Shirley M. Taylor

Exploitation of the Apoptosis-Primed State of MYCN-Amplified Neuroblastoma to Develop a Potent and Specific Targeted Therapy Combination

Summary Fewer than half of children with high-risk neuroblastoma survive. Many of these tumors harbor high-level amplification of MYCN, which correlates with poor disease outcome. Using data from our large drug screen we predicted, and subsequently demonstrated, that MYCN-amplified neuroblastomas are sensitive to the BCL-2 inhibitor ABT-199. This sensitivity occurs in part through low anti-apoptotic BCL-xL expression, high pro-apoptotic NOXA expression, and paradoxical, MYCN-driven upregulation of NOXA. Screening for enhancers of ABT-199 sensitivity in MYCN-amplified neuroblastomas, we demonstrate that the Aurora Kinase A inhibitor MLN8237 combines with ABT-199 to induce widespread apoptosis. In diverse models of MYCN-amplified neuroblastoma, including a patient-derived xenograft model, this combination uniformly induced tumor shrinkage, and in multiple instances led to complete tumor regression.

Epigenetic Modifications of the PGC-1α Promoter during Exercise Induced Expression in Mice

The transcriptional coactivator, PGC-1α, is known for its role in mitochondrial biogenesis. Although originally thought to exist as a single protein isoform, recent studies have identified additional promoters which produce multiple mRNA transcripts. One of these promoters (promoter B), approximately 13.7kb upstream of the canonical PGC-1α promoter (promoter A), yields alternative transcripts present at levels much lower than the canonical PGC-1α mRNA transcript. In skeletal muscle, exercise resulted in a substantial, rapid increase of mRNA of these alternative PGC-1α transcripts. Although the β2-adrenergic receptor was identified as a signaling pathway that activates transcription from PGC-1α promoter B, it is not yet known what molecular changes occur to facilitate PGC-1α promoter B activation following exercise. We sought to determine whether epigenetic modifications were involved in this exercise response in mouse skeletal muscle. We found that DNA hydroxymethylation correlated to increased basal mRNA levels from PGC-1α promoter A, but that DNA methylation appeared to play no role in the exercise-induced activation of PGC-1α promoter B. The level of the activating histone mark H3K4me3 increased with exercise 2–4 fold across PGC-1α promoter B, but remained unaltered past the canonical PGC-1α transcriptional start site. Together, these data show that epigenetic modifications partially explain exercise-induced changes in the skeletal muscle mRNA levels of PGC-1α isoforms.

Mammalian Mitochondrial and Cytosolic Folylpolyglutamate Synthetase Maintain the Subcellular Compartmentalization of Folates

Background: Folylpolyglutamates are in the cytosol and mitochondria and the enzyme that makes these compounds, folylpolyglutamate synthetase (FPGS) is in both compartments. Results: Folylpolyglutamates cannot traverse mitochondrial membranes in either direction. Conclusion: Subcellular isoforms of FPGS are required to establish and maintain subcellular folate compartmentalization and function. Significance: Mitochondrial folates are a separate metabolic pool not in equilibrium with cytosol. Folylpoly-γ-glutamate synthetase (FPGS) catalyze the addition of multiple glutamates to tetrahydrofolate derivatives. Two mRNAs for the fpgs gene direct isoforms of FPGS to the cytosol and to mitochondria in mouse and human tissues. We sought to clarify the functions of these two compartmentalized isoforms. Stable cell lines were created that express cDNAs for the mitochondrial and cytosolic isoforms of human FPGS under control of a doxycycline-inducible promoter in the AUXB1 cell line. AUXB1 are devoid of endogenous FPGS activity due to a premature translational stop at codon 432 in the fpgs gene. Loss of folates was not measurable from these doxycycline-induced cells or from parental CHO cells over the course of three CHO cell generations. Likewise, there was no detectable transfer of folate polyglutamates either from the cytosol to mitochondria, or from mitochondria to the cytosol. The cell line expressing cytosolic FPGS required exogenous glycine but not thymidine or purine, whereas cells expressing the mitochondrial isoform required exogenous thymidine and purine but not glycine for optimal growth and survival. We concluded that mitochondrial FPGS is required because folate polyglutamates are not substrates for transport across the mitochondrial membrane in either direction and that polyglutamation not only traps folates in the cytosol, but also in the mitochondrial matrix.

p53 Deletion or Hotspot Mutations Enhance mTORC1 Activity by Altering Lysosomal Dynamics of TSC2 and Rheb

