Hypocretin (orexin; Hcrt)-containing neurons of the hypothalamus are essential for the normal regulation of sleep and wake behaviors and have been implicated in feeding, anxiety, depression, and reward. The absence of these neurons causes narcolepsy in humans and model organisms. However, little is known about the molecular phenotype of these cells; previous attempts at comprehensive profiling had only limited sensitivity or were inaccurate. We generated a Hcrt translating ribosome affinity purification (bacTRAP) line for comprehensive translational profiling of all ribosome-bound transcripts in these neurons in vivo. From this profile, we identified >6000 transcripts detectably expressed above background and 188 transcripts that are highly enriched in these neurons, including all known markers of the cells. Blinded analysis of in situ hybridization databases suggests that 60% of these are expressed in a Hcrt marker-like pattern. Fifteen of these were confirmed with double labeling and microscopy, including the transcription factor Lhx9. Ablation of this gene results in a >30% loss specifically of Hcrt neurons, without a general disruption of hypothalamic development. Polysomnography and activity monitoring revealed a profound hypersomnolence in these mice. These data provide an in-depth and accurate profile of Hcrt neuron gene expression and suggest that Lhx9 may be important for specification or survival of a subset of these cells.[Keywords: translational profiling; hypocretin; orexin; bacTRAP; Lhx9; narcolepsy] Supplemental material is available for this article. Narcolepsy is a profound disorder of sleep regulation, characterized by excessive daytime sleepiness, sleep attacks, cataplexy, and sleep-onset REM (rapid eye movement) periods. Studies indicate that narcolepsy, especially when accompanied by cataplexy, is due to the absence of hypocretinergic neurons and signaling (Nishino et al. 2000;Thannickal et al. 2000;Bourgin et al. 2008). The hypocretins (Hcrts; also called orexins) are peptidergic neurotransmitters used by a discrete population of neurons in the lateral hypothalamus Sakurai et al. 1998). In model organisms, disruption of the gene encoding the Hcrt peptides or a Hcrt receptor (Chemelli et al. 1999;Lin et al. 1999) or, alternatively, the ablation of the neurons producing Hcrt (Gerashchenko et al. 2001;Hara et al. 2001;Zhang et al. 2007) recapitulates features of narcolepsy. In humans, there are two variations of the disorder: narcolepsy with cataplexy (NC) and narcolepsy without cataplexy (NwoC). NC is strongly linked and associated with the HLA DQB1*0602 allele, a T-cell receptor locus, and P2RY11, a receptor that can regulate immune cell survival (Hallmayer et al. 2009;Kornum et al. 2011). This strongly suggests involvement of the immune system in the etiology of NC, while the involvement in NwoC is less clear. Cold Spring Harbor Laboratory Press on May 13, 2018 -Published by genesdev.cshlp.org Downloaded from

Platelet derived growth factor receptor alpha (PDGFRA)-positive oligodendrocyte progenitor cells (OPC) located within the mature central nervous system may remain quiescent, proliferate, or differentiate into oligodendrocytes. Human glioblastoma multiforme tumors (GBM) often contain rapidly proliferating oligodendrocyte lineage transcription factor 2 (Olig2)-positive cells that resemble OPCs. In this study, we sought to identify candidate pathways that promote OPC differentiation or quiescence rather than proliferation. Gene expression profiling performed in both normal murine OPCs and highly proliferative Olig2-positive glioma cells identified all the transcripts associated with the highly proliferative state of these cells and demonstrated that, among the various cell types found within the brain, Olig2-positive tumor cells are most similar to OPCs. We then subtracted OPC transcripts found in tumor samples from those found in normal brain samples and identified 28 OPC transcripts as candidates for promoting differentiation or quiescence. Systematic analysis of human glioma data revealed that these genes have similar expression profiles in human tumors, and were significantly enriched in genomic deletions, suggesting an anti-proliferative role. Treatment of primary murine glioblastoma cells with agonists of one candidate gene, Gpr17, resulted in a decreased number of neurospheres. Together, our findings demonstrate that comparison of the molecular phenotype of progenitor cells in tumors to the equivalent cells in the normal brain represents a novel approach for the identification of targeted therapies.

