Human chromosomal translocations at CpG sites and a theoretical basis for their lineage and stage specificity.

We have assembled, annotated, and analyzed a database of over 1700 breakpoints from the most common chromosomal rearrangements in human leukemias and lymphomas. Using this database, we show that although the CpG dinucleotide constitutes only 1% of the human genome, it accounts for 40%-70% of breakpoints at pro-B/pre-B stage translocation regions-specifically, those near the bcl-2, bcl-1, and E2A genes. We do not observe CpG hotspots in rearrangements involving lymphoid-myeloid progenitors, mature B cells, or T cells. The stage specificity, lineage specificity, CpG targeting, and unique breakpoint distributions at these cluster regions may be explained by a lesion-specific double-strand breakage mechanism involving the RAG complex acting at AID-deaminated methyl-CpGs.

Pathogenic B-cell receptor signaling in lymphoid malignancies: New insights to improve treatment.

Signals emanating from the B-cell receptor (BCR) promote proliferation and survival in diverse forms of B-cell lymphoma. Precision medicine strategies targeting the BCR pathway have been generally effective in treating lymphoma, but often fail to produce durable responses in diffuse large B-cell lymphoma (DLBCL), a common and aggressive cancer. New insights into DLBCL biology garnered from genomic analyses and functional proteogenomic studies have identified novel modes of BCR signaling in this disease. Herein, we describe the distinct roles of antigen-dependent and antigen-independent BCR signaling in different subtypes of DLBCL. We highlight mechanisms by which the BCR cooperates with TLR9 and mutant isoforms of MYD88 to drive sustained NF-κB activity in the activated B-cell-like (ABC) subtype of DLBCL. Finally, we discuss progress in detecting and targeting oncogenic BCR signaling to improve the survival of patients with lymphoma.

Detection of Myosin 1g Overexpression in Pediatric Leukemia by Novel Monoclonal Antibodies.

Myosin 1g (Myo1g) is a mechanoenzyme associated with actin filaments, expressed exclusively in hematopoietic cells, and involved in various cellular functions, including cell migration, adhesion, and membrane trafficking. Despite the importance of Myo1g in distinct functions, there is currently no monoclonal antibody (mAb) against Myo1g. mAbs are helpful tools for the detection of specific antigens in tumor cells and other tissues. The development of mAbs against targeted dysregulated molecules in cancer cells remains a crucial tool for aiding in the diagnosis and the treatment of patients. Using hybridoma technology, we generated a panel of hybridomas specific for Myo1g. ELISA, immunofluorescence, and Western blot assay results revealed the recognition of Myo1g by these novel monoclonal antibodies in normal and transformed T and B cells. Here, we report the development and application of new monoclonal antibodies against Myo1g for their potential use to detect its overexpression in acute lymphoblastic leukemia (ALL) patients.

VLX1570 induces apoptosis through the generation of ROS and induction of ER stress on leukemia cell lines.

A novel proteasome deubiquitinase inhibitor, VLX1570, has been highlighted as a promising therapeutic agent mainly for lymphoid neoplasms and solid tumors. We examined in vitro effects of VLX1570 on eight myeloid and three lymphoid leukemia cell lines. From cell culture studies, 10 out of 11 cell lines except K562 were found to be susceptible to VLX1570 treatment and it inhibited cell growth mainly by apoptosis. Next, to identify the signaling pathways associated with apoptosis, we performed gene expression profiling using HL-60 with or without 50 nmol/L of VLX1570 for 3 hours and demonstrated that VLX1570 induced the genetic pathway involved in "heat shock transcription factor 1 (HSF1) activation", "HSF1 dependent transactivation", and "Regulation of HSF1 mediated heat shock response". VLX1570 increased the amount of high molecular weight polyubiquitinated proteins and the expression of HSP70 as the result of the suppression of ubiquitin proteasome system, the expression of heme oxygenase-1, and the amount of phosphorylation in JNK and p38 associated with the generation of reactive oxygen species (ROS) induced apoptosis and the amount of phosphorylation in eIF2α, inducing the expression of ATF4 and endoplasmic reticulum (ER) stress dependent apoptosis protein, CHOP, and the amount of phosphorylation slightly in IRE1α, leading to increased expression of XBP-1s in leukemia cell lines. In the present study, we demonstrate that VLX1570 induces apoptosis and exerts a potential anti-leukemic effect through the generation of ROS and induction of ER stress in leukemia cell lines.

