Background and objectivesSeveral autoantibodies against proteins located at the node of Ranvier has been identified in patients with chronic inflammatory demyelinating polyneuropathy (CIDP) in the last few years. Then a new concept, autoimmune nodo-paranodopathies was proposed. Cases of Caspr1 autoantibodies are the most rare. Here we describe an anti-Caspr1 nodopathy patient, summarized his clinical, physiological and pathological features. Case presentationWe present the case of a 56-year-old male patient with proprioceptive loss, ataxia, coarse tremor and distal limb weakness without any painess and cranial involvement. Electrophysiological studies showed prolonged distal motor latencies, conduction slowing and reduced amplitude distal compound muscle action potential (CMAP) amplitude. Antibodies against the nodes of Ranvier in serum samples revealed a positive finding for the anti-Caspr1 antibody (1:10).Myelinated fiber loss could be revealed in nerve biopsy. Longitudinal ultrathin sections of the nodal region was discovered in electron microscope, the paranodal/nodal architecture was destructed. It was lack of transverse bands and enlargement of the space between the axon and the paranodal loops was seen. The patient improved obviously after three times immunoadsorption(IA) therapy. ConclusionAnti-Caspr1 nodopathy patient may present atypical symptoms without any neuropathic pain and cranial palsy. The destruction of paranodal/nodal architecture could be observed in nerve biopsy, which may be caused by the lost of axoglial complex formed by NF155, CNTN1 and Caspr1. Antibodies detection is important for the diagnosis, while IA therapy could be regarded as an option for the patients allergic to rituximab (RTX).

Aggressive NK-cell leukemia (ANKL) is a rare form of NK cell neoplasm that is more prevalent among people from Asia and Central and South America. Patients usually die within days to months, even after receiving prompt therapeutic management. Here we performed the first comprehensive study of ANKL by integrating whole genome, transcriptome and targeted sequencing, cytokine array as well as functional assays. Mutations in the JAK-STAT pathway were identified in 48% (14/29) of ANKL patients, while the extracellular STAT3 stimulator IL10 was elevated by an average of 56-fold (P < 0.0001) in the plasma of all patients examined. Additional frequently mutated genes included TP53 (34%), TET2 (28%), CREBBP (21%) and MLL2 (21%). Patient NK leukemia cells showed prominent activation of STAT3 phosphorylation, MYC expression and transcriptional activities in multiple metabolic pathways. Functionally, STAT3 activation and MYC expression were critical for the proliferation and survival of ANKL cells. STAT signaling regulated the MYC transcription program, and both STAT signaling and MYC transcription were required to maintain the activation of nucleotide synthesis and glycolysis. Collectively, the JAK-STAT pathway represents a major target for genomic alterations and IL10 stimulation in ANKL. This newly discovered JAK/STAT-MYC-biosynthesis axis may provide opportunities for the development of novel therapeutic strategies in treating this subtype of leukemia.

Cervical cancer is the third most common cancer and the fourth leading cause of cancer deaths among women in the world. The discovery of vital diagnostic and therapeutic markers against cervical squamous cell carcinoma (CSCC) would broaden our understanding on the molecular basis of CSCC. In this study, we thoroughly analyzed the transcriptome of CSCC and matched adjacent nontumor (ATN) tissue. RNA sequencing was performed to screen the differentially expressed genes (DEGs) of three pairs of CSCC and ATN tissues. Functional enrichment analysis was used to uncover the biological functions of DEGs. Protein interaction network was carried out to reveal interaction of DEGs. Quantitative real-time PCR was conducted to validate the expression of DEGs. Immunohistochemistry was used to detect the relationship between clinicopathological parameters of CSCC and DEGs. There were a total of 347 significantly common DEGs in the three paired examples, including 104 consistent upregulated and 148 consistent downregulated DEGs. The 347 DEGs were categorized into 73 functional categories by Gene Ontology (GO) analysis. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis suggested six significantly signal pathways. The protein interaction network uncovered three important DEGs, including retinol dehydrogenase 12 (RDH12), ubiquitin D (UBD), and serum amyloid A1 (SAA1). We found that RDH12 expression was decreased in 74.5 % of CSCC tissues. RDH12 expression was negatively associated with tumor size and depth of cervical invasion. The UBD was overexpressed in 61.7 % of CSCC tissues and was positively related with tumor size and lymphatic metastasis. The SAA1 protein was overexpressed in 57.4 % of CSCC tissues and was positively related with clinicopathological parameters of tumor size, lymphatic metastasis, and depth of cervical invasion. The RDH12, UBD, and SAA1 genes might participate in the progression of CSCC.

