Development Of Hematopoietic Malignancies Research Articles

Screening of proliferation related lncRNAs in leukemia cell lines by lentivirus shRNA library combined with second-generation sequencing

Long non-coding RNA (lncRNA) has become an important regulator of many cellular processes, including cell proliferation. Although studies have shown that a variety of lncRNAs play an important role in the occurrence and development of hematopoietic malignancies, a more comprehensive and unbiased method to study the function of lncRNAs in leukemia cell lines is lacking. Here, we used short hairpin RNA (shRNA) library combined with high-throughput sequencing to screen lncRNAs that may affect the proliferation of leukemia cell lines, and identified lncRNA C20orf204-203 among 74 candidate lncRNAs in this study. Further experiments showed that C20orf204-203 was localized in the cytoplasm in both K562 and THP-1 cell lines. C20orf204-203 knockdown decreased the proliferation of K562 and THP-1 cell lines accompanied with the increased proportion of early apoptotic cells. We observed the increased mRNA level of BAD gene while decreased protein level of TP53 and BCL2. The expression of Caspase 3 decreased and Caspase 3-cleaved protein increased in THP-1 cell line. However, their changes were inconsistent in the two cell lines. Our experimental results showed that knockdown of lncRNA C20orf204-203 in leukemia cell lines affected cell proliferation although the mechanism of action in different cell lines may differ. Importantly, our research demonstrated the feasibility of using shRNA library combined with high-throughput sequencing to study the role of lncRNA in leukemia cell lines on a large scale.

Germline CHEK2 and ATM Variants in Myeloid and Other Hematopoietic Malignancies

An intact DNA damage response is crucial to preventing cancer development, including in myeloid and lymphoid malignancies. Deficiencies in the homologous recombination (HR) pathway can lead to defective DNA damage responses, and this can occur through inherited germline mutations in HR pathway genes, such as CHEK2 and ATM. We now understand that germline mutations can be identified frequently (~ 5-10%) in patients with myeloid and lymphoid malignancies, and among the most common of these are CHEK2 and ATM. We review the role that deleterious germline CHEK2 and ATM variants play in the development of hematopoietic malignancies, and how this influences clinical practice, including cancer screening, hematopoietic stem cell transplantation, and therapy choice. In recent large cohorts of patients diagnosed with myeloid or lymphoid malignancies, deleterious germline loss of function variants in CHEK2 and ATM are among the most common identified. Germline CHEK2 variants predispose to a range of myeloid malignancies, most prominently myeloproliferative neoplasms and myelodysplastic syndromes (odds ratio range: 2.1-12.3), and chronic lymphocytic leukemia (odds ratio 14.83). Deleterious germline ATM variants have been shown to predispose to chronic lymphocytic leukemia (odds ratio range: 1.7-10.1), although additional studies are needed to demonstrate the risk they confer for myeloid malignancies. Early studies suggest there may also be associations between deleterious germline CHEK2 and ATM variants and development of clonal hematopoiesis. Identifying CHEK2 and ATM variants is crucial for the optimal management of patients and families affected by hematopoietic malignancies. OPENING CLINICAL CASE: "A 45year-old woman presents to your clinic with a history of triple-negative breast cancer diagnosed five years ago, treated with surgery, radiation, and chemotherapy. About six months ago, she developed cervical lymphadenopathy, and a biopsy demonstrated small lymphocytic leukemia. Peripheral blood shows a small population of lymphocytes with a chronic lymphocytic leukemia immunophenotype, and FISH demonstrates a complex karyotype: gain of one to two copies of IGH and FGFR3; gain of two copies of CDKN2C at 1p32.3; gain of two copies of CKS1B at 1q21; tetrasomy for chromosome 3; trisomy and tetrasomy for chromosome 7; tetrasomy for chromosome 9; tetrasomy for chromosome 12; gain of one to twocopies of ATM at 11q22.3; deletion of chromosome 13 deletion positive; gain of one to two copies of TP53 at 17p13.1). Given her history of two cancers, you arrange for germline genetic testing using DNA from cultured skin fibroblasts, which demonstrates pathogenic variants in ATM [c.1898 + 2T > G] and CHEK2 [p.T367Metfs]. Her family history is significant for multiple cancers. (Fig.1)." Fig. 1 Representative pedigree from a patient with germline pathogenic ATM and CHEK2 variants who was affected by early onset breast cancer and chronic lymphocytic leukemia. Arrow indicates proband. Colors indicate cancer type/disease: purple, breast cancer; blue, lymphoma; brown, melanoma; yellow, colon cancer; and green, autoimmune disease.

