Phosphatidylinositol 3-kinase-related Kinase Research Articles

Transcriptional Elongation Requires DNA Double Stand Break and Repair

TRIM28 plays a key role in transcriptional regulation by controlling RNA polymerase II entry from promoter-proximal pausing into processive elongation. We have shown that transcriptional activation involves TRIM28 phosphorylation by members of the phosphatidylinositol 3-kinase-related kinases (PIKKs) such as DNA-PK and ATM at HSPA1B. Here, we show that DNA double strand break (DSB) and repair proteins such as Topoisomerase IIB, phosphorylated TRIM28 (S824), phosphorylated DNA-PK (T2609), Ku70, and gH2AX, were rapidly accumulated at activated paused genes in vivo. Transcriptional activation-induced DSB was indicated by enrichment of γH2AX and activated DNA repair enzymes through in vivo cell based and genome-wide analyses and visualized through in vitro biochemical analysis. Our data reveal a novel transcriptional activation mechanism involving rapid DSB and repair.

The role of epigenomics in the neurodegeneration of ataxia-telangiectasia.

Ataxia-telangiectasia (A-T) is a multisystem disease characterized by neurodegeneration in the CNS [1–5]. The earliest and most profound neuropathology involves the Purkinje and granule cells of the cerebellum. A-T is caused by mutation of the A-T mutated (ATM) gene, which is ubiquitously expressed throughout development and encodes a serine/threonine protein kinase of the phosphatidylinositol-3 kinase-related kinase family [6]. A-T is classified as a rare neurodegenerative disease, mainly impacting on cerebellum integrity and functioning, resulting in a progressive deterioration of motor functional capabilities [5]. Moreover, since there is currently no effective treatment to cure or even slow down the rate of cerebellar neurodegeneration, A-T significantly decreases life quality of those suffering from it. The best-known function of ATM is to ensure the integrity of the genome. After DNA damage, ATM activates cell cycle checkpoints, arresting the cycle until DNA repair is complete [7]. Its role in maintaining the health and survival of neurons is complex. Consistent with its function in DNA damage repair [8], ATM protects postmitotic neurons from degeneration by suppressing the cell cycle [9–11]. While non-neurological phenotypes are also found, including immune system defects, germ cell defects, hypersensitivity to ionizing radiation and increased susceptibility to, it is the origins of the CNS motor phenotypes of A-T that are the most poorly understood. Yet the pathway that leads from these defects to neuronal cell loss and the other classical neurological phenotypes remains unknown. Epigenetic systems, heritable changes in gene expression that do not involve coding sequence modifications, include DNA methylation, histone modifications and chromatin remodeling, and noncoding RNA regulation. These types of changes have been proposed to be responsible for controlling the expression and function of genes and have emerged as important mediators of development and aging [12–19]. Several diseases are known to have a multifactorial origin, depending not only on genetic but also on environmental factors. They are called ‘complex disorders’ and include cardiovascular disease, cancer, diabetes, and neuropsychiatric and neurodegenerative diseases [20–26]. The neurological disorder most intensely studied with regard to epigenetic changes is Rett syndrome; patients with Rett syndrome have neurodevelopmental defects associated with mutations in MeCP2, which encodes the methyl CpG binding protein 2, that binds to methylated DNA. Other mental retardation disorders are also linked to the disruption of genes involved in epigenetic mechanisms; such disorders include α thalassaemia/mental retardation X-linked syndrome, Rubinstein–Taybi syndrome, and Coffin–Lowry syndrome [26]. Due to epigenetic variability among different neuronal cell types and epigenetic instability during development and maturation of neurons, whether specific epigenetic system is involved in the vulnerability of ATM-deficient Purkinje cell with a large and pale nuclei will be helpful to understand A-T neurodegeneration. In this review, we place emphasis both on addressing nuclear accumulation of histone The role of epigenomics in the neurodegeneration of ataxia-telangiectasia

Substrate recognition and function of the R2TP complex in response to cellular stress.

The R2TP complex is a HSP90 co-chaperone, which consists of four subunits: PIH1D1, RPAP3, RUVBL1, and RUVBL2. It is involved in the assembly of large protein or protein–RNA complexes such as RNA polymerase, small nucleolar ribonucleoproteins (snoRNPs), phosphatidylinositol 3 kinase-related kinases (PIKKs), and their complexes. While RPAP3 has a HSP90 binding domain and the RUVBLs comprise ATPase activities important for R2TP functions, PIH1D1 contains a PIH-N domain that specifically recognizes phosphorylated substrates of the R2TP complex. In this review we provide an overview of the current knowledge of the R2TP complex with the focus on the recently identified structural and mechanistic features of the R2TP complex functions. We also discuss the way R2TP regulates cellular response to stress caused by low levels of nutrients or by DNA damage and its possible exploitation as a target for anti-cancer therapy.

