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Supplementary Table from P2RX7 Enhances Tumor Control by CD8+ T Cells in Adoptive Cell Therapy

Expression of the purinergic receptor P2RX7 by CD8+ T cells promotes the generation of memory populations following acute infections. However, data suggest that P2RX7 may limit the efficacy of antitumor responses. Herein, we show that P2RX7 is beneficial for optimal melanoma control in a mouse CD8+ T-cell adoptive transfer model. Tumor-specific P2rx7–/– CD8+ T cells exhibited impaired mitochondrial maintenance and function but did not display signs of overt exhaustion early in the antitumor response. However, as the tumor burden increased, the relative frequency of P2RX7-deficient CD8+ T cells declined within the tumor; this correlated with reduced proliferation, increased apoptosis, and mitochondrial dysfunction. Extending these studies, we found that the transient in vitro stimulation of P2RX7 using the ATP analogue BzATP led to enhanced B16 melanoma control by CD8+ T cells. These findings are in keeping with the concept that extracellular ATP (eATP) sensing by P2RX7 on CD8+ T cells is required for their ability to efficiently eliminate tumors by promoting mitochondrial fitness and underscore the potential for P2RX7 stimulation as a novel therapeutic treatment to enhance tumor immunotherapy.

Supplementary Figure from P2RX7 Enhances Tumor Control by CD8+ T Cells in Adoptive Cell Therapy

PHLPP2 is a member of the PHLPP family of phosphatases, known to suppress cell growth by inhibiting proliferation or promoting apoptosis. Oncogenic kinases Akt, S6K, and PKC, and pro-apoptotic kinase Mst1, have been recognized as functional targets of the PHLPP family. However, we observed that, in T-leukemia cells subjected to metabolic stress from glucose limitation, PHLPP2 specifically targets the energy-sensing AMP-activated protein kinase, pAMPK, rather than Akt or S6K. PHLPP2 dephosphorylates pAMPK in several other human cancer cells as well. PHLPP2 and pAMPK interact with each other, and the pleckstrin homology (PH) domain on PHLPP2 is required for their interaction, for dephosphorylating and inactivating AMPK, and for the apoptotic response of the leukemia cells to glucose limitation. Silencing PHLPP2 protein expression prolongs the survival of leukemia cells subjected to severe glucose limitation by promoting a switch to AMPK-mediated fatty acid oxidation for energy generation. Thus, this study reveals a novel role for PHLPP2 in suppressing a survival response mediated through AMPK signaling. Given the multiple ways in which PHLPP phosphatases act to oppose survival signaling in cancers and the pivotal role played by AMPK in redox homeostasis via glucose and fatty acid metabolism, the revelation that AMPK is a target of PHLPP2 could lead to better therapeutics directed both at cancer and at metabolic diseases.

Leucine-rich alpha-2-glycoprotein-1 (LRG1) has been shown to compete with apoptosis activating factor-1 (Apaf-1) for binding cytochrome c (Cyt c) and could play a role in inhibition of apoptosis. Employing MCF-7 breast cancer cells, we report that intracellular LRG1 does protect against apoptosis. Thus, cells transfected with the lrg1 gene and expressing higher levels of LRG1 were more resistant to hydrogen peroxide-induced apoptosis than parental cells, while cells in which LRG mRNA was knocked down by short hairpin (sh) RNA-induced degradation were more sensitive. The amount of Cyt c co-immunoprecipitated with Apaf-1 from the cytosol of apoptotic cells was inversely related to the level of LRG1 expression. In lrg1-transfected cells partially-glycosylated LRG1 was found in the cytosol and there was an increase in cytosolic Cyt c in live lrg1-transfected cells relative to parental cells. However, apoptosis was not spontaneously induced because Cyt c was bound to LRG1 and not to Apaf-1. Cyt c was the only detectable protein co-immunoprecipitated with LRG1. Following hydrogen peroxide treatment degradation of LRG1 allowed for induction of apoptosis. We propose that intracellular LRG1 raises the threshold of cytoplasmic Cyt c required to induce apoptosis and, thus, prevents onset of the intrinsic pathway in cells where Cyt c release from mitochondria does not result from committed apoptotic signaling. This mechanism of survival afforded by LRG1 is likely to be distinct from its extracellular survival function that has been reported by several research groups.

Suppressive regulatory T cell (Treg) differentiation is controlled by diverse immunometabolic signaling pathways and intracellular metabolites. Here we show that cell-permeable α-ketoglutarate (αKG) alters the DNA methylation profile of naive CD4 T cells activated under Treg polarizing conditions, markedly attenuating FoxP3+ Treg differentiation and increasing inflammatory cytokines. Adoptive transfer of these T cells into tumor-bearing mice results in enhanced tumor infiltration, decreased FoxP3 expression, and delayed tumor growth. Mechanistically, αKG leads to an energetic state that is reprogrammed toward a mitochondrial metabolism, with increased oxidative phosphorylation and expression of mitochondrial complex enzymes. Furthermore, carbons from ectopic αKG are directly utilized in the generation of fatty acids, associated with lipidome remodeling and increased triacylglyceride stores. Notably, inhibition of either mitochondrial complex II or DGAT2-mediated triacylglyceride synthesis restores Treg differentiation and decreases the αKG-induced inflammatory phenotype. Thus, we identify a crosstalk between αKG, mitochondrial metabolism and triacylglyceride synthesis that controls Treg fate. ispartof: CELL REPORTS vol:37 issue:5 ispartof: location:United States status: published

Expression of the purinergic receptor P2RX7 by CD8+ T cells promotes the generation of memory populations following acute infections. However, data suggest that P2RX7 may limit the efficacy of antitumor responses. Herein, we show that P2RX7 is beneficial for optimal melanoma control in a mouse CD8+ T-cell adoptive transfer model. Tumor-specific P2rx7–/– CD8+ T cells exhibited impaired mitochondrial maintenance and function but did not display signs of overt exhaustion early in the antitumor response. However, as the tumor burden increased, the relative frequency of P2RX7-deficient CD8+ T cells declined within the tumor; this correlated with reduced proliferation, increased apoptosis, and mitochondrial dysfunction. Extending these studies, we found that the transient in vitro stimulation of P2RX7 using the ATP analogue BzATP led to enhanced B16 melanoma control by CD8+ T cells. These findings are in keeping with the concept that extracellular ATP (eATP) sensing by P2RX7 on CD8+ T cells is required for their ability to efficiently eliminate tumors by promoting mitochondrial fitness and underscore the potential for P2RX7 stimulation as a novel therapeutic treatment to enhance tumor immunotherapy.

Regulatory T cells (Tregs) are critical for maintaining immune homeostasis. However, current Treg immunotherapies do not optimally treat inflammatory diseases in patients. Understanding the cellular processes that control Treg function may allow for the augmentation of therapeutic efficacy. In contrast to activated conventional T cells, in which protein kinase C-θ (PKC-θ) localizes to the contact point between T cells and antigen-presenting cells, in human and mouse Tregs, PKC-θ localizes to the opposite end of the cell in the distal pole complex (DPC). Here, using a phosphoproteomic screen, we identified the intermediate filament vimentin as a PKC-θ phospho target and show that vimentin forms a DPC superstructure on which PKC-θ accumulates. Treatment of mouse Tregs with either a clinically relevant PKC-θ inhibitor or vimentin siRNA disrupted vimentin and enhanced Treg metabolic and suppressive activity. Moreover, vimentin-disrupted mouse Tregs were significantly better than controls at suppressing alloreactive T cell priming in graft-versus-host disease (GVHD) and GVHD lethality, using a complete MHC-mismatch mouse model of acute GVHD (C57BL/6 donor into BALB/c host). Interestingly, vimentin disruption augmented the suppressor function of PKC-θ-deficient mouse Tregs. This suggests that enhanced Treg activity after PKC-θ inhibition is secondary to effects on vimentin, not just PKC-θ kinase activity inhibition. Our data demonstrate that vimentin is a key metabolic and functional controller of Treg activity and provide proof of principle that disruption of vimentin is a feasible, translationally relevant method to enhance Treg potency.

