A Novel Peptide that Disrupts the Lck-IP3R Protein-Protein Interaction Induces Widespread Cell Death in Leukemia and Lymphoma.

There is increasing evidence that the T-cell protein, Lck, is involved in the pathogenesis of chronic lymphocytic leukemia (CLL) as well as other leukemias and lymphomas. We previously discovered that Lck binds to domain 5 of inositol 1,4,5-trisphosphate receptors (IP3R) to regulate Ca2+ homeostasis. Using bioinformatics, we targeted a region within domain 5 of IP3R-1 predicted to facilitate protein-protein interactions (PPIs). We generated a synthetic 21 amino acid peptide, KKRMDLVLELKNNASKLLLAI, which constitutes a domain 5 sub-domain (D5SD) of IP3R-1 that specifically binds Lck via its SH2 domain. With the addition of an HIV-TAT sequence to enable cell permeability of D5SD peptide, we observed wide-spread, Ca2+-dependent, cell killing of hematological cancer cells when the Lck-IP3R PPI was disrupted by TAT-D5SD. All cell lines and primary cells were sensitive to D5SD peptide, but malignant T-cells were less sensitive compared with B-cell or myeloid malignancies. Mining of RNA-seq data showed that LCK was expressed in primary diffuse large B-cell lymphoma (DLBCL) as well as acute myeloid leukemia (AML). In fact, LCK shows a similar pattern of expression as many well-characterized AML oncogenes and is part of a protein interactome that includes FLT3-ITD, Notch-1, and Kit. Consistent with these findings, our data suggest that the Lck-IP3R PPI may protect malignant hematopoietic cells from death. Importantly, TAT-D5SD showed no cytotoxicity in three different non-hematopoietic cell lines; thus its ability to induce cell death appears specific to hematopoietic cells. Together, these data show that a peptide designed to disrupt the Lck-IP3R PPI has a wide range of pre-clinical activity in leukemia and lymphoma.

A novel peptide that disrupts the Lck-IP3R protein-protein interaction induces widespread cell death in leukemia and lymphoma.

There is increasing evidence that the T-cell protein, Lck, is involved in the pathogenesis of chronic lymphocytic leukemia (CLL) as well as other leukemias and lymphomas. We previously discovered that Lck binds to domain 5 of inositol 1,4,5-trisphosphate receptors (IP3R) to regulate Ca2+ homeostasis. Using bioinformatics, we targeted a region within domain 5 of IP3R-1 predicted to facilitate protein-protein interactions (PPIs). We generated a synthetic 21 amino acid peptide, KKRMDLVLELKNNASKLLLAI, which constitutes a domain 5 sub-domain (D5SD) of IP3R-1 that specifically binds Lck via its SH2 domain. With the addition of an HIV-TAT sequence to enable cell permeability of D5SD peptide, we observed wide-spread, Ca2+-dependent, cell killing of hematological cancer cells when the Lck-IP3R PPI was disrupted by TAT-D5SD. All cell lines and primary cells were sensitive to D5SD peptide, but malignant T-cells were less sensitive compared with B-cell or myeloid malignancies. Mining of RNA-seq data showed that LCK was expressed in primary diffuse large B-cell lymphoma (DLBCL) as well as acute myeloid leukemia (AML). In fact, LCK shows a similar pattern of expression as many well-characterized AML oncogenes and is part of a protein interactome that includes FLT3-ITD, Notch-1, and Kit. Consistent with these findings, our data suggest that the Lck-IP3R PPI may protect malignant hematopoietic cells from death. Importantly, TAT-D5SD showed no cytotoxicity in three different non-hematopoietic cell lines; thus its ability to induce cell death appears specific to hematopoietic cells. Together, these data show that a peptide designed to disrupt the Lck-IP3R PPI has a wide range of pre-clinical activity in leukemia and lymphoma.

Retinoblastoma Expression and Targeting by CDK4/6 Inhibitors in Small Cell Lung Cancer.

