Hsp90 is required for the normal activity of steroid receptors, and in steroid receptor complexes it is typically bound to one of the immunophilin-related cochaperones: the peptidylprolyl isomerases FKBP51, FKBP52 or CyP40, or the protein phosphatase PP5. The physiological roles of the immunophilins in regulating steroid receptor function have not been well de®ned, and so we examined in vivo the in¯uences of immunophilins on hormone-dependent gene activation in the Saccharomyces cerevisiae model for glucocorticoid receptor (GR) function. FKBP52 selectively potentiates hormone-dependent reporter gene activation by as much as 20-fold at limiting hormone concentrations, and this potentiation is readily blocked by co-expression of the closely related FKBP51. The mechanism for potentiation is an increase in GR hormone-binding af®nity that requires both the Hsp90-binding ability and the prolyl isomerase activity of FKBP52.

Transcription complexes that assemble on tRNA genes in a crude Saccharomyces cerevisiae cell extract extend over the entire transcription unit and approximately 40 base pairs of contiguous 5'-flanking DNA. We show here that the interaction with 5'-flanking DNA is due to a protein that copurifies with transcription factor TFIIIB through several steps of purification and shares characteristic properties that are normally ascribed to TFIIIB: dependence on prior binding of TFIIIC and great stability once the TFIIIC-TFIIIB-DNA complex is formed. SUP4 gene (tRNATyr) DNA that was cut within the 5'-flanking sequence (either 31 or 28 base pairs upstream of the transcriptional start site) was no longer able to stably incorporate TFIIIB into a transcription complex. The TFIIIB-dependent 5'-flanking DNA protein interaction was predominantly not sequence specific. The extension of the transcription complex into this DNA segment does suggest two possible explanations for highly diverse effects of flanking-sequence substitutions on tRNA gene transcription: either (i) proteins that are capable of binding to these upstream DNA segments are also potentially capable of stimulating or interfering with the incorporation of TFIIIB into transcription complexes or (ii) 5'-flanking sequence influences the rate of assembly of TFIIIB into stable transcription complexes.Specific transcription by RNA polymerase III (Pol III) requires the participation of multiple transcription factors. With the exception of the U6 and 7SK RNA genes (7, 14, 17. 41. 50, 53), the DNA-binding sites that anchor these factors are located within transcription units. It is a remarkable property of Pol III that it can transcribe through the bulky transcription complexes that are built up on these internal promoters (also called internal control regions) without dispersing them (69).Specific initiation of transcription at tRNA genes, with which this paper primarily deals, requires factors TFIIIB and -C. Although the yeast analog of TFIIIC (also called T) has been relatively highly purified as a single component (54: see below), HeLa TFIIIC splits into two components upon purification: C2 is the primary DNA-binding determinant, and Cl either modifies the binding properties of C2 or binds to DNA in a C2-dependent manner (12,18,71). Both HeLa C2 factor and yeast TFIIIC (T) are large (12,54,64). The Bonbvx moni tRNA gene transcription factors can also be separated into three fractions, whose relationship to TFIIIB, Cl, and C2 remains to be established (51).The first promoter dissections of Pol III genes identified essential gene-internal elements (10,19,25,55) and implied that flanking DNA sequence was almost without effect on promoter strength. The general situation with regard to the effects of flanking sequence on promoter strength of Pol III genes is, however, diverse, and this fact has only gradually been recognized (3, 4, 20, 29, 52, 62, 63; tions with very substantial effects have been reported. Flanking sequences affecting promoter strength are located within ...

Molecular chaperones mediate multiple aspects of steroid receptor function, but the physiological importance of most receptor-associated cochaperones has not been determined. To help fill this gap, we targeted for disruption the mouse gene for the 52-kDa FK506 binding protein, FKBP52, a 90-kDa heat shock protein (Hsp90)-binding immunophilin found in steroid receptor complexes. A mouse line lacking FKBP52 (52KO) was generated and characterized. Male 52KO mice have several defects in reproductive tissues consistent with androgen insensitivity; among these defects are ambiguous external genitalia and dysgenic prostate. FKBP52 and androgen receptor (AR) are coexpressed in prostate epithelial cells of wild-type mice. However, FKBP52 and AR are similarly coexpressed in testis even though testis morphology and spermatogenesis in 52KO males are usually normal. Molecular studies confirm that FKBP52 is a component of AR complexes, and cellular studies in yeast and human cell models demonstrate that FKBP52 can enhance AR-mediated transactivation. AR enhancement requires FKBP52 peptidylprolyl isomerase activity as well as Hsp90-binding ability, and enhancement probably relates to an affect of FKBP52 on AR-folding pathways. In the presence of FKBP52, but not other cochaperones, the function of a minimally active AR point mutant can be dramatically restored. We conclude that FKBP52 is an AR folding factor that has critically important physiological roles in some male reproductive tissues.

