ALBERT

Description: Endocannabinoid signaling regulates feeding and metabolic processes and has been linked to obesity development. Several hormonal signals, such as glucocorticoids and ghrelin, regulate feeding and metabolism by engaging the endocannabinoid system. Similarly, studies have suggested that leptin interacts with the endocannabinoid system, yet the mechanism and functional relevance of this interaction remain elusive. Therefore, we explored the interaction between leptin and endocannabinoid signaling with a focus on fatty acid amide hydrolase (FAAH), the primary degradative enzyme for the endocannabinoid N-arachidonoylethanolamine (anandamide; AEA). Mice deficient in leptin exhibited elevated hypothalamic AEA levels and reductions in FAAH activity while leptin administration to WT mice reduced AEA content and increased FAAH activity. Following high fat diet exposure, mice developed resistance to the effects of leptin administration on hypothalamic AEA content and FAAH activity. At a functional level, pharmacological inhibition of FAAH was sufficient to prevent leptin-mediated effects on body weight and food intake. Using a novel knock-in mouse model recapitulating a common human polymorphism (FAAH C385A; rs324420), which reduces FAAH activity, we investigated whether human genetic variance in FAAH affects leptin sensitivity. While WT (CC) mice were sensitive to leptin-induced reductions in food intake and body weight gain, low-expressing FAAH (AA) mice were unresponsive. These data demonstrate that FAAH activity is required for leptin’s hypophagic effects and, at a translational level, suggest that a genetic variant in the FAAH gene contributes to differences in leptin sensitivity in human populations.

Description: Article A risk variant located at 9p21.3 is associated with cancer risk in pediatric B-cell precursor acute lymphoblastic leukaemia. Here, the authors show that this variant affects the gene expression of the tumour suppressor gene Cdkn2b . Nature Communications doi: 10.1038/ncomms10635 Authors: Eric A. Hungate, Sapana R. Vora, Eric R. Gamazon, Takaya Moriyama, Timothy Best, Imge Hulur, Younghee Lee, Tiffany-Jane Evans, Eva Ellinghaus, Martin Stanulla, Jéremie Rudant, Laurent Orsi, Jacqueline Clavel, Elizabeth Milne, Rodney J. Scott, Ching-Hon Pui, Nancy J. Cox, Mignon L. Loh, Jun J. Yang, Andrew D. Skol, Kenan Onel

Description: Few clades of plants have proven as difficult to classify as cacti. One explanation may be an unusually high level of convergent and parallel evolution (homoplasy). To evaluate support for this phylogenetic hypothesis at the molecular level, we sequenced the genomes of four cacti in the especially problematic tribe Pachycereeae,...

Description: Introduction: Overactivity of osteoclasts resulting in bone destruction is a hallmark of multiple myeloma (MM). Receptor for activation of NF-kB ligand (RANKL) and monocyte colony stimulating factor (MCSF) signaling pathways both promote proliferation and survival of the precursors of the osteoclast lineage, and have been widely investigated in MM. The third pathway involved in osteoclast differentiation is the immunoreceptor tyrosine-based activation motif (ITAM) with c-Fms signaling. ITAM and its inhibitor ITIM provide the basis for two opposed signaling modules that duel for control of osteoclast formation. Human monocyte/macrophage expresses the low-affinity FcγRIIb and high-affinity Fcε receptor 1 (FcεRI). Both receptors mediate Syk phosphorylation to activate or inactivate downstream ITAM or ITIM signaling molecules. In this study, we determined the effects of an IgG(CH2-CH3) and IgE(CH2-CH3-CH4) fusion protein that activates the ITIM inhibitory pathway on downstream signaling of Syk and osteoclast formation in monocytes from MM patients. Methods: We constructed IgG(CH2-CH3) with an IgE(CH2-CH3-CH4) fusion protein using standard cloning techniques. We evaluated the fusion protein on osteoclast formation using cells from either human monocytes isolated from MM patients' peripheral blood mononuclear cells (PBMCs) or bone marrow (BM) MCs with an anti-CD14 micro-bead affinity column and magnetic bead selection (Miltenyi Biotec, Auburn, CA). The monocytes were cultured on slide-culture dishes (2 X 105 cells/well). The cells were treated with the fusion protein or with IgE or IgG and subsequently treated with 50ng/ml RANKL (receptor for activation of nuclear factor kB and 10ng/ml MCSF (monocyte colony stimulating factor) in order to stimulate osteoclast formation