An immune-sympathetic neuron communication axis guides adipose tissue browning in cancer-associated cachexia.

Cancer-associated cachexia (CAC) is a hypermetabolic syndrome characterized by unintended weight loss due to the atrophy of adipose tissue and skeletal muscle. A phenotypic switch from white to beige adipocytes, a phenomenon called browning, accelerates CAC by increasing the dissipation of energy as heat. Addressing the mechanisms of white adipose tissue (WAT) browning in CAC, we now show that cachexigenic tumors activate type 2 immunity in cachectic WAT, generating a neuroprotective environment that increases peripheral sympathetic activity. Increased sympathetic activation, in turn, results in increased neuronal catecholamine synthesis and secretion, ß-adrenergic activation of adipocytes, and induction of WAT browning. Two genetic mouse models validated this progression of events. 1) Interleukin-4 receptor deficiency impeded the alternative activation of macrophages, reduced sympathetic activity, and restrained WAT browning, and 2) reduced catecholamine synthesis in peripheral dopamine ß-hydroxylase (DBH)-deficient mice prevented cancer-induced WAT browning and adipose atrophy. Targeting the intraadipose macrophage-sympathetic neuron cross-talk represents a promising therapeutic approach to ameliorate cachexia in cancer patients.

Adverse cardiac remodeling augments adipose tissue ß-adrenergic signaling and lipolysis counteracting diet-induced obesity.

Cardiac triacylglycerol accumulation is a common characteristic of obesity and type 2 diabetes and strongly correlates with heart morbidity and mortality. We have previously shown that cardiomyocyte-specific perilipin 5 overexpression (Plin5-Tg) provokes significant cardiac steatosis via lowering cardiac lipolysis and fatty acid (FA) oxidation. In strong contrast to cardiac steatosis and lethal heart dysfunction in adipose triglyceride lipase deficiency, Plin5-Tg mice do not develop heart dysfunction and show a normal life span on chow diet. This finding prompted us to study heart function and energy metabolism in Plin5-Tg mice fed high-fat diet (HFD). Plin5-Tg mice showed adverse cardiac remodeling on HFD with heart function only being compromised in one-year-old mice, likely due to reduced cardiac FA uptake, thereby delaying deleterious cardiac lipotoxicity. Notably, Plin5-Tg mice were less obese and protected from glucose intolerance on HFD. Changes in cardiac energy catabolism in Plin5-Tg mice increased ß-adrenergic signaling, lipolytic, and thermogenic protein expression in adipose tissue ultimately counteracting HFD-induced obesity. Acute cold exposure further augmented ß-adrenergic signaling in Plin5-Tg mice, whereas housing at thermoneutrality did not protect Plin5-Tg mice from HFD-induced obesity albeit blood glucose and insulin levels remained low in transgenic mice. Overall, our data suggest that the limited capacity for myocardial FA oxidation on HFD increases cardiac stress in Plin5-Tg mice, thereby stimulating adipose tissue ß-adrenergic signaling, triacylglycerol catabolism, and thermogenesis. However, long-term HFD-mediated metabolic stress causes contractile dysfunction in Plin5-Tg mice, which emphasizes the importance of a carefully controlled dietary regime in patients with cardiac steatosis and hypertrophy.

Fine-Tuning Cardiac Insulin-Like Growth Factor 1 Receptor Signaling to Promote Health and Longevity.

BACKGROUND: The insulin-like growth factor 1 (IGF1) pathway is a key regulator of cellular metabolism and aging. Although its inhibition promotes longevity across species, the effect of attenuated IGF1 signaling on cardiac aging remains controversial. METHODS: We performed a lifelong study to assess cardiac health and lifespan in 2 cardiomyocyte-specific transgenic mouse models with enhanced versus reduced IGF1 receptor (IGF1R) signaling. Male mice with human IGF1R overexpression or dominant negative phosphoinositide 3-kinase mutation were examined at different life stages by echocardiography, invasive hemodynamics, and treadmill coupled to indirect calorimetry. In vitro assays included cardiac histology, mitochondrial respiration, ATP synthesis, autophagic flux, and targeted metabolome profiling, and immunoblots of key IGF1R downstream targets in mouse and human explanted failing and nonfailing hearts, as well. RESULTS: Young mice with increased IGF1R signaling exhibited superior cardiac function that progressively declined with aging in an accelerated fashion compared with wild-type animals, resulting in heart failure and a reduced lifespan. In contrast, mice with low cardiac IGF1R signaling exhibited inferior cardiac function early in life, but superior cardiac performance during aging, and increased maximum lifespan, as well. Mechanistically, the late-life detrimental effects of IGF1R activation correlated with suppressed autophagic flux and impaired oxidative phosphorylation in the heart. Low IGF1R activity consistently improved myocardial bioenergetics and function of the aging heart in an autophagy-dependent manner. In humans, failing hearts, but not those with compensated hypertrophy, displayed exaggerated IGF1R expression and signaling activity. CONCLUSIONS: Our findings indicate that the relationship between IGF1R signaling and cardiac health is not linear, but rather biphasic. Hence, pharmacological inhibitors of the IGF1 pathway, albeit unsuitable for young individuals, might be worth considering in older adults.

