First-hit SETBP1 mutations cause a myeloproliferative disorder with bone marrow fibrosis.

ABSTRACT: SETBP1 mutations are found in various clonal myeloid disorders. However, it is unclear whether they can initiate leukemia, because SETBP1 mutations typically appear as later events during oncogenesis. To answer this question, we generated a mouse model expressing mutated SETBP1 in hematopoietic tissue: this model showed profound alterations in the differentiation program of hematopoietic progenitors and developed a myeloid neoplasm with megakaryocytic dysplasia, splenomegaly, and bone marrow fibrosis, prompting us to investigate SETBP1 mutations in a cohort of 36 triple-negative primary myelofibrosis (TN-PMF) cases. We identified 2 distinct subgroups, one carrying SETBP1 mutations and the other completely devoid of somatic variants. Clinically, a striking difference in disease aggressiveness was noted, with patients with SETBP1 mutation showing a much worse clinical course. In contrast to myelodysplastic/myeloproliferative neoplasms, in which SETBP1 mutations are mostly found as a late clonal event, single-cell clonal hierarchy reconstruction in 3 patients with TN-PMF from our cohort revealed SETBP1 to be a very early event, suggesting that the phenotype of the different SETBP1+ disorders may be shaped by the opposite hierarchy of the same clonal SETBP1 variants.

JAK2V617F, CALR, and MPL Mutations and Bone Marrow Histology in Patients with Essential Thrombocythaemia.

INTRODUCTION: Mutations in the JAK2, CALR, and MPL genes have been shown to have prognostic value in essential thrombocythaemia (ET), but no clear association with morphological changes has been reported so far. We investigated the possible correlation between gene mutations and histopathological features in bone marrow (BM) biopsies of patients with ET. METHODS: Marrow cellularity, fibrosis, and the number of total and dysmorphic megakaryocytes and clusters of megakaryocytes were compared to gene mutations in 90 cases of ET at diagnosis. RESULTS: The JAK2V617F mutation was found in 58.9%, CALR in 28.9%, and MPL in 4.4% of the cases, and 7.8% were triple-negative. JAK2V617F-mutated ET showed a high BM cellularity, the lowest number of clusters of megakaryocytes and the highest number of dysmorphic megakaryocytes; CALR-mutated ET showed a reduced BM cellularity, many clusters of large megakaryocytes, and very few dysmorphic megakaryocytes; MPL-mutated ET showed the lowest BM cellularity, the highest number of clustered and large megakaryocytes, and the lowest number of dysmorphic megakaryocytes. Triple-negative ET cases had the highest BM cellularity. CONCLUSIONS: Distinct morphological patterns were associated with gene mutations in ET, supporting the classification of ET into different subtypes.

Survival improvement of poor-prognosis AML/MDS patients by maintenance treatment with low-dose chemotherapy and differentiating agents.

We evaluated a maintenance, post-remission treatment with low-dose chemotherapy plus differentiating agents on poor-prognosis acute myeloid leukemia (AML)/myelodysplastic syndrome (MDS) patients ineligible to allografting. Patients had either age over 60 and/or secondary AML, therapy-related AML, previous relapse, high-risk MDS. Forty-five patients received the maintenance therapy based on two alternated schedules: (a) 6-thioguanine + 13-cis retinoic acid + dihydroxylated vitamin D3 and (b) low-dose cytarabine + 6-mercaptopurine + all-trans retinoic acid + dihydroxylated vitamin D3. We compared their outcome, at a median follow-up of 52 months, to that of a matched population of 49 patients who stopped treatments after consolidation. Maintenance group had a lower relapse incidence (70.3 vs. 86.4 % at 5 years p = 0.007) and a longer disease-free survival (median 21.2 vs. 8.7 months, p = 0.017). The relapse reduction improved overall survival: median 40.4 months (35.9 % at 5 years) for maintenance group vs. 15.8 (14.2 % at 5 years) for controls (p = 0.005). At multivariate Cox analysis, both cytogenetic and maintenance therapies resulted independent outcome predictors for overall survival. Maintenance treatment also reduced minimal residual disease (detected by WT1 and CBFß-MYH11) in five of eight evaluable patients. The present results suggest that our strategy of maintenance therapy might improve the outcome of poor-risk AML/MDS patients.