Depletion of ATR selectively sensitizes ATM-deficient human mammary epithelial cells to ionizing radiation and DNA-damaging agents

DNA damage response (DDR) to double strand breaks is coordinated by 3 phosphatidylinositol 3-kinase-related kinase (PIKK) family members: the ataxia-telangiectasia mutated kinase (ATM), the ATM and Rad3-related (ATR) kinase and the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs). ATM and ATR are central players in activating cell cycle checkpoints and function as an active barrier against genome instability and tumorigenesis in replicating cells. Loss of ATM function is frequently reported in various types of tumors, thus placing more reliance on ATR for checkpoint arrest and cell survival following DNA damage. To investigate the role of ATR in the G2/M checkpoint regulation in response to ionizing radiation (IR), particularly when ATM is deficient, cell lines deficient of ATM, ATR, or both were generated using a doxycycline-inducible lentiviral system. Our data suggests that while depletion of ATR or ATM alone in wild-type human mammary epithelial cell cultures (HME-CCs) has little effect on radiosensitivity or IR-induced G2/M checkpoint arrest, depletion of ATR in ATM-deficient cells causes synthetic lethality following IR, which correlates with severe G2/M checkpoint attenuation. ATR depletion also inhibits IR-induced autophagy, regardless of the ATM status, and enhances IR-induced apoptosis particularly when ATM is deficient. Collectively, our results clearly demonstrate that ATR function is required for the IR-induced G2/M checkpoint activation and subsequent survival of cells with ATM deficiency. The synthetic lethal interaction between ATM and ATR in response to IR supports ATR as a therapeutic target for improved anti-cancer regimens, especially in tumors with a dysfunctional ATM pathway.

Abstract 5485: Induction of proliferation sensitizes chronic lymphocytic leukemia cells to apoptosis mediated by the ATR inhibitor AZD6738

Abstract ATR (Ataxia Telangiectasia and RAD3-related) is a PI3K-related kinase involved in repair of DNA damage incurred through the genotoxic stress of cellular replication. During cell division, ATR is recruited and activated at sites of single strand DNA breaks, such as those that occur at stalled replication forks, and helps initiate DNA repair. Tumor cells may develop an enhanced dependency on ATR to maintain genomic stability and survival, making ATR an attractive target for anticancer therapy. AZD6738 is a first-in-class small molecule that inhibits ATR at sub micromolar (uM) concentrations. We examined the leukemic cells from patients with chronic lymphocytic leukemia (CLL) for expression of ATR and sensitivity to this specific ATR inhibitor. For this, we examined both resting CLL cells and CLL cells induced to undergo replication, as assessed by flow cytometry of CLL cells stained with Carboxyfluorescein succinimidyl ester (CFSE). We found that conditions that induced of leukemic cell proliferation (eg: CD40 ligation in the context of interleukin(IL)4 and IL10) also induced expression of ATR. Examination of both resting and proliferating leukemic cells for sensitivity to AZD6738 revealed that those leukemic cells that expressed ATR were highly sensitive to the cytotoxic effects of this ATR inhibitor in a time and dose dependent fashion. As such, concentrations of AZD6738 as low as 1 uM were cytotoxic for proliferating CLL cells. In contrast, the IC50 of resting CLL cells was approximately 30 uM, indicating that resting CLL cells are relatively insensitive to the activity of this drug. AZD6738 may therefore be particularly effective in eliminating the leukemic cells that are being stimulated in the leukemia microenvironment found in lymphoid tissues, which provides survival/growth signals that ordinarily mitigate the activity of standard cytotoxic drugs. ATR inhibition also may be particularly effective in tumors lacking ATM (Ataxia Telangiectasia Mutated). Such cells are even more dependent on the activity of ATR to repair DNA damage that occurs during replication or genotoxic stress, such as that incurred by cancer chemotherapy. To this end, we are examining differential sensitivity to ATR inhibition of leukemic cells that lack functional ATM, which is often times associated with deletions of the long arm of chromosome 11 (Del 11q22). An ongoing phase 1 clinical trial testing the safety of AZD6738 in patients with relapsed or refractory CLL with Del11q also will examine the activity of this compound for the treatment of patients with CLL. Citation Format: Michael Y. Choi, Jessie-Farah Fecteau, Jeff Brown, Alan Lau, Thomas J. Kipps. Induction of proliferation sensitizes chronic lymphocytic leukemia cells to apoptosis mediated by the ATR inhibitor AZD6738. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5485. doi:10.1158/1538-7445.AM2014-5485

Ursolic Acid-Regulated Energy Metabolism-Reliever or Propeller of Ultraviolet-Induced Oxidative Stress and DNA Damage?

