Tumor development is characterized by an initial phase of rapid expansion, followed by a period of slowed growth as the proliferating malignant cells outstrip the local supply of oxygen and nutrients. In the absence of a dedicated blood supply, early-stage tumors attain steady-state volumes of only a few cubic millimeters, at which time the rate of cell death, due to oxygen and nutrient depletion, equals the rate of cell division (19). To resume growth, these microtumors must adapt to hypoxic stress through alterations in cellular metabolism and the stimulation of neovascularization, which provides the additional blood needed to sustain cellular proliferation. Accordingly, cellular adaptation to growth during hypoxic stress contributes to malignant progression and is correlated with a poor clinical outcome in several types of cancer (3, 4, 18). Two hallmark features of hypoxic adaptation are increased rates of anaerobic glycolysis and the secretion of proangiogenic factors, such as vascular endothelial growth factors (VEGFs) (28, 39). The molecular mechanisms that underlie cellular responses to hypoxic stress are therefore of considerable relevance to cancer biology and therapy.A key regulator of the cellular response to oxygen deprivation is the transcription factor, hypoxia-inducible factor 1 (HIF-1). Originally identified as an oxygen-responsive activator of erythropoietin gene transcription, HIF-1 is now known to play a central role in the maintenance of oxygen homeostasis in virtually all bodily tissues (42, 43). The predominant form of HIF-1 is a heterodimer consisting of HIF-1␣ and HIF-1␤ subunits, both of which are members of the basic helix-loop-helix family of transcription factors. Although HIF-1␤ is a constitutively expressed nuclear protein, the expression of the HIF-1␣ subunit is tightly coupled to the ambient oxygen tension. Under normoxic conditions, the HIF-1␣ gene is continuously transcribed and translated; however, the HIF-1␣ protein is expressed at very low levels due to rapid destruction via the ubiquitin-proteasome pathway. In addition to its DNA-binding and transactivating motifs, HIF-1␣ contains a stretch of ca. 200 amino acids, termed the oxygen-dependent degradation (ODD) domain. As its name implies, the ODD domain mediates the interaction between HIF-1␣ and the E3 ubiquitin ligase complex that mediates continuous poly ubiquitination of HIF-1␣ in normoxic cells.The oxygen-dependent turnover of HIF-1␣ is governed by a novel family of prolyl 4-hydroxylases (PHDs) that specifically modify HIF-1␣ at two conserved proline residues (Pro-402 and Pro-564), both located in the ODD domain (5,15,27,41). Prolyl hydroxylation triggers the recognition of HIF-1␣ by the product of the VHL tumor suppressor gene, which serves as the targeting subunit of an E3 ubiquitin ligase complex (20). Although the exact mechanism remains unclear, a decrease in ambient oxygen tension leads to a correlative decrease in HIF-1␣ prolyl hydroxylation, which in turn leads to decreased rates of HIF-1␣ polyubiquitination and...

Summary The dynamic and reversible acetylation of proteins catalyzed by histone acetyltransferases (HATs) and histone deacetylases (HDACs) is a major epigenetic regulatory mechanism of gene transcription 1 associated with multiple diseases. While HDAC inhibitors are approved to treat certain cancers, progress on the development of drug-like HAT inhibitors has lagged 2. The HAT paralogs p300 and CBP (p300/CBP) are key transcriptional co-activators essential for a multitude of cellular processes and also implicated in human pathological conditions, including cancer 3. Current p300/CBP HAT domain inhibitors including natural products, 4 bi-substrate analogs (Lys-CoA) 5 and the widely utilized C646 6, 7 lack potency or selectivity. Here, we describe A-485, a potent, selective and drug-like p300/CBP catalytic inhibitor. We show the first high resolution (1.95Å) co-crystal structure of a small molecule bound to the catalytic active site of p300 and demonstrate that A-485 is acetyl-CoA competitive. A-485 selectively inhibited proliferation across lineage-specific tumor types, including several hematological malignancies and androgen receptor-positive prostate cancer. A-485 inhibited the androgen receptor transcriptional program in both androgen sensitive and castrate resistant prostate cancer and inhibited tumor growth in a castration resistant xenograft model. These results demonstrate the feasibility of selectively targeting the catalytic activity of histone acetyltransferases.