Involvement of Grb2 adaptor protein in nucleophosmin-anaplastic lymphoma kinase (NPM-ALK)-mediated signaling and anaplastic large cell lymphoma growth.

Most anaplastic large cell lymphomas (ALCL) express oncogenic fusion proteins derived from chromosomal translocations or inversions of the anaplastic lymphoma kinase (ALK) gene. Frequently ALCL carry the t(2;5) translocation, which fuses the ALK gene to the nucleophosmin (NPM1) gene. The transforming activity mediated by NPM-ALK fusion induces different pathways that control proliferation and survival of lymphoma cells. Grb2 is an adaptor protein thought to play an important role in ALK-mediated transformation, but its interaction with NPM-ALK, as well as its function in regulating ALCL signaling pathways and cell growth, has never been elucidated. Here we show that active NPM-ALK, but not a kinase-dead mutant, bound and induced Grb2 phosphorylation in tyrosine 160. An intact SH3 domain at the C terminus of Grb2 was required for Tyr(160) phosphorylation. Furthermore, Grb2 did not bind to a single region but rather to different regions of NPM-ALK, mainly Tyr(152-156), Tyr(567), and a proline-rich region, Pro(415-417). Finally, shRNA knockdown experiments showed that Grb2 regulates primarily the NPM-ALK-mediated phosphorylation of SHP2 and plays a key role in ALCL cell growth.

The enzymatic activity of 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase is enhanced by NPM-ALK: new insights in ALK-mediated pathogenesis and the treatment of ALCL.

Anaplastic large cell lymphoma represents a subset of neoplasms caused by translocations that juxtapose the anaplastic lymphoma kinase (ALK) to dimerization partners. The constitutive activation of ALK fusion proteins leads to cellular transformation through a complex signaling network. To elucidate the ALK pathways sustaining lymphomagenesis and tumor maintenance, we analyzed the tyrosine-kinase protein profiles of ALK-positive cell lines using 2 complementary proteomic-based approaches, taking advantage of a specific ALK RNA interference (RNAi) or cell-permeable inhibitors. A well-defined set of ALK-associated tyrosine phosphopeptides, including metabolic enzymes, kinases, ribosomal and cytoskeletal proteins, was identified. Validation studies confirmed that vasodilator-stimulated phosphoprotein and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/inosine monophosphate cyclohydrolase (ATIC) associated with nucleophosmin (NPM)-ALK, and their phosphorylation required ALK activity. ATIC phosphorylation was documented in cell lines and primary tumors carrying ALK proteins and other tyrosine kinases, including TPR-Met and wild type c-Met. Functional analyses revealed that ALK-mediated ATIC phosphorylation enhanced its enzymatic activity, dampening the methotrexate-mediated transformylase activity inhibition. These findings demonstrate that proteomic approaches in well-controlled experimental settings allow the definition of informative proteomic profiles and the discovery of novel ALK downstream players that contribute to the maintenance of the neoplastic phenotype. Prediction of tumor responses to methotrexate may justify specific molecular-based chemotherapy.

Bone marrow morphological features and therapy in patients with Philadelphia-negative neoplasms.