Ultraviolet (UV) light is a leading cause of diseases, such as skin cancers and cataracts. A main process mediating UV-induced pathogenesis is the production of reactive oxygen species (ROS). Excessive ROS levels induce the formation of DNA adducts (e.g., pyrimidine dimers) and result in stalled DNA replication forks. In addition, ROS promotes phosphorylation of tyrosine kinase-coupled hormone receptors and alters downstream energy metabolism. With respect to the risk of UV-induced photocarcinogenesis and photodamage, the antitumoral and antioxidant functions of natural compounds become important for reducing UV-induced adverse effects. One important question in the field is what determines the differential sensitivity of various types of cells to UV light and how exogenous molecules, such as phytochemicals, protect normal cells from UV-inflicted damage while potentiating tumor cell death, presumably via interaction with intracellular target molecules and signaling pathways. Several endogenous molecules have emerged as possible players mediating UV-triggered DNA damage responses. Specifically, UV activates the PIKK (phosphatidylinositol 3-kinase-related kinase) family members, which include DNA-PKcs, ATM (ataxia telangiectasia mutated) and mTOR (mammalian target of rapamycin), whose signaling can be affected by energy metabolism; however, it remains unclear to what extent the activation of hormone receptors regulates PIKKs and whether this crosstalk occurs in all types of cells in response to UV. This review focuses on proteomic descriptions of the relationships between cellular photosensitivity and the phenotypic expression of the insulin/insulin-like growth receptor. It covers the cAMP-dependent pathways, which have recently been shown to regulate the DNA repair machinery through interactions with the PIKK family members. Finally, this review provides a strategic illustration of how UV-induced mitogenic activity is modulated by the insulin sensitizer, ursolic acid (UA), which results in the metabolic adaptation of normal cells against UV-induced ROS, and the metabolic switch of tumor cells subject to UV-induced damage. The multifaceted natural compound, UA, specifically inhibits photo-oxidative DNA damage in retinal pigment epithelial cells while enhancing that in skin melanoma. Considering the UA-mediated differential effects on cell bioenergetics, this article reviews the disparities in glucose metabolism between tumor and normal cells, along with (peroxisome proliferator-activated receptor-γ coactivator 1α)-dependent mitochondrial metabolism and redox (reduction-oxidation) control to demonstrate UA-induced synthetic lethality in tumor cells.

Radiosensitization of human leukemic HL-60 cells by ATR kinase inhibitor (VE-821): phosphoproteomic analysis.

DNA damaging agents such as ionizing radiation or chemotherapy are frequently used in oncology. DNA damage response (DDR)—triggered by radiation-induced double strand breaks—is orchestrated mainly by three Phosphatidylinositol 3-kinase-related kinases (PIKKs): Ataxia teleangiectasia mutated (ATM), DNA-dependent protein kinase (DNA-PK) and ATM and Rad3-related kinase (ATR). Their activation promotes cell-cycle arrest and facilitates DNA damage repair, resulting in radioresistance. Recently developed specific ATR inhibitor, VE-821 (3-amino-6-(4-(methylsulfonyl)phenyl)-N-phenylpyrazine-2-carboxamide), has been reported to have a significant radio- and chemo-sensitizing effect delimited to cancer cells (largely p53-deficient) without affecting normal cells. In this study, we employed SILAC-based quantitative phosphoproteomics to describe the mechanism of the radiosensitizing effect of VE-821 in human promyelocytic leukemic cells HL-60 (p53-negative). Hydrophilic interaction liquid chromatography (HILIC)-prefractionation with TiO2-enrichment and nano-liquid chromatography—tandem mass spectrometry (LC-MS/MS) analysis revealed 9834 phosphorylation sites. Proteins with differentially up-/down-regulated phosphorylation were mostly localized in the nucleus and were involved in cellular processes such as DDR, all phases of the cell cycle, and cell division. Moreover, sequence motif analysis revealed significant changes in the activities of kinases involved in these processes. Taken together, our data indicates that ATR kinase has multiple roles in response to DNA damage throughout the cell cycle and that its inhibitor VE-821 is a potent radiosensitizing agent for p53-negative HL-60 cells.