Genetic deletion of MMP12 ameliorates cardiometabolic disease by improving insulin sensitivity, systemic inflammation, and atherosclerotic features in mice.

BACKGROUND: Matrix metalloproteinase 12 (MMP12) is a macrophage-secreted protein that is massively upregulated as a pro-inflammatory factor in metabolic and vascular tissues of mice and humans suffering from cardiometabolic diseases (CMDs). However, the molecular mechanisms explaining the contributions of MMP12 to CMDs are still unclear. METHODS: We investigated the impact of MMP12 deficiency on CMDs in a mouse model that mimics human disease by simultaneously developing adipose tissue inflammation, insulin resistance, and atherosclerosis. To this end, we generated and characterized low-density lipoprotein receptor (Ldlr)/Mmp12-double knockout (DKO) mice fed a high-fat sucrose- and cholesterol-enriched diet for 16-20 weeks. RESULTS: DKO mice showed lower cholesterol and plasma glucose concentrations and improved insulin sensitivity compared with LdlrKO mice. Untargeted proteomic analyses of epididymal white adipose tissue revealed that inflammation- and fibrosis-related pathways were downregulated in DKO mice. In addition, genetic deletion of MMP12 led to alterations in immune cell composition and a reduction in plasma monocyte chemoattractant protein-1 in peripheral blood which indicated decreased low-grade systemic inflammation. Aortic en face analyses and staining of aortic valve sections demonstrated reduced atherosclerotic plaque size and collagen content, which was paralleled by an improved relaxation pattern and endothelial function of the aortic rings and more elastic aortic sections in DKO compared to LdlrKO mice. Shotgun proteomics revealed upregulation of anti-inflammatory and atheroprotective markers in the aortas of DKO mice, further supporting our data. In humans, MMP12 serum concentrations were only weakly associated with clinical and laboratory indicators of CMDs. CONCLUSION: We conclude that the genetic deletion of MMP12 ameliorates obesity-induced low-grade inflammation, white adipose tissue dysfunction, biomechanical properties of the aorta, and the development of atherosclerosis. Therefore, therapeutic strategies targeting MMP12 may represent a promising approach to combat CMDs.

Whole Exome Sequencing reveals NOTCH1 mutations in anaplastic large cell lymphoma and points to Notch both as a key pathway and a potential therapeutic target.

Patients diagnosed with Anaplastic Large Cell Lymphoma (ALCL) are still treated with toxic multi-agent chemotherapy and as many as 25-50% of patients relapse. To understand disease pathology and to uncover novel targets for therapy, Whole-Exome Sequencing (WES) of Anaplastic Lymphoma Kinase (ALK)+ ALCL was performed as well as Gene-Set Enrichment Analysis. This revealed that the T-cell receptor (TCR) and Notch pathways were the most enriched in mutations. In particular, variant T349P of NOTCH1, which confers a growth advantage to cells in which it is expressed, was detected in 12% of ALK+ and ALK- ALCL patient samples. Furthermore, we demonstrate that NPM-ALK promotes NOTCH1 expression through binding of STAT3 upstream of NOTCH1. Moreover, inhibition of NOTCH1 with γ-secretase inhibitors (GSIs) or silencing by shRNA leads to apoptosis; co-treatment in vitro with the ALK inhibitor Crizotinib led to additive/synergistic anti-tumour activity suggesting this may be an appropriate combination therapy for future use in the circumvention of ALK inhibitor resistance. Indeed, Crizotinib-resistant and sensitive ALCL were equally sensitive to GSIs. In conclusion, we show a variant in the extracellular domain of NOTCH1 that provides a growth advantage to cells and confirm the suitability of the Notch pathway as a second-line druggable target in ALK+ ALCL.