Introduction Chronic myeloproliferative neoplasm (MPNs) are clonal malignant bone marrow (BM) diseases, arising from a hematopoietic stem cell. All therapies for these neoplasms have peculiar effects on the bone marrow, but little evidence has been described in the literature.Areas covered This review examines BM morphological changes following the main treatments in Philadelphia-negative MPNs. Hydroxyurea can reduce the cellularity of the erythroid and megakaryocyte lineages but has minimal impact on fibrotic evolution. There is general agreement on its dysplastic effects, with a high incidence of acute myeloid leukemia and myelodysplastic syndrome. Interferon treatment can reduce or normalize BM cellularity, improve erythropoiesis, and reduce the number and atypicality of megakaryocytes. Most data describe reduction or complete resolution of marrow fibrosis; dysplastic effects are not reported. Anagrelide may induce an increase in the number of BM megakaryocytes, especially immature megakaryocytes or precursors, and a worsening of marrow fibrosis or increased transformation of essential thrombocythemia into myelofibrosis. Ruxolitinib can improve or stabilize BM fibrosis and reduces the frequency and dense clustering of megakaryocytes.Expert opinion Since previous therapy can modify BM features, it is essential to obtain information on previous or current therapies and to collect complete clinical information.

Stat3 is required for anchorage-independent growth and metastasis but not for mammary tumor development downstream of the ErbB-2 oncogene.

The oncogenic transcription factor Stat3 is constitutively active in a high percentage of human tumors including mammary adenocarcinomas and is reported to participate in the ErbB-2 oncogene signaling. In order to assess the role of signal transducer and activator of transcription 3 (Stat3) in mammary tumorigenesis downstream of ErbB-2, we generated mice expressing the activated rat ErbB-2 (neu) but lacking Stat3 in the mammary epithelium. Stat3 is apparently not required for neu-driven mammary tumorigenesis as tumors developed similarly in both Stat3-sufficient and Stat3-deficient glands. However, short hairpin RNA (shRNA)-mediated Stat3 silencing in a neu-overexpressing tumor-derived cell line completely abolished both neu-driven anchorage-independent growth and lung metastasis. Our data suggest that Stat3 might be a useful therapeutic target in breast tumors showing amplification and/or overexpression of the ErbB-2 oncogene, which normally display aggressive, metastatic behavior.

Can pegylated interferon improve the outcome of polycythemia vera patients?

Pegylated interferon (peg-IFN) was proven by phase II trials to be effective in polycythemia vera (PV); however, it is not clear whether it could improve patient outcome compared to hydroxyurea (HU). Here, we present an observational study on 65 PV patients aged 65 years or younger, who received either peg-IFN (30) or HU (35) according to the physician choice. Median follow-up was 75 months. The two cohorts were comparable for patient and disease characteristics. Eighty-seven percent of the patients treated with peg-INF responded, with a CR rate of 70% as compared to 100 and 49% with HU, respectively. Discontinuation rate was similar in the two groups (20% in peg-IFN vs 17% in HU). JAK2 allele burden was monitored in peg-INF arm only, and a reduction was observed in 88% of the patients. No thrombotic events were observed during peg-IFN treatment compared to three on HU. Disease progression to myelofibrosis or acute myeloid leukemia occurred to a patient only in peg-INF, compared to three in HU. Overall, three second malignancies were observed during the study, two in patients who received HU only, and one in a patient largely treated HU who received also peg-IFN for 3 months. Overall survival was significantly better for peg-IFN patients compared to HU, p = 0.027. Our study, albeit limited by small patient and event number and lack of randomization, confirms the efficacy of peg-INF in PV and shows a significant survival advantage for peg-INF-treated patients. Waiting for confirming data from the ongoing phase III trials, our study can support peg-INF as a first-line treatment option for PV, at least for younger patients.

Post-remissional and pre-transplant role of minimal residual disease detected by WT1 in acute myeloid leukemia: A retrospective cohort study.