Insight into Bortezomib Focusing on Its Efficacy against P-gp-Positive MDR Leukemia Cells.

In this paper, we compared the effects of bortezomib on L1210 (S) cells with its effects on P-glycoprotein (P-gp)-positive variant S cells, which expressed P-gp either after selection with vincristine (R cells) or after transfection with a human gene encoding P-gp (T cells). Bortezomib induced the death-related effects in the S, R, and T cells at concentrations not exceeding 10 nM. Bortezomib-induced cell cycle arrest in the G2/M phase was more pronounced in the S cells than in the R or T cells and was related to the expression levels of cyclins, cyclin-dependent kinases, and their inhibitors. We also observed an increase in the level of polyubiquitinated proteins (via K48-linkage) and a decrease in the gene expression of some deubiquitinases after treatment with bortezomib. Resistant cells expressed higher levels of genes encoding 26S proteasome components and the chaperone HSP90, which is involved in 26S proteasome assembly. After 4 h of preincubation, bortezomib induced a more pronounced depression of proteasome activity in S cells than in R or T cells. However, none of these changes alone or in combination sufficiently suppressed the sensitivity of R or T cells to bortezomib, which remained at a level similar to that of S cells.

Protein ubiquitination in lymphoid malignancies.

Human lymphoid malignancies inherit gene expression networks from their normal B-cell counterpart and co-opt them for their own oncogenic purpose, which is usually governed by transcription factors and signaling pathways. These transcription factors and signaling pathways are precisely regulated at multiple steps, including ubiquitin modification. Protein ubiqutination plays a role in almost all cellular events and in many human diseases. In the past few years, multiple studies have expanded the role of ubiquitination in the genesis of diverse lymphoid malignancies. Here, we discuss our current understanding of both proteolytic and non-proteolytic functions of the protein ubiquitination system and describe how it is involved in the pathogenesis of human lymphoid cancers. Lymphoid-restricted ubiquitination mechanisms, including ubiquitin E3 ligases and deubiquitinating enzymes, provide great opportunities for the development of targeted therapies for lymphoid cancers.

Affecting NF-κB cell signaling pathway in chronic lymphocytic leukemia by dendrimers-based nanoparticles.

The complex genetic diversity of chronic lymphocytic leukemia (CLL) makes it difficult to determine the effective and durable therapy beneficial to patients. During the several past years' significant insights in the biology of the disease and its treatment have been made, allowing for the identification of promising novel therapeutic agents. The investigation of signaling pathways to understand the biological character of CLL together with the development of molecular profiling is key in personalized approach in therapy for this disease. As it was already proven, maltotriose (M3) modified fourth generation poly(propylene imine) dendrimers (PPI-G4) modulate BCR, TRAIL and WNT signaling pathway gene expression in CLL cells and strongly influence their survival by inducing apoptosis and inhibiting proliferation. The aim of this study was to evaluate the influence of PPI-G4-M3 dendrimers on NFκB pathway gene expression in CLL (MEC-1) cells with 60 K microarray, as it is one of the major factors in the pathogenesis of B-cell neoplasms. The findings were compared with those obtained with Fludarabine (FA) and the results indicate that PPI-G4-M3 dendrimers affect the expression of the examined genes and exert comparable effect on the CLL cells to FA. Dendrimers are one of the most potent groups of nanometer-sized macromolecules for closing the gap between the present ineffective treatment and the future effective personalized therapy due to their potential versatile biological properties.

Molecular Regulation of Differentiation in Early B-Lymphocyte Development.

B-lymphocyte differentiation is one of the best understood developmental pathways in the hematopoietic system. Our understanding of the developmental trajectories linking the multipotent hematopoietic stem cell to the mature functional B-lymphocyte is extensive as a result of efforts to identify and prospectively isolate progenitors at defined maturation stages. The identification of defined progenitor compartments has been instrumental for the resolution of the molecular features that defines given developmental stages as well as for our understanding of the mechanisms that drive the progressive maturation process. Over the last years it has become increasingly clear that the regulatory networks that control normal B-cell differentiation are targeted by mutations in human B-lineage malignancies. This generates a most interesting link between development and disease that can be explored to improve diagnosis and treatment protocols in lymphoid malignancies. The aim of this review is to provide an overview of our current understanding of molecular regulation in normal and malignant B-cell development.

