Ronald K. Blackman

Complementary whole-genome technologies reveal the cellular response to proteasome inhibition by PS-341

Although the biochemical targets of most drugs are known, the biological consequences of their actions are typically less well understood. In this study, we have used two whole-genome technologies in Saccharomyces cerevisiae to determine the cellular impact of the proteasome inhibitor PS-341. By combining population genomics, the screening of a comprehensive panel of bar-coded mutant strains, and transcript profiling, we have identified the genes and pathways most affected by proteasome inhibition. Many of these function in regulated protein degradation or a subset of mitotic activities. In addition, we identified Rpn4p as the transcription factor most responsible for the cells ability to compensate for proteasome inhibition. Used together, these complementary technologies provide a general and powerful means to elucidate the cellular ramifications of drug treatment.

Ganetespib, a unique triazolone-containing Hsp90 inhibitor, exhibits potent antitumor activity and a superior safety profile for cancer therapy

Targeted inhibition of the molecular chaperone Hsp90 results in the simultaneous blockade of multiple oncogenic signaling pathways and has, thus, emerged as an attractive strategy for the development of novel cancer therapeutics. Ganetespib (formerly known as STA-9090) is a unique resorcinolic triazolone inhibitor of Hsp90 that is currently in clinical trials for a number of human cancers. In the present study, we showed that ganetespib exhibits potent in vitro cytotoxicity in a range of solid and hematologic tumor cell lines, including those that express mutated kinases that confer resistance to small-molecule tyrosine kinase inhibitors. Ganetespib treatment rapidly induced the degradation of known Hsp90 client proteins, displayed superior potency to the ansamycin inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG), and exhibited sustained activity even with short exposure times. In vivo, ganetespib showed potent antitumor efficacy in solid and hematologic xenograft models of oncogene addiction, as evidenced by significant growth inhibition and/or regressions. Notably, evaluation of the microregional activity of ganetespib in tumor xenografts showed that ganetespib was efficiently distributed throughout tumor tissue, including hypoxic regions >150 μm from the microvasculature, to inhibit proliferation and induce apoptosis. Importantly, ganetespib showed no evidence of cardiac or liver toxicity. Taken together, this preclinical activity profile indicates that ganetespib may have broad application for a variety of human malignancies, and with select mechanistic and safety advantages over other first- and second-generation Hsp90 inhibitors. Mol Cancer Ther; 11(2); 475–84. ©2011 AACR.

Synergistic activity of the Hsp90 inhibitor ganetespib with taxanes in non-small cell lung cancer models

SummarySystemic chemotherapy using two-drug platinum-based regimens for the treatment of advanced stage non-small cell lung cancer (NSCLC) has largely reached a plateau of effectiveness. Accordingly, efforts to improve survival and quality of life outcomes have more recently focused on the use of molecularly targeted agents, either alone or in combination with standard of care therapies such as taxanes. The molecular chaperone heat shock protein 90 (Hsp90) represents an attractive candidate for therapeutic intervention, as its inhibition results in the simultaneous blockade of multiple oncogenic signaling cascades. Ganetespib is a non-ansamycin inhibitor of Hsp90 currently under clinical evaluation in a number of human malignancies, including NSCLC. Here we show that ganetespib potentiates the cytotoxic activity of the taxanes paclitaxel and docetaxel in NSCLC models. The combination of ganetespib with paclitaxel, docetaxel or another microtubule-targeted agent vincristine resulted in synergistic antiproliferative effects in the H1975 cell line in vitro. These benefits translated to improved efficacy in H1975 xenografts in vivo, with significantly enhanced tumor growth inhibition observed in combination with paclitaxel and tumor regressions seen with docetaxel. Notably, concurrent exposure to ganetespib and docetaxel improved antitumor activity in 5 of 6 NSCLC xenograft models examined. Our data suggest that the improved therapeutic indices are likely to be mechanistically multifactorial, including loss of pro-survival signaling and direct cell cycle effects resulting from Hsp90 modulation by ganetespib. Taken together, these findings provide preclinical evidence for the use of this combination to treat patients with advanced NSCLC.

