Development Of Tumor Models Resistant To The Novel Microtubule Destabilizer Bal27862 (Active Moiety Of The Prodrug Bali 01553)

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Background: The synthetic small molecule [BAL27862][1], active component of the prodrug BAL101553, destabilizes microtubules, potently inducing apoptosis in cancer cells. [BAL27862][1] elicits a broad antitumor activity and BAL101553/BAL27862 can be administered orally and intravenously. A unique microtubule phenotype suggests a novel mechanism of action (MoA), which is associated with activity in tumor models refractory to microtubule stabilizers (e.g. taxanes; epothilones) and destabilizers (e.g. Vinca-alkaloids such as vinblastine [VBL]). To further investigate the MoA and provide a platform for the identification of patient stratification biomarkers, [BAL27862][1]-resistant tumor models were developed. Methods: Drug-resistant tumor lines (Hela, H460, A549, SKOV3, Jurkat) were selected in vitro by incubation with increasing [BAL27862][1] concentrations. Proliferation was measured by crystal violet assay, apoptosis by flow cytometry. Tubulin polymerization was assessed by immunoblotting or electron microscopy (EM). Tubulin binding experiments used a spin column and [3H]-VBL and [14C]-BAL27862. Results: Selection for [BAL27862][1]-resistant tumor cells in vitro proved slow, requiring 8 – 12 months to culture lines with resistance factors (RF) ranging from 4.5 – 16. As an example, the EC50 for induction of apoptosis following [BAL27862][1] treatment in the parental (‘Par’) Jurkat line was 17 nM, whereas the resistant line (‘Res’) had an EC50 of 127 nM. Consistent with [BAL27862][1] not being a Pgp efflux pump substrate, Pgp up regulation could not account for resistance development in any line. Moreover, tubulin polymerization assays indicated a higher proportion of tubulin in the polymerized fraction in ‘Res’ lines, indicative rather of microtubule stabilization. Furthermore, the microtubule network in ‘Res’ lines was significantly less sensitive to the depolymerizing activity of [BAL27862][1] than in the ‘Par’ lines. The Jurkat ‘Res’ line was as sensitive as the ‘Par’ line to VBL treatment (EC50 for induction of apoptosis: 2 nM in both lines), whereas ‘Res’ H460 were equally resistant to [BAL27862][1] (RF: 5.5) and VBL (RF: 4.2). Both Jurkat and H460 ‘Res’ lines had a slightly increased sensitivity to paclitaxel treatment (2-3 fold). Drug cross-resistance in H460 could not be simply due to overlapping binding sites, as [BAL27862][1] and VBL bound independently to tubulin heterodimers. Furthermore, microtubule disassembly analysis by EM showed that [BAL27862][1] did not induce the small oligomeric structures associated with VBL treatment. Conclusion: Selection for [BAL27862][1] resistance in vitro requires prolonged culturing, is associated with more stable microtubules and may be mediated through diverse mechanisms. Results from work in progress to define the molecular changes associated with [BAL27862][1] resistance could facilitate patient stratification during clinical development of the prodrug BAL101553. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 743. doi:10.1158/1538-7445.AM2011-743 [1]: /lookup/external-ref?link_type=GENPEPT&access_num=BAL27862&atom=%2Fcanres%2F71%2F8_Supplement%2F743.atom