Lifestyle factors and risk of myeloproliferative neoplasms in the NIH-AARP diet and health study.

The etiology of Philadelphia chromosome-negative myeloproliferative neoplasms (MPN) is largely unknown. We assessed potential associations between lifestyle factors and MPN risk in the NIH-AARP Diet and Health Study. In this prospective cohort with 463,049 participants aged 50-71 years at baseline (1995-1996) and a median follow-up of 15.5 years, we identified 490 MPN cases, including 190 with polycythemia vera (PV) and 146 with essential thrombocythemia (ET). Multivariable Cox proportional hazards regression models were used to estimate hazard ratios (HRs) and 95% confidence intervals (CIs). Smoking was not associated with MPN risk in the overall cohort, but analyses stratified by sex suggested that smoking increased the risk of MPN in women (former smoker vs. nonsmokers, HR = 1.43, 95% CI: 1.03-2.00, p = 0.03; current smokers vs. nonsmokers, HR = 1.71, 95% CI: 1.08-2.71, p = 0.02). Coffee consumption was inversely associated with the risk of PV (high vs. low intake, HR = 0.53, 95% CI: 0.33-0.84, p-trend < 0.01), but not the risk of ET or MPN overall. Further analysis revealed an inverse association between the amount of caffeine intake and PV risk (high vs. low intake, HR = 0.55, 95% CI: 0.39-0.79, p-trend < 0.01). While the consumption of caffeinated coffee appeared to confer a protective effect against PV, the consumption of decaffeinated coffee did not. This large prospective study identified smoking as a risk factor for MPN in women and suggests that caffeine intake is associated with a lower risk of PV.

Epigenetic therapy restores normal hematopoiesis in a zebrafish model of NUP98-HOXA9-induced myeloid disease.

Acute myeloid leukemia (AML) occurs when multiple genetic aberrations alter white blood cell development, leading to hyperproliferation and arrest of cell differentiation. Pertinent animal models link in vitro studies with the use of new agents in clinical trials. We generated a transgenic zebrafish expressing human NUP98-HOXA9 (NHA9), a fusion oncogene found in high-risk AML. Embryos developed a preleukemic state with anemia and myeloid cell expansion, and adult fish developed a myeloproliferative neoplasm (MPN). We leveraged this model to show that NHA9 increases the number of hematopoietic stem cells, and that oncogenic function of NHA9 depends on downstream activation of meis1, the PTGS/COX pathway and genome hypermethylation through the DNA methyltransferase, dnmt1. We restored normal hematopoiesis in NHA9 embryos with knockdown of meis1 or dnmt1, as well as pharmacologic treatment with DNA (cytosine-5)-methyltransferase (DNMT) inhibitors or cyclo-oxygenase (COX) inhibitors. DNMT inhibitors reduced genome methylation to near normal levels. Strikingly, we discovered synergy when we combined sub-monotherapeutic doses of a histone deacetylase inhibitor plus either a DNMT inhibitor or COX inhibitor to block the effects of NHA9 on zebrafish blood development. Our work proposes novel drug targets in NHA9-induced myeloid disease, and suggests rational therapies by combining minimal doses of known bioactive compounds.

Mechanistic insights into activation and SOCS3-mediated inhibition of myeloproliferative neoplasm-associated JAK2 mutants from biochemical and structural analyses.