Structures of SMG1-UPFs Complexes: SMG1 Contributes to Regulate UPF2-Dependent Activation of UPF1 in NMD

SMG1, a PI3K-related kinase, plays a critical role in nonsense-mediated mRNA decay (NMD) in mammals. SMG1-mediated phosphorylation of the UPF1 helicase is an essential step during NMD initiation. Both SMG1 and UPF1 are presumably activated by UPF2, but this regulation is incompletely understood. Here we reveal that SMG1C (a complex containing SMG1, SMG8, and SMG9) contributes to regulate NMD by recruiting UPF1 and UPF2 to distinct sites in the vicinity of the kinase domain. UPF2 binds SMG1 in an UPF1-independent manner invivo, and the SMG1C-UPF2 structure shows UPF2 recognizes the FRB domain, a region that regulates the related mTOR kinase. The molecular architectures of several SMG1C-UPFs complexes, obtained by combining electron microscopy with invivo and invitro interaction analyses, competition experiments, and mutations, suggest that UPF2 can be transferred to UPF1 within SMG1C, inducing UPF2-dependent conformational changes required to activate UPF1 within an SMG1C-UPF1-UPF2 complex.

DNA-PK phosphorylation of RPA32 Ser4/Ser8 regulates replication stress checkpoint activation, fork restart, homologous recombination and mitotic catastrophe.

Genotoxins and other factors cause replication stress that activate the DNA damage response (DDR), comprising checkpoint and repair systems. The DDR suppresses cancer by promoting genome stability, and it regulates tumor resistance to chemo- and radiotherapy. Three members of the phosphatidylinositol 3-kinase-related kinase (PIKK) family, ATM, ATR, and DNA-PK, are important DDR proteins. A key PIKK target is replication protein A (RPA), which binds single-stranded DNA and functions in DNA replication, DNA repair, and checkpoint signaling. An early response to replication stress is ATR activation, which occurs when RPA accumulates on ssDNA. Activated ATR phosphorylates many targets, including the RPA32 subunit of RPA, leading to Chk1 activation and replication arrest. DNA-PK also phosphorylates RPA32 in response to replication stress, and we demonstrate that cells with DNA-PK defects, or lacking RPA32 Ser4/Ser8 targeted by DNA-PK, confer similar phenotypes, including defective replication checkpoint arrest, hyper-recombination, premature replication fork restart, failure to block late origin firing, and increased mitotic catastrophe. We present evidence that hyper-recombination in these mutants is ATM-dependent, but the other defects are ATM-independent. These results indicate that DNA-PK and ATR signaling through RPA32 plays a critical role in promoting genome stability and cell survival in response to replication stress.

Inositol Pyrophosphates Mediate the DNA-PK/ATM-p53 Cell Death Pathway by Regulating CK2 Phosphorylation of Tti1/Tel2

The apoptotic actions of p53 require its phosphorylation by a family of phosphoinositide-3-kinase-related-kinases (PIKKs), which include DNA-PKcs and ATM. These kinases are stabilized by the TTT (Tel2, Tti1, Tti2) cochaperone family, whose actions are mediated by CK2 phosphorylation. The inositol pyrophosphates, such as 5-diphosphoinositol pentakisphosphate (IP7), are generated by a family of inositol hexakisphosphate kinases (IP6Ks), of which IP6K2 has been implicated in p53-associated cell death. In the present study we report an apoptotic signaling cascade linking CK2, TTT, the PIKKs, and p53. We demonstrate that IP7, formed by IP6K2, binds CK2 to enhance its phosphorylation of the TTT complex, thereby stabilizing DNA-PKcs and ATM. This process stimulates p53 phosphorylation at serine 15 to activate the cell death program in human cancer cells and in murine B cells.

