Somatic inactivation of Nf1 in hematopoietic cells results in a progressive myeloproliferative disorder

The NF1 tumor suppressor gene encodes a guanosine triphosphotase (GTPase)-activating protein that negatively regulates Ras signaling and is inactivated in a subset of juvenile myelomonocytic leukemias (JMMLs). Adoptive transfer of fetal liver cells from Nf1 mutant mice models JMML; however, this system has important limitations as a platform for performing biologic and preclinical studies. We have exploited the interferon-inducible Mx1-Cre transgene to ablate a conditional mutant Nf1 allele in hematopoietic cells. Somatic inactivation of Nf1 induces a myeloproliferative disorder with 100% penetrance that is associated with a sub-acute clinical course, tissue infiltration by myeloid cells, hypersensitivity to granulocyte-macrophage colony stimulating factor, hyperproliferation, and resistance to apoptosis. These Mx1-Cre, Nf1(flox/flox) mice establish a tractable experimental model for testing therapeutics and for identifying mutations that cooperate with hyperactive Ras in myeloid leukemogenesis.     

Endogenous oncogenic Nras mutation initiates hematopoietic malignancies in a dose- and cell type-dependent manner

Both monoallelic and biallelic oncogenic NRAS mutations are identified in human leukemias, suggesting a dose-dependent role of oncogenic NRAS in leukemogenesis. Here, we use a hypomorphic oncogenic Nras allele and a normal oncogenic Nras allele (Nras G12D(hypo) and Nras G12D, respectively) to create a gene dose gradient ranging from 25% to 200% of endogenous Nras G12D/+. Mice expressing Nras G12D(hypo)/G12D(hypo) develop normally and are tumor-free, whereas early embryonic expression of Nras G12D/+ is lethal. Somatic expression of Nras G12D/G12D but not Nras G12D/+ leads to hyperactivation of ERK, excessive proliferation of myeloid progenitors, and consequently an acute myeloproliferative disease. Using a bone marrow transplant model, we previously showed that ～ 95% of animals receiving Nras G12D/+ bone marrow cells develop chronic myelomonocytic leukemia (CMML), while ～ 8% of recipients develop acute T-cell lymphoblastic leukemia/lymphoma [TALL] (TALL-het). Here we demonstrate that 100% of recipients transplanted with Nras G12D/G12D bone marrow cells develop TALL (TALL-homo). Although both TALL-het and -homo tumors acquire Notch1 mutations and are sensitive to a γ-secretase inhibitor, endogenous Nras G12D/+ signaling promotes TALL through distinct genetic mechanism(s) from Nras G12D/G12D. Our data indicate that the tumor transformation potential of endogenous oncogenic Nras is both dose- and cell type-dependent.     

Retroviral transduction of TLS-ERG initiates a leukemogenic program in normal human hematopoietic cells

Many chimeric oncogenes have been identified by virtue of the association between chromosomal translocation and specific human leukemias. However, the biological mechanism by which these oncogenes disrupt the developmental program of normal human hematopoietic cells during the initiation of the leukemogenic process is poorly understood due to the absence of an appropriate experimental system to study their function. Here, we report that retroviral transduction of TLS-ERG, a myeloid leukemia-associated fusion gene, to human cord blood cells results in altered myeloid and arrested erythroid differentiation and a dramatic increase in the proliferative and self-renewal capacity of transduced myeloid progenitors. Thus, TLS-ERG expression alone induced a leukemogenic program that exhibited similarities to the human disease associated with this translocation. These results provide an experimental examination of the early stages of the human leukemogenic process induced by a single oncogene and establish a paradigm to functionally assay putative leukemogenic genes in normal human hematopoietic cells.     

