Induced pluripotent stem cells and hematological malignancies: A powerful tool for disease modeling and drug development.

The derivation of human pluripotent stem cell (iPSC) lines by in vitro reprogramming of somatic cells revolutionized research: iPSCs have been used for disease modeling, drug screening and regenerative medicine for many disorders, especially when combined with cutting-edge genome editing technologies. In hematology, malignant transformation is often a multi-step process, that starts with either germline or acquired genetic alteration, followed by progressive acquisition of mutations combined with the selection of one or more pre-existing clones. iPSCs are an excellent model to study the cooperation between different genetic alterations and to test relevant therapeutic drugs. In this review, we will describe the use of iPSCs for pathophysiological studies and drug testing in inherited and acquired hematological malignancies.

The cell division control protein 42-Src family kinase-neural Wiskott-Aldrich syndrome protein pathway regulates human proplatelet formation.

Essentials The role of the cytoskeleton during megakaryocyte differentiation was examined. Human megakaryocytes are derived from in vitro cultured CD34+ cells. Cell division control protein 42 (CDC42) positively regulates proplatelet formation (PPF). Neural Wiskott-Aldrich syndrome protein, the main effector of CDC42 with Src positively regulates PPF. SUMMARY: Background Cytoskeletal rearrangements are essential for platelet release. The RHO small GTPase family, as regulators of the actin cytoskeleton, play an important role in proplatelet formation (PPF). In the neuronal system, CDC42 is involved in axon formation, a process that combines elongation and branching as for PPF. Objective To analyze the role of CDC42 and its effectors of the Wiskott-Aldrich syndrome protein (WASP) family in PPF. Methods Human megakaryocytes (MKs) were obtained from CD34+ cells. Inhibition of CDC42 in MKs was performed with the chemical inhibitor CASIN or with an active or a dominant-negative form of CDC42. The knock-down of N-WASP was obtained with a small hairpin RNA strategy Results Herein, we show that CDC42 activity increased during MK differentiation. The use of the chemical inhibitor CASIN or of an active or a dominant-negative form of CDC42 demonstrated that CDC42 positively regulated PPF in vitro. We determined that N-WASP, but not WASP, regulated PPF. We found that N-WASP knockdown led to a marked decrease in PPF, owing to a defect in the demarcation membrane system (DMS). This was associated with RHOA activation, and a concomitant augmentation in the phosphorylation of mysosin light chain 2. Phosphorylation of N-WASP, creating a primed form of N-WASP, increased during MK differentiation. Phosphorylation inhibition by two Src family kinase inhibitors decreased PPF. Conclusions We conclude that N-WASP positively regulates DMS development and PPF, and that the Src family kinases in association with CDC42 regulate PPF through N-WASP.

Mechanisms underlying platelet function defect in a pedigree with familial platelet disorder with a predisposition to acute myelogenous leukemia: potential role for candidate RUNX1 targets.

BACKGROUND: Familial platelet disorder with a predisposition to acute myelogenous leukemia (FPD/AML) is an inherited platelet disorder caused by a germline RUNX1 mutation and characterized by thrombocytopenia, a platelet function defect, and leukemia predisposition. The mechanisms underlying FPD/AML platelet dysfunction remain incompletely clarified. We aimed to determine the contribution of platelet structural abnormalities and defective activation pathways to the platelet phenotype. In addition, by using a candidate gene approach, we sought to identify potential RUNX1-regulated genes involved in these defects. METHODS: Lumiaggregometry, α-granule and dense granule content and release, platelet ultrastructure, αIIb ß3 integrin activation and outside-in signaling were assessed in members of one FPD/AML pedigree. Expression levels of candidate genes were measured and luciferase reporter assays and chromatin immunoprecipitation were performed to study NF-E2 regulation by RUNX1. RESULTS: A severe decrease in platelet aggregation, defective αIIb ß3 integrin activation and combined αδ storage pool deficiency were found. However, whereas the number of dense granules was markedly reduced, α-granule content was heterogeneous. A trend towards decreased platelet spreading was found, and ß3 integrin phosphorylation was impaired, reflecting altered outside-in signaling. A decrease in the level of transcription factor p45 NF-E2 was shown in platelet RNA and lysates, and other deregulated genes included RAB27B and MYL9. RUNX1 was shown to bind to the NF-E2 promoter in primary megakaryocytes, and wild-type RUNX1, but not FPD/AML mutants, was able to activate NF-E2 expression. CONCLUSIONS: The FPD/AML platelet function defect represents a complex trait, and RUNX1 orchestrates platelet function by regulating diverse aspects of this process. This study highlights the RUNX1 target NF-E2 as part of the molecular network by which RUNX1 regulates platelet biogenesis and function.