Hematopoietic cells represent an attractive starting cell type for induced pluripotent stem (iPS) cells induction, yet the molecular mechanisms in hematopoietic reprogramming are poorly defined. In this study, we showed that long-term hematopoietic stem cells are more amenable for iPS cells induction among several hematopoietic stem and progenitor cell (HSPC) populations, and that this is accompanied by an earlier induction of the transcriptional program that is involved in the promotion of macromolecule metabolism and cell proliferation. Notably, we identified multiple signaling pathways that exhibited distinct expression patterns in HSPCs compared to that of fibroblasts, which is the most commonly used model for probing the somatic reprogramming process. We further experimentally confirmed the differential requirements of the Wnt/β-catenin and transforming growth factor-beta (TGF-β) signaling pathways in these two cell types. These data demonstrate that hematopoietic cells have a cell-type specific transcriptional program and possess unique signaling requirements in the early phase of reprogramming.

Background: Alternative splicing (AS) is a key regulatory mechanism in protein synthesis and proteome diversity. In this study, we identified alternative splicing events in four pairs of cervical squamous cell carcinoma (CSCC) and adjacent nontumor tissues using RNA sequencing.Methods: The transcripts of the four paired samples were thoroughly analyzed by RNA sequencing. SpliceMap software was used to detect the splicing junctions. Kyoto Encyclopedia of Genes and Genomes pathway analysis was conducted to detect the alternative spliced genes-related signal pathways. The alternative spliced genes were validated by reverse transcription- polymerase chain reaction (RT-PCR).Results: There were 35 common alternative spliced genes in the four CSCC samples; they were novel and CSCC specific. Sixteen pathways were significantly enriched (P<0.05). One novel 5' AS site in the KLHDC7B gene, encoding kelch domain-containing 7B, and an exonskipping site in the SYCP2 gene, encoding synaptonemal complex 2, were validated by RTPCR. The KLHDC7B gene with 5' AS was found in 67.5% (27/ 40) of CSCC samples and was significantly related with cellular differentiation and tumor size. The exon-skipping site of the SYCP2 gene was found in 35.0% (14/ 40) of CSCC samples and was significantly related with depth of cervical invasion.Conclusion: The KLHDC7B and the SYCP2 genes with alternative spliced events might be involved in the development and progression of CSCC and could be used as biomarkers in the diagnosis and prognosis of CSCC.

Acute leukemia characterized by chromosomal rearrangements requires additional molecular disruptions to develop into full-blown malignancy, yet the cooperative mechanisms remain elusive. Using whole-genome sequencing of a pair of monozygotic twins discordant for MLL (also called KMT2A) gene-rearranged leukemia, we identified a transforming MLL-NRIP3 fusion gene and biallelic mutations in SETD2 (encoding a histone H3K36 methyltransferase). Moreover, loss-of-function point mutations in SETD2 were recurrent (6.2%) in 241 patients with acute leukemia and were associated with multiple major chromosomal aberrations. We observed a global loss of H3K36 trimethylation (H3K36me3) in leukemic blasts with mutations in SETD2. In the presence of a genetic lesion, downregulation of SETD2 contributed to both initiation and progression during leukemia development by promoting the self-renewal potential of leukemia stem cells. Therefore, our study provides compelling evidence for SETD2 as a new tumor suppressor. Disruption of the SETD2-H3K36me3 pathway is a distinct epigenetic mechanism for leukemia development.

Aberrant transcriptional programs play a critical role in the development of acute myeloid leukemias (AMLs). Although persistent over-expression of MEIS1 and HOXA9 has been shown to be essential for the initiation and maintenance of MLL-associated leukemia, it is still poorly understood what additional transcriptional regulators, independent of the MLL fusion-driven MEIS/HOX pathway, dictate the development of MLL leukemia. Considering that AMLs with MLL translocation are typically associated with the monocytic lineage (FAB M4 and M5), we explored the potential role of the monocytic lineage-specific transcriptional program in MLL leukemia.

