Human genetics indicate that kisspeptin and neurokinin B (NKB) signaling are necessary for generating pulsatile LH release and therefore for initiation of puberty and maintaining gonadal function. In the present study, male monkeys were employed to examine 1) whether activation of the NKB receptor (NK3R) is associated with GnRH release, and 2) hypothalamic localization of these peptides using immunofluorescence histochemistry. Agonadal juveniles, in which pituitary responsiveness to GnRH was heightened by GnRH priming, were employed to indirectly examine GnRH-releasing actions of NK3R and kisspeptin receptor agonists by tracking LH after their i.v. injection. Castrated adults were used for immunohistochemistry. Single i.v. injections of NKB or senktide (an NK3R agonist) elicited robust LH discharges that were abolished by GnRH receptor antagonism (acyline) confirming the ligands' hypothalamic action. Intermittent infusion of senktide (1-min pulse every hour for 4 h), in contrast to that of kisspeptin, failed to sustain pulsatile GnRH release. Repetitive senktide injections did not compromise the GnRH-releasing action of kisspeptin. NKB and kisspeptin were colocalized in perikarya of the arcuate nucleus and in axonal projections to the median eminence, confirming earlier findings in sheep. These results are consistent with the human genetics, and indicate that although brief activation of NK3R stimulates GnRH release, repetitive stimulation of this pathway, in contrast to that of kisspeptin receptor, fails to sustain pulsatile GnRH release. In addition, the data provide a platform for future elucidation of the interactions between NKB and kisspeptin that are required for generating pulsatile GnRH release in primates.

• FL-associated STAT6 mutations hyperactivate the IL-4/JAK/STAT6 axis.Follicular lymphoma (FL) is the second most common non-Hodgkin lymphoma in the Western world. FL cell-intrinsic and cell-extrinsic factors influence FL biology and clinical outcome. To further our understanding of the genetic basis of FL, we performed whole-exome sequencing of 23 highly purified FL cases and 1 transformed FL case and expanded findings to a combined total of 114 FLs. We report recurrent mutations in the transcription factor STAT6 in 11% of FLs and identified the STAT6 amino acid residue 419 as a novel STAT6 mutation hotspot (p.419D/G, p.419D/A, and p.419D/H). FL-associated STAT6 mutations were activating, as evidenced by increased transactivation in HEK293T cell-based transfection/luciferase reporter assays, heightened interleukin-4 (IL-4) -induced activation of target genes in stable STAT6 transfected lymphoma cell lines, and elevated baseline expression levels of STAT6 target genes in primary FL B cells harboring mutant STAT6. Mechanistically, FL-associated STAT6 mutations facilitated nuclear residency of STAT6, independent of IL-4-induced STAT6-Y641 phosphorylation. Structural modeling of STAT6 based on the structure of the STAT1-DNA complex revealed that most FL-associated STAT6 mutants locate to the STAT6-DNA interface, potentially facilitating heightened interactions. The genetic and functional data combined strengthen the recognition of the IL-4/JAK/STAT6 axis as a driver of FL pathogenesis. (Blood. 2015;125(4):668-679)

Purpose: Chronic lymphocytic leukemia (CLL)-associated gene mutations that influence CLL cell fitness and chemotherapy resistance should increase in clonal representation when measured before therapy and at relapse.Experimental Design: To uncover mutations associated with CLL relapse, we have performed whole-exome sequencing in a discovery cohort of 61 relapsed CLL patients identifying 86 recurrently mutated genes. The variant allele fractions (VAF) of 19 genes with mutations in !3 of 61 cases were measured in 53 paired pre-and posttreatment CLL samples sorted to purity using panel-based deep resequencing or by droplet digital PCR.Results: We identify mutations in TP53 as the dominant subclonal gene driver of relapsed CLL often demonstrating substantial increases in VAFs. Subclonal mutations in SAMHD1 also recurrently demonstrated increased VAFs at relapse. Mutations in ATP10A, FAT3, FAM50A, and MGA, although infrequent, demonstrated enrichment in !2 cases each. In contrast, mutations in NOTCH1, SF3B1, POT1, FBXW7, MYD88, NXF1, XPO1, ZMYM3, or CHD2 were predominantly already clonal prior to therapy indicative of a pretreatment pathogenetic driver role in CLL. Quantitative analyses of clonal dynamics uncover rising, stable, and falling clones and subclones without clear evidence that gene mutations other than in TP53 and possibly SAMHD1 are frequently selected for at CLL relapse.Conclusions: Data in aggregate support a provisional categorization of CLL-associated recurrently mutated genes into three classes (i) often subclonal before therapy and strongly enriched after therapy, or, (ii) mostly clonal before therapy or without further enrichments at relapse, or, (iii) subclonal before and after therapy and enriching only in sporadic cases.