JAK2 (Janus kinase 2) initiates the intracellular signalling cascade downstream of cell surface receptor activation by cognate haemopoietic cytokines, including erythropoietin and thrombopoietin. The pseudokinase domain (JH2) of JAK2 negatively regulates the catalytic activity of the adjacent tyrosine kinase domain (JH1) and mutations within the pseudokinase domain underlie human myeloproliferative neoplasms, including polycythaemia vera and essential thrombocytosis. To date, the mechanism of JH2-mediated inhibition of JH1 kinase activation as well as the susceptibility of pathological mutant JAK2 to inhibition by the physiological negative regulator SOCS3 (suppressor of cytokine signalling 3) have remained unclear. In the present study, using recombinant purified JAK2JH1-JH2 proteins, we demonstrate that, when activated, wild-type and myeloproliferative neoplasm-associated mutants of JAK2 exhibit comparable enzymatic activity and inhibition by SOCS3 in in vitro kinase assays. SAXS (small-angle X-ray scattering) showed that JAK2JH1-JH2 exists in an elongated configuration in solution with no evidence for interaction between JH1 and JH2 domains in cis. Collectively, these data are consistent with a model in which JAK2's pseudokinase domain does not influence the activity of JAK2 once it has been activated. Our data indicate that, in the absence of the N-terminal FERM domain and thus cytokine receptor association, the wild-type and pathological mutants of JAK2 are enzymatically equivalent and equally susceptible to inhibition by SOCS3.

Role of SHP2 phosphatase in KIT-induced transformation: identification of SHP2 as a druggable target in diseases involving oncogenic KIT.

Intracellular mechanism(s) that contribute to promiscuous signaling via oncogenic KIT in systemic mastocytosis and acute myelogenous leukemia are poorly understood. We show that SHP2 phosphatase is essential for oncogenic KIT-induced growth and survival in vitro and myeloproliferative disease (MPD) in vivo. Genetic disruption of SHP2 or treatment of oncogene-bearing cells with a novel SHP2 inhibitor alone or in combination with the PI3K inhibitor corrects MPD by disrupting a protein complex involving p85α, SHP2, and Gab2. Importantly, a single tyrosine at position 719 in oncogenic KIT is sufficient to develop MPD by recruiting p85α, SHP2, and Gab2 complex to oncogenic KIT. Our results demonstrate that SHP2 phosphatase is a druggable target that cooperates with lipid kinases in inducing MPD.

Growth factor independence 1 protects hematopoietic stem cells against apoptosis but also prevents the development of a myeloproliferative-like disease.

The regulation of gene transcription is elementary for the function of hematopoietic stem cells (HSCs). The transcriptional repressor growth factor independence 1 (Gfi1) restricts HSC proliferation and is essential to maintain their self-renewal capacity and multipotency after transplantation. In addition, Gfi1(-/-) HSCs are severely compromised in their ability to compete with wild-type (wt) HSCs after transplantation. We now report that Gfi1 protects HSCs against stress-induced apoptosis, probably, by repressing the proapoptotic target gene Bax, since irradiated Gfi1(-/-) HSCs display higher expression of Bax and show a higher rate of apoptosis than wt HSCs. This protective function of Gfi1 appears to be functionally relevant since Gfi1(-/-) HSCs that express Bcl-2, which antagonizes the effects of Bax, regain their ability to self renew and to initiate multilineage differentiation after transplantation. Surprisingly, Gfi1(-/-) xBcl-2 transgenic mice also show a strong, systemic expansion of Mac-1(+) Gr-1(-) myeloid cells in bone marrow and peripheral lymphoid organs. These cells express high levels of the proleukemogenic transcription factor Hoxa9 and, in older mice, appear as atypical monocytoid-blastoid cells in the peripheral blood. As a result of this massive expansion of myeloid cells, all Gfi1(-/-) xBcl-2 mice eventually succumb to a myeloproliferative-like disease resembling a preleukemic state. In summary, our data demonstrate that Gfi1's ability to protect against apoptosis is essential for HSC function. In addition, our finding show that Gfi1 prevents the development of myeloproliferative diseases and provides evidence how Gfi1 deficiency could be linked to myeloid leukemia.

NCI first international workshop on the biology, prevention, and treatment of relapse after allogeneic hematopoietic stem cell transplantation: report from the committee on disease-specific methods and strategies for monitoring relapse following allogeneic stem cell transplantation. part II: chronic leukemias, myeloproliferative neoplasms, and lymphoid malignancies.