Increased glucose consumption is a hallmark of cancer cells. The increased consumption and subsequent metabolism of glucose during proliferation creates the need for a constant supply of NAD, a co-factor in glycolysis. Regeneration of the NAD required to support enhanced glycolysis has been attributed to the terminal glycolytic enzyme, lactate dehydrogenase (LDH). However, loss of glucose carbons to biosynthetic pathways early in glycolysis reduces the carbon supply to LDH. Thus, alternative routes for NAD regeneration must exist to support the increased glycolytic rate while allowing for the diversion of glucose to generate biomass and support proliferation. Here we demonstrate, using a variety of cancer cell lines as well as activated primary T cells, that cytosolic malate dehydrogenase 1 (MDH1) is an alternative to LDH as a supplier of NAD. Moreover, our results indicate that MDH1 generates malate with carbons derived from glutamine, thus enabling utilization of glucose carbons for glycolysis and for biomass. Amplification of MDH1 occurs at an impressive frequency in human tumors and correlates with poor prognosis. Together, our findings suggest that proliferating cells rely on both MDH1 and LDH to replenish cytosolic NAD, and that therapies designed at targeting glycolysis must consider both dehydrogenases.

PHLPP2, a member of the PH-domain leucine-rich repeat protein phosphatase (PHLPP) family, which targets oncogenic kinases, has been actively investigated as a tumor suppressor in solid tumors. Little is known, however, regarding its regulation in hematological malignancies. We observed that PHLPP2 protein expression, but not its mRNA, was suppressed in late differentiation stage acute myeloid leukemia (AML) subtypes. MicroRNAs (miR or miRNAs) from the miR-17-92 cluster, oncomir-1, were shown to inhibit PHLPP2 expression and these miRNAs were highly expressed in AML cells that lacked PHLPP2 protein. Studies showed that miR-17-92 cluster regulation was, surprisingly, independent of transcription factors c-MYC and E2F in these cells; instead all-trans-retinoic acid (ATRA), a drug used for terminally differentiating AML subtypes, markedly suppressed miR-17-92 expression and increased PHLPP2 protein levels and phosphatase activity. Finally, we demonstrate that the effect of ATRA on miR-17-92 expression is mediated through its target, transcription factor C/EBPβ, which interacts with the intronic promoter of the miR-17-92 gene to inhibit transactivation of the cluster. These studies reveal a novel mechanism for upregulation of the phosphatase activity of PHLPP2 through C/EBPβ-mediated repression of the miR-17-92 cluster in terminally differentiating myeloid cells.

High-resolution characterization of the structure and dynamics of intrinsically disordered proteins (IDPs) remains a challenging task. Consequently, a detailed understanding of the structural and functional features of IDPs remains limited, as very few full-length disordered proteins have been structurally characterized. We have performed microsecond-long molecular dynamics (MD) simulations of Noxa, the smallest member of the large Bcl-2 family of apoptosis regulating proteins, to characterize in atomic-level detail the structural features of a disordered protein. A 2.5 μs MD simulation starting from an unfolded state of the protein revealed the formation of a central antiparallel β-sheet structure flanked by two disordered segments at the N- and C-terminal ends. This topology is in reasonable agreement with protein disorder predictions and available experimental data. We show that this fold plays an essential role in the intracellular function and regulation of Noxa. We demonstrate that unbiased MD simulations in combination with a modern force field reveal structural and functional features of disordered proteins at atomic-level resolution.

Extracellular ATP (eATP) is an ancient 'danger signal' used by eukaryotes to detect cellular damage1. In mice and humans, the release of eATP during inflammation or injury stimulates both innate immune activation and chronic pain through the purinergic receptor P2RX72-4. It is unclear, however, whether this pathway influences the generation of immunological memory, a hallmark of the adaptive immune system that constitutes the basis of vaccines and protective immunity against re-infection5,6. Here we show that P2RX7 is required for the establishment, maintenance and functionality of long-lived central and tissue-resident memory CD8+ T cell populations in mice. By contrast, P2RX7 is not required for the generation of short-lived effector CD8+ T cells. Mechanistically, P2RX7 promotes mitochondrial homeostasis and metabolic function in differentiating memory CD8+ T cells, at least in part by inducing AMP-activated protein kinase. Pharmacological inhibitors of P2RX7 provoked dysregulated metabolism and differentiation of activated mouse and human CD8+ T cells in vitro, and transient P2RX7 blockade in vivo ameliorated neuropathic pain but also compromised production of CD8+ memory T cells. These findings show that activation of P2RX7 by eATP provides a common currency that both alerts the nervous and immune system to tissue damage, and promotes the metabolic fitness and survival of the most durable and functionally relevant memory CD8+ T cell populations.

The rapid development of drug resistance is a major obstacle in the treatment of cancer. Multidrug resistance (MDR) is a common form of acquired drug resistance in which cancer cells, upon exposure to a single chemotherapeutic drug, develop resistance to a variety of drugs that are structurally and mechanistically unrelated. Due to the rapid development of MDR, many patients do not respond well to chemotherapy and often relapse with a more aggressive form of cancer. Therefore, there is a need for new anticancer agents that can selectively target MDR cancers and ideally re-sensitize them to standard therapies. Cancer cells can acquire MDR through various mechanisms. One major mechanism is the over-expression of ATP-binding cassette (ABC) transporter proteins, such as p-glycoprotein (P-gp).(1–7) Tumor cells with elevated ABC transporter proteins can decrease the intracellular concentration of anticancer drugs through ABC transporter-mediated enhanced efflux, leading to MDR. The other major mechanism is through altering the apoptotic pathway. Apoptosis is a programmed cell death process regulated by a variety of cellular signals. The two main apoptotic pathways are the intrinsic and extrinsic pathways.(8–10) The intrinsic pathway is of particular interests because a majority of chemotherapeutic drugs are believed to rely on this pathway to induce programmed cell death among cancer cells.(11–16) B-cell lymphoma-2 (Bcl-2) family proteins play an important role in the intrinsic apoptosis, consisting of anti-apoptotic members, such as Mcl-1, Bcl-2, and Bcl-XL, and pro-apoptotic members, including Bax, Bad, Bak, Bid, Bim and Noxa. The anti-apoptotic and pro-apoptotic members of the Bcl-2 family proteins antagonize each other to control cytochrome c release from mitochondria and to determine the fate of a cell – death or survival.(17) As key regulators that prevent programmed cell death, the anti-apoptotic Bcl-2 family proteins are over-expressed in 60–90% of all cancers,(18, 19) being considered as one general mechanism by which cancer cells gain resistance against standard therapies. Results of recent studies have revealed that the Bcl-2 family proteins also localize on other cellular compartments besides mitochondria, including the nuclear envelope and endoplasmic reticulum (ER).(20–22) Although the function of the Bcl-2 family proteins on the nuclear envelope is not fully understood, there have been some insights into their role on the ER membrane. Calcium release from the ER can act as a secondary messenger for cell death signaling, which involves multiple communication loops between ER and mitochondria that help regulate apoptosis.(23, 24) The ER membrane-localized Bcl-2 family proteins regulate calcium homeostasis and subsequently, programmed cell death, by interacting with various ER calcium transporters. For instance, Bcl-XL and Bax can interact with and regulate the inositol triphosphate receptors (IP3Rs), which release ER Ca2+ into the cytosol.(25, 26) Some reports also suggest that Bcl-2 interacts with and regulates the function of sarco-endoplasmic reticulum ATPase (SERCA) pumps, which transport cytosolic Ca2+ into the ER.(27–29) Given that the over-expression of the anti-apoptotic Bcl-2 family proteins and dysregulation in Ca2+ homeostasis have both been reported to be responsible for MDR in cancer,(30–33) simultaneously regulating both pathways may provide an effective strategy to overcome drug resistance in cancer. We previously reported the discovery of sHA 14-1 (Figure 1), a dual inhibitor of Bcl-2 and SERCA.(34) Recently, we developed ethyl 2-amino-6-(3,5-dimethoxyphenyl)-4-(2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate (CXL017) (Figure 1), a more potent analog of sHA 14-1, which demonstrates increased cytotoxicity towards a variety of MDR resistant cancer cell lines as well as synergism with a wide range of standard therapies.(35, 36) The improved potency observed in MDR cancer cell lines, also known as collateral sensitivity (CS), is a relatively rare phenomenon in cancer therapy and the mechanisms leading to CS are largely unknown. Given its synergistic and CS properties, CXL017 harbors the potential to improve the treatment response of cancer patients. Figure 1 The chemical structures of sHA 14-1 and CXL017. In this report, we show that CXL017 demonstrates increased efficacy towards tumors inoculated from HL60/MX2, an MDR cell line, in a xenograft mouse model. Moreover, HL60/MX2 cell line fails to develop stable resistance to CXL017 whereas it develops stable resistance (>2000-fold) to cytarabine (Ara-C) and ABT-737 (a representative Bcl-2 inhibitor). More significantly, upon long-term exposure to CXL017, the HL60/MX2 cell line becomes hypersensitive to standard therapies, such as cytarabine, doxorubicin, mitoxantrone, and etoposide. Molecular characterization of CXL017 treated HL60/MX2 cell lines reveals significant changes among several Bcl-2 family proteins, SERCA proteins, and ER calcium content. These changes may be responsible for CXL017-induced re-sensitization of drug resistant cancer cell lines.

