UDP glucuronosyltransferase 1A expression levels determine the response of colorectal cancer cells to the heat shock protein 90 inhibitor ganetespib

HSP90 inhibition represents a promising route to cancer therapy, taking advantage of cancer cell-inherent proteotoxic stress. The HSP90-inhibitor ganetespib showed benefit in advanced clinical trials. This raises the need to identify the molecular determinants of treatment response. We tested the efficacy of ganetespib on a series of colorectal cancer (CRC)-derived cell lines and correlated their sensitivities with comprehensive gene expression analysis. Notably, the drug concentration required for 50% growth inhibition (IC₅₀) varied up to 70-fold (from 36 to 2500 nM) between different cell lines. Correlating cell line-specific IC₅₀s with the corresponding gene expression patterns revealed a strong association between ganetespib resistance (IC₅₀>500 nM) and high expression of the UDP glucuronosyltransferase 1A (UGT1A) gene cluster. Moreover, CRC tumor samples showed a comparable distribution of UGT1A expression levels. The members of the UGT1A gene family are known as drug-conjugating liver enzymes involved in drug excretion, but their function in tumor cells is hardly understood. Chemically unrelated HSP90 inhibitors, for example, 17-N-allylamino-17-demethoxygeldanamycin (17-AAG), did not show correlation of drug sensitivities with UGT1A levels, whereas the ganetespib-related compound NVP-AUY922 did. When the most ganetespib-resistant cell line, HT29, was treated with ganetespib, the levels of HSP90 clients were unaffected. However, HT29 cells became sensitized to the drug, and HSP90 client proteins were destabilized by ganetespib upon siRNA-mediated UGT1A knockdown. Conversely, the most ganetespib-sensitive cell lines HCT116 and SW480 became more tolerant toward ganetespib upon UGT1A overexpression. Mechanistically, ganetespib was rapidly glucuronidated and excreted in resistant but not in sensitive CRC lines. We conclude that CRC cell-expressed UGT1A inactivates ganetespib and other resorcinolic Hsp90 inhibitors by glucuronidation, which renders the drugs unable to inhibit Hsp90 and thereby abrogates their biological activity. UGT1A levels in tumor tissues may be a suitable predictive biomarker to stratify CRC patients for ganetespib treatment.Constant proteotoxic stress is a frequent occurrence in cancer cells and is derived from an adverse external microenvironment (hypoxia, acidosis) and internally from conformationally aberrant oncoproteins, high reactive oxygen species (ROS) levels, genomic instability, and stoichiometric imbalances in multi-protein machines. This stress condition raises the need for massive heat-shock chaperone support, especially from the heat-shock protein 90 (HSP90) system, to prevent protein aggregation and illicit interactions and promote tumor cell survival. Cancer-associated factors, such as mutant p53,^{1, 2} ErbB2,³ AKT,⁴ and macrophage migration inhibitory factor (MIF),^{5, 6} among others, represent HSP90 clients and require HSP90 for their stabilization in tumors. Hence, the multi-component HSP90 chaperone is highly upregulated and activated specifically in cancer cells as an adaptive response to malignancy.⁷HSP90 inhibitors have emerged as a highly promising class of anti-cancer compounds because of their ability to interfere with broadly active molecular networks, rather than a narrowly defined signaling pathway^{8, 9} and they enhance proteotoxic stress.¹⁰ Geldanamycin-based compounds represented the mainstay of HSP90 inhibition for the last 20 years.⁸ Clinically, however, these compounds proved to be of limited value due to their inherent liver and ocular toxicity coupled with only modest potency in vivo.¹¹ Major advances came with recently developed second-generation synthetic inhibitors such as the resorcinol containing compounds ganetespib (STA-9090)¹² and NVP-AUY922¹³ that are considerably more potent and less toxic. These compounds are currently being tested in phase II/III trials for their efficacy against various cancer types. Anaplastic lymphoma kinase (ALK)-driven NSCLC cancers showed particular clinical responsiveness to ganetespib.^{14, 15}Colorectal carcinoma (CRC) represents one of the most frequent malignancies worldwide, with a correspondingly high death toll. Moreover, the identification of predictive markers for patient stratification has proven to be difficult.¹⁶ HSP90 inhibition might be an attractive strategy for therapeutic improvement. However, this requires studies on how HSP90 inhibitors act on tumor cells in this cancer type.One approach to identify genes that determine cancer drug response was provided by highly parallel analysis of many cancer cell lines, specifically comparing their response with a large variety of small compounds with their gene expression patterns.^{17, 18} This strategy led to the identification of genes with expression patterns correlating with drug sensitivity. Notably, for many anticancer drugs this approach did not yield strong candidates, but in the case of the HSP90 inhibitor 17-N-allylamino-17-demethoxygeldanamycin (17-AAG) a strong correlation was seen between drug sensitivity and the expression of functional NAD(P)H dehydrogenase quinone 1 (NQO1), an enzyme that can metabolize and activate 17-AAG.^{19, 20} Hence, correlating drug sensitivity and gene expression patterns in cell lines can identify mechanisms that determine drug response.Drugs are subjected to metabolic turnover, and a major route of excretion from the body consists in conjugation with a hydrophilic sugar moiety within the liver parenchyma, followed by secretion into the bile. A major group of enzymes that carry out such conjugations are the UDP glucuronosyltransferases (UGTs).^{21, 22, 23} These enzymes are the products of gene clusters that cover various substrate specificities. UGT substrates include bilirubin, amines, and phenol structures.²⁴ The existence of such mechanisms for drug conjugation in the liver raises the question if and under what circumstances they can be found directly in tumor cells, and presumably cause drug resistance when highly expressed.Here, we show that human CRC-derived cell lines fall into ganetespib-sensitive and -resistant groups. While the majority of CRC lines were sensitive, two lines were highly resistant. Importantly, resistant cancer cells show a high expression of the UGT1A gene, and high levels of UGT1A were shown to be critical for ganetespib turnover, drug inactivation, and cell resistance. Thus, UDP glucuronosyl conjugation detoxifies ganetespib not only in the liver but also in a subset of CRC cells, representing a potential predictive biomarker for ganetespib response in CRC and possibly other tumor types.