Relapse has become the major cause of treatment failure after allogeneic hematopoietic stem cell transplantation. Outcome of patients with clinical relapse after transplantation generally remains poor, but intervention prior to florid relapse improves outcome for certain hematologic malignancies. To detect early relapse or minimal residual disease, sensitive methods such as molecular genetics, tumor-specific molecular primers, fluorescence in situ hybridization (FISH), and multiparameter flow cytometry (MFC) are commonly used after allogeneic stem cell transplantation to monitor patients, but not all of them are included in the commonly employed disease-specific response criteria. The highest sensitivity and specificity can be achieved by molecular monitoring of tumor- or patient-specific markers measured by polymerase chain reaction-based techniques, but not all diseases have such targets for monitoring. Similar high sensitivity can be achieved by determination of recipient-donor chimerism, but its specificity regarding detection of relapse is low and differs substantially among diseases. Here, we summarize the current knowledge about the utilization of such sensitive monitoring techniques in chronic leukemias, myeloproliferative neoplasms, and lymphoid malignancies based on tumor-specific markers and cell chimerism and how these methods might augment the standard definitions of posttransplant remission, persistence, progression, relapse, and the prediction of relapse. Critically important is the need for standardization of the different residual disease techniques and to assess the clinical relevance of minimal residual disease and chimerism surveillance in individual diseases, which in turn must be followed by studies to assess the potential impact of specific interventional strategies.

JunB protects against myeloid malignancies by limiting hematopoietic stem cell proliferation and differentiation without affecting self-renewal.

Loss of the JunB/AP-1 transcription factor induces a myeloproliferative disease (MPD) arising from the hematopoietic stem cell (HSC) compartment. Here, we show that junB inactivation deregulates the cell-cycle machinery and increases the proliferation of long-term repopulating HSCs (LT-HSCs) without impairing their self-renewal or regenerative potential in vivo. We found that JunB loss destabilizes a complex network of genes and pathways that normally limit myeloid differentiation, leading to impaired responsiveness to both Notch and TGF-beta signaling due in part to transcriptional deregulation of the Hes1 gene. These results demonstrate that LT-HSC proliferation and differentiation are uncoupled from self-renewal and establish some of the mechanisms by which JunB normally limits the production of myeloid progenitors, hence preventing initiation of myeloid malignancies.

Developing a telephone follow-up service for myeloproliferative disorders.

Each year in the NHS there are 37 million follow-up appointments. A significant proportion of these are clinically unnecessary, create inconvenience and anxiety for patients, and waste valuable resources. The nature of haematological disease means that some patients are followed-up on a long-term basis as outpatients at varying degrees of frequency ranging from monthly to annually. These patients often attend hospital for a review and are then advised that their disease is stable and a further follow-up appointment is scheduled. This frequently means that the patient has to travel to the hospital, pay to park, wait for a blood test, wait for the test result and then wait to see the doctor for a very short consultation. This article discusses the development and early evaluation of a nurse-led telephone follow-up service for patients with stable haematological disease. Responses to an initial patient satisfaction survey identified that the majority of patients found the system to be effective and convenient. From this experience it would appear that telephone follow-up positively impacts on the patient and the service. Patients receive appropriate and timely care in the right setting and this in turn has created additional capacity in the outpatient setting for those who require it.

Beta common receptor inactivation attenuates myeloproliferative disease in Nf1 mutant mice.