Novel small-molecule inhibitors of RNA polymerase III

ABSTRACT A genetic approach utilizing the yeast Saccharomyces cerevisiae was used to identify the target of antifungal compounds. This analysis led to the identification of small molecule inhibitors of RNA polymerase (Pol) III from Saccharomyces cerevisiae. Three lines of evidence show that UK-118005 inhibits cell growth by targeting RNA Pol III in yeast. First, a dominant mutation in the g domain of Rpo31p, the largest subunit of RNA Pol III, confers resistance to the compound. Second, UK-118005 rapidly inhibits tRNA synthesis in wild-type cells but not in UK-118005 resistant mutants. Third, in biochemical assays, UK-118005 inhibits tRNA gene transcription in vitro by the wild-type but not the mutant Pol III enzyme. By testing analogs of UK-118005 in a template-specific RNA Pol III transcription assay, an inhibitor with significantly higher potency, ML-60218, was identified. Further examination showed that both compounds are broad-spectrum inhibitors, displaying activity against RNA Pol III transcription systems derived from Candida albicans and human cells. The identification of these inhibitors demonstrates that RNA Pol III can be targeted by small synthetic molecules.

Abstract C11: The oxidative stress inducer elesclomol requires copper chelation for its anticancer activity

Introduction: Elesclomol [N‐malonyl‐bis (N′‐methyl‐N′‐thiobenzoyl hydrazide)] is a first‐in‐class investigational drug, believed to exert anticancer activity through the elevation of reactive oxygen species (ROS) levels leading to the activation of the mitochondrial apoptosis pathway. The mechanism of ROS induction by elesclomol was previously unknown. Data presented here shows that ROS is generated via chelation and redox cycling of copper. Methods: Formation of a Cu chelate of elesclomol was analyzed by LCMS and single crystal X‐ray diffraction. ROS was measured using DCF‐DA and cytotoxicity assessed using a WST‐8 assay primarily in Ramos human B cell lymphoma and M14 human melanoma cell lines. Cellular levels of free elesclomol and elesclomol‐Cu were determined by LCMS. Redox potential was analyzed by cyclic voltammetry. Results: Elesclomol readily formed a Cu chelate and strongly preferred Cu over zinc, iron or manganese in competition assays. Cu bound elesclomol in a 1:1 molar ratio and the chelate formed a flat rigid structure. The donation of four lone‐pair electrons from elesclomol to Cu(II) yields higher hydrophobicity, which may facilitate greater cell permeability relative to free elesclomol. We evaluated the effect of Cu on both cellular uptake and cytotoxic activity of elesclomol on cultured cancer cells. Elesclomol lost cytotoxicity when applied to cells under Cu‐starved conditions. The presence of Cu was required for elesclomol entry into cells. The cell membrane impermeable Cu chelator BCP blocked both uptake of elesclomol and cytotoxicity, indicating that elesclomol obtains Cu outside the cell and requires it for cellular entry and cytotoxicity. Elesclomol was able to obtain Cu from serum as well as from purified ceruloplasmin, the primary Cu‐binding protein in blood. Elesclomol had poor activity against densely‐plated cancer cells in culture, but became highly potent when extra Cu was added to the medium, indicating that Cu may be limiting under dense culture conditions. We next evaluated the impact of Cu on ROS generation. A cellfree assay system showed that elesclomol‐Cu(II) was capable of efficient generation of ROS via the reduction of Cu(II) to Cu(I). A correlation was observed between redox potential and ROS production for Cu chelates of elesclomol and its analogs. Elesclomol also chelated nickel. However, while elesclomol‐Ni was highly cell permeable, it was inactive for ROS production and cancer cell cytotoxicity. The lack of activity of elesclomol‐Ni can be explained by its low redox potential. The redox potential of elesclomol‐Cu(II) was −333mV, while that of elesclomol‐Ni was −1100mV. Conclusion: Elesclomol chelates Cu outside of cells and enters cells as elesclomol‐Cu(II). At present, our data support the hypothesis that elesclomol generates ROS by redox cycling of Cu(II) to Cu(I), and that this process is necessary for its anticancer activity. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C11.

Abstract 4545: Anticancer activity of elesclomol correlates with low LDH levels and active mitochondrial respiration

