There is high morbidity associated with local recurrence of rectal cancer. However, the adjuvant therapies given to prevent such recurrences also have significant side effects and associated risks. The ability to select patients with the highest risk of recurrence and greatest therapeutic response will improve rectal cancer care.

SUMMARY

AMP-activated protein kinase (AMPK) is activated when the AMP/ATP ratio in cells is elevated due to energy stress. Here we describe a biosensor, AMPKAR, which exhibits enhanced fluorescence resonance energy transfer (FRET) in response to phosphorylation by AMPK, allowing spatio-temporal monitoring of AMPK activity in single cells. We show that this reporter responds to a variety of stimuli that are known to induce energy stress and that the response is dependent on AMPK α1 & α2 and on the upstream kinase, LKB1. Interestingly we found that AMPK activation is confined to the cytosol in response to energy stress but can be observed in both the cytosol and nucleus in response to calcium elevation. Finally, using this probe with U2OS cells in a microfluidics device, we observed a very high cell-to-cell variability in the amplitude and time course of AMPK activation and recovery in response to pulses of glucose deprivation.

Despite their homology, IκB kinase α (IKKα) and IKKβ have divergent roles in NF-κB signaling. IKKβ strongly activates NF-κB while IKKα can downregulate NF-κB under certain circumstances. Given this, identifying independent substrates for these kinases could help delineate their divergent roles. Peptide substrate array technology followed by bioinformatic screening identified TRAF4 as a substrate for IKKα. Like IKKα, TRAF4 is atypical within its family because it is the only TRAF family member to negatively regulate innate immune signaling. IKKα's phosphorylation of serine-426 on TRAF4 was required for this negative regulation. Binding to the Crohn's disease susceptibility protein, NOD2, is required for TRAF4 phosphorylation and subsequent inhibition of NOD2 signaling. Structurally, serine-426 resides within an exaggerated β-bulge in TRAF4 that is not present in the other TRAF proteins, and phosphorylation of this site provides a structural basis for the atypical function of TRAF4 and its atypical role in NOD2 signaling.

SUMMARY

The energy-sensing AMP-activated protein kinase (AMPK) is activated by low nutrient levels. Functions of AMPK, other than its role in cellular metabolism, are just beginning to emerge. Here we use a chemical genetics screen to identify direct substrates of AMPK in human cells. We find that AMPK phosphorylates 28 previously unidentified substrates, several of which are involved in mitosis and cytokinesis. We identify the residues phosphorylated by AMPK in vivo in several substrates, including protein phosphatase 1 regulatory subunit 12C (PPP1R12C) and p21 -activated protein kinase (PAK2). AMPK-induced phosphorylation is necessary for PPP1R12C interaction with 14-3-3 and phosphorylation of myosin regulatory light chain. Both AMPK activity and PPP1R12C phosphorylation are increased in mitotic cells and are important for mitosis completion. These findings suggest that AMPK coordinates nutrient status with mitosis completion, which may be critical for the organism’s response to low nutrients during development, or in adult stem and cancer cells.

Summary

The activities of both mTORC1 and mTORC2 are negatively regulated by their endogenous inhibitor, DEPTOR. As such, the abundance of DEPTOR is a critical determinant in the activity status of the mTOR network. DEPTOR stability is governed by the 26S-proteasome through a largely unknown mechanism. Here we describe an mTOR-dependent phosphorylation-driven pathway for DEPTOR destruction via SCFβ-TRCP. DEPTOR phosphorylation by mTOR in response to growth signals, and in collaboration with casein kinase I (CKI), generates a phosphodegron that binds β-TRCP. Failure to degrade DEPTOR through either degron mutation or β-TRCP depletion leads to reduced mTOR activity, reduced S6 kinase activity, and activation of autophagy to reduce cell growth. This work expands the current understanding of mTOR regulation by revealing a positive feedback loop involving mTOR and CKI-dependent turnover of its inhibitor, DEPTOR, suggesting that misregulation of the DEPTOR destruction pathway might contribute to aberrant activation of mTOR in disease.

Control of intracellular reactive oxygen species (ROS) concentrations is critical for cancer cell survival. We show that, in human lung cancer cells, acute increases in intracellular concentrations of ROS caused inhibition of the glycolytic enzyme pyruvate kinase M2 (PKM2) through oxidation of Cys358. This inhibition of PKM2 is required to divert glucose flux into the pentose phosphate pathway and thereby generate sufficient reducing potential for detoxification of ROS. Lung cancer cells in which endogenous PKM2 was replaced with the Cys358 to Ser358 oxidation-resistant mutant exhibited increased sensitivity to oxidative stress and impaired tumor formation in a xenograft model. Besides promoting metabolic changes required for proliferation, the regulatory properties of PKM2 may confer an additional advantage to cancer cells by allowing them to withstand oxidative stress.