The canonical model of "small cell lung cancer" (SCLC) depicts tumors arising from dual inactivation of TP53 and RB1. However, many genomic studies have persistently identified tumors with no RB1 mutations. Here, we examined RB1 protein expression and function in SCLC. RB1 expression was examined by IHC analysis of 62 human SCLC tumors. These studies showed that ∼14% of SCLC tumors expressed abundant RB1 protein, which is associated with neuroendocrine gene expression and is enriched in YAP1 expression, but no other lineage proteins that stratify SCLC. SCLC cells and xenograft tumors with RB1 protein expression were sensitive to growth inhibition by the CDK4/6 inhibitor palbociclib, and this inhibition was shown to be dependent on RB1 expression by CRISPR knockout. Furthermore, a patient with biopsy-validated wild-type RB1 SCLC who received the CDK4/6 inhibitor abemaciclib demonstrated a dramatic decrease in mutant TP53 ctDNA allelic fraction from 62.1% to 0.4% and decreased tumor mass on CT scans. Importantly, IHC of the diagnostic biopsy specimen showed RB1 positivity. Finally, we identified a transcriptomics-based RB1 loss-of-function signature that discriminates between SCLC cells with or without RB1 protein expression and validated it in the patient who was responsive to abemaciclib, suggesting its potential use to predict CDK4/6 inhibitor response in patients with SCLC. Our study demonstrates that RB1 protein is an actionable target in a subgroup of SCLC, a cancer that exhibits no currently targetable mutations.

A non-canonical role for pyruvate kinase M2 as a functional modulator of Ca2+ signalling through IP3 receptors.

Pyruvate kinase isoform M2 (PKM2) is a rate-limiting glycolytic enzyme that is widely expressed in embryonic tissues. The expression of PKM2 declines in some tissues following embryogenesis, while other pyruvate kinase isozymes are upregulated. However, PKM2 is highly expressed in cancer cells and is believed to play a role in supporting anabolic processes during tumour formation. In this study, PKM2 was identified as an inositol 1,4,5-trisphosphate receptor (IP3R)-interacting protein by mass spectrometry. The PKM2:IP3R interaction was further characterized by pull-down and co-immunoprecipitation assays, which showed that PKM2 interacted with all three IP3R isoforms. Moreover, fluorescence microscopy indicated that both IP3R and PKM2 localized at the endoplasmic reticulum. PKM2 binds to IP3R at a highly conserved 21-amino acid site (corresponding to amino acids 2078-2098 in mouse type 1 IP3R isoform). Synthetic peptides (denoted 'TAT-D5SD' and 'D5SD'), based on the amino acid sequence at this site, disrupted the PKM2:IP3R interaction and potentiated IP3R-mediated Ca2+ release both in intact cells (TAT-D5SD peptide) and in a unidirectional 45Ca2+ flux assay on permeabilized cells (D5SD peptide). The TAT-D5SD peptide did not affect the enzymatic activity of PKM2. Reducing PKM2 protein expression using siRNA increased IP3R-mediated Ca2+ signalling in intact cells without altering the ER Ca2+ content. These data identify PKM2 as an IP3R-interacting protein that inhibits intracellular Ca2+ signalling. The elevated expression of PKM2 in cancer cells is therefore not solely connected to its canonical role in glycolytic metabolism, rather PKM2 also has a novel non-canonical role in regulating intracellular signalling.

Methodology and implementation of the WHO European Childhood Obesity Surveillance Initiative (COSI).

Establishment of the WHO European Childhood Obesity Surveillance Initiative (COSI) has resulted in a surveillance system which provides regular, reliable, timely, and accurate data on children's weight status-through standardized measurement of bodyweight and height-in the WHO European Region. Additional data on dietary intake, physical activity, sedentary behavior, family background, and school environments are collected in several countries. In total, 45 countries in the European Region have participated in COSI. The first five data collection rounds, between 2007 and 2021, yielded measured anthropometric data on over 1.3 million children. In COSI, data are collected according to a common protocol, using standardized instruments and procedures. The systematic collection and analysis of these data enables intercountry comparisons and reveals differences in the prevalence of childhood thinness, overweight, normal weight, and obesity between and within populations. Furthermore, it facilitates investigation of the relationship between overweight, obesity, and potential risk or protective factors and improves the understanding of the development of overweight and obesity in European primary-school children in order to support appropriate and effective policy responses.

