Acquired mutations in BAX confer resistance to BH3-mimetic therapy in acute myeloid leukemia.

Randomized trials in acute myeloid leukemia (AML) have demonstrated improved survival by the BCL-2 inhibitor venetoclax combined with azacitidine in older patients, and clinical trials are actively exploring the role of venetoclax in combination with intensive chemotherapy in fitter patients with AML. As most patients still develop recurrent disease, improved understanding of relapse mechanisms is needed. We find that 17% of patients relapsing after venetoclax-based therapy for AML have acquired inactivating missense or frameshift/nonsense mutations in the apoptosis effector gene BAX. In contrast, such variants were rare after genotoxic chemotherapy. BAX variants arose within either leukemic or preleukemic compartments, with multiple mutations observed in some patients. In vitro, AML cells with mutated BAX were competitively selected during prolonged exposure to BCL-2 antagonists. In model systems, AML cells rendered deficient for BAX, but not its close relative BAK, displayed resistance to BCL-2 targeting, whereas sensitivity to conventional chemotherapy was variable. Acquired mutations in BAX during venetoclax-based therapy represent a novel mechanism of resistance to BH3-mimetics and a potential barrier to the long-term efficacy of drugs targeting BCL-2 in AML.

Adipocyte mesenchymal transition contributes to mammary tumor progression.

Obesity is associated with increased cancer incidence and progression. However, the relationship between adiposity and cancer remains poorly understood at the mechanistic level. Here, we report that adipocytes from tumor-invasive mammary fat undergo de-differentiation to fibroblast-like precursor cells during tumor progression and integrate into the tumor microenvironment. Single-cell sequencing reveals that these de-differentiated adipocytes lose their original identities and transform into multiple cell types, including myofibroblast- and macrophage-like cells, with their characteristic features involved in immune response, inflammation, and extracellular matrix remodeling. The de-differentiated cells are metabolically distinct from tumor-associated fibroblasts but exhibit comparable effects on tumor cell proliferation. Inducing de-differentiation by Xbp1s overexpression promotes tumor progression despite lower adiposity. In contrast, promoting lipid-storage capacity in adipocytes through MitoNEET overexpression curbs tumor growth despite greater adiposity. Collectively, the metabolic interplay between tumor cells and adipocytes induces adipocyte mesenchymal transition and contributes to reconfigure the stroma into a more tumor-friendly microenvironment.

Preclinical characterization of [18F]T-008, a novel PET imaging radioligand for cholesterol 24-hydroxylase.

PURPOSE: Cholesterol 24-hydroxylase (CH24H) is a brain-specific enzyme that plays a major role in brain cholesterol homeostasis by converting cholesterol into 24S-hydroxycholesterol. The selective CH24H inhibitor soticlestat (TAK-935) is being pursued as a drug for treatment of seizures in developmental and epileptic encephalopathies. Herein, we describe the successful discovery and the preclinical validation of the novel radiolabeled CH24H ligand (3-[18F]fluoroazetidin-1-yl){1-[4-(4-fluorophenyl)pyrimidin-5-yl]piperidin-4-yl}methanone ([18F]T-008) and its tritiated analog, [3H]T-008. METHODS: In vitro autoradiography (ARG) studies in the CH24H wild-type (WT) and knockout (KO) mouse brain sections were conducted using [3H]T-008. PET imaging was conducted in two adult rhesus macaques using [18F]T-008. Each macaque received two test-retest baseline scans and a series of two blocking doses of soticlestat administered prior to [18F]T-008 to determine the CH24H enzyme occupancy. PET data were analyzed with Logan graphical analysis using plasma input. A Lassen plot was applied to estimate CH24H enzyme occupancy by soticlestat. RESULTS: In ARG studies, binding of [3H]T-008 was specific to CH24H in the mouse brain sections, which was not observed in CH24H KO or in wild-type mice after pretreatment with soticlestat. In rhesus PET studies, the rank order of [18F]T-008 uptake was striatum > cortical regions > cerebellum, which was consistent with CH24H distribution in the brain. Pre-blocking with soticlestat reduced the maximum uptake and increased the washout in all brain regions in a dose-dependent manner. Calculated global occupancy values for soticlestat at a dose of 0.89 mg/kg were 97-98%, indicating maximum occupancy. CONCLUSION: The preclinical in vitro and in vivo evaluation of labeled T-008 demonstrates that [18F]T-008 is suitable for imaging CH24H in the brain and warrants further studies in humans.