In acute myeloid leukemia (AML), the detection of minimal residual disease (MRD) is still under investigation. The aim of the present retrospective study was to assess the role of Wilms tumor gene 1 (WT1) overexpression in a large monocentric cohort of AML patients. Among 255 enrolled patients, MRD was investigated in those in complete remission (CR) with an available WT1 at baseline (>250 copies) and at two further time-points: after induction (n=117) and prior allogeneic hematopoietic cell transplantation (allo-HCT), n=65. Baseline BM WT1 overexpression was not associated with response to induction (p=0.244). Median overall survival (OS) and disease-free survival (DFS) were significantly shorter in patients with >350 WT1 copies after induction compared to those with ≤350 (HR for mortality 2.13; 95% CI 1.14-3.97, p=0.018 and HR for relapse 2.81; 95% CI 1.14-6.93, p=0.025). Patients with WT1>150 copies pre allo-HCT had a significantly higher 2-year cumulative incidence of relapse (CIR) compared to those with WT1≤150 (HR 4.61; 95% CI 1.72-12.31, p=0.002). The prognostic role of WT1 overexpression resulted independent from other well-established risk factors. According to these results, WT1 overexpression might represent an additional MRD tool for risk stratification in patients classified nowadays in CR.

ALK-dependent control of hypoxia-inducible factors mediates tumor growth and metastasis.

Rearrangements involving the anaplastic lymphoma kinase (ALK) gene are defining events in several tumors, including anaplastic large-cell lymphoma (ALCL) and non-small cell lung carcinoma (NSCLC). In such cancers, the oncogenic activity of ALK stimulates signaling pathways that induce cell transformation and promote tumor growth. In search for common pathways activated by oncogenic ALK across different tumors types, we found that hypoxia pathways were significantly enriched in ALK-rearranged ALCL and NSCLC, as compared with other types of T-cell lymphoma or EGFR- and K-RAS-mutated NSCLC, respectively. Consistently, in both ALCL and NSCLC, we found that under hypoxic conditions, ALK directly regulated the abundance of hypoxia-inducible factors (HIF), which are key players of the hypoxia response in normal tissues and cancers. In ALCL, the upregulation of HIF1α and HIF2α in hypoxic conditions required ALK activity and its downstream signaling proteins STAT3 and C/EBPß. In vivo, ALK regulated VEGFA production and tumor angiogenesis in ALCL and NSCLC, and the treatment with the anti-VEGFA antibody bevacizumab strongly impaired ALCL growth in mouse xenografts. Finally, HIF2α, but not HIF1α, was required for ALCL growth in vivo whereas the growth and metastasis potential of ALK-rearranged NSCLC required both HIF1α and HIF2α. In conclusion, we uncovered an ALK-specific regulation of the hypoxia response across different ALK(+) tumor types and propose HIFs as a powerful specific therapeutic target in ALK-rearranged ALCL and NSCLC.

Core binding factor acute myeloid leukaemia and c-KIT mutations.

Core binding factor (CBF) acute myeloid leukaemia (AML) represents 5-8% of all AMLs and has a relatively favourable prognosis. However, activating c-KIT mutations are reported to be associated with higher risk of relapse and shorter survival. To verify the incidence and prognostic value of c-KIT mutations in CBF AML, we retrospectively analysed bone marrow samples of 23 consecutive adult patients with de novo CBF AML [14 inv(16) and 9 t(8;21)] treated at a single institution from 2000 to 2011. All patients received standard induction chemotherapy with cytarabine, idarubicin and etoposide; 13 underwent allogeneic stem cell transplantation. c-KIT mutations in exons 8, 9, 10, 11, 13, 14 and 17 were assessed by PCR amplification in combination with direct sequencing. c-KIT mutations (3 in exon 10 and 4 in exon 17) were detected in 7/23 (30.4%) patients, 3 with t(8;21) and 4 with inv(16). No difference in c-KIT mutation status was observed between cases with inv(16) or t(8;21) alone and cases with additional cytogenetic abnormalities. No association between gender, age, white blood cell and platelet count, peripheral blood and bone marrow blast cells at diagnosis, achievement of complete remission, cytogenetic risk groups and Wilms tumour gene 1 (WT1) levels was found. On the contrary, lactate dehydrogenase (LDH) values were higher in mutated than in non-mutated patients (p=0.01). Overall survival (OS) rates were longer in CBF compared to the other types of AML and disease-free survival (DFS) was longer in inv(16) than in t(8;21) AML. OS and DFS were similar in mutated and non-mutated CBF AML patients. Our results confirm a better prognosis for CBF AML than all other AML categories, and for inv(16) than t(8;21) AML. However, no prognostic value for c-KIT mutational status was found in our series. The association between LDH levels and c-KIT mutation would indicate a more active proliferation for mutated CBF AML.