Ischemic preconditioning is the phenomenon whereby brief periods of sublethal ischemia protect against a subsequent, more prolonged, ischemic insult. In remote ischemic preconditioning (RIPC), ischemia to one organ protects others organs at a distance. We created mouse models to ask if inhibition of the alpha-ketoglutarate (αKG)-dependent dioxygenase Egln1, which senses oxygen and regulates the hypoxia-inducible factor (HIF) transcription factor, could suffice to mediate local and remote ischemic preconditioning. Using somatic gene deletion and a pharmacological inhibitor, we found that inhibiting Egln1 systemically or in skeletal muscles protects mice against myocardial ischemia-reperfusion (I/R) injury. Parabiosis experiments confirmed that RIPC in this latter model was mediated by a secreted factor. Egln1 loss causes accumulation of circulating αKG, which drives hepatic production and secretion of kynurenic acid (KYNA) that is necessary and sufficient to mediate cardiac ischemic protection in this setting., Broad Institute of MIT and Harvard. SPARC Program, Burroughs Wellcome Fund

Recent studies have implicated multipotential mesenchymal stem cells (MSCs) as an aid to breast cancer cell proliferation and metastasis, partly as a result of the MSCs secretome. As the tumor gets beyond 2 mm in diameter, the stromal cells could undergo starvation due to the lack of sufficient nutrients in solid tumor microenvironment. In this study, we investigated the survival mechanisms used by stressed stromal cells in breast cancers. We used serum-deprived mesenchymal stem cells (SD-MSCs) and MCF-7 breast cancer cells as model system with a hypothesis that stromal cells in the nutrient-deprived core utilize survival mechanisms for supporting surrounding cells. We tested this hypothesis using in vivo tumor xenografts in immunodeficient mice, which indicated that SD-MSCs supported MCF-7 tumor growth by protection from apoptosis. Histochemical assays showed that SD-MSCs-injected tumors exhibited higher cellularity, decreased apoptosis and decreased differentiation. Beclin-1 staining indicated autophagic areas surrounded by actively proliferating cells. Furthermore, in vitro studies demonstrate that SD-MSCs survive using autophagy and secrete paracrine factors that support tumor cells following nutrient/serum deprivation. Western blot and immunocytochemistry analysis of SD-MSCs demonstrated upregulation and perinuclear relocation of autophagy key regulators such as beclin-1, ATG10, ATG12, MAP-LC3 and lysosomes. Electron microscopic analysis detected a time-dependent increase in autophagosome formation and HDAC6 activity assays indicated the upregulation of autophagy. Taken together, these data suggest that under nutrient-deprived conditions that can occur in solid tumors, stromal cells utilize autophagy for survival and also secrete anti-apoptotic factors that can facilitate solid tumor survival and growth.

Summary The BH3-only protein, Noxa, is induced in response to apoptotic stimuli, such as DNA damage, hypoxia, and proteasome inhibition in most human cells. Noxa is constitutively expressed in proliferating cells of hematopoietic lineage and required for apoptosis in response to glucose stress. We show that Noxa is phosphorylated on a serine residue (S 13 ) in the presence of glucose. Phosphorylation promotes its cytosolic sequestration and suppresses its apoptotic function. We identify Cdk5 as the Noxa kinase and show that Cdk5 knockdown or expression of a Noxa S 13 to A mutant increases sensitivity to glucose starvation, confirming that the phosphorylation is protective. Both glucose deprivation and Cdk5 inhibition promote apoptosis by dephosphorylating Noxa. Paradoxically, Noxa stimulates glucose consumption and may enhance glucose turnover via the pentose phosphate pathway rather than through glycolysis. We propose that Noxa plays both growth-promoting and proapoptotic roles in hematopoietic cancers with phospho-S 13 as the glucose-sensitive toggle switch controlling these opposing functions.

Endostatin is a well-characterized endogenous inhibitor of angiogenesis that affects cell proliferation and migration by inhibiting integrin and Wnt-mediated signalling pathways. Here, we show that endothelial cells treated with native and P125A-endostatin activate autophagy. Because autophagy can either be protective or induce programmed cell death, experiments were carried out to understand the signalling pathways leading to autophagy in endothelial cells. P125A-endostatin treatment increased the levels of Beclin 1, a crucial molecule in vesicle nucleation and autophagy. The treatment also reduced the levels of Bcl-2, Bcl-x(L) and beta-catenin; however, progressively increasing amounts of Bcl-2 and Bcl-x(L) were found to be complexed with Beclin 1. Increased beta-catenin and Wnt-mediated signalling reduced Beclin 1 levels and rescued endothelial cells from endostatin-induced autophagy. Finally, knocking down Beclin 1 levels by RNA interference decreased autophagy and accelerated caspase activation in endostatin-treated cells. These studies suggest that endothelial cells may initiate autophagy as a survival response to limit the effects of angiogenesis inhibitors. Thus, interfering with autophagy can potentiate the effects of endostatin by promoting a switch to apoptosis.

Previous studies from our laboratory had indicated that cytochrome c-independent processing and activation of caspase-9 by caspase-8 contributed to early amplification of the caspase cascade in tumor necrosis factor (TNF)-α-treated murine cells. Here we show that murine caspase-9 is phosphorylated by casein kinase 2 (CK2) on a serine near the site of caspase-8 cleavage. CK2 has been shown to regulate cleavage of the pro-apoptotic Bid protein by phosphorylating serine residues near its caspase-8 cleavage site. Similarly, CK2 modification of Ser348 on caspase-9 appears to render the protease refractory to cleavage by active caspase-8. This phosphorylation did not affect the ability of caspase-9 to autoprocess. Substitution of Ser348 abolished phosphorylation but not cleavage, and a phospho-site mutant promoted apoptosis in TNF-α-treated caspase-9 knock-out mouse embryo fibroblasts. Furthermore, inhibition of CK2 activity and RNA interference-mediated knockdown of the kinase accelerated caspase-9 activation, whereas phosphatase inhibition delayed both caspase-9 activation and death in response to TNF receptor occupation. Taken together, these studies show that TNF receptor cross-linking promotes dephosphorylation of caspase-9, rendering it susceptible to processing by activated caspase-8 protein. Thus, our data suggest that modification of procaspase-9 to protect it from inappropriate cleavage and activation is yet another mechanism by which the oncogenic kinase CK2 promotes survival.

Angiogenesis, the process by which new blood vessels are formed is critical for embryonic development and physiological functioning of normal tissues. Angiogenesis also plays a critical role in the pathology of many diseases including cancer, wherein the supply and demand for blood vessels determines the rate of cancer growth. A number of therapeutic strategies are being developed to inhibit pathological angiogenesis. Kringle domains of plasminogen such as kringle 5 (K5) and a proteolytic fragment of collagen type XVIII (endostatin) are well-characterized, potent angiogenesis inhibitors. These inhibitors activate different intracellular signaling pathways to induce apoptosis and inhibit cell proliferation. Recent studies from our group have shown that K5 and endostatin can also induce autophagy in addition to apoptosis in endothelial cells. A common feature of the two treatments was the upregulation of Beclin 1 levels leading to alterations in the Beclin 1-Bcl-2 complex. Angiogenesis inhibitor-induced autophagy in endothelial cells was independent of nutritional or hypoxic stress and initiated even in the presence of endothelial-specific survival factors such as vascular endothelial growth factor (VEGF). Interfering with the autophagic response by knocking down Beclin 1 levels dramatically increased apoptosis of endothelial cells. These findings identify the autophagic response as a novel target for enhancing the therapeutic efficacy of angiogenesis inhibitors.