L1210 Cells Overexpressing ABCB1 Drug Transporters Are Resistant to Inhibitors of the N- and O-glycosylation of Proteins.

Overexpression of P-glycoprotein (P-gp, drug transporter) in neoplastic cells is the most frequently observed molecular cause of multidrug resistance. Here, we show that the overexpression of P-gp in L1210 cells leads to resistance to tunicamycin and benzyl 2-acetamido-2-deoxy-α-d-galactopyranoside (GalNAc-α-O-benzyl). Tunicamycin induces both glycosylation depression and ubiquitination improvement of P-gp. However, the latter is not associated with large increases in molecular mass as evidence for polyubiquitination. Therefore, P-gp continues in maturation to an active membrane efflux pump rather than proteasomal degradation. P-gp-positive L1210 cells contain a higher quantity of ubiquitin associated with cell surface proteins than their P-gp-negative counterparts. Thus, P-gp-positive cells use ubiquitin signaling for correct protein folding to a higher extent than P-gp-negative cells. Elevation of protein ubiquitination after tunicamycin treatment in these cells leads to protein folding rather than protein degradation, resulting at least in the partial lack of cell sensitivity to tunicamycin in L1210 cells after P-gp expression. In contrast to tunicamycin, to understand why P-gp-positive cells are resistant to GalNAc-α-O-benzyl, further research is needed.

Role of the CARMA1/BCL10/MALT1 complex in lymphoid malignancies.

PURPOSE OF REVIEW: The CARMA1/BCL10/MALT1 (CBM) complex is a multimeric signaling complex controlling several important aspects of lymphocyte activation. Gain-of-function mutations in the genes encoding CBM proteins or their upstream regulators are associated with lymphoid malignancies, whereas loss-of-function mutations lead to immunodeficiency. This review reports on recent findings advancing our understanding of how CBM proteins contribute to malignant and nonmalignant hematological diseases in humans. RECENT FINDINGS: Somatic gain-of-function mutations of CARMA1 (also known as CARD11), originally described for patients with diffuse large B-cell lymphoma, have recently been identified in patients with acute T-cell leukemia/lymphoma or Sézary syndrome, and in patients with a B-cell lymphoproliferative disorder known as BENTA. Loss-of-function mutations of CARMA1 and MALT1, on the other hand, have been reported to underlie human immunodeficiency. Lately, it has become clear that CBM-dependent signaling promotes lymphomagenesis not only via NF-κB activation, but also via the AP-1 family of transcription factors. The identification of new substrates of the protease MALT1 and the characterization of mice expressing catalytically inactive MALT1 have deepened our understanding of how the CBM complex controls lymphocyte proliferation through promoting MALT1's protease activity. SUMMARY: The discovery of CARMA1 gain-of-function mutations in T-cell malignancies and BENTA patients, as well as the association of CARMA1 and MALT1 mutations with human immunodeficiency highlight the importance of CBM proteins in the regulation of lymphocyte functions, and suggest that the protease activity of MALT1 might be targeted to treat specific lymphoid malignancies.

'Trained immunity': consequences for lymphoid malignancies.

In hematological malignancies complex interactions exist between the immune system, microorganisms and malignant cells. On one hand, microorganisms can induce cancer, as illustrated by specific infection-induced lymphoproliferative diseases such as Helicobacter pylori-associated gastric mucosa-associated lymphoid tissue lymphoma. On the other hand, malignant cells create an immunosuppressive environment for their own benefit, but this also results in an increased risk of infections. Disrupted innate immunity contributes to the neoplastic transformation of blood cells by several mechanisms, including the uncontrolled clearance of microbial and autoantigens resulting in chronic immune stimulation and proliferation, chronic inflammation, and defective immune surveillance and anti-cancer immunity. Restoring dysfunction or enhancing responsiveness of the innate immune system might therefore represent a new angle for the prevention and treatment of hematological malignancies, in particular lymphoid malignancies and associated infections. Recently, it has been shown that cells of the innate immune system, such as monocytes/macrophages and natural killer cells, harbor features of immunological memory and display enhanced functionality long-term after stimulation with certain microorganisms and vaccines. These functional changes rely on epigenetic reprogramming and have been termed 'trained immunity'. In this review the concept of 'trained immunity' is discussed in the setting of lymphoid malignancies. Amelioration of infectious complications and hematological disease progression can be envisioned to result from the induction of trained immunity, but future studies are required to prove this exciting new hypothesis.