Rapidly fatal myeloproliferative disorders in mice with deletion of Casitas B-cell lymphoma (Cbl) and Cbl-b in hematopoietic stem cells

Casitas B-cell lymphoma (Cbl)-family E3 ubiquitin ligases are negative regulators of tyrosine kinase signaling. Recent work has revealed a critical role of Cbl in the maintenance of hematopoietic stem cell (HSC) homeostasis, and mutations in CBL have been identified in myeloid malignancies. Here we show that, in contrast to Cbl or Cbl-b single-deficient mice, concurrent loss of Cbl and Cbl-b in the HSC compartment leads to an early-onset lethal myeloproliferative disease in mice. Cbl, Cbl-b double-deficient bone marrow cells are hypersensitive to cytokines, and show altered biochemical response to thrombopoietin. Thus, Cbl and Cbl-b play redundant but essential roles in HSC regulation, whose breakdown leads to hematological abnormalities that phenocopy crucial aspects of mutant Cbl-driven human myeloid malignancies.     

Photosensitivity and acute liver injury in myeloproliferative disorder secondary to late-onset protoporphyria caused by deletion of a ferrochelatase gene in hematopoietic cells

Late-onset erythropoietic protoporphyria (EPP) is a rare complication of myelodysplastic syndrome (MDS) but has not been described in association with a myeloproliferative disorder (MPD). EPP is normally an inherited disorder characterized by photosensitivity that starts in early childhood and results from overproduction of protoporphyrin secondary to ferrochelatase (FECH) deficiency. Severe liver disease occurs in 1% to 2% of patients. Here we report that severe photosensitivity and cholestatic liver disease in a patient with a myeloproliferative disorder was caused by excess protoporphyrin production from a clone of hematopoietic cells in which one FECH allele had been deleted. Our observations suggest that the usual explanation for the association of late-onset EPP with MPD and MDS is acquired somatic mutation of one FECH allele in bone marrow and show for the first time that the consequent overproduction of protoporphyrin may be severe enough to cause acute liver damage.     

FLT3 expression initiates in fully multipotent mouse hematopoietic progenitor cells

Lymphoid-primed multipotent progenitors with down-regulated megakaryocyte-erythroid (MkE) potential are restricted to cells with high levels of cell-surface FLT3 expression, whereas HSCs and MkE progenitors lack detectable cell-surface FLT3. These findings are compatible with FLT3 cell-surface expression not being detectable in the fully multipotent stem/progenitor cell compartment in mice. If so, this process could be distinct from human hematopoiesis, in which FLT3 already is expressed in multipotent stem/progenitor cells. The expression pattern of Flt3 (mRNA) and FLT3 (protein) in multipotent progenitors is of considerable relevance for mouse models in which prognostically important Flt3 mutations are expressed under control of the endogenous mouse Flt3 promoter. Herein, we demonstrate that mouse Flt3 expression initiates in fully multipotent progenitors because in addition to lymphoid and granulocyte-monocyte progenitors, FLT3(-) Mk- and E-restricted downstream progenitors are also highly labeled when Flt3-Cre fate mapping is applied.     

Differentiation of blast cells from a Down's syndrome patient with transient myeloproliferative disorder

A male neonate with Down's syndrome and congenital myeloproliferative disorder was studied. His blood picture showed the unique coexistence of leukocytosis with matured cells and a large number of blast cells. The in vitro proliferation and differentiation of blast cells into various lineages in the presence of phytohemagglutinin-stimulated leukocyte conditioned medium (PHA-LCM) was examined by using a liquid culture and a methylcellulose culture system. The differentiation of blast cells into myeloid cells was confirmed by specific cytochemical stainings, electron microscopy, and an immunologic study. No specific factors in the plasma of the patient promoted the proliferation or differentiation of blast cells. The cellular composition of colonies grown in methylcellulose culture from single blast cells was studied by a micromanipulation technique. High plating efficiency was observed. Of 136 cultures, 78 showed colony growth. Half of the blast cells were colony-forming cells that could proliferate and differentiate into basophils, neutrophils, eosinophils, macrophages, and erythrocytes in the presence of PHA-LCM. Using the blast cells with a high differentiation capacity to the basophil pathway, we studied the effect of recombinant granulocyte-macrophage colony-stimulating factor (GM- CSF). Recombinant GM-CSF support neutrophils, eosinophils, and macrophages but not typical basophils. These findings of the cell differentiation of blast cells into various kinds of cells in vitro were in agreement with the finding of neutrophilia, eosinophilia, basophilia, and thrombocythemia in this patient.     