Purpose Ibrutinib, a Bruton’s tyrosine kinase (BTK) inhibitor, is approved for the treatment of relapsed CLL and CLL with del17p. Mechanistically, ibrutinib interferes with BCR signaling as well as multiple CLL cell to microenvironment interactions. Given the importance of ibrutinib in the management of CLL, a deeper understanding of factors governing sensitivity and resistance is warranted. Experimental Design We studied 48 longitudinally sampled paired CLL samples, 42 of which were procured before and after standard CLL chemotherapies, and characterized them for well-studied CLL molecular traits as well as by whole exome sequencing and SNP 6.0 array profiling. We exposed these samples to 0.25 μM – 5 μM of ibrutinib ex vivo and measured apoptosis fractions as well as BCR signaling by immunoblotting. We disrupted TP53 in HG3, PGA1 and PG-EBV cell lines and measured BCR signaling and ibrutinib responses. Results CLL samples demonstrated a surprisingly wide range of ex vivo sensitivities to ibrutinib with IC50 values ranging from 0.4 μM – 9.7 μM. Unmutated IGVH status, elevated ZAP70 expression and trisomy 12 were associated with heightened sensitivity to ibrutinib treatment. Five CLL samples were substantially more resistant to ibrutinib following relapse from chemotherapy; of these, three had acquired a del17p/TP53 mutated status. A validation sample of 15 CLL carrying TP53 mutations, of which 13 carried both del17p and a TP53 mutation confirmed substantially less sensitivity to ibrutinib-induced apoptosis. Conclusions This study identifies that CLL harboring del17p/TP53 mutated cells are substantially less sensitive to ibrutinib-induced apoptosis than del17p/TP53 wild type cells.

Reactive oxygen and nitrogen species have been implicated in the pathogenesis of noise-induced hearing loss. In this case-control study, we investigated the oxidative and antioxidative status of erythrocytes from workers in noisy workplace. Blood samples of 127 workers in noisy workplace (WNW) and 117 workers in non-noisy workplace (WNNW) from the same company were taken into tubes with potassium EDTA as anticoagulant in order to obtain hemolysate. Total superoxide dismutase (SOD) and catalase (CAT) activities as the enzymes of antioxidative defense mechanism in the erythrocytes together with malondialdehyde (MDA) as the lipid peroxidation index and total nitric oxide (NO) as an index for nitrogen species analyses were performed by spectrophotometric methods. SOD activity was found to be 450.0±106.4 U/g Hb in WNW and 443.1±83.1 U/g Hb in WNNW. The difference between two groups were not statistically significant (p= 0.582). CAT activity was found to be 426.0±98.0 k/g Hb in WNW and 432.6±109.0 k/g Hb in WNNW showing statistically insignificant difference (p= 0.621). MDA levels in erythrocytes from WNW was significantly higher than WNNW (39.28±10.22 nmol/g Hb and 32.51±10.73 nmol/g Hb, respectively and p= 0.0001). On the other hand, NO levels were found to be significantly reduced in WNW (0.275±0.187 µmol/g Hb) compared to WNNW (0.382±0.284 µmol/g Hb) (p= 0.001). When we analyzed the hematological parameters, all the cell counts increased in WNW except monocytes and platelets compared to WNNW (p= 0.0001). Related to this changes, hemoglobin, MCHC, and hematocrit also increased in WNW (p= 0.0001). The oxidative stress, which is possibly propagated by the physical environment, seems to have an important pathophysiological role in hearing loss and lipid peroxidative cellular changes in all of the workers who work in noisy occupations.Key words: Noise, Worker, Oxidative stress, Superoxide dismutase, Catalase, Malondialdehyde, Nitric oxide ÖZET Gürültünün ‹nsan Eritrositlerindeki Oksidan ve Antioksidan Dengeye EtkileriReaktif oksijen ve nitrojen türleri, yüksek ses kaynakl› duyma kay›plar›nda önemli bir patolojik faktör olarak kabul görmekte-dir. Bu vaka kontrollü çal›flmam›zda yüksek sesli ortamlarda çal›flan iflçilerin eritrositlerinde oksidan ve antioksidan dengeyi araflt›rd›k. Yüksek sesli ortamda çal›flan (YSOÇ) 127 iflçi ile normal ortamda çal›flan (NOÇ) 117 iflçinin kanlar› hemolizat elde etmek üzere içinde antikoagulan olarak potasyum EDTA bulunan tüplere al›nd›.