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Positional cloning of hereditary deafness genes is a direct approach to identify molecules and mechanisms underlying auditory function. Here we report a locus for dominant deafness, DFNA36, which maps to human chromosome 9q13–21 in a region overlapping the DFNB7/B11 locus for recessive deafness. We identified eight mutations in a new gene, transmembrane cochlear-expressed gene 1 (TMC1), in a DFNA36 family and eleven DFNB7/B11 families. We detected a 1.6-kb genomic deletion encompassing exon 14 of Tmc1 in the recessive deafness (dn) mouse mutant, which lacks auditory responses and has hair-cell degeneration1,2. TMC1 and TMC2 on chromosome 20p13 are members of a gene family predicted to encode transmembrane proteins. Tmc1 mRNA is expressed in hair cells of the postnatal mouse cochlea and vestibular end organs and is required for normal function of cochlear hair cells.

The c-Mos serine/threonine protein kinase is an essential component of cytostatic factor (CSF), which is required for metaphase II arrest of eggs in vertebrates. Previously, we showed that c-Mos residue Ser-16 is phosphorylated in the ts110 Mo-MuSV-encoded Gag-Mos fusion protein. Here we provide evidence that Mos is phosphorylated at Ser-16 in transfected COS-1 cells. To investigate the role of this phosphorylation, Ser-16 was substituted with alanine or glutamic acid in full-length v-Mos (an Env-Mos fusion protein that contains 31 additional amino acids at the amino terminus of c-Mos), its mouse c-Mos equivalent version (v-Mos residues 32-374, hereafter referred to as Mos), and mouse c-Mos. Constructs expressing mutant versions of Mos were transfected into COS-1 and NIH3T3 cells in a transient and stable manner, respectively. Synthesis and proteolysis of Mos were evaluated by pulse-chase analysis of 35S-methionine-labeled proteins. Our findings indicate that the S16A mutant of Mos was highly unstable. It accumulated to approximately 10% of the level of wild-type Mos or its S16E mutant. In addition, the S16A mutation but not the S16E mutation inhibited Mos interaction with a cellular protein, p35, suggesting that phosphorylation at Ser-16 may promote Mos interaction with p35. As expected from its destabilizing effect, the S16A mutation caused a dramatic decrease in the cellular transforming activity of Mos (determined by soft-agar colony-formation assay with the stably transfected NIH3T3 cells), which is known to correlate with its CSF function. Efficient ubiquitin-mediated proteolysis of c-Mos requires proline as the second residue from the amino-terminus. In contrast to Mos, neither the stability nor protein kinase activity of v-Mos (in which c-Mos residue Pro-2 becomes Pro-33) was affected by the S16A mutation. To provide further proof that, similar to c-Mos, the S16A mutant is recognized by the proteolysis system through Pro-2, we show that the effect of the S16A mutation is reversed by the Pro-2-Ala mutation. Thus, our results indicate that Ser-16 has an important role in the regulation of c-Mos and that phosphorylation at Ser-16 may inhibit proteolysis of c-Mos.