Sustained PU.1 levels balance cell-cycle regulators to prevent exhaustion of adult hematopoietic stem cells.

To provide a lifelong supply of blood cells, hematopoietic stem cells (HSCs) need to carefully balance both self-renewing cell divisions and quiescence. Although several regulators that control this mechanism have been identified, we demonstrate that the transcription factor PU.1 acts upstream of these regulators. So far, attempts to uncover PU.1's role in HSC biology have failed because of the technical limitations of complete loss-of-function models. With the use of hypomorphic mice with decreased PU.1 levels specifically in phenotypic HSCs, we found reduced HSC long-term repopulation potential that could be rescued completely by restoring PU.1 levels. PU.1 prevented excessive HSC division and exhaustion by controlling the transcription of multiple cell-cycle regulators. Levels of PU.1 were sustained through autoregulatory PU.1 binding to an upstream enhancer that formed an active looped chromosome architecture in HSCs. These results establish that PU.1 mediates chromosome looping and functions as a master regulator of HSC proliferation.

Hepatocyte-specific deletion of lysosomal acid lipase leads to cholesteryl ester but not triglyceride or retinyl ester accumulation.

Lysosomal acid lipase (LAL) hydrolyzes cholesteryl ester (CE) and retinyl ester (RE) and triglyceride (TG). Mice globally lacking LAL accumulate CE most prominently in the liver. The severity of the CE accumulation phenotype progresses with age and is accompanied by hepatomegaly and hepatic cholesterol crystal deposition. In contrast, hepatic TG accumulation is much less pronounced in these mice, and hepatic RE levels are even decreased. To dissect the functional role of LAL for neutral lipid ester mobilization in the liver, we generated mice specifically lacking LAL in hepatocytes (hep-LAL-ko). On a standard chow diet, hep-LAL-ko mice exhibited increased hepatic CE accumulation but unaltered TG and RE levels. Feeding the hep-LAL-ko mice a vitamin A excess/high-fat diet (VitA/HFD) further increased hepatic cholesterol levels, but hepatic TG and RE levels in these mice were lower than in control mice. Performing in vitro activity assays with lysosome-enriched fractions from livers of mice globally lacking LAL, we detected residual acid hydrolytic activities against TG and RE. Interestingly, this non-LAL acid TG hydrolytic activity was elevated in lysosome-enriched fractions from livers of hep-LAL-ko mice upon VitA/HFD feeding. In conclusion, the neutral lipid ester phenotype in livers from hep-LAL-ko mice indicates that LAL is limiting for CE turnover, but not for TG and RE turnovers. Furthermore, in vitro hydrolase activity assays revealed the existence of non-LAL acid hydrolytic activities for TG and RE. The corresponding acid lipase(s) catalyzing these reactions remains to be identified.

Transcriptomic analysis of human primary breast cancer identifies fatty acid oxidation as a target for metformin.

BACKGROUND: Epidemiological studies suggest that metformin may reduce the incidence of cancer in patients with diabetes and multiple late phase clinical trials assessing the potential of repurposing this drug are underway. Transcriptomic profiling of tumour samples is an excellent tool to understand drug bioactivity, identify candidate biomarkers and assess for mechanisms of resistance to therapy. METHODS: Thirty-six patients with untreated primary breast cancer were recruited to a window study and transcriptomic profiling of tumour samples carried out before and after metformin treatment. RESULTS: Multiple genes that regulate fatty acid oxidation were upregulated at the transcriptomic level and there was a differential change in expression between two previously identified cohorts of patients with distinct metabolic responses. Increase in expression of a mitochondrial fatty oxidation gene composite signature correlated with change in a proliferation gene signature. In vitro assays showed that, in contrast to previous studies in models of normal cells, metformin reduces fatty acid oxidation with a subsequent accumulation of intracellular triglyceride, independent of AMPK activation. CONCLUSIONS: We propose that metformin at clinical doses targets fatty acid oxidation in cancer cells with implications for patient selection and drug combinations. CLINICAL TRIAL REGISTRATION: NCT01266486.

Loss of RAF kinase inhibitor protein is involved in myelomonocytic differentiation and aggravates RAS-driven myeloid leukemogenesis.