Neurofibromatosis type 1 (NF1) syndrome is caused by germline mutations in the NF1 tumor suppressor, which encodes neurofibromin, a GTPase activating protein for Ras. Children with NF1 are predisposed to juvenile myelomonocytic leukemia (JMML) and lethally irradiated mice given transplants with homozygous Nf1 mutant (Nf1-/-) hematopoietic stem cells develop a fatal myeloproliferative disorder (MPD) that models JMML. We investigated the requirement for signaling through the GM-CSF receptor to initiate and sustain this MPD by generating Nf1 mutant hematopoietic cells lacking the common beta chain (Beta c) of the GM-CSF receptor. Mice reconstituted with Nf1-/-, beta c-/- stem cells did not develop evidence of MPD despite the presence of increased number of immature hematopoietic progenitors in the bone marrow. Interestingly, when the Mx1-Cre transgene was used to inactivate a conditional Nf1 mutant allele in hematopoietic cells, concomitant loss of beta c-/- reduced the severity of the MPD, but did not abrogate it. Whereas inhibiting GM-CSF signaling may be of therapeutic benefit in JMML, our data also demonstrate aberrant proliferation of Nf1-/-myeloid progenitors that is independent of signaling through the GM-CSF receptor.

Identification of 12/15-lipoxygenase as a suppressor of myeloproliferative disease.

Though Abl inhibitors are often successful therapies for the initial stages of chronic myelogenous leukemia (CML), refractory cases highlight the need for novel molecular insights. We demonstrate that mice deficient in the enzyme 12/15-lipoxygenase (12/15-LO) develop a myeloproliferative disorder (MPD) that progresses to transplantable leukemia. Although not associated with dysregulation of Abl, cells isolated from chronic stage 12/15-LO-deficient (Alox15) mice exhibit increased activation of the phosphatidylinositol 3-kinase (PI3-K) pathway, as indicated by enhanced phosphorylation of Akt. Furthermore, the transcription factor interferon consensus sequence binding protein (ICSBP) is hyperphosphorylated and displays decreased nuclear accumulation, translating into increased levels of expression of the oncoprotein Bcl-2. The ICSBP defect, exaggerated levels of Bcl-2, and prolonged leukemic cell survival associated with chronic stage Alox15 MPD are all reversible upon treatment with a PI3-K inhibitor. Remarkably, the evolution of Alox15 MPD to leukemia is associated with additional regulation of ICSBP on an RNA level, highlighting the potential usefulness of the Alox15 model for understanding the transition of CML to crisis. Finally, 12/15-LO expression suppresses the growth of a human CML-derived cell line. These data identify 12/15-LO as an important suppressor of MPD via its role as a critical upstream effector in the regulation of PI3-K-dependent ICSBP phosphorylation.

Vitamin E and L-carnitine, separately or in combination, in the prevention of radiation-induced oral mucositis and myelosuppression: a controlled study in a rat model.

The aim of this study was to determine the effects of vitamin E (VE) and L-carnitine (LC) supplementation, separately or in combination, on radiation-induced oral mucositis and myelosuppression. Group 1 received no treatment (control). Group 2 received 15 Gray of 60Co gamma irradiation as a single dose to total cranium (IR). Group 3, 4, and 5 received irradiation plus 40 mg/kg/day VE (IR+VE) or 200 mg/kg/day LC (IR+LC) or in combination (IR+VE+LC) respectively. Clinically and histopathologically, assessments of mucosal reactions were performed by two independent experts in Radiation Oncology and Pathology, respectively. Hematologic analyses and antioxidant enzyme evaluations were also performed. Irradiation significantly increased oral mucositis, and decreased thrombocyte and White Blood Cell counts. A significant increase in malondialdehyde (MDA) levels and decrease in superoxide dismutase (SOD) and catalase (CAT) activities in plasma were found in the IR group. VE and LC administration, separately, plus irradiation significantly delayed the starting day, and reduced the severity of, oral mucositis. This administration also reduced a fall in the numbers of thrombocyte and WBC caused by irradiation, and decreased the MDA level, and increased the activity of SOD and CAT enzymes in the plasma. VE and LC, in combination, plus irradiation did not provide a superior radioprotection against radiation-induced toxicities.

Myeloproliferative stem cell disorders by deregulated Rap1 activation in SPA-1-deficient mice.