C‐5 Aryl Substituted Azaspirooxindolinones Derivatives: Synthesis and Biological Evaluation as Potential Inhibitors of Tec Family Kinases

AbstractThe interleukin‐2‐inducible kinase (ITK) and Bruton tyrosine kinase (BTK) are two crucial Tec family kinase members with important roles in the development of hematopoietic malignancies, autoimmune disorders and other diseases in humans. Thus, ITK and BTK are key targets for drug development. Spirooxindoles are important scaffolds for the synthesis of small molecules with broad and potent biological activities. In this study, we performed a structure‐activity relationship study of a new series of 5′‐(benzo[d][1,3]dioxol‐5‐yl)spiro[piperidine‐4,3′‐pyrrolo[2,3–b]pyridin]‐2′(1′H)‐one linked with N‐acyl and C‐5 aryl‐substituted scaffolds in a panel of ITK and BTK cancer cell lines. Four compounds 11, 12, 14, and 15 showed high antiproliferative activity against ITK and BTK cell lines. Compounds 11 and 12 with a C‐5 benzodioxole group and gem‐dialkyl group attached to carbonyl on piperidine were highly effective in ITK‐high Jurkat and CEM cell lines, and compound 14, a biotin analogue, was identified as a good inhibitor of BTK‐high RAMOS cells. Compound 15 with cyclopropyl group attached to carbonyl on piperidine also showed good activity in ITK and BTK cell lines.

The Nlrp3 inflammasome - the evolving story of its positive and negative effects on hematopoiesis.

Hematopoiesis is co-regulated by innate immunity, which is an ancient evolutionary defense mechanism also involved in the development and regeneration of damaged tissues. This review seeks to shed more light on the workings of the Nlrp3 inflammasome, which is an intracellular innate immunity pattern recognition receptor and sensor of changes in the hematopoietic microenvironment, and focus on its role in hematopoieisis. Hematopoietic stem progenitor cells (HSPCs) are exposed to several external mediators of innate immunity. Moreover, since hemato/lymphopoietic cells develop from a common stem cell, their behavior and fate are coregulated by intracellular innate immunity pathways. Therefore, the Nlrp3 inflammasome is functional both in immune cells and in HSPCs and affects hematopoiesis in either a positive or negative way, depending on its activity level. Specifically, while a physiological level of activation regulates the trafficking of HSPCs and most likely maintains their pool in the bone marrow, hyperactivation may lead to irreversible cell damage by pyroptosis and HSPC senescence and contribute to the origination of myelodysplasia and hematopoietic malignancies. Modulation of the level of Nrp3 inflammasome activation will enable improvements in HSPC mobilization, homing, and engraftment strategies. It may also control pathological activation of this protein complex during HSPC senescence, graft-versus-host disease, the induction of cytokine storms, and the development of hematopoietic malignancies.

Abstract IA15: Genetic predisposition to hematopoietic malignancies

Abstract There is a growing appreciation for the impact of baseline genetic variants in the development of hematopoietic malignancies, and the germline risk factors that make an individual more susceptible to developing bone marrow-derived cancers are being defined through highly informative families and next-generation sequencing with increasing frequency. At the University of Chicago, we use a rigorous pipeline for clinical testing of individuals comprising next-generation sequencing-based molecular testing to detect point mutations as well as microarrays to detect chromosomal rearrangements. Overall, we are now able to identify the causative germline mutation in about 20% of cases, a level commensurate with other cancer risk clinics, but hinting that additional predisposition alleles remain to be discovered. Many of our patients/families also contribute to research by banking samples for future studies designed to identify new predisposition syndromes. This presentation will include a discussion of the new insights into hematopoietic biology that can be appreciated by studying these emerging predisposition syndromes. Citation Format: Lucy A. Godley. Genetic predisposition to hematopoietic malignancies [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr IA15.

Desynchronization of Circadian Clocks in Cancer: A Metabolic and Epigenetic Connection.

Circadian clocks are innate oscillators that drive daily rhythms in metabolism, physiology, and behavior. 24-h rhythms in gene expression, driven by core clock transcription factors, reflect the epigenetic state of the cell, which in turn is dictated by the metabolic environment. Cancer cells alter their metabolic state and gene expression and therefore are likely to tweak circadian clock function in their favor. Over the past decade, we have witnessed an extraordinary increase in systems-level studies that suggest intricate mechanistic links between the cellular metabolome and the circadian epigenome. In parallel, reprogramming of cellular clock function in cancers is increasingly evident and the role of clock genes in the development of hematological tumors, as well as their pathophysiological effects on tissues distal to the tumor, has been described. Furthermore, the interplay between components of the circadian clock, metabolic enzymes, and oncogenes is starting to be better understood, such as the close association between overexpression of the Myc oncogene and perturbation of circadian and metabolic rhythms, thus opening new avenues to treat cancers. This review article explores current knowledge on the circadian metabolome and the molecular pathways they control, with a focus on their involvement in the development of hematopoietic malignancies.

