UCH-L1 bypasses mTOR to promote protein biosynthesis and is required for MYC-driven lymphomagenesis in mice.

The mechanistic target of rapamycin (mTOR) is a central regulator of cellular proliferation and metabolism. Depending on its binding partners, mTOR is at the core of 2 complexes that either promote protein biosynthesis (mTOR complex 1; mTORC1) or provide survival and proliferation signals (mTORC2). Protein biosynthesis downstream of mTORC1 plays an important role in MYC-driven oncogenesis with translation inhibitors garnering increasing therapeutic attention. The germinal center B-cell oncogene UCHL1 encodes a deubiquitinating enzyme that regulates the balance between mTOR complexes by disrupting mTORC1 and promoting mTORC2 assembly. While supporting mTORC2-dependent growth and survival signals may contribute to its role in cancer, the suppression of mTORC1 activity is enigmatic, as its phosphorylation of its substrate 4EBP1 promotes protein biosynthesis. To address this, we used proximity-based proteomics to identify molecular complexes with which UCH-L1 associates in malignant B cells. We identified a novel association of UCH-L1 with the translation initiation complex eIF4F, the target of 4EBP1. UCH-L1 associates with and promotes the assembly of eIF4F and stimulates protein synthesis through a mechanism that requires its catalytic activity. Because of the importance of mTOR in MYC-driven oncogenesis, we used novel mutant Uchl1 transgenic mice and found that catalytic activity is required for its acceleration of lymphoma in the Eµ-myc model. Further, we demonstrate that mice lacking UCH-L1 are resistant to MYC-induced lymphomas. We conclude that UCH-L1 bypasses the need for mTORC1-dependent protein synthesis by directly promoting translation initiation, and that this mechanism may be essential for MYC in B-cell malignancy.

Lymphoma diagnostics: getting more from less.

In the modern era, clinicians and pathologists increasingly make challenging diagnoses in patients with suspected lymphoma using minimal amounts of diagnostic material. The increase in utilization of minimally invasive procedures, such as fine needle aspiration or needle core biopsies, lead to challenges in our ability to make accurate histopathological assessments of disease, including the integration of new diagnostic and prognostic testing, with smaller amounts of material. The trend towards minimally invasive diagnostics is also often in conflicting interest with researchers seeking to study tissue specimens to better understand the biology and genetics of these diseases to move the field forward. Thankfully, there are emerging fields which seek to extract large amounts of diagnostic and prognostic data out of material that is circulating in the blood of patients with lymphoma. Here we will review recent exciting data regarding the use of circulating tumour cells, circulating tumour DNA, and the detection and utility of circulating exosomes and how it can assist in diagnosis, prognosis and therapeutic monitoring. These advances hold the promise to enable continued safe patient care while also advancing discovery, translational and clinical research.

UCH-L1 is induced in germinal center B cells and identifies patients with aggressive germinal center diffuse large B-cell lymphoma.

Gene expression profiling has identified 2 major subclasses of diffuse large B-cell lymphoma (DLBCL). Cases resembling germinal center (GC) B cells (GCB-DLBCL) generally occur in younger patients, have a distinct molecular pathophysiology, and have improved outcomes compared with those similar to activated post-GC cells (activated B-cell DLBCL). We previously found that the ubiquitin hydrolase UCH-L1 is frequently overexpressed in mature B-cell malignancies and is a potent oncogene in mice. The cause for its overexpression in lymphoma, and whether it impacts the outcome of patients with DLBCL is unknown. Here, we show that UCH-L1 reflects GC lineage in lymphoma and is an oncogenic biomarker of aggressive GCB-DLBCL. We find that UCH-L1 is specifically induced in GC B cells in mice and humans, and that its expression correlates highly with the GCB subtype in DLBCL. We also find that UCH-L1 cooperates with BCL6 in a mouse model of GC B-cell lymphoma, but not with the development of multiple myeloma derived from post-GC cells. Despite the typically good outcomes of GCB-DLBCL, increased UCHL1 identifies a subgroup with early relapses independent of MYC expression, suggesting biological diversity in this subset of disease. Consistent with this, forced Uchl1 overexpression had a substantial impact on gene expression in GC B cells including pathways of cell cycle progression, cell death and proliferation, and DNA replication. These data demonstrate a novel role for UCH-L1 outside of the nervous system and suggest its potential use as a biomarker and therapeutic target in DLBCL.