Elesclomol is a first-in-class investigational drug that exerts anticancer activity through the elevation of reactive oxygen species (ROS). Our previous studies revealed that elesclomol selectively chelates copper and generates ROS via reduction of Cu(II) to Cu(I). In a Phase 3 trial in metastatic melanoma, the level of baseline lactate dehydrogenase (LDH) in patients emerged as an important prognostic factor for treatment outcomes with elesclomol. Additional investigations were undertaken to evaluate the relation between elesclomol activity and LDH levels. Because the copper redox reactions necessary for elesclomol activity can be influenced by changes in cellular metabolic properties, we investigated whether elesclomol activity varies between normoxic conditions (active mitochondrial respiration, low LDH) and hypoxic conditions (decreased mitochondrial respiration, high LDH). To assess the influence of hypoxic conditions, we evaluated elesclomol activity under several situations in which cells express high levels of Hypoxia Inducible Factor-1α (HIF1α), a transcription factor that induces a complete set of glycolytic enzymes, including LDH, and switches off glycolytic carbon flow to mitochondria. First, an increase in the level of HIF1α protein was observed in M14 melanoma cells at low oxygen level or high cell density. Elesclomol showed decreased cytotoxicity under both of these conditions. Second, we evaluated lines within the same cancer phenotype but showing distinct levels of HIF1α, and found that the high-HIF1α-expressing Caki-2 renal cancer line was resistant to elesclomol, while lower-HIF1α-expressing renal cancer cell lines were sensitive to elesclomol. Third, we assessed HIF1α levels and elesclomol activity in cells treated with CoCl 2 , a chemical mimetic of hypoxia. High levels of HIF1α induction were present in M14 cells treated with CoCl 2 , and elesclomol was significantly less active in these cells. The addition of oxamate, which selectively inhibits LDHA and activates pyruvate entry into mitochondria, restored the activity of elesclomol in the CoCl 2 -treated cells. These results support the hypothesis that elesclomol is more active under normoxic conditions (low LDH) and less active under hypoxic conditions (high LDH), consistent with the clinical findings. The results may be important for identifying patient populations for future clinical development of elesclomol. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4545.

Abstract 1638: Potent anticancer actions of the Hsp90 inhibitor STA-9090 in wild-type EGFR models of lung cancer

Non-small cell lung cancer (NSCLC) is a heterogeneous disease that can be sub-classified based on the specific alterations in oncogenes that drive it. While EGFR and KRAS are most often implicated in the molecular epidemiology of NSCLC, aberrations in several other genes have been shown to contribute to oncogenesis. These include mutation and/or amplification of MET, mutation in BRAF or chromosomal rearrangements involving ALK. Targeted therapy against these kinases has shown signs of therapeutic success; however, acquired drug resistance universally develops. Heat Shock Protein 90 (Hsp90) is a molecular chaperone that mediates the post-translational stability of its protein substrates, many of which are validated oncogenes. Hsp90 is emerging as an important target in cancer therapy because its inactivation results in the abrogation of multiple signaling pathways simultaneously, irrespective of the mutational status of its substrate. STA-9090 is a second-generation, synthetic, small-molecule Hsp90 inhibitor that has shown potent and selective activity preclinically and is currently in Phase 2 trials in a number of indications. We show here that in the presence of STA-9090, upregulation of the MET pathway, either through transient stimulation by its ligand, HGF, or through amplification of MET itself, is incapable of maintaining survival in EGFR-inhibitor-resistant NSCLC. To identify additional genetic lesions sensitive to Hsp90 inhibition, we screened a panel of wild-type EGFR NSCLC cell lines for viability in the presence of STA-9090. All the cell lines assayed, driven by mutations in genes such as PDGFRα, BRAF, PI3K and EML4-ALK or amplification of wild-type EGFR, were sensitive to STA-9090, with IC 50 values between 10 and 150 nM. Further analysis demonstrated that STA-9090 potently destabilized the oncogenic driver for each cell line. In vivo, STA-9090 showed strong single-agent activity in xenograft models of human NSCLC carrying either a BRAF mutation or EML4-ALK fusion, in accordance with the sensitivity of these client proteins to the effects of STA-9090 action. Inhibition of Hsp90 activity therefore presents a promising approach for combating NSCLC induced by mutations in genes other than EGFR, as well as by compensatory pathways upregulated in the context of EGFR-inhibitor resistance. 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 1638. doi:10.1158/1538-7445.AM2011-1638

Abstract B199: In vitro and in vivo efficacy of the novel Hsp90 inhibitor STA‐9090 and its synergy with paclitaxel