We showed previously that phosphorylation of Noxa, a 54-residue Bcl-2 protein, at serine 13 (Ser13) inhibited its ability to promote apoptosis through interactions with canonical binding partner, Mcl-1. Using EPR spectroscopy, molecular dynamics (MD) simulations and binding assays, we offer evidence that a structural alteration caused by phosphorylation partially masks Noxa’s BH3 domain, inhibiting the Noxa-Mcl-1 interaction. EPR of unphosphorylated Noxa, with spin-labeled amino acid TOAC incorporated within the BH3 domain, revealed equilibrium between ordered and dynamically disordered states. Mcl-1 further restricted the ordered component for non-phosphorylated Noxa, but left the pSer13 Noxa profile unchanged. Microsecond MD simulations indicated that the BH3 domain of unphosphorylated Noxa is housed within a flexible loop connecting two antiparallel β-sheets, flanked by disordered N- and C-termini and Ser13 phosphorylation creates a network of salt-bridges that facilitate the interaction between the N-terminus and the BH3 domain. EPR showed that a spin label inserted near the N-terminus was weakly immobilized in unphosphorylated Noxa, consistent with a solvent-exposed helix/loop, but strongly constrained in pSer13 Noxa, indicating a more ordered peptide backbone, as predicted by MD simulations. Together these studies reveal a novel mechanism by which phosphorylation of a distal serine inhibits a pro-apoptotic BH3 domain and promotes cell survival.

We showed previously that phosphorylation of Noxa, a 54-residue Bcl-2 protein, at serine 13 (Ser13) inhibited its ability to promote apoptosis through interactions with canonical binding partner, Mcl-1. Using EPR spectroscopy, molecular dynamics (MD) simulations and binding assays, we offer evidence that a structural alteration caused by phosphorylation partially masks Noxa’s BH3 domain, inhibiting the Noxa-Mcl-1 interaction. EPR of unphosphorylated Noxa, with spin-labeled amino acid TOAC incorporated within the BH3 domain, revealed equilibrium between ordered and dynamically disordered states. Mcl-1 further restricted the ordered component for non-phosphorylated Noxa, but left the pSer13 Noxa profile unchanged. Microsecond MD simulations indicated that the BH3 domain of unphosphorylated Noxa is housed within a flexible loop connecting two antiparallel β-sheets, flanked by disordered N- and C-termini and Ser13 phosphorylation creates a network of salt-bridges that facilitate the interaction between the N-terminus and the BH3 domain. EPR showed that a spin label inserted near the N-terminus was weakly immobilized in unphosphorylated Noxa, consistent with a solvent-exposed helix/loop, but strongly constrained in pSer13 Noxa, indicating a more ordered peptide backbone, as predicted by MD simulations. Together these studies reveal a novel mechanism by which phosphorylation of a distal serine inhibits a pro-apoptotic BH3 domain and promotes cell survival.

Early signaling in camptothecin-treated MCF-7 cells followed an intrinsic pathway, but death was delayed and late events exhibited few hallmarks of apoptosis. BH3-only proteins, such as Noxa, Puma and BimEL, were activated and localized to mitochondrial sites within 24 h following drug exposure. However, caspase activity was low and death was unaffected by caspase inhibition. Transmission electron micrographs showed the presence of large vacuoles in drug-treated cells. An autophagic survival response has been attributed to MCF-7 cells following nutrient starvation or exposure to tamoxifen. Here, we show that autophagy also plays an important role in the delayed DNA damage response. Confocal microscopy revealed colocalization of mitochondria with large autophagic vacuoles and inhibitors of autophagy increased mitochondrial depolarization and caspase-9 activity, and accelerated cell death. Furthermore, downregulation of autophagy proteins, Beclin 1 and Atg7, unmasked a caspase-dependent, apoptotic response to DNA damage. We propose that a post-mitochondrial caspase cascade is delayed as a result of early disposal of damaged mitochondria within autophagosomes. Our data also suggest that the use of autophagy as a means of delaying apoptosis or prolonging survival may be characteristic of noninvasive breast tumor cells. These studies underscore a potential role for autophagy inhibitors in combination with conventional chemotherapeutic drugs in early breast cancer therapy.

Release of cytochrome c from mitochondria is a central event in apoptotic signaling. In this study, we utilized a cytochrome c fusion that binds fluorescent biarsenical ligands (cytochrome c-4CYS (cyt. c-4CYS)) as well as cytochrome c-green fluorescent protein (cyt. c-GFP) to measure its release from mitochondria in different cell types during apoptosis. In single cells, the kinetics of cyt. c-4CYS release was indistinguishable from that of cyt. c-GFP in apoptotic cells expressing both molecules. Lowering the temperature by 7 degrees C did not affect this corelease, but further separated cytochrome c release from the subsequent decrease in mitochondrial membrane potential (DeltaPsi(m)). Cyt. c-GFP rescued respiration in cells lacking endogenous cytochrome c, and the duration of cytochrome c release was approximately 5 min in a variety of cell types induced to die by various forms of cellular stress. In addition, we could observe no evidence of caspase-dependent amplification of cytochrome c release or changes in DeltaPsi(m) preceding the release of cyt. c-GFP. We conclude that there is a general mechanism responsible for cytochrome c release that proceeds in a single step that is independent of changes in DeltaPsi(m).

The Bcl-2 family of apoptotic regulators comprises both pro- and anti-apoptotic proteins. Pro-apoptotic BH3-only members of the Bcl-2 family are subject to stringent control in normal proliferating cells, but can be activated by a variety of mechanisms in response to diverse apoptotic signals. To facilitate the simultaneous identification of BH3-only targets that are regulated at the RNA level, we have designed and assembled multi-probe ribonuclease protection assay templates and demonstrated their ability to detect multiple BH3-only transcripts in a variety of human cell lines and primary tissues. Following this, we have determined the modulation of BH3-only transcripts to a variety of apoptotic stimuli, in selected primary and transformed cell lines. Early experiments, showing that a given cell type upregulates a unique subset of BH3-only RNAs in response to different death-inducing stimuli, indicate that the multi-probe BH3-only RPA template sets could serve as useful diagnostic and analytical tools in cancer research.

Lats2, also known as Kpm, is the second mammalian member of the novel Lats tumor suppressor gene family. Recent studies have demonstrated that Lats2 negatively regulates the cell cycle by controlling G1/S and/or G2/M transition. To further understand the role of Lats2 in the control of human cancer development, we have expressed the protein in human lung cancer cells by transduction of a replication-deficient adenovirus expressing human Lats2 (Ad-Lats2). Using a variety of techniques, including Annexin V uptake, cleavage of PARP, and DNA laddering, we have demonstrated that the ectopic expression of human Lats2 induced apoptosis in two lung cancer cell lines, A549 and H1299. Caspases-3, 7, 8, and 9 were processed in the Ad-Lats2-transduced cells; however, it was active caspase-9, not caspase-8, that initiated the caspase cascade. Inhibitors specific to caspase-3 and 9 delayed the onset of Lats2-mediated apoptosis. Western blot analysis revealed that anti-apoptotic proteins, BCL-2 and BCL-xL, but not the pro-apoptotic protein, BAX, were downregulated in Ad-Lats2-transduced human lung cancer cells. Overexpression of either Bcl-2 or Bcl-xL in these cells lead to the suppression of Lats2-mediated caspase cleavage and apoptosis. These results show that Lats2 induces apoptosis through downregulating anti-apoptotic proteins, BCL-2 and BCL-xL, in human lung cancer cells.

In past studies, we showed that T cells transduced with retroviral diphtheria immunotoxin (IT) target genes could serve as vehicles for delivering IT to tumors in vivo. We took advantage of the observation that antigen-specific T cells are able to penetrate tumors to design an approach delivering combined cellular and humoral therapy directly to the tumor site. To improve tumor specificity, we selected interleukin (IL)-3 as a ligand because its receptor is selectively overexpressed on myeloid leukemia progenitors. Because Bcl-2 family proteins show structural similarity to diphtheria toxin (DT), we constructed a unique retroviral IT using Bax, a proapoptotic member of the Bcl-2 family, in place of DT. Bax was chosen because several studies showed that its transduction induces lethal apoptosis in different cancers. The retroviral construct for gene therapy included IL-3 positioned downstream of its 80 amino acid leader, and permitted cotranslational protein synthesis of hybrid IL-3/human Bax fusion protein. Other vectors were constructed with IL-3 fused to DT or Pseudomonas exotoxin. Retroviral vectors were used to transiently transduce C8, a CD4(+) T cell clone that specifically recognized FBL-3, a lethal myeloid leukemia. Supernatants collected from transduced cells showed proapoptotic activity and selectively inhibited FBL-3 cells in vitro. Intraperitoneal injection of transduced but not nontransduced C8 into mice with subcutaneous tumors or systemic cancer significantly inhibited tumor growth. These results indicate that retroviral IT made with IL-3 and various toxic proteins may be useful in patients with acute myelogenous leukemia (AML). Furthermore, the Bax construct may be particularly useful as a nonimmunogenic substitute for bacterial toxins in retIT.