Intact TP-53 function is essential for sustaining durable responses to BH3-mimetic drugs in leukemias.

Selective targeting of BCL-2 with the BH3-mimetic venetoclax has been a transformative treatment for patients with various leukemias. TP-53 controls apoptosis upstream of where BCL-2 and its prosurvival relatives, such as MCL-1, act. Therefore, targeting these prosurvival proteins could trigger apoptosis across diverse blood cancers, irrespective of TP53 mutation status. Indeed, targeting BCL-2 has produced clinically relevant responses in blood cancers with aberrant TP-53. However, in our study, TP53-mutated or -deficient myeloid and lymphoid leukemias outcompeted isogenic controls with intact TP-53, unless sufficient concentrations of BH3-mimetics targeting BCL-2 or MCL-1 were applied. Strikingly, tumor cells with TP-53 dysfunction escaped and thrived over time if inhibition of BCL-2 or MCL-1 was sublethal, in part because of an increased threshold for BAX/BAK activation in these cells. Our study revealed the key role of TP-53 in shaping long-term responses to BH3-mimetic drugs and reconciled the disparate pattern of initial clinical response to venetoclax, followed by subsequent treatment failure among patients with TP53-mutant chronic lymphocytic leukemia or acute myeloid leukemia. In contrast to BH3-mimetics targeting just BCL-2 or MCL-1 at doses that are individually sublethal, a combined BH3-mimetic approach targeting both prosurvival proteins enhanced lethality and durably suppressed the leukemia burden, regardless of TP53 mutation status. Our findings highlight the importance of using sufficiently lethal treatment strategies to maximize outcomes of patients with TP53-mutant disease. In addition, our findings caution against use of sublethal BH3-mimetic drug regimens that may enhance the risk of disease progression driven by emergent TP53-mutant clones.

7-Fluorosialyl Glycosides Are Hydrolysis Resistant but Readily Assembled by Sialyltransferases Providing Easy Access to More Metabolically Stable Glycoproteins.

The maintenance of therapeutic glycoproteins within the circulatory system is associated, in large part, with the integrity of sialic acids as terminal sugars on the glycans. Glycoprotein desialylation, either by spontaneous cleavage or through host sialidases, leads to protein clearance, mainly through the liver. Thus, the installation of minimally modified sialic acids that are hydrolysis-resistant yet biologically equivalent should lead to increased circulatory half-lives and improved pharmacokinetic profiles. Here we describe the chemoenzymatic synthesis of CMP-sialic acid sugar donors bearing fluorine atoms at the 7-position, starting from the corresponding 4-deoxy-4-fluoro-N-acetylhexosamine precursors. For the derivative with natural stereochemistry we observe efficient glycosyl transfer by sialyltransferases, along with improved stability of the resultant 7-fluorosialosides toward spontaneous hydrolysis (3- to 5-fold) and toward cleavage by GH33 sialidases (40- to 250-fold). Taking advantage of the rapid transfer of 7-fluorosialic acid by sialyltransferases, we engineered the O-glycan of Interferon α-2b and the N-glycans of the therapeutic glycoprotein α1-antitrypsin. Studies of the uptake of the glyco-engineered α1-antitrypsin by HepG2 liver cells demonstrated the bioequivalence of 7-fluorosialic acid to sialic acid in suppressing interaction with liver cell lectins. In vivo pharmacokinetic studies reveal enhanced half-life of the protein decorated with 7-fluorosialic acid relative to unmodified sialic acid in the murine circulatory system. 7-Fluorosialylation therefore offers considerable promise as a means of prolonging circulatory half-lives of glycoproteins and may pave the way toward biobetters for therapeutic use.