Heat shock protein 90 (Hsp90) is a molecular chaperone that regulates the post‐translational folding of its protein substrates (“client proteins”). Cancer cells contain elevated levels of active Hsp90 and, because many client proteins play critical oncogenic roles, cancer cells are especially sensitive to Hsp90 inhibition. Here we report on the initial characterization of STA‐9090, a highly potent Hsp90 inhibitor that is currently in multiple Phase 1/2 clinical trials in solid tumor and hematological malignancies. STA‐9090 is a small molecule drug that is structurally unrelated to the ansamycin Hsp90 inhibitor 17‐AAG and binds the N‐terminal ATP‐binding pocket of the chaperone. In vitro, treatment with STA‐9090 rapidly induced the degradation of known Hsp90 client proteins, such as HER2 and KIT, and growth inhibition IC50 values typically ranged from 1 to 100 nM. STA‐9090 demonstrated, on average, ∼30‐fold greater potency than 17‐AAG for the ∼60 hematological and solid tumor cell lines tested. STA‐9090 also retained its potency against cell lines expressing mutated kinases that confer resistance to kinase inhibitors such as erlotinib and imatinib. In vivo, STA‐9090 demonstrated single‐agent activity in a wide variety of human tumor cell line subcutaneous xenograft models in mice, including those representing solid tumor malignancies such as gastric carcinoma, non‐small cell lung cancer, prostate carcinoma and melanoma, and hematological malignancies such as acute myeloid leukemia, B‐cell lymphoma, chronic myeloid leukemia and multiple myeloma. In a mouse leukemia model in which wild type BCR‐ABL or imatinib/dasatinib‐resistant BCR‐ABLT315I was introduced into mouse bone marrow cells and then transplanted into host mice to induce the development of B‐cell acute lymphoblastic leukemia, STA‐ 9090 prolonged average survival from 27 to 37 days for BCR‐ABL and from 29 to 57 days for BCR‐ABLT315I. STA‐9090 also accumulated in tumors, with a half‐life of 58 hr in tumors versus 3–5 hr in plasma and non‐tumor tissues. To examine the potential for therapeutic synergy, STA‐9090 was combined with the microtubule stabilizer paclitaxel. STA‐9090 dramatically synergized with paclitaxel in in vitro cytotoxicity assays, particularly when paclitaxel treatment preceded treatment with STA‐9090. Similarly, STA‐9090 enhanced the activity of paclitaxel in the erlotinib‐resistant NCI‐H1975 lung cancer xenograft model, although this enhancement depended less on the order of dosing than was observed in vitro. No significant pharmacokinetic interactions were observed between the two agents. In conclusion, STA‐9090 is a highly potent Hsp90 inhibitor that in vitro and in vivo rapidly induces the degradation of Hsp90 client proteins and apoptosis in a broad range of cancer types, including those resistant to targeted kinase inhibitors. It also demonstrates synergy with paclitaxel. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B199.

Microarray approach for cloned library quality assessment and the comprehensive identification of positives from library screens

coding regions represented in the library. We chose to label the library DNA using random-primed synthesis that incorporated biotinylated nucleotides directly. Alternatively, we could have chosen PCR using biotinylated primers or nucleotides, but this approach might have selected against some classes of inserts (e.g., long inserts) and has other complications (see below). The library DNA (400 ng) was labeled in a 100-μL reaction using the BioPrime® DNA Labeling System (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocol, except that we labeled for 2 h and used only 4 μL of stop buffer at the end of the reaction. We ran 5 μL of the material on a 4% polyacrylamide gel to monitor the DNA labeling and confirm the manufacturer’s estimate of 2–5 μg of synthesized product. After ethidium bromide staining, the newly synthesized DNA appeared as a strongly stained smear between 40 and 140 bp. An added benefit of using the random-primed labeling protocol is that these fragments are appropriately sized to provide a good population of targets for the microarray hybridization (3). This could be a significant issue if intact (i.e., much larger) labeled PCR products are used directly. For the chip hybridization, 80 μL of the labeling reaction were brought to a total volume of 305 μL in the standard hybridization solution recommended by Affymetrix. (In the preparation of the hybridization cocktail, we considered the 80 μL BioPrime reaction as if it were contributing only water to the mixture). The U133A GeneChip was pretreated, hybridized (45°C for 16 h), and washed using the manufacturer’s procedure typically used for RNA profiling analysis (3). The chip was scanned, and the data was processed by the MAS 5 software (Affymetrix) using the default settings for the analysis. For the spleen library hybridization shown in Figure 1, the software called one-third (33.7%) of the genes represented on the chip as “P” (present). While this is a respectable representation, it is somewhat lower than the percentages obtained from typical tissue-specific RNA profiling experiments (4). However, the overall signals we obtained in our hybridizations are also somewhat lower, indicating that our protocol using a few micrograms of double-stranded DNA for Microarray approach for cloned library quality assessment and the comprehensive identification of positives from library screens