Physical inactivity in nine European and Central Asian countries: an analysis of national population-based survey results.

BACKGROUND: Physical inactivity is a major risk factor for non-communicable diseases. However, recent and systematically obtained national-level data to guide policy responses are often lacking, especially in countries in Eastern Europe and Central Asia. This article describes physical inactivity patterns among adults in Armenia, Azerbaijan, Belarus, Georgia, Kyrgyzstan, Republic of Moldova, Tajikistan, Turkey and Uzbekistan. METHODS: Data were collected using the Global Physical Activity Questionnaire drawing nationally representative samples of adults in each country. The national prevalence of physical inactivity was calculated as well as the proportional contribution to total physical activity (PA) during work, transport and leisure-time. An adjusted logistic regression model was applied to analyze the association of age, gender, education, household status and income with physical inactivity. RESULTS: National prevalence of physical inactivity ranged from 10.1% to 43.6%. The highest proportion of PA was registered during work or in the household in most countries, whereas the lowest was during leisure-time in all countries. Physical inactivity was more likely with older age in eight countries, with female gender in three countries, and with living alone in three countries. There was no clear pattern of association with education and income. CONCLUSION: Prevalence of physical inactivity is heterogeneous across the region. PA during leisure-time contributes minimally to total PA in all countries. Policies and programs that increase opportunities for active travel and leisure-time PA, especially for older adults, women and people living alone will be an essential part of strategies to increase overall population PA.

Mobilizing governments and society to combat obesity: Reflections on how data from the WHO European Childhood Obesity Surveillance Initiative are helping to drive policy progress.

To meet the need for regular and reliable data on the prevalence of overweight and obesity among children in Europe, the World Health Organization (WHO) European Childhood Obesity Surveillance Initiative (COSI) was established in 2007. The resulting robust surveillance system has improved understanding of the public health challenge of childhood overweight and obesity in the WHO European Region. For the past decade, data from COSI have helped to inform and drive policy action on nutrition and physical activity in the region. This paper describes illustrative examples of how COSI data have fed into national and international policy, but the real scope of COSI's impact is likely to be much broader. In some countries, there are signs that policy responses to COSI data have helped halt the rise in childhood obesity. As the countries of the WHO European Region commit to pursuing United Action for Better Health in Europe in WHO's new European Programme of Work, COSI provides an excellent example of such united action in practice. Further collaborative action will be key to tackling this major public health challenge which affects children throughout the region.

Identification of RUNX1T1 as a potential epigenetic modifier in small-cell lung cancer.

Small-cell lung cancer (SCLC) can be subgrouped into common 'pure' and rare 'combined' SCLC (c-SCLC). c-SCLC features a mixed tumor histology of both SCLC and non-small-cell lung cancer (NSCLC). We performed targeted exome sequencing on 90 patients with SCLC, including two with c-SCLC, and discovered RUNX1T1 amplification specific to small cell tumors of both patients with c-SCLC, but in only 2 of 88 'pure' SCLC patients. RUNX1T1 was first identified in the fusion transcript AML1/ETO, which occurs in 12%-15% of acute myelogenous leukemia (AML). We further show higher expression of RUNX1T1 in the SCLC component of another c-SCLC tumor by in situ hybridization. RUNX1T1 expression was enriched in SCLC compared with all other cancers, including NSCLC, in both cell lines and tumor specimens, as shown by mRNA level and western blotting. Transcriptomic analysis of hallmark genes decreased by stable RUNX1T1 overexpression revealed a significant change in E2F targets. Validation experiments in multiple lung cancer cell lines showed that RUNX1T1 overexpression consistently decreased CDKN1A (p21) expression and increased E2F transcriptional activity, which is commonly altered in SCLC. Chromatin immunoprecipitation (ChIP) in these overexpressing cells demonstrated that RUNX1T1 interacts with the CDKN1A (p21) promoter region, which displayed parallel reductions in histone 3 acetylation. Furthermore, reduced p21 expression could be dramatically restored by HDAC inhibition using Trichostatin A. Reanalysis of ChIP-seq data in Kasumi-1 AML cells showed that knockdown of the RUNX1T1 fusion protein was associated with increased global acetylation, including the CDKN1A (p21) promoter. Thus, our study identifies RUNX1T1 as a biomarker and potential epigenetic regulator of SCLC.