The study of normal mammalian cell growth and the defects that contribute to disease pathogenesis constitutes a fundamental avenue of research that links metabolism to cell growth. Here we visit several aspects of this metabolism, emphasizing recent advances in our understanding of how alterations in glucose metabolism affect cytosolic and mitochondrial redox potential and ATP generation. These alterations drive cell growth not only through supporting biosynthesis, energy metabolism, and maintaining redox potential but also through initiating signaling mechanisms that are still poorly characterized. The evolutionary basis of these additional layers of growth control is also discussed.

PI3K is activated in some cancers by direct mutation, but it is activated more commonly in cancer by mutation of upstream acting receptor tyrosine kinases (TKs). At present, there is no systematic method to determine which TK signaling cascades activate PI3K in certain cancers, despite the likely utility of such information to help guide selection of tyrosine kinase inhibitor (TKI) drug strategies for personalized therapy. Here we present a quantitative tandem mass spectrometry (LC/MS/MS) approach that identifies upstream activators of PI3K both in vitro and in vivo. Using non-small cell lung carcinoma (NSCLC) to illustrate this approach, we demonstrate a correct identification of the mechanism of PI3K activation in several models, thereby identifying the most appropriate TKI to down-regulate PI3K signaling. This approach also determined the molecular mechanisms and adaptors required for PI3K activation following inhibition of the mTOR kinase TORC1. We further validated the approach in breast cancer cells with mutational activation of PIK3CA, where tandem mass spectrometry detected and quantitatively measured the abundance of a helical domain mutant (E545K) of PIK3CA connected to PI3K activation. Overall, our findings establish a mass spectrometric approach to identify functional interactions that govern PI3K regulation in cancer cells. Using this technique to define the pathways which activate PI3K signaling in a given tumor could help inform clinical decision making by helping guide personalized therapeutic strategies for different patients.

GTP-loaded Ras induces multiple signaling pathways by binding to its numerous effectors such as Raf and PI3K. Ras activity can be influenced by activation of Ras-GEFs that stimulate GDP release and GTP loading or by inhibition of Ras-GAPs that stimulate GTP hydrolysis. Here, we report that monoubiquitination of Lys147 within the G domain of wild-type K-Ras, the Ras gene most frequently mutated in cancer, leads to enhanced GTP loading. Furthermore, ubiquitination increases the ability of the oncogenic Gly-12-Val mutant of K-Ras to bind the downstream effectors PI3K and Raf. These results indicate that monoubiquitination both enhances GTP loading on K-Ras and increases its affinity for specific downstream effectors, providing a previously unidentified mechanism for Ras activation.

Phosphopeptide binding domains mediate the directed and localized assembly of protein complexes essential to intracellular kinase signaling. To identify phosphopeptide binding proteins, we developed a proteomic screening method using immobilized partially-degenerate phosphopeptide mixtures combined with SILAC and microcapillary LC/MS/MS. The method was used to identify proteins that specifically bound to phosphorylated peptide library affinity matrices, including pTyr, and the motifs pSer/pThr-Pro, pSer/pThr-X-X-X-pSer/pThr, pSer/pThr-Glu/Asp or pSer/pThr-pSer/pThr in degenerate sequence contexts. Heavy and light SILAC lysates were flowed over columns containing these phosphorylated and non-phosphorylated (control) peptide libraries respectively, and bound proteins were eluted, combined, digested and analyzed by LC/MS/MS using a hybrid quadrupole-TOF mass spectrometer. Heavy:light peptide ion ratios were calculated, and peptides that yielded ratios greater than ~3:1 were considered as being from potential phosphopeptide binding proteins since this ratio represents the lowest ratio from a known positive control. Many of those identified were known phosphopeptide-binding proteins, including the SH2 domain containing p85 subunit of PI3K bound to pTyr, 14-3-3 bound to pSer/pThr-Asp/Glu, polo-box domain containing PLK1 and Pin1 bound to pSer/pThr-Pro and pyruvate kinase M2 binding to pTyr. Approximately half of the hits identified by the peptide library screens were novel. Protein domain enrichment analysis revealed that most pTyr hits contain SH2 domains, as expected and to lesser extent SH3, C1, STAT, Tyr phosphatase, Pkinase, C2 and PH domains, however, pSer/pThr motifs did not reveal enriched domains across hits.