Hypereosinophilia in a monosomy 7 myeloproliferative disorder in childhood

Hyperosinophilia in association with a monosomy 7 myeloproliferative disorder is described in an eight-year-old boy. Antileukemic treatment resulted in a decrease in the eosinophilia, but did not appear to affect the blast cell population or the presence of monosomy 7 cells in the bone marrow. These findings suggest that bone marrow karyotyping may be useful in differentiating pre-malignant causes of eosinophilia from those with a more benign etiology.     

Dysregulated Mpl-Ligand production by hemopoietic cellsinduces a fatal myeloproliferative syndrome in mice

To evaluate the effects of long-term high-dose exposure to Mpl-ligand also called thrombopoietin (TPO), C57BL/6J murine marrow cells were infected with a retrovirus carrying the murine TPO gene. Mice were treated 4 days by 5-FU and marrow cells were then infected by coculture using a MPZen vector containing the murine TPO cDNA. Non adherent marrow cells were transplanted into lethally irradiated recipients. A majority of hematopoietic cells in the marrow, spleen, thymus and blood was transduced by the retroviral vector, one and three months after reconstitution. Plasma TPO activity in transplanted mice was extremely high (10⁴ U/ml). A disease with two distinct steps was observed. During the two first months after transplantation, platelet (plt) and white blood cell (WBC) counts increased 4- and 10-fold, respectively. Abnormal platelet size and granules were observed. Spleen weight increased 4-fold and marrow cellularity decreased 5-fold. Histology revealed hyperplasia of the megakaryocytic and myeloid cells. Total numbers of CFU-MK and CFU-GM increased. In contrast, the hematocrit progressively fell accompanied by a decrease in the erythroblasts and CFU-E numbers. Beginning two months after transplantation, plt and WBC numbers also declined. Thrombocytopenia was noted 5 months after transplantation. The Hcts continued to decrease. Few cells were isolated from the marrow cavities and spleens. Histology revealed fibrosis of the marrow and spleen and significant osteosclerosis of the marrow. An extramedullary hematopoiesis was observed in numerous organs such as the liver or the kidney. Total numbers of progenitors were very low in hematopoietic organs. Mice died 7 months after transplantation with severe pancytopenia. Two early deaths were observed with a marked increase in blast cells. This disorder was transplantable into secondary recipients who developed an attenuated form of the disease similar to the one previously described [Yan et al (1995) Blood 86: 4025]. In conclusion, dysregulated TPO production by hemopoietic cells in mice results in a fatal myeloproliferative disease which mimics the clinical evolution of idiopathic myelofibrosis observed in man.     

Forced expression of Nanog in hematopoietic stem cells results in a gammadeltaT-cell disorder

Nanog is a key molecule involved in the maintenance of the self-renewal of undifferentiated embryonic stem (ES) cells. In this work we investigate whether Nanog can enhance self-renewal in hematopoietic stem cells. Contrary to our expectation, no positive effect of Nanog transduction was detected in bone marrow reconstitution assays. However, recipients of Nanog-transduced (Nanog) hematopoietic stem cells (HSCs) invariantly develop a unique disorder typified by an atrophic thymus occupied by Nanog-expressing gammadeltaT-cell receptor-positive (TCR(+)) cells (Nanog T cells). All thymi are eventually occupied by Nanog T cells with CD25(+)CD44(+) surface phenotype that home selectively to the thymus on transfer and suppress normal thymocyte development, which is partly ascribed to destruction of the microenvironment in the thymus cortex. Moreover, this initial disorder invariantly develops to a lymphoproliferative disorder, in which Nanog T cells undergo unlimited proliferation in the peripheral lymphoid tissues and eventually kill the host. This invariable end result suggests that Nanog is a candidate oncogene for gammadeltaT-cell malignancy.     