Implementation of WHO Recommended Policies and Interventions on Healthy Diet in the Countries of the Eastern Mediterranean Region: From Policy to Action.

Non-communicable diseases (NCDs) are responsible for almost two-thirds of the deaths in the 22 countries and territories of the WHO Eastern Mediterranean Region and unhealthy diets are a major contributor. Prevalence of overweight and obesity has increased among adults, adolescents and older children in recent decades. Among countries with the highest prevalence there are signs that the increase is slowing down or even that prevalence is declining. There has been no increase in the prevalence rate in younger children, although the absolute number of children under five years affected by overweight has increased. This review summarizes prevalence data and examines current implementation of regulatory, fiscal and voluntary measures to promote healthy diet across the Region. The last decade has seen a step up in such action. Ten of the Region's countries have policies relating to trans-fatty acids and they are increasingly implementing specific regulatory measures. Thirteen countries had fully or partially implemented national salt reduction policies by 2019. Only four countries had adopted policies relating to aspects of marketing food to children by 2019, and concrete action in this area is still lacking. Eight countries have introduced taxes-sometimes at a rate of 50%-on carbonated or sugar-sweetened beverages. In order to meet the agreed global and regional goals relating to nutrition and diet-related NCDs, countries will need to build on this progress and scale up action across the Region while intensifying efforts in areas where concrete action is lacking.

Tackling Childhood Stunting in the Eastern Mediterranean Region in the Context of COVID-19.

Over 20 million children under 5 years old in the WHO Eastern Mediterranean Region have stunted growth, as a result of chronic malnutrition, with damaging long-term consequences for individuals and societies. This review extracted and analyzed data from the UNICEF, WHO and the World Bank malnutrition estimates to present an overall picture of childhood stunting in the region. The number of children under 5 in the region who are affected by stunting has dropped from 24.5 million (40%) in 1990 to 20.6 million (24.2%) in 2019. The reduction rate since the 2012 baseline is only about two fifths of that required and much more rapid progress will be needed to reach the internationally agreed targets by 2025 and 2030. Prevalence is highest in low-income countries and those with a lower Human Development Index. The COVID-19 pandemic threatens to undermine efforts to reduce stunting, through its impact on access and affordability of safe and nutritious foods and access to important health services. Priority areas for action to tackle stunting as part of a comprehensive, multisectoral nutrition strategy are proposed. In light of the threat that COVID-19 will exacerbate the already heavy burden of malnutrition in the Eastern Mediterranean Region, implementation of such strategies is more important than ever.

Improving Nutrition Information in the Eastern Mediterranean Region: Implementation of Front-Of-Pack Nutrition Labelling.

The provision of simplified nutrition information, in a prominent place on the front of food packages, is recommended as an important element of comprehensive strategies to tackle the burden of death and disease caused by unhealthy diets. There is growing evidence that front-of-pack nutrition labels are preferred by consumers, are more likely to be looked at or noticed than nutrition labelling on the back or side of packages and can help consumers to better identify healthier and less healthy products. This review summarizes current implementation of front-of-pack nutrition labelling policies in the countries of the WHO Eastern Mediterranean Region. Implementation of front-of-pack nutrition labelling in the Eastern Mediterranean Region remains limited, but three types of scheme were identified as having been implemented or at an advanced stage of development by governments in six countries. Through a review of reviews of existing research and evidence from country implementation, the authors suggest some pointers for implementation for other countries in the Region deciding to implement front-of-pack nutrition labelling policies.