The mammalian target of rapamycin (mTOR) coordinates cell growth with the growth factor and nutrient/ energy status of the cell. The phosphatidylinositol 3-kinase-AKT pathway is centrally involved in the transmission of mitogenic signals to mTOR. Previous studies have shown that mTOR is a direct substrate for the AKT kinase and identified Ser-2448 as the AKT target site in mTOR. In this study, we demonstrate that rapamycin, a specific inhibitor of mTOR function, blocks serum-stimulated Ser-2448 phosphorylation and that this drug effect is not explained by the inhibition of AKT. Furthermore, the phosphorylation of Ser-2448 was dependent on mTOR kinase activity, suggesting that mTOR itself or a protein kinase downstream from mTOR was responsible for the modification of Ser-2448. Here we show that p70S6 kinase phosphorylates mTOR at Ser-2448 in vitro and that ectopic expression of rapamycin-resistant p70S6 kinase restores Ser-2448 phosphorylation in rapamycin-treated cells. In addition, we show that cellular amino acid status, which modulates p70S6 kinase (S6K1) activity via the TSC/Rheb pathway, regulates Ser-2448 phosphorylation. Finally, small interfering RNA-mediated depletion of p70S6 kinase reduces Ser-2448 phosphorylation in cells. Taken together, these results suggest that p70S6 kinase is a major effector of mTOR phosphorylation at Ser-2448 in response to both mitogen-and nutrient-derived stimuli.The mammalian target of rapamycin (mTOR) 1 is a member of the phosphatidylinositol 3 (PI-3)-kinase-related kinase family (PIKK), which includes ATM, ATR, hSMG-1, and DNA-PK (1-3). These large Ser/Thr protein kinases play essential roles in cellular responses to growth factors and stress. In particular, mTOR plays a critical role in coordinating cell growth with growth factor inputs as well as cellular nutrient and energy status.The bacterially derived macrolide ester, rapamycin, is clinically approved as an immunosuppressant and shows promising anti-tumor activity. Rapamycin, when complexed with FKBP-12 (FK506-binding protein, 12 kDa), binds specifically to mTOR at a conserved stretch of ϳ100 amino acids termed the FKBP-12⅐ra-pamycin binding domain (4). Mutation of a critical serine residue (Ser-2035) in the FKBP-12⅐rapamycin binding domain to a more bulky amino acid, such as isoleucine, abrogates FKBP-12⅐rapa-mycin binding to mTOR, and generates a rapamycin-resistant form of mTOR (4, 5). The mechanism by which rapamycin inhibits mTOR function remains poorly understood.Recent studies have provided significant insights into the growth factor and nutrient signaling pathway(s) upstream of mTOR. Stimulation of many growth factor receptors leads to activation of the PI-3 kinase-AKT pathway, and it appears that this pathway is centrally involved in the coupling of mitogenic stimuli to mTOR. Moreover, loss of the tumor suppressor PTEN provides a powerful signal for tumor progression and simultaneously confers increased sensitivity to the anti-proliferative effect of rapamycin (6, 7). Nutrient/energy status also regulat...

The Chk1 kinase is a major effector of S phase checkpoint signaling during the cellular response to genotoxic stress. Here, we report that replicative stress induces the polyubiquitination and degradation of Chk1 in human cells. This response is triggered by phosphorylation of Chk1 at Ser-345, a known target site for the upstream activating kinase ATR. The ubiquitination of Chk1 is mediated by E3 ligase complexes containing Cul1 or Cul4A. Treatment of cells with the anticancer agent camptothecin (CPT) triggers Chk1 destruction, which blocks recovery from drug-induced S phase arrest and leads to cell death. These findings indicate that ATR-dependent phosphorylation of Chk1 delivers a signal that both activates Chk1 and marks this protein for proteolytic degradation. Proteolysis of activated Chk1 may promote checkpoint termination under normal conditions, and may play an important role in the cytotoxic effects of CPT and related anticancer drugs.

Metabolic rewiring is an established hallmark of cancer, but the details of this rewiring at a systems level are not well characterized. Here we acquire this insight in a melanoma cell line panel by tracking metabolic flux using isotopically labeled nutrients. Metabolic profiling and flux balance analysis were used to compare normal melanocytes to melanoma cell lines in both normoxic and hypoxic conditions. All melanoma cells exhibited the Warburg phenomenon; they used more glucose and produced more lactate than melanocytes. Other changes were observed in melanoma cells that are not described by the Warburg phenomenon. Hypoxic conditions increased fermentation of glucose to lactate in both melanocytes and melanoma cells (the Pasteur effect). However, metabolism was not strictly glycolytic, as the tricarboxylic acid (TCA) cycle was functional in all melanoma lines, even under hypoxia. Furthermore, glutamine was also a key nutrient providing a substantial anaplerotic contribution to the TCA cycle. In the WM35 melanoma line glutamine was metabolized in the "reverse" (reductive) direction in the TCA cycle, particularly under hypoxia. This reverse flux allowed the melanoma cells to synthesize fatty acids from glutamine while glucose was primarily converted to lactate. Altogether, this study, which is the first comprehensive comparative analysis of metabolism in melanoma cells, provides a foundation for targeting metabolism for therapeutic benefit in melanoma.Metabolism in cancer cells differs from that of normal nonproliferative cells. Perhaps the most common variation from the norm in cancer metabolism is "aerobic glycolysis" or the Warburg effect. Under the Warburg effect, metabolism of glucose is largely fermentative rather than respiratory, with increased production of lactate, in normal atmospheric oxygen conditions (1). This is also associated with increased uptake of glucose, a common characteristic of cancers detectable in tumors in patients via 18 F-deoxyglucose-PET (2). However, the extent to which the Warburg effect represents a rebalancing of metabolism (increasing fermentation while decreasing respiration) versus an amplification of metabolism (increasing fermentation while maintaining, or even increasing, respiration) is the subject of debate (3, 4). The Warburg effect contrasts with the Pasteur effect, in that the latter describes the switch from fermentation to respiration when oxygen is plentiful, and its reversal when oxygen is limiting (5), while the Warburg effect describes fermentative activity of cancer cells irrespective of oxygen. In the progression of tumors, cancer cells are subject to a range of oxygen concentrations, and low oxygen induces hypoxia-inducible factor (HIF), 2 which leads to a metabolic rewiring of cancer cells, resulting in a more glycolytic metabolism (6). Therefore, cancer cells may potentially demonstrate both Warburg and Pasteur effects. Furthermore, beyond glycolysis, altered oncogene expression has strong effects on other branches of central carbon metabolism. For insta...