RAS-signaling mutations induce the myelomonocytic differentiation and proliferation of hematopoietic stem and progenitor cells. Moreover, they are important players in the development of myeloid neoplasias. RAF kinase inhibitor protein (RKIP) is a negative regulator of RAS-signaling. As RKIP loss has recently been described in RAS-mutated myelomonocytic acute myeloid leukemia, we now aimed to analyze its role in myelomonocytic differentiation and RAS-driven leukemogenesis. Therefore, we initially analyzed RKIP expression during human and murine hematopoietic differentiation and observed that it is high in hematopoietic stem and progenitor cells and lymphoid cells but decreases in cells belonging to the myeloid lineage. By employing short hairpin RNA knockdown experiments in CD34+ umbilical cord blood cells and the undifferentiated acute myeloid leukemia cell line HL-60, we show that RKIP loss is indeed functionally involved in myelomonocytic lineage commitment and drives the myelomonocytic differentiation of hematopoietic stem and progenitor cells. These results could be confirmed in vivo, where Rkip deletion induced a myelomonocytic differentiation bias in mice by amplifying the effects of granulocyte macrophage-colony-stimulating factor. We further show that RKIP is of relevance for RAS-driven myelomonocytic leukemogenesis by demonstrating that Rkip deletion aggravates the development of a myeloproliferative disease in NrasG12D -mutated mice. Mechanistically, we demonstrate that RKIP loss increases the activity of the RAS-MAPK/ERK signaling module. Finally, we prove the clinical relevance of these findings by showing that RKIP loss is a frequent event in chronic myelomonocytic leukemia, and that it co-occurs with RAS-signaling mutations. Taken together, these data establish RKIP as novel player in RAS-driven myeloid leukemogenesis.

N-acetylaspartate availability is essential for juvenile survival on fat-free diet and determines metabolic health.

N-acetylaspartate (NAA) is synthesized by aspartate N-acetyltransferase (gene: Nat8l) from acetyl-coenzyme A and aspartate. In the brain, NAA is considered an important energy metabolite for lipid synthesis. However, the role of NAA in peripheral tissues remained elusive. Therefore, we characterized the metabolic phenotype of knockout (ko) and adipose tissue-specific (ako) Nat8l-ko mice as well as NAA-supplemented mice on various diets. We identified an important role of NAA availability in the brain during adolescence, as 75% of Nat8l-ko mice died on fat-free diet (FFD) after weaning but could be rescued by NAA supplementation. In adult life, NAA deficiency promotes a beneficial metabolic phenotype, as Nat8l-ko and Nat8l-ako mice showed reduced body weight, increased energy expenditure, and improved glucose tolerance on chow, high-fat, and FFDs. Furthermore, Nat8l-deficient adipocytes exhibited increased mitochondrial respiration, ATP synthesis, and an induction of browning. Conversely, NAA-treated wild-type mice showed reduced adipocyte respiration and lipolysis and increased de novo lipogenesis, culminating in reduced energy expenditure, glucose tolerance, and insulin sensitivity. Mechanistically, our data point to a possible role of NAA as modulator of pancreatic insulin secretion and suggest NAA as a critical energy metabolite for adipocyte and whole-body energy homeostasis.-Hofer, D. C., Zirkovits, G., Pelzmann, H. J., Huber, K., Pessentheiner, A. R., Xia, W., Uno, K., Miyazaki, T., Kon, K., Tsuneki, H., Pendl, T., Al Zoughbi, W., Madreiter-Sokolowski, C. T., Trausinger, G., Abdellatif, M., Schoiswohl, G., Schreiber, R., Eisenberg, T., Magnes, C., Sedej, S., Eckhardt, M., Sasahara, M., Sasaoka, T., Nitta, A., Hoefler, G., Graier, W. F., Kratky, D., Auwerx, J., Bogner-Strauss, J. G. N-acetylaspartate availability is essential for juvenile survival on fat-free diet and determines metabolic health.

Tumor Macroenvironment: An Update.