c-Mos is a germ cell-specific MAP kinase kinase kinase (MAPKKK) that plays an essential role during meiotic divisions of oocytes. c-Mos is a key component of an activity, cytostatic factor, required for metaphase II arrest of unfertilized eggs in vertebrates. To understand the regulation of c-Mos, we are investigating c-Mos-interacting proteins. We provide evidence that mouse c-Mos binds to Hsp70, a molecular chaperone. Hsp70 was found to associate with Mos ectopically expressed in COS-1 cells. Mos-Hsp70 complexes could be immunoprecipitated with both Mos and Hsp70 antibodies. Despite a low-abundance of Mos, the Hsp70 antibody immunoprecipitated Mos as the major protein. Of importance, the Mos protein present in anti-Hsp70 immunoprecipitates functioned as an active MAPKKK indicating that it is not grossly misfolded. It is known that c-Mos protein kinase activity in cell extracts of transfected COS-1 or NIH3T3 cells is labile. We found that the inclusion of adenosine triphosphate (ATP) in cell extracts protected against the loss of Mos kinase activity. In the absence of ATP from cell extracts, protein kinase activity of Mos was lost within 6 h on ice even though the Mos protein was not degraded and remained bound to Hsp70. Based on our identification of c-Mos-Hsp70 interaction, one of the roles of ATP may be to assist the regulation of c-Mos via ATP involvement in the protein-folding function of Hsp70 and possibly other molecular chaperones. We also detected by coimmunoprecipitation a physical association between endogenous c-Mos and Hsp70 in Xenopus eggs. To provide further evidence for the functional significance of Hsp70 interaction to Mos function, we show that the residue serine 3 in Mos, which is important for the regulation of protein kinase activity of Mos is also important for Hsp70 association.

Mos is a germ cell-specific serine/threonine protein kinase that plays an important role during meiotic divisions of oocytes. Upon expression in somatic cells, Mos causes cell cycle perturbations leading to neoplastic transformation. Mos activates the MAP kinase pathway in both oocytes and transformed somatic cells. To determine the mechanism of cell cycle perturbation in mos-transformed cells, we examined the status of some key regulators of G1 phase. We provide evidence that Mos causes an elevation in the level of cyclin D1 in NIH/3T3 cells. As expected from the increased cyclin D1 level, mos transformation of NIH/3T3 cells caused an increase in the protein kinase activities of cyclin D1-Cdk4 and cyclin E-Cdk2 and induced hyperphosphorylation of the retinoblastoma protein. Of importance, the level of cyclin D1 was also elevated in eye lens of the c-mos-transgenic mice compared to normal mice. Our results indicate that the mechanism of cellular transformation by Mos involves an elevation in the level of cyclin D1 in somatic cells.

Halogenated organic compounds can represent a useful class of intermediates as they could act as precursors to a number of organometallic species and suitable substrates for metal-catalyzed cross coupling reactions. Here we present an interesting means of functionalizing graphene substrates based on a Suzuki coupling reaction. Firstly, brominated chemically converted graphene sheets were obtained by treating the graphene with ultrasonic assisted bromination. Next, the brominated graphene were modified via the formation of C–C bond between the brominated graphene and the employed aryl boronic. 4-Hexylphenyl boronic acid and 7-bromo-9,9-dihexyl-fluorene-2-ylboronic acids were chosen to show the utility of this reaction scheme in functionalizing graphene. The solubility and optical properties of the grafted material was improved dramatically by the modification. This will open a new interesting filed for the exploitation and application of graphene based materials.

We investigated the effect of cyclic AMP-dependent protein kinase (PKA ) on v-Mos kinase activity. Increase in PKA activity in vivo brought about either by forskolin treatment or by overexpression of PKA catalytic subunit resulted in a significant inhibition of v-Mos kinase activity. The purified PKA catalytic subunit was able to phosphorylate recombinant p37v-mos in vitro, suggesting that the mechanism of in vivo inhibition of v-Mos kinase involves direct phosphorylation by PKA. Combined tryptic phosphopeptide two-dimensional mapping analysis and in vitro mutagenesis studies indicated that Ser-56 is the major in vivo phosphorylation site on v-Mos. In vivo phosphorylation at Ser-56 correlated with slower migration of the v-Mos protein during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, even though Ser-56 was phosphorylated by PKA, this phosphorylation was not involved in the inhibition of v-Mos kinase. The alanine-for-serine substitution at residue 56 did not affect the ability of v-Mos to autophosphorylate in vitro or, more importantly, to activate MEK1 in transformed NIH 3T3 cells. We identified Ser-263 phosphorylation, the Ala-263 mutant of v-Mos was not inhibited by forskolin treatment. From our results, we propose that the known inhibitory role of PKA in the initiation of oocyte maturation in mice could be explained at least in part by its inhibition of Mos kinase.