Reverse-engineering the genetic circuitry of a cancer cell with predicted intervention in chronic lymphocytic leukemia.

Cellular behavior is sustained by genetic programs that are progressively disrupted in pathological conditions--notably, cancer. High-throughput gene expression profiling has been used to infer statistical models describing these cellular programs, and development is now needed to guide orientated modulation of these systems. Here we develop a regression-based model to reverse-engineer a temporal genetic program, based on relevant patterns of gene expression after cell stimulation. This method integrates the temporal dimension of biological rewiring of genetic programs and enables the prediction of the effect of targeted gene disruption at the system level. We tested the performance accuracy of this model on synthetic data before reverse-engineering the response of primary cancer cells to a proliferative (protumorigenic) stimulation in a multistate leukemia biological model (i.e., chronic lymphocytic leukemia). To validate the ability of our method to predict the effects of gene modulation on the global program, we performed an intervention experiment on a targeted gene. Comparison of the predicted and observed gene expression changes demonstrates the possibility of predicting the effects of a perturbation in a gene regulatory network, a first step toward an orientated intervention in a cancer cell genetic program.

Pathogenetic importance and therapeutic implications of NF-κB in lymphoid malignancies.

Derangement of the nuclear factor κB (NF-κB) pathway initiates and/or sustains many types of human cancer. B-cell malignancies are particularly affected by oncogenic mutations, translocations, and copy number alterations affecting key components the NF-κB pathway, most likely owing to the pervasive role of this pathway in normal B cells. These genetic aberrations cause tumors to be 'addicted' to NF-κB, which can be exploited therapeutically. Since each subtype of lymphoid cancer utilizes different mechanisms to activate NF-κB, several different therapeutic strategies are needed to address this pathogenetic heterogeneity. Fortunately, a number of drugs that block signaling cascades leading to NF-κB are in early phase clinical trials, several of which are already showing activity in lymphoid malignancies.

Oncogene iASPP enhances self-renewal of hematopoietic stem cells and facilitates their resistance to chemotherapy and irradiation.

iASPP is a member of the apoptosis-stimulating proteins of p53 (ASPP) family and negatively regulates the apoptotic function of p53. In a hematopoietic system, overexpression of iASPP results in blockage of apoptosis, which may play a role in regulating hematopoietic stem cell (HSC) numbers. To address this, we first analyzed the expression of iASPP in patients with acute leukemia (AL) and found it was highly expressed in patients with AL. We further established a transgenic mouse model in which human iASPP was specifically expressed in hematopoietic cells. Overexpression of iASPP led to an increase in the proportion of long-term HSCs, short-term HSCs, multipotent progenitors, and common myeloid progenitor. HSCs from iASPP transgenic mice had an advantage in long-term reconstitution potential. In addition, the hematopoietic cells from iASPP transgenic mice exhibited a significantly lower level of p53 dependent apoptosis. After irradiation damage, hematopoietic cells of iASPP transgenic mice had a higher level of γ-H2AX expression, which lasted for a longer time. These results provide the first evidence that the iASPP can increase HSC populations and reconstitution capacity. Interestingly, in response to cell damage stimuli, hematopoietic cells can be protected against apoptosis by iASPP; meanwhile these apoptosis-resistant cells would have more mutation accumulation, which might be the potential risk for malignant transformation.-Jia, Y., Peng, L., Rao, Q., Xing, H., Huai, L., Yu, P., Chen, Y., Wang, C., Wang, M., Mi, Y., Wang, J. Oncogene iASPP enhances self-renewal of hematopoietic stem cells and facilitates their resistance to chemotherapy and irradiation.

Molecular deregulation of signaling in lymphoid tumors.