at the beginning of the culture and during a medium change after 3 days with the same amount of growth factors added. The cells were fixed for tartrate resistant acid phosphatase (TRAP)-staining assay on day 21. To investigate ITIM signaling pathway we determined Syk phosphorylation of monocytes treated or without treated with fusion protein by Western blot analysis. Results: We found that in a concentration-dependent fashion, the fusion protein inhibited osteoclast cell formation from CD14+ MCs from PB or BM exposed to RANKL and MCSF. We further analyzed the effects on the FcγRIIb-SHIP signaling pathway in monocytes induced with 50ng/ml RANKL and 10ng/ml MCSF following exposure to fusion protein or control IgG or IgE. The results showed that the monocytes showed markedly lower Syk phosphorylation following exposure to the fusion protein (100-200ng/ml). There was no change of Syk phosphorylationl in monocytes treated with IgG or IgE or IgG with IgE. Conclusions: The results of our study show that intact human IgG or IgE does not affect the ITAM or ITIM signaling pathways. However, a fusion protein consisting of IgG(CH2-CH3) with IgE(CH2-CH3-CH4) showed the ability to activate the ITIM inhibition pathway through FcγRIIb to reduce osteoclast formation. Thus, blockage of ITAM may be treating novel treatment for preventing bone loss for MM patients. Disclosures No relevant conflicts of interest to declare.

Description: Introduction Rivaroxaban, an oral anti-Xa inhibitor, was approved in 2008 in Canada for prevention of venous thromboembolism (VTE) post elective total hip replacement (THR). Studies completed in elective THR patients showed superiority or noninferiority of rivaroxaban over injectable low molecular weight heparin (LMWH) for prevention of VTE, with no difference in trial defined major bleeding. Many centres use rivaroxaban for VTE prevention post THR as standard of care. Our centre introduced rivaroxaban for THR patients in May 2009. As there is limited data on real world experience with this agent, beyond bleeding and VTE events, we evaluated the change in VTE prophylaxis for this indication. Methods A retrospective cohort study was completed comparing the change in use of a LMWH, enoxaparin, to rivaroxaban for VTE prophylaxis for elective THR patients ≥ 19 years not requiring therapeutic anticoagulation. The setting was a regional health authority servicing a population of 290 000, and the timeframe was 18 months before and 18 months after the introduction of rivaroxaban, allowing a 3 month period in between for incorporation of rivaroxaban into routine care. Patients with THR completed pre introduction of rivaroxaban were in the enoxaparin (E) group and patients with THR completed post introduction were in either the enoxaparin and rivaroxaban (E+R, given sequentially) group, or the rivaroxaban (R) group. Data were abstracted from computerized and paper records using a standardized form. Data collected included demographics, medical and medication history, risk factors for bleeding and VTE, type of anesthesia, VTE prophylaxis details, post-discharge community health (CH) nursing visits and hospital visits (emergency room (ER) and inpatient admissions), and major bleeding or VTE events. Hospital visits and VTE were assessed up to 30 days, and major bleeding up to 2 days, after the last presumed dose of medication. Primary outcomes were differences between E, E+R, and R in hospital visits, as well as referrals to, and reasons for, post discharge CH nursing visits. Results There were 246 THR procedures in E, 43 in E+R, and 190 in R. Baseline characteristics were similar between the three groups. For E, E+R, R, 52%, 49% and 53% were female with a mean age of 64, 62, 64 years and a mean BMI of 31, 32, 31 kg/m2. There was no difference in surgery duration (mean 92, 97, 91 min for E, E+R, R), or number of patients transfused (19, 16, 14% for E, E+R, R). More patients (74%) in E+R received regional anesthesia exclusively, compared to E (57%) and R (52%), p=0.01. There was no difference in post-surgery time to VTE prophylaxis initiation (mean of 22, 22, 23 hours for E, E+R, R). There was a difference in the length of stay (mean 7.1, 8.4, 6.3 days for E, E+R, R, p=0.005), as well as total duration of prophylaxis (mean 10.4, 16.1, 14.5 days for E, E+R, R, p