Anaplastic lymphoma kinase in human cancer.

The receptor tyrosine kinases (RTKs) play a critical role, controlling cell proliferation, survival, and differentiation of normal cells. Their pivotal function has been firmly established in the pathogenesis of many cancers as well. The anaplastic lymphoma kinase (ALK), a transmembrane RTK, originally identified in the nucleophosmin (NPM)-ALK chimera of anaplastic large cell lymphoma, has emerged as a novel tumorigenic player in several human cancers. In this review, we describe the expression of the ALK-RTK, its related fusion proteins, and their molecular mechanisms of activation. Novel tailored strategies are briefly illustrated for the treatment of ALK-positive neoplasms.

Description of a novel Janus kinase 3 P132A mutation in acute megakaryoblastic leukemia and demonstration of previously reported Janus kinase 3 mutations in normal subjects.

Gain-of-function (GOF) mutations of Janus kinase 2 (JAK2) are frequently seen in myeloproliferative disorders (MPDs). Meanwhile, JAK3 activating substitutions have been found in a few megakaryocytic cell lines and in primary myeloid leukemia (AMKL). Here, we sought to discover novel leukemogenetic mutations in de novo acute myeloid leukemia of non-Down syndrome (N-DS) by DNA sequencing. A total of 191 normal Caucasian individuals were studied to define single nucleotide polymorphisms (SNPs) within the JH2 and JH6 domains. Although known activating substitutions were observed in rare cases of acute myeloid leukemia (AML) (V722I [2/134] or P132T [1/119]), all samples were wild-type (WT) for the oncogenic A572V (119/119). Interestingly, a novel homozygous mutation (P132A) was discovered in a patient with acute megakaryoblastic leukemia and in vivo studies demonstrated that its ectopic expression was oncogenic in a mouse xenotransplant model. This study defines a novel JAK3 mutation among patients with N-DS AML and demonstrates that normal individuals can also display germline JAK3 substitutions, previously proven to have oncogenic properties, in vitro and in vivo. The discovery of these substitutions in normal donors encourages future studies to define new risk factors among patients with MPDs.

NPM-ALK oncogenic tyrosine kinase controls T-cell identity by transcriptional regulation and epigenetic silencing in lymphoma cells.

Transformed cells in lymphomas usually maintain the phenotype of the postulated normal lymphocyte from which they arise. By contrast, anaplastic large cell lymphoma (ALCL) is a T-cell lymphoma with aberrant phenotype because of the defective expression of the T-cell receptor and other T-cell-specific molecules for still undetermined mechanisms. The majority of ALCL carries the translocation t(2;5) that encodes for the oncogenic tyrosine kinase NPM-ALK, fundamental for survival, proliferation, and migration of transformed T cells. Here, we show that loss of T-cell-specific molecules in ALCL cases is broader than reported previously and involves most T-cell receptor-related signaling molecules, including CD3epsilon, ZAP70, LAT, and SLP76. We further show that NPM-ALK, but not the kinase-dead NPM-ALK(K210R), downregulated the expression of these molecules by a STAT3-mediated gene transcription regulation and/or epigenetic silencing because this downregulation was reverted by treating ALCL cells with 5-aza-2-deoxycytidine or by knocking down STAT3 through short hairpin RNA. Finally, NPM-ALK increased the methylation of ZAP70 intron 1-exon 2 boundary region, and both NPM-ALK and STAT3 regulated the expression levels of DNA methyltransferase 1 in transformed T cells. Thus, our data reveal that oncogene-deregulated tyrosine kinase activity controls the expression of molecules that determine T-cell identity and signaling.

p130Cas mediates the transforming properties of the anaplastic lymphoma kinase.