Cisplatin associated with LY294002 increases cytotoxicity and induces changes in transcript profiles of glioblastoma cells

Glioblastoma, one of the deadliest forms of brain tumor, responds poorly to available therapies. This highlights the intense search for new treatment approaches, and an emerging strategy is based on molecular targets. In the present work, we aimed to study whether glioblastoma cells can be sensitized by cisplatin combined with LY294002 (LY), which is an inhibitor of PI3K-related family (ATM, ATR, DNA-PK). We observed that cisplatin caused a pronounced reduction in cell proliferation in U343 and U87 cells, and LY significantly increased the cytotoxic effects caused by cisplatin under these conditions. Differently of U343, U87 cells did not show a significant induction of apoptosis. The phosphorylation level of damage response proteins was analyzed after drug-treatment either with/without LY. The presence of γH2AX foci and phosphorylation of TP53(ser15) and CHK1(ser317) were shown in U343 cells, compatible with cisplatin-induced DNA damage. Similarly, the level of ATR phosphorylation (ser428) was also increased (24 h). The transcript expression profiles of drug-treated compared with untreated U343 cells showed significant changes in the expression of 108 genes, while 274 genes were modulated by cisplatin+LY. The combined treatment caused a high proportion of down-regulated genes, which were mainly involved with DNA repair, cell death and cell cycle control/proliferation, metabolism, transcription regulation and cellular adhesion. Altogether, the present results indicate that most probably, PI3K-related kinases may play an important role in the resistance of glioblastomas cells to cisplatin, and the combination with LY can, at least in part, sensitize these cells to drug treatment.

SMG1 Identified as a Regulator of Parkinson’s Disease-Associated alpha-Synuclein through siRNA Screening

Synucleinopathies are a broad class of neurodegenerative disorders characterized by the presence of intracellular protein aggregates containing α-synuclein protein. The aggregated α-synuclein protein is hyperphosphorylated on serine 129 (S129) compared to the unaggregated form of the protein. While the precise functional consequences of S129 hyperphosphorylation are still being clarified, numerous in vitro and in vivo studies suggest that S129 phosphorylation is an early event in α-synuclein dysfunction and aggregation. Identifying the kinases and phosphatases that regulate this critical phosphorylation event may ultimately prove beneficial by allowing pharmacological mitigation of synuclein dysfunction and toxicity in Parkinson’s disease and other synucleinopathies. We report here the development of a high-content, fluorescence-based assay to quantitate levels of total and S129 phosphorylated α-synuclein protein. We have applied this assay to conduct high-throughput loss-of-function screens with siRNA libraries targeting 711 known and predicted human kinases and 206 phosphatases. Specifically, knockdown of the phosphatidylinositol 3-kinase related kinase SMG1 resulted in significant increases in the expression of pS129 phosphorylated α-synuclein (p-syn). Moreover, SMG1 protein levels were significantly reduced in brain regions with high p-syn levels in both dementia with Lewy bodies (DLB) and Parkinson’s disease with dementia (PDD). These findings suggest that SMG1 may play an important role in increased α-synuclein pathology during the course of PDD, DLB, and possibly other synucleinopathies.

1-Substituted (Dibenzo[b,d]thiophen-4-yl)-2-morpholino-4H-chromen-4-ones Endowed with Dual DNA-PK/PI3-K Inhibitory Activity

Analogues of (dibenzo[b,d]thiophen-4-yl)-2-morpholino-4H-chromen-4-one (NU7441), a potent inhibitor of DNA-dependent protein kinase (DNA-PK; IC50 = 42 ± 2 nM), have been synthesized in which water-solubilizing groups [NHCO(CH₂)nNR¹R², where n = 1 or 2 and the moiety R¹R²N was derived from a library of primary and secondary amines, e.g., morpholine] were placed at the 1-position. Several of the newly synthesized compounds exhibited high potency against DNA-PK and potentiated the cytotoxicity of ionizing radiation (IR) in vitro 10-fold or more (e.g., 2-(4-ethylpiperazin-1-yl)-N-(4-(2-morpholino-4-oxo-4H-chromen-8-yl)dibenzo[b,d]thio-phen-1-yl)acetamide, 39; DNA-PK IC₅₀ = 5.0 ± 1 nM, IR dose modification ratio = 13). Furthermore, 39 was shown to potentiate not only IR in vitro but also DNA-inducing cytotoxic anticancer agents, both in vitro and in vivo. Counter-screening against other members of the phosphatidylinositol 3-kinase (PI-3K) related kinase (PIKK) family unexpectedly revealed that some of the compounds were potent mixed DNA-PK and PI-3K inhibitors.

ERK kinases modulate the activation of PI3 kinase related kinases (PIKKs) in DNA damage response.