The Effects of Galactic Cosmic Radiation Exposure on Hematopoietic Stem Cell Dysfunction and Oncogenesis

Natural sources of radiation in space include galactic cosmic rays (GCR), solar energetic particles (SPE) and trapped energetic particles in a planetary magnetic field. These different sources of space radiation consist of protons of various energies, particle nuclei of high energy and charge (HZE) and neutrons of different energies. These sources are difficult to shield because of their high energies and dense ionization patterns, thus posing significant health risks to astronauts on long term inter-planetary missions. Efforts to protect astronauts from harmful cosmic radiation require a deeper understanding of the effects of GCR on human health. In particular, very little is known about the effects of GCR exposure on the hematopoietic stem cell (HSC) population and whether disruptions in genetic stability in HSCs could result in the development of hematopoietic malignancies in astronauts on deep space missions.The average age of shuttle crew has risen above 46 years, and our work and others have shown that HSCs display diminished function with age. Recent data from our group has demonstrated that middle-aged individuals show frequent defects in DNA mismatch repair (MMR) in HSCs. MMR corrects DNA mismatches generated by DNA polymerase during replication which prevents mutations from becoming permanent in dividing cells. Thus, MMR plays a crucial role in the DNA damage response pathway to prevent short-term mutagenesis and long-term tumorigenesis. Several human MMR proteins have been identified as MutS and MutL homologues consisting of MSH2 and MLH1 heterodimers that functions in DNA mismatch/damage recognition, endonuclease activity and termination of mismatch-provoked excision. Our group has shown that humans accumulate microsatellite instability (MSI) with acquired loss of MLH1 protein in hematopoietic stem and progenitor cells as a function of age. Therefore, we employed a DNA mismatch repair deficient mouse model (MLH1+/- and MLH1-/-) to study the effects of different radiation sources including 56Fe, 28Si, 4He, 1H and γ-rays on HSCs to examine HSCs of potential astronaut population under GCR conditions. The complete blood count (CBC) data after 5 months and 9 months of whole body irradiation with different ions showed a slight dose-dependent decrease in all blood counts but absence of any significant difference in CBC of MLH1+/+ vs MLH1+/- mice. In addition, CFU and competitive repopulation data demonstrated a radiation quality effects on HSC function, but not an MLH1 effect. These results demonstrate that hematopoietic stem cell function is normal and that a MLH1 defect does not differentiate progenitor and mature effector cells following HZE radiation.To study long term effects of different ions on the potential for disease progression in a MLH1 dependent manner, we performed whole body irradiation with 56Fe, 28Si, 1H and γ-rays on MLH1+/+ and MLH1+/- mice and followed them up to 18 months post exposure. We observed that MLH1+/- mice show dramatic increases in lymphomagenesis 10-12 months after 56Fe irradiation compared to wild type mice, with greater than 60 % of MLH1+/- mice developed lymphomas at doses 10 cGy and 100 cGy compared to less than 10 % of wild type. For comparison, roughly 10 % of MLH1+/- mice developed lymphomas when mice were treated with whole body sparsely ionizing γ-rays at 100 cGy compared to none of the control. Thus the date show that MMR defects in HSCs lead to sensitization to radiation induced hematopoietic malignancy and that radiation quality effects exacerbate the sensitivity. The findings could have profound effects on astronaut screening, as well as lead to important questions regarding safety of ion therapy and development of second malignancies for cancer patients who remain on Earth. DisclosuresNo relevant conflicts of interest to declare.

Kinome-Wide shRNA Screen Reveals Functional Connections Between FANCA, Mitotic Centrosomes and the Spindle Assembly Checkpoint Tumor Suppressor Pathways