Design, Synthesis, and Evaluation of (2-Aminocyclopropyl)phenyl Derivatives as Novel Positron Emission Tomography Imaging Agents for Lysine-Specific Demethylase 1 in the Brain.

Dysregulation of histone H3 lysine 4 (H3K4) methylation is implicated in the pathogenesis of neurodevelopmental disorders. Lysine-specific demethylase 1 (LSD1) determines the methylation status of H3K4 through flavin adenine dinucleotide (FAD)-mediated histone demethylation. Therefore, LSD1 inhibition in the brain can be a novel therapeutic option for treating these disorders. Positron emission tomography (PET) imaging of LSD1 allows for investigating LSD1 expression levels under normal and disease conditions and validating target engagement of therapeutic LSD1 inhibitors. This study designed and synthesized (2-aminocyclopropyl)phenyl derivatives with irreversible binding to LSD1 as PET imaging agents for LSD1 in the brain. We optimized lipophilicity of the lead compound to minimize the risk of nonspecific binding and identified 1e with high selectivity over monoamine oxidase A and B, which are a family of FAD-dependent enzymes homologous to LSD1. PET imaging in a monkey showed a high uptake of [18F]1e to regions enriched with LSD1, indicating its specific binding to LSD1.

Dopamine D1 Receptor Agonist PET Tracer Development: Assessment in Nonhuman Primates.

Non-catechol-based high-affinity selective dopamine D1 receptor (D1R) agonists were recently described, and candidate PET ligands were selected on the basis of favorable properties. The objective of this study was to characterize in vivo in nonhuman primates 2 novel D1R agonist PET radiotracers, racemic 18F-MNI-800 and its more active atropisomeric (-)-enantiomer, 18F-MNI-968. Methods: Ten brain PET experiments were conducted with 18F-MNI-800 on 2 adult rhesus macaques and 2 adult cynomolgus macaques, and 8 brain PET experiments were conducted with 18F-MNI-968 on 2 adult rhesus macaques and 2 adult cynomolgus macaques. PET data were analyzed with both plasma-input-based methods and reference-region-based methods. Whole-body PET images were acquired with 18F-MNI-800 from 2 adult rhesus macaques for radiation dosimetry estimates. Results:18F-MNI-800 and 18F-MNI-968 exhibited regional uptake consistent with D1R distribution. Specificity and selectivity were demonstrated by dose-dependent blocking with the D1 antagonist SCH-23390. 18F-MNI-968 showed a 30% higher specific signal than 18F-MNI-800, with a nondisplaceable binding potential of approximately 0.3 in the cortex and approximately 1.1 in the striatum. Dosimetry radiation exposure was favorable, with an effective dose of about 0.023 mSv/MBq. Conclusion:18F-MNI-968 has significant potential as a D1R agonist PET radiotracer, and further characterization in human subjects is warranted.

Characterizing Lymphangiogenesis and Concurrent Inflammation in Adipose Tissue in Response to VEGF-D.