Modes of tumor cell interaction include autocrine stimulation, secretion of paracrine growth factors and inhibitors, as well as interaction with the tumor macroenvironment. This evolving concept in tumor pathobiology describes the interaction of a malignant tumor with its host as an extension and addition to its local interaction with tumor cells and surrounding nontransformed cells, the tumor microenvironment. Angiogenesis, which is considered part of the tumor microenvironment, also allows reciprocal interactions between cancer cells and other organs and systems. Well-known examples of tumor endocrine signaling are the paraneoplastic syndromes. In addition, cachexia, a severe complication of tumor growth, results from the systemic reprogramming of the host metabolism as a result of tumor growth and progression. Moreover, recent reports indicate that cancer cells may secrete factors that might play a role in forming premetastatic niches at distant sites. In addition, cancer cells seem to be able to secrete factors influencing and resetting endogenous circadian organizers. The importance of understanding the whole complex interaction of a malignant tumor and its host - the tumor macroenvironment - is of great importance for the better management and treatment of cancer patients.

MiR-1287-5p inhibits triple negative breast cancer growth by interaction with phosphoinositide 3-kinase CB, thereby sensitizing cells for PI3Kinase inhibitors.

BACKGROUND: Non-coding RNAs and especially microRNAs have been discovered to act as master regulators of cancer initiation and progression. The aim of our study was to discover and characterize the function of yet functionally uncharacterized microRNAs in human breast carcinogenesis. METHODS: In an unbiased approach, we utilized an established model system for breast cancer (BC) stem cell formation ("mammosphere assay") to identify whole miRNome alterations in breast carcinogenesis. Clinical samples of BC patients were used to evaluate the human relevance of the newly identified miRNA candidates. One promising candidate, miR-1287-5p, was further explored on its impact on several hallmarks of cancer. The molecular mode of action was characterized by whole transcriptome analysis, in silico prediction tools, miRNA-interaction assays, pheno-copy assays, and drug sensitivity assays. RESULTS: Among several other microRNAs, miR-1287-5p was significantly downregulated in mammospheres and human BC tissue compared to normal breast tissue (p < 0.0001). Low expression levels were significantly associated with poor prognosis in BC patients. MiR-1287-5p significantly decreased cellular growth, cells in S phase of cell cycle, anchorage-independent growth, and tumor formation in vivo. In addition, we identified PIK3CB as a direct molecular interactor of miR-1287-5p and a novel prognostic factor in BC. Finally, PI3Kinase pathway chemical inhibitors combined with miR-1287-5p mimic increased the pharmacological growth inhibitory potential in triple negative BC cells. CONCLUSION: Our data identified for the first time the involvement of miR-1287-5p in human BC and suggest a potential for therapeutic interventions in difficult to treat triple negative BC.

Hedgehog pathway proteins SMO and GLI expression as prognostic markers in head and neck squamous cell carcinoma.

AIMS: Because the hedgehog signalling pathway plays a major role in many types of cancer and can nowadays be targeted by specific compounds, we aimed to investigate the role of this pathway in squamous cell carcinoma of the head and neck. METHODS AND RESULTS: Ninety-eight treatment-naive head and neck cancer specimens were immunohistologically stained for SMO, GLI-1, p53 and p16 expression and correlated with clinicopathological factors. Immunoreactivity for SMO and GLI-1 was found in 20 (20.4%) and 52 (53.1%) cases of tumours, respectively. SMO expression correlated with GLI-1 expression (ρ = 0.258, P = 0.010) in univariate and multivariate analysis (P = 0.007, t = 2.81). In univariate analysis, high SMO expression was associated with shorter overall survival (HR = 0.56; 95% CI = 0.32-0.98; P = 0.044) and disease-free survival (HR = 0.53; 95% CI = 0.30-0.95; P = 0.034). In multivariate cox regression analysis SMO expression showed a trend towards an independent predictor for shorter overall survival (HR = 0.57; 95% CI = 0.30-1.05; P = 0.072) and disease-free survival (HR = 0.53; 95% CI = 0.28-1.02; P = 0.056). In head and neck cancer patients with low tumour p16 expression, SMO expression was an independent factor for overall survival (HR = 0.49; 95% CI = 0.24-0.98; P = 0.043) and disease-free survival (HR = 0.45; 95% CI = 0.22-0.96; P = 0.037). CONCLUSION: Although it needs to be confirmed in larger cohorts, our results suggest that targeting SMO might be a potentially therapeutic option in patients with head and neck cancer. In line, molecular pathological analyses including mutation analysis in the hedgehog pathway might point to additional therapeutic leads.

Neoplastic cell percentage estimation in tissue samples for molecular oncology: recommendations from a modified Delphi study.