Targeting childhood, adolescent and young adult non-Hodgkin lymphoma: therapeutic horizons.

Non-Hodgkin lymphoma (NHL) is the third most common malignancy in children, adolescents and young adults (CAYA). NHL is a diverse set of diseases that arise at key regulatory checkpoints during B or T cell development in the bone marrow, germinal centre or thymus. While advances in the use of conventional cytotoxic agents have led to dramatic improvements in survival, these cures are associated with significant acute and long-term toxicities. Moreover, the prognosis for CAYA patients with relapsed or refractory NHL remains dismal, with the vast majority dying of their disease. Thanks to a large number of candidate-based biological studies, together with large-scale sequencing efforts, there has been an explosion of knowledge regarding the molecular pathophysiology of B- and T-NHL. This has ushered development of a flurry of novel therapeutic approaches that may simultaneously provide new hope for relapsed patients and an opportunity to reduce the therapeutic burden in newly diagnosed CAYA. Here we review a selection of the most promising new therapeutic approaches to these diseases. While the vast majority of these agents are untested in children, on-going work from many cooperative groups will soon explore their use in paediatric disease, in hope of further improving outcomes while maximizing quality of life.

USP44 overexpression drives a MYC-like gene expression program in neuroblastoma through epigenetic reprogramming.

Neuroblastoma is an embryonic cancer that contributes disproportionately to death in young children. Sequencing data have uncovered few recurrently mutated genes in this cancer, although epigenetic pathways have been implicated in disease pathogenesis. We used an expression-based computational screen that examined the impact of deubiquitinating enzymes on patient survival to identify potential new targets. We identified the histone H2B deubiquitinating enzyme USP44 as the enzyme with the greatest impact on survival in patients with neuroblastoma. High levels of USP44 significantly correlate with metastatic disease, unfavorable histology, advanced patient age, and MYCN-amplification. The subset of patients with tumors expressing high levels of USP44 had a significantly worse survival, including those with tumors lacking MYCN amplification. We showed experimentally that USP44 regulates neuroblastoma cell proliferation, migration, invasion, and neuronal development. Depletion of the histone H2B ubiquitin ligase subunit RNF20 resulted in similar findings, strongly implicating this histone mark as the target of USP44 activity in this disease. Integration of transcriptome and epigenome in analyses demonstrates a distinct set of genes that is regulated by USP44, including those in Hallmark MYC target genes in both murine embryonic fibroblasts and the SH-SY5Y neuroblastoma cell line. We conclude that USP44 is a novel epigenetic regulator that promotes aggressive features and may be a novel target in neuroblastoma. Implications: This study identifies a new genetic marker of aggressive neuroblastoma and identifies the mechanisms by which its overactivity contributes to pathophysiology in this disease.

Leucine biosynthesis regulates cytoplasmic iron-sulfur enzyme biogenesis in an Atm1p-independent manner.

Fe-S clusters (ISCs) are versatile cofactors utilized by many mitochondrial, cytoplasmic, and nuclear enzymes. Whereas mitochondria can independently initiate and complete ISC synthesis, other cellular compartments are believed to assemble ISCs from putative precursors exported from the mitochondria via an ATP binding cassette (ABC) transporter conserved from yeast (Atm1p) to humans (ABCB7). However, the regulatory interactions between mitochondrial and extramitochondrial ISC synthesis are largely unknown. In yeast, we found that mitochondrial ISC synthesis is regulated by the leucine biosynthetic pathway, which among other proteins involves an abundant cytoplasmic [4Fe-4S] enzyme, Leu1p. Enzymatic blocks in the pathway (i.e. leu1Δ or leu2Δ gene deletion mutations) induced post-transcriptional up-regulation of core components of mitochondrial ISC biosynthesis (i.e. the sulfur donor Nfs1p, the iron donor Yfh1p, and the ISC scaffold Isu1p). In leu2Δ cells, transcriptional mechanisms also led to dramatic up-regulation of Leu1p with concomitant down-regulation of mitochondrial aconitase (Aco1p), a [4Fe-4S] enzyme in the tricarboxylic acid cycle. Accordingly, the leu2Δ deletion mutation exacerbated Aco1p inactivation in cells with mutations in Yfh1p. These data indicate that defects in leucine biosynthesis promote the biogenesis of enzymatically active Leu1p at the expense of Aco1p activity. Surprisingly, this effect is independent of Atm1p; previous reports linking the loss of Leu1p activity to Atm1p depletion were confounded by the fact that LEU2 was used as a selectable marker to create Atm1p-depleted cells, whereas a leu2Δ allele was present in Atm1p-repleted controls. Thus, still largely unknown transcriptional and post-transcriptional mechanisms control ISC distribution between mitochondria and other cellular compartments.