Abstract 1470 To discover oncogenic pathways that are characteristically deregulated in T-cell acute lymphoblastic leukemia (T-ALL), we performed RNA interference screens both in T-ALL cell lines and primary specimens. We found that the JAK tyrosine kinase family member, TYK2, and its downstream effector, STAT1, are each required for the survival of T-ALL cells. To identify the effector molecules downstream of the TYK2-STAT1 pathway in T-ALL, we analyzed global gene expression profiles in TYK2-dependent T-ALL cell lines after silencing of TYK2 or STAT1. As expected, gene set enrichment analysis revealed that genes downregulated by TYK2 knockdown were generally also downregulated by knockdown of STAT1. Importantly, we found that expression of the anti-apoptotic gene BCL2 was significantly downregulated after silencing of both TYK2 and STAT1. Analysis by quantitative PCR of additional T-ALL cell lines revealed that silencing of TYK2 resulted in significant reductions of BCL2 mRNA expression in multiple TYK2-dependent cell lines. Expression of the wild-type but not the kinase-dead TYK2 protein was sufficient to rescue BCL2 protein expression and to prevent apoptosis after knockdown of endogenous TYK2, indicating that the tyrosine kinase activity of TYK2 is required for BCL2 upregulation. Similarly, expression of the shRNA-resistant wild-type STAT1A protein partially rescued BCL2 protein expression and prevented apoptosis, while a variant of STAT1A (Y701F) that is incapable of becoming phosphorylated on a requisite tyrosine residue did not rescue BCL2 levels. Taken together, our findings indicate that aberrant activation of a TYK2-STAT1 pathway upregulates BCL2 expression in T-ALL cells, and that the T-ALL cells develop pathway dependence, in that they require these sustained high levels BCL2 expression for survival. Disclosures: No relevant conflicts of interest to declare.

