Jason C. Wong

Domain-selective small-molecule inhibitor of histone deacetylase 6 (HDAC6)-mediated tubulin deacetylation

Protein acetylation, especially histone acetylation, is the subject of both research and clinical investigation. At least four small-molecule histone deacetylase inhibitors are currently in clinical trials for the treatment of cancer. These and other inhibitors also affect microtubule acetylation. A multidimensional, chemical genetic screen of 7,392 small molecules was used to discover “tubacin,” which inhibits α-tubulin deacetylation in mammalian cells. Tubacin does not affect the level of histone acetylation, gene-expression patterns, or cell-cycle progression. We provide evidence that class II histone deacetylase 6 (HDAC6) is the intracellular target of tubacin. Only one of the two catalytic domains of HDAC6 possesses tubulin deacetylase activity, and only this domain is bound by tubacin. Tubacin treatment did not affect the stability of microtubules but did decrease cell motility. HDAC6 overexpression disrupted the localization of p58, a protein that mediates binding of Golgi elements to microtubules. Our results highlight the role of α-tubulin acetylation in mediating the localization of microtubule-associated proteins. They also suggest that small molecules that selectively inhibit HDAC6-mediated α-tubulin deacetylation, a first example of which is tubacin, might have therapeutic applications as antimetastatic and antiangiogenic agents.

The Synergistic Combination of the Farnesyl Transferase Inhibitor Lonafarnib and Paclitaxel Enhances Tubulin Acetylation and Requires a Functional Tubulin Deacetylase

Farnesyl transferase (FT) inhibitors (FTI) are anticancer agents developed to target oncogenic Ras proteins by inhibiting Ras farnesylation. FTIs potently synergize with paclitaxel and other microtubule-stabilizing drugs; however, the mechanistic basis underlying this synergistic interaction remains elusive. Here we show that the FTI lonafarnib affects the microtubule cytoskeleton resulting in microtubule bundle formation, increased microtubule stabilization and acetylation, and suppression of microtubule dynamics. Notably, treatment with the combination of low doses of lonafarnib with paclitaxel markedly enhanced tubulin acetylation (a marker of microtubule stability) as compared with either drug alone. This synergistic effect correlated with FT inhibition and was accompanied by a synergistic increase in mitotic arrest and cell death. Mechanistically, we show that the combination of lonafarnib and paclitaxel inhibits the in vitro deacetylating activity of the only known tubulin deacetylase, histone deacetylase 6 (HDAC6). In addition, the lonafarnib/taxane combination is synergistic only in cells lines expressing the wild-type HDAC6, but not a catalytic-mutant HDAC6, revealing that functional HDAC6 is required for the synergy of lonafarnib with taxanes. Furthermore, tubacin, a specific HDAC6 inhibitor, synergistically enhanced tubulin acetylation in combination with paclitaxel, similar to the combination of lonafarnib and paclitaxel. Taken together, these data suggest a relationship between FT inhibition, HDAC6 function, and cell death, providing insight into the putative molecular basis of the lonafarnib/taxane synergistic antiproliferative combination.

Chemical Genetic Modifier Screens: Small Molecule Trichostatin Suppressors as Probes of Intracellular Histone and Tubulin Acetylation

Histone deacetylase (HDAC) inhibitors are being developed as new clinical agents in cancer therapy, in part because they interrupt cell cycle progression in transformed cell lines. To examine cell cycle arrest induced by HDAC inhibitor trichostatin A (TSA), a cytoblot cell-based screen was used to identify small molecule suppressors of this process. TSA suppressors (ITSAs) counteract TSA-induced cell cycle arrest, histone acetylation, and transcriptional activation. Hydroxamic acid-based HDAC inhibitors like TSA and suberoylanilide hydroxamic acid (SAHA) promote acetylation of cytoplasmic alpha-tubulin as well as histones, a modification also suppressed by ITSAs. Although tubulin acetylation appears irrelevant to cell cycle progression and transcription, it may play a role in other cellular processes. Small molecule suppressors such as the ITSAs, available from chemical genetic suppressor screens, may prove to be valuable probes of many biological processes.