AIMS: Results from external quality assessment revealed considerable variation in neoplastic cell percentages (NCP) estimation in samples for biomarker testing. As molecular biology tests require a minimal NCP, overestimations may lead to false negative test results. We aimed to develop recommendations to improve the NCP determination in a prototypical entity - colorectal carcinoma - that can be adapted for other cancer types. METHODS AND RESULTS: A modified Delphi study was conducted to reach consensus by 10 pathologists from 10 countries with experience in determining the NCP for colorectal adenocarcinoma. This study included two online surveys and a decision-making meeting. Consensus was defined a priori as an agreement of > 80%. All pathologists completed both surveys. Consensus was reached for 8 out of 19 and 2 out of 13 questions in the first and second surveys, respectively. Remaining issues were resolved during the meeting. Twenty-four recommendations were formulated. Major recommendations resulted as follows: only pathologists should conduct the morphological evaluation; nevertheless molecular biologists/technicians may estimate the NCP, if specific training has been performed and a pathologist is available for feedback. The estimation should be determined in the area with the highest density of viable neoplastic cells and lowest density of inflammatory cells. Other recommendations concerned: the determination protocol itself, needs for micro- and macro-dissection, reporting and interpreting, referral practices and applicability to other cancer types. CONCLUSION: We believe these recommendations may lead to more accurate NCP estimates, ensuring the correct interpretation of test results, and might help in validating digital algorithms in the future.

APMAP interacts with lysyl oxidase-like proteins, and disruption of Apmap leads to beneficial visceral adipose tissue expansion.

Adipocyte plasma membrane-associated protein (APMAP) has been described as an adipogenic factor in 3T3-L1 cells with unknown biochemical function; we therefore aimed to investigate the physiologic function of APMAP in vivo We generated Apmap-knockout mice and challenged them with an obesogenic diet to investigate their metabolic phenotype. We identified a novel truncated adipocyte-specific isoform of APMAP in mice that is produced by alternative transcription. Mice lacking the full-length APMAP protein, the only isoform that is expressed in humans, have an improved metabolic phenotype upon diet-induced obesity, indicated by enhanced insulin sensitivity, preserved glucose tolerance, increased respiratory exchange ratio, decreased inflammatory marker gene expression, and reduced adipocyte size. At the molecular level, APMAP interacts with the extracellular collagen cross-linking matrix proteins lysyl oxidase-like 1 and 3. On a high-fat diet, the expression of lysyl oxidase-like 1 and 3 is strongly decreased in Apmap-knockout mice, paralleled by reduced expression of profibrotic collagens and total collagen content in epididymal white adipose tissue, indicating decreased fibrotic potential. Together, our data suggest that APMAP is a novel regulator of extracellular matrix components, and establish that APMAP is a potential target to mitigate obesity-associated insulin resistance.-Pessentheiner, A. R., Huber, K., Pelzmann, H. J., Prokesch, A., Radner, F. P. W., Wolinski, H., Lindroos-Christensen, J., Hoefler, G., Rülicke, T., Birner-Gruenberger, R., Bilban, M., Bogner-Strauss, J. G. APMAP interacts with lysyl oxidase-like proteins, and disruption of Apmap leads to beneficial visceral adipose tissue expansion.

Residual disease detection using targeted parallel sequencing predicts relapse in cytogenetically normal acute myeloid leukemia.

Despite achieving complete remission after intensive therapy, most patients with cytogenetically normal (CN) AML relapse due to the persistence of submicroscopic residual disease. In this pilot study, we hypothesized that detection of leukemia-specific mutations following consolidation treatment using a targeted parallel sequencing approach predicts relapse. We included 34 AML patients of whom diagnostic material and remission bone marrow slides after at least one cycle of consolidation were available. Isolated DNA was screened for mutations in 19 genes using an Ion Torrent sequencing platform. Furthermore, the variant allelic frequency of distinct mutations was validated by digital PCR and sequencing using a barcoding approach. Twenty-seven out of 34 patients could be analyzed for mutation clearance. We identified 68 somatic mutations at diagnosis (median, 3 mutations per patient; range 1-5) and 22 of these were still detected in 16 patients after consolidation therapy with a reliable sensitivity of 0.5% (median, 1 mutation; range 0-3). The most frequent noncleared mutations were found in DNMT3A. However, as persistence of these mutations has recently been shown to be without any impact on relapse risk, we performed survival and relapse risk analysis excluding DNMT3A mutations. Importantly, persistence of non-DNMT3A mutations was associated with a higher risk of AML relapse (7/8 pts versus 6/19 pts; P = .013) and with a shorter relapse-free survival (333 days vs. not reached; log-rank P = .0219). Detection of residual disease by routine targeted parallel sequencing proved feasible and effective as persistence of somatic mutations other than DNMT3A were prognostic for relapse in CN AML.