Fanconi anemia (FA) is a genetic disorder characterized by progressive bone marrow failure, congenital abnormalities and predilection towards development of hematopoietic malignancies, including acute myeloid leukemia (AML). Congenital biallelic disruption of the FA/BRCA signaling network causes Fanconi anemia and somatic mutations within the same genes are increasingly identified in a variety of malignancies in non-FA individuals, consistent with the critical role of this signaling pathway in FA and in the general population.The FA/BRCA tumor suppressor network orchestrates interphase DNA-damage repair (DDR) and DNA replication to maintain genomic stability. Additionally, we and others have demonstrated that the genome housekeeping function of FA/BRCA signaling extends beyond interphase: loss of FA/BRCA signaling perturbs execution of mitosis, including the spindle assembly checkpoint (SAC), centrosome maintenance, cytokinesis and resolution of anaphase DNA bridges. Interphase errors exacerbate mitotic abnormalities and mitotic failure promotes interphase mutagenesis. Consequently, we had demonstrated that primary FA patients' cells accumulate genomic abnormalities consistent with a dual mechanism of impaired interphase DDR/replication and defective mitosis. Previous detailed studies had elucidated multiple mechanisms of interphase DDR-dependent assembly and activation of the FA complex at DNA damage sites to arrest the cell cycle and repair DNA lesions. However, the signaling cross-talk nodes between the FA and mitotic checkpoint pathways remain to be discovered.In this study, we identified functionally relevant mitotic signaling defects resulting from FANCA deficiency via a synthetic lethal kinome-wide pooled shRNA screen in primary patient-derived FANCA -deficient cells compared to isogenic FANCA -corrected cell line. Bioinformatics analysis of our screen results followed by secondary validation of selected hits with alternative shRNAs and small-molecule inhibitors revealed conserved mitotic signal transduction pathways regulating the SAC and centrosome maintenance. Our super-resolution structured illumination (SR-SIM) microscopy coupled with deconvolution imaging revealed that a fraction of FANCA co-localizes with key SAC kinases at mitotic centrosomes and kinetochores, consistent with the role of FANCA in centrosome maintenance and the SAC. Co-immunoprecipitation assays identified the biochemical interaction between FANCA and an essential SAC kinase whose loss is synthetic lethal with FANCA deficiency, providing first insights into the interactions between FA signaling and the canonical SAC network.Together, our study has unraveled functional and biochemical connections between FANCA and the centrosome/SAC kinases, consistent with the essential role of FANCA in cell division. Our ongoing work is aimed at mechanistically dissecting molecular links between these two key tumor suppressor signaling pathways in more detail. We hypothesize that impaired FANCA/SAC cross-talk may contribute to genomic instability in FA-deficient cells and provide opportunities to selectively kill FANCA-/- cells. DisclosuresNo relevant conflicts of interest to declare.

The Chemokine Receptor Profile As Distinctive Criterion Between Normal B-Cell Subsets and As Potential Discriminative Marker to Identify the Cell of Origin in Patients with Chronic Lymphocytic Leukemia and Richter Syndrome

Chemokine receptors are G-protein-coupled cell surface receptors, which dissociate upon activation by their ligands and cause downstream signaling. Several studies have revealed the crucial contribution of chemokine receptors and their ligands in normal B-cell differentiation and development of hematopoietic malignancies. The Richter syndrome (RS) represents the clinico-pathologic transformation of chronic lymphocytic leukaemia (CLL) to an aggressive lymphoma, most commonly diffuse large B-cell lymphoma (DLBCL). Due to the lack of knowledge on the chemokine receptor, we aimed to investigate their expression profile in patients with CLL and Richter syndrome. Therefore, we investigated the mRNA expression levels of 18 known chemokine receptors (CCR1-CCR9, CXCR1-CXCR7, XCR1, CX3CR1) by using semi-quantitative real-time PCR on seven samples of paired (CLL and transformed DLBCL) RS samples, additionally four CLL samples -all of them subsequently transformed into DLBCL-, and eight transformed DLBCL samples originating from CLL. Additionally, 30 samples of de-novo DLBCL, including 10 germinal center B-cell (GCB) lymphomas, 12 non-germinal center B-cell lymphomas (non-GCB), and 8 unclassified DLBCL were included. Four samples of naïve B-cells (CD5 neg), CD5+ naïve B-cells and CD27+ memory B-cells (n=12) served as non-neoplastic controls.No differences in the chemokine receptor profile were detected between CD5+ and negative naïve B-cells. When comparing CD27+ memory B-cells to naïve B-cells a significant lower expression level was found for CCR7 (7-fold), CXCR4 (4-fold), and CXCR5 (1.5 fold). CCR7 (5-fold) and CXCR4 (5-fold) were also lower expressed in CD27+ memory B-cells compared to CD5+ naïve B-cells.Five out of 18 chemokine receptors were differentially expressed comparing the distinct normal B-cell subsets with RS samples. Comparing CLL samples and RS samples to CD5+ naïve B-cells, CXCR4 (12-fold for CLLs and 10-fold for RS samples) and CXCR5 (2-fold for CLLs and 2.4-fold for RS samples) were lower expressed, whereas CXCR3 (10-fold for CLLs and 8.5-fold for the transformed samples) was higher expressed and CCR5 de-novo expressed. Compared to naïve B-cells, the same chemokine receptors were deregulated: CXCR4 (10-fold for CLLs and 8.5-fold for the RS samples) and CXCR5 (2-fold for CLLs and 2.4-fold for the transformed samples) were lower expressed, CXCR3 (45-fold for CLLs and 30-fold for the transformed samples) was higher expressed and CCR5 was de-novo expressed. Comparing CLL samples and transformed RS samples to CD27+ memory B-cells, CCR5 (5.1-fold for CLLs and 4.3-fold for the RS samples) and CCR7 (8.7-fold for CLLs and 10-fold for the transformed samples) were higher expressed in both malignancies.Only one chemokine receptor was found to be differentially expressed in our seven paired RS samples: CCR6 showed a trend of a higher expression (1.4-fold) in CLL components.Considering RS and GCB DLBCL, CCR1, CCR5, and CXCR6 were found to be significantly down-regulated in RS (at least 4-fold), in contrast to CCR7 and CXCR4, which showed higher expression levels in RS (6-fold). CCR1 and CCR5 were lower expressed comparing RS and non-GCB DLBCL (25-fold and 8-fold), whereas CCR7 again, together with CXCR7, was higher expressed (3- fold and 6-fold respectively).Our data indicate a difference in the chemokine receptor profile within normal B-cell subsets. These differences are also reflected in the different expression profile of low and high aggressive component of CLL/RS compared to the distinct B cell subtypes. Hence, in future these multiple deregulated CC and CXC receptors might serve as a further hint in identifying the cell of origin of different B-cell malignancies. DisclosuresNo relevant conflicts of interest to declare.

