HLH and TET2 Mutation Presenting after First Cycle of CLL Treatment.

Here we report development of hemophagocytic lymphohistiocytosis (HLH), along with unmasking of a TET2-mutated myeloid neoplasm, after initial doses of bendamustine and rituximab for longstanding chronic lymphocytic leukemia (CLL). After many years of CLL showing minimally progressive lymphocytosis, the patient's white blood cell count began to decline in parallel with neutrophil count, hemoglobin, and platelet count. Bone marrow biopsy showed partial CLL involvement; bendamustine+rituximab therapy was augmented with granulocyte colony-stimulating factor (g-CSF) and romiplostim to mitigate worsening pancytopenia, without response. Laboratory evaluation revealed a pattern supportive of the clinical impression of HLH, while bone marrow biopsy showed persistent CLL, new reticulin fibrosis, megakaryocytic proliferation, and 32% mutated TET2, but no compelling morphologic evidence of hemophagocytosis. The patient recovered with dexamethasone and g-CSF support.

Pulmonary MALT Lymphoma in Patients with Sjögren's Syndrome.

To describe clinical features and outcomes of seven patients with pulmonary mucosa-associated lymphoid tissue (MALT) lymphoma in the setting of underlying primary Sjögren's syndrome from a single center, we reviewed medical records of consecutive patients with pulmonary MALT lymphoma evaluated at our facility from January 1, 1999 to December 31, 2015 for clinical features, laboratory, pathologic and radiographic findings, management, and outcomes. Out of 13 patients with pulmonary MALT lymphoma, 7 (54 %) met the criteria for Sjögren's syndrome. The mean age at lymphoma diagnosis was 66 years; male-female ratio was 1:6. One-third of patients were asymptomatic at the time lymphoma was discovered. When symptomatic, patients reported nonspecific pulmonary complaints such as cough and dyspnea. All patients had positive antinuclear antibody and anti-SSA/Ro antibody. Rheumatoid factor was positive in six cases. A monoclonal gammopathy was present in three patients; the remaining four had polyclonal hypergammaglobulinemia. The radiologic, morphologic, and immunohistochemical features of primary Sjögren's syndrome-associated pulmonary MALT lymphomas did not differ significantly from pulmonary MALT lymphoma cases in general. All treatment modalities used resulted in complete and sustained response. One patient died 11 years after initial diagnosis with no lymphoma but of another cause. The remaining six patients are still alive and disease-free to date. The present series confirms the favorable course of pulmonary MALT lymphoma in Sjögren's patients. The overall imaging and pathologic features are in accordance with pulmonary MALT lymphoma not associated with primary Sjögren's syndrome. Further studies should be carried out in order to better understand pulmonary MALT lymphomagenesis, treatment, and outcomes in Sjögren's patients.

Identification and characterization of Hoxa9 binding sites in hematopoietic cells.

The clustered homeobox proteins play crucial roles in development, hematopoiesis, and leukemia, yet the targets they regulate and their mechanisms of action are poorly understood. Here, we identified the binding sites for Hoxa9 and the Hox cofactor Meis1 on a genome-wide level and profiled their associated epigenetic modifications and transcriptional targets. Hoxa9 and the Hox cofactor Meis1 cobind at hundreds of highly evolutionarily conserved sites, most of which are distant from transcription start sites. These sites show high levels of histone H3K4 monomethylation and CBP/P300 binding characteristic of enhancers. Furthermore, a subset of these sites shows enhancer activity in transient transfection assays. Many Hoxa9 and Meis1 binding sites are also bound by PU.1 and other lineage-restricted transcription factors previously implicated in establishment of myeloid enhancers. Conditional Hoxa9 activation is associated with CBP/P300 recruitment, histone acetylation, and transcriptional activation of a network of proto-oncogenes, including Erg, Flt3, Lmo2, Myb, and Sox4. Collectively, this work suggests that Hoxa9 regulates transcription by interacting with enhancers of genes important for hematopoiesis and leukemia.

Structural basis of multimer-mediated mayhem.

Oligomerization of AML1-ETO contributes to leukemogenesis through obscure mechanisms. In this issue of Cancer Cell, Bushweller and colleagues show the crystal structure of the ETO NHR2 domain to be a tetramer. Tetramer formation is important for maturation arrest and self-renewal, and gene expression is altered in the absence of self-association. Loss of oligomer formation disrupts interactions between AML1-ETO and members of the ETO corepressor family, but not other corepressor molecules posited to be important for leukemogenesis. The findings clarify the role of oligomer formation in AML1-ETO function and suggest a possible therapeutic strategy of targeting ETO-corepressor interactions.

Vimentin is secreted by activated macrophages.