Retinal microangiopathy and rapidly fatal cerebral edema in a patient with adult-onset Still's disease and concurrent macrophage activation syndrome

Hemophagocytic lymphohistiocytosis (HLH) is a complex inflammatory disease with multiple diagnostic and therapeutic pitfalls. The congenital form, referred to as familial hemophagocytic lymphohistiocytosis (FHL), is often associated with cerebromeningeal involvement, whereas neurological complications are not characteristic of the adult form of secondary HLH (sHLH). Here we report the case of a 20-year-old woman with adult-onset Still's disease (AOSD), retinal microangiopathy and concurrent macrophage activation syndrome (MAS), in the context of sHLH. Following treatment with etanercept, ibuprofen, methylprednisolone, and phenylbutazone for 3 weeks, MAS deteriorated and fatal cerebral edema occurred within only 24 h. The clinical signs and neuropathological findings are discussed with special emphasis on possible relationships between the aggravation of MAS and therapeutic interventions for AOSD. In conclusion, even the slightest sign of mental decline in a patient with AOSD must be considered central nervous system MAS which can be rapidly fatal.     

STING activation of tumor endothelial cells initiates spontaneous and therapeutic antitumor immunity

Spontaneous CD8 T-cell responses occur in growing tumors but are usually poorly effective. Understanding the molecular and cellular mechanisms that drive these responses is of major interest as they could be exploited to generate a more efficacious antitumor immunity. As such, stimulator of IFN genes (STING), an adaptor molecule involved in cytosolic DNA sensing, is required for the induction of antitumor CD8 T responses in mouse models of cancer. Here, we find that enforced activation of STING by intratumoral injection of cyclic dinucleotide GMP-AMP (cGAMP), potently enhanced antitumor CD8 T responses leading to growth control of injected and contralateral tumors in mouse models of melanoma and colon cancer. The ability of cGAMP to trigger antitumor immunity was further enhanced by the blockade of both PD1 and CTLA4. The STING-dependent antitumor immunity, either induced spontaneously in growing tumors or induced by intratumoral cGAMP injection was dependent on type I IFNs produced in the tumor microenvironment. In response to cGAMP injection, both in the mouse melanoma model and an ex vivo model of cultured human melanoma explants, the principal source of type I IFN was not dendritic cells, but instead endothelial cells. Similarly, endothelial cells but not dendritic cells were found to be the principal source of spontaneously induced type I IFNs in growing tumors. These data identify an unexpected role of the tumor vasculature in the initiation of CD8 T-cell antitumor immunity and demonstrate that tumor endothelial cells can be targeted for immunotherapy of melanoma.Metastatic melanoma is a highly aggressive cancer with a fast increasing incidence worldwide. Unless diagnosed early and surgically resected, the disease becomes metastatic and life threatening. Both chemotherapy and irradiation are ineffective. Novel therapies that target oncogenic drivers have brought some improvements, but tumor cells escape regularly (1). Melanoma is a prototypical immunogenic tumor, as shown by the occurrence of spontaneous CD8 T-cell responses that drive tumor regressions and by the identification of CD8 T cells that recognize melanoma antigens (2, 3). Although many immunotherapeutic strategies have been developed to induce such responses, clinical efficacies have been poor. More recently, “checkpoint blockade” therapies that target T-cell–inhibitory pathways mediated by CTLA4 (4) and PD1 (5) have yielded encouraging clinical results and demonstrated that spontaneous CD8 T-cell responses in tumors can be boosted to treat melanoma.The mechanisms that drive spontaneous antitumor immune responses are poorly understood. Type I IFNs (IFN-α and IFN-β) may play a role as the expression type I IFN-related genes in primary melanoma has been associated with spontaneous tumor regressions (6) and correlated to the tumor infiltration by specific CD8+ T cells (6, 7). Furthermore, the lack of type I IFN signaling or IFN-β expression inhibited the generation of tumor-specific CD8 T cells and accelerated tumor growth in a murine melanoma model (7–9).Type I IFNs are typically induced upon recognition of nucleic acids in intracellular compartments. Several cytosolic DNA receptors have been identified and include DNA-dependent activator of IFN-regulatory factors (DAI) (10), gamma-interferon-inducible protein-16 (IFI16) (11), and the helicase DEAD (Asp-Glu-Ala-Asp) box protein 41 (DDX41) (12). These cytosolic receptors trigger IFN-β production via a signaling cascade that involves the master adaptor molecule stimulator of IFN genes (STING), which binds to tank-binding kinase 1 (TBK1) and induces phosphorylation of the interferon regulatory factor 3 (IRF3) (13). STING associates weakly to dsDNA (14) but strongly binds the endogenous cyclic dinucleotide GMP-AMP (cGAMP) synthesized by the cGMP-AMP synthase (cGAS) (15, 16).Recently, spontaneous STING activation was found to be required for the spontaneous induction of antitumor immunity (17). Activation of STING occurred via tumor DNA-dependent cGAS activation and generation of endogenous cGAMP (17, 18). However, the cellular mechanism underlying this response and whether this mechanism could be exploited to generate more efficient antitumor immune responses is currently unknown.Here, we show that intratumoral injection of exogenous cGAMP enhanced STING activation and strongly promoted the generation of antitumor CD8 T-cell responses leading to efficient growth control of injected and contralateral tumors. This response was dependent on IFN-β produced by tumor endothelial cells. In fact, endothelial cells were found to be the principal producers of type I IFN in response to both spontaneous and enforced STING activation, suggesting a role of the tumor vasculature in the initiation of antitumor immunity.     