Description: Introduction: The proteasome inhibitor (PI) bortezomib is a novel anticancer drug that shows activity especially for treating patients with multiple myeloma (MM). However, its clinical efficacy has been hampered by the emergence of drug-resistance phenomena, the genetic and molecular basis of which remains elusive. We propose that the resistance to bortezomib treatment may be related to a polymorphism in a gene known to be a target for bortezomib's activity which has been identified as CIP2A which is a tumor suppressor PP2A inhibitor. We have used a high resolution melting curve (HRM) approach to screen several CIP2A SNPs containing significant genetic changes that may be different between bortezomib sensitive and resistant MM patients. In this study, we evaluated SNPs in this gene in healthy subjects and MM patients who were sensitive or resistant to bortezomib-based therapy. Methods: A panel of DNA samples was purified from MM patients (N=73) and healthy subjects (N=95). The samples were divided into three groups: bortezomib sensitive MM patients (N=61), bortezomib resistant MM patients (N=12), and healthy subjects. A single nucleotide polymorphism (SNP) of the bortezomib target genes CIP2A was selected. The PCR prime pairs for each SNP marker were designed with the software "Primer Expression 3" (Thermo Fisher Scientific). The genotype of each DNA sample was determined with the high resolution melt curve (HRM) technology on a real-time PCR instrument, StepOne Plus (Thermo Fisher Scientific). Results: The frequency of heterozygosity and homozygosity of CIP2A, SNP rs34172460(S258A) was 0.049 (3 out of 61 cases) in the bortezomib sensitive group, while it was 0.25 (3 out of 12 cases) in the bortezomib resistant group, a 5-fold increase in the frequency of this SNP. The frequency of heterozygosity and homozygosity of this same SNP in the healthy subjects was only 0.03 (3 out of 95 samples).The minor allele frequency (MAF) in the normal population is 0.046, according to the SNP data base, compatible with what we observed in both our healthy subjects and the bortezomib sensitive MM patients. Conclusion: This preliminary data suggests that heterozygotic and homozygotic mutants of rs34172460(S258A) may have an impact on resistance to bortezomib among patients with multiple myeloma. Disclosures No relevant conflicts of interest to declare.

Description: Introduction: The bone marrow (BM) microenvironment plays an important role in multiple myeloma (MM). The BM niche is composed of multiple cell types including macrophages. Macrophages polarize into pro-inflammatory macrophage-1 (M1) or alternative M2 states that promote tumor growth and metastasis. We evaluated the proportion of M2 macrophages in BM from MM pts either showing complete response (CR) or progressive disease (PD), the effects of MM cells on M1 and M2 differentiation, and the role of Trib1 in M2 differentiation in MM BM. Since the JAK-STAT signaling pathway plays key roles in macrophages, we also evaluated the effects of the JAK2 inhibitor ruxolitinib (RUX) on M2 polarization in MM. Methods: Using immunofluorescence (IFC), we determined the proportion of M1 and M2 macrophages in BM biopsies and aspirates from MM pts with PD or CR. The BM biopsy samples were stained with antibodies directed against human iNOS and CD86 for M1 and arginase 1(ARG1) and CD36 for M2 cells. MM BM aspirates were also examined using flow cytometric analysis (FCA). Human monocytes isolated from healthy subjects or the THP1 monocyte cell line were co-cultured with MM cell lines (RPMI8226 and U266) or primary MM tumor cells. The effects of RUX at low concentrations (IC20) on M2 polarization were determined. The percentages of M1 and M2 macrophages were determined using FCA. Total RNA was extracted from monocytes. Quantitative PCR was measured with TaqMan technology. For the in vivo studies, human MM tumors (LAGκ-2) were surgically implanted into the left superficial gluteal muscle of SCID mice and tumor volume measured on a weekly basis. Results: The proportion of M2 macrophages (CD36+/ARG1+) was markedly increased in BM biopsies or mononuclear cells from MM pts with PD compared with those in CR using IFC staining. FCA also showed the percentage of M2 macrophages in BM was significantly increased in MM pts with PD (n=25) compared to those in CR (n=10; P=0.005) whereas there was no difference in the percentage of M1 (CD86+/iNOS+) macrophages in BM derived from MM pts with PD compared to those in CR. Trib1 gene mRNA levels were higher among pts with PD compared to those in CR whereas the gene expression of Trib2 and Trib3 was not different. Next, we co-cultured MM cell lines (U266) or fresh MM BMMCs with