SPA-1 (signal-induced proliferation-associated gene-1) is a principal Rap1 GTPase-activating protein in hematopoietic progenitors. SPA-1-deficient mice developed a spectrum of myeloid disorders that resembled human chronic myelogenous leukemia (CML) in chronic phase, CML in blast crisis, and myelodysplastic syndrome as well as anemia. Preleukemic SPA-1-deficient mice revealed selective expansion of marrow pluripotential hematopoietic progenitors, which showed abnormal Rap1GTP accumulation. Overexpression of an active form of Rap1 promoted the proliferation of normal hematopoietic progenitors, while SPA-1 overexpression markedly suppressed it. Furthermore, restoring SPA-1 gene in a SPA-1-deficient leukemic blast cell line resulted in the dissolution of Rap1GTP accumulation and concomitant loss of the leukemogenicity in vivo. These results unveiled a role of Rap1 in myeloproliferative stem cell disorders and a tumor suppressor function of SPA-1.

Proapoptotic BID is required for myeloid homeostasis and tumor suppression.

The proper expansion and contraction of hematopoietic cells requires tight regulation of cell death. BID, a "BH3-only" molecule, amplifies death receptor signals connecting the extrinsic to intrinsic pathways by triggering the mitochondrial pathway of apoptosis. Bid-deficient mice, as they age, spontaneously develop a myeloproliferative disorder, which progresses from myeloid hyperplasia to a fatal, clonal malignancy closely resembling chronic myelomonocytic leukemia (CMML). Thus, an apoptotic defect can result in myeloid leukemogenesis. Premalignant Bid-/- myeloid precursor cells are resistant to death receptor-induced apoptosis. Furthermore, a competitive reconstitution assay demonstrates that Bid-deficient long-term repopulating cells give rise to expanded myelomonocytic cells in vivo. Surprisingly, a single BH3-only molecule operating in the extrinsic death receptor pathway proved essential in vivo for physiologic cell death required to maintain myeloid homeostasis. Moreover, progression to CMML indicates that an upstream BH3-only molecule, BID, is required to suppress tumorigenesis.

Expression of interferon consensus sequence binding protein (ICSBP) is downregulated in Bcr-Abl-induced murine chronic myelogenous leukemia-like disease, and forced coexpression of ICSBP inhibits Bcr-Abl-induced myeloproliferative disorder.

Chronic myelogenous leukemia (CML) is a clonal myeloproliferative disorder resulting from the neoplastic transformation of a hematopoietic stem cell. The majority of cases of CML are associated with the (9;22) chromosome translocation that generates the bcr-abl chimeric gene. Alpha interferon (IFN-alpha) treatment induces hematological remission and prolongs life in 75% of CML patients in the chronic phase. It has been shown that mice deficient in interferon consensus sequence binding protein (ICSBP), a member of the interferon regulatory factor family, manifest a CML-like syndrome. We have shown that expression of Bcr-Abl in bone marrow (BM) cells from 5-fluorouracil (5-FU)-treated mice by retroviral transduction efficiently induces a myeloproliferative disease in mice resembling human CML. To directly test whether icsbp can function as a tumor suppressor gene, we examined the effect of ICSBP on Bcr-Abl-induced CML-like disease using this murine model for CML. We found that expression of the ICSBP protein was significantly decreased in Bcr-Abl-induced CML-like disease. Forced coexpression of ICSBP inhibited the Bcr-Abl-induced colony formation of BM cells from 5-FU-treated mice in vitro and Bcr-Abl-induced CML-like disease in vivo. Interestingly, coexpression of ICSBP and Bcr-Abl induced a transient B-lymphoproliferative disorder in the murine model of Bcr-Abl-induced CML-like disease. Overexpression of ICSBP consistently promotes rather than inhibits Bcr-Abl-induced B lymphoproliferation in a murine model where BM cells from non-5-FU-treated donors were used, indicating that ICSBP has a specific antitumor activity toward myeloid neoplasms. We also found that overexpression of ICSBP negatively regulated normal hematopoiesis. These data provide direct evidence that ICSBP can act as a tumor suppressor that regulates normal and neoplastic proliferation of hematopoietic cells.

