Abstract 4507: Small molecule inhibitor of MDM2 induces p53-dependent HMGB1 secretion followed by apoptosis in cancer cells and incomplete senescence in normal fibroblasts

Abstract

Abstract An attractive therapeutic approach against cancers with wild-type (wt) p53 is the emergent class of small molecule MDM2 inhibitors, including the imidazoline nutlin-3 and more potent spiro-oxindole MI-63 (Ascenta Therapeutics). Both can rapidly increase intracellular p53wt levels and induce apoptosis or a senescence growth arrest, depending on the neoplastic status of the target cell. A critical question in the clinical development of such MDM2 inhibitors is their potential for normal tissue toxicity. In the absence of MDM2, p53 activation can cause fatal normal tissue pathology; and MDM2 inhibition can induce p53-dependent cell senescence in mouse fibroblasts. To assess the effects of MI-63 effects in human cells, we compared treatment (2.5-10 uM, 1-7 d) responses in p53wt malignant human mammary epithelial cells (MCF7) with those of p53wt normal human foreskin fibroblasts (HCA2). As expected, MI-63 had little effect on p53mut human cancer cells (MDA-231). However, against p53wt cancer cells and fibroblasts, MI-63 produced a dose-dependent increase in both nuclear p53 levels and reporter gene (p21) expression within 24 h. MCF7 cells responded with a dose- and time-dependent increase in apoptosis after 2 d exposure (vital dye uptake, cleaved PARP). By contrast, HCA2 cells responded with partially reversible growth arrest and a senescent-like phenotype (increased ROS, SOD2, and SA-βgal) within 3 days. A common early (24 h) MI-63 response by both the malignant and normal cells was nuclear loss of high-mobility group box-1 protein (HMGB1) associated with its active extracellular secretion. Acetylation of this damage-reporting cytokine, thought to be required for its secretion, was detected by western and mass spectrometry analyses. Although HMGB1 is generally pro-inflammatory and may be a harbinger of the senescence-associated secretory phenotype (SASP), MI-63 treated fibroblasts failed to exhibit induction of DNA damage foci or other features characteristic of SASP including IL-6 secretion. Surprisingly, MI-63 treatment of HCA2 beginning 24 or 48 h after a senescence-inducing dose of ionizing radiation (10 Gy) prevented enlarged cell morphology and produced a >80% reduction in SASP associated IL-6 secretion. These findings indicate that upregulation of p53wt in proliferating fibroblasts by MI-63, in the absence of DNA damage or other cell stresses, induces an incomplete senescence like growth arrest without a SASP response, consistent with our finding that p53 restrains SASP. In contrast, MI-63 upregulation of p53wt in malignant epithelial cells caused apoptotic cell death. The early release of HMGB1 from these dying cancer cells may modulate host innate immune response and/or recruit tumor infiltrating inflammatory cells. 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 4507. doi:10.1158/1538-7445.AM2011-4507