686 Background DLBCL has two molecular subtypes, termed activated B cell-like (ABC) and germinal center B cell-like (GCB), with ABC DLBCL being less curable with current therapy. The survival of ABC but not GCB DLBCL cell lines is sustained by “chronic active” B cell receptor (BCR) signaling. Gain-of-function mutations affecting the BCR subunit CD79B occur in 21% of ABC but only 5% of GCB DLBCL tumors. ABC DLBCL cell lines also depend upon a second pathway for survival that is coordinated by MYD88, an adapter for Toll-like receptors. A constitutively active MYD88 mutant (L265P) is frequent in ABC DLBCL tumors (29%) but rare in GCB DLBCL. CD79B and MYD88 L265P mutations often coexist in ABC DLBCL tumors, suggesting oncogenic collaboration, but can also occur alone. Here, we present interim results of a phase 2 study in relapsed/refractory DLBCL of ibrutinib, a first in class inhibitor of BTK, a kinase in the BCR pathway. We tested the hypothesis that ibrutinib would be more active in ABC than GCB DLBCL due to their different addiction pathways, and we assessed the association of CD79B and MYD88 mutations with response. Methods Subjects with relapsed/refractory de novo DLBCL received ibrutinib 560 mg PO QD. Gene expression profiling (GEP) of formalin-fixed paraffin-embedded biopsy tissues using Affymetrix arrays was used to identify the DLBCL subtype (ABC, GCB, unclassifiable) or were not arrayed (unknown). Sanger sequencing was used to identify CD79B and MYD88 mutations. Subjects underwent CT and PET scanning pre-treatment and every 2 cycles. The primary objective of the study was overall response rate (ORR) categorized by molecular subtype. Response was investigator determined using the revised International Working Group Criteria for NHL. Subjects Seventy subjects were enrolled; median age 63 yrs (28–92); male 71%; stage IV 63%; HI-I/HI IPI 59%; disease ≥10 cm 23%; median prior systemic therapies 3 (1–7); relapsed (27%), refractory (54%) and unknown (19%); prior stem cell transplant 23%; and median time from diagnosis 19 months. Results Safety data are available for 68 subjects who received ≥ 1 dose of ibrutinib. Ibrutinib was well tolerated, with treatment-emergent AEs consistent with data reported in other ibrutinib studies. No new safety signals were identified. Sixty subjects were evaluable per protocol for response (≥ 1 dose of ibrutinib and at least one response assessment). Four subjects in the ABC cohort were not evaluable for response (1 death at study day 12, 1 PD at study day 65, and 2 remain on study treatment but have not had their first response assessment at this analysis). One subject in the GCB cohort was not evaluable for response (death at study day 41). In the ABC subtype, per protocol ORR was 40% (10/25, 95% CI: 21–61%), CR 8% (2/25) and PR 32% (8/25). The median PFS at the time of this analysis is 5.5 months in ABC responders with 60% having not progressed (5 remain on treatment and 1 responder proceeded to transplant). Only one PR was observed in the GCB subtype and none in unclassifiable cases. Thus, ibrutinib showed preferential response activity in ABC versus GCB DLBCL (p=0.0126, Fisher's exact test). Responses occurred in ABC DLBCL tumors with CD79B mutations (60%; 3/5), but also in those with wild type CD79B (37%; 7/19), suggesting that ibrutinib sensitivity does not require a BCR mutation. All cases with both CD79B and MYD88 L265P mutations (n=4) responded, showing that the MYD88 pathway does not prevent ibrutinib activity. In comparison, tumors with only a MYD88 L265P mutation (n=4) did not respond (p=0.0286, Fisher's exact test), suggesting a MYD88-dependent but BCR-independent pathogenesis for some ABC DLBCL cases. | | ABC subtype (N=29) | GCB subtype (N=20) | Unclassifiable[1][1] (N=16) | Unknown[2][2] (N=5) | Total (N=70) | |:-------------------------- | ------------------ | ------------------ | --------------------------- | ------------------- | ------------ | | Not Evaluable for Response | 4 | 1 | 3 | 2 | 10 | | PP ORR[4][3] (CR + PR) | 10 (40%) | 1 (5.3%) | | 2 (66.7%) | 13 (21.7%) | | Complete Response (CR) | 2 (8%) | | | 1 (33.3%) | 3 (5%) | | Partial Response (PR) | 8 (32%) | 1 (5.3%) | | 1 (33.3%) | 10 (16.7%) | | PFS (months) | 2.5 | 1.28 | 0.95 | NR[3][4] | 1.64 | * [↵][5]1 GEP performed, but not assignable to ABC or GCB subtypes. * [↵][6]2 GEP not yet performed or tissue not available. * [↵][7]3 Not reached. * [↵][8]4 PP = per protocol. Efficacy Data Conclusions Ibrutinib showed a clinically meaningful response rate in relapsed/refractory ABC DLBCL, but not in other molecular subtypes. These results are consistent with an essential role of BCR signaling in ABC DLBCL and indicate that future clinical trials of ibrutinib in DLBCL should enroll patients with this subtype. Disclosures: Goy: Pharmacyclics: Research Funding. de Vos: Pharmacyclics: Research Funding. Kenkre: Pharmacyclics: Research Funding. Blum: Pharmacyclics: Research Funding. Advani: Phramacyclics, Inc: Research Funding. Kunkel: Pharmacyclics, Inc: Employment, Equity Ownership. McGreivy: Pharmacyclics, Inc.: Employment, Equity Ownership. Balasubramanian: Pharmacyclics, Inc.: Employment, Equity Ownership. Cheng: Pharmacyclics, Inc.: Employment, Equity Ownership. Moussa: Pharmacyclics, Inc.: Employment, Equity Ownership. Buggy: Pharmacyclics, Inc.: Employment, Equity Ownership. 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