Distinct Chemokine Receptor Profile In Chronic Lymphocytic Leukaemia and Richter Transformed Diffuse Large B Cell Lymphomas Compared To Germinal Center B Cells and De Novo Diffuse Large B Cell Lymphomas

Chemokine receptors are G-protein-coupled cell surface receptors, which dissociate upon activation by their ligands and cause downstream signaling. Recently, several studies have revealed the crucial contribution of chemokine receptors and their ligands in normal B-cell differentiation and development of hematopoietic malignancies. The Richter syndrome (RS) represents the clinico-pathologic transformation of chronic lymphocytic leukaemia (CLL) to an aggressive lymphoma, most commonly diffuse large B-cell lymphoma (DLBCL). Due to the lack of knowledge on the chemokine receptors in RS, we aimed to investigate their expression profile in Richter syndrome patients. Therefore, we investigated the mRNA expression levels of 18 known chemokine receptors (CCR1-CCR9, CXCR1-CXCR7, XCR1, CX3CR1) by using semi-quantitative real time PCR on seven samples of paired (CLL and transformed DLBCL) RS samples, and six samples of de novo DLBCL, additionally four CLL samples –all of them transformed into DLBCL-, and eight transformed DLBCL samples –all of them originated from CLL- were included. Four samples of peripheral B cells and four samples of germinal center B cells (GCB) served as non-neoplastic controls.11 out of 18 chemokine receptors were differentially expressed in CLL (RL) and transformed DLBCL originated from CLL (RH) compared to GCBs. RL exhibited an at least 15-fold higher expression of CCR2, CCR4, CCR7, CXCR3, and XCR1, as well as a de novo expression of CCR3, CCR8, CXCR1, CXCR2, and CX3CR1 compared to GCB. Additionally to these chemokine receptors, CCR1 also showed significant higher expression levels comparing GCB and RH samples. Only one chemokine receptor was found to be differentially expressed in our seven paired RS samples: CCR6 showed a trend of up-regulation in CLL components. Interestingly, the transformed DLBCL originated from CLL were characterized by a significant up-regulation of six chemokine receptors (CCR3, CCR7, CXCR1, CXCR3, CXCR4, and CX3CR1) and a down-regulation of CCR5 compared to DLBCL.Our data indicate that the chemokine receptor expression profile of CLL and transformed DLBCL, originated from CLL samples, differs substantially from those of non neoplastic germinal center B cells and de novo DLBCL, suggesting other cellular origin and an impact on more aggressive clinical course of Richter syndrome patients. Hence, these multiple deregulated CC and CXC receptor might serve as useful prognostic tool to separate high malignant Richter syndrome from de novo DLBCL. Disclosures:No relevant conflicts of interest to declare.