Upon binding to thalidomide and other immunomodulatory drugs, the E3 ligase substrate receptor cereblon (CRBN) promotes proteosomal destruction by engaging the DDB1-CUL4A-Roc1-RBX1 E3 ubiquitin ligase in human cells but not in mouse cells, suggesting that sequence variations in CRBN may cause its inactivation. Therapeutically, CRBN engagers have the potential for broad applications in cancer and immune therapy by specifically reducing protein expression through targeted ubiquitin-mediated degradation. To examine the effects of defined sequence changes on CRBN's activity, we performed a comprehensive study using complementary theoretical, biophysical, and biological assays aimed at understanding CRBN's nonprimate sequence variations. With a series of recombinant thalidomide-binding domain (TBD) proteins, we show that CRBN sequence variants retain their drug-binding properties to both classical immunomodulatory drugs and dBET1, a chemical compound and targeting ligand designed to degrade bromodomain-containing 4 (BRD4) via a CRBN-dependent mechanism. We further show that dBET1 stimulates CRBN's E3 ubiquitin-conjugating function and degrades BRD4 in both mouse and human cells. This insight paves the way for studies of CRBN-dependent proteasome-targeting molecules in nonprimate models and provides a new understanding of CRBN's substrate-recruiting function.

Protein acetylation is an important contributor to cancer initiation. Histone deacetylase 6 (HDAC6) controls JAK2 translation and protein stability and has been implicated in JAK2-driven diseases best exemplified by myeloproliferative neoplasms (MPNs). By using novel classes of highly selective HDAC inhibitors and genetically deficient mouse models, we discovered that HDAC11 rather than HDAC6 is necessary for the proliferation and survival of oncogenic JAK2-driven MPN cells and patient samples. Notably, HDAC11 is variably expressed in primitive stem cells and is expressed largely upon lineage commitment. Although Hdac11is dispensable for normal homeostatic hematopoietic stem and progenitor cell differentiation based on chimeric bone marrow reconstitution, Hdac11 deficiency significantly reduced the abnormal megakaryocyte population, improved splenic architecture, reduced fibrosis, and increased survival in the MPLW515L-MPN mouse model during primary and secondary transplantation. Therefore, inhibitors of HDAC11 are an attractive therapy for treating patients with MPN. Although JAK2 inhibitor therapy provides substantial clinical benefit in MPN patients, the identification of alternative therapeutic targets is needed to reverse MPN pathogenesis and control malignant hematopoiesis. This study establishes HDAC11 as a unique type of target molecule that has therapeutic potential in MPN.

Previously we reported that lipopolysaccharide (LPS) treatment of murine mammary carcinomas resulted in decreased growth of the tumors. Here we show the decreased growth following LPS treatment was mediated through effects downstream of TLR4 and Myd88. Perhaps more notably, simply reducing TLR4 or Myd88 levels was sufficient to slow tumor growth rates. Moreover, reduced levels of Myd88 correlated with a significant reduction in lung metastasis as well as decreased CCL2 and CCL5 expression. To determine whether inhibiting Myd88 function could also alter tumor growth and chemokine expression we used a Myd88 homodimerization inhibitory peptide. Indeed, inhibiting Myd88 function in four different murine mammary carcinomas as well as the human breast cancer cell line MDA-MB-231 led to decreased growth as well as CCL2 and CCL5 expression. These data imply that Myd88 is important for growth and metastasis of breast cancer, and expression of at least two proinflammatory chemokines.