The activity of mammalian target of rapamycin complex 1 (mTORC1) is frequently enhanced in carcinomas, an effect thought to contribute to the malignant phenotype. Here, it is demonstrated that either deletion or mutation of TP53 in colon or lung carcinoma cells substantially enhances mTORC1 kinase activity by an effect downstream of and independent of AMPK. Mechanistically, it was determined that loss or mutation of p53 decreased expression of TSC2 and Sestrin2 (SESN2). Complementation of p53 null cells with TSC2 or Sestrin2 reduced mTORC1 activity to levels found in p53 wild-type (wt) cells, whereas their genetic depletion enhanced mTORC1 activity in p53 wt cells. However, the primary causal event in enhanced mTORC1 activity upon loss of p53 appeared to be a diminished distribution of TSC2 to lysosomal membranes containing mTOR. Subsequently, there was increased Rheb in the lysosomal compartment, and a higher mTOR association with Raptor. Transfection of TSC2 into p53 null cells replaced TSC2 and diminished Rheb at the lysosome, recapitulating cells with wt p53. In contrast, transfection of Sestrin2 decreased mTOR in lysosomes, but the lower levels of Sestrin2 in p53 null cells did not change lysosomal mTOR. In summary, loss of the transcriptional activity of p53, either by deletion or by key mutations in the DNA-binding domain, diminishes expression of TSC2 and Sestrin2, thus, shifting membrane-bound TSC2 out of lysosomal membranes, increasing lysosomal Rheb and increasing the kinase activity of mTORC1. Implications: This study establishes that loss of p53 function decreases lysosomal TSC2 and increases lysosomal Rheb resulting in hyperactive mTORC1, findings that are consistent with a more malignant phenotype. Mol Cancer Res; 14(1); 66–77. ©2015 AACR.

Epithelial-to-Mesenchymal Transition Antagonizes Response to Targeted Therapies in Lung Cancer by Suppressing BIM

Purpose: Epithelial-to-mesenchymal transition (EMT) confers resistance to a number of targeted therapies and chemotherapies. However, it has been unclear why EMT promotes resistance, thereby impairing progress to overcome it. Experimental Design: We have developed several models of EMT-mediated resistance to EGFR inhibitors (EGFRi) in EGFR-mutant lung cancers to evaluate a novel mechanism of EMT-mediated resistance. Results: We observed that mesenchymal EGFR-mutant lung cancers are resistant to EGFRi-induced apoptosis via insufficient expression of BIM, preventing cell death despite potent suppression of oncogenic signaling following EGFRi treatment. Mechanistically, we observed that the EMT transcription factor ZEB1 inhibits BIM expression by binding directly to the BIM promoter and repressing transcription. Derepression of BIM expression by depletion of ZEB1 or treatment with the BH3 mimetic ABT-263 to enhance “free” cellular BIM levels both led to resensitization of mesenchymal EGFR-mutant cancers to EGFRi. This relationship between EMT and loss of BIM is not restricted to EGFR-mutant lung cancers, as it was also observed in KRAS-mutant lung cancers and large datasets, including different cancer subtypes. Conclusions: Altogether, these data reveal a novel mechanistic link between EMT and resistance to lung cancer targeted therapies. Clin Cancer Res; 24(1); 197–208. ©2017 AACR.

Humanizing mouse folate metabolism: conversion of the dual-promoter mouse folylpolyglutamate synthetase gene to the human single-promoter structure

The mouse is extensively used to model human folate metabolism and therapeutic outcomes with antifolates. However, the folylpoly‐γ‐glutamate synthetase (fpgs) gene, whose product determines folate/antifolate intracellular retention and antifolate antitumor activity, displays a pronounced species difference. The human gene uses only a single promoter, whereas the mouse uses two: P2, akin to the human promoter, at low levels in most tissues; and P1, an upstream promoter used extensively in liver and kidney. We deleted the mouse P1 promoter through homologous recombination to study the dual‐promoter mouse system and to create a mouse with a humanized fpgs gene structure. Despite the loss of the predominant fpgs mRNA species in liver and kidney (representing 95 and 75% of fpgs transcripts in these tissues, respectively), P1‐knockout mice developed and reproduced normally. The survival of these mice was explained by increased P2 transcription due to relief of transcriptional interference, by a 3‐fold more efficient translation of P2‐derived than P1‐derived transcripts, and by 2‐fold higher stability of P2‐derived FPGS. In combination, all 3 effects reinstated FPGS function, even in liver. By eliminating mouse P1, we created a mouse model that mimicked the human housekeeping pattern of fpgs gene expression.—Yang, C., Xie, L.Y., Windle, J. J., Taylor, S. M., Moran, R. G. Humanizing mouse folate metabolism: conversion of the dual‐promoter mouse folylpolyglutamate synthetase gene to the human single‐promoter structure. FASEB J. 28, 1998–2008 (2014). www.fasebj.org

