Laura Saunders

Suppression of Oxidative Stress by β-Hydroxybutyrate, an Endogenous Histone Deacetylase Inhibitor

Stress Protector During prolonged fasting, the oxidation of fatty acids leads to increased accumulation of d-β-hydroxybutyrate (βOHB) in the bloodstream. Such increased concentrations of βOHB inhibit class I histone deacetylases. Histone acetylation in turn influences transcriptional activity at various genes. Shimazu et al. (p. 211, published online 6 December; see the Perspective by Sassone-Corsi) found that among the genes showing increased transcription in animals treated with high concentrations of βOHB were two genes implicated in cellular responses to oxidative stress. When treated ahead of time with βOHB, mice were protected from the toxic effects of the oxidative stress causing poison paraquat. Ketone bodies, metabolites that accumulate during fasting, change gene expression by inhibiting histone deacetylases. [Also see Perspective by Sassone-Corsi] Concentrations of acetyl–coenzyme A and nicotinamide adenine dinucleotide (NAD+) affect histone acetylation and thereby couple cellular metabolic status and transcriptional regulation. We report that the ketone body d-β-hydroxybutyrate (βOHB) is an endogenous and specific inhibitor of class I histone deacetylases (HDACs). Administration of exogenous βOHB, or fasting or calorie restriction, two conditions associated with increased βOHB abundance, all increased global histone acetylation in mouse tissues. Inhibition of HDAC by βOHB was correlated with global changes in transcription, including that of the genes encoding oxidative stress resistance factors FOXO3A and MT2. Treatment of cells with βOHB increased histone acetylation at the Foxo3a and Mt2 promoters, and both genes were activated by selective depletion of HDAC1 and HDAC2. Consistent with increased FOXO3A and MT2 activity, treatment of mice with βOHB conferred substantial protection against oxidative stress.

Destabilization of ERBB2 transcripts by targeting 3' untranslated region messenger RNA associated HuR and histone deacetylase-6.

In addition to repressing ERBB2 promoter function, histone deacetylase (HDAC) inhibitors induce the accelerated decay of mature ERBB2 transcripts; the mechanism mediating this transcript destabilization is unknown but depends on the 3′ untranslated region (UTR) of ERBB2 mRNA. Using ERBB2-overexpressing human breast cancer cells (SKBR3), the mRNA stability factor HuR was shown to support ERBB2 transcript integrity, bind and endogenously associate with a conserved U-rich element within the ERBB2 transcript 3′ UTR, coimmunoprecipitate with RNA-associated HDAC activity, and colocalize with HDAC6. HDAC6 also coimmunoprecipitates with HuR in an RNA-dependent manner and within 6 hours of exposure to a pan-HDAC inhibitor dose, that does not significantly alter cytosolic HuR levels or HuR binding to ERBB2 mRNA. Cellular ERBB2 transcript levels decline while remaining physically associated with HDAC6. Knockdown of HDAC6 protein by small interfering RNA partially suppressed the ERBB2 transcript decay induced by either pan-HDAC or HDAC6-selective enzymatic inhibitors. Three novel hydroxamates, ST71, ST17, and ST80 were synthesized and shown to inhibit HDAC6 with 14-fold to 31-fold greater selectivity over their binding and inhibition of HDAC1. Unlike more potent pan-HDAC inhibitors, these HDAC6-selective inhibitors produced dose-dependent growth arrest of ERBB2-overexpressing breast cancer cells by accelerating the decay of mature ERBB2 mRNA without repressing ERBB2 promoter function. In sum, these findings point to the therapeutic potential of HuR and HDAC6-selective inhibitors, contrasting ERBB2 stability effects induced by HDAC6 enzymatic inhibition and HDAC6 protein knockdown, and show that ERBB2 transcript stability mechanisms include exploitable targets for the development of novel anticancer therapies. (Mol Cancer Res 2008;6(7):1250–8)

Pyridylalanine-Containing Hydroxamic Acids as Selective HDAC6 Inhibitors

Pyridylalanine inhibitors of histone deacetylase (HDAC) have been synthesized that show selectivity for the isoform HDAC6 over HDAC1 in vitro. This selectivity was also identified in cancer cells by analyzing tubulin versus histone acetylation. The compounds show decreased intrinsic cytotoxicity relative to pan‐HDAC inhibitors, but show antiproliferative synergy with the proteasome inhibitor bortezomib.