FL5.12 pro-B lymphoma cells utilize the mitochondrial pathway to apoptosis in response to tumor necrosis factor (TNF) receptor occupation, yet high levels of the Bcl-2 family antiapoptotic protein, Bcl-x(L), fail to protect these cells against TNF-receptor-activated death. Bcl-x(L) expression delays, but does not totally block, the release of mitochondrial cytochrome c (cyt c) in these cells in response to TNFalpha-induced apoptosis and caspase-9 is processed prior to mitochondrial cyt c release under these circumstances. Early processing of caspase-9 also occurred in Apaf-1 knockout murine fibroblasts in response to TNF-receptor occupation. A caspase-9-specific inhibitor was more effective in delaying the progression of apoptosis in the FL5.12 Bcl-x(L) cells than was an inhibitor specific to caspase-3. Furthermore, downregulation of caspase-9 levels by RNA interference resulted in partial protection of these cells against TNF-receptor-activated apoptosis, indicating that caspase-9 activation contributed to early amplification of the caspase cascade. Consistent with this, proteolytic processing of caspase-9 was observed prior to processing by caspase-3, suggesting that caspase-3 was not responsible for early caspase-9 activation. We show that murine caspase-9 is efficiently processed by active caspase-8 at SEPD, the motif at which caspase-9 autoprocesses following its recruitment to the apoptosome. Our results suggest that, in addition to processing procaspase-3 and the BH3 protein Bid, active caspase-8 can cleave and activate procaspase-9 in response to TNF receptor crosslinking in murine cells.

The mechanisms by which cancer cell-intrinsic CYP monooxygenases promote tumor progression are largely unknown. CYP3A4 was unexpectedly associated with breast cancer mitochondria and synthesized arachidonic acid (AA)-derived epoxyeicosatrienoic acids (EETs), which promoted the electron transport chain/respiration and inhibited AMPKα. CYP3A4 knockdown activated AMPKα, promoted autophagy, and prevented mammary tumor formation. The diabetes drug metformin inhibited CYP3A4-mediated EET biosynthesis and depleted cancer cell-intrinsic EETs. Metformin bound to the active site heme of CYP3A4 in a co-crystal structure, establishing CYP3A4 as a biguanide target. Structure-based design led to discovery of N1-hexyl-N5-benzyl-biguanide (HBB), which bound to the CYP3A4 heme with higher affinity than metformin. HBB potently and specifically inhibited CYP3A4 AA epoxygenase activity. HBB also inhibited growth of established ER+ mammary tumors and suppressed intratumoral mTOR. CYP3A4 AA epoxygenase inhibition by biguanides thus demonstrates convergence between eicosanoid activity in mitochondria and biguanide action in cancer, opening a new avenue for cancer drug discovery.

Granulysin is an antimicrobial and tumoricidal molecule expressed in granules of CTL and NK cells. In this study, we show that granulysin damages cell membranes based upon negative charge, disrupts the transmembrane potential (Δψ) in mitochondria, and causes release of cytochrome c. Granulysin-induced apoptosis is blocked in cells overexpressing Bcl-2. Despite the release of cytochrome c, procaspase 9 is not processed. Nevertheless, activation of caspase 3 is observed in granulysin-treated cells, suggesting that granulysin activates a novel pathway of CTL- and NK cell-mediated death distinct from granzyme- and death receptor-induced apoptosis.

Thymic regulatory T cells (tTreg) or in vitro induced Tregs (iTregs) adoptive transfer suppresses murine acute graft-versus-host disease (GVHD). Previously we demonstrated that plasmacytoid dendritic cell (pDC) indoleamine 2,3-dioxygenase (IDO) fosters the development of human iTregs through tryptophan (Trp) depletion and production of catabolites including kynurenines (Kyn). IDO is an immunosuppressive enzyme implicated in maternal-fetal acceptance, tumor immunity, and autoimmunity. IDO is highly up-regulated in the colon of mice and patients with GVHD; in rodents, the lack of IDO leads to increased colon pathology and accelerated lethality. Because pDCs are rare and difficult to purify and expand, we sought to determine whether an optimally suppressive iTreg could be generated using clinically amenable conditions that mimicked pDCs support (i.e. Low Trp+Kyn). Naïve human CD4+ T cells purified with cGMP-compatible reagents and expanded in vitro using an anti-CD3-loaded artificial APC expressing CD86 under conditions of low Trp + kynurenines suppress in vitro T cell proliferation equivalently to tTreg (A) (Fig 1). The calculated yield of Low Trp+Kyn iTreg (~1x1010) produced from an apheresis product is ~10-fold greater than similarly expanded cord blood-derived tTreg that decreased GVHD severity in the clinic (B). Low Trp+Kyn iTreg have a phenotype distinct from tTreg, including: higher CD25 expression, lower Foxp3 expression, and no Helios expression (consistent with the non-thymus origin of iTregs) (C). Since Trp depletion was important for iTreg generation, we determined whether iTregs were under metabolic stress. Surprisingly, bioenergetics profiling demonstrated that Low Trp+Kyn iTreg maintain a remarkably high metabolic rate, with both oxidative phosphorylation and glycolytic pathways higher than that observed in expanded Teffectors (Teff) and tTregs (D). High metabolic rates had functional consequences, as Low Trp+Kyn iTreg were exquisitely dependent on IL-2, which drives the expression of genes needed for glycolytic metabolism. In addition, high basal oxidative phosphorylation rates correlated with increased mitochondrial membrane potential and ROS production (not shown) and decreased viability. A xenogeneic model of GVHD was used to assess the in vivo efficacy of Low Trp+Kyn iTreg. Because Low Trp+Kyn iTreg were so sensitive to IL-2 withdrawal in vitro, mice were also treated ± low dose IL-2 for 9 days (IP, 25kU/day). While low dose IL-2 prolonged in vivo Low Trp+Kyn iTreg persistence and improved survival compared to no IL-2, survival was not significantly increased compared to GVHD mice receiving only PBMC (E). To determine whether increasing the duration of IL-2 therapy would further increase Low Trp+Kyn iTreg persistence and efficacy, the experiment was repeated (with the addition of a cohort of mice receiving PBMC + IL-2 only), and IL-2 treatment was continued indefinitely (F). However, prolonged IL-2 therapy enhanced Teff expansion, and all animals receiving exogenous IL-2 (either PBMC only or with Low Trp+Kyn iTreg) were deceased by day 18. Studies are in progress to determine whether lower doses of IL2 (i.e. 2.5 or 10k U/day) can preferentially foster iTreg expansion and efficacy in suppressing xenogeneic GVHD over Teff expansion and exacerbation of disease. Figure 1. tTreg and naïve CD4+ T-cells were purified from apheresis products using magnetic beads, stimulated with a cell line expressing CD86 and CD64 (KT64/86) to which anti-CD3 mAb is bound, and cultured for 14 days. Low Trp+Kyn iTreg (and Teff) were re-stimulated on day 7. (A) Suppressive function was determined using a CFSE-based proliferation assay at 1:16 (expanded cell:PBMC) and stimulated with anti-CD3 mAb beads for 4 days. (B) Calculated yield of tTreg and Low Trp+Kyn iTreg from a 12 billion cell apheresis product. (C) Representative expression of CD25, Foxp3, and Helios. (D) Representative metabolic analysis showing oxygen consumption rate and extracellular acidification rate for tTreg Low Trp+Kyn iTreg and Teff. (E) Kaplan-Meier survival curves for mice receiving expanded tTreg, and Low Trp+Kyn iTregs and an equal number of HLA-mismatched PBMC. Low dose IL-2 was administered to a cohort of mice receiving iTreg. Mice were bled on day 6 and tTreg and iTreg in circulation quantitated by flow. (F) As in E, but IL-2 given to cohorts of mice receiving PBMC only or PBMC + iTreg for 18 days (n=8 per cohort). Figure 1. tTreg and naïve CD4+ T-cells were purified from apheresis products using magnetic beads, stimulated with a cell line expressing CD86 and CD64 (KT64/86) to which anti-CD3 mAb is bound, and cultured for 14 days. Low Trp+Kyn iTreg (and Teff) were re-stimulated on day 7. (A) Suppressive function was determined using a CFSE-based proliferation assay at 1:16 (expanded cell:PBMC) and stimulated with anti-CD3 mAb beads for 4 days. (B) Calculated yield of tTreg and Low Trp+Kyn iTreg from a 12 billion cell apheresis product. (C) Representative expression of CD25, Foxp3, and Helios. (D) Representative metabolic analysis showing oxygen consumption rate and extracellular acidification rate for tTreg Low Trp+Kyn iTreg and Teff. (E) Kaplan-Meier survival curves for mice receiving expanded tTreg, and Low Trp+Kyn iTregs and an equal number of HLA-mismatched PBMC. Low dose IL-2 was administered to a cohort of mice receiving iTreg. Mice were bled on day 6 and tTreg and iTreg in circulation quantitated by flow. (F) As in E, but IL-2 given to cohorts of mice receiving PBMC only or PBMC + iTreg for 18 days (n=8 per cohort). Disclosures Hippen: University of Minnesota: Other: holds patents for the production and use of iTregs for clinical trials. Riley:Tmunity Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; University of Pennsylvania: Patents & Royalties: hold patents for the production and use of iTregs for clinical trials. June:Pfizer: Honoraria; Celldex: Consultancy, Equity Ownership; Tmunity: Equity Ownership, Other: Founder, stockholder ; Johnson & Johnson: Research Funding; Novartis: Honoraria, Patents & Royalties: Immunology, Research Funding; Immune Design: Consultancy, Equity Ownership; University of Pennsylvania: Patents & Royalties. Kean:Juno Therapeutics, Inc: Research Funding. Miller:Fate Therapeutics: Consultancy, Research Funding; Oxis Biotech: Consultancy, Other: SAB.

