IgM paraprotein-associated peripheral neuropathy: small CD20-positive B-cell clones may predict a monoclonal gammopathy of neurological significance and rituximab responsiveness

IgM paraprotein‐associated peripheral neuropathy (PN) in patients without overt evidence of lymphoma is a recognised clinical entity of unknown aetiology. Interrogating the bone marrow B‐cell or plasma cell clones underlying paraproteinemic neuropathies may improve our understanding of both pathogenesis and treatment options. This retrospective observational analysis of IgM paraprotein‐associated PN identified five patients with small pathological MYD88 L265P and CD20‐positive B‐cell clones in their bone marrow using multi‐parametric flow cytometry, who have shown durable neurological response to rituximab. We posit that multi‐parametric flow cytometry may be instrumental in identifying the cellular source of the paraprotein in IgM paraprotein‐associated PN, and thus directing appropriate immunomodulatory therapy. Further understanding of these small pathological B‐cell clones may also provide additional insight into mechanisms of monoclonal gammopathy of clinical significance overall

Allele-specific transcriptional elongation regulates monoallelic expression of the IGF2BP1 gene

<p>Abstract</p> <p>Background</p> <p>Random monoallelic expression contributes to phenotypic variation of cells and organisms. However, the epigenetic mechanisms by which individual alleles are randomly selected for expression are not known. Taking cues from chromatin signatures at imprinted gene loci such as the insulin-like growth factor 2 gene 2 (<it>IGF2</it>), we evaluated the contribution of CTCF, a zinc finger protein required for parent-of-origin-specific expression of the <it>IGF2 </it>gene, as well as a role for allele-specific association with DNA methylation, histone modification and RNA polymerase II.</p> <p>Results</p> <p>Using array-based chromatin immunoprecipitation, we identified 293 genomic loci that are associated with both CTCF and histone H3 trimethylated at lysine 9 (H3K9me3). A comparison of their genomic positions with those of previously published monoallelically expressed genes revealed no significant overlap between allele-specifically expressed genes and colocalized CTCF/H3K9me3. To analyze the contributions of CTCF and H3K9me3 to gene regulation in more detail, we focused on the monoallelically expressed <it>IGF2BP1 </it>gene. <it>In vitro </it>binding assays using the CTCF target motif at the <it>IGF2BP1 </it>gene, as well as allele-specific analysis of cytosine methylation and CTCF binding, revealed that CTCF does not regulate mono- or biallelic <it>IGF2BP1 </it>expression. Surprisingly, we found that RNA polymerase II is detected on both the maternal and paternal alleles in B lymphoblasts that express <it>IGF2BP1 </it>primarily from one allele. Thus, allele-specific control of RNA polymerase II elongation regulates the allelic bias of <it>IGF2BP1 </it>gene expression.</p> <p>Conclusions</p> <p>Colocalization of CTCF and H3K9me3 does not represent a reliable chromatin signature indicative of monoallelic expression. Moreover, association of individual alleles with both active (H3K4me3) and silent (H3K27me3) chromatin modifications (allelic bivalent chromatin) or with RNA polymerase II also fails to identify monoallelically expressed gene loci. The selection of individual alleles for expression occurs in part during transcription elongation.</p

Editing of hnRNP K protein mRNA in colorectal adenocarcinoma and surrounding mucosa

The heterogeneous nuclear ribonucleoprotein K (hnRNP K) protein is an RNA-binding protein involved in many processes that compose gene expression. K protein is upregulated in the malignant processes and has been shown to modulate the expression of genes involved in mitogenic responses and tumorigenesis. To explore the possibility that there are alternative isoforms of K protein expressed in colon cancer, we amplified and sequenced K protein mRNA that was isolated from colorectal cancers as well as from normal tissues surrounding the tumours. Sequencing revealed a single G-to-A base substitution at position 274 that was found in tumours and surrounding mucosa, but not in individuals that had no colorectal tumour. This substitution most likely reflects an RNA editing event because it was not found in the corresponding genomic DNAs. Sequencing of RNA from normal colonic mucosa of patients with prior resection of colorectal cancer revealed only the wild-type K protein transcript, indicating that G274A isoform is tumour related. To our knowledge, this is the first example of an RNA editing event in cancer and its surrounding tissue, a finding that may offer a new diagnostic and treatment marker