Abstract 1213: Endogenous p53 affinity tagging with CRISPR

Several antimetabolites used for cancer treatment, including hydroxyurea (HU) and pemetrexed (PTX), stabilize tumor suppressor protein p53, but fail to induce the transcription of p53 downstream target genes. To understand the underlying mechanism for the deficiency of stabilized p53, we are quantitatively characterizing p53 posttranslational modification and its interaction partners with mass spectrometry (MS). To facilitate the purification of endogenous p53, we established an HCT116 human colon carcinoma cell line, in which one allele of endogenous p53 was tagged with a classic Tandem Affinity Tag (TAP) at the C-terminus, using adenovirus associated virus (AAV) mediated homologous recombination (HR). The purification of p53 from this cell exhibited severe losses at each step, compromising analysis by MS. Because of this limited yield from TAP purification, we performed a systematic tagging of p53 with 3xFLAG, Strep-II, and HALO tags, and their combinations, at both N- and C-termini. The latest CRISPR (clustered regularly interspaced short palindromic repeats)/cas9 nuclease system provides major advantages for endogenous gene modification through HR. Efficient site-specific protein tagging (or mutation) requires a DNA break point in the immediate vicinity, limiting the choice of guide DNA (gDNA) positions possible. By extending the complementary sequence between CRISPR targeting RNA (crRNA) and trans-activating crRNA (tracrRNA) in the chimeric single guide RNA (sgRNA), we expanded the essential Protospacer Adjacent Motif (PAM) sequence of the S. pyogenes CRISPR II system from NGG to NAG with equal targeting efficiency. Cas9 nickase (both D10A, and H840A-N854A-N863A) generated more favorable HR rates compared to non-homologous end joining (NHEJ), while mitigating off-target effects, in comparison to wild type cas9. Utilizing our conditions, we achieved 2-8% (for different tags) endogenous p53 tagging in the background of 10-60% NHEJ from wild type cas9 and single sgRNA. With cas9 nickase and two sgRNA, we consistently achieved 0.2-2% specific p53 tagging in the background of 0.01-8% NHEJ, with higher efficiency for 5′ than 3′ single strand overhangs generated. We are currently generating single cell clones, selecting those tags most faithful to natural p53 expression levels, and testing p53 purification efficiencies with different tags. In conclusion, we optimized the CRISPR system, extensively characterized the individual steps in generating single cell clones for endogenous gene tagging, and will apply the optimal tag to purify p53 and its co-eluted binding partners for quantitative MS analysis. Citation Format: Chen Yang, Cortney L. Lawrence, Charles E. Lyons, Shirley M. Taylor, Richard G. Moran. Endogenous p53 affinity tagging with CRISPR. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1213. doi:10.1158/1538-7445.AM2015-1213

Abstract 4803: A novel mitochondrial isoform of mammalian DNA methyltransferase 1

Cancer cells, in contrast to normal cells and tissues, fulfill their energy requirements by increased glycolysis, with a distinct downregulation of oxidative phosphorylation in the mitochondria. Regulation of enzymes of the glycolytic pathway under the anoxic conditions prevalent in human solid tumors by stabilization of HIF1α have been intensely investigated; however, the mechanisms regulating transcription of the mitochondrial genome, and its coordinate regulation with the nuclear genome are less well understood. Eukaryotic mitochondrial DNA carries a low but significant level of cytosine methylation, but molecular mechanisms responsible for the generation of this modification and its role in mitochondrial function has not been studied. The mitochondrial genome encodes 13 polypeptides involved in oxidative phosphorylation, as well as the tRNAs and rRNAs required for mitochondrial translation. All other proteins required for mitochondrial function are encoded by the nuclear genome and translocated to the mitochondria using mitochondrial targeting sequences frequently located at the N-terminus. We have identified a novel isoform of mammalian DNA methyltransferase 1 (DNMT1) that translocates to the mitochondrion, using a conserved N-terminal mitochondrial targeting sequence; the mRNA encoding this isoform is expressed from an upstream transcription start site. De novo methyltransferases DNMT3a and DNMT3b are not found in mammalian mitochondria. Expression of the mitochondrial DNMT1 isoform (mtDNMT1) is regulated by nuclear respiratory factor 1 (NRF1) and coactivator PGC1α, which together upregulate several nuclear-encoded mitochondrial genes involved in the electron transport chain, following hypoxic stress. The mitochondrial isoform (mtDNMT1) is also preferentially upregulated following loss of p53 function, leading to specific effects on mitochondrial gene expression and potentially affecting mitochondrial function. We therefore suggest that mitochondrial DNA methylation, established and maintained by mtDNMT1, plays a role in coordination of mitochondrial gene expression and that the expression of the mtDNMT1 isoform is under the control of p53. 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 4803.