The phox homology (PX) domain is a phosphoinositide-binding module that typically binds phosphatidylinositol 3-phosphate. Out of 47 mammalian proteins containing PX domains, more than 30 are denoted sorting nexins and several of these have been implicated in internalization of cell surface proteins to the endosome, where phosphatidylinositol-3-phosphate is concentrated. Here we investigated a multimodular protein termed PXK, composed of a PX domain, a protein kinase-like domain, and a WASP homology 2 domain. We show that the PX domain of PXK localizes this protein to the endosomal membrane via binding to phosphatidylinositol 3-phosphate. PXK expression in COS7 cells accelerated the ligand-induced internalization and degradation of epidermal growth factor receptors by a mechanism requiring phosphatidylinositol 3-phosphate binding but not involving the WASP homology 2 domain. Conversely, depletion of PXK using RNA interference decreased the rate of epidermal growth factor receptor internalization and degradation. Ubiquitination of epidermal growth factor receptor by the ligand stimulation was enhanced in PXK-expressing cells. These results indicate that PXK plays a critical role in epidermal growth factor receptor trafficking through modulating ligand-induced ubiquitination of the receptor.

In this issue of Science Translational Medicine, Wallin et al. have identified a subset of breast and ovarian cancer cell lines that show synergistic response to the combination of doxorubicin and GDC-0941, a class IA phosphatidylinositol 3-kinase (PI3K) inhibitor. Here, we discuss the potential implications of these data on the clinical development of PI3K pathway inhibitors as cancer therapeutics.

Summary

Previous experiments suggest a connection between the N-alpha-acetylation of proteins and the sensitivity of cells to apoptotic signals. Here, we describe a novel biochemical assay to detect the acetylation status of proteins and demonstrate that protein N-alpha-acetylation is regulated by the availability of acetyl-CoA. Because the anti-apoptotic protein Bcl-xL is known to influence mitochondrial metabolism, we reasoned that Bcl-xL may provide a link between protein N-alpha-acetylation and apoptosis. Indeed, Bcl-xL overexpression leads to a reduction in levels of acetyl-CoA and N-alpha-acetylated proteins in the cell. This effect is independent of Bax and Bak, the known binding partners of Bcl-xL. Increasing cellular levels of acetyl-CoA by addition of acetate or citrate restores protein N-alpha-acetylation in Bcl-xL-expressing cells and confers sensitivity to apoptotic stimuli. We conclude that acetyl-CoA serves as a signaling molecule that couples apoptotic sensitivity to metabolism by regulating protein N-alpha-acetylation.

IKKε and TBK1 are noncanonical IKK family members which regulate inflammatory signaling pathways and also play important roles in oncogenesis. However, few inhibitors of these kinases have been identified. While the substrate specificity of IKKε has recently been described, the substrate specificity of TBK1 is unknown, hindering the development of high-throughput screening technologies for inhibitor identification. Here, we describe the optimal substrate phosphorylation motif for TBK1, and show that it is identical to the phosphorylation motif previously described for IKKε. This information enabled the design of an optimal TBK1/IKKε substrate peptide amenable to high-throughput screening and we assayed a 6,006 compound library that included 4,727 kinase-focused compounds to discover in vitro inhibitors of TBK1 and IKKε. 227 compounds in this library inhibited TBK1 at a concentration of 10 µM, while 57 compounds inhibited IKKε. Together, these data describe a new high-throughput screening assay which will facilitate the discovery of small molecule TBK1/IKKε inhibitors possessing therapeutic potential for both inflammatory diseases and cancer.

ATP citrate lyase (ACL) catalyzes the conversion of cytosolic citrate to acetyl-CoA and oxaloacetate. A definitive role for ACL in tumorigenesis has emerged from ACL RNAi and chemical inhibitor studies, showing that ACL inhibition limits tumor cell proliferation and survival and induces differentiation in vitro. In vivo, it reduces tumor growth leading to a cytostatic effect and induces differentiation. However, the underlying molecular mechanisms are poorly understood and agents that could enhance the efficacy of ACL inhibition have not been identified. Our studies focus on non-small cell lung cancer (NSCLC) lines, which show phosphatidylinositol 3-kinase (PI3K)/AKT activation secondary to a mutation in the K-Ras gene or the EGFR gene. Here we show that ACL knockdown promotes apoptosis and differentiation, leading to the inhibition of tumor growth in vivo. Moreover, in contrast to most studies, which elucidate how activation/ suppression of signaling pathways can modify metabolism, we show that inhibition of a metabolic pathway “reverse signals” and attenuates PI3K/AKT signaling. Additionally, we find that statins, inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, which act downstream of ACL in the cholesterol synthesis pathway, dramatically enhance the anti-tumor effects of ACL inhibition, even regressing established tumors. With statin treatment, both PI3K/AKT and the MAPK pathways are affected. Moreover, this combined treatment is able to reduce the growth of EGF receptor resistant tumor cell types. Given the essential role of lipid synthesis in numerous cancers, this work may impact therapy in a broad range of tumors.