Abstract Abstract 781 While serving as an initiating event in the disease etiology, chromosomal translocation alone may not be sufficient to drive a particular phenotype of leukemia. Acute leukemia characterized by rearrangements of the histone methyltransferase gene MLL (mixed lineage leukemia) requires additional mutations to develop the full blown malignancy, yet the molecular basis of cooperating events remains under-studied. We reasoned that monozygotic (MZ) twin pairs discordant for human leukemia are well-matched for both inherited genetic background and tissue-specific events, and thereby somatic mutations that arise in the disease twin may play a more prominent role in leukemogenesis. Using whole genome sequencing in a pair of 3-year old monozygotic twins discordant for MLL leukemia, we identified a MLL-NRIP3 fusion gene and mutations in histone H3 lysine 36 methyltransferase SETD2 in the leukemia twin. Retrovirus-mediated ectopic expression of MLL-NRIP3 in mouse hematopoietic cells was able to induce the same type of myeloid leukemia as the patient's in a transplant mouse model. A relative delay in disease onset suggested the occurrence of cooperating events in addition to the initial hit of MLL-NRIP3, in the development of induced leukemia. SETD2 mutations were recurrent (5.4%) in 241 acute leukemia patients, particularly in those with MLL-rearranged myeloid leukemia (22.2%). The identified SETD2 mutations are loss-of-function in nature, characterized by biallelic and truncating mutations, and accompanied by a global loss of trimethylation of H3K36 (H3K36me3) in the patient leukemic blasts. These data suggest that SETD2 acts as tumor suppressor gene in leukemia development. Functionally, transfection of SETD2 shRNA in MLL-AF9 knockin bone marrow cells resulted in decreased levels of SETD2 expression and H3K36 trimethylation. Notably, SETD2 knockdown significantly accelerated the development of MLL-AF9 leukemia in the mouse bone marrow transplantation experiment. Moreover, SETD2 knockdown yielded a significantly higher number of total colonies through the second and third rounds of serial replating of colony-forming cell (CFC) assay, suggesting that loss of SETD2 increased the self-renewal and proliferation potential of MLL leukemia-initiating cells. Finally, we showed that SETD2 deficiency was able to activate gene expression in MAPK, Jak-STAT and mTOR signaling, and dysregulates multiple metabolic and DNA repair pathways that are known to directly contribute to leukemogenesis. This comprehensive study provides compelling evidence for SETD2 as a novel tumor suppressor for leukemia, and suggests that the disruption of distinct histone modifying enzymes, MLL and SETD2, synergistically promotes the development of human leukemia. In addition, our study illustrates that whole-genome sequencing of phenotypically discordant monozygotic twins provides an effective approach, in combination with mutational analysis in patients and functional assays using experimental models, to uncover disease-causal genes. Disclosures: No relevant conflicts of interest to declare.

MLL encodes a histone methyltransferase that is critical in maintaining gene expression during embryonic development and hematopoiesis. 11q23 translocations result in the formation of chimeric MLL fusion proteins that act as potent drivers of acute leukemia. However, it remains unclear what portion of the leukemic genome is under the direct control of MLL fusions. By comparing patient-derived leukemic cell lines, we find that MLL fusion-bound genes are a small subset of that recognized by wild-type MLL. In an inducible MLL-ENL model, MLL fusion protein binding and changes in H3K79 methylation are limited to a specific portion of the genome, whereas wild-type MLL distributes to a much larger set of gene loci. Surprisingly, among 223 MLL-ENL-bound genes, only 12 demonstrate a significant increase in mRNA expression on induction of the fusion protein. In addition to Hoxa9 and Meis1, this includes Eya1 and Six1, which comprise a heterodimeric transcription factor important in several developmental pathways. We show that Eya1 has the capacity to immortalize hematopoietic progenitor cells in vitro and collaborates with Six1 in hematopoietic transformation assays. Altogether, our data suggest that MLL fusions contribute to the development of acute leukemia through direct activation of a small set of target genes.