Hormone-dependent transactivation by several of the steroid hormone receptors is potentiated by the Hsp90-associated cochaperone FKBP52, although not by the closely related FKBP51. Here we analyze the mechanisms of potentiation and the functional differences between FKBP51 and FKBP52. While both have peptidyl-prolyl isomerase activity, this is not required for potentiation, as mutations abolishing isomerase activity did not affect potentiation. Genetic selection in Saccharomyces cerevisiae for gain of potentiation activity in a library of randomly mutated FKBP51 genes identified a single residue at position 119 in the N-terminal FK1 domain as being a critical difference between these two proteins. In both the yeast model and mammalian cells, the FKBP51 mutation L119P, which is located in a hairpin loop overhanging the catalytic pocket and introduces the proline found in FKBP52, conferred significant potentiation activity, whereas the converse P119L mutation in FKBP52 decreased potentiation. A second residue in this loop, A116, also influences potentiation levels; in fact, the FKBP51-A116V L119P double mutant potentiated hormone signaling as well as wild-type FKBP52 did. These results suggest that the FK1 domain, and in particular the loop overhanging the catalytic pocket, is critically involved in receptor interactions and receptor activity.Multiple cellular factors influence hormone-dependent activation of steroid receptors and cellular responses to hormone exposure. Our interest has focused on molecular chaperones that assemble with steroid receptors and alter receptor activity. More than a dozen chaperone and cochaperone proteins have been identified in steroid receptor complexes (23, 28); some chaperones are restricted to different stages of receptor assembly, and others compete for common assembly sites in the receptor complex. In vitro studies have identified five chaperones that are minimally necessary for efficient maturation and maintenance of the ability of the receptor to bind hormone (9,11,17). These are the major heat shock proteins Hsp40, Hsp70, and Hsp90 plus the cochaperone Hop, which can act as an adaptor by simultaneously binding both Hsp70 and Hsp90 (3,30), and the cochaperone p23, which stabilizes the association of Hsp90 with receptor (15, 16). Of the multiple other cochaperones observed in receptor complexes, some have unknown functions, although others are involved in the proteolytic stability of the receptor and yet others have been shown to modulate the receptor response to hormone. Most notable of the latter are two Hsp90 cochaperones in the FK506 binding protein (FKBP) family of peptidyl-prolyl isomerase (PPIase) that have been shown to alter hormonal potency (5, 24, 25). FKBP51 was identified as a cellular factor contributing to glucocorticoid resistance in cells from New World primates (24) by inhibiting glucocorticoid receptor (GR) response to hormone (8). In contrast, FKBP52 was found to enhance GR response to hormone (25) and to similarly enhance the receptors for androgens (AR) ...

MYC locus rearrangements – often complex combinations of translocations, insertions, deletions, and inversions - in multiple myeloma (MM) were thought to be a late progression event, which often did not involve immunoglobulin genes. Yet germinal center activation of MYC expression has been reported to cause progression to MM in an MGUS prone mouse strain. Although previously detected in 16% of MM, we find MYC rearrangements in nearly 50% of MM, including smoldering MM, and they are heterogeneous in some cases. Rearrangements reposition MYC near a limited number of genes associated with conventional enhancers, but mostly with super-enhancers (e.g., IGH, IGL, IGK, NSMCE2, TXNDC5, FAM46C, FOXO3, IGJ, PRDM1). MYC rearrangements are associated with a significant increase of MYC expression that is monoallelic, but MM tumors lacking a rearrangement have bi-allelic MYC expression at significantly higher levels than in MGUS. We also show that germinal center activation of MYC does not cause MM in a mouse strain that rarely develops spontaneous MGUS. It appears that increased MYC expression at the MGUS/MM transition usually is bi-allelic, but sometimes can be mono-allelic if there is a MYC rearrangement. Our data suggests that MYC rearrangements, regardless of when they occur during MM pathogenesis, provide one event that contributes to tumor autonomy.

The cellular inhibitor of apoptosis cIAP1 and −2 are amplified in about 3% of cancers, and were identified in multiple malignancies as potential therapeutic targets due to their role in evasion of apoptosis. Consequently, small molecule IAP antagonists, like LCL161, have entered clinical trials for their ability to induce TNF-mediated apoptosis of cancer cells. However, cIAP1 and −2 are recurrently homozygously deleted in multiple myeloma resulting in constitutive activation of the non-canonical NFkB pathway. It was therefore counterintuitive to observe a robust in vivo anti-myeloma activity of LCL161 in a transgenic myeloma mouse model and patients with relapsed-refractory myeloma, where addition of cyclophosphamide resulted in a median progression free survival of 10 months. This effect is not due to direct induction of tumor cell death, but rather to upregulation of a tumor cell autonomous type I interferon signaling and a strong inflammatory response with activation of macrophages and dendritic cells resulting in phagocytosis of tumor cells. Treatment with LCL161 established long-term anti-tumor protection and cure in a fraction of transgenic Vk*MYC mice. Remarkably, combination of LCL161 with the immune-checkpoint inhibitor anti-PD1 was curative in all treated mice.