A Promoting Effect Of Spleen On Disseminated Leukemia Cells

Abnormal environment not only takes part in the malignant transformation of hematopoietic cells but also has great impact on the development of hematopoietic malignancies. Leukemia cells arise from a specific environment and compete with normal cells from the beginning of leukemogenesis. Then they induce leukemic environment favorable for the outgrowth of themselves. Both bone marrow (BM) and spleen are involved and other organs are also infiltrated by leukemia cells at late stage. Splenomegaly is more frequently observed in T cell acute lymphoblastic leukemia (T-ALL), and is related to the poor prognosis of patients. However, the mechanism is not clear. In this study, we used the non-radiated Notch1-induced mouse T-ALL model to study the effects of spleen and BM environment on the early residing, malignant phenotype and dissemination of leukemia cells.The gross change of organs during the development of leukemia was analyzed and the earliest, sustained and a final 12.25±0.6802-time increase in weight could be observed in spleen. Though leukemia cells could be detected in all organs analyzed at late stage, the earliest and sustained increase was detected in spleen but not in BM. Furthermore, two-photon fluorescence microscopy analysis showed that more leukemia cells were found in spleens but not in BM within 3 days post-transplantation. These results suggested that spleen may be more important for the initiation and early development of Notch1 induced T-ALL than any other organs. In vitro transwell experiment showed that more leukemia cells migrated to lower chambers with normal spleen cells than with normal BM cells. The concentration of a panel of cytokines/chemokines in BM, spleen and peripheral blood was screened pre-transplantation or within 3 days post-transplantation. Notably, the concentration of MIP-3β was higher in spleen sample at day 0, and it increased rapidly and kept at high levels within 3 days. Further evidence showed that leukemia cells expressed basal level of CCR7 and MIP-3β, and higher level of them could be observed when leukemia cells were co-cultured with spleen cells but not BM cells. Moreover, transwell experiment confirmed that MIP-3β promoted the migration of leukemia cells. Then we study the effects of spleen environment on leukemia cells. Spleen cells were more potent to stimulate the proliferation of leukemia cells than BM cells. Leukemia cells co-cultured with spleen cells were more potent to migrate in the in vitro migration assay than those co-cultured with BM cells. Leukemia cells freshly isolated from spleen expressed higher level of both MIP-3β and CCR7. When same amount of leukemia cells were injected to mice, spleen-origin leukemia cells caused shorter life span than BM-origin leukemia cells. Therefore, spleen environment promotes the malignant phenotype of leukemia cells. In vivo experiments were then carried out to see whether splenectomy had effect on the survival of leukemia mice. The survival time of mice in either group (splenectomy pre- or post-injection of leukemia cells) was significantly longer than that in the sham group. Furthermore, distinct effects could be observed in mice groups underwent splenectomy at both early stage and late stage post-injection of leukemia cells. Moreover, less severe invasion and destruction of organ structures in thymus, lymph nodes and BM could be observed in two splenectomy groups.In summary, our work demonstrates that spleen has a promoting effect on disseminated leukemia cells. The highly expressed MIP-3β in the spleen environment recruits leukemia cells. Subsequently, spleen environment promotes the proliferation of leukemia cells, stimulates the expression of CCR7, and enhances the migration ability of leukemia cells. The removal of spleen could prolong the survival of leukemia mice. Therefore, this study provides evidence for an organ specific effect on emerging leukemia cells in vivo and has implications for developing new treatments for leukemia patients. Disclosures:No relevant conflicts of interest to declare.

A Special Linker between Macrophage and Hematopoietic Malignant Cells: Membrane Form of Macrophage Colony-Stimulating Factor

The membrane form of macrophage colony-stimulating factor (mM-CSF) is an alternative splicing variant of this cytokine. Although its high expression was detected in hematopoietic malignancies, its physiologic and pathologic roles in hematopoietic system have not been established. In this report, stable transfectant clones expressing mM-CSF (Namalwa-M and Ramos-M) were obtained, which showed reduced proliferation potential in vitro. Moreover, the in vivo study showed that Namalwa-M and Ramos-M exhibited enhanced oncogenicity in tumor size in nude mice model, which could be inhibited by M-CSF monoclonal antibody. A remarkable increase in infiltrating macrophage and the vessel densities was found in tumor tissues formed by lymphoma cell lines that stably expressed mM-CSF, which suggested the involvement of macrophages in this process. The in vitro results using coculture system showed that macrophages could promote Namalwa-M and Ramos-M proliferation and activate extracellular signal-regulated kinase/mitogen-activated protein kinase signal pathway. In addition, the expression of murine origin vascular endothelial growth factor, basic fibroblast growth factor, and hepatocyte growth factor was elevated in Namalwa-M formed tumor tissues. These results suggested that mM-CSF should be a positive regulator in the development of hematopoietic malignancies by abnormally activating infiltrating macrophages, which in turn promote the malignant development. Thus, mM-CSF may be a critical linker between macrophages and malignant cells in the development of hematopoietic malignancies.