Vimentin is a widely expressed intermediate filament protein thought to be involved mainly in structural processes, such as wound healing. We now demonstrate that activated human macrophages secrete vimentin into the extracellular space. The maturation of blood-derived monocytes into macrophages involves several signalling pathways. We show that secretion of vimentin, which is phosphorylated at serine and threonine residues, is enhanced by the phosphatase inhibitor okadaic acid and blocked by the specific protein kinase C inhibitor GO6983. These findings are consistent with previous observations that phosphorylation of vimentin affects its intracellular localization and that vimentin is a substrate for protein kinase C (PKC). We also show that the anti-inflammatory cytokine interleukin-10 (IL-10), which inhibits PKC activity, blocks secretion of vimentin. In contrast, the pro-inflammatory cytokine tumour necrosis factor alpha (TNF-alpha) can trigger secretion of vimentin. Finally, we found that extracellular vimentin is involved in bacterial killing and the generation of oxidative metabolites, two important functions of activated macrophages. These data establish that vimentin is secreted by macrophages in response to pro-inflammatory signalling pathways and is probably involved in immune function.

The DEK nuclear autoantigen is a secreted chemotactic factor.

The nuclear DNA-binding protein DEK is an autoantigen that has been implicated in the regulation of transcription, chromatin architecture, and mRNA processing. We demonstrate here that DEK is actively secreted by macrophages and is also found in synovial fluid samples from patients with juvenile arthritis. Secretion of DEK is modulated by casein kinase 2, stimulated by interleukin-8, and inhibited by dexamethasone and cyclosporine A, consistent with a role as a proinflammatory molecule. DEK is secreted in both a free form and in exosomes, vesicular structures in which transcription-modulating factors such as DEK have not previously been found. Furthermore, DEK functions as a chemotactic factor, attracting neutrophils, CD8+ T lymphocytes, and natural killer cells. Therefore, the DEK autoantigen, previously described as a strictly nuclear protein, is secreted and can act as an extracellular chemoattractant, suggesting a direct role for DEK in inflammation.

YY1 and NF-Y binding sites regulate the transcriptional activity of the dek and dek-can promoter.

The mammalian protein DEK has been implicated in multiple cellular processes, including transcriptional regulation, mRNA processing, and chromatin remodeling, and is associated with a number of clinical autoimmune and neoplastic conditions. The connection between DEK and cancer exists at multiple levels: (a) the t(6;9) chromosomal translocation that characterizes a subtype of acute myelogenous leukemia cases results in the formation of a DEK-CAN fusion oncoprotein; (b) a fragment of dek cDNA is capable of partially reversing the radiation-sensitive phenotype of fibroblasts cultured from ataxia-telangiectasia patients; and (c) increased levels of dek mRNA have been found to be associated with hepatocellular carcinoma, glioblastoma, and melanoma. Despite the growing list of cancer subtypes with a connection to DEK, the factors that mediate its expression have yet to be characterized. Here we undertake the analysis of DEK regulation by mapping the discrete elements within the proximal promoter that are responsible for constitutive transcription of dek in transformed cells. We find that functional elements include an inverted CCAAT box and a YY1 consensus binding site, and the introduction of point mutations into these sites markedly diminishes transcriptional activity. In addition, we identify the transcriptional activator NF-Y as a member of the CCAAT-binding complex, and verify binding of the transcription factor YY1 at its consensus site in the dek promoter. The discovery of NF-Y and YY1 as regulatory determinants of DEK expression is consistent with the well-documented roles of these two factors in cellular proliferation and transformation.

Role of Endothelin-1 in a Syndrome of Myelofibrosis and Osteosclerosis.

CONTEXT: Primary myelofibrosis is one of the chronic myeloproliferative disorders characterized by bone marrow fibrosis associated with extramedullary hematopoiesis and osteosclerosis. Endothelin-1 (ET1) is a potent vasoconstrictor that is also a key mediator of osteoblastic bone metastases by stimulating osteoblast proliferation and new bone formation. CASE DESCRIPTION: We report laboratory, radiographic, bone densitometry, and bone histology data of a patient presenting with newly diagnosed, biopsy-proven myelofibrosis and osteosclerosis. We were able to demonstrate abundant ET1 signaling in the bones of our patient. CONCLUSIONS: We believe that ET1 is responsible for the osteosclerosis that develops with advanced myelofibrosis and suggest that ET1 signaling may play a role in other osteosclerotic settings as well.

Minireview: DEK and gene regulation, oncogenesis and AIDS.

A brief review summarizing the relationships that exist between the DEK protein and gene regulation, oncogenesis and AIDS.

Gelatinous bone marrow transformation secondary to unusual eating habits and drastic weight loss.

Gelatinous bone marrow transformation (GMT), also known as starvation bone marrow, has been reported in a number of chronic illnesses, eating disorders (anorexia nervosa) and malignancies. We report the case of a 37-year-old man with a history of bipolar disorder and obesity (weighing >300 pounds) who presented due to recently developing a deep yellow colour to his skin. Over the past 2 years, through diet and exercise, he lost over 150 pounds. He reported running 6-8 miles per day and eating 'lots of squash'. We made the diagnosis of starvation hepatitis and bone marrow degeneration, and referred the patient to a dietician and haematologist/oncologist, where improvements were observed at 4 weeks follow-up.

The PAF complex synergizes with MLL fusion proteins at HOX loci to promote leukemogenesis.