Normal and Friedreich ataxia cells express different isoforms of frataxin with complementary roles in iron-sulfur cluster assembly.

Friedreich ataxia (FRDA) is an autosomal recessive degenerative disease caused by insufficient expression of frataxin (FXN), a mitochondrial iron-binding protein required for Fe-S cluster assembly. The development of treatments to increase FXN levels in FRDA requires elucidation of the steps involved in the biogenesis of functional FXN. The FXN mRNA is translated to a precursor polypeptide that is transported to the mitochondrial matrix and processed to at least two forms, FXN(42-210) and FXN(81-210). Previous reports suggested that FXN(42-210) is a transient processing intermediate, whereas FXN(81-210) represents the mature protein. However, we find that both FXN(42-210) and FXN(81-210) are present in control cell lines and tissues at steady-state, and that FXN(42-210) is consistently more depleted than FXN(81-210) in samples from FRDA patients. Moreover, FXN(42-210) and FXN(81-210) have strikingly different biochemical properties. A shorter N terminus correlates with monomeric configuration, labile iron binding, and dynamic contacts with components of the Fe-S cluster biosynthetic machinery, i.e. the sulfur donor complex NFS1·ISD11 and the scaffold ISCU. Conversely, a longer N terminus correlates with the ability to oligomerize, store iron, and form stable contacts with NFS1·ISD11 and ISCU. Monomeric FXN(81-210) donates Fe(2+) for Fe-S cluster assembly on ISCU, whereas oligomeric FXN(42-210) donates either Fe(2+) or Fe(3+). These functionally distinct FXN isoforms seem capable to ensure incremental rates of Fe-S cluster synthesis from different mitochondrial iron pools. We suggest that the levels of both isoforms are relevant to FRDA pathophysiology and that the FXN(81-210)/FXN(42-210) molar ratio should provide a useful parameter to optimize FXN augmentation and replacement therapies.

USP24 Is a Cancer-Associated Ubiquitin Hydrolase, Novel Tumor Suppressor, and Chromosome Instability Gene Deleted in Neuroblastoma.

Deubiquitinating enzymes are increasingly recognized to play important roles in cancer, with many acting as oncogenes or tumor suppressors. In this study, we employed a bioinformatics approach to screen for enzymes from this family involved in cancer and found USP24 as a potent predictor of poor outcomes in neuroblastoma, an aggressive childhood cancer. USP24 resides in a region commonly deleted in neuroblastoma, yet was independently associated with poor outcomes in this disease. Deletion of Usp24 in a murine model resulted in degradation of collapsin response mediator protein 2 (CRMP2), a regulator of axon growth, guidance, and neuronal polarity. Cells lacking USP24 had significant increases in spindle defects, chromosome missegregation, and aneuploidy, phenotypes that were rescued by the restoration of CRMP2. USP24 prevented aneuploidy by maintaining spindle-associated CRMP2, which is required for mitotic accuracy. Our findings further indicate that USP24 is a tumor suppressor that may play an important role in the pathogenesis of neuroblastoma. SIGNIFICANCE: This study identifies the chromosome instability gene USP24 as frequently deleted in neuroblastoma and provides important insight into the pathogenesis of this aggressive childhood cancer.

A cysteine near the C-terminus of UCH-L1 is dispensable for catalytic activity but is required to promote AKT phosphorylation, eIF4F assembly, and malignant B-cell survival.