Transient myeloproliferative disorder (TMD) with transiently increased tetrasomy-21 cells in a phenotypically normal newborn

We reported a case of TMD with transient increase of tetrasomy-21 cells in a phenotypically normal newborn. The patient was admitted to the St. Marianna University Hospital due to hepatosplenomegaly on the 7th days after birth. Hematological findings on admission revealed remarkable leukocytosis (168,300/microliters) with 79% blasts. Immunological studies of the blasts showed a positive reaction for platelet associated antigens, KOR-P77, AN50, TP80 and a pan-T antigen, TP40. Cytochemically blasts were strongly positive for acid phosphatase, positive for alpha NAE and weakly positive for PAS. The platelet peroxidase reaction was observed in rough endoplasmic reticulum of blast cells. Both immunological and cytochemical findings suggested that the blasts were of megakaryocyte lineage. Chromosomal analysis of the blasts showed 48, XX, + 21, +21 (21 tetrasomy). After chemotherapy with PSL and 6MP, bone marrow showed a complete remission. But we thought it was spontaneous remission because PSL and 6MP were not effective to acute megakaryocytic leukemia (AMKL). Bone marrow cells were karyotypically normal on the 67th day of life when abnormal blasts were not observed in the bone marrow.     

The SKI proto-oncogene enhances the in vivo repopulation of hematopoietic stem cells and causes myeloproliferative disease

The proto-oncogene SKI is highly expressed in human myeloid leukemia and also in murine hematopoietic stem cells. However, its operative relevance in these cells remains elusive. We have over-expressed SKI to define its intrinsic role in hematopoiesis and myeloid neoplasms, which resulted in a robust competitive advantage upon transplantation, a complete dominance of the stem and progenitor compartments, and a marked enhancement of myeloid differentiation at the expense of other lineages. Accordingly, enforced expression of SKI induced a gene signature associated with hematopoietic stem cells and myeloid differentiation, as well as hepatocyte growth factor signaling. Here we demonstrate that, in contrast to what has generally been assumed, the significant impact of SKI on hematopoiesis is independent of its ability to inhibit TGF-beta signaling. Instead, myeloid progenitors expressing SKI are partially dependent on functional hepatocyte growth factor signaling. Collectively our results demonstrate that SKI is an important regulator of hematopoietic stem cell activity and its overexpression leads to myeloproliferative disease.     

Latent period of 9 years in the presentation of a myeloproliferative disorder

A woman developed a leukaemic infiltrate at the site of a smallpox vaccination; the lesion subsequently regressed spontaneously. 9 years later she developed a generalised myeloproliferative disorder.