MLL is involved in chromosomal rearrangements that generate fusion proteins with deregulated transcriptional activity. The mechanisms of MLL fusion protein-mediated transcriptional activation are poorly understood. Here we show MLL interacts directly with the polymerase associated factor complex (PAFc) through sequences flanking the CxxC domain. PAFc interacts with RNA polymerase II and stimulates posttranslational histone modifications. PAFc augments MLL and MLL-AF9 mediated transcriptional activation of Hoxa9. Conversely, knockdown of PAFc disrupts MLL fusion protein-mediated transcriptional activation and MLL recruitment to target loci. PAFc gene expression is downregulated during hematopoiesis and likely serves to regulate MLL function. Deletions of MLL that abolish interactions with PAFc also eliminate MLL-AF9 mediated immortalization indicating an essential function for this interaction in leukemogenesis.

Persistence of leukemia-initiating cells in a conditional knockin model of an imatinib-responsive myeloproliferative disorder.

Despite remarkable responses to the tyrosine kinase inhibitor imatinib, CML patients are rarely cured by this therapy perhaps due to imatinib refractoriness of leukemia-initiating cells (LICs). Evidence for this is limited because of poor engraftment of human CML-LICs in NOD-SCID mice and nonphysiologic expression of oncogenes in retroviral transduction mouse models. To address these challenges, we generated mice bearing conditional knockin alleles of two human oncogenes: HIP1/PDGFbetaR (H/P) and AML1-ETO (A/E). Unlike retroviral transduction, physiologic expression of H/P or A/E individually failed to induce disease, but coexpression of both H/P and A/E led to rapid onset of a fully penetrant, myeloproliferative disorder, indicating cooperativity between these two alleles. Although imatinib dramatically decreased disease burden, LICs persisted, demonstrating imatinib refractoriness of LICs.

HOX proteins and leukemia.

HOX and three amino acid loop extension (TALE) proteins cooperate to induce transformation in mouse leukemia models, and are dysregulated in a variety of human leukemias. Despite decades of research, the mechanism of action for Hox proteins in embryogenesis and hematopoiesis remains unclear. Recent studies on the roles of Hoxa9 and Meis1 in leukemia has led to a wealth of new data, but their molecular mechanisms of action and synergy remain obscure. Advances in genome-wide technologies offer new avenues for understanding how homeodomain-containing transcription factors exert their programs in normal and neoplastic development.

p300/CBP-associated factor drives DEK into interchromatin granule clusters.

DEK is a mammalian protein that has been implicated in the pathogenesis of autoimmune diseases and cancer, including acute myeloid leukemia, melanoma, glioblastoma, hepatocellular carcinoma, and bladder cancer. In addition, DEK appears to participate in multiple cellular processes, including transcriptional repression, mRNA processing, and chromatin remodeling. Sub-nuclear distribution of this protein, with the attendant functional ramifications, has remained a controversial topic. Here we report that DEK undergoes acetylation in vivo at lysine residues within the first 70 N-terminal amino acids. Acetylation of DEK decreases its affinity for DNA elements within the promoter, which is consistent with the involvement of DEK in transcriptional repression. Furthermore, deacetylase inhibition results in accumulation of DEK within interchromatin granule clusters (IGCs), sub-nuclear structures that contain RNA processing factors. Overexpression of P/CAF acetylase drives DEK into IGCs, and addition of a newly developed, synthetic, cell-permeable P/CAF inhibitor blocks this movement. To our knowledge, this is the first reported example of acetylation playing a direct role in relocation of a protein to IGCs, and this may explain how DEK can function in multiple pathways that take place in distinct sub-nuclear compartments. These findings also suggest that DEK-associated malignancies and autoimmune diseases might be amenable to treatment with agents that alter acetylation.

DEK binding to class II MHC Y-box sequences is gene- and allele-specific.

Using electrophoretic mobility shift assays, we examined sequence-specific binding of DEK, a potential autoantigen in juvenile rheumatoid arthritis, to conserved Y-box regulatory sequences in class II MHC gene promoters. Nuclear extracts from several cell lines of different phenotypes contained sequence-specific binding activity recognizing DRA, DQA1*0101, and DQA1*0501 Y-box sequences. Participation of both DEK and NF-Y in the DQA1 Y-box binding complex was confirmed by 'supershifting' with anti-DEK and anti-NF-Y antibodies. Recombinant DEK also bound specifically to the DQA1*0101 Y box and to the polymorphic DQA1*0501 Y box, but not to the consensus DRA Y box. Measurement of the apparent dissociation constants demonstrated a two- to fivefold difference in DEK binding to the DQA1 Y-box sequence in comparison with other class II MHC Y-box sequences. Residues that are crucial for DEK binding to the DQA1*0101 Y box were identified by DNase I footprinting. The specific characteristics of DEK binding to these related sequences suggests a potential role for DEK in differential regulation of class II MHC expression, and thus in the pathogenesis of juvenile rheumatoid arthritis and other autoimmune diseases.