The metabolic consequences of obesity arise from local inflammation within expanding adipose tissue. In pre-clinical studies targeting various inflammatory factors, systemic metabolism can be improved through reduced adipose inflammation. Lymphatic vessels are a critical regulator of inflammation through roles in fluid and macromolecule transport and immune cell trafficking and immunomodulation. Lymphangiogenesis, the expansion of the lymphatic network, is often a necessary step in restoring tissue homeostasis. Using Adipo-VD mice, a model of adipocyte-specific, inducible overexpression of the potent lymphangiogenic factor vascular endothelial growth factor-D (VEGF-D), we previously identified that dense de novo adipose lymphatics reduced immune accumulation and improved glucose homeostasis in obesity. On chow diet, however, Adipo-VD mice demonstrated increased adipose tissue immune cells, fibrosis, and inflammation. Here, we characterize the time course of resident macrophage accumulation and lymphangiogenesis in male and female Adipo-VD mice fed chow and high fat diets, examining multiple adipose depots over 4 months. We find that macrophage infiltration occurs early, but resolves with concurrent lymphatic expansion that begins robustly after 1 month of VEGF-D overexpression in white adipose tissue. In obesity, female Adipo-VD mice exhibit reduced lymphangiogenesis and maintain a more glycolytic metabolism compared to Adipo-VD males and their littermates. Adipose lymphatic structures appear to expand by a lymphvasculogenic mechanism involving lymphatic endothelial cell proliferation and organization with a cell source we that failed to identify; hematopoietic cells afford minimal structural contribution. While a net positive effect occurs in Adipo-VD mice, adipose tissue lymphangiogenesis demonstrates a dichotomous, and time-dependent, inflammatory tissue remodeling response.

A Novel Model of Diabetic Complications: Adipocyte Mitochondrial Dysfunction Triggers Massive ß-Cell Hyperplasia.

Obesity-associated type 2 diabetes mellitus (T2DM) entails insulin resistance and loss of ß-cell mass. Adipose tissue mitochondrial dysfunction is emerging as a key component in the etiology of T2DM. Identifying approaches to preserve mitochondrial function, adipose tissue integrity, and ß-cell mass during obesity is a major challenge. Mitochondrial ferritin (FtMT) is a mitochondrial matrix protein that chelates iron. We sought to determine whether perturbation of adipocyte mitochondria influences energy metabolism during obesity. We used an adipocyte-specific doxycycline-inducible mouse model of FtMT overexpression (FtMT-Adip mice). During a dietary challenge, FtMT-Adip mice are leaner but exhibit glucose intolerance, low adiponectin levels, increased reactive oxygen species damage, and elevated GDF15 and FGF21 levels, indicating metabolically dysfunctional fat. Paradoxically, despite harboring highly dysfunctional fat, transgenic mice display massive ß-cell hyperplasia, reflecting a beneficial mitochondria-induced fat-to-pancreas interorgan signaling axis. This identifies the unique and critical impact that adipocyte mitochondrial dysfunction has on increasing ß-cell mass during obesity-related insulin resistance.

Preclinical In Vitro and In Vivo Characterization of Synaptic Vesicle 2A-Targeting Compounds Amenable to F-18 Labeling as Potential PET Radioligands for Imaging of Synapse Integrity.

PURPOSE: Current synaptic vesicle 2A (SV2A) positron emission tomography (PET) imaging agents include the nanomolar affinity probes [11C]UCB-J and [18F]UCB-H derived from the anti-epileptic drug levitaracetam (Keppra®). An industry-utilized "de-risking" approach was used to carry out initial pharmacological characterization and to assess potential next-generation candidates amenable to F-18 radiolabeling for preliminary evaluation. PROCEDURES: Radioligand binding methods were employed in mammalian brain homogenates to determine the SV2A affinity (Kd) and maximal binding capacity (Bmax) of [3H]UCB-J. Novel leads were then screened to identify compounds minimally with comparable binding affinities with UCB-J in order to select a F-18-labeled candidate for subsequent in vivo assessment in rat. In parallel, mammalian brain tissue section autoradiography was performed to assess specific SV2A distribution. RESULTS: [3H]UCB-J bound with high affinity to a single population of sites in the rat brain (Kd = 2.6 ± 0.25 nM; Bmax = 810 ± 25 fmol/mg protein) and control human cortex (Kd = 2.9 ± 0.54 nM; Bmax = 10,000 ± 640 fmol/mg protein). Distribution of specific SV2A binding was shown to be homogeneous throughout the rodent brain and primarily in gray matter regions of rodent and human brain sections. Analog screening identified MNI-1038, MNI-1126/SDM-8, and SDM-2 as having comparable binding affinities with the currently available PET ligands. Subsequent [18F]MNI-1126/[18F]SDM-8 dynamic micro-PET imaging in rats revealed in vivo uptake and accumulation in the brain with favorable kinetics. Chase studies using 30 mg/kg levetiracetam confirmed that in vivo brain uptake of [18F]MNI-1126/[18F]SDM-8 was reversible. CONCLUSIONS: Taken together, these data suggest [18F]MNI-1126/[18F]SDM-8 (since renamed as [18F]SynVesT-1) characterized via an in vitro screening cascade provided a measurable in vivo SV2A specific signal in the rodent brain. This tracer as well as the close analog [18F]SDM-2 (since renamed as [18F]SynVesT-2) is currently undergoing further evaluation in preclinical and clinical studies.