Heterogeneous nuclear ribonucleoprotein K is over expressed, aberrantly localised and is associated with poor prognosis in colorectal cancer

Heterogeneous ribonucleoprotein K (hnRNP K) is a member of the hnRNP family which has several different cellular roles including transcription, mRNA shuttling, RNA editing and translation. Several reports implicate hnRNP K having a role in tumorigenesis, for instance hnRNP K increases transcription of the oncogene c-myc and hnRNP K expression is regulated by the p53/MDM 2 pathway. In this study comparing normal colon to colorectal cancer by proteomics, hnRNP K was identified as being overexpressed in this type of cancer. Immunohistochemistry with a monoclonal antibody to hnRNP K (which we developed) on colorectal cancer tissue microarray, confirmed that hnRNP K was overexpressed in colorectal cancer (P<0.001) and also showed that hnRNP K had an aberrant subcellular localisation in cancer cells. In normal colon hnRNP K was exclusively nuclear whereas in colorectal cancer the protein localised both in the cytoplasm and the nucleus. There were significant increases in both nuclear (P=0.007) and cytoplasmic (P=0.001) expression of hnRNP K in Dukes C tumours compared with early stage tumours. In Dukes C patient's good survival was associated with increased hnRNP K nuclear expression (P=0.0093). To elaborate on the recent observation that hnRNP K is regulated by p53, the expression profiles of these two proteins were also analysed. There was no correlation between hnRNP K and p53 expression, however, patients who presented tumours that were positive for hnRNP K and p53 had a poorer survival outcome (P=0.045)

Comprehensive Analysis of the Palindromic Motif TCTCGCGAGA: A Regulatory Element of the HNRNPK Promoter

Definitive identification of promoters, their cis-regulatory motifs, and their trans-acting proteins requires experimental analysis. To define the HNRNPK promoter and its cognate DNA–protein interactions, we performed a comprehensive study combining experimental approaches, including luciferase reporter gene assays, chromatin immunoprecipitations (ChIP), electrophoretic mobility shift assays (EMSA), and mass spectrometry (MS). We discovered that out of the four potential HNRNPK promoters tested, the one containing the palindromic motif TCTCGCGAGA exhibited the highest activity in a reporter system assay. Although further EMSA and MS analyses, performed to uncover the identity of the palindrome-binding transcription factor, did identify a complex of DNA-binding proteins, neither method unambiguously identified the pertinent direct trans-acting protein(s). ChIP revealed similar chromatin states at the promoters with the palindromic motif and at housekeeping gene promoters. A ChIP survey showed significantly higher recruitment of PARP1, a protein identified by MS as ubiquitously attached to DNA probes, within heterochromatin sites. Computational analyses indicated that this palindrome displays features that mark nucleosome boundaries, causing the surrounding DNA landscape to be constitutively open. Our strategy of diverse approaches facilitated the direct characterization of various molecular properties of HNRNPK promoter bearing the palindromic motif TCTCGCGAGA, despite the obstacles that accompany in vitro methods

Heterogeneous nuclear ribonucleoprotein K: altered pattern of expression associated with diagnosis and prognosis of prostate cancer