Mapping Chemical Space Using Molecular Descriptors and Chemical Genetics: Deacetylase Inhibitors

An objective of chemical genetics is to understand the relationships between the structures of small molecules and their phenotypic effects in intact living systems. We present here the results of a global analysis of a molecular descriptor space constructed using structural descriptors of an aryl 1,3-dioxane-based diversity-oriented synthesis-derived library containing structural biasing elements directed at inhibiting protein deacetylases. Using principal component analysis and three-dimensional visualization, we generated metric space maps with morphological features contributed by different diversity elements within the library. Filtering these maps using phenotypic descriptors derived from measurements of small-molecule activities in an array of cell-based assays revealed different densities of biological activity within specific subspaces. These results provide evidence that certain structural features may be important for conferring potency and selectivity on deacetylase inhibitors with respect to tubulin and histone acetylation. Moreover, these results highlight an example of the importance of using functional measures to assess molecular diversity. Similar analyses of other chemical spaces and activity classes promise to facilitate the development of chemical genetics.

0231 Acute inflammatory response to secondhand smoke exposure among non-smoking construction workers: a repeated measures study0231 Acute inflammatory response to secondhand smoke exposure among non-smoking construction workers: a repeated measures study

Objectives This study aimed to characterise the cardiovascular inflammatory response to secondhand smoke (SHS) exposure among non-smoking construction workers. Method Non-smoking workers (n = 27) were recruited from a local union and monitored inside a union hall while exposed to SHS over approximately 6 h. Using a repeated measures study design, blood samples were taken before SHS exposure (baseline), immediately following SHS exposure (post) and the morning following SHS exposure (next morning). Inflammatory markers including acute phase proteins (SAA, CRP), adhesion molecules (s-ICAM, s-VCAM), and inflammatory cytokines (IL-1, IL-2, IL-6, IL-8, IL-10, TNF-alpha, VEGF) were analysed. Linear mixed effects regression models were used to examine within-person changes in inflammatory markers at post and next morning compared to baseline. Exposure-response relationships with TWA PM2.5 were also examined using mixed effects models. All models were adjusted for age, BMI and circadian variation. Results There was a decrease in SAA (baseline = 2322 ng/ml, post = 1949 ng/ml, p = 0.04) and TNF-alpha (baseline = 9.6 pg/ml, post = 8.4 pg/ml, p < 0.01) post exposure, as compared to baseline. There was a decrease in IL-10 (baseline = 5.9 pg/ml, next morning = 6.5 pg/ml, p < 0.01) next morning compared to baseline. Increases in SHS-related PM2.5 exposures were associated with significant (p < 0.01) increases in next morning CRP, s-ICAM, and s-VCAM levels. Conclusions Our results indicate that exposure to SHS can lead to a cardiovascular inflammatory response approximately 18 h following SHS exposures, further supporting a pathway between SHS exposure and adverse cardiovascular outcomes.

Abstract C27: Evaluation of the telomere QPCR assay for use in gauging the effect of storage conditions on telomere length

Telomere length has been reported as potentially biomarker and prognostic indicator in cancer. Our goal was to evaluate whether DNA storage/handling or sample source would result in fragmented telomere DNA using an established QPCR‐based telomere assay that quantifies the length of telomeres (Cawthon 2002) Because the effect of storage conditions on telomere length has not been thoroughly evaluated, we were concerned that the QPCR could misclassify telomere length and lead to misleading epidemiological results. Genomic DNA samples for this preliminary study were gathered from either The Nurses Health Study, healthy volunteers, the MDA‐MB‐231 cell line (human breast cancer), and a commercial source (Promega). To create a model system of degraded DNA, the DNA were either subjected to multiple freeze thaws (FT, Due to the repetitive nature of the telomeric sequence, we expected to see discrete amplicon sizes starting at 76 Bp reaching over hundreds of Bps. Bioanalyzer electropherograms indicated that eight to twelve major products of ∼8 Bp iterations are commonly produced up to 150 Bp with some minor products In summary, under the current experimental conditions, the QPCR assay does not generate sufficient variable length amplicons as to present a measurable metric using the Bioanalyzer. Elevated Ct values do seem to correlate with the presence of degraded DNA, and therefore suggest under the conditions used multiple FTs and sonication do not damage the telomeric sequences. However this assay design cannot currently determine the relative contribution of full length vs. degraded telomeric sequence to the Ct value. Further investigation is warranted before this assay becomes clinically diagnostic or prognostic. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C27.