Vascular Endothelial Growth Factor-D (VEGF-D) Overexpression and Lymphatic Expansion in Murine Adipose Tissue Improves Metabolism in Obesity.

Obese adipose tissue expansion is an inflammatory process that results in dysregulated lipolysis, increased circulating lipids, ectopic lipid deposition, and systemic insulin resistance. Lymphatic vessels provide a route of fluid, macromolecule, and immune cell clearance, and lymphangiogenesis increases this capability. Indeed, inflammation-associated lymphangiogenesis is critical in resolving acute and chronic inflammation, but it is largely absent in obese adipose tissue. Enhancing adipose tissue lymphangiogenesis could, therefore, improve metabolism in obesity. To test this hypothesis, transgenic mice with doxycycline-inducible expression of murine vascular endothelial growth factor (VEGF)-D under a tightly controlled Tet-On promoter were crossed with adipocyte-specific adiponectin-reverse tetracycline-dependent transactivator mice (Adipo-VD) to stimulate adipose tissue-specific lymphangiogenesis during 16-week high-fat diet-induced obesity. Adipose VEGF-D overexpression induced de novo lymphangiogenesis in murine adipose tissue, and obese Adipo-VD mice exhibited enhanced glucose clearance, lower insulin levels, and reduced liver triglycerides. On ß-3 adrenergic stimulation, Adipo-VD mice exhibited more rapid and increased glycerol flux from adipose tissue, suggesting that the lymphatics are a potential route of glycerol clearance. Resident macrophage crown-like structures were scarce and total F4/80+ macrophages were reduced in obese Adipo-VD s.c. adipose tissue with evidence of increased immune trafficking from the tissue. Augmenting VEGF-D signaling and lymphangiogenesis specifically in adipose tissue, therefore, reduces obesity-associated immune accumulation and improves metabolic responsiveness.

Acute loss of adipose tissue-derived adiponectin triggers immediate metabolic deterioration in mice.

AIM/HYPOTHESIS: Adiponectin (APN), a circulating hormone secreted by mature adipocytes, has been extensively studied because it has beneficial metabolic effects. While many studies have focused on the congenital loss of APN and its effects on systemic body glucose and lipid metabolism, little is known about the effects triggered by acute loss of APN in the adult mouse. We anticipated that genetically induced acute depletion of APN in adult mice would have a more profound effect on systemic metabolic health than congenital deletion of Adipoq, the gene encoding APN, with its associated potential for adaptive responses that may mask the phenotypes. METHODS: Mice carrying loxP-flanked regions of Adipoq were generated and bred to the Adipoq (APN) promoter-driven reverse tetracycline-controlled transactivator (rtTA) (APN-rtTA) gene and a tet-responsive Cre line (TRE-Cre) to achieve acute depletion of APN. Upon acute removal of APN in adult mice, systemic glucose and lipid homeostasis were assessed under basal and insulinopenic conditions. RESULTS: The acute depletion of APN results in more severe systemic insulin resistance and hyperlipidaemia than in mice with congenital loss of APN. Furthermore, the acute depletion of APN in adult mice results in a much more dramatic reduction in survival rate, with 50% of inducible knockouts dying in the first 5 days under insulinopenic conditions compared with 0% of congenital Adipoq knockout mice under similar conditions. CONCLUSIONS/INTERPRETATION: Acute systemic removal of APN results in a much more negative metabolic phenotype compared with congenital knockout of Adipoq. Specifically, our data demonstrate that acute depletion of APN is especially detrimental to lipid homeostasis, both under basal and insulinopenic conditions. This suggests that compensatory mechanisms exist in congenital knockout mice that offset some of the metabolic actions covered by APN.