The abnormal proplatelet formation in MYH9-related macrothrombocytopenia results from an increased actomyosin contractility and is rescued by myosin IIA inhibition.

BACKGROUND: Mutations in the MYH9 gene cause autosomal dominant MYH9-related diseases (MYH9-RD) that associate macrothrombocytopenia with various other clinical conditions. The mechanisms giving rise to giant platelets remain poorly understood. OBJECTIVES/PATIENTS: To study the proplatelet formation (PPF) derived from megakaryocytes (MKs) generated in vitro from 11 patients with MYH9-RD with different mutations, compared with controls. METHODS: Proplatelet formation from cultured patients' MKs was evaluated with or without blebbistatin or the ROCK inhibitor Y27632. Myosin IIA and actin distribution were studied in spreading MKs on different surfaces by immunoconfocal analysis. Kinetic studies of contractility were performed on spreading MKs and the impact of blebbistatin on the maturation of the patients' MKs was evaluated by electron microscopy. RESULTS AND CONCLUSIONS: We show that in vitro MKs of 11 patients formed significantly fewer proplatelets than controls. MKs from MYH9-RD displayed an abnormal spreading on polylysine, fibronectin and collagen, with a disorganized actin network and a marked increase in stress fiber formation. Traction force microscopy studies demonstrated an elevated level of contractile forces in adherent mutated MKs. The myosin II inhibitor blebbistatin and the ROCK inhibitor Y27632 both rescued the proplatelet formation defect and normalized the ultrastructural characteristics of MYH9-RD MKs. Altogether, our results show that in MYH9-RD, mutations modify the overall MYH9 function and provoke a proplatelet defect through an excess of actomyosin contractility in spreading MKs. These results may promote new therapeutic strategies aimed at reducing this actomyosin contractility.

Thrombocytopenia induced by the histone deacetylase inhibitor abexinostat involves p53-dependent and -independent mechanisms.

Abexinostat is a pan histone deacetylase inhibitor (HDACi) that demonstrates efficacy in malignancy treatment. Like other HDACi, this drug induces a profound thrombocytopenia whose mechanism is only partially understood. We have analyzed its effect at doses reached in patient plasma on in vitro megakaryopoiesis derived from human CD34(+) cells. When added at day 0 in culture, abexinostat inhibited CFU-MK growth, megakaryocyte (MK) proliferation and differentiation. These effects required only a short incubation period. Decreased proliferation was due to induction of apoptosis and was not related to a defect in TPO/MPL/JAK2/STAT signaling. When added later (day 8), the compound induced a dose-dependent decrease (up to 10-fold) in proplatelet (PPT) formation. Gene profiling from MK revealed a silencing in the expression of DNA repair genes with a marked RAD51 decrease at protein level. DNA double-strand breaks were increased as attested by elevated γH2AX phosphorylation level. Moreover, ATM was phosphorylated leading to p53 stabilization and increased BAX and p21 expression. The use of a p53 shRNA rescued apoptosis, and only partially the defect in PPT formation. These results suggest that HDACi induces a thrombocytopenia by a p53-dependent mechanism along MK differentiation and a p53-dependent and -independent mechanism for PPT formation.

Developmental changes in human megakaryopoiesis.

