Daniel E. Bauer

Growth Factor Regulation of Autophagy and Cell Survival in the Absence of Apoptosis

In animals, cells are dependent on extracellular signals to prevent apoptosis. However, using growth factor-dependent cells from Bax/Bak-deficient mice, we demonstrate that apoptosis is not essential to limit cell autonomous survival. Following growth factor withdrawal, Bax-/-Bak-/- cells activate autophagy, undergo progressive atrophy, and ultimately succumb to death. These effects result from loss of the ability to take up sufficient nutrients to maintain cellular bioenergetics. Despite abundant extracellular nutrients, growth factor-deprived cells maintain ATP production from catabolism of intracellular substrates through autophagy. Autophagy is essential for maintaining cell survival following growth factor withdrawal and can sustain viability for several weeks. During this time, cells respond to growth factor readdition by rapid restoration of the ability to take up and metabolize glucose and by subsequent recovery of their original size and proliferative potential. Thus, growth factor signal transduction is required to direct the utilization of sufficient exogenous nutrients to maintain cell viability.

Akt stimulates aerobic glycolysis in cancer cells.

Cancer cells frequently display high rates of aerobic glycolysis in comparison to their nontransformed counterparts, although the molecular basis of this phenomenon remains poorly understood. Constitutive activity of the serine/threonine kinase Akt is a common perturbation observed in malignant cells. Surprisingly, although Akt activity is sufficient to promote leukemogenesis in nontransformed hematopoietic precursors and maintenance of Akt activity was required for rapid disease progression, the expression of activated Akt did not increase the proliferation of the premalignant or malignant cells in culture. However, Akt stimulated glucose consumption in transformed cells without affecting the rate of oxidative phosphorylation. High rates of aerobic glycolysis were also identified in human glioblastoma cells possessing but not those lacking constitutive Akt activity. Akt-expressing cells were more susceptible than control cells to death after glucose withdrawal. These data suggest that activation of the Akt oncogene is sufficient to stimulate the switch to aerobic glycolysis characteristic of cancer cells and that Akt activity renders cancer cells dependent on aerobic glycolysis for continued growth and survival.

ATP citrate lyase is an important component of cell growth and transformation

Cell proliferation requires a constant supply of lipids and lipid precursors to fuel membrane biogenesis and protein modification. Cytokine stimulation of hematopoietic cells directly stimulates glucose utilization in excess of bioenergetic demand, resulting in a shift from oxidative to glycolytic metabolism. A potential benefit of this form of metabolism is the channeling of glucose into biosynthetic pathways. Here we report that glucose supports de novo lipid synthesis in growing hematopoietic cells in a manner regulated by cytokine availability and the PI3K/Akt signaling pathway. The net conversion of glucose to lipid is dependent on the ability of cells to produce cytosolic acetyl CoA from mitochondria-derived citrate through the action of ATP citrate lyase (ACL). Stable knockdown of ACL leads to a significant impairment of glucose-dependent lipid synthesis and an elevation of mitochondrial membrane potential. Cells with ACL knockdown display decreased cytokine-stimulated cell proliferation. In contrast, these cells resist cell death induced by either cytokine or glucose withdrawal. However, ACL knockdown significantly impairs Akt-mediated tumorigenesis in vivo. These data suggest that enzymes involved in the conversion of glucose to lipid may be targets for the treatment of pathologic cell growth.

An Erythroid Enhancer of BCL11A Subject to Genetic Variation Determines Fetal Hemoglobin Level

BCL11A Variants Recent chromatin mapping data have suggested that trait-associated variants often mark regulatory DNA. However, there has been little rigorous experimental investigation of regulatory variation. Bauer et al. (p. 253; see the Perspective by Hardison and Blobel) performed an in-depth study of the BCL11A fetal hemoglobin-associated locus. The trait-associated variants revealed a chromatin signature that enhanced erythroid development. The enhancer was required for erythroid expression of BCL11A and thus for globin gene expression. Fine-mapping reveals a promising therapeutic target for genome engineering in the β-hemoglobinopathies. [Also see Perspective by Hardison and Blobel] Genome-wide association studies (GWASs) have ascertained numerous trait-associated common genetic variants, frequently localized to regulatory DNA. We found that common genetic variation at BCL11A associated with fetal hemoglobin (HbF) level lies in noncoding sequences decorated by an erythroid enhancer chromatin signature. Fine-mapping uncovers a motif-disrupting common variant associated with reduced transcription factor (TF) binding, modestly diminished BCL11A expression, and elevated HbF. The surrounding sequences function in vivo as a developmental stage–specific, lineage-restricted enhancer. Genome engineering reveals the enhancer is required in erythroid but not B-lymphoid cells for BCL11A expression. These findings illustrate how GWASs may expose functional variants of modest impact within causal elements essential for appropriate gene expression. We propose the GWAS-marked BCL11A enhancer represents an attractive target for therapeutic genome engineering for the β-hemoglobinopathies.