DNA damage response (DDR) is the critical surveillance mechanism in maintaining genome integrity. The mechanism activates checkpoints to prevent cell cycle progression in the presence of DNA lesions, and mediates lesion repair. DDR is coordinated by three apical PI3 kinase related kinases (PIKKs), including ataxia-telangiectasia mutated (ATM), ATM- and Rad3-related (ATR), and DNA-PKcs (the catalytic subunit of the DNA dependent protein kinase). These kinases are activated in response to specific DNA damage or lesions, resulting in checkpoint activation and DNA lesion repair. While it is clear that the pathways of ATM, ATR, and DNA-PK are the core components of DDR, there is accumulating evidence revealing the involvement of other cellular pathways in regulating DDR; this is in line with the concept that in addition to being a nuclear event DDR is also a cellular process. One of these pathways is the extracellular signal-regulated kinase (ERK) MAPK (mitogen-activated protein kinase) pathway. ERK is a converging point of multiple signal transduction pathways involved in cell proliferation, differentiation, and apoptosis. Adding to this list of pathways is the recent development of ERK in DDR. The ERK kinases (ERK1 and ERK2) contribute to the proper execution of DDR in terms of checkpoint activation and the repair of DNA lesions. This review summarizes the contributions of ERK to DDR with emphasis on the relationship of ERK kinases with the activation of ATM, ATR, and DNA-PKcs.

NMR- and Circular Dichroism-monitored Lipid Binding Studies Suggest a General Role for the FATC Domain as Membrane Anchor of Phosphatidylinositol 3-Kinase-related Kinases (PIKK)

The FATC domain is shared by all members of the family of phosphatidylinositol-3 kinase-related kinases (PIKKs). It has been shown that the FATC domain plays an important role for the regulation of each PIKK. However, other than an involvement in protein-protein interactions, a common principle for the action of the FATC domain has not been detected. A detailed characterization of the structure and lipid binding properties of the FATC domain of the Ser/Thr kinase target of rapamycin (TOR) revealed that it contains a redox-sensitive membrane anchor in its C terminus. Because the C-terminal regions of the FATC domains of all known PIKKs are rather hydrophobic and especially rich in aromatic residues, we examined whether the ability to interact with lipids and membranes might be a general property. Here, we present the characterization of the interactions with lipids and different membrane mimetics for the FATC domains of human DNA-PKcs, human ATM, human ATR, human SMG-1, and human TRRAP by NMR and CD spectroscopy. The data indicate that all of these can interact with different membrane mimetics and may have different preferences only for membrane properties such as surface charge, curvature, and lipid packing. The oxidized form of the TOR FATC domain is well structured overall and forms an α-helix that is followed by a disulfide-bonded loop. In contrast, the FATC domains of the other PIKKs are rather unstructured in the isolated form and only significantly populate α-helical secondary structure upon interaction with membrane mimetics.

The role of mechanistic target of rapamycin (mTOR) complexes signaling in the immune responses.

The mechanistic Target of Rapamycin (mTOR) is an evolutionarily conserved serine/threonine kinase which is a member of the PI3K related kinase (PIKK) family. mTOR emerged as a central node in cellular metabolism, cell growth, and differentiation, as well as cancer metabolism. mTOR senses the nutrients, energy, insulin, growth factors, and environmental cues and transmits signals to downstream targets to effectuate the cellular and metabolic response. Recently, mTOR was also implicated in the regulation of both the innate and adaptive immune responses. This paper will summarize the current knowledge of mTOR, as related to the immune microenvironment and immune responses.

Abstract B22: Combined disruption of ATM and CHK1 functionalities reveals redundancies in the DNA damage response pathways and results in synthetic growth inhibition following γ-irradiation

