Nadia M. Atallah

Abscisic acid controlled sex before transpiration in vascular plants

Significance Since the dawn of land plants, the phytohormone abscisic acid (ABA) has played a critical role in regulating plant responses to water availability. Here we seek to explain the origins of the core ABA signaling pathway found in modern seed plants. Using the characterization of mutants and gene silencing in a fern species, we find that the same hormone signaling components are used in sex determination of ferns as are used for the control of seed dormancy and transpiration in seed plants. Ferns are shown to lack downstream functionality of stomatal components, suggesting that the origins of the core ABA signaling pathway in seed plants may lie in the sexual differentiation of ferns. Sexual reproduction in animals and plants shares common elements, including sperm and egg production, but unlike animals, little is known about the regulatory pathways that determine the sex of plants. Here we use mutants and gene silencing in a fern species to identify a core regulatory mechanism in plant sexual differentiation. A key player in fern sex differentiation is the phytohormone abscisic acid (ABA), which regulates the sex ratio of male to hermaphrodite tissues during the reproductive cycle. Our analysis shows that in the fern Ceratopteris richardii, a gene homologous to core ABA transduction genes in flowering plants [SNF1-related kinase2s (SnRK2s)] is primarily responsible for the hormonal control of sex determination. Furthermore, we provide evidence that this ABA–SnRK2 signaling pathway has transitioned from determining the sex of ferns to controlling seed dormancy in the earliest seed plants before being co-opted to control transpiration and CO2 exchange in derived seed plants. By tracing the evolutionary history of this ABA signaling pathway from plant reproduction through to its role in the global regulation of plant–atmosphere gas exchange during the last 450 million years, we highlight the extraordinary effect of the ABA–SnRK2 signaling pathway in plant evolution and vegetation function.

Reproduction and the pheromonal regulation of sex type in fern gametophytes

The fern life cycle includes a haploid gametophyte that is independent of the sporophyte and functions to produce the gametes. In homosporous ferns, the sex of the gametophyte is not fixed but can vary depending on its social environment. In many species, the sexual phenotype of the gametophyte is determined by the pheromone antheridiogen. Antheridiogen induces male development and is secreted by hermaphrodites once they become insensitive to its male-inducing effect. Recent genetic and biochemical studies of the antheridiogen response and sex-determination pathway in ferns, which are highlighted here, reveal many similarities and interesting differences to GA signaling and biosynthetic pathways in angiosperms.

Abscisic acid (ABA) and key proteins in its perception and signaling pathways are ancient, but their roles have changed through time

ABSTRACT Homologs of the Arabidopsis core abscisic acid (ABA) signaling component OPEN STOMATA1 (OST1) are best known for their role in closing stomata in angiosperm species. We recently characterized a fern OST1 homolog, GAMETOPHYTES ABA INSENSITIVE ON ANTHERDIOGEN 1 (GAIA1), which is not required for stomatal closure in ferns, consistent with physiologic evidence that shows the stomata of these plants respond passively to changes in leaf water status. Instead, gaia1 mutants reveal a critical role in ABA signaling for spore dormancy and sex determination, in a system regulated by antagonism between ABA and the gibberellin (GA)-derived fern hormone antheridiogen (ACE). ABA and key proteins, including ABA receptors from the PYR/PYL/RCAR family and negative regulators of ABA-signaling from Group A of the type-2C protein phosphatases (PP2Cs), in addition to OST1 homologs, can be found in all terrestrial land plant lineages, ranging from liverworts that lack stomata, to angiosperms. As land plants have evolved and diversified over the past 450 million years, so too have the roles of this important plant hormone and the genes involved in its signaling and perception.

APE1/Ref‐1 knockdown in pancreatic ductal adenocarcinoma – characterizing gene expression changes and identifying novel pathways using single‐cell RNA sequencing

Apurinic/apyrimidinic endonuclease 1/redox factor‐1 (APE1/Ref‐1 or APE1) is a multifunctional protein that regulates numerous transcription factors associated with cancer‐related pathways. Because APE1 is essential for cell viability, generation of APE1‐knockout cell lines and determining a comprehensive list of genes regulated by APE1 has not been possible. To circumvent this challenge, we utilized single‐cell RNA sequencing to identify differentially expressed genes (DEGs) in relation to APE1 protein levels within the cell. Using a straightforward yet novel statistical design, we identified 2837 genes whose expression is significantly changed following APE1 knockdown. Using this gene expression profile, we identified multiple new pathways not previously linked to APE1, including the EIF2 signaling and mechanistic target of Rapamycin pathways and a number of mitochondrial‐related pathways. We demonstrate that APE1 has an effect on modifying gene expression up to a threshold of APE1 expression, demonstrating that it is not necessary to completely knockout APE1 in cells to accurately study APE1 function. We validated the findings using a selection of the DEGs along with siRNA knockdown and qRT‐PCR. Testing additional patient‐derived pancreatic cancer cells reveals particular genes (ITGA1, TNFAIP2, COMMD7, RAB3D) that respond to APE1 knockdown similarly across all the cell lines. Furthermore, we verified that the redox function of APE1 was responsible for driving gene expression of mitochondrial genes such as PRDX5 and genes that are important for proliferation such as SIPA1 and RAB3D by treating with APE1 redox‐specific inhibitor, APX3330. Our study identifies several novel genes and pathways affected by APE1, as well as tumor subtype specificity. These findings will allow for hypothesis‐driven approaches to generate combination therapies using, for example, APE1 inhibitor APX3330 with other approved FDA drugs in an innovative manner for pancreatic and other cancer treatments.