The enzyme UCH-L1 is a neuro-endocrine and germinal center B-cell marker that contributes to the development and aggressive behavior of mature B-cell malignancies. While mutations in this enzyme have been associated with Parkinson's disease, relatively little is known about the molecular features associated with the biochemical activities of UCH-L1. Here we use a survival-based complementation assay and site-directed mutagenesis and identify a novel role for the C-terminus of UCH-L1 in supporting cell survival. The C220 residue is required for UCH-L1 to promote the assembly of mTOR complex 2 and phosphorylation of the pro-survival kinase AKT. While this residue was previously described as a potential farnesylation site, destruction of the putative CAAX motif by adding a C-terminal epitope tag did not interfere with cell survival, indicating an alternate mechanism. We used proximity-based proteomics comparing the proteomes of wild-type and C220S UCH-L1 and identified a selective loss of association with RNA-binding proteins including components of the translation initiation machinery. As a consequence, the C220S mutant did not promote the assembly of the eIF4F complex. These data identify a novel role for the C-terminus of UCH-L1 in supporting pro-survival and metabolic activities in malignant B-cells. This finding may lead to the development of therapeutics with selective activity towards malignancy that potentially avoid neuronal toxicities.

UCHL1 is a biomarker of aggressive multiple myeloma required for disease progression.

The success of proteasome inhibition in multiple myeloma highlights the critical role for the ubiquitin-proteasome system (UPS) in this disease. However, there has been little progress in finding more specific targets within the UPS involved in myeloma pathogenesis. We previously found the ubiquitin hydrolase UCH-L1 to be frequently over-expressed in B-cell malignancies, including myeloma, and showed it to be a potent oncogene in mice. Here we show that UCH-L1 is a poor prognostic factor that is essential for the progression of myeloma. We found high levels of UCHL1 to predict early progression in newly diagnosed patients; a finding reversed by the inclusion of bortezomib. We also found high UCHL1 levels to be a critical factor in the superiority of bortezomib over high-dose dexamethasone in relapsed patients. High UCHL1 partially overlaps with, but is distinct from, known genetic risks including 4p16 rearrangement and 1q21 amplification. Using an orthotopic mouse model, we found UCH-L1 depletion delays myeloma dissemination and causes regression of established disease. We conclude that UCH-L1 is a biomarker of aggressive myeloma that may be an important marker of bortezomib response, and may itself be an effective target in disseminated disease.

Partial conservation of functions between eukaryotic frataxin and the Escherichia coli frataxin homolog CyaY.

Frataxin is a mitochondrial protein structurally conserved from bacteria to humans. Eukaryotic frataxins are known to be involved in the maintenance of mitochondrial iron balance via roles in iron delivery and iron detoxification. The prokaryotic frataxin homolog, CyaY, has been shown to bind and donate iron for the assembly of [2Fe-2S] clusters in vitro. However, in contrast to the severe phenotypes associated with the partial or complete loss of frataxin in humans and other eukaryotes, deletion of the cyaY gene does not cause any obvious alteration of iron balance in bacterial cells, an effect that probably reflects functional redundancy between CyaY and other bacterial proteins. To study CyaY function in a nonredundant setting, we have expressed a mitochondria-targeted form of CyaY in a Saccharomyces cerevisiae strain depleted of the endogenous yeast frataxin protein (yfh1Delta). We show that in this strain CyaY complements to a large extent the loss of iron-sulfur cluster enzyme activities and heme synthesis, and thereby maintains a nearly normal respiratory growth. In addition, CyaY effectively protects yfh1Delta from oxidative damage during treatment with hydrogen peroxide but is less efficient in detoxifying excess labile iron during aerobic growth.

Biogenesis of cytosolic ribosomes requires the essential iron-sulphur protein Rli1p and mitochondria.

Mitochondria perform a central function in the biogenesis of cellular iron-sulphur (Fe/S) proteins. It is unknown to date why this biosynthetic pathway is indispensable for life, the more so as no essential mitochondrial Fe/S proteins are known. Here, we show that the soluble ATP-binding cassette (ABC) protein Rli1p carries N-terminal Fe/S clusters that require the mitochondrial and cytosolic Fe/S protein biogenesis machineries for assembly. Mutations in critical cysteine residues of Rli1p abolish association with Fe/S clusters and lead to loss of cell viability. Hence, the essential character of Fe/S clusters in Rli1p explains the indispensable character of mitochondria in eukaryotes. We further report that Rli1p is associated with ribosomes and with Hcr1p, a protein involved in rRNA processing and translation initiation. Depletion of Rli1p causes a nuclear export defect of the small and large ribosomal subunits and subsequently a translational arrest. Thus, ribosome biogenesis and function are intimately linked to the crucial role of mitochondria in the maturation of the essential Fe/S protein Rli1p.