Inhibition of Hsp90 via 17-DMAG induces apoptosis in a p53-dependent manner to prevent medulloblastoma

Elevated expression of HSP90 is observed in many tumor types and is associated with a limited clinical response. Targeting HSP90 using inhibitors such as 17-DMAG (17-desmethoxy-17-N,N-dimethylaminoethylaminogeldanamycin) has shown limited therapeutic success. HSP90 regulates the function of several proteins implicated in tumorigenesis although the precise mechanism through which 17-DMAG regulates tumor cell survival remains unclear. We observed a requirement for p53 in mediating 17-DMAG-induced cell death. The sensitivity of primary mouse embryonic fibroblasts and tumor cells to 17-DMAG-induced apoptosis depended on the p53 status. Wild-type MEFs underwent 17-DMAG-induced caspase-dependent cell death, whilst those lacking p53 failed to do so. Interestingly p53-dependent cell death occurred independently of Atm or Arf. Primary tumor cells derived from two models of murine medulloblastoma (Ptch1^+/−;Ink4c^−/− and p53^FL/FL;Nestin-Cre⁺; Ink4c^−/−) that retain and lack p53 function, respectively, displayed a dependence on functional p53 to engage 17-DMAG-induced apoptosis. Strikingly, 17-DMAG treatment in an allograft model of Ptch1^+/−;Ink4c^−/− but not p53^FL/FL;Nestin-Cre⁺; Ink4c^−/− tumor cells prevented tumor growth in vivo. Our data suggest that p53 status is a likely predictor of the sensitivity of tumors to 17-DMAG.Acquisition of inactivating p53 mutations or aberrant expression of signaling molecules that engage p53 are extremely common in tumors and can render them refractive to conventional therapies (1). Because the tumor suppressor activity of p53 is mediated largely by its ability to engage apoptosis, its inactivation provides tolerance to the tumor microenvironment (1) and is analogous to the survival promoting effects of heat shock proteins (HSPs) that, in response to stresses including hypoxia and nutrient deprivation and in collaboration with co-chaperone proteins, regulate the refolding and repair of damaged proteins (2). By doing so, they preserve protein function and maintain cellular survival in part by preventing apoptosis (3). The expression of several HSPs, including HSP90 is increased in tumors (4), suggesting that elevated HSP expression may contribute to aberrant tumor survival. New clinical strategies aim to exploit this weakness by targeting components of the stress pathway (5).HSP90 displays the unique ability to selectively associate with signaling molecules implicated in the aberrant survival of tumor cells (4). These include mutant (6, 7) and wild-type (wt) p53 (8), Raf-1 (9), and Akt (10). HSP90 is ubiquitously expressed in both normal and malignant tissues, but its altered ‘high-affinity’ conformation in tumor cells confers 100-fold selectivity for HSP90 inhibitors (11). Consequently, several HSP90 inhibitors derived from the ansamycin antibiotic geldanamycin (GA) are in clinical trials for the treatment of cancer (5, 12, 13). Ansamycin compounds bind tightly to the ATP-binding pocket of HSP90 to prevent its stable interaction with substrates and to target them for proteasomal degradation (14, 15). HSP90 inhibitors have shown promising but limited signs of clinical activity (5, 12, 13). It therefore remains important to understand how 17-DMAG acts as an effective anti-tumor agent and if its efficacy is likely to be challenged by features of tumor cells that confer resistance to conventional therapies.Elevated expression of HSP90 in human medulloblastoma (16) suggests that it may represent a candidate for therapeutic intervention in this disease. Here, we sought to preclinically evaluate if the HSP90 inhibitor, 17-DMAG, affects the growth of medulloblastoma, a form of pediatric cancer arising in the cerebellum that develops largely after birth due to the failure of granule neuron precursors (GNPs) to exit the cell cycle and differentiate (17). This aberrant process has been linked to human medulloblastomas involving TP53 inactivation (10% of human cases), defective Sonic Hedgehog/PATCHED (SHH/PTCH) signaling (27% of human cases), lesions in the WNT signaling pathway (15% of human cases), as well as the persistent expression of pro-proliferative genes (18).Several murine models for medulloblastoma that recapitulate causative genetic lesions identified in human medulloblastoma (19) are characterized by activation of the Shh/Ptch signaling pathway, two of which were used in our studies (20). The first (denoted throughout as Ptch1^+/−;Ink4c^−/−) was generated through a germline deletion of one copy of the Patched gene (Ptch1), the receptor for Shh (21), which, when combined with the deletion of Ink4c (22), induce medulloblastomas with an approximate 60% incidence (23). Importantly, all tumors retain functional p53 (20, 23) but lose expression of the wt Ptch1 allele (20). The second model (denoted throughout as p53^FL/FL;Ink4c^−/−) is generated by the conditional deletion of floxed p53 in the cerebellum using Cre recombinase under the control of the Nestin promoter (24) which, when combined with germ line deletion of Ink4c and irradiation of postnatal day 7 mice displays complete penetrance of medulloblastomas (20). Importantly, tumors arising in p53-deficient mice are characterized by the homozygous deletion of Ptch1 (20). Therefore, both the Ptch1^+/−;Ink4c^−/− and p53^FL/FL;Ink4c^−/− models of medulloblastoma are characterized by the constitutive activation of Shh/Ptch signaling, regardless of their founding mutations, with the only difference being the presence or absence of functional p53.Using in vitro assays in primary wt and p53-null mouse embryonic fibroblasts (MEFs) and purified GNP-like tumor cells, we show that 17-DMAG induced apoptosis in a p53- and caspase-dependent manner that required Puma or Bax/Bak, but was independent of p19^Arf and Atm signaling. Transfer of tumor cells derived from each of the murine models into immunocompromised recipients demonstrated that 17-DMAG efficiently prevented medulloblastoma tumor formation and growth in vivo but only when p53 was functional.Our studies establish a relationship between Hsp90 and p53 activity in vivo and provide evidence that the Hsp90 inhibitor, 17-DMAG requires an intact p53 response to exert its anti-tumorigenic effect. Although the relevance of these findings in a clinical setting remains to be examined, they predict that HSP90 inhibitors may be an effective treatment option for human medulloblastoma, a tumor type in which a significant percentage of tumors retain functional p53.