purified healthy human monocytes for one week. The percentage of M2 cells markedly increased and the proportion of M1 cells decreased. Trib1 gene expression increased during co-culture whereas there was no change in expression of the other two Tribs. When direct cell-to-cell contact occurred between the MM tumor cells and the monocytes, the percentage of M2 macrophages markedly increased. We investigated the effects of the JAK2 inhibitor RUX on M2 differentiation induced with MM tumor cells. After exposure to a low concentration of RUX, the percentage of M2 cells decreased when the monocytes were co-cultured with MM tumor cells. Trib1 gene expression of the monocytes treated with RUX was also notably reduced compared with cells not treated with the JAK2 inhibitor. Using our human MM xenograft model LAGκ-2, RUX (1.5mg/kg) reduced tumor growth and decreased the proportion of M2 macrophages in the tumor tissue of MM tumor-bearing SCID mice. Conclusion: M2 cells are present at high levels in BM derived from MM pts with PD compared to those in CR, MM cells induce monocytes to become M2 macrophages and increase Trib1 gene expression. This induces monocyte differentiation into M2 macrophages that support MM tumor cell growth.. Notably, the JAK2 inhibitor RUX inhibits both M2 macrophage polarization and Trib1 gene expression in MM, and reduces tumor growth in SCID mice bearing human MM. These results suggest that RUX may be effective for treating MM pts. Disclosures No relevant conflicts of interest to declare.

Description: Background: Familial predisposition to myeloid malignancies is more common than previously appreciated. 15-20% of acute leukemia patients have at least 1 additional first-degree relative with leukemia predisposition. Germline predisposition to myeloid neoplasms was incorporated in the WHO 2016 classification of myeloid neoplasms and acute leukemia. The clinical guidelines now include testing for inherited susceptibility as a critical element of patient diagnostics. Identification of germline predisposition syndrome can significantly impact treatment decisions, screening of potential sibling donors for allogeneic stem cell transplantation, and patient and family surveillance. Here we developed a framework to guide routine identification of potential pathogenic germline mutations for further characterization and aid in identification of individuals who are at high risk of developing leukemia so that these individual can be closely monitored for disease management and treatment interventions. Methods: As part of the Beat AML consortium, we performed deep whole-exome sequencing of 424 tumor samples from 378 AML patients with matched skin biopsies (Tyner, et al, Nature 2018). High confidence germline variants called by VarScan2 with Exac and gnoMD frequency of less than 1% and normal variant allele frequency greater than 40%, were annotated if they appeared in a curated list of 244 genes that have been previously associated with hematological malignancies and leukemia predisposition. Putative novel variants were nominated based on review of other databases (Exac, gnoMD, dbSNPOSMIC, ClinVAR, Varsome). The mutants were classified as pathogenic/ likely pathogenic using Clinvar and ACMG/AMP guidelines. In silico curation were made using computational predictions of pathogenicity and functional impact (e.g. Sift, Polyphen, Revel, DANN, CADD) with emphasis on the Revel scores. All patients with available family history data were evaluated for evidence of familial clustering of hematological malignancies in 1st, 2nd or 3rd degree relatives. Results: The overall frequency of pathogenic and likely pathogenic germline variants using Clinvar and ACMG/AMP criteria is 11.1% (42/378) with 38 variants in 29 leukemia predisposition genes. The most frequent variants were DDX41 (6), CHEK2 (5), and FANC complex (10) with the remaining patients having single observations in a diversity of leukemia predisposition genes. Recognizing the concerns about tumor contamination within the skin biopsy, we validated a subset of variants from 12 cultured stromal samples for matched patients. Overall, in 378 primary AML patients, we identified 55 novel germline variants in 44 leukemia predisposition genes with the potential to be pathogenic by in silico prediction [Revel 〉 0.5 or DANN 〉 0.9 or CADD 〉 19]. Interestingly, evaluation of the family history found 16 out of 172 patients with the available data who have a 1st degree relative with a hematologic malignancy, 9 patients with an affected 2nd degree relative, and 5 patients with a 3rd degree affected relative. All of these patients with familial clustering have germline variants in leukemia predisposition genes. 