Shafiq U. Ahmed

Glutamine synthetase activity fuels nucleotide biosynthesis and supports growth of glutamine-restricted glioblastoma.

L-Glutamine (Gln) functions physiologically to balance the carbon and nitrogen requirements of tissues. It has been proposed that in cancer cells undergoing aerobic glycolysis, accelerated anabolism is sustained by Gln-derived carbons, which replenish the tricarboxylic acid (TCA) cycle (anaplerosis). However, it is shown here that in glioblastoma (GBM) cells, almost half of the Gln-derived glutamate (Glu) is secreted and does not enter the TCA cycle, and that inhibiting glutaminolysis does not affect cell proliferation. Moreover, Gln-starved cells are not rescued by TCA cycle replenishment. Instead, the conversion of Glu to Gln by glutamine synthetase (GS; cataplerosis) confers Gln prototrophy, and fuels de novo purine biosynthesis. In both orthotopic GBM models and in patients, 13C–glucose tracing showed that GS produces Gln from TCA-cycle-derived carbons. Finally, the Gln required for the growth of GBM tumours is contributed only marginally by the circulation, and is mainly either autonomously synthesized by GS-positive glioma cells, or supplied by astrocytes.

JNK2-dependent regulation of SIRT1 protein stability

Mammalian SIRT1 is an NAD-dependent deacetylase with critical roles in the maintenance of homeostasis and cell survival. Elevated levels of SIRT1 protein are evident in cancer in which SIRT1 can function as a cancer-specific survival factor. Here we demonstrate that elevated SIRT1 protein in human cells is not attributable to increased SIRT1 mRNA levels but, instead, reflects SIRT1 protein stability. RNAi-mediated depletion of JNK2 reduced the half-life of SIRT1 protein from > 9h to < 2h and this correlated with lack of SIRT1 protein phosphorylation at serine 27. In contrast, depletion of JNK1 had no effect upon SIRT1 protein stability and SIRT1 phosphorylation at serine 47 showed no correlation with SIRT1 protein stability. Thus we show that JNK2 is linked, directly or indirectly, with SIRT1 protein stability and that this function is coupled with SIRT1 phosphorylation at serine 27. Our observations identify a route for therapeutic modulation of SIRT1 protein levels in SIRT1-linked diseases including cancer, neurodegeneration and diabetes.

Limited Mitochondrial Permeabilization Causes DNA Damage and Genomic Instability in the Absence of Cell Death

Summary During apoptosis, the mitochondrial outer membrane is permeabilized, leading to the release of cytochrome c that activates downstream caspases. Mitochondrial outer membrane permeabilization (MOMP) has historically been thought to occur synchronously and completely throughout a cell, leading to rapid caspase activation and apoptosis. Using a new imaging approach, we demonstrate that MOMP is not an all-or-nothing event. Rather, we find that a minority of mitochondria can undergo MOMP in a stress-regulated manner, a phenomenon we term “minority MOMP.” Crucially, minority MOMP leads to limited caspase activation, which is insufficient to trigger cell death. Instead, this caspase activity leads to DNA damage that, in turn, promotes genomic instability, cellular transformation, and tumorigenesis. Our data demonstrate that, in contrast to its well-established tumor suppressor function, apoptosis also has oncogenic potential that is regulated by the extent of MOMP. These findings have important implications for oncogenesis following either physiological or therapeutic engagement of apoptosis.

Isoindolinone Inhibitors of the Murine Double Minute 2 (MDM2)-p53 Protein-Protein Interaction: Structure-Activity Studies Leading to Improved Potency

Inhibition of the MDM2-p53 interaction has been shown to produce an antitumor effect, especially in MDM2 amplified tumors. The isoindolinone scaffold has proved to be versatile for the discovery of MDM2-p53 antagonists. Optimization of previously reported inhibitors, for example, NU8231 (7) and NU8165 (49), was guided by MDM2 NMR titrations, which indicated key areas of the binding interaction to be explored. Variation of the 2-N-benzyl and 3-alkoxy substituents resulted in the identification of 3-(4-chlorophenyl)-3-((1-(hydroxymethyl)cyclopropyl)methoxy)-2-(4-nitrobenzyl)isoindolin-1-one (74) as a potent MDM2-p53 inhibitor (IC(50) = 0.23 ± 0.01 μM). Resolution of the enantiomers of 74 showed that potent MDM2-p53 activity primarily resided with the (+)-R-enantiomer (74a; IC(50) = 0.17 ± 0.02 μM). The cellular activity of key compounds has been examined in cell lines with defined p53 and MDM2 status. Compound 74a activates p53, MDM2, and p21 transcription in MDM2 amplified cells and shows moderate selectivity for wild-type p53 cell lines in growth inhibition assays.