Post-transcriptional regulation of PIAS3 expression by miR-18a in malignant mesothelioma.

Protein inhibitor of activated STAT3 (PIAS3) is an endogenous suppressor of signal transducer and activator of transcription 3 (STAT3) signaling. By directly interacting with phosphorylated STAT3, PIAS3 can block the downstream transcriptional activity of STAT3, which is hyper-activated in various cancers. We previously reported that in malignant mesothelioma (MM), low PIAS3 expression is associated with increased STAT3 activation and correlates with poor patient survival, yet the regulatory mechanism(s) governing PIAS3 expression in MM remain unclear. Here, we demonstrate that PIAS3 protein expression does not correlate with its mRNA level in MM cell lines, indicating that PIAS3 expression is regulated at a post-transcriptional level. Inhibition of proteasomal degradation with MG132 (10 µm) or bortezomib (1 µm), alone and in combination, did not increase PIAS3 protein levels; furthermore, inhibition of protein synthesis by cycloheximide treatment did not decrease PIAS3 levels within 48 h, suggesting that PIAS3 expression is not actively regulated at a post-translational level. To determine whether miRNA (miRs) can translationally regulate PIAS3 expression, we combined miR microarray analysis with bioinformatic screening to identify candidate miRs, in MM cell lines with low PIAS3 expression, followed by luciferase reporter assays to validate miR regulation of the PIAS3 3'UTR. We identified miR-18a as a suppressor of PIAS3 expression that is upregulated in MM cells and whose inhibition can increase PIAS3 expression and suppress STAT3 activity. Moreover, we showed that miR-18a inhibition can decrease MM cell viability and that its expression is negatively correlated with MM patient survival. Taken together, these results suggest that targeting miR-18a may have therapeutic benefit in MM.

Reciprocal expression of INSM1 and YAP1 defines subgroups in small cell lung cancer.

The majority of small cell lung cancer (SCLC) patients demonstrate initial chemo-sensitivity, whereas a distinct subgroup of SCLC patients, termed chemo-refractory, do not respond to treatment. There is little understanding of how to distinguish these patients prior to disease treatment. Here we used gene expression profiling to stratify SCLC into subgroups and characterized a molecular phenotype that may identify, in part, chemo-refractive SCLC patients. Two subgroups of SCLC were identified in both cell lines and tumors by the reciprocal expression of two genes; INSM1, a neuroendocrine transcription factor, and YAP1, a key mediator of the Hippo pathway. The great majority of tumors expressed INSM1, which was prognostic for increased progression-free survival and associated with chemo-sensitivity in cell lines. YAP1 is expressed in a minority of SCLC tumors and was shown in cell lines to be downstream of the retinoblastoma protein (RB1) and associated with decreased drug sensitivity. RB1 expression in SCLC cell lines sensitizes them to CDK4/6 inhibitors. Wild-type RB1 mutation status, used as a surrogate marker of YAP1 expression, was prognostic for decreased patient survival and increased chemo-refractory tumor response. Thus, the reciprocal expression of INSM1 and YAP1 appears to stratify SCLC into distinct subgroups and may be useful, along with RB1 mutation status, to identify chemo-refractory SCLC patients.

A synthetic peptide targeting the BH4 domain of Bcl-2 induces apoptosis in multiple myeloma and follicular lymphoma cells alone or in combination with agents targeting the BH3-binding pocket of Bcl-2.