T(14;18) Translocations and Risk of Follicular Lymphoma

The chromosomal translocation t(14;18)(q32;q21) is characteristic of follicular lymphoma and a frequent abnormality in other types of non-Hodgkin lymphoma (NHL). In healthy individuals, the same translocation may also be found in a small fraction of peripheral blood lymphocytes, the biological significance of which is beginning to be explored. Translocation prevalence and frequency are potential risk factors for developing NHL. Here, we review the published data and describe recent and ongoing work on this promising biomarker. We have a series of studies in four major areas: 1) t(14;18) prevalence and frequency in healthy individuals; 2) maturation of translocation-harboring cells; 3) effect of rituximab treatment on t(14;18) carriage; and 4) predictive and clonotypic relationship between t(14;18) and follicular lymphoma or other NHL. Further studies are warranted to increase understanding of this crucial molecular event in the development of hematopoietic malignancies. Potential applications include determination of elevated risk for lymphoma, early detection of disease, and identification of molecular targets for preventive interventions.

Mitochondria Damage Leading to High Frequency of Lymphoma and Myeloproliferative Disorder in Transgenic Mice Overexpressing a Human 8-Oxoguanine DNA-Glycosylase Gene.

Alterations of nuclear genes in human lymphoma and leukemias have been well investigated in past several decades and established a predominant role in the pathogenesis. However, the relationship of mitochondrial genome alteration or dysfunction and human lymphoma and leukemias remains large unknown. Mitochondria are dynamic organelles that play critical roles in oxidative phosphorylation, energy metabolism, cell growth and apoptosis. We have successfully generated a novel transgenic (TG) mouse model of mitochondrial disorder by overexpressing human hOGG1, a base excision DNA repair gene, in the mitochondria of a wide variety of tissues in mice. We reported here the high frequency of essential thrombocythemia (ET)-like myeloproliferative disorder and lymphoma in the TG mice. TG mice were generated via pronuclear microinjection of a mammalian expression construct of a mitochondrial isoform of hOGG under the control of a mouse metallothionein-1 promoter. Peripheral blood smears were prepared from TG and non-TG control mice for platelet counts and morphologic evaluation. TG mice were sacrificed and various organs were harvested for histologic, biochemical and molecular studies. Two of the male founder mice successfully transmitted the transgene to their offspring at an expected frequency of 50%. All the female mice failed to reproduction. All TG mice expressed high levels of hOGG mRNA in their liver by RT-PCR and direct DNA sequencing. Over-expression of this gene produced a wide range of adverse biological phenotype, manifesting early-onset obesity, metabolic disturbance, female infertility and high frequency of ET-like myeloproliferative disorder and lymphoma (>90%) in nodal and extranodal sites. Eight TG mice (ranging from 1 to 2 years) become moribund and subsequently sacrificed. Upon histologic examination, the TG mice displayed moderate to severe splenomegaly. The spleen weights were 2-, 4- and 10 times respectively in the 3 TG founder mice as compared to wild type, age-matched mice. Extensive abdominal lymphadenopathy and numerous enlarged nodules involving liver, spleen, peritoneum, lung and diaphragm were identified. Microscopically, the red pulp in the enlarged spleens is markedly expanded with aggregates of large, mature but dysplastic-appearing megakaryocytes, focally disrupting the normal splenic structure. Various lymphomas ranged from low-grade lymphoma to high-grade Burkitt-like or lymphoblastic lymphomas were seen. Peripheral blood from these aged TG mice showed marked thrombocytosis and platelet clumping. In bone marrow, the aged TG mice displayed marked myeloid and megakaryocytic hyperplasia with relative erythroid hypoplasia in the absence of marrow fibrosis, indicative of myeloid and megakaryocytic proliferation. Bone marrow involvement by B-cell lymphoma was also seen. Of note, none of the above phenotype was seen in younger TG mice (4 months), indicating that the full development of this pathologic process is age-dependent. The molecular basis for this process is currently under investigation. Our preliminary data showed that mitochondrial DNA deletion or decrease in DNA copy numbers due to overexpressed hOGG1 and imbalance of base excision repair resulted in defects in mitochondrial respiration and increased ROS production. We thus hypothesize that oxidative stress caused by mitochondrial malfunction may play important part in the development of hematopoietic malignancies.

Tumor angiogenesis in the bone marrow of multiple myeloma patients and its alteration by thalidomide treatment.