Directing visceral white adipocyte precursors to a thermogenic adipocyte fate improves insulin sensitivity in obese mice.

Visceral adiposity confers significant risk for developing metabolic disease in obesity whereas preferential expansion of subcutaneous white adipose tissue (WAT) appears protective. Unlike subcutaneous WAT, visceral WAT is resistant to adopting a protective thermogenic phenotype characterized by the accumulation of Ucp1+ beige/BRITE adipocytes (termed 'browning'). In this study, we investigated the physiological consequences of browning murine visceral WAT by selective genetic ablation of Zfp423, a transcriptional suppressor of the adipocyte thermogenic program. Zfp423 deletion in fetal visceral adipose precursors (Zfp423loxP/loxP; Wt1-Cre), or adult visceral white adipose precursors (PdgfrbrtTA; TRE-Cre; Zfp423loxP/loxP), results in the accumulation of beige-like thermogenic adipocytes within multiple visceral adipose depots. Thermogenic visceral WAT improves cold tolerance and prevents and reverses insulin resistance in obesity. These data indicate that beneficial visceral WAT browning can be engineered by directing visceral white adipocyte precursors to a thermogenic adipocyte fate, and suggest a novel strategy to combat insulin resistance in obesity.

Kinetic Modeling of the Tau PET Tracer 18F-AV-1451 in Human Healthy Volunteers and Alzheimer Disease Subjects.

18F-AV-1451 is currently the most widely used of several experimental tau PET tracers. The objective of this study was to evaluate 18F-AV-1451 binding with full kinetic analysis using a metabolite-corrected arterial input function and to compare parameters derived from kinetic analysis with SUV ratio (SUVR) calculated over different imaging time intervals. Methods:18F-AV-1451 PET brain imaging was completed in 16 subjects: 4 young healthy volunteers (YHV), 4 aged healthy volunteers (AHV), and 8 Alzheimer disease (AD) subjects. Subjects were imaged for 3.5 h, with arterial blood samples obtained throughout. PET data were analyzed using plasma and reference tissue-based methods to estimate the distribution volume, binding potential (BPND), and SUVR. BPND and SUVR were calculated using the cerebellar cortex as a reference region and were compared across the different methods and across the 3 groups (YHV, AHV, and AD). Results: AD demonstrated increased 18F-AV-1451 retention compared with YHV and AHV based on both invasive and noninvasive analyses in cortical regions in which paired helical filament tau accumulation is expected in AD. A correlation of R2 > 0.93 was found between BPND (130 min) and SUVR-1 at all time intervals. Cortical SUVR curves reached a relative plateau around 1.0-1.2 for YHV and AHV by approximately 50 min, but increased in AD by up to approximately 20% at 110-130 min and approximately 30% at 160-180 min relative to 80-100 min. Distribution volume (130 min) was lower by 30%-35% in the YHV than AHV. Conclusion: Our data suggest that although 18F-AV-1451 SUVR curves do not reach a plateau and are still increasing in AD, an SUVR calculated over an imaging window of 80-100 min (as currently used in clinical studies) provides estimates of paired helical filament tau burden in good correlation with BPND, whereas SUVR sensitivity to regional cerebral blood changes needs further investigation.

