Highlights d LSD1 represses master epidermal transcription factors that promote differentiation d LSD1 inhibition activates the epidermal differentiation transcriptional program d LSD1 inhibition represses invasion in a model of cutaneous squamous cell carcinoma

Hematopoietic stem cell (HSC) homeostasis is controlled by cytokine receptor–mediated Janus kinase 2 (JAK2) signaling. We previously found that JAK2 is promptly ubiquitinated upon cytokine stimulation. Whether a competing JAK2 deubiquitination activity exists is unknown. LNK is an essential adaptor protein that constrains HSC expansion through dampening thrombopoietin (TPO)–induced JAK2 signaling. We show here that a LNK-associated lysine-63 (K63)–deubiquitinating enzyme complex, Brcc36 isopeptidase complex (BRISC), attenuates HSC expansion through control of JAK2 signaling. We pinpoint a direct interaction between the LNK SH2 domain and a phosphorylated tyrosine residue in KIAA0157 (Abraxas2), a unique and defining BRISC component. Kiaa0157 deficiency in mice led to an expansion of phenotypic and functional HSCs. Endogenous JAK2 and phospho-JAK2 were rapidly K63-ubiquitinated upon TPO stimulation, and this action was augmented in cells depleted of the BRISC core components KIAA0157, MERIT40, or BRCC36. This increase in JAK2 ubiquitination after BRISC knockdown was associated with increased TPO-mediated JAK2 activation and protein levels, and increased MPL receptor presence at the cell surface. In addition, BRISC depletion promoted membrane proximal association between the MPL receptor and pJAK2/JAK2, thus enhancing activated JAK2/MPL at the cell membrane. These findings define a novel pathway by which K63-ubiquitination promotes JAK2 stability and activation in a proteasome-independent manner. Moreover, mutations in BRCC36 are found in clonal hematopoiesis in humans. This research may shed light on the mechanistic understanding of a potential role of BRCC36 in human HSCs.

Neuroblastoma is a malignancy of the developing sympathetic nervous system that accounts for 12% of childhood cancer deaths. Like many childhood cancers, neuroblastoma shows a relative paucity of somatic single-nucleotide variants (SNVs) and small insertions and deletions (indels) compared to adult cancers. Here, we assessed the contribution of somatic structural variation (SV) in neuroblastoma using a combination of whole-genome sequencing (WGS) of tumor-normal pairs (n = 135) and single-nucleotide polymorphism (SNP) genotyping of primary tumors (n = 914). Our study design allowed for orthogonal validation and replication across platforms. SV frequency, type, and localization varied significantly among highrisk tumors. MYCN nonamplified high-risk tumors harbored an increased SV burden overall, including a significant excess of tandem duplication events across the genome. Genes disrupted by SV breakpoints were enriched in neuronal lineages and associated with phenotypes such as autism spectrum disorder (ASD). The postsynaptic adapter protein-coding gene, SHANK2, located on Chromosome 11q13, was disrupted by SVs in 14% of MYCN nonamplified high-risk tumors based on WGS and 10% in the SNP array cohort. Expression of SHANK2 was low across human-derived neuroblastoma cell lines and high-risk neuroblastoma tumors. Forced expression of SHANK2 in neuroblastoma cells resulted in significant growth inhibition (P = 2.6 × 10 −2 to 3.4 × 10 −5) and accelerated neuronal differentiation following treatment with all-trans retinoic acid (P = 3.1 × 10 −13 to 2.4 × 10 −30). These data further define the complex landscape of somatic structural variation in neuroblastoma and suggest that events leading to deregulation of neurodevelopmental processes, such as inactivation of SHANK2, are key mediators of tumorigenesis in this childhood cancer.

Neuroblastoma, like many childhood cancers, exhibits a relative paucity of somatic single nucleotide variants (SNVs). Here, we assess the contribution of structural variation (SV) in neuroblastoma using a combination of whole genome sequencing (WGS; n=135) and single nucleotide polymorphism (SNP) genotyping (n=914) of matched tumor-normal pairs. Our study design provided means for orthogonal validation of SVs as well as validation across genomic platforms. SV frequency, type, and localization varied significantly among high-risk tumors, with MYCN non-amplified tumors harboring an increased SV burden overall (P=1.12x10 -5 ). Genes disrupted by SV breakpoints were enriched in neuronal lineages and autism spectrum disorder. The postsynaptic adapter protein-coding gene SHANK2, located on chromosome 11q13, was disrupted by SVs in 14% and 10% of MYCN non-amplified highrisk tumors based on WGS and SNP array cohorts, respectively. Forced expression of SHANK2 in neuroblastoma cell models resulted in significant growth inhibition (P=2.62x10 -2 to 3.4x10 -5 ) and accelerated neuronal differentiation following treatment with all-trans retinoic acid (P=3.08x10 -13 to 2.38x10 -30 ). These data further define the complex landscape of structural variation in neuroblastoma and suggest that events leading to deregulation of neurodevelopmental processes, such as inactivation of SHANK2, are key mediators of tumorigenesis.Neuroblastoma is a cancer of the developing sympathetic nervous system that most commonly affects children under 5 years of age, with a median age at diagnosis of 17 months 1 . Approximately 50% of cases present with disseminated disease at the time of diagnosis, and despite intense multi-modal therapy, the survival rate for this high-risk subset remains less than 50% 1 . Recent whole genome and exome sequencing studies of neuroblastoma have revealed relatively few recurrent protein-coding somatic mutations including single nucleotide variations (SNVs) and small (<50b) insertion/deletions (indels) 2-5 .Large-scale structural variations (SVs) such as deletions, insertions, inversions, tandem duplications and translocations can arise from mutational processes that alter chromosome structure and evade innate mechanisms of maintaining genomic stability. These diverse SVs are commonly acquired somatically and act as driver mutations 6 .A plethora of approaches have been applied to detect SVs across large cancer datasets 6-9 .First, methods that identify copy number variations (CNVs) can be applied to intensity log R ratios from genotyping and comparative genomic hybridization (CGH) arrays as well as read-depth measures from next generation sequencing. Different segmentation algorithms have been applied to either platform in order to obtain copy number gain and loss calls, which range from a few hundred base-pair size to whole chromosomal alterations. These methods are dosage sensitive, allowing numerical quantification of amplifications and homozygous deletions. Analysis of CNVs in neuroblastoma primary tumor and matched...