Angiogenesis in solid tumors is important to tumor growth, invasion and metastasis. Recently, it has been suggested that angiogenesis plays a certain role in the development of hematopoietic malignancies, including leukemia and multiple myeloma. We evaluated tumor angiogenesis in the bone marrow (BM) of multiple myeloma (MM) patients by calculating microvessel density (MVD) in needle-biopsy specimens obtained from 51 cases of untreated MM or monoclonal gammopathy of undetermined significance (MGUS). The MVD in the BM of donors for transplantation and patients with non-hematological diseases was calculated as a control. There was an obvious increase in MVD in the BM of MM patients, and the MVD correlated with the grade of myeloma cell invasion of the BM in the untreated MM cases. It was recently reported that thalidomide might be effective for the treatment of MM. We assessed the effect of thalidomide on angiogenesis in BM treatment of 11 patients with refractory MM. The concentration of M-protein in the serum or urine of seven of the 11 patients was reduced by at least 30% after thalidomide treatment, and MVD in the BM decreased in three of these seven cases in response to thalidomide. Increased plasma concentrations of basic fibroblast growth factor (FGF-2) and vascular endothelial growth factor (VEGF) were observed in all 11 cases before thalidomide administration and both levels were reduced after treatment with thalidomide. Augmented angiogenesis in the bone marrow of MM patients was confirmed in the present study. It seems that thalidomide is effective in the treatment of MM through the impairment of angiogenesis by decreasing FGF-2 and VEGF production. This is the first report on pathological evidence in the bone marrow of MM before and after thalidomide treatment, in Japan.

Early hematopoietic zinc finger protein (EHZF), the human homolog to mouse Evi3, is highly expressed in primitive human hematopoietic cells

AbstractComparison of the gene expression repertoire in human hematopoietic progenitors and mature leukocytes led to identification of a transcript expressed in CD34+cells and undetectable in differentiated cells. Sequencing of the cDNA (termed EHZF: early hematopoietic zinc finger) revealed 30 zinc fingers with 96% homology to mouse Evi3, a recently identified gene associated with the retroviral integration site in AKXD-27 B-cell lymphomas. EHZF and Evi3 share high homology with the transcription cofactor OAZ, implicated in the control of olfactory epithelium and B-lymphocyte differentiation and in the bone morphogenic protein (BMP) signal transduction. Here we show that (1) EHZF expression is abundant in human CD34+ progenitors and declines rapidly during cytokine-driven differentiation; (2) significant mRNA levels are found in most acute myelogenous leukemias; (3) in response to BMPs EHZF complexes SMADs 1 and 4, binds to, and enhances the transcriptional activity of, a BMP2/4 responsive element; (4) EHZF inhibits the transcriptional activity of early B-cell factor (EBF), a transcription factor essential for specification of the B-cell lineage. Taken together, our data suggest that EHZF is likely to play a relevant role in the control of human hematopoiesis and might be implicated in the development of hematopoietic malignancies.

Cell cycle regulation: Mechanisms and therapeutics

Cyclin-dependent kinase 4 (Cdk4) is an important regulator of G1/S cell cycle progression of mammalian cells. To understand the role of Cdk4in vivo we generated strains of mice that either lack Cdk4 or express an activated form of this enzyme (Cdk4R24C), which cannot interact with the cycli kinase inhibitor, p16INK4A. Cdk4R24C mutations that abolish the interaction of pl6INK4A and CDK predispose humans to familial melanoma. Cdk4-/- mutant mice are viable but express defects in growth, fertility and are diabetic due to loss of insulin-producing islet cells. In contrast, CDK4 (R24C/R24C) mice exhibit growth advantage and their fibroblasts have a shorter cycle with high proliferative rates. The Cdk4 (R24C/R24C) fibroblasts fail to undergo senescence and are capable long-term growth in culture. Moreover, approximate 80% of Cdk4 (R24C/R24C) mice developed spontaneous tumors of varying cellular origin such as breast, pituitary, brain, skin, pancreas, liver, lung, testes, ovarian and hematopoietic tumors by 12 month. In addition, cdk4(R24C/R24C) mice are highly susceptible to TPA or DMBA carcinogen-induced tumors and Cdk4(R24C/R24C) mice developed skin tumors with average latency of 10–12 weeks. In contrast, wild type mice had a reduced frequency and latency of tumor formation. These studies indicated that the cdk4(R24C) mutation could exacerbate de-regulated Ras-pathways and accelerate tumorigenesisIn addition, Cdk4 (R24C/R24C) mice exhibit severe hyperplasia of the immune system and are highly susceptible to the development of hematopoietic malignancies. These results suggest that Cdk4 is an important modulator of cell cycle progression and abnormalities associated with this gene results in the development of abnormalities in multiple organs including the hematopoietic compartment.