An Effective Epigenetic-PARP Inhibitor Combination Therapy for Breast and Ovarian Cancers Independent of BRCA Mutations.

Purpose: PARP inhibitors (PARPi) are primarily effective against BRCA1/2-mutated breast and ovarian cancers, but resistance due to reversion of mutated BRCA1/2 and other mechanisms is common. Based on previous reports demonstrating a functional role for DNMT1 in DNA repair and our previous studies demonstrating an ability of DNA methyltransferase inhibitor (DNMTi) to resensitize tumors to primary therapies, we hypothesized that combining a DNMTi with PARPi would sensitize PARPi-resistant breast and ovarian cancers to PARPi therapy, independent of BRCA status.Experimental Design: Breast and ovarian cancer cell lines (BRCA-wild-type/mutant) were treated with PARPi talazoparib and DNMTi guadecitabine. Effects on cell survival, ROS accumulation, and cAMP levels were examined. In vivo, mice bearing either BRCA-proficient breast or ovarian cancer cells were treated with talazoparib and guadecitabine, alone or in combination. Tumor progression, gene expression, and overall survival were analyzed.Results: Combination of guadecitabine and talazoparib synergized to enhance PARPi efficacy, irrespective of BRCA mutation status. Coadministration of guadecitabine with talazoparib increased accumulation of ROS, promoted PARP activation, and further sensitized, in a cAMP/PKA-dependent manner, breast and ovarian cancer cells to PARPi. In addition, DNMTi enhanced PARP "trapping" by talazoparib. Guadecitabine plus talazoparib decreased xenograft tumor growth and increased overall survival in BRCA-proficient high-grade serous ovarian and triple-negative breast cancer models.Conclusions: The novel combination of the next-generation DNMTi guadecitabine and the first-in-class PARPi talazoparib inhibited breast and ovarian cancers harboring either wild-type- or mutant-BRCA, supporting further clinical exploration of this drug combination in PARPi-resistant cancers. Clin Cancer Res; 24(13); 3163-75. ©2018 AACR.

Protein kinase A-mediated phosphorylation regulates STAT3 activation and oncogenic EZH2 activity.

Polycomb repressive complex 2 (PRC2) member enhancer of zeste homolog 2 (EZH2) trimethylates histone H3 lysine 27 (H3K27me3), alters chromatin structure and contributes to epigenetic regulation of gene expression in normal and disease processes. Phosphorylation of EZH2 augmented EZH2 oncogenic activity in cancer but observations have been limited to threonine 350 (T350) and serine 21 (S21) residues by cyclin-dependent kinase 1 and protein kinase B, respectively. In addition, phosphorylation of the evolutionarily conserved T372 motif of EZH2 by p38 resulted in EZH2 interaction with Ying Yang 1 and promoted muscle stem cell differentiation. In the present study, we used epithelial ovarian cancer (OC) cells as a model to demonstrate that phosphorylation of EZH2 at T372 by protein kinase A (PKA) induced a dominant-negative EZH2 phenotype, inhibited OC cell proliferation and migration in vitro and decreased ovarian xenograft tumor growth in vivo. Phosphorylation of T372 by PKA enhanced the interaction between EZH2 and signal transducer and activator of transcription 3 (STAT3), and STAT3 binding to pT372-EZH2 reduced cellular levels of pSTAT3 and downregulated interleukin 6 receptor expression in OC. Furthermore, PKA-mediated pT372-EZH2 decreased ATP levels and altered mitochondrial gene expression, resulting in mitochondrial dysfunction and reduced OC cell growth. These findings demonstrate that PKA-mediated T372 phosphorylation reduces oncogenic EZH2 activity and reveal a novel role for pT372 in regulating EZH2 in OC and possibly other cancers.

Therapeutic targeting using tumor specific peptides inhibits long non-coding RNA HOTAIR activity in ovarian and breast cancer.

