Proteasome complexes experience profound structural and functional rearrangements throughout mammalian spermatogenesis

International audienceSignificanceThe proteasome is responsible for the homeostasis of intracellular proteins. Here, we describe structural and functional aspects of a poorly characterized proteasome subtype found exclusively in germ cells. The spermatoproteasome was recently shown to be essential for spermatogenesis, a process requiring intense proteolysis. It differs from the constitutive proteasome by only one subunit, α4s, a subunit that replaces its α4 ubiquitous counterpart. In this work, we show how the shift from α4 to α4s regulates proteasome composition, dynamics, interactome, and activity. We reveal a regulation process more complex than previously suggested, which provides the basis for structural and functional studies of the spermatoproteasome

Integrated Genomic Characterization Reveals Novel, Therapeutically Relevant Drug Targets in FGFR and EGFR Pathways in Sporadic Intrahepatic Cholangiocarcinoma

<div><p>Advanced cholangiocarcinoma continues to harbor a difficult prognosis and therapeutic options have been limited. During the course of a clinical trial of whole genomic sequencing seeking druggable targets, we examined six patients with advanced cholangiocarcinoma. Integrated genome-wide and whole transcriptome sequence analyses were performed on tumors from six patients with advanced, sporadic intrahepatic cholangiocarcinoma (SIC) to identify potential therapeutically actionable events. Among the somatic events captured in our analysis, we uncovered two novel therapeutically relevant genomic contexts that when acted upon, resulted in preliminary evidence of anti-tumor activity. Genome-wide structural analysis of sequence data revealed recurrent translocation events involving the <i>FGFR2</i> locus in three of six assessed patients. These observations and supporting evidence triggered the use of FGFR inhibitors in these patients. In one example, preliminary anti-tumor activity of pazopanib (<i>in vitro</i> FGFR2 IC₅₀≈350 nM) was noted in a patient with an <i>FGFR2-TACC3</i> fusion. After progression on pazopanib, the same patient also had stable disease on ponatinib, a pan-FGFR inhibitor (<i>in vitro</i>, FGFR2 IC₅₀≈8 nM). In an independent non-FGFR2 translocation patient, exome and transcriptome analysis revealed an allele specific somatic nonsense mutation (E384X) in <i>ERRFI1</i>, a direct negative regulator of <i>EGFR</i> activation. Rapid and robust disease regression was noted in this <i>ERRFI1</i> inactivated tumor when treated with erlotinib, an EGFR kinase inhibitor. <i>FGFR2</i> fusions and <i>ERRFI</i> mutations may represent novel targets in sporadic intrahepatic cholangiocarcinoma and trials should be characterized in larger cohorts of patients with these aberrations.</p></div

Fusion events.

<p>Fusion events.</p

Copy number changes and structural rearrangements.

<p>Whole genome data was utilized to determine copy number alterations and structural rearrangements in the genome for Patients 1–5. WGS was not conducted for patient 6. Red indicates copy number gain, green copy number loss and blue lines indicate structural rearrangements. Significant variability between samples was observed for both copy number changes and structural rearrangements. Patient 5 presented with numerous copy number changes and structural rearrangements contrasting with patient 4 who had minimal structural rearrangements and much smaller regions of copy number changes. Patient 3 is characterized by a large number of structural rearrangements with almost no copy number alterations; in contrast, Patient 1 has a moderate number of structural variations, but has large regions of copy number gain and loss. Patient 2 has a moderate number of structural rearrangements with multiple focal amplifications across the genome.</p

Immunohistochemistry demonstrating FGFR2 and FGFR3 expression.

<p><b>A</b>) Tumor stained with FGFR2 antibody. Patient 1 demonstrates moderate cytoplasmic positivity (solid arrows); background fibro-inflammatory tissue is negative (empty arrows). Patient 2 demonstrates moderate cytoplasmic expression for FGFR2; tumor nuclei are negative. Patient 3 demonstrates tumor cells with negative nuclear and weak cytoplasmic expression of FGFR2 (solid arrows) with cells demonstrating moderate basolateral or complete membranous staining as well. Patient 4 demonstrates weak/moderate cytoplasmic positivity with multi-focal weak/moderate membranous expression (solid arrows); background fibro-inflammatory tissue demonstrates negative/weak staining (empty arrows). Patient 5 demonstrates weak/moderate cytoplasmic positivity with multi-focal moderate/strong membranous expression (solid arrows); background fibro-inflammatory tissue is negative (empty arrows). Patient 6 demonstrates moderate/strong cytoplasmic positivity (solid arrows); background lymphocytes are negative (empty arrows). <b>B</b>) Tumor stained with FGFR3 antibody. Patient 1 demonstrates strong cytoplasmic positivity, variable nuclear expression and occasional moderate/strong membranous expression (solid arrows); background fibrous tissue is negative (empty arrows). Patient 2 demonstrates negatively staining background neutrophils (focally intraepithelial-far right) (empty arrows) and tumor cells with strong nuclear expression and moderate cytoplasmic positivity (solid arrows). Patient 3 demonstrates negatively staining background inflammation (empty arrows) and tumor cells with weak nuclear expression and moderate cytoplasmic positivity (solid arrows). Patient 4 demonstrates weak/moderate cytoplasmic positivity and variable nuclear expression; background fibro-inflammatory tissue demonstrates negative/weak positivity (empty arrows). Patient 5 demonstrates moderate cytoplasmic positivity, variable nuclear expression and strong multi-focal membranous expression (solid arrows); background fibrous tissue is negative. Patient 6 demonstrates diffuse/moderate/strong cytoplasmic and membranous positivity and variable nuclear expression (solid arrows); background lymphocytes are negative (empty arrows).</p