Drugs that target novel surfaces on the androgen receptor (AR) and/or novel AR regulatory mechanisms are promising alternatives for the treatment of castrate-resistant prostate cancer. The 52 kDa FK506 binding protein (FKBP52) is an important positive regulator of AR in cellular and whole animal models and represents an attractive target for the treatment of prostate cancer. We used a modified receptor-mediated reporter assay in yeast to screen a diversified natural compound library for inhibitors of FKBP52-enhanced AR function. The lead compound, termed MJC13, inhibits AR function by preventing hormone-dependent dissociation of the Hsp90-FKBP52-AR complex, which results in less hormonebound receptor in the nucleus. Assays in early and late stage human prostate cancer cells demonstrated that MJC13 inhibits AR-dependent gene expression and androgen-stimulated prostate cancer cell proliferation.immunophilin | FKBP4 | steroid hormone receptor A ndrogens are a major stimulator of prostate tumor growth, and all current therapies act as classic antagonists by competing with androgens for binding the androgen receptor (AR) hormone binding pocket. This mechanism of action exploits the dependence of AR on hormone activation, but current treatment options become ineffective in castrate-resistant prostate cancer (CRPC), although CRPC remains ligand/AR-dependent. Thus, drugs that target novel surfaces on AR and/or novel AR regulatory mechanisms may provide promising alternatives for the treatment of CRPC (reviewed in ref. 1).The maturation of cytoplasmic steroid hormone receptors (SHR) to a mature hormone binding conformation is a highly ordered, dynamic process that involves multiple chaperone and cochaperone components (reviewed in ref. 2), all of which present potential opportunities for therapeutic intervention. The final mature complex in which the receptor is capable of high affinity hormone binding includes heat shock protein 90 (Hsp90), a 23 kDa cochaperone (p23), and one of a class of proteins (termed FKBPs) characterized by their Hsp90-binding tetratricopeptide repeat (TPR) domain. The 52 kDa FK506 binding protein (FKBP52) associates with receptor-Hsp90 complexes by way of a C-terminal TPR domain and is a specific positive regulator of AR, glucocorticoid receptor (GR), and progesterone receptor (PR) signaling (3-5). FKBP52 is required for normal male sexual differentiation and development in mice as the fkbp52-deficient mice (52KO) display characteristics of partial androgen insensitivity syndrome including dysgenic prostate (4, 6). FKBP proteins are validated targets of immunosuppressive drugs. FK506 (Tacrolimus) is used clinically to suppress the immune system following organ transplantation. FK506 binds within the peptidyl-prolyl isomerase (PPIase) catalytic pocket of a related family member, FKBP12. The chemical groups of FK506 that project out from the PPIase pocket allow the FKBP12-drug complex to bind tightly to and inhibit calcineurin, which ultimately leads to immunosupression (7). Although FK506 binding ...

Multiple myeloma (MM) is a plasma cell malignancy preceded by a premalignant stage, named monoclonal gammopathy of undetermined significance (MGUS), and often a smoldering phase (SMM). 1, 2 Primary events, which include recurrent translocations of the IgH locus and hyperdiploidy, occur early in pathogenesis, and are followed by the acquisition of secondary genetic events such as MYC structural variants (SV), mutations that activate the RAS or NFkB pathways, mutations of DIS3 or FAM46C that drive precursor stages of disease toward MM. [3][4][5][6] Whole exome sequencing (WES) studies comparing serial MGUS/SMM and MM samples indicate clonal stability, and no significant increase in mutational load in patients that progress rapidly to MM. 7 In contrast, in 33 unselected MGUS patients single-nucleotide variants (SNVs) were less frequent, and no MYC translocations identified. 8 To study the role of MYC in myeloma we performed an integrated genomic analysis of 612 newly diagnosed myeloma (NDMM) patients enrolled in the CoMMpass study, as well as Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:http://www.nature.com/authors/editorial_policies/license.html#terms * shared first co-authors Authorship Contributions PLB, WMK and KM originated concept and design of investigation, KMK EB developed custom capture panel, SVW FISH analysis, GA primary samples, YA GD WMK PLB developed additional bioinformatics methods, NK KM CKS PLB WMK performed analyses, and NK KM CKS WMK PLB composed manuscript. We thank JK and MMRF research network for their work on CoMMpass. All authors read and approved of final manuscript. Conflict of Interest DisclosuresRafael Fonseca works as a consultant for AMGEN, BMS, Celgene, Takeda, Bayer, Jansen, Pharmacyclics, Merck, Sanofi, Kite and Juno. He is on the board of Scientific Advisory Board: Adaptive Biotechnologies. Mayo Clinic and Rafael Fonseca hold a patent for prognosticating myeloma using FISH.