BACKGROUND: The molecular bases of the cellular changes that occur during human megakaryocyte (MK) ontogeny remain unknown, and may be important for understanding the significance of MK differentiation from human embryonic stem cells (hESCs) METHODS: We optimized the differentiation of MKs from hESCs, and compared these with MKs obtained from primary human hematopoietic tissues at different stages of development. RESULTS: Transcriptome analyses revealed a close relationship between hESC-derived and fetal liver-derived MKs, and between neonate-derived and adult-derived MKs. Major changes in the expression profiles of cell cycle and transcription factors (TFs), including MYC and LIN28b, and MK-specific regulators indicated that MK maturation progresses during ontogeny towards an increase in MK ploidy and a platelet-forming function. Important genes, including CXCR4, were regulated by an on-off mechanism during development. DISCUSSION: Our analysis of the pattern of TF network and signaling pathways was consistent with a growing specialization of MKs towards hemostasis during ontogeny, and support the idea that MKs derived from hESCs reflect primitive hematopoiesis.

Primary hematopoietic cells from DBA patients with mutations in RPL11 and RPS19 genes exhibit distinct erythroid phenotype in vitro.

Diamond-Blackfan anemia (DBA) is caused by aberrant ribosomal biogenesis due to ribosomal protein (RP) gene mutations. To develop mechanistic understanding of DBA pathogenesis, we studied CD34⁺ cells from peripheral blood of DBA patients carrying RPL11 and RPS19 ribosomal gene mutations and determined their ability to undergo erythroid differentiation in vitro. RPS19 mutations induced a decrease in proliferation of progenitor cells, but the terminal erythroid differentiation was normal with little or no apoptosis. This phenotype was related to a G0/G1 cell cycle arrest associated with activation of the p53 pathway. In marked contrast, RPL11 mutations led to a dramatic decrease in progenitor cell proliferation and a delayed erythroid differentiation with a marked increase in apoptosis and G0/G1 cell cycle arrest with activation of p53. Infection of cord blood CD34⁺ cells with specific short hairpin (sh) RNAs against RPS19 or RPL11 recapitulated the two distinct phenotypes in concordance with findings from primary cells. In both cases, the phenotype has been reverted by shRNA p53 knockdown. These results show that p53 pathway activation has an important role in pathogenesis of DBA and can be independent of the RPL11 pathway. These findings shed new insights into the pathogenesis of DBA.

Regulation of megakaryocyte maturation and platelet formation.

Each day in every human, approximately 1 x 10(11) platelets are produced by the cytoplasmic fragmentation of megakaryocytes (MK), their marrow precursor cells. Platelets are the predominating factor in the process of hemostasis and thrombosis. Recent studies have shown that platelets also play a hitherto unsuspected role in several other processes such as inflammation, innate immunity, neoangiogenesis and tumor metastasis. The late phases of MK differentiation identified by polyploidization, maturation and organized fragmentation of the cytoplasm leading to the release of platelets in the blood stream represent a unique model of differentiation. The molecular and cellular mechanisms regulating platelet biogenesis are better understood and may explain several platelet disorders. This review focuses on MK polyploidization, and platelet formation, and discusses their alteration in some platelet disorders.

Occurrence of human papillomavirus 16 and 18 in smears from the two cervix regions of onco-gynecological patients in Slovakia.

We evaluated the relevance of tests for Human papillomavirus 16 and 18 (HPV-16, HPV-18) in two cervix regions (exocervical and endocervical) separately. The total of 142 cervical smears obtained from 91 women in Slovakia attending onco-gynecological outpatient care were examined for the presence of HPVs by PCR with the general primers GP5 and GP6 (GP5/6). The HPV-positive smears were examined for the presence of HPV-16 and HPV-18 and the results compared with cytological assessment. In 73 HPV-positive smears, the number of cases with detected HPV-16 was about three times higher in exocervix and about two times higher in endocervix in comparison with number of cases with detected HPV-18. In the smears considered as normal by cytology, two times higher occurrence of HPV-18 in endocervical smears was found in comparison with exocervical ones. Eight patients were double-infected with HPV-16 and HPV-18, but no patient was infected with these HPVs in both cervical regions. This finding emphasized the importance of examination of both cervical regions separately. Overlooking of the endocervical canal for the close examination by cytology and PCR might increase the failure to detect HPVs associated with adenocarcinoma.

p210(BCR-ABL) reprograms transformed and normal human megakaryocytic progenitor cells into erythroid cells and suppresses FLI-1 transcription.