Zmynd15 Encodes a Histone Deacetylase-dependent Transcriptional Repressor Essential for Spermiogenesis and Male Fertility

Spermatogenesis is a complex process through which male germ line stem cells undergo a multi-step differentiation program and sequentially become spermatogonia, spermatocytes, spermatids, and eventually spermatozoa. In this process, transcription factors act as switches that precisely regulate the expression of genes that in turn control the developmental program of male germ cells. Transcription factors identified to be essential for normal haploid gene expression all display transcription-activating effects and thus serve as the “on” switch for haploid gene expression. Here, we report that ZMYND15 acts as a histone deacetylase-dependent transcriptional repressor and controls normal temporal expression of haploid cell genes during spermiogenesis. Inactivation of Zmynd15 results in early activation of transcription of numerous important haploid genes including Prm1, Tnp1, Spem1, and Catpser3; depletion of late spermatids; and male infertility. ZMYND15 represents the first transcriptional repressor identified to be essential for sperm production and male fertility.

Ornithine decarboxylase activity in tumor cell lines correlates with sensitivity to cell death induced by histone deacetylase inhibitors

Inhibitors of histone deacetylases (HDAC) show significant promise as targeted anticancer agents against a variety of hematologic and solid tumors. HDAC inhibitors arrest the growth of primary cells, but they induce apoptosis or differentiation of tumor cells. Although the precise mechanism is unknown, differences in cell cycle checkpoints and chromatin structure may be responsible. Cellular polyamines regulate both cell cycle progression and chromatin structure. In tumors, polyamines are abundantly produced because of increased activity of the rate-limiting enzyme in polyamine synthesis, ornithine decarboxylase (ODC). To determine if polyamines contribute to the cellular response to HDAC inhibitors, we inhibited ODC activity with α-difluoromethylornithine. Polyamine depletion increased resistance to apoptosis induced by HDAC inhibitors. In addition, we found that ODC activity levels correlated with sensitivity to HDAC inhibitors in a panel of tumor cell lines. We conclude that polyamines participate in the cellular response to HDAC inhibitors and that ODC activity correlates with sensitivity to HDAC inhibitor–induced apoptosis. Thus, elevated polyamine levels might be a biomarker for tumor sensitivity to HDAC inhibitor–induced apoptosis. These findings warrant clinical evaluation of tumor samples to determine if high ODC activity levels predict sensitivity to HDAC inhibitors. [Mol Cancer Ther 2006;5(11):2777–85]

Abstract 3093: Expression of DLL3 in metastatic melanoma, glioblastoma and high-grade extrapulmonary neuroendocrine carcinomas as potential indications for rovalpituzumab tesirine (Rova-T; SC16LD6.5), a delta-like protein 3 (DLL3)-targeted antibody drug conjugate (ADC)