Abstract Exposure of proliferating cells to genotoxic stresses activates a cascade of signaling events termed the DNA damage response (DDR). The DDR preserves genetic stability by detecting DNA lesions, activating cell cycle checkpoints, and promoting DNA damage repair. The phosphoinositide 3-kinase related kinases (PIKKs) Ataxia Telangiectasia-Mutated (ATM), ATM and Rad 3-related kinase (ATR), and DNA-dependent Protein Kinase (DNA-PK) are crucial for lesion sensing and signal transduction. The checkpoint kinase 1 (CHK1) is a traditional ATR target involved in DDR and normal cell cycle progression and represents a pharmacological target for anti-cancer regimens. We hypothesized that ATM-deficient cells depend on signaling through CHK1 for survival after genotoxic exposures. The purpose of this study was to develop isogenic cell lines that are stably depleted for CHK1, ATM, or both and examine cross-talk and compensatory effects on cellular growth in general and the G2/M checkpoint in particular following treatment with γ-irradiation (IR). Human epithelial cell cultures (HME-CC) were stably transduced with either control or CHK1-targeting lentiviral shRNA particles in a wild-type or ATM-deficient background. Cell line validation and characterization was performed with cellular assays in conjunction with real-time quantitative PCR and Western blotting. Cell cycle profiling was done by flow cytometry. In addition, pharmacological approaches were used to functionally disrupt the proteins of interest for this study. We show that individual depletion of CHK1 and ATM results in augmented cellular proliferation as reflected in shortened cell population doubling times. Notably, a high level of depletion of CHK1 (HEK293 and HME-CC cells) or ATM (HME-CC) renders cells radiosensitive without abrogating their IR-mediated G2/M checkpoint arrest. Our data indicates that following IR, CHK1-deficient cells display enhanced ATM phosphorylation compared to their wild-type counterparts and conversely, ATM-depleted cells exhibit enhanced CHK1 phosphorylation, thus revealing a redundancy in the G2/M checkpoint enforcement after DNA damage. We provide evidence that this ATM-CHK1 cooperation is mediated at least in part by a feedback regulatory loop with the phosphatase PP2A. CHK1-depleted cells exhibit a lower nuclear abundance of the PP2A catalytic subunit, and a concurrent increase in the inhibitory phosphorylated state of the PP2A catalytic subunit compared to CHK1-proficient cells. Intriguingly, stable depletion of CHK1 in an ATM-deficient background showed only a 50% reduction from wild-type CHK1 protein expression levels, suggesting that loss of CHK1 by ATM-deficient cells may be synthetically lethal. Combined ATM depletion (10% of wild-type level) and CHK1-deficiency (50%) resulted in an additive attenuation of the G2/M checkpoint response compared to the individual knockdown, whereas pharmacological ATM inhibition in conjunction with 90% CHK1 depletion abrogated the early G2/M checkpoint and precluded the cells from mounting an efficient compensatory response to IR at later time-points. Furthermore, at 48 h post-IR the individually-depleted cell lines continued to proliferate, whereas the double-deficient cell line ATM-CHK1 halted proliferation. We conclude that dual targeting of ATM and CHK1 functionalities disrupts the compensatory response to DNA damage, abrogates the checkpoint arrest, and leads to synthetic growth inhibition. A combined targeting of ATM and CHK1 functionalities merits further investigation for developing more efficient anti-neoplastic treatments. Citation Format: Stela S. Palii, Richard S. Paules. Combined disruption of ATM and CHK1 functionalities reveals redundancies in the DNA damage response pathways and results in synthetic growth inhibition following γ-irradiation. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr B22.

Abstract 4358: Involvement of mTOR pathway in metastatic brain tumors.

Abstract Metastatic dissemination to the brain occurs in almost forty percent of all cancers, and is ten times more common than primary brain tumors. Mammalian target of Rapamycin (mTOR), an atypical PI3K related kinase, exists in two distinct multiprotein complexes (mTORC1 and mTORC2). mTORC1/2 regulate various cellular functions and are shown to be activated in primary brain tumors such as glioblastoma and medulloblastoma. In addition, metastatic tumor cells possess a unique reprogramming resulting in a stem cell like phenotype with invasive, migratory and proliferative potential similar to that seen in glioblastoma, where mTOR appears to play an essential role. However, the involvement of the mTOR pathway in metastatic brain tumors remains to be elucidated. This study aimed to test the hypothesis that the mTOR pathway plays a critical role in metastatic brain tumors. In order to achieve our goals, expression of mTOR and its components (Raptor and Rictor) were studied in metastatic brain tumors using immunohistochemical analysis. Further, the function of mTOR in relation to proliferation, migration, and cell spreading was studied in a metastatic breast cancer cell line (MBA-MD 231). The expression of epithelial marker E-Cadherin and meschenchymal marker Vimentin following the inhibition of mTOR using pharmacological inhibitors (Rapamycin and PP242) as well as short interfering RNA (mTOR, Raptor and Rictor) was examined by immunofluorescence analysis. Results demonstrated that a significant number of metastatic brain tumors expressed mTOR, Raptor, and Rictor; 50% expressing all three components. The metastatic potential showed that migration of cancer cells was inhibited significantly by mTORC1 inhibition (Rapamycin) and mTORC1/2 inhibition (PP242) by 29% and 64%, respectively. Utilizing the EdU incorporation technique, S-phase cell cycle entry analysis rendered a 52% decline in proliferation after Rapamycin treatment. Cell spreading/attachment analysis exhibited total attachment of control cells within 20 minutes of plating. This effect was prevented by pretreatment with Rapamycin or PP242. Immunoflourescence analysis showed that E-cadherin underwent forced nuclear localization following acute inhibition of mTOR, using Rapamycin or siRNA treatments. These results were substantiated by Western blotting, suggesting its role in cellular reprogramming, namely Epithelial-Mesenchymal Transition or its counterparts. However, Vimentin expression remained unaltered. In conclusion, these results provide evidence that mTOR is enhanced in metastatic brain tumors, suggesting its critical role in achieving a metastatic potential to the brain. Importantly, these findings underscore the therapeutic value of mTOR inhibition in the management of patients with metastatic brain tumors. Citation Format: Amanda Kwasnicki, Dhruve Jeevan, Anita Goyal, Alex Braun, Raj Murali, Meena Jhanwar-Uniyal. Involvement of mTOR pathway in metastatic brain tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4358. doi:10.1158/1538-7445.AM2013-4358

