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.

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.

To determine the role of CD8+ T cells in controlling simian immunodeficiency virus (SIV) replication in vivo, we examined the effect of depleting this cell population using an anti-CD8 monoclonal antibody, OKT8F. There was on average a 99.9% reduction of CD8 cells in peripheral blood in six infected Macaca mulatta treated with OKT8F. The apparent CD8 depletion started 1 h after antibody administration, and low CD8 levels were maintained until day 8. An increase in plasma viremia of one to three orders of magnitude was observed in five of the six macaques. The injection of a control antibody to an infected macaque did not induce a sustained viral load increase, nor did it significantly reduce the number of CD8+ T cells. These results demonstrate that CD8 cells play a crucial role in suppressing SIV replication in vivo.

Many tumors display a high rate of glucose utilization, as evidenced by 18-F-2-deoxyglucose PET imaging. One potential advantage of catabolizing glucose through glycolysis at a rate that exceeds bioenergetic need is that the growing cell can redirect the excess glycolytic end product pyruvate toward lipid synthesis. Such de novo lipid synthesis is necessary for membrane production and lipid-based posttranslational modification of proteins. A key enzyme linking glucose metabolism to lipid synthesis is ATP citrate lyase (ACL), which catalyzes the conversion of citrate to cytosolic acetyl-CoA. ACL inhibition by RNAi or the chemical inhibitor SB-204990 limits in vitro proliferation and survival of tumor cells displaying aerobic glycolysis. The same treatments also reduce in vivo tumor growth and induce differentiation.

Summary Enhancers, critical determinants of cellular identity, are commonly identified by correlative chromatin marks and gain-of-function potential, though only loss-of-function studies can demonstrate their requirement in the native genomic context. Previously we identified an erythroid enhancer of BCL11A, subject to common genetic variation associated with fetal hemoglobin (HbF) level, whose mouse ortholog is necessary for erythroid BCL11A expression. Here we develop pooled CRISPR-Cas9 guide RNA libraries to perform in situ saturating mutagenesis of the human and mouse enhancers. This approach reveals critical minimal features and discrete vulnerabilities of these enhancers. Despite conserved function of the composite enhancers, their architecture diverges. The crucial human sequences appear primate-specific. Through editing of primary human progenitors and mouse transgenesis, we validate the BCL11A erythroid enhancer as a target for HbF reinduction. The detailed enhancer map will inform therapeutic genome editing. The screening approach described here is generally applicable to functional interrogation of noncoding genomic elements.

Necrosis has been considered a passive form of cell death in which the cell dies as a result of a bioenergetic catastrophe imposed by external conditions. However, in response to alkylating DNA damage, cells undergo necrosis as a self-determined cell fate. This form of death does not require the central apoptotic mediators p53, Bax/Bak, or caspases and actively induces an inflammatory response. Necrosis in response to DNA damage requires activation of the DNA repair protein poly(ADP-ribose) polymerase (PARP), but PARP activation is not sufficient to determine cell fate. Cell death is determined by the effect of PARP-mediated ␤-nicotinamide adenine dinucleotide (NAD) consumption on cellular metabolism. Cells using aerobic glycolysis to support their bioenergetics undergo rapid ATP depletion and death in response to PARP activation. In contrast, cells catabolizing nonglucose substrates to maintain oxidative phosphorylation are resistant to ATP depletion and death in response to PARP activation. Because most cancer cells maintain their ATP production through aerobic glycolysis, these data may explain the molecular basis by which DNA-damaging agents can selectively induce tumor cell death independent of p53 or Bcl-2 family proteins.

Applications of adenine base editors (ABEs) have been constrained by the limited compatibility of the deoxyadenosine deaminase component with Cas homologs other than SpCas9. We evolved the deaminase component of ABE7.10 using phage-assisted non-continuous and continuous evolution (PANCE and PACE), resulting in ABE8e. ABE8e contains eight additional mutations that increase activity (k app ) 590-fold compared with ABE7.10. ABE8e offers substantially improved editing efficiencies when paired with a variety of Cas9 or Cas12 homologs. ABE8e is more processive than ABE7.10, which could benefit screening, disrupting regulatory regions and multiplex base editing applications. A modest increase in Cas9-dependent and -independent DNA off-target editing, and in transcriptome-wide RNA off-target editing can be ameliorated by introducing additional mutations in the TadA-8e domain. Finally, we show that ABE8e can efficiently edit natural mutations in a GATA1 binding site in the BCL11A enhancer or the HBG promoter in human cells, targets which were poorly edited with ABE7.10. ABE8e broadens the effectiveness and applicability of adenine base editing.

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 NMC patients 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 NMC patients 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% CI of 1%–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 appears 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.