Genomic studies have led to a significant impact both on the pace and the nature of understanding the molecular and biological bases of a variety of lymphoid tumors. An increasingly emerging aspect from genomic studies is that malignant lymphoid cells manipulate signaling pathways that are central to the homeostasis of their normal counterpart, including B- and T-cell receptor signaling, NF-κB signaling, Toll-like receptor signaling, cytokine signaling, MAP kinase signaling, and NOTCH signaling. This review aims at covering the signaling pathways that are affected by mutations in lymphoid tumors, and how genetic alteration of these pathways may contribute to disease pathogenesis and management.

miR-155 targets histone deacetylase 4 (HDAC4) and impairs transcriptional activity of B-cell lymphoma 6 (BCL6) in the Eµ-miR-155 transgenic mouse model.

Multiple studies have established that microRNAs (miRNAs) are involved in the initiation and progression of cancer. Notably, miR-155 is one of the most overexpressed miRNAs in several solid and hematological malignancies. Ectopic miR-155 expression in mice B cells (Eµ-miR-155 transgenic mice) has been shown to induce pre-B-cell proliferation followed by high-grade lymphoma/leukemia. Loss of miR-155 in mice resulted in impaired immunity due to defective T-cell-mediated immune response. Here we provide a mechanistic insight into miR-155-induced leukemogenesis in the Eµ-miR-155 mouse model through genome-wide transcriptome analysis of naïve B cells and target studies. We found that a key transcriptional repressor and proto-oncogene, Bcl6 is significantly down-regulated in Eµ-miR-155 mice. The reduction of Bcl6 subsequently leads to de-repression of some of the known Bcl6 targets like inhibitor of differentiation (Id2), interleukin-6 (IL6), cMyc, Cyclin D1, and Mip1α/ccl3, all of which promote cell survival and proliferation. We show that Bcl6 is indirectly regulated by miR-155 through Mxd1/Mad1 up-regulation. Interestingly, we found that miR-155 directly targets HDAC4, a corepressor partner of BCL6. Furthermore, ectopic expression of HDAC4 in human-activated B-cell-type diffuse large B-cell lymphoma (DLBCL) cells results in reduced miR-155-induced proliferation, clonogenic potential, and increased apoptosis. Meta-analysis of the diffuse large B-cell lymphoma patient microarray data showed that miR-155 expression is inversely correlated with Bcl6 and Hdac4. Hence this study provides a better understanding of how miR-155 causes disruption of the BCL6 transcriptional machinery that leads to up-regulation of the survival and proliferation genes in miR-155-induced leukemias.

Targeting CXCR4 with cell-penetrating pepducins in lymphoma and lymphocytic leukemia.

The chemokine receptor CXCR4, which normally regulates stromal stem cell interactions in the bone marrow, is highly expressed on a variety of malignant hematologic cells, including lymphoma and lymphocytic leukemias. A new treatment concept has arisen wherein CXCR4 may be an effective therapeutic target as an adjunct to treatment of hematologic neoplasms with chemo- and immunotherapy. In the present study, we developed pepducins, cell-penetrating lipopeptide antagonists of CXCR4, to interdict CXCL12-CXCR4 transmembrane signaling to intracellular G-proteins. We demonstrate that pepducins targeting the first (i1) or third (i3) intracellular loops of CXCR4 completely abrogate CXCL12-mediated cell migration of lymphocytic leukemias and lymphomas. Stromal-cell coculture protects lymphoma cells from apoptosis in response to treatment with the CD20-targeted Ab rituximab. However, combination treatment with CXCR4 pepducins and rituximab significantly increases the apoptotic effect of rituximab. Furthermore, treatment of mice bearing disseminated lymphoma xenografts with pepducins alone or in combination with rituximab significantly increased their survival. These data demonstrate that CXCL12-CXCR4 signaling can be effectively inhibited by cell-penetrating pepducins, which represents a potential new treatment strategy for lymphoid malignancies.

Glycogen synthase kinase 3 in MLL leukaemia maintenance and targeted therapy.