Bcl-2 inhibits apoptosis by two distinct mechanisms but only one is targeted to treat Bcl-2-positive malignancies. In this mechanism, the BH1-3 domains of Bcl-2 form a hydrophobic pocket, binding and inhibiting pro-apoptotic proteins, including Bim. In the other mechanism, the BH4 domain mediates interaction of Bcl-2 with inositol 1,4, 5-trisphosphate receptors (IP3Rs), inhibiting pro-apoptotic Ca2+ signals. The current anti-Bcl-2 agents, ABT-263 (Navitoclax) and ABT-199 (Venetoclax), induce apoptosis by displacing pro-apoptotic proteins from the hydrophobic pocket, but do not inhibit Bcl-2-IP3R interaction. Therefore, to target this interaction we developed BIRD-2 (Bcl-2 IP3 Receptor Disruptor-2), a decoy peptide that binds to the BH4 domain, blocking Bcl-2-IP3R interaction and thus inducing Ca2+-mediated apoptosis in chronic lymphocytic leukemia, multiple myeloma, and follicular lymphoma cells, including cells resistant to ABT-263, ABT-199, or the Bruton's tyrosine kinase inhibitor Ibrutinib. Moreover, combining BIRD-2 with ABT-263 or ABT-199 enhances apoptosis induction compared to single agent treatment. Overall, these findings provide strong rationale for developing novel therapeutic agents that mimic the action of BIRD-2 in targeting the BH4 domain of Bcl-2 and disrupting Bcl-2-IP3R interaction.

PIAS3 expression in squamous cell lung cancer is low and predicts overall survival.

Unlike lung adenocarcinoma, little progress has been made in the treatment of squamous cell lung carcinoma (SCC). The Cancer Genome Atlas (TCGA) has recently reported that receptor tyrosine kinase signaling pathways are altered in 26% of SCC tumors, validating the importance of downstream Signal Transducers and Activators of Transcription 3 (STAT3) activity as a prime therapeutic target in this cancer. In the present report we examine the status of an endogenous inhibitor of STAT3, called Protein Inhibitor of Activated STAT3 (PIAS3), in SCC and its potential role in this disease. We examine PIAS3 expression in SCC tumors and cell lines by immunohistochemistry of a tissue microarray and western blotting. PIAS3 mRNA expression and survival data are analyzed in the TCGA data set. SCC cell lines are treated with curcumin to regulate PIAS3 expression and cell growth. PIAS3 protein expression is decreased in a majority of lung SCC tumors and cell lines. Analysis of PIAS3 mRNA transcript levels demonstrated that low PIAS3 levels predicted poor survival; Cox regression analysis revealed a hazard ratio of 0.57 (95% CI: 0.37-0.87), indicating a decrease in the risk of death by 43% for every unit elevation in PIAS3 gene expression. Curcumin treatment increased endogenous PIAS3 expression and decreased cell growth and viability in Calu-1 cells, a model of SCC. Our results implicate PIAS3 loss in the pathology of lung SCC and raise the therapeutic possibility of upregulating PIAS3 expression as a single target that can suppress signaling from the multiple receptor tyrosine kinase receptors found to be amplified in SCC.

PIAS3 activates the intrinsic apoptotic pathway in non-small cell lung cancer cells independent of p53 status.

Protein inhibitor of activated signal transducer and activator of transcription 3 (STAT3) (PIAS3) is an endogenous inhibitor of STAT3 that negatively regulates STAT3 transcriptional activity and cell growth and demonstrates limited expression in the majority of human squamous cell carcinomas of the lung. In this study, we sought to determine whether PIAS3 inhibits cell growth in non-small cell lung cancer cell lines by inducing apoptosis. Our results demonstrate that overexpression of PIAS3 promotes mitochondrial depolarization, leading to cytochrome c release, caspase 9 and 3 activation and poly (ADP-ribose) polymerase cleavage. This intrinsic pathway activation was associated with decreased Bcl-xL expression and increased Noxa expression and was independent of p53 status. Furthermore, PIAS3 inhibition of STAT3 activity was also p53 independent. Microarray experiments were performed to discover STAT3-independent mediators of PIAS3-induced apoptosis by comparing the apoptotic gene expression signature induced by PIAS3 overexpression with that induced by STAT3 siRNA. The results showed that a subset of apoptotic genes was uniquely expressed only after PIAS3 expression. Thus, PIAS3 may represent a promising lung cancer therapeutic target because of its p53-independent efficacy and its potential to synergize with Bcl-2 targeted inhibitors.

Acidosis Sensing Receptor GPR65 Correlates with Anti-Apoptotic Bcl-2 Family Member Expression in CLL Cells: Potential Implications for the CLL Microenvironment.