Translocations of the anaplastic lymphoma kinase (ALK) gene have been described in anaplastic large-cell lymphomas (ALCLs) and in stromal tumors. The most frequent translocation, t(2;5), generates the fusion protein nucleophosmin (NPM)-ALK with intrinsic tyrosine kinase activity. Along with transformation, NPM-ALK induces morphologic changes in fibroblasts and lymphoid cells, suggesting a direct role of ALK in cell shaping. In this study, we used a mass-spectrometry-based proteomic approach to search for proteins involved in cytoskeleton remodeling and identified p130Cas (p130 Crk-associated substrate) as a novel interactor of NPM-ALK. In 293 cells and in fibroblasts as well as in human ALK-positive lymphoma cell lines, NPM-ALK was able to bind p130Cas and to induce its phosphorylation. Both of the effects were dependent on ALK kinase activity and on the adaptor protein growth factor receptor-bound protein 2 (Grb2), since no binding or phosphorylation was found with the kinase-dead mutant NPM-ALK(K210R) or in the presence of a Grb2 dominant-negative protein. Phosphorylation of p130Cas by NPM-ALK was partially independent from Src (tyrosine kinase pp60c-src) kinase activity, as it was still detectable in Syf-/- cells. Finally, p130Cas-/- (also known as Bcar1-/-) fibroblasts expressing NPM-ALK showed impaired actin filament depolymerization and were no longer transformed compared with wild-type cells, indicating an essential role of p130Cas activation in ALK-mediated transformation.

Human cytomegalovirus requires cellular deoxycytidylate deaminase for replication in quiescent cells.

We have previously observed that the expression of two thymidylate biosynthesis enzymes, dihydrofolate reductase and thymidylate synthase (TS), is upregulated in quiescent human fibroblasts infected with human cytomegalovirus (HCMV). Here, we have demonstrated that HCMV increases expression of the cellular deoxycytidylate deaminase (dCMP deaminase), which provides the substrate for TS by converting dCMP to dUMP. We observed an increase in dCMP deaminase protein levels, whereas deoxyuridine triphosphatase (dUTPase), another cellular enzyme that may provide dUMP by hydrolysing dUTP, was undetectable. The essential requirement of cellular dCMP deaminase for productive HCMV replication was further emphasized by showing that a precursor of a potent dCMP deaminase inhibitor, zebularine, suppressed virus replication and DNA synthesis. These results suggest that HCMV exploits the host's dCMP deaminase activity to replicate in quiescent cells.

Evidence that the human cytomegalovirus 46-kDa UL72 protein is not an active dUTPase but a late protein dispensable for replication in fibroblasts.

The Human Cytomegalovirus (HCMV) UL72 gene is considered to be the equivalent of the dUTPase gene of the Alpha- and Gamma-herpesviruses. To characterize its function, the expression profiles of UL72 at both the RNA and the protein level were determined. The gene is expressed with a late kinetics and the corresponding UL72 46-kDa protein accumulates late during infection in the cytoplasm of infected cells. The pUL72 was expressed in E. coli and the purified recombinant protein did not display a detectable dUTPase activity. The viral yields of reconstituted HCMV RVDeltaUL72 viruses carrying a deletion within the UL72 ORF demonstrated a moderate growth defect following low MOI infections, whereas their DNA synthesis profiles were not significantly different from those of the parental HCMV RVAD169. These results demonstrate that the UL72 gene product is not a dUTPase and is not essential for replication in human fibroblasts.