Abstract 3466 Down-regulation of transcription factor PU.1, a key regulator of hematopoiesis, induces myeloid leukemia in mice, demonstrating a role of PU.1 as tumor suppressor. Recent studies, however, have also suggested that PU.1 is required for repopulation/self-renewal capacity of normal hematopoietic stem cells (HSCs), and presence of PU.1 activity may be necessary to favor growth of myeloid leukemia stem cells. To explore whether PU.1 could possibly act as an oncogene in the development of certain type of myeloid leukemia, we set to look for differential up-regulation of PU.1 among AML patients with distinct cytogenetic and genetic alterations in public databases. Consistent with recent molecular studies showing suppression of PU.1 expression by AML1-ETO and PML-RARa fusion proteins, PU.1 expresses at a significant lower level in AML patients with t(8;21) and t(15;17) translocations. In contrast, PU.1 expression level in MLL leukemia patients is significantly higher than that of other subgroups of AML. In addition, we found that a set of PU.1 direct target genes, as defined by genome wide location analysis of this factor, expresses at higher level in MLL leukemia patients comparing with those with t(8;21) and t(15;17) translocations, supporting an increased PU.1 activity in this subgroup of leukemia. In our effort to characterize the functional consequence of high expression of PU.1 in AML, we found that PU.1 plays an essential role in activation of MEIS1 , an oncogene essential for MLL leukemia stem cell potential, and in development of MLL fusion leukemia. MEIS1 , as PU.1 , is differentially up-regulated in MLL leukemia patients, and expresses at a significant lower level in AML patients with t(8;21) and t(15;17) translocations. Among AML patients with higher level MEIS1 expression, a positive correlation was observed between expression of PU.1 and that of MEIS1 . Using promoter reporter assay, electro mobility shift assay (EMSA) and chromatin immunoprecipiation (ChIP) analysis, we found that PU.1 directly binds to and activates MEIS1 promoter in vitro and in vivo . Analysis of a hypomorphic PU.1 mouse model indicated that PU.1 is required to maintain Meis1 expression in murine HSCs and progenitors, and knockdown of PU.1 in patient-derived MLL leukemia cell lines resulted in lower enrichment of PU.1 protein at MEIS1 promoter, accompanied by down-regulation of MEIS1 expression and decreased proliferation and survival of these cells. We are now examining whether the ability of MLL-AF9 fusion protein to drive leukemia is compromised in PU.1-deficient mouse HSC/HPCs, and whether introduction of exogenous Meis1 can compensate for the loss of PU.1 in the development of MLL-AF9 leukemia in mouse bone marrow transplantation model. Finally, we are also testing knock-down of PU.1 as a therapeutic approach to primary AMLs isolated from MLL leukemia patients. Collectively, our data indicate that PU.1 is required for the pathogenesis of MLL associated leukemia, at least partially, through direct activation of MEIS1 . In veiw of the dependency of MEIS1 in MLL leukemic transformation, targeting PU.1 mediated MEIS1 gene activation could be an alternative or synergistic approach for MLL leukemia therapies aimed at inhibition of DOT1L and HOXA9. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 2507 The MLL (mixed-lineage leukemia) gene encodes a histone methyltransferase that is critical in maintaining gene expression during hematopoiesis. Chromosomal translocations disrupting MLL often leads to the creation of MLL fusion genes that act as potent drivers of acute leukemia. MLL fusion proteins are oncogenic transcription factors that activate the expression of downstream target genes. Expression profiling on patient primary samples and established mouse models has revealed hundreds of protein coding genes which are either up-regulated or down-regulated in MLL associated leukemias. Persistent coexpression of two of those genes, HoxA9 and Meis1, is essential for the initiation and maintenance of MLL leukemia. Our studies have also shown strong association of a microRNA (miRNA) expression signature with MLL- rearranged leukemia, and the expression of several miRNAs were under the control of MLL wild type and fusion proteins. Although profiling of miRNA expression has been reported, the mechanisms underlying deregulated miRNA expression in MLL associated leukemia are poorly understood. Given the role of miRNA as a global suppressor of mRNA gene expression, we hypothesized that the expression of miRNAs could be directly activated by MLL fusion and/or wild type proteins upon MLL gene rearrangement and subsequently down-regulate pertinent target mRNAs to contribute to leukemogenesis. To test our hypothesis in a systematic way, we examined an inducible MLL-ENL-ER transformed mouse cell line, which grow as myeloblastic cells in the presence of MLL-ENL, and differentiate into neutrophils upon inactivation of the fusion protein. Using chromatin immunoprecipitation assay followed by next generation sequencing (ChIP-Seq), we determined whole genome MLL binding pattern in this cellular model. Upon activation of MLL-ENL, 24 miRNAs showed a significant increase in the level of MLL binding (FDR<0.25), suggesting that those genes are directly bound by the MLL-ENL fusion protein. To explore the impact of MLL fusion protein on miRNA and mRNA gene regulation, we performed whole genome expression analysis using Affymetrix mouse microarray in the presence or absence of MLL-ENL. Upon induction of MLL-ENL, the expression levels of 38 miRNAs (out of 609 tiled on the array) were increased, and 57 of 7858 expressed protein-coding genes were down-regulated. An integrative analysis of MLL binding and mRNA/miRNA expression profiling data showed that transcription of three miRNAs were activated upon binding of MLL-ENL, and ten protein coding genes are potential targets of these miRNAs. We are currently exploring the role of these three miRNAs and their respective mRNA target genes in leukemogenesis using in vitro and in vivo models. Taken together, our data suggest that MLL fusion protein may play an important role in leukemogenesis by promoting miRNA transcription, which subsequently inhibit the expression of critical mRNA target genes. Our study provides a basis to further explore the regulatory network involving MLL fusion protein and its key miRNA target genes in the leukemic genome. Disclosures: No relevant conflicts of interest to declare.