The phosphoinositide-3-kinase (PI3K) family of lipid kinases has been well conserved from yeast to mammals. In this evolutionary perspective on the PI3K family, we discuss the prototypical properties of PI3Ks: 1) the utilization of sparse but specifically localized lipid substrates; 2) the nucleation signaling complexes at membrane-targeted sites; and 3) the integration of intracellular signaling with extracellular cues. Together, these three core properties serve to establish order within the entropic environment of the cell. Many human diseases, including cancer and diabetes, are the direct result of loss or defects in one or more of these core properties, putting much hope in the clinical use of PI3K inhibitors singly and in combination to restore order within diseased tissues.

Summary

Background

Cell-to-cell variability in populations has been widely observed in mammalian cells. This heterogeneity can result from random stochastic events or can be deliberately maintained through regulatory processes. In the latter case, heterogeneity should confer a selective advantage that benefits the entire population.

Results

Using multicolor flow cytometry, we have uncovered robust heterogeneity in PI3K activity in MCF10A cell populations, which had been previously masked by techniques that only measure population averages. We show that AKT activity is bimodal in response to EGF stimulation and correlates with PI3K protein level, such that only cells with high PI3K protein can activate AKT. We further show that heterogeneity in PI3K protein levels is invariably maintained in cell populations through a degradation/re-synthesis cycle that can be regulated by cell density.

Conclusions

Given that the PI3K pathway is one of the most frequently upregulated pathways in cancer, we propose that heterogeneity in PI3K activity is beneficial to normal tissues by restricting PI3K activation to only a subset of cells. This may serve to protect the population as a whole from over-activating the pathway, which can lead to cellular senescence or cancer. Consistent with this, we show that oncogenic mutations in p110α (H1047R and E545K) partially evade this negative regulation, resulting in increased AKT activity in the population.

Phosphoinositide-3-OH kinases (PI3K) are critical regulators of cell metabolism, growth, and survival. In a recent publication in Nature, Jia et al. (2008) identify specific functions of the p110β isoform of PI3K in glucose metabolism, cellular proliferation, and tumorigenesis.

We demonstrate that phosphatidylinositol 3-kinase (PI3K) pathway aberrations occur in >80% of endometrioid endometrial cancers, with coordinate mutations of multiple PI3K pathway members being more common than predicted by chance. PIK3R1 (p85α) mutations occur at a higher rate in endometrial cancer than in any other tumor lineage, and PIK3R2 (p85β), not previously demonstrated to be a cancer gene, is also frequently mutated. The dominant activation event in the PI3K pathway appears to be PTEN protein loss. However, in tumors with retained PTEN protein, PI3K pathway mutations phenocopy PTEN loss, resulting in pathway activation. KRAS mutations are common in endometrioid tumors activating independent events from PI3K pathway aberrations. Multiple PIK3R1 and PIK3R2 mutations demonstrate gain of function including disruption of a novel mechanism of pathway regulation wherein p85α dimers bind and stabilize PTEN. Taken together, the PI3K pathway represents a critical driver of endometrial cancer pathogenesis and a novel therapeutic target.

Double drugs are obtained when two pharmacologically active entities are covalently joined to improve potency. We conjugated the viridin Wm with a self-activating linkage to cetuximab and demonstrated the retention of immunoreactivity by the conjugate. Though cetuximab lacked a growth inhibitory activity against A549 cells, the Wmcetuximab conjugate had an anti-proliferative IC50 of 155 nM in vitro. The chemistry of attaching a self-releasing Wm to clinically approved antibodies is general and, in selected instances, may yield antibody-based double drugs with improved efficacy.

Summary

Tremendous advances in technologies have allowed the attainment of powerful insights into the molecular and genetic determinants that drive human cancers. However, this acquired knowledge has been translated into effective therapeutics very slowly, in part due to difficulty in predicting which drug or drug combination is likely to be effective in the complex mutational background of human cancers. To address this difficulty we have proposed and initiated the “co-clinical trial” project, in which we exploit mouse models that faithfully replicate the variety of mutational events observed in human cancers, to conduct preclinical trials that parallel ongoing human phase I/II clinical trials. Here, we focus on concepts relevant to the application of this novel paradigm and the essential components required for its implementation to ultimately achieve the rational and rapid development of new therapeutic treatments.

Cancer cells have distinct metabolic needs that are different from normal cells and can be exploited for development of anti-cancer therapeutics. Activation of the tumor specific M2 form of pyruvate kinase (PKM2) is a potential strategy for returning cancer cells to a metabolic state characteristic of normal cells. Here, we describe activators of PKM2 based upon a substituted thieno[3,2-b]pyrrole[3,2-d]pyridazinone scaffold. The synthesis of these agents, structure activity relationships, analysis of activity at related targets (PKM1, PKR and PKL) and examination of aqueous solubility are investigated. These agents represent the second reported chemotype for activation of PKM2.