The tumor microenvironment is generally an acidic environment, yet the effect of extracellular acidosis on chronic lymphocytic leukemia (CLL) is not well established. Here we are the first to report that the extracellular acid sensing G-protein coupled receptor, GPR65, is expressed in primary CLL cells where its level correlate strongly with anti-apoptotic Bcl-2 family member levels. GPR65 expression is found normally within the lymphoid lineage and has not been previously reported in CLL. We demonstrate a wide range of GPR65 mRNA expression among CLL 87 patient samples. The correlation between GPR65 mRNA levels and Bcl-2 mRNA levels is particularly strong (r=0.8063, p= <0.001). The correlation extends to other anti-apoptotic Bcl-2 family members, Mcl-1 (r=0.4847, p=0.0010) and Bcl-xl (r=0.3411, p=0.0252), although at lower levels of significance. No correlation is detected between GPR65 and levels of the pro-apoptotic proteins BIM, PUMA or NOXA. GPR65 expression also correlates with the favorable prognostic marker of 13q deletion. The present findings suggest the acid sensing receptor GPR65 may be of significance to allow CLL tolerance of extracellular acidosis. The correlation of GPR65 with Bcl-2 suggests a novel cytoprotective mechanism that enables CLL cell adaptation to acidic extracellular conditions. These findings suggest the potential value of targeting GPR65 therapeutically.

Acidosis blocks CCAAT/enhancer-binding protein homologous protein (CHOP)- and c-Jun-mediated induction of p53-upregulated mediator of apoptosis (PUMA) during amino acid starvation.

Cancer cells must avoid succumbing to a variety of noxious conditions within their surroundings. Acidosis is one such prominent feature of the tumor microenvironment that surprisingly promotes tumor survival and progression. We recently reported that acidosis prevents apoptosis of starved or stressed lymphoma cells through regulation of several Bcl-2 family members (Ryder et al., JBC, 2012). Mechanistic studies in that work focused on the acid-mediated upregulation of anti-apoptotic Bcl-2 and Bcl-xL, while additionally showing inhibition of glutamine starvation-induced expression of pro-apoptotic PUMA by acidosis. Herein we report that amino acid (AA) starvation elevates PUMA, an effect that is blocked by extracellular acidity. Knockdown studies confirm that PUMA induction during AA starvation requires expression of both CHOP and c-Jun. Interestingly, acidosis strongly attenuates AA starvation-mediated c-Jun expression, which correlates with PUMA repression. As c-Jun exerts a tumor suppressive function in this and other contexts, its inhibition by acidosis has broader implications for survival of cancer cells in the acidic tumor milieu.

Acidosis promotes Bcl-2 family-mediated evasion of apoptosis: involvement of acid-sensing G protein-coupled receptor Gpr65 signaling to Mek/Erk.

Acidosis arises in solid and lymphoid malignancies secondary to altered nutrient supply and utilization. Tumor acidosis correlates with therapeutic resistance, although the mechanism behind this effect is not fully understood. Here we show that incubation of lymphoma cell lines in acidic conditions (pH 6.5) blocks apoptosis induced by multiple cytotoxic metabolic stresses, including deprivation of glucose or glutamine and treatment with dexamethasone. We sought to examine the role of the Bcl-2 family of apoptosis regulators in this process. Interestingly, we found that acidic culture causes elevation of both Bcl-2 and Bcl-xL, while also attenuating glutamine starvation-induced elevation of p53-up-regulated modulator of apoptosis (PUMA) and Bim. We confirmed with knockdown studies that these shifts direct survival decisions during starvation and acidosis. Importantly, the promotion of a high anti- to pro-apoptotic Bcl-2 family member ratio by acidosis renders cells exquisitely sensitive to the Bcl-2/Bcl-xL antagonist ABT-737, suggesting that acidosis causes Bcl-2 family dependence. This dependence appears to be mediated, in part, by the acid-sensing G protein-coupled receptor, GPR65, via a MEK/ERK pathway.