UV radiation (UVR) causing DNA damage is a well-documented risk factor for nonmelanoma skin cancer. Although poorly understood, UVR may also indirectly contribute to carcinogenesis by promoting immune evasion. To our knowledge, we report the first epidemiological study designed to investigate the association between quantitative measures of UVR, obtained using a spectrophotometer, and circulating T regulatory (Treg) cells. In addition to total Treg cells, the proportion of functionally distinct Treg cell subsets defined by CD45RA and CD27 phenotypic markers, graded expression of FOXP3 and CD25, and those expressing cutaneous lymphocyte–associated Ag and the chemokine receptor CCR4 were enumerated in 350 individuals undergoing routine skin cancer screening exams and determined not to have prevalent skin cancer. No associations were identified for UVR exposure or the overall proportion of circulating Treg cells; however, Treg cell subpopulations with an activation-associated phenotype, CD45RA−/CD27−, and those expressing cutaneous homing receptors were significantly positively associated with UVR. These subpopulations of Treg cells also differed by age, sex, and race. After stratification by natural skin tone, and adjusting for age and sex, we found that spectrophotometer-based measures of UVR exposure, but not self-reported measures of past sun exposure, were positively correlated with the highest levels of these Treg cell subpopulations, particularly among lighter-skinned individuals. Findings from this large epidemiologic study highlight the diversity of human Treg cell subpopulations associated with UVR, thus raising questions about the specific coordinated expression of CD45RA, CD27, CCR4, and cutaneous lymphocyte–associated Ag on Treg cells and the possibility that UVR contributes to nonmelanoma skin cancer carcinogenesis through Treg cell–mediated immune evasion.

Clonal hematopoiesis of indeterminate potential (CHIP) is a recently identified process where older patients accumulate distinct subclones defined by recurring somatic mutations in hematopoietic stem cells. CHIP’s implications for stem cell transplantation have been harder to identify due to the high degree of mutational heterogeneity that is present within the genetically distinct subclones. In order to gain a better understanding of CHIP and the impact of clonal dynamics on transplantation outcomes, we created a mathematical model of clonal competition dynamics. Our analyses highlight the importance of understanding competition intensity between healthy and mutant clones. Importantly, we highlight the risk that CHIP poses in leading to dominance of precancerous mutant clones and the risk of donor derived leukemia. Furthermore, we estimate the degree of competition intensity and bone marrow niche decline in mice during aging by using our modeling framework. Together, our work highlights the importance of better characterizing the ecological and clonal composition in hematopoietic donor populations at the time of stem cell transplantation.

Immunomodulatory drugs, such as thalidomide and related compounds, potentiate T-cell effector functions. Cereblon (CRBN), a substrate receptor of the DDB1-cullin-RING E3 ubiquitin ligase complex, is the only known molecular target for this drug class, where drug-induced ubiquitin-dependent degradation of "neo-substrates" such as IKAROS, AIOLOS, and CK1α accounts for their biological activity. Far less clear is whether these Cereblon E3 ligase modulating compounds disrupt the endogenous functions of CRBN. Strikingly, here we report that CRBN functions in a feedback loop that harnesses antigen-specific CD8+ T-cell effector responses. Specifically, Crbn deficiency in murine CD8+ T-cells augments their central metabolism manifest as elevated bioenergetics, with supraphysiological levels of polyamines secondary to enhanced glucose and amino acid transport, and with increased expression of metabolic enzymes, including the polyamine biosynthetic enzyme ornithine decarboxylase. Importantly, treatment with Celeblon-modulatingcompounds similarly augments central metabolism of human CD8+T-cell. Notably, the metabolic control of CD8+ T-cells by modulating compounds or Crbn deficiency is linked to increased and sustained expression of the master metabolic regulator MYC. Finally, Crbn deficient T-cells have augmented antigen-specific cytolytic activity versus melanoma tumor cells ex vivo and in vivo and drive accelerated and highly aggressive graft-versus-host disease. Therefore, CRBN functions to harness the activation of activated CD8+ T-cells and this phenotype can be exploited by treatment with drugs.