Combined inhibition of PI3K-related DNA damage response kinases and mTORC1 induces apoptosis in MYC-driven B-cell lymphomas

AbstractPharmacological strategies capable of directly targeting MYC are elusive. Previous studies have shown that MYC-driven lymphomagenesis is associated with mammalian target of rapamycin (mTOR) activation and a MYC-evoked DNA damage response (DDR) transduced by phosphatidylinositol-3-kinase (PI3K)-related kinases (DNA-PK, ATM, and ATR). Here we report that BEZ235, a multitargeted pan-PI3K/dual-mTOR inhibitor, potently killed primary Myc-driven B-cell lymphomas and human cell lines bearing IG-cMYC translocations. Using pharmacologic and genetic dissection of PI3K/mTOR signaling, dual DDR/mTORC1 inhibition was identified as a key mediator of apoptosis. Moreover, apoptosis was initiated at drug concentrations insufficient to antagonize PI3K/mTORC2-regulated AKT phosphorylation. p53-independent induction of the proapoptotic BH3-only protein BMF was identified as a mechanism by which dual DDR/mTORC1 inhibition caused lymphoma cell death. BEZ235 treatment induced apoptotic tumor regressions in vivo that correlated with suppression of mTORC1-regulated substrates and reduced H2AX phosphorylation and also with feedback phosphorylation of AKT. These mechanistic studies hold important implications for the use of multitargeted PI3K inhibitors in the treatment of hematologic malignancies. In particular, the newly elucidated role of PI3K-related DDR kinases in response to PI3K inhibitors offers a novel therapeutic opportunity for the treatment of hematologic malignancies with an MYC-driven DDR.

Dissecting cellular responses to irradiation via targeted disruptions of the ATM-CHK1-PP2A circuit

Exposure of proliferating cells to genotoxic stresses activates a cascade of signaling events termed the DNA damage response (DDR). The DDR preserves genetic stability by detecting DNA lesions, activating cell cycle checkpoints and promoting DNA damage repair. The phosphoinositide 3-kinase-related kinases (PIKKs) ataxia telangiectasia-mutated (ATM), ATM and Rad 3-related kinase (ATR) and DNA-dependent protein kinase (DNA-PK) are crucial for sensing lesions and signal transduction. The checkpoint kinase 1 (CHK1) is a traditional ATR target involved in DDR and normal cell cycle progression and represents a pharmacological target for anticancer regimens. This study employed cell lines stably depleted for CHK1, ATM or both for dissecting cross-talk and compensatory effects on G₂/M checkpoint in response to ionizing radiation (IR). We show that a 90% depletion of CHK1 renders cells radiosensitive without abrogating their IR-mediated G₂/M checkpoint arrest. ATM phosphorylation is enhanced in CHK1-deficient cells compared with their wild-type counterparts. This correlates with lower nuclear abundance of the PP2A catalytic subunit in CHK1-depleted cells. Stable depletion of CHK1 in an ATM-deficient background showed only a 50% reduction from wild-type CHK1 protein expression levels and resulted in an additive attenuation of the G₂/M checkpoint response compared with the individual knockdowns. ATM inhibition and 90% CHK1 depletion abrogated the early G₂/M checkpoint and precluded the cells from mounting an efficient compensatory response to IR at later time points. Our data indicates that dual targeting of ATM and CHK1 functionalities disrupts the compensatory response to DNA damage and could be exploited for developing efficient anti-neoplastic treatments.