Using proteomic analysis of the nuclear matrix (NM), we found that heterogeneous nuclear ribonucleoprotein K (hnRNP K), a member of the hnRNP family with pleiotropic functions, was differentially expressed in prostate cancer (PCa) tissues. This study aimed to characterise the expression of hnRNP K and its subcellular localisation in PCa, utilising immunohistochemical and quantitative western blot techniques. Furthermore, the hnRNP K expression was studied in human PCa cell lines in order to determine its modulation by bicalutamide, the anti-androgen widely used in PCa therapy. Immunohistochemical staining of paraffin-embedded tissues showed that hnRNP K was overexpressed in PCa, where it was localised both in the cytoplasm and in the nucleus. Staining of non-tumour tissues showed exclusively nuclear localisation and a less intense or absent signal. Immunoblot analysis demonstrated that the hnRNP K level within the NM was higher in PCa compared with non-tumour tissues and closely correlated with Gleason score (P=0.008). Higher expression within the NM was significantly (P=0.032) associated with poor prognosis. In two-dimensional western blot analysis hnRNP K presented several isoforms; the one with pI 5.1 was the most differently expressed between non-tumour and PCa tissues. Preliminary results indicate that hnRNP K can be modulated in vitro by a non-steroidal anti-androgen. Taken together, our findings suggest that hnRNP K has potential implications at the diagnostic, prognostic and therapeutic levels in PCa

A prognostic index predicting survival in transformed Waldenström macroglobulinemia

Histological transformation into diffuse large B-cell lymphoma is a rare complication in patients with Waldenström macroglobulinemia (WM) usually associated with a poor prognosis. The objective of this study was to develop and validate a prognostic index for survival in transformed WM patients. Through this multicenter, international collaborative effort, we developed a scoring system based on data from 133 patients with transformed WM who were evaluated between 1995 and 2016 (training cohort). Univariate and multivariate analyses were used to propose a prognostic index with 2-year survival after transformation as an end-point. For external validation, a data set of 67 patients was used to evaluate the performance of the model (validation cohort). By multivariate analysis, three adverse covariates were identified as independent predictors of 2-year survival after transformation: elevated serum LDH (2 points), platelet count < 100 x 109/L (1 point) and any previous treatment for WM (1 point). Three risk groups were defined: low-risk (0-1 point, 24% of patients), intermediate-risk (2-3 points, 59%, hazard ratio (HR) = 3.4) and high-risk (4 points, 17%, HR = 7.5). Two-year survival rates were 81%, 47%, and 21%, respectively (P < 0.0001). This model appeared to be a better discriminant than the International Prognostic Index (IPI) and the revised IPI (R-IPI). We validated this model in an independent cohort. This easy-to-compute scoring index is a robust tool that may allow identification of groups of transformed WM patients with different outcomes and could be used for improving the development of risk-adapted treatment strategies

Heterogeneous Nuclear Ribonucleoprotein K Interacts with Abi-1 at Postsynaptic Sites and Modulates Dendritic Spine Morphology

BACKGROUND: Abelson-interacting protein 1 (Abi-1) plays an important role for dendritic branching and synapse formation in the central nervous system. It is localized at the postsynaptic density (PSD) and rapidly translocates to the nucleus upon synaptic stimulation. At PSDs Abi-1 is in a complex with several other proteins including WASP/WAVE or cortactin thereby regulating the actin cytoskeleton via the Arp 2/3 complex. PRINCIPAL FINDINGS: We identified heterogeneous nuclear ribonucleoprotein K (hnRNPK), a 65 kDa ssDNA/RNA-binding-protein that is involved in multiple intracellular signaling cascades, as a binding partner of Abi-1 at postsynaptic sites. The interaction with the Abi-1 SH3 domain is mediated by the hnRNPK-interaction (KI) domain. We further show that during brain development, hnRNPK expression becomes more and more restricted to granule cells of the cerebellum and hippocampal neurons where it localizes in the cell nucleus as well as in the spine/dendritic compartment. The downregulation of hnRNPK in cultured hippocampal neurons by RNAi results in an enlarged dendritic tree and a significant increase in filopodia formation. This is accompanied by a decrease in the number of mature synapses. Both effects therefore mimic the neuronal morphology after downregulation of Abi-1 mRNA in neurons. CONCLUSIONS: Our findings demonstrate a novel interplay between hnRNPK and Abi-1 in the nucleus and at synaptic sites and show obvious similarities regarding both protein knockdown phenotypes. This indicates that hnRNPK and Abi-1 act synergistic in a multiprotein complex that regulates the crucial balance between filopodia formation and synaptic maturation in neurons