bcl-x is a member of the bcl-2 family of genes. The major protein product, Bcl-x(L), is a 233-amino-acid protein which has antiapoptotic properties. In contrast, one of the alternatively spliced transcripts of the bcl-x gene codes for the protein Bcl-x(S), which lacks 63 amino acids present in Bcl-x(L) and has proapoptotic activity. Unlike other proapoptotic Bcl-2 family members, such as Bax and Bak, Bcl-x(S) does not seem to induce cell death in the absence of an additional death signal. However, Bcl-x(S) does interfere with the ability of Bcl-x(L) to antagonize Bax-induced death in transiently transfected 293 cells. Mutational analysis of Bcl-x(S) was conducted to identify the domains necessary to mediate its proapoptotic phenotype. Deletion mutants of Bcl-x(S) which still contained an intact BH3 domain retained the ability to inhibit survival through antagonism of Bcl-x(L). Bcl-x(S) was able to form heterodimers with Bcl-x(L) in mammalian cells, and its ability to inhibit survival correlated with the ability to heterodimerize with Bcl-x(L). Deletion mutants of Bax and Bcl-2, which lacked BH1 and BH2 domains but contained a BH3 domain, were able to antagonize the survival effect conferred by Bcl-x(L). The results suggest that BH3 domains from both pro- and antiapoptotic Bcl-2 family members, while lacking an intrinsic ability to promote programmed cell death, can be potent inhibitors of Bcl-x(L) survival function.

Bcl-2-related proteins have come to occupy a prominent position in the realm of programmed cell death. Members of this fast-growing family are highly related in one or more specific regions, commonly referred to as Bcl-2 homology (BH) domains. BH domains contribute at multiple levels to the function of these proteins in cell death and survival. Particularly intriguing is the emergence of the BH3 domain as a potent 'death domain' and of a growing subclass of pro-apoptotic proteins with no similarity to Bcl-2 beyond their BH3 homology. Here, the authors classify proteins of the Bcl-2 family on the basis of function and domain organization, discuss the importance of the BH3 domain in protein-protein interactions and in cell death and provide possible explanations for the perceived redundancy in the expression of this subclass of death promoters.

Regulatory T-cells (Tregs) prevent autoimmune and alloimmune reactions. Augmenting Treg function may enhance Treg therapies for these diseases. Treg-specific inhibition of protein kinase C-theta (PKC-θ) enhances Treg function. However, it is unclear whether PKC-θ inhibition can boost Treg function in systemic inflammatory conditions. In a mouse model of acute GVHD, we found that Tregs treated with the PKC-θ inhibitor AEB071 reduced GVHD mortality and severity significantly better than DMSO treated Tregs. Compared to DMSO, AEB071 treated Tregs significantly reduced conventional T-cells (Tcon) proliferation on D4 after transplant. Multi-photon microscopy showed that AEB071 treated Tregs significantly increased Tcon velocity and displacement compared to DMSO. Mechanistically, AEB071 augments expression of Neuropilin-1 (Nrp1) and Lymphocyte activation gene 3 (Lag3). Antibody blockade of Nrp1 and Lag3 in transwell suppression assays reduced the effect of AEB071 on Treg function. PKC-θ inhibition also reduces phosphorylation of mTORC2 targets FoxO3a and Akt phospho-site S473, but not mTORC1 targets S6, 4E-BP1 or Akt phospho-site T308. Compared to DMSO, AEB071 treatment significantly increased fatty acid uptake and oxygen consumption rate (OCR). Phosphoproteomic analysis identified a significant alteration in the interaction between PKC-θ and the intermediate filament vimentin after AEB071 treatment, which was confirmed by confocal. Vimentin siRNA treatment also significantly reduces PKC-θ/vimentin interaction, increases Treg function, Nrp1 expression and OCR. In summary, PKC-θ and vimentin modulate multiple aspects of Treg function, and altering these molecules may enhance the efficacy of Treg therapeutics.

Regulatory T-cells (Tregs) play a critical role in preventing autoimmune and alloimmune reactions, including graft-versus-host disease (GVHD). Two recent clinical trials demonstrated that in patients undergoing hematopoietic stem cell transplantation, adoptive transfer of Tregs significantly reduced the incidence of grades II-IV GVHD. While Tregs significantly reduced GVHD severity, they did not eliminate GVHD. One potential way to augment Treg-mediated inhibition of GVHD is to increase Treg suppressive potency. We showed previously that Treg-specific inhibition of protein kinase C-theta (PKC-θ) enhances Treg function (Science 328:372, 2010). However, it is unclear whether PKC-θ inhibition can boost Treg function in a systemic inflammatory condition like GVHD. Furthermore, the mechanism by which PKC-θ inhibition augments Treg function is unknown. In this study, we address these unanswered questions. Using a mouse MHC class I/II disparate acute GVHD model, we found that freshly isolated Tregs treated for 30 minutes with 10uM of the clinically available PKC-θ inhibitor AEB071 suppressed GVHD mortality (Fig 1A) and severity significantly better than DMSO treated Tregs. As Tregs exert much of their protective effect against GVHD early in the course of the disease, we analyzed proliferation of GVHD-causing conventional T-cells (Tcon) on D4 after transplant. We observed a significant reduction in Tcon proliferation in mice given AEB071 treated Tregs compared to DMSO treated Tregs. We then performed multi-photon microscopy on D4 after transplant using TEα-GFP Tcon, CD11c-eYFP antigen presenting cells (APCs) and wild-type Tregs. Compared to DMSO, AEB071 treated Tregs significantly increased Tcon velocity and displacement from APCs. Increased velocity and displacement are indicative of decreased Tcon-APC interactions, suggesting reduced priming when AEB071 Tregs are present. Mechanistically, AEB071 vs DMSO treatment of Tregs resulted in augmented expression of the suppressive molecules Neuropilin-1 (Nrp1) and Lymphocyte activation gene 3 (Lag3) after in vitro activation (Fig 1B, C) and in Tregs isolated from acute GVHD mice. Antibody blockade of Nrp1 and Lag3 in in vitro transwell suppression assays reduced the effect of AEB071 treatment, suggesting that these molecules may play a role in enhancing Treg function after PKC-θ inhibition. Flow cytometry analysis of phosphorylated proteins in activated Tregs revealed that PKC-θ inhibition resulted in reduced phosphorylation of the mTORC2 target FoxO3a, but not mTORC1 targets S6 and 4E-BP1. In addition, the mTORC2-specific phosphorylation site on Akt, serine 473, was reduced, whereas the mTORC1-specific site, threonine 308, was unaltered. Together, these data suggest reduced mTORC2 activity. Reduced phosphorylation increases Foxo3a nuclear translocation, which may result in increased Nrp1 and Lag3 expression, since Foxo3a has binding sites in both gene promoters. As both mTORC1 and 2 are involved in T-cell metabolism, we investigated the effect of AEB071 treatment on Treg oxygen consumption rate (OCR). Compared to DMSO, AEB071 treatment significantly increased Treg baseline and maximal OCRs after activation (Fig 1D). Increased OCR has been associated with increased Treg function. To identify additional alterations in phosphorylated proteins after PKC-θ inhibition, we performed a phosphoproteomic screen using in vitro expanded human Tregs treated with AEB701 or DMSO. We identified significant alterations in phosphorylation sites on 72 proteins, including reduced phosphorylation of an adaptor molecule that links PKC-θ to the intermediate filament vimentin. We found that vimentin is highly upregulated in Tregs compared to Tcon and that in Tregs, vimentin interacts with PKC-θ after activation. AEB071 treatment reduced the interaction between vimentin and PKC-θ. As with AEB071 treatment, Vimentin siRNA significantly increased Treg suppression in vitro compared to control transfected Tregs (Fig 1E), and augmented expression of Nrp1 and Lag3. AEB071 treatment of vimentin siRNA transfected Tregs did not further augment Treg function, suggesting an overlapping mechanism. In summary, our data demonstrate that PKC-θ interacts with mTORC2 and vimentin to modulate multiple aspects of Treg function, and that a brief incubation of Tregs with a PKC-θ inhibitor may be a viable method to enhance the efficacy of Treg therapeutics. Disclosures No relevant conflicts of interest to declare.