Glycogen synthase kinase 3 (GSK3) is a multifunctional serine/threonine kinase that participates in numerous signalling pathways involved in diverse physiological processes. Several of these pathways are implicated in disease pathogenesis, which has prompted efforts to develop GSK3-specific inhibitors for therapeutic applications. However, before now, there has been no strong rationale for targeting GSK3 in malignancies. Here we report pharmacological, physiological and genetic studies that demonstrate an oncogenic requirement for GSK3 in the maintenance of a specific subtype of poor prognosis human leukaemia, genetically defined by mutations of the MLL proto-oncogene. In contrast to its previously characterized roles in suppression of neoplasia-associated signalling pathways, GSK3 paradoxically supports MLL leukaemia cell proliferation and transformation by a mechanism that ultimately involves destabilization of the cyclin-dependent kinase inhibitor p27(Kip1). Inhibition of GSK3 in a preclinical murine model of MLL leukaemia provides promising evidence of efficacy and earmarks GSK3 as a candidate cancer drug target.

C-KIT mutation cooperates with full-length AML1-ETO to induce acute myeloid leukemia in mice.

The full-length AML1-ETO (AE) fusion gene resulting from t(8;21)(q22;q22) in human acute myeloid leukemia (AML) is not sufficient to induce leukemia in animals, suggesting that additional mutations are required for leukemogenesis. We and others have identified activating mutations of C-KIT in nearly half of patients with t(8;21) AML. To test the hypothesis that activating C-KIT mutations cooperate with AE to cause overt AML, we generated a murine transduction and transplantation model with both mutated C-KIT and AE. To overcome the intracellular transport block of human C-KIT in murine cells, we engineered hybrid C-KIT (HyC-KIT) by fusing the extracellular and transmembrane domains of the murine c-Kit in-frame to the intracellular signaling domain of human C-KIT. We showed that tyrosine kinase domain mutants HyC-KIT N822K and D816V, as well as juxtamembrane mutants HyC-KIT 571+14 and 557-558Del, could transform murine 32D cells to cytokine-independent growth. The protein tyrosine kinase inhibitor dasatinib inhibited the proliferation of 32D cells expressing these C-KIT mutants, with potency in the low nanomolar range. In mice, HyC-KIT N822K induced a myeloproliferative disease, whereas HyC-KIT 571+14 induces both myeloproliferative disease and lymphocytic leukemia. Interestingly, coexpression of AE and HyC-KIT N822K led to fatal AML. Our data have further enriched the two-hit model that abnormalities of both transcription factor and membrane/cytosolic signaling molecule are required in AML pathogenesis. Furthermore, dasatinib prolonged lifespan of mice bearing AE and HyC-KIT N822K-coexpressing leukemic cells and exerted synergic effects while combined with cytarabine, thus providing a potential therapeutic for t(8;21) leukemia.

Structural basis for WDR5 interaction (Win) motif recognition in human SET1 family histone methyltransferases.

Translocations and amplifications of the mixed lineage leukemia-1 (MLL1) gene are associated with aggressive myeloid and lymphocytic leukemias in humans. MLL1 is a member of the SET1 family of histone H3 lysine 4 (H3K4) methyltransferases, which are required for transcription of genes involved in hematopoiesis and development. MLL1 associates with a subcomplex containing WDR5, RbBP5, Ash2L, and DPY-30 (WRAD), which together form the MLL1 core complex that is required for sequential mono- and dimethylation of H3K4. We previously demonstrated that WDR5 binds the conserved WDR5 interaction (Win) motif of MLL1 in vitro, an interaction that is required for the H3K4 dimethylation activity of the MLL1 core complex. In this investigation, we demonstrate that arginine 3765 of the MLL1 Win motif is required to co-immunoprecipitate WRAD from mammalian cells, suggesting that the WDR5-Win motif interaction is important for the assembly of the MLL1 core complex in vivo. We also demonstrate that peptides that mimic SET1 family Win motif sequences inhibit H3K4 dimethylation by the MLL1 core complex with varying degrees of efficiency. To understand the structural basis for these differences, we determined structures of WDR5 bound to six different naturally occurring Win motif sequences at resolutions ranging from 1.9 to 1.2 Å. Our results reveal that binding energy differences result from interactions between non-conserved residues C-terminal to the Win motif and to a lesser extent from subtle variation of residues within the Win motif. These results highlight a new class of methylation inhibitors that may be useful for the treatment of MLL1-related malignancies.