Abstract B164: Novel natural withanolides inhibit Philadelphia-chromosome-positive acute lymphoblastic leukemia cells through downregulation of BCR-ABL and synergize with imatinib in vitro.
Authors: Buseman Jennifer L. , Timmermann Barbara N. , Cohen Mark S. 
Published: June 2012

Abstract
Introduction: BCR-ABL plays a key role in the pathogenesis of chronic myeloid leukemia (CML) and 30% of acute lymphoblastic leukemia (ALL). Imatinib mesylate is a tyrosine kinase inhibitor (TKI) specific for the abl tyrosine kinase. While imatinib results in high remission rates, Philadelphia-chromosome positive (Ph+) ALL responds poorly compared with CML, and relapse is common. While second generation TKIs can overcome certain mutations, others such as T315I render resistance to all available TKIs. Therefore, there is a critical need for novel pharmacologic therapies for Ph+ ALL. Heat Shock Protein 90 (HSP90) is a molecular chaperone that facilitates folding and promotes stability of client proteins, including BCR-ABL, and its expression is increased in leukemic cells. HSP90 inhibitors have been shown in solid tumors as well as in CML to be effective at inducing apoptosis and inhibiting growth of cells in vitro and tumor growth in vivo. Withanolides, including the steroidal-lactone Withaferin A (WA), that have been isolated from the Solanaceae plant family have been shown to downregulate HSP90 activity. Our hypothesis is that novel Solanaceae-derived withanolides will inhibit Ph+ ALL cell proliferation through down-regulation of BCR-ABL activity and may represent a novel therapeutic strategy for this difficult disease.
Methods: We investigated novel withanolides X001 and X003, isolated from the Physalis plant, as well as WA for anti-leukemic effects in Ph+ ALL cells. Cell proliferation was analyzed using MTS assay and Trypan Blue exclusion, and apoptosis and cell cycle modulation were examined using flow cytometry. Modulation of BCR-ABL and downstream effector proteins was analyzed using western blot. Syngery between withanolides and TKIs was evaluated using MTS viability to calculate combination indices.
Results: All Solanaceae-derived withanolides tested selectively inhibited growth and proliferation of Ph+ ALL cells in vitro after 72 hr treatment with IC50 values ranging from 35–350 nM. After 48 hr treatment with each withanolide, Ph+ ALL cells showed 55–65% apoptosis at 2–3× IC50 concentrations (p&lt;0.01 compared to controls) with shift of an additional 25–45% of the cell population into G2/M cell cycle arrest (p&lt;0.01 compared to controls) by flow cytometry. With 24 hr treatment of Ph+ ALL cells with withanolides at IC50 concentrations, western blot analysis demonstrated &gt;50% downregulation of phospho-BCR-ABL protein levels and confirmed apoptosis with caspase-3 activation and PARP cleavage. Finally, novel withanolides X001 and X003 showed strong synergy with imatinib mesylate at all concentration combinations tested, with combination indicies (CI) of .511 and .136, respectively, while WA only showed synergy at select few concentration combinations with CI &gt; 1.
Conclusion: Novel Solanaceae-derived withanolides X001 and X003 demonstrate potent anti-leukemic effects against Ph+ ALL cells in vitro through down-regulation of BCR-ABL activity with resulting induction of apoptosis and cell-cycle shift. These drugs have excellent synergy with imatinib and represent a novel therapeutic strategy for further translational applications to determine their clinical potential in patients with Ph+ ALL.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B164.