A Novel Sterol-Signaling Pathway Governs Azole Antifungal Drug Resistance and Hypoxic Gene Repression in Saccharomyces cerevisiae

During antifungal drug treatment and hypoxia, genetic and epigenetic changes occur to maintain sterol homeostasis and cellular function. In this study, we show that SET domain-containing epigenetic factors govern drug efficacy to the medically relevant azole class of antifungal drugs. Upon this discovery, we determined that Set4 is induced when Saccharomyces cerevisiae are treated with azole drugs or grown under hypoxic conditions; two conditions that deplete cellular ergosterol and increase sterol precursors. Interestingly, Set4 induction is controlled by the sterol-sensing transcription factors, Upc2 and Ecm22. To determine the role of Set4 on gene expression under hypoxic conditions, we performed RNA-sequencing analysis and showed that Set4 is required for global changes in gene expression. Specifically, loss of Set4 led to an upregulation of nearly all ergosterol genes, including ERG11 and ERG3, suggesting that Set4 functions in gene repression. Furthermore, mass spectrometry analysis revealed that Set4 interacts with the hypoxic-specific transcriptional repressor, Hap1, where this interaction is necessary for Set4 recruitment to ergosterol gene promoters under hypoxia. Finally, an erg3Δ strain, which produces precursor sterols but lacks ergosterol, expresses Set4 under untreated aerobic conditions. Together, our data suggest that sterol precursors are needed for Set4 induction through an Upc2-mediated mechanism. Overall, this new sterol-signaling pathway governs azole antifungal drug resistance and mediates repression of sterol genes under hypoxic conditions.

Inhibition of the Wnt/β-catenin pathway overcomes resistance to enzalutamide in castration-resistant prostate cancer

Enzalutamide is a second-generation nonsteroidal antiandrogen clinically approved for the treatment of castration-resistant prostate cancer (CRPC), yet resistance to endocrine therapy has limited its success in this setting. Although the androgen receptor (AR) has been associated with therapy failure, the mechanisms underlying this failure have not been elucidated. Bioinformatics analysis predicted that activation of the Wnt/β-catenin pathway and its interaction with AR play a major role in acquisition of enzalutamide resistance. To validate the finding, we show upregulation of β-catenin and AR in enzalutamide-resistant cells, partially due to reduction of β-TrCP-mediated ubiquitination. Although activation of the Wnt/β-catenin pathway in enzalutamide-sensitive cells led to drug resistance, combination of β-catenin inhibitor ICG001 with enzalutamide inhibited expression of stem-like markers, cell proliferation, and tumor growth synergistically in various models. Analysis of clinical datasets revealed a molecule pattern shift in different stages of prostate cancer, where we detected a significant correlation between AR and β-catenin expression. These data identify activation of the Wnt/β-catenin pathway as a major mechanism contributing to enzalutamide resistance and demonstrate the potential to stratify patients with high risk of said resistance.Significance: Wnt/β-catenin inhibition resensitizes prostate cancer cells to enzalutamide. Cancer Res; 78(12); 3147-62. ©2018 AACR.

Sex Determination in Ceratopteris richardii Is Accompanied by Transcriptome Changes That Drive Epigenetic Reprogramming of the Young Gametophyte

The fern Ceratopteris richardii is an important model for studies of sex determination and gamete differentiation in homosporous plants. Here we use RNA-seq to de novo assemble a transcriptome and identify genes differentially expressed in young gametophytes as their sex is determined by the presence or absence of the male-inducing pheromone called antheridiogen. Of the 1,163 consensus differentially expressed genes identified, the vast majority (1,030) are up-regulated in gametophytes treated with antheridiogen. GO term enrichment analyses of these DEGs reveals that a large number of genes involved in epigenetic reprogramming of the gametophyte genome are up-regulated by the pheromone. Additional hormone response and development genes are also up-regulated by the pheromone. This C. richardii gametophyte transcriptome and gene expression dataset will prove useful for studies focusing on sex determination and differentiation in plants.