16 patients with familial clustering have 6 novel variants and 35 reported variants with previously unpredicted clinical significance that show potential to be deleterious by in silico prediction. For these 16 patients with familial clustering, age at the diagnosis of AML ranged from 36-83 years. Ten of these patients have overlapping somatic variants in FLT3 (5), NPM1 (4), DNMT3A (5), and CEBPA (4). The remaining 6 patients have isolated cases of somatic variants in genes such as RAS, IDH2, U2AF1, EZH2, TET2, etc. Overall, novel germline variants are candidates for functional validation to understand their contribution in disease pathogenicity. Conclusions: Exome sequencing and in silico prediction identified ~11% of AML patients in the Beat AML cohort have pathogenic/likely pathogenic germline variants with leukemia predisposition genes. In the cohort, 9.3% of the patients had evidence of familial clustering of hematological malignancies. Combining identification of novel germline variants in leukemia predisposition genes, with emphasis on those seen in patients with familial clustering with functional validation can improve in silico prediction efforts and help to guide patient monitoring and clinical disease management. Disclosures Borate: Novartis: Consultancy; Takeda: Consultancy; Pfizer: Consultancy; Daiichi Sankyo: Consultancy; AbbVie: Consultancy. Druker:Bristol-Myers Squibb: Patents & Royalties, Research Funding; Pfizer: Research Funding; Aileron Therapeutics: #2573, Constructs and cell lines harboring various mutations in TNK2 and PTPN11, licensing fees , Membership on an entity's Board of Directors or advisory committees; ALLCRON: Membership on an entity's Board of Directors or advisory committees; Amgen: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Aptose Biosciences: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Beta Cat: Membership on an entity's Board of Directors or advisory committees, Other: Stock options; Blueprint Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Burroughs Wellcome Fund: Membership on an entity's Board of Directors or advisory committees; Cepheid: Consultancy, Honoraria; GRAIL: Equity Ownership, Other: former member of Scientific Advisory Board; Patient True Talk: Consultancy; The RUNX1 Research Program: Membership on an entity's Board of Directors or advisory committees; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees, Other: Stock options; Beat AML LLC: Other: Service on joint steering committee; CureOne: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy; Gilead Sciences: Other: former member of Scientific Advisory Board; ICON: Other: Scientific Founder of Molecular MD, which was acquired by ICON in Feb. 2019; Monojul: Other: former consultant; Novartis: Other: PI or co-investigator on clinical trial(s) funded via contract with OHSU., Patents & Royalties: Patent 6958335, Treatment of Gastrointestinal Stromal Tumors, exclusively licensed to Novartis, Research Funding; Bristol-Myers Squibb: Other: PI or co-investigator on clinical trial(s) funded via contract with OHSU., Research Funding; Pfizer: Other: PI or co-investigator on clinical trial(s) funded via contract with OHSU., Research Funding; Merck & Co: Patents & Royalties: Dana-Farber Cancer Institute license #2063, Monoclonal antiphosphotyrosine antibody 4G10, exclusive commercial license to Merck & Co; Dana-Farber Cancer Institute (antibody royalty): Patents & Royalties: #2524, antibody royalty; OHSU (licensing fees): Patents & Royalties: #2573, Constructs and cell lines harboring various mutations in TNK2 and PTPN11, licensing fees . Tyner:Takeda: Research Funding; Incyte: Research Funding; Gilead: Research Funding; Janssen: Research Funding; Syros: Research Funding; Array: Research Funding; Genentech: Research Funding; Constellation: Research Funding; Agios: Research Funding; Array: Research Funding; AstraZeneca: Research Funding; Agios: Research Funding; Janssen: Research Funding; Petra: Research Funding; Petra: Research Funding; Seattle Genetics: Research Funding; Aptose: Research Funding; Genentech: Research Funding; Gilead: Research Funding; Seattle Genetics: Research Funding; Syros: Research Funding; Takeda: Research Funding; Aptose: Research Funding; Incyte: Research Funding; AstraZeneca: Research Funding; Constellation: Research Funding.