Selective inhibition of parallel DNA damage response pathways optimizes radiosensitization of glioblastoma stem-like cells

Glioblastoma is the most common form of primary brain tumor in adults and is essentially incurable. Despite aggressive treatment regimens centered on radiotherapy, tumor recurrence is inevitable and is thought to be driven by glioblastoma stem-like cells (GSC) that are highly radioresistant. DNA damage response pathways are key determinants of radiosensitivity but the extent to which these overlapping and parallel signaling components contribute to GSC radioresistance is unclear. Using a panel of primary patient-derived glioblastoma cell lines, we confirmed by clonogenic survival assays that GSCs were significantly more radioresistant than paired tumor bulk populations. DNA damage response targets ATM, ATR, CHK1, and PARP1 were upregulated in GSCs, and CHK1 was preferentially activated following irradiation. Consequently, GSCs exhibit rapid G2-M cell-cycle checkpoint activation and enhanced DNA repair. Inhibition of CHK1 or ATR successfully abrogated G2-M checkpoint function, leading to increased mitotic catastrophe and a modest increase in radiation sensitivity. Inhibition of ATM had dual effects on cell-cycle checkpoint regulation and DNA repair that were associated with greater radiosensitizing effects on GSCs than inhibition of CHK1, ATR, or PARP alone. Combined inhibition of PARP and ATR resulted in a profound radiosensitization of GSCs, which was of greater magnitude than in bulk populations and also exceeded the effect of ATM inhibition. These data demonstrate that multiple, parallel DNA damage signaling pathways contribute to GSC radioresistance and that combined inhibition of cell-cycle checkpoint and DNA repair targets provides the most effective means to overcome radioresistance of GSC.

Abrogation of radioresistance in glioblastoma stem-like cells by inhibition of ATM kinase

Resistance to radiotherapy in glioblastoma (GBM) is an important clinical problem and several authors have attributed this to a subpopulation of GBM cancer stem cells (CSCs) which may be responsible for tumour recurrence following treatment. It is hypothesised that GBM CSCs exhibit upregulated DNA damage responses and are resistant to radiation but the current literature is conflicting. We investigated radioresistance of primary GBM cells grown in stem cell conditions (CSC) compared to paired differentiated tumour cell populations and explored the radiosensitising effects of the ATM inhibitor KU‐55933.

SIRT1 Undergoes Alternative Splicing in a Novel Auto-Regulatory Loop with p53

Background The NAD-dependent deacetylase SIRT1 is a nutrient-sensitive coordinator of stress-tolerance, multiple homeostatic processes and healthspan, while p53 is a stress-responsive transcription factor and our paramount tumour suppressor. Thus, SIRT1-mediated inhibition of p53 has been identified as a key node in the common biology of cancer, metabolism, development and ageing. However, precisely how SIRT1 integrates such diverse processes remains to be elucidated. Methodology/Principal Findings Here we report that SIRT1 is alternatively spliced in mammals, generating a novel SIRT1 isoform: SIRT1-ΔExon8. We show that SIRT1-ΔExon8 is expressed widely throughout normal human and mouse tissues, suggesting evolutionary conservation and critical function. Further studies demonstrate that the SIRT1-ΔExon8 isoform retains minimal deacetylase activity and exhibits distinct stress sensitivity, RNA/protein stability, and protein-protein interactions compared to classical SIRT1-Full-Length (SIRT1-FL). We also identify an auto-regulatory loop whereby SIRT1-ΔExon8 can regulate p53, while in reciprocal p53 can influence SIRT1 splice variation. Conclusions/Significance We characterize the first alternative isoform of SIRT1 and demonstrate its evolutionary conservation in mammalian tissues. The results also reveal a new level of inter-dependency between p53 and SIRT1, two master regulators of multiple phenomena. Thus, previously-attributed SIRT1 functions may in fact be distributed between SIRT1 isoforms, with important implications for SIRT1 functional studies and the current search for SIRT1-activating therapeutics to combat age-related decline.