The glucose dependence of Akt-transformed cells can be reversed by pharmacologic activation of fatty acid β -oxidation

Activation of the oncogenic kinase Akt stimulates glucose uptake and metabolism in cancer cells and renders these cells susceptible to death in response to glucose withdrawal. Here we show that 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) reverses the sensitivity of Akt-expressing glioblastoma cells to glucose deprivation. AICARs protection depends on the activation of AMPK, as expression of a dominant-negative form of AMPK abolished this effect. AMPK is a cellular energy sensor whose activation can both block anabolic pathways such as protein synthesis and activate catabolic reactions such as fatty acid oxidation to maintain cellular bioenergetics. While rapamycin treatment mimicked the effect of AICAR on inhibiting markers of cap-dependent translation, it failed to protect Akt-expressing cells from death upon glucose withdrawal. Compared to control cells, Akt-expressing cells were impaired in the ability to induce fatty acid oxidation in response to glucose deprivation unless stimulated with AICAR. Stimulation of fatty acid oxidation was sufficient to maintain cell survival as activation of fatty acid oxidation with bezafibrate also protected Akt-expressing cells from glucose withdrawal-induced death. Conversely, treatment with a CPT-1 inhibitor to block fatty acid import into mitochondria prevented AICAR from stimulating fatty acid oxidation and promoting cell survival in the absence of glucose. Finally, cell survival did not require reversal of Akts effects on either protein translation or lipid synthesis as the addition of the cell penetrant oxidizable substrate methyl-pyruvate was sufficient to maintain survival of Akt-expressing cells deprived of glucose. Together, these data suggest that activation of Akt blocks the ability of cancer cells to metabolize nonglycolytic bioenergetic substrates, leading to glucose addiction.

Characterization of Genomic Deletion Efficiency Mediated by Clustered Regularly Interspaced Palindromic Repeats (CRISPR)/Cas9 Nuclease System in Mammalian Cells

Background: CRISPR/Cas9-directed cleavages may result in genomic deletion. Results: CRISPR/Cas9-produced genomic deletion frequency is inversely related to deletion size, with large deletions and inversions practicable and biallelic deletions exceeding probabilistic expectation. Conclusion: Biallelic, large genomic deletions are efficiently engineered in mammalian cells by CRISPR/Cas9. Significance: CRISPR/Cas9-mediated genomic deletion represents a robust method for loss-of-function studies in mammalian cells. The clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 nuclease system has provided a powerful tool for genome engineering. Double strand breaks may trigger nonhomologous end joining repair, leading to frameshift mutations, or homology-directed repair using an extrachromosomal template. Alternatively, genomic deletions may be produced by a pair of double strand breaks. The efficiency of CRISPR/Cas9-mediated genomic deletions has not been systematically explored. Here, we present a methodology for the production of deletions in mammalian cells, ranging from 1.3 kb to greater than 1 Mb. We observed a high frequency of intended genomic deletions. Nondeleted alleles are nonetheless often edited with inversions or small insertion/deletions produced at CRISPR recognition sites. Deleted alleles also typically include small insertion/deletions at predicted deletion junctions. We retrieved cells with biallelic deletion at a frequency exceeding that of probabilistic expectation. We demonstrate an inverse relationship between deletion frequency and deletion size. This work suggests that CRISPR/Cas9 is a robust system to produce a spectrum of genomic deletions to allow investigation of genes and genetic elements.