Expression of DLL3 was examined in additional tumor types, as it was found to be highly expressed in tumor-initiating cells (TIC) in small cell lung cancer (SCLC), where a DLL3-targeted antibody drug conjugate (ADC), rovalpituzumab tesirine (Rova-T; SC16LD6.5; Saunders et al. 2015 Sci Transl Med 7:302ra136)) exerted clinically meaningful anti-tumor effects in a phase I trial (Spigel et al. 2016 Lancet Oncology; In Press). DLL3 expression was profiled by qRT-PCR, ELISA and immunohistochemistry in multiple tumor types. Patient-derived xenografts (PDX) from melanoma and ovarian small cell carcinoma were established and used for efficacy studies to determine the ability of Rova-T to impact tumor growth and TIC frequency. DLL3 expression was seen in metastatic melanoma (55%), low grade gliomas (90%), glioblastoma (70%), medullary thyroid cancer (65%), carcinoids (33%), dispersed neuroendocrine tumors in the pancreas (9%), bladder (57%) and prostate (24%), testicular cancer (90%), and lung adenocarcinomas with neuroendocrine features (80%). Unlike SCLC, where DLL3 does not predict clinical outcome on standard therapies, DLL3 expression negatively correlates with overall survival in melanoma and small cell bladder cancer. In mice bearing DLL3 positive melanoma PDX, treatment with a single dose of Rova-T resulted in effective and durable responses (>100 days), which correlated with a significant impact on TIC frequency. Similarly, in mice bearing DLL3-positive ovarian small cell PDX, a single dose of Rova-T resulted in effective and durable responses (>100 days). Our results show that DLL3 is expressed in many neuroendocrine tumors (lung, ovarian, prostate, bladder, etc), metastatic melanoma, medullary thyroid cancer, low-grade gliomas and glioblastoma. Given pre-clinical results showing efficacy of Rova-T in melanoma and ovarian small cell carcinoma, as well as encouraging clinical data with Rova-T in patients with recurrent/refractory SCLC, clinical evaluation of Rova-T in DLL3-positive melanoma, glioblastoma, medullary thyroid cancer and other high-grade neuroendocrine carcinomas is warranted. A “basket” trial enrolling patients with DLL3-positive solid tumors is now recruiting patients (NCT02709889). Citation Format: Laura R. Saunders, Samuel A. Williams, Sheila Bheddah, Kumiko Isse, Sarah Fong, Marybeth A. Pysz, Himisha Beltran, Loredana Puca, Verena Sailer, Juan M. Mosquera, Yu Yin, Jiaoti Huang, Andrew J. Armstrong, Jorge Garcia, Cristina Magi-Galluzzi, Vadim Koshkin, Petros Grivas, Farhad Kosari, John Cheville, Justin C. Moser, Thomas J. Flotte, Thorvardur Halfdanarson, Aaron Mansfield, Konstantinos N. Leventakos, Julian R. Molina, Douglas W. Ball, Barry D. Nelkin, Jill E. Shea, Courtney L. Scaife, Scott J. Dylla. Expression of DLL3 in metastatic melanoma, glioblastoma and high-grade extrapulmonary neuroendocrine carcinomas as potential indications for rovalpituzumab tesirine (Rova-T; SC16LD6.5), a delta-like protein 3 (DLL3)-targeted antibody drug conjugate (ADC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3093. doi:10.1158/1538-7445.AM2017-3093

Transcriptomic and protein analysis of small cell bladder cancer (SCBC) identifies prognostic biomarkers and DLL3 as a relevant therapeutic target

Purpose: Transcriptomic profiling can shed light on the biology of small-cell bladder cancer (SCBC), nominating biomarkers, and novel therapeutic targets. Experimental Design: Sixty-three patients with SCBC had small-cell histology confirmed and quantified by a genitourinary pathologist. Gene expression profiling was performed for 39 primary tumor samples, 1 metastatic sample, and 6 adjacent normal urothelium samples (46 total) from the same cohort. Protein levels of differentially expressed therapeutic targets, DLL3 and PDL1, and also CD56 and ASCL1, were confirmed by IHC. A SCBC PDX model was utilized to assess in vivo efficacy of DLL3-targeting antibody–drug conjugate (ADC). Results: Unsupervised hierarchical clustering of 46 samples produced 4 clusters that correlated with clinical phenotypes. Patients whose tumors had the most “normal-like” pattern of gene expression had longer overall survival (OS) compared with the other 3 clusters while patients with the most “metastasis-like” pattern had the shortest OS (P = 0.047). Expression of DLL3, PDL1, ASCL1, and CD56 was confirmed by IHC in 68%, 30%, 52%, and 81% of tissue samples, respectively. In a multivariate analysis, DLL3 protein expression on >10% and CD56 expression on >30% of tumor cells were both prognostic of shorter OS (P = 0.03 each). A DLL3-targeting ADC showed durable antitumor efficacy in a SCBC PDX model. Conclusions: Gene expression patterns in SCBC are associated with distinct clinical phenotypes ranging from more indolent to aggressive disease. Overexpression of DLL3 mRNA and protein is common in SCBC and correlates with shorter OS. A DLL3-targeted ADC demonstrated in vivo efficacy superior to chemotherapy in a PDX model of SCBC.