Basal cancer cell survival involves JNK2 suppression of a novel JNK1/c-Jun/Bcl-3 apoptotic network.

Background The regulation of apoptosis under basal (non-stress) conditions is crucial for normal mammalian development and also for normal cellular turnover in different tissues throughout life. Deficient regulation of basal apoptosis, or its perturbation, can result in impaired development and/or disease states including cancer. In contrast to stress-induced apoptosis the regulation of apoptosis under basal conditions is poorly understood. To address this issue we have compared basal- and stress-induced apoptosis in human epithelial cells of normal and cancerous origins. For this purpose we focussed our study on the opposing pro-apoptotic JNK/anti-apoptotic NFκB pathways. Methodology/Principal Findings Combinatorial RNAi plus gene knockout were employed to access and map basal regulatory pathways of apoptosis. Follow-on, in-depth analyses included exogenous expression of phosphorylation mutants and chromatin immunoprecipitation. We demonstrate that basal apoptosis is constitutively suppressed by JNK2 in a range of human cancer cell lines. This effect was not observed in non-cancer cells. Silencing JNK2 by RNAi resulted in JNK1-dependent apoptosis of cancer cells via up-regulation of the AP-1 factor c-Jun. Unexpectedly we discovered that JNK1 and c-Jun promote basal apoptosis in the absence of “activating phosphorylations” typically induced by stress. Hypo-phosphorylated c-Jun accumulated to high levels following JNK2 silencing, auto-regulated its own expression and suppressed expression of Bcl-3, an unusual IκB protein and regulator of NFκB. Basal apoptosis was mediated by components of the TNFα response pathway but was mechanistically distinct from TNFα-induced apoptosis. Conclusions/Significance Our results demonstrate that mechanistically distinct pathways operate to regulate apoptosis in mammalian cells under basal (physiological) versus stress-induced conditions. We also describe a novel apoptotic network which governs the basal survival of cancer cells. Such information is crucial for understanding normal cellular turnover during mammalian development and subsequently throughout life. This information also opens new avenues for therapeutic intervention in human proliferative disease states including cancer.

A Deacetylase-Deficient SIRT1 Variant Opposes Full-Length SIRT1 in Regulating Tumor Suppressor p53 and Governs Expression of Cancer-Related Genes

ABSTRACT SIRT1 is an NAD-dependent deacetylase and epigenetic regulator essential for normal mammalian development and homeostasis. Here we describe a human SIRT1 splice variant, designated SIRT1-Δ2/9, in which the deacetylase coding sequence is lost due to splicing between exons 2 and 9. This work aimed to determine if SIRT1-Δ2/9 is a novel functional product of the SIRT1 gene. Endogenous SIRT1-Δ2/9 protein was identified in human cell lysate by immunoblotting and splice variant-specific RNA interference (RNAi). SIRT1-Δ2/9 mRNA is bound by CUGBP2, which downregulates its translation. Using pulldown assays, we demonstrate that SIRT1-Δ2/9 binds p53 protein. SIRT1-Δ2/9 maintains basal p53 protein levels and supports p53 function in response to DNA damage, as evidenced by RNAi-mediated depletion of SIRT1-Δ2/9 prior to damage. In turn, basal p53 downregulates SIRT1-Δ2/9 RNA levels, while stress-activated p53 eliminates SIRT1-Δ2/9. Loss of wild-type (wt) p53 has been correlated with overexpression of SIRT1-Δ2/9 in a range of human cancers. Exogenous SIRT1-Δ2/9 protein associates with specific promoters in chromatin and can regulate cancer-related gene expression, as evidenced by chromatin immunoprecipitation analysis and RNAi/genomic array data. SIRT1 is of major therapeutic importance, and potential therapeutic drugs are screened against SIRT1 deacetylase activity. Our discovery of SIRT1-Δ2/9 identifies a new, deacetylase-independent therapeutic target for SIRT1-related diseases, including cancer.

Diaryl- and triaryl-pyrrole derivatives: inhibitors of the MDM2–p53 and MDMX–p53 protein–protein interactions

Triarylpyrroles e.g. 4c and 4s inhibit the MDM2–p53 and MDMX–p53 protein–protein interactions.