Early Loss of Forkhead Transcription Factor, O Subgroup, Member 1 Protein in the Development of Pancreatic Ductal Adenocarcinoma.

OBJECTIVES: Forkhead transcription factor, O subgroup, member 1 (FOXO1) is a regulatory protein that plays an essential role in cellular homeostasis. A biological function as a tumor suppressor has been proposed. Here, we examined FOXO1 expression in human pancreatic ductal adenocarcinoma (PDAC) and its precursor lesions. METHODS: We immunohistochemically labeled tissue samples from 47 patients with PDAC for FOXO1 protein. In addition, we extracted data from the Cancer Genome Atlas and the Cancer Cell Line Encyclopedia and studied a potential association with well-established genetic variants. A publicly available microarray dataset of 102 PDAC samples was used to explore the influence of FOXO1 expression on patients' clinical outcome. RESULTS: Normal ductal epithelium universally expressed nuclear and cytoplasmic FOXO1. Reduced expression was observed in PanIN lesions and PDAC of all cases. Analysis of several datasets showed that the FOXO1 gene transcript levels do not correlate with KRAS, TP53, SMAD4, or CDKN2A mutation status, but positively correlate with patients' outcomes. CONCLUSIONS: Loss of FOXO1 protein is identified as an early event during PDAC development and may be independent of the top 4 mutated cancer genes. Because of its strong expression in normal ductal cells, immunohistochemical detection of FOXO1 can function as a valuable test to establish the diagnosis of transformation and malignancy in pancreatic tissues.

Novel role of a triglyceride-synthesizing enzyme: DGAT1 at the crossroad between triglyceride and cholesterol metabolism.

Acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) is a key enzyme in triacylglycerol (TG) biosynthesis. Here we show that genetic deficiency and pharmacological inhibition of DGAT1 in mice alters cholesterol metabolism. Cholesterol absorption, as assessed by acute cholesterol uptake, was significantly decreased in the small intestine and liver upon DGAT1 deficiency/inhibition. Ablation of DGAT1 in the intestine (I-DGAT1(-/-)) alone is sufficient to cause these effects. Consequences of I-DGAT1 deficiency phenocopy findings in whole-body DGAT1(-/-) and DGAT1 inhibitor-treated mice. We show that deficiency/inhibition of DGAT1 affects cholesterol metabolism via reduced chylomicron size and increased trans-intestinal cholesterol excretion. These effects are independent of cholesterol uptake at the apical surface of enterocytes but mediated through altered dietary fatty acid metabolism. Our findings provide insight into a novel role of DGAT1 and identify a pathway by which intestinal DGAT1 deficiency affects whole-body cholesterol homeostasis in mice. Targeting intestinal DGAT1 may represent a novel approach for treating hypercholesterolemia.

Expansion of BCR/ABL1+ cells requires PAK2 but not PAK1.

The p21-activated kinases (PAKs) are key nodes in oncogenic signalling pathways controlling growth, survival, and motility of cancer cells. Their activity is increased in many human cancers and is associated with poor prognosis. To date, PAK deregulation has mainly been studied in solid tumours, where PAK1 and PAK4 are the main isoforms deregulated. We show that PAK1 and PAK2 are the critical isoforms in a BCR/ABL1+ haematopoietic malignancy. In suspension, leukaemic cells deficient for PAK1 and PAK2 undergo apoptosis, while the loss of either protein is well tolerated. Transfer of medium conditioned by shPAK2- but not shPAK1-expressing leukaemic cells interferes with endothelial cell growth. We found that leukaemic cells produce exosomes containing PAK2. Transfer of isolated exosomes supports endothelial cell proliferation. In parallel, we found that leukaemic cells explicitly require PAK2 to grow towards an extracellular matrix. PAK2-deficient cells fail to form colonies in methylcellulose and to induce lymphomas in vivo. PAK2 might therefore be the critical isoform in leukaemic cells by controlling tumour growth in a dual manner: vascularization via exosome-mediated transfer to endothelial cells and remodelling of the extracellular matrix. This finding suggests that the PAK2 isoform represents a promising target for the treatment of haematological diseases.