The purpose of this study was to determine the sphingolipid (SL) profile in cells defective in autophagy protein ATG-7 and overall cell death after photodynamic therapy (PDT) with the photosensitizer Pc 4. MCF-7 human breast cancer cells with downregulated ATG-7 and their scrambled controls (Scr) were used. Exposure of ATG-7 knockdown cells to PDT led to increased cell killing. PDT evoked an early (2 h) greater global increase in ceramides in ATG-7 defective cells compared to Scr cells. The total increases in dihydroceramide (DHceramide) were significant at 2 h and 24 h in both cell types post-PDT. The levels of sphingosine-1-phosphate (S1P) and sphingosine were decreased below resting levels at both time points irrespective of the cell type. The data imply that ceramide might be a marker of ATG-7 deficiency in cells sensitized to PDT.

Previously we reported that serum leucine-rich alpha-2-glycoprotein-1 (LRG) binds cytochrome c (Cyt c; Cummings et al., Apoptosis 11:1121-1129, 2009). Here we show that LRG binding to Cyt c is similar to that of Apaf-1. LRG and Apaf-1 share partial amino acid sequences, compete for binding Cyt c, and are inhibited by modification at lysine 72 in Cyt c. However, in contrast to Apaf-1, LRG acts as a survival factor in vitro rather than a pro-apoptotic factor. By depleting LRG from culture medium we found that LRG protects against a toxic effect of exogenous Cyt c on lymphocytes that would otherwise result in an apoptotic phenotype. LRG, as well as antibodies specific for Cyt c, increased cell viability in the absence of added Cyt c indicating that Cyt c released by dying cells in the cultures is itself toxic. Protection from extracellular Cyt c-induced lymphotoxicity appears to involve an active mechanism rather than steric hindrance of Cyt c. Thus, serum LRG when bound to extracellular Cyt c that is released from apoptotic cells acts as a survival factor for lymphocytes and possibly other cells that are susceptible to the toxic effect of extracellular Cyt c.

The objective of this study was to determine the sphingolipid (SL) profile in autophagy-defective cells and overall cell death after PDT with Pc 4 (PDT). Human breast cancer MCF-7 cells with downregulated autophagy protein ATG-7 and their scrambled controls (Scr) were used. Exposure of ATG-7 knockdown cells to PDT led to defective processing of the autophagy marker LC3, and increased overall cell killing. In both cell types PDT evoked an early (2 h) increase in ceramides and dihydroceramides (DHceramides). When the two cell types were compared regarding time (2 and 24 h) and treatment conditions (with and without PDT), the levels of several ceramides and DHceramides were reduced, whereas the concentrations of C14-ceramide, C16-ceramide and C12-DHceramide were higher in ATG-7 knockdown cells. The data imply that the SL profile might be a marker of autophagy-deficiency in cells sensitized to PDT.

Glucose uptake and utilization are growth factor-stimulated processes that are frequently upregulated in cancer cells and that correlate with enhanced cell survival. The mechanism of metabolic protection from apoptosis, however, has been unclear. Here we identify a novel signaling pathway initiated by glucose catabolism that inhibited apoptotic death of growth factor-deprived cells. We show that increased glucose metabolism protected cells against the proapoptotic Bcl-2 family protein Bim and attenuated degradation of the antiapoptotic Bcl-2 family protein Mcl-1. Maintenance of Mcl-1 was critical for this protection, as glucose metabolism failed to protect Mcl-1-deficient cells from apoptosis. Increased glucose metabolism stabilized Mcl-1 in both cell lines and primary lymphocytes via inhibitory phosphorylation of glycogen synthase kinase 3alpha and 3beta (GSK-3alpha/beta), which otherwise promoted Mcl-1 degradation. While a number of kinases can phosphorylate and inhibit GSK-3alpha/beta, we provide evidence that protein kinase C may be stimulated by glucose-induced alterations in diacylglycerol levels or distribution to phosphorylate GSK-3alpha/beta, maintain Mcl-1 levels, and inhibit cell death. These data provide a novel nutrient-sensitive mechanism linking glucose metabolism and Bcl-2 family proteins via GSK-3 that may promote survival of cells with high rates of glucose utilization, such as growth factor-stimulated or cancerous cells.

Inhibition of endothelial cell proliferation and angiogenesis is emerging as an important strategy in cancer therapeutics. Kringle 5 (K5) of human plasminogen is a potent angiogenesis inhibitor. Previous studies have shown K5 exposure promotes caspase activity and apoptosis in endothelial cells. Here we report that K5 treatment evokes an autophagic response in endothelial cells that is specific and initiated even in the absence of nutritional stress. Endothelial cells exposed to K5 up-regulated Beclin 1 levels within a few hours. Furthermore, progressively increasing amounts of antiapoptotic Bcl-2 were found to be complexed with Beclin 1, although total levels of Bcl-2 remained unchanged. Prolonged exposure to K5 ultimately led to apoptosis via mitochondrial membrane depolarization and caspase activation in endothelial cells. Knocking down Beclin 1 levels by RNA interference decreased K5 induced autophagy but accelerated K5-induced apoptosis. These studies suggest that interfering with the autophagic survival response can potentiate the antiangiogenic effects of Kringle 5 in endothelial cells.

Apoptosis evasion and altered nutrient metabolism are recognized as major contributors to cancer development. However, the degree to which apoptosis and metabolism are integrated in malignant cells remains poorly understood. Previously our group observed alterations in mitochondrial function mediated by expression of the pro-apoptotic member of the Bcl-2 family Noxa. Here, we report the presence of a large 650 kilodalton multi-protein complex specific to the mitochondria of Jurkat leukemia cells that contains Noxa. We also detect a similar mitochondrial protein complex in cells expressing Noxa with a mutated BH3 domain, suggestive of an apoptosis-independent metabolic function for this Bcl-2 family member. Lastly, we present here the successful isolation of this complex aimed at identifying other complex components via mass spectrometry. This unique strategy included purifying the complex by gel-filtration chromatography, and then resolving the complex into an acrylamide gel by either size or charge, followed by separating the individual complex components by size. Future studies are geared towards identifying the other components of the complex, looking for presence of this complex in other cancers, and understanding how this complex may function in maintaining mitochondrial homeostasis in cancer. In summary, the data presented here strongly suggests a non-canonical function for Noxa in regulating mitochondrial function independent of apoptosis in leukemia, and possibly other malignancies. Note: This abstract was not presented at the meeting. Citation Format: Jeffrey S. Gaynes, Eric A. Hanse, Ameeta Kelekar. Discovery of a novel mitochondrial protein complex containing pro-apoptotic Noxa in leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3043. doi:10.1158/1538-7445.AM2015-3043