The BCR-ABL oncoprotein exhibits deregulated protein tyrosine kinase activity and is implicated in the pathogenesis of Philadelphia chromosome (Ph)-positive human leukemias. Here, we report that ectopic expression of p210(BCR-ABL) in the megakaryoblastic Mo7e cell line and in primary human CD34(+) progenitors trigger erythroid differentiation at the expense of megakaryocyte (MK) differentiation. Clonal culture of purified CD41(+)CD42(-) cells, a population highly enriched in MK progenitors, combined with the conditional expression of p210(BCR-ABL) tyrosine kinase activity by imatinib identified a true lineage reprogramming. In both Mo7e or CD41(+)CD42(-) cells transduced with p210(BCR-ABL), lineage switching was associated with a downregulation of the friend leukemia Integration 1 (FLI-1) transcription factor. Re-expression of FLI-1 in p210(BCR-ABL)-transduced Mo7e cells rescued the megakaryoblastic phenotype. Altogether, these results demonstrate that alteration of signal transduction via p210(BCR-ABL) reprograms MK cells into erythroid cells by a downregulation of FLI-1. In addition, our findings underscore the role of kinases in lineage choice and infidelity in pathology and suggest that downregulation of FLI-1 may have important implications in CML pathogenesis.

Susceptibility of mouse mammary glands to murine gammaherpesvirus 72 (MHV-72) infection: evidence of MHV-72 transmission via breast milk.

Murine gammaherpesvirus 72 (MHV-72) is a virus of wild rodents and serves as a convenient small animal model to understand the pathogenesis of Epstein-Barr virus (EBV) and human herpesvirus 8 (HHV8) infection. In laboratory mice MHV-72 causes an acute infection of lung epithelial cells and establishes the latency in B lymphocytes. In this study, we investigated athymic nude and immunocompetent mice for distribution of virus in organs after infection with MHV-72. Ten days following subcutaneous dorsal injection of nude mice, virus replicated in lungs, lymphoid organs, salivary glands and also in mammary glands. The virus titre decreased by day 21 post-infection in former tissues, but increased in mammary glands. Presence of virus DNA sequences was detected in the lymphoid and non-lymphoid tissues until the death of the animals (about 1 month post-infection). Infection of immunocompetent mice with MHV-72 induced replication of virus up to 42 days post-infection in mammary glands reaching the highest level of infectious virus at day 8 post-infection. These data show that there is latent infection in mice never detected before. Moreover, virus DNA was detected using nested PCR (by amplification of a portion of gp150 gene sequence) in the mammary glands and the milk of mouse mothers infected with MHV-72 2 days before delivery. We demonstrated the presence of virus DNA also in the milk removed from the stomach of non-infected newborn mice, which were nourished by infected mothers (wet-nurses) for 1 or 2 days. The failure to detect the virus DNA in newborn mice lungs confirmed that they did not become infected from wet-nurses by the intranasal route. This suggests that MHV may be naturally transmitted to newborn mice via breast milk.

Immunophenotypic study of atypical lymphocytes. Generated in peripheral blood and spleen of nude mice After MHV-72 infection.

Inbred athymic nude mice (BALB/c) were injected subcutaneously with the wild-type murine gammaherpesvirus 72 (MHV-72), which has been shown to induce the infectious mononucleosis (IM)-like syndrome in immunocompetent mice. The mice were also injected with UV-irradiated MHV-72. We studied the pattern of acute and chronic infection in the blood cells of the nude mice and detected viral DNA sequences in the infected leukocytes by polymerase chain reaction (PCR) technique up to when the animal died, close to 1 month postinfection. Using the UV-irradiated virus that induces an increase in mouse survival time, the viral sequences were present in the blood up to 3 months postinfection, then disappeared. We detected atypical lymphocytes in the blood of mice infected with both wt and UV-irradiated viruses. These atypical cells were similar in shape to those present in the blood of patients with IM induced by Epstein-Barr virus (EBV). Via Unscheduled DNA Synthesis (UDS), DNA synthesis was demonstrated in the atypical cells whose phenotype is identical to that of B cells, as shown with a panel of monoclonal antibodies. By double immunofluorescence staining, using an hyperimmune anti-MHV-72 serum and an anti-IgG + IgM + IgA monoclonal antibody, we demonstrated that these atypical B cells express some viral antigens. Contrary to the immunocompetent mice, the nude mice did not develop splenomegaly after infection with wt virus, probably due to the lack of T cell subsets. However, we observed an increase of nude mice B cells in the spleen. The nude mice died 1 month postinfection showing a high frequency (40%) of atypical lymphoblast-like B-cells in the blood; the increase in natural killer (NK) cell number was not detected after infection. Such findings suggest that NK cells probably did not play an important role in immune response to the MHV infection in nude mice. Finally, this mouse model could play an important role in antigammaherpesviral therapy of immunocompromised patients.