Acute arterial thrombosis in a patient with breast cancer after chemotherapy with 5-fluorouracil, doxorubicin, leucovorin, cyclophosphamide, and interleukin-3.

BACKGROUND: Interleukin-3 (IL-3) is an experimental agent used to ameliorate neutropenia in patients receiving chemotherapy. Arterial thrombotic episodes after use of IL-3 have not been reported previously. METHODS: The case of a patient with Stage III adenocarcinoma of the breast who developed hypotension and acute cerebellar artery and superior mesenteric artery thrombosis after receiving chemotherapy and treatment with IL-3 is reported. RESULTS: To the authors' knowledge, this is the first patient with arterial thrombosis reported after treatment with IL-3. CONCLUSIONS: Interleukin-3 may be associated with increased propensity for thrombosis.

Phase I study of mitozantrone, methotrexate and mitomycin with granulocyte colony-stimulating factor (filgrastim) in patients with advanced breast cancer.

The combination of mitozantrone, methotrexate and mitomycin (3M) gives a response rate of around 50% in patients with advanced breast cancer. The predominant toxicity is haematological. In this study, previously untreated patients were given 3M with increasing doses of mitozantrone (7-14 mg m-2) with recombinant human granulocyte colony-stimulating factor (metHuG-CSF) (filgrastim) to prevent marrow toxicity. Doses administered were 7 mg m-2 mitomycin i.v. 6 weekly, methotrexate i.v. 35 mg m-2 (maximum 50 mg) 3 weekly and mitozantrone i.v. 3 weekly as follows: 7 mg m-2, six patients (group 1); 10 mg m-2, six patients (group 2); 12 mg m-2, six patients (group 3); 14 mg m-2, six patients (group 4); all on day 1 for six cycles at the assigned dose. All patients received filgrastim (Amgen 0.3 mg ml-1) at a dose of 5 micrograms kg-1 subcutaneously daily on days 4-17 of each cycle. All treatment was given on an out-patient basis. A total of 24 patients were entered into the study. The median age was 63 years (range 48-75). ECOG performance status was 0 in ten, 1 in 11 patients and 2 in three patients. Locoregional disease alone was present in seven patients. The remainder had one or more sites of metastases. The actual dose administered to the 24 patients was as follows. The six patients in group 1 all completed six courses of treatment as per protocol. In group 2, three patients completed six courses, two stopped because of toxicity after one and four courses and one had progressive disease after one course. In group 3, three patients completed and three stopped early because of progressive disease. In group 4, two patients completed, one progressed after four courses and three responding patients stopped treatment because of toxicity. The maximum tolerated dose of mitozantrone in the 3M combination was 12 mg m-2. The use of filgrastim with increasing doses of chemotherapy prevents neutropenia, but other toxicities, namely thrombocytopenia and lethargy, then become dose limiting.

bcl-2 gene enables rescue from in vitro myelosuppression (bone marrow cell death) induced by chemotherapy.

Recent studies have shown that the use of cytokines such as granulocyte colony-stimulating factor (G-CSF) to ameliorate chemotherapy-induced myelosuppression may enhance the viability of tumour cells with functional receptors for these cytokines. In this study, therefore, we used murine bone marrow (BM) cells in an in vitro model in an attempt to determine whether topoisomerase inhibitors (camptothecin, etoposide and doxorubicin) induce myelosuppression (BM cell death) and whether novel treatments other than the administration of G-CSF can be used for rescue from myelosuppression. DNA fragmentation assay, ultrastructural analysis and cell cycle analysis demonstrated that these chemotherapeutic agents induced apoptosis in BM cells. We demonstrated in addition that enforced expression of the bcl-2 gene in BM cells by MPZenNeo (bcl-2) retroviral gene transfer increased resistance to the apoptosis induced by these agents. These findings suggest the possibility that enforced expression of the bcl-2 gene in BM cells using gene transfer techniques may enable rescue from chemotherapy-induced myelosuppression.