While cytochrome P450 enzymes (CYPs) are implicated in tumor angiogenesis through biosynthesis of epoxyeicosatrienoic acids (EETs), little is known about breast cancer cell-intrinsic CYPs that exhibit epoxygenase activity, such as CYP3A4. In an orthotopic breast cancer model, silencing of epithelial CYP3A4 suppressed angiogenesis-related escape of ER+ breast tumors from dormancy. While the diabetes drug metformin inhibits mitochondrial complex I and inhibits tumor growth, how it does so is unknown. Metformin inhibited CYP epoxygenase activity and co-crystallized in the active site of CYP3A4, hydrogen bonding with arginine 212, allowing the development of hexyl-benzyl-biguanide (HBB) as a CYP3A4 inhibitor using molecular modeling. HBB exhibited more than 10-fold greater potency than metformin for inhibition of ER+ mammary tumor growth and inhibited associated tumor angiogenesis. HBB inhibited EET biosynthesis ∼40-fold more potently than metformin and was ∼40-fold more potent for activation of AMPK phosphorylation. EETs suppressed and CYP silencing promoted AMPK phosphorylation, linking CYPs with AMPK regulation in breast cancer. HBB depolarized mitochondria, reduced oxygen consumption rates and suppressed the Warburg effect, while EETs restored the mitochondrial membrane potential. CYP3A4 silencing and HBB treatment increased reactive oxygen species (ROS) production, suggesting that CYPs suppress cancer cell death, in part, through suppression of ROS. CYP3A4 silencing sensitized breast cancer cells to hormonal therapy and chemotherapy, abrogated by EETs. Because EETs are autocrine, paracrine and endocrine, these results implicate CYPs in tumor growth, in part, through cell-cell mediation of mitochondrial homeostasis and demonstrate the potential of CYP3A4 as a therapeutic target in breast cancer. Citation Format: Zhijun Guo, Irina F. Sevrioukova, Eric Hanse, Ilia Denisov, Xia Zhang, Ting-Lan Chiu, Daniel Swedien, Justin Stamschror, Juan Alvarez, William Marerro Ortiz, Monique Morgan, Michael Maher, Kathryn J. Chavez, Dafydd Thomas, Young Kyung Bae, Jonathan Henriksen, Beverly Norris, Robert J. Schumacher, Henry Wang, Robin Bliss, Haitao Chu, Rebecca Cuellar, Thomas L. Poulos, Stephen G. Sligar, William Atkins, Stephen Schmechel, Jorge Capdevila, John Falck, Ian Blair, Jeffrey P. Jones, Gunda Georg, Kalpna Gupta, Ameeta Kelekar, Elizabeth Amin, David A. Potter. CYP3A4 epoxygenase activity mediates ER+ mammary tumor growth and angiogenesis, in part, through EET biosynthesis and is inhibited by biguanides. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3568. doi:10.1158/1538-7445.AM2015-3568

Metformin is a widely used biguanide diabetes drug that is associated with decreased breast cancer risk and is currently being studied for treatment and prevention of breast cancer. While metformin and biguanides buformin and phenformin exhibit inhibitory activity against breast cancer in vitro and in vivo, they lack potency (IC50=5-20 mM) and their mechanisms of action remain unclear. More potent biguanides may provide insights into biguanide anti-cancer activity and we therefore studied the novel biguanide N1-hexyl-N5-benzyl-biguanide mesylate (HBB), which potently inhibits the MCF-7 and MDA-MB-231 breast cancer lines (IC50=20 uM for both lines). HBB induces AMPK phosphorylation in both lines at 10 uM concentration, whereas similarly dosed metformin, buformin or phenformin exhibits no activity. HBB also inhibits STAT3 phosphorylation at 10 uM concentration, whereas metformin dosed at 10 uM exhibits no activity. HBB reduced the mitochondrial membrane potential of both lines, but the effect was more prominent in the MDA-MB-231 line. HBB also induced ROS within 2.5 hours of exposure in the MCF-7 and MDA-MB-231 lines and caused rapid necrosis, but not apoptosis. N-acetylcysteine provides partial protection from HBB for MDA-231 line, but not the MCF-7 line. HBB provides proof of principle that highly potent biguanides can be synthesized with at least 250-fold greater potency than metformin, which can provide insights into the cancer inhibitory mechanisms of biguanide drugs. R01 CA113570, Randy Shaver Foundation, CTSI University of Minnesota Citation Format: Zhijun Guo, Kathryn J. Chavez, Juan Alvarez, Xia Zhang, Beverly Norris, Michael Maher, Monique Morgan, Robert J. Schumacher, Rebecca Cuellar, Irina F. Sevrioukova, Thomas L. Poulos, Ilia Denisov, Stephen G. Sligar, Kalpna Gupta, Ian A. Blair, Jorge Capdevila, Ameeta Kelekar, Elizabeth Amin, Gunda Georg, David A. Potter. Breast cancer inhibition by a novel and potent biguanide, N1-hexyl-N5-benzyl-biguanide. [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 2689. doi:10.1158/1538-7445.AM2014-2689

Upon activation, T cells undergo changes to meet the metabolic demands of proliferation, including the upregulation of aerobic glycolysis. The expression of the Bcl-2 protein Mcl-1 and paradoxically that of its pro-apoptotic binding partner Noxa is also upregulated. Studies showing Noxa is post-translationally modified in both primary and leukemic human T cells, such that its death function is masked, suggest that Noxa plays a direct role in T cell proliferation and survival. Our data show that Noxa also regulates metabolic flux in Jurkat T cells. Here we evaluate Noxa’s role in early proliferation and metabolic reprogramming of human primary T cells. Surprisingly glutamine, but not glucose, was essential for Noxa induction following stimulation. T cells stimulated in the absence of glucose or glutamine exhibit an activated phenotype comparable to controls although cell viability was low in the absence of glucose. The high oxygen consumption and low extracellular acidification rates (OCR and ECAR) of glucose-deprived cells pointed to an early switch to glutamine for mitochondrial respiration during activation. Conversely, glutamine-deprived cells decreased OCR but increased glycolysis to maintain an elevated ECAR, reminiscent of the Warburg effect. We also traced the path of [13C] glucose and glutamine during this metabolic switch. Our data demonstrate for the first time how human primary T cells reprogram metabolism upon activation and how Noxa contributes to these pathways.

Transgenic mice that overexpress the anti-apoptotic gene bcl-xL under the control of the keratin 14 promoter have significantly shorter hair than non-transgenic littermates. The deficit in hair length correlated with a decrease in the duration of anagen, the growth phase of the hair cycle. A prolongation in telogen, the resting phase of the hair cycle, was also observed in adult animals. In the developing hair bulb, bcl-xL transgene expression was observed exclusively in the outer root sheath (ORS) cells. Bcl-xL expression enhanced the survival of ORS cells treated with apoptotic stimuli. The results suggest that preventing the apoptotic death of ORS cells during anagen leads to a more rapid termination of progenitor cell commitment/proliferation, while the increased survival of ORS cells during telogen delays the initiation of a new hair cycle. ORS cells produce fibroblast growth factor-5 (FGF-5), which acts in a paracrine fashion to terminate precursor cell division during anagen. The short hair phenotype of bcl-xL transgenic mice was substantially reversed in FGF-5-deficient mice. Thus, the production of growth inhibitory factors by ORS cells may provide a mechanism through which the hair-growth cycle is regulated by cell survival.

A hydrophobic cleft formed by the BH1, BH2 and BH3 domains of Bcl-xL is responsible for interactions between Bcl-xL and BH3-containing death agonists. Mutants were constructed which did not bind to Bax but retained anti-apoptotic activity. Since Bcl-xL can form an ion channel in synthetic lipid membranes, the possibility that this property has a role in heterodimerization-independent cell survival was tested by replacing amino acids within the predicted channel-forming domain with the corresponding amino acids from Bax. The resulting chimera showed a reduced ability to adopt an open conductance state over a wide range of membrane potentials. Although this construct retained the ability to heterodimerize with Bax and to inhibit apoptosis, when a mutation was introduced that rendered the chimera incapable of heterodimerization, the resulting protein failed to prevent both apoptosis in mammalian cells and Bax-mediated growth defect in yeast. Similar to mammalian cells undergoing apoptosis, yeast cells expressing Bax exhibited changes in mitochondrial properties that were inhibited by Bcl-xL through heterodimerization-dependent and -independent mechanisms. These data suggest that Bcl-xL regulates cell survival by at least two distinct mechanisms; one is associated with heterodimerization and the other with the ability to form a sustained ion channel.