Abstract 4802: Combination therapy in PDAC involving blockade of the APE1/Ref-1 signaling pathway: An investigation into drug synthetic lethality and anti-neuropathy therapeutic approach

Pancreatic ductal adenocarcinoma (PDAC) is the 4th leading cause of cancer-related mortality in the US. Most patients present with advanced disease and ~93% die within five years, with most surviving less than six months. Combination therapies including Gemcitabine (GemzarTM) and sustained release, nab-paclitaxel (AbraxaneTM) and FOLFIRINOX (5-FU/leucovorin/irinotecan/oxaliplatin) offer modest improvement in survival, albeit at an increase in side effects including chemotherapy-induced peripheral neuropathy. Data is presented on Apurinic/apyrimidinic endonuclease/redox factor-1 (APE1/Ref-1 or APE1) and redox-specific APE1 inhibitor, APX3330 and its effects on tumor cell growth and sensory neuron function. APE1 is a multifunctional protein involved in repairing DNA damage via endonuclease activity and in redox regulation of transcription factors such as HIF-1α, NFkB and STAT3. High expression levels of APE1 indicate decreased survival in PDAC as well as other cancers. Because APE1 is essential for cell viability, generation of APE1 knockout cell lines and determining a comprehensive list of genes regulated by APE1 has been difficult. To circumvent this, we performed single cell RNA-Sequencing on PDAC cells following APE1 knockdown under normoxia and hypoxia to identify differentially expressed genes and further explore APE19s effects on HIF-1α and STAT3 signaling under both conditions. Proteomic analysis on PDAC cells following APE1 knockdown in normoxia and hypoxia revealed changes in signaling downstream of APE1, complementing the transcriptomic data and providing a more complete understanding of pathways affected by APE1. We used the newly identified APE1 targets and pathways along with drug sensitivity data of cancer cell lines from the Cancer Cell Line Encyclopedia (CCLE) to generate potential combination therapies of FDA approved drugs and the APE1 redox inhibitor, APX3330 and next generation analogs. These combinations were tested using an ex vivo 3D tumor-stroma model system using patient derived cells from the tumor as well as cancer-associated fibroblasts. We identified synergy with agents such as Napabucasin and Entinostat. We also tested APX3330 in combination with drugs that are part of PDAC standard of care. In vivo studies combining APX3330 with Gemcitabine showed significantly decreased tumor volume. Combining oxaliplatin (part of FOLFIRINOX) with APX3330 caused a significant reduction in oxaliplatin-induced DNA damage in sensory neurons from a KPC orthotopic graft model, without hindering its anti-cancer activity. With the phase I clinical trial for APX3330 underway (IND 125360), the potential for APE1 targeted therapy enhancing tumor efficacy while providing neuroprotective effects in the sensory neurons provides a win-win scenario. Citation Format: Fenil L. Shah, Nadia Atallah, Michelle Grimard, Chunlu Guo, Chi Zhang, Jill Fehrenbacher, Mark R. Kelley, Melissa Fishel. Combination therapy in PDAC involving blockade of the APE1/Ref-1 signaling pathway: An investigation into drug synthetic lethality and anti-neuropathy therapeutic approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4802.

Wnt9a Can Influence Cell Fates and Neural Connectivity across the Radial Axis of the Developing Cochlea

Vertebrate hearing organs manifest cellular asymmetries across the radial axis that underlie afferent versus efferent circuits between the inner ear and the brain. Therefore, understanding the molecular control of patterning across this axis has important functional implications. Radial axis patterning begins before the cells become postmitotic and is likely linked to the onset of asymmetric expression of secreted factors adjacent to the sensory primordium. This study explores one such asymmetrically expressed gene, Wnt9a, which becomes restricted to the neural edge of the avian auditory organ, the basilar papilla, by embryonic day 5 (E5). Radial patterning is disrupted when Wnt9a is overexpressed throughout the prosensory domain beginning on E3. Sexes were pooled for analysis and sex differences were not studied. Analysis of gene expression and afferent innervation on E6 suggests that ectopic Wnt9a expands the neural-side fate, possibly by re-specifying the abneural fate. RNA sequencing reveals quantitative changes, not only in Wnt-pathway genes, but also in genes involved in axon guidance and cytoskeletal remodeling. By E18, these early patterning effects are manifest as profound changes in cell fates [short hair cells (HCs) are missing], ribbon synapse numbers, outward ionic currents, and efferent innervation. These observations suggest that Wnt9a may be one of the molecules responsible for breaking symmetry across the radial axis of the avian auditory organ. Indirectly, Wnt9a can regulate the mature phenotype whereby afferent axons predominantly innervate neural-side tall HCs, resulting in more ribbon synapses per HC compared with abneural-side short HCs with few ribbons and large efferent synapses. SIGNIFICANCE STATEMENT Wnts are a class of secreted factors that are best known for stimulating cell division in development and cancer. However, in certain contexts during development, Wnt-expressing cells can direct neighboring cells to take on specific fates. This study suggests that the Wnt9a ligand may play such a role in the developing hearing organ of the bird cochlea. This was shown through patterning defects that occur in response to the overexpression of Wnt9a. This manipulation increased one type of sensory hair cell (tall HCs) at the expense of another (short HCs) that is usually located furthest from the Wnt9a source. The extraneous tall HCs that replaced short HCs showed some physiological properties and neuronal connections consistent with a fate switch.