Synthesis and in†vivo Evaluation of Fluorine-18 and Iodine-123 Pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine Derivatives as PET and SPECT Radiotracers for Mapping A2A Receptors.

Imaging agents that target adenosine type 2A (A2A ) receptors play an important role in evaluating new pharmaceuticals targeting these receptors, such as those currently being developed for the treatment of movement disorders like Parkinson's disease. They are also useful for monitoring progression and treatment efficacy by providing a noninvasive tool to map changes in A2A receptor density and function in neurodegenerative diseases. We previously described the successful evaluation of two A2A -specific radiotracers in both nonhuman primates and in subsequent human clinical trials: [(123) I]MNI-420 and [(18) F]MNI-444. Herein we describe the development of both of these radiotracers by selection from a series of A2A ligands, based on the pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine core of preladenant. Each of this series of 16 ligands was found to bind to recombinant human A2A receptor in the low nanomolar range, and of these 16, six were radiolabeled with either fluorine-18 or iodine-123 and evaluated in nonhuman primates. These initial in vivo results resulted in the identification of 7-(2-(4-(4-(2-[(18) F]fluoroethoxy)phenyl)piperazin-1-yl)ethyl)-2-(furan-2-yl)-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine ([(18) F]MNI-444) and 7-(2-(4-(2-fluoro-4-[(123) I]iodophenyl)piperazin-1-yl)ethyl)-2-(furan-2-yl)-7H-imidazo[1,2-c]pyrazolo[4,3-e]pyrimidin-5-amine ([(123) I]MNI-420) as PET and SPECT radiopharmaceuticals for mapping A2A receptors in brain.

Paraneoplastic cerebellar degeneration as a marker of endometrial cancer recurrence.

An 84-year-old woman developed a cerebellar syndrome having undergone a total abdominal hysterectomy and bilateral salpingo-oophorectomy for endometrial cancer 1 year previously. She was found to be anti-Yo antibody positive and was diagnosed with paraneoplastic cerebellar degeneration (PCD). A subsequent positron emission tomography scan and lymph node biopsy identified recurrence of her endometrial cancer. This case illustrates how PCD can be an indicator of cancer recurrence, underlines the significance of PCD as a prompt to search for underlying malignancy, and highlights the difficulties PCD poses to the clinician in terms of diagnosis and management.

Identification and in vivo evaluation of a fluorine-18 rolipram analogue, [(18) F]MNI-617, as a radioligand for PDE4 imaging in mammalian brain.

Phosphodiesterase (PDE) 4 is the most prevalent PDE in the central nervous system (CNS) and catalyzes hydrolysis of intracellular cAMP, a secondary messenger. By therapeutic inhibition of PDE4, intracellular cAMP levels can be stabilized, and the symptoms of psychiatric and neurodegenerative disorders including depression, memory loss and Parkinson's disease can be ameliorated. Radiotracers targeting PDE4 can be used to study PDE4 density and function, and evaluate new PDE4 therapeutics, in vivo in a non-invasive way, as has been shown using the carbon-11 labeled PDE4 inhibitor R-(-)-rolipram. Herein we describe a small series of rolipram analogs that contain fluoro- or iodo-substituents that could be used as fluorine-18 PET or iodine-123 SPECT PDE4 radiotracers. This series was evaluated with an in vitro binding assay and a 4-(fluoromethyl) derivative of rolipram, MNI-617, was identified, with a five-fold increase in affinity for PDE4 (Kd = 0.26 nM) over R-(-)-rolipram (Kd = 1.6 nM). A deutero-analogue d2 -[(18) F]MNI-617 was radiolabeled and produced in 23% yield with high (>5 Ci/µmol) specific activity and evaluated in non-human primate, where it rapidly entered the brain, with SUVs between 4 and 5, and with a distribution pattern consistent with that of PDE4.