Long non-coding RNAs (lncRNAs) play key roles in human diseases, including cancer. Functional studies of the lncRNA HOTAIR (HOX transcript antisense RNA) provide compelling evidence for therapeutic targeting of HOTAIR in cancer, but targeting lncRNAs in vivo has proven to be difficult. In the current study, we describe a peptide nucleic acids (PNA)-based approach to block the ability of HOTAIR to interact with EZH2 and subsequently inhibit HOTAIR-EZH2 activity and resensitize resistant ovarian tumors to platinum. Treatment of HOTAIR-overexpressing ovarian and breast cancer cell lines with PNAs decreased invasion and increased chemotherapy sensitivity. Furthermore, the mechanism of action correlated with reduced nuclear factor-kappaB (NF-κB) activation and decreased expression of NF-κB target genes matrix metalloprotease 9 and interleukin 6. To deliver the anti-lncRNA to the acidic (pH approximately 6) tumor microenvironment, PNAs were conjugated to pH-low insertion peptide (pHLIP). Treatment of mice harboring platinum-resistant ovarian tumor xenografts with pHLIP-PNA constructs suppressed HOTAIR activity, reduced tumor formation and improved survival. This first report on pHLIP-PNA lncRNA targeting solid tumors in vivo suggests a novel cancer therapeutic approach.

Real-time fluorescence assays to monitor duplex unwinding and ATPase activities of helicases.

Many physiological functions of helicases are dependent on their ability to unwind nucleic acid duplexes in an ATP-dependent fashion. Determining the kinetic frameworks of these processes is crucial to understanding how these proteins function. We recently developed a fluorescence assay to monitor RNA duplex unwinding by DEAD-box helicases in real time. In this assay, two fluorescently modified short reporter oligonucleotides are annealed to an unmodified RNA loading strand of any length so that the fluorescent moieties of the two reporters find themselves in close proximity to each other and fluorescence is quenched. One reporter is modified with cyanine 3 (Cy3), whereas the other is modified with a spectrally paired black-hole quencher (BHQ). As the helicase unwinds the loading strand, the enzyme displaces the Cy3-modified reporter, which will bind to a capture or competitor DNA strand, permanently separating it from the BHQ-modified reporter. Complete separation of the Cy3-modified reporter strand is thus detected as an increase in total fluorescence. This assay is compatible with reagentless biosensors to monitor ATPase activity so that the coupling between ATP hydrolysis and duplex unwinding can be determined. With the protocol described, obtaining data and analyzing results of unwinding and ATPase assays takes ∼4 h.

Duplex unwinding and ATPase activities of the DEAD-box helicase eIF4A are coupled by eIF4G and eIF4B.

Eukaryotic initiation factor (eIF) 4A is a DEAD-box helicase that stimulates translation initiation by unwinding mRNA secondary structure. The accessory proteins eIF4G, eIF4B, and eIF4H enhance the duplex unwinding activity of eIF4A, but the extent to which they modulate eIF4A activity is poorly understood. Here, we use real-time fluorescence assays to determine the kinetic parameters of duplex unwinding and ATP hydrolysis by these initiation factors. To ensure efficient duplex unwinding, eIF4B and eIF4G cooperatively activate the duplex unwinding activity of eIF4A. Our data reveal that eIF4H is much less efficient at stimulating eIF4A unwinding activity than eIF4B, implying that eIF4H is not able to completely substitute for eIF4B in duplex unwinding. By monitoring unwinding and ATPase assays under identical conditions, we demonstrate that eIF4B couples the ATP hydrolysis cycle of eIF4A with strand separation, thereby minimizing nonproductive unwinding events. Using duplex substrates with altered GC contents but similar predicted thermal stabilities, we further show that the rate of formation of productive unwinding complexes is strongly influenced by the local stability per base pair, in addition to the stability of the entire duplex. This finding explains how a change in the GC content of a hairpin is able to influence translation initiation while maintaining the overall predicted thermal stability.