activity in preclinical model of pancreatic cancer. A rapid assay was rationally developed that not only identified cases sensitive to ON 01910.Na, but also anticipated the pharmacodynamic events occurring after in vivo exposure.

Figure 1

(a) Comparison of the average growth inhibition of the 12 cell lines to ON 01910.Na and gemcitabine, where they showed equivalent potency. Error bars represent s.d. (experiments conducted in triplicate three times). (b) In vivo tumor growth inhibition of the sensitive (HS766T) and resistant (MiaPaCa2) cell lines after being xenografted on nude mice. HS766T tumors treated with ON 01910.Na intraperitoneally (■) decreased in size by 43% compared with controls (♦; P=0.025 by t-test means comparison). Mice lived 8.4 days more than untreated controls (P=0.05 by log-rank). In addition, ON 01910.Na-treated tumors tended to ulcerate less and later in time. In MiaPaCa2 tumors, no biologic activity was observed in terms of differences in tumor growth or survival. Error bars represent s.e.(n=10 tumors per group); asterisk indicates P<0.05 (Student's t-test) compared with control.

Figure 2

Pharmacodynamic marker evaluation in two sensitive (Panc 1 and HS766T) and two resistant (MiaPaca2 and L36PL) cell lines after ON-01910 treatment. In both sensitive strains, pCDC25C decreases after treatment, whereas in the resistant strains, it increases. Also, cyclin B1, both by western blot and RT–PCR, increases in a dose-dependent manner upon exposure to ON 01910.Na in the resistant strains.

Figure 3

(a) The ex vivo approach. A small sample of tissue is acquired either from a patient's tumor or from a tumor xenografted on mice, and is plated and exposed to the drug for a short period of time. The cells are then collected and analysed by the assay that is specifically coupled to that drug, and can be mRNA expression by reverse transcriptase PCR (RT–PCR), and/or protein assessment by western blot or immunohistochemistry (IHC). (b) Western blot analysis of pCDC25C in vitro, ex vivo and in vivo. Upon treatment with ON 01910.Na in the sensitive HS766t, there is a decrease, whereas in the resistant MiaPaCa2, there is a clear increase in the activation status of CDC25C, both in vitro and in vivo. However, the ex vivo analysis rendered largely inconclusive results, partly because of the high detection threshold for the tested end point. (c) Western blot and RT–PCR analysis of cyclin B1 in vitro, ex vivo and in vivo. Upon treatment with ON 01910.Na in the sensitive HS766T, there is a mild decrease, whereas in the resistant MiaPaCa2, there is a clear increase in cyclin B1 levels, both at the protein and mRNA levels. The ex vivo assay faithfully correlated with the pharmacodynamic events that occurred in the xenografted tumors (in vivo). (d) Four tumors per treatment group were examined by IHC for cyclin B1 expression, confirming the western blot analyses. In HS766T, there was a mild downregulation of cyclin B1, whereas in all ON 01910.Na-treated MiaPaCa2 tumors, there was an upregulation in cyclin B1.

Figure 4

(a) Cyclin B1 evaluation after gemcitabine in highly sensitive (Panc1 and HS766T) and resistant (MiaPaca2 and L36PL) cell lines after ON 01910.Na treatment. No change in cyclin B1 is observed after gemcitabine treatment in sensitive or resistant cells. (b and c) Small interference RNA (siRNA) of cyclin B1 and Plk1 decreased both mRNA and protein levels in Hs766T and MiaPaca2. Downregulation of polo-like kinase 1 (Plk1) induced increases in cyclin B1 mRNA also in both cell lines, but the effects at the protein level were more evident in HS766T. After assessing the effects of siRNA, cyclin B1 and Plk1 in conjunction with treatment with gemcitabine and ON 01910.Na, it can be concluded that cyclin B1 had no role in determining sensitivity status, nor did have an effect of its own. However, Plk1 downregulation had an effect in HS766T, but not in MiaPaca2, confirming that in the former cell line, cell growth is Plk1-dependent. There was also some additive effect when Plk1 siRNA and ON 01910.Na were given together. Error bars represent s.d.; asterisk indicates Po0.05 (Student's t-test) compared with control.

Figure 5

Evaluation of the ex vivo assay in the pancreatic cancer xenograft bank. (a) Western blot and reverse transcriptase PCR (RT–PCR) analysis of cyclin B1 ex vivo in four of the nine selected cases that were prospectively assessed. The ex vivo assay faithfully correlated with the efficacy of the drug after it was administered to the xenografted tumors. Below, growth curves of two selected examples. (b) Plot of the nuclear cyclin B1-staining index (calculated as intensity of staining (0–3) multiplied by the percentage cells staining positive). (c) Correlation between the relative tumor growth (in T/C) and the cyclin B1 protein expression by immunohistochemistry (IHC; in % normalized to control). There was a significant direct correlation between tumor growth inhibition and changes in cyclin B1; cases with higher tumor inhibition after 28 days of ON 01910.Na showed decreases in nuclear cyclin B1. Error bars represent s.e.; asterisk indicates P<0.05 (Student's t-test) compared with control.