The bystander effect mediated by the new murine gammaherpesvirus 72--thymidine kinase/5'-fluoro-2'-deoxyuridine (MHV72-TK/5-FUdR) system in vitro.

To investigate the potential of murine gamma-herpesvirus 72 thymidine kinase (MHV-72-TK) to act as a suicide gene, we used a mammalian expression vector on rat fibroblastoid cells deficient in the cellular TK gene. Substrate specificity was assessed in vitro in cells with stable expression of MHV-72-TK. The Herpes simplex virus 1-TK (HSV-1-TK) was used as a reference suicide gene. Unlike HSV-1-TK modified cells, which were sensitive to ganciclovir (GCV) (IC50=9.7 microM), cells modified by MHV-72-TK did not show sensitivity to this drug. The use of 3'-azido-3'-deoxythymidine (AZT) and (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) did not affect the growth of cells expressing either MHV-72-TK or HSV-1-TK in the range of concentration used for AZT (0-375 microM) and for BVDU (0-50 microM). In contrast, 5'-fluoro-2'-deoxyuridine (5-FUdR) was extremely cytotoxic and effectively killed MHV-72-TK expressing cells (IC50 value 2.1 microM). This value was 16 times lower than that required to kill cells expressing HSV1-TK. To test whether the bystander effect between two heterologous cell types could be mediated by the MHV-72-TK/5-FUdR system in vitro, cells expressing MHV-72-TK were co-cultured with the tumour fibroblastoid cell line NAD for 48 hours before the drug (10.8 microM) was added. The cell mixtures contained various ratios of cells expressing MHV-72-TK (0 to 50% of total cells). Only 1% of MHV-72-TK-expressing cells were needed to enhance mouse tumour cell killing and to decrease the survival rate to 25.6%. The bystander effect was more pronounced when 10% of cells expressing MHV-72-TK were used, decreasing survival to 17.4%. In parallel, the same concentration of 5-FUdR dose only marginally inhibited tumour cell growth in the absence of exogenous TK activity (84% survival). These results demonstrate the efficiency of MHV-72-TK as a suicide gene when 5-FUdR is used as a prodrug. When sequenced, MHV-72-TK proved to be identical to MHV-68 strain TK.

A murine gammaherpesvirus.

In 1976, within a project on isolation of herpesviruses from small rodents in former Czechoslovakia, the mouse herpesvirus strain 68 (MHV-68) was isolated (Blaskovic et al., 1980). This virus was accepted by The International Committee on Taxonomy of Viruses (ICTV) as a new, so far unassigned species (member) of the Gammaherpesvirinae subfamily of the Herpesviridae family (Murphy et al., 1995). Besides MHV-68, four more isolates (MHV-60, MHV-72, MHV-76, and MHV-78) similar to MHV-68 were obtained in that field experiment in Slovakia. Later, three more isolates (MHV-Sumava from Bohemia and MHV-4556 and MHV-5682 from Slovakia) were obtained in other field experiments. All these isolates are in some properties similar but in others different from each other. Nevertheless, as their comparative genome sequence analysis is not yet available, we propose at present to regard all the abovementioned isolates as different isolates of the same virus and strain, i.e. MHV-68. It is not excluded that a more detailed characterization of these isolates in the future will lead to proposals of designating some of these isolates as new strains of the virus of concern. This review summarizes the up to date knowledge of various biological and physico-chemical properties of MHV-68. At least three isolates, MHV-68, MHV-72 and MHV-Sumava seem to be involved in malignant neoplasm development in mice. It should be stressed that the pathogenesis of the induced lymphoproliferative disease in mice is similar to that caused by Epstein-Barr virus (EBV) in humans.