Change in PDE10 across early Huntington disease assessed by [18F]MNI-659 and PET imaging.

OBJECTIVE: To evaluate whether striatal [(18)F]MNI-659 PET imaging of phosphodiesterase 10A (PDE10) serves as a sensitive and reliable biomarker of striatal neurodegeneration in a longitudinal cohort of participants with early Huntington disease (HD). METHODS: A cohort of participants with HD, including both participants premanifest or manifest with motor signs, underwent clinical assessments, genetic determination, and 2 [(18)F]MNI-659 PET imaging sessions approximately 1 year apart. Eleven healthy control (HC) participants underwent clinical assessments and [(18)F]MNI-659 PET imaging once. Striatal binding potentials (BPnd) were estimated for brain regions of interest, specifically within the basal ganglia, and compared between baseline and follow-up imaging. Clinical measures of HD severity were assessed at each visit. RESULTS: Eight participants with HD (6 manifest; 2 premanifest) participated. Of those with manifest HD, all had relatively early stage disease (stage 1, n = 2; stage 2, n = 4) and a Unified Huntington's Disease Rating Scale total motor score <45. As expected, the HD cohort as a whole had a reduction in the basal ganglia BPnd to approximately 50% of that seen in HC. On follow-up scans, [(18)F]MNI-659 uptake declined in the putamen and caudate nucleus in all 8 participants. The mean annualized rates of decline in signal in the caudate, putamen, and globus pallidus and the putamen were 16.6%, 6.9%, and 5.8%, respectively. In HC, the annualized reduction in signal in striatal regions was less than 1%. CONCLUSION: Longitudinal data in this small cohort of participants with early HD support [(18)F]MNI-659 PET imaging of PDE10 as a useful biomarker to track HD disease progression.

Pdgfrß+ Mural Preadipocytes Contribute to Adipocyte Hyperplasia Induced by High-Fat-Diet Feeding and Prolonged Cold Exposure in Adult Mice.

The expansion of white adipose tissue (WAT) in obesity involves de novo differentiation of new adipocytes; however, the cellular origin of these cells remains unclear. Here, we utilize Zfp423(GFP) reporter mice to characterize adipose mural (Pdgfrß(+)) cells with varying levels of the preadipocyte commitment factor Zfp423. We find that adipose tissue contains distinct mural populations, with levels of Zfp423 distinguishing adipogenic from inflammatory-like mural cells. Using our "MuralChaser" lineage tracking system, we uncover adipose perivascular cells as developmental precursors of adipocytes formed in obesity, with adipogenesis and precursor abundance regulated in a depot-dependent manner. Interestingly, Pdgfrß(+) cells do not significantly contribute to the initial cold-induced recruitment of beige adipocytes in WAT; it is only after prolonged cold exposure that these cells differentiate into beige adipocytes. These results provide genetic evidence for a mural cell origin of white adipocytes in obesity and suggest that beige adipogenesis may originate from multiple sources.

Selective enhancement of insulin sensitivity in the mature adipocyte is sufficient for systemic metabolic improvements.

Dysfunctional adipose tissue represents a hallmark of type 2 diabetes and systemic insulin resistance, characterized by fibrotic deposition of collagens and increased immune cell infiltration within the depots. Here we generate an inducible model of loss of function of the protein phosphatase and tensin homologue (PTEN), a phosphatase critically involved in turning off the insulin signal transduction cascade, to assess the role of enhanced insulin signalling specifically in mature adipocytes. These mice gain more weight on chow diet and short-term as well as long-term high-fat diet exposure. Despite the increase in weight, they retain enhanced insulin sensitivity, show improvements in oral glucose tolerance tests, display reduced adipose tissue inflammation and maintain elevated adiponectin levels. These improvements also lead to reduced hepatic steatosis and enhanced hepatic insulin sensitivity. Prolonging insulin action selectively in the mature adipocyte is therefore sufficient to maintain normal systemic metabolic homeostasis.