Cytokine stimulation of aerobic glycolysis in hematopoietic cells exceeds proliferative demand

The relationship between growth factor‐dependent cell growth and proliferation and the up‐ regulation of cellular metabolism required to support these processes remains poorly defined. Here, we demonstrate that cell growth, proliferation, and glucose metabolism are coordinately regulated by interleukin‐3 (IL‐3) in cytokine‐dependent cells. Surprisingly, glycolytic activity is stimulated to a greater extent than would be expected based on the rate of cell growth or proliferation. IL‐3 signaling exerts a direct effect on glycolytic commitment independent of cell growth control. These results are not restricted to IL‐3 as the cytokines IL‐7 and IL‐2 have similar effects on glucose metabolism when assayed in factor‐dependent cell lines or primary lymphocytes, respectively. Growth factor stimulation leads cells to consume less oxygen and produce more lactate per glucose, indicative of conversion from oxidative to glycolytic metabolism. The enforced rate of glucose metabolism is in excess of that required to support cell growth; accordingly, if extracellular glucose is reduced, cells retain the ability to grow and proliferate by derepressing oxidative metabolism. These data suggest that the high rate of glycolysis observed in response to growth factor stimulation is a primary effect rather than a homeostatic response to increased cell growth.

Reawakening fetal hemoglobin: prospects for new therapies for the β-globin disorders.

The level of fetal hemoglobin (HbF) modifies the severity of the common β-globin disorders. Knowledge of the normal mechanisms that repress HbF in the adult stage has remained limited until recently despite nearly 3 decades of molecular investigation, in part because of imperfect model systems. Recent studies have provided new insights into the developmental regulation of globin genes and identified specific transcription factors and epigenetic regulators responsible for physiologic silencing of HbF. Most prominent among these regulators is BCL11A, a transcriptional repressor that inhibits adult-stage HbF expression. KLF1 and c-Myb are additional critical HbF-regulating erythroid transcription factors more broadly involved in erythroid gene expression programs. Chromatin modifiers, including histone deacetylases and DNA methyltransferases, also play key roles in orchestrating appropriate globin gene expression. Taken together, these discoveries present novel therapeutic targets for further consideration. Although substantial hurdles remain, opportunities are now rich for the rational design of HbF inducers.

Clinicopathologic Features and Long-Term Outcomes of NUT Midline Carcinoma

Purpose: NUT midline carcinoma (NMC) is a poorly differentiated squamous cancer characterized by rearrangement of the NUT gene. Research advances have provided opportunities for targeted therapy in NMC, yet the clinical features of this rare disease have not been systematically characterized. We report on a large population of such patients to identify the disease characteristics and treatments, correlate them with outcome, and to consider clinical recommendations. Experimental Design: A clinical database was established using retrospective demographic and outcomes data available on all known cases of NMC. Questionnaires were completed by treating physicians. Pathologic, demographic, and clinical variables were assessed for 63 patients, the largest cohort of patients with NMC studied to date. Outcome data from 54 patients were available for survival analyses. Results: The diagnosis of NMC has increased annually since 2007. Since 2009, there has been an observed increase in the age at diagnosis (P < 0.05). Geographic distribution of patients with NMC has been concentrated in the United States (n = 41, 65%). The median overall survival for patients with NMC was 6.7 months. The 2-year progression-free survival (PFS) was 9% with a 95% confidence interval (CI) of 1% to 17% [1-year PFS 15% (5–24%) and 2-year overall survival (OS) was 19% with a 95% CI of 7%–31% (1-year OS: 30% (27–34%)]. Multivariate analysis suggested that extent of surgical resection and initial radiotherapy were independent predictors of PFS and OS. Notably, no chemotherapeutic regimen was associated with improved outcome. Conclusions: NMC portends a poor prognosis among all squamous cell neoplasms and seems to be frequently unrecognized. The finding that conventional chemotherapy has been inadequate indicates a pressing need for the development of targeted therapeutics. Intensive local therapies such as gross total resection and radiotherapy might be associated with enhanced survival. Clin Cancer Res; 18(20); 5773–9. ©2012 AACR.

Combinatorial Assembly of Developmental Stage-Specific Enhancers Controls Gene Expression Programs during Human Erythropoiesis

Gene-distal enhancers are critical for tissue-specific gene expression, but their genomic determinants within a specific lineage at different stages of development are unknown. Here we profile chromatin state maps, transcription factor occupancy, and gene expression profiles during human erythroid development at fetal and adult stages. Comparative analyses of human erythropoiesis identify developmental stage-specific enhancers as primary determinants of stage-specific gene expression programs. We find that erythroid master regulators GATA1 and TAL1 act cooperatively within active enhancers but confer little predictive value for stage specificity. Instead, a set of stage-specific coregulators collaborates with master regulators and contributes to differential gene expression. We further identify and validate IRF2, IRF6, and MYB as effectors of an adult-stage expression program. Thus, the combinatorial assembly of lineage-specific master regulators and transcriptional coregulators within developmental stage-specific enhancers determines gene expression programs and temporal regulation of transcriptional networks in a mammalian genome.