Contribution of cytokines to the etiology and progression of Primary Myelofibrosis

Primary Myelofibrosis (PMF) is the end-stage of Philadelphia-negative myeloproliferative neoplasms (MPN) and is characterized by fibrosis and hematopoietic failure in bone marrow, with a consequential migration of the malignant hematopoietic stem cells (HSC) in the spleen where they induce ineffective haematopoiesis. To date, available therapies for PMF are still palliative and do not halt the progression of this neoplasm. During my PhD years, our laboratory investigated the factors promoting the onset and progression of PMF. In our PMF mice model, Gata1low mouse, we studied the role of the interaction of HSC niche with megakaryocytes and HSC localization in the bone marrow during their division and cycle. We observed the inflammation and the main protagonists (LNC-2, CXCL1, and TGF-β) of this process and how their level changes before and after the onset of the disease. We investigated the different megakaryocyte populations in the fibrotic environment in different organs (lung and bone marrow) to define the megakaryocytes implicated in this process. In human samples, we described different ultrastructural abnormalities of megakaryocytes from the bone marrow and the spleen, identifying a possible different metabolism in those two populations. In conclusion, we highlighted the intricated crosstalk between the megakaryocytes, the niche and HSC in PMF. We identified megakaryocytes-dependent cytokines altering the homeostasis of the niche and HSC. Those cytokines could be used as alternative therapeutic targets. Furthermore, we observed different megakaryocytic populations in different organs, providing new prospective on the role of megakaryocytes in different microenvironments.

Deregulation of apoptosis-related genes is associated with PRV1 overexpression and JAK2 V617F allele burden in Essential Thrombocythemia and Myelofibrosis

Background: Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF) are Chronic Myeloproliferative Neoplasms (MPN) characterized by clonal myeloproliferation/myeloaccumulation without cell maturation impairment. The JAK2 V617F mutation and PRV1 gene overexpression may contribute to MPN physiopathology. We hypothesized that deregulation of the apoptotic machinery may also play a role in the pathogenesis of ET and PMF. In this study we evaluated the apoptosis-related gene and protein expression of BCL2 family members in bone marrow CD34(+) hematopoietic stem cells (HSC) and peripheral blood leukocytes from ET and PMF patients. We also tested whether the gene expression results were correlated with JAK2 V617F allele burden percentage, PRV1 overexpression, and clinical and laboratory parameters. Results: By real time PCR assay, we observed that A1, MCL1, BIK and BID, as well as A1, BCLW and BAK gene expression were increased in ET and PMF CD34(+) cells respectively, while pro-apoptotic BAX and anti-apoptotic BCL2 mRNA levels were found to be lower in ET and PMF CD34(+) cells respectively, in relation to controls. In patients' leukocytes, we detected an upregulation of anti-apoptotic genes A1, BCL2, BCL-XL and BCLW. In contrast, pro-apoptotic BID and BIMEL expression were downregulated in ET leukocytes. Increased BCL-XL protein expression in PMF leukocytes and decreased BID protein expression in ET leukocytes were observed by Western Blot. In ET leukocytes, we found a correlation between JAK2 V617F allele burden and BAX, BIK and BAD gene expression and between A1, BAX and BIK and PRV1 gene expression. A negative correlation between PRV1 gene expression and platelet count was observed, as well as a positive correlation between PRV1 gene expression and splenomegaly. Conclusions: Our results suggest the participation of intrinsic apoptosis pathway in the MPN physiopathology. In addition, PRV1 and JAK2 V617F allele burden were linked to deregulation of the apoptotic machinery.

Microenvironment and therapeutic modulation of myelofibrosis in an experimental mouse model.

Primary myelofibrosis(PMF) is the most severe form of Philadelphia-negative myeloproliferative neoplasms(MPNs), characterized by splenomegaly, extramedullary hematopoiesis and bone marrow(BM) fibrosis, with disease progression to leukemia and low survival. The best therapy currently available includes treatment with a JAK inhibitor(Ruxolitinib), which only ameliorates symptoms. Unfortunately, the pathogenesis of the disease is still poorly understood. It has been hypothesized that its progression may be determined by the presence of inflammatory cytokines produced by the bone marrow microenvironment that promote fibrosis. The three aims of this PhD thesis, using the Gata1low mouse model of myelofibrosis, were: 1. Investigate the presence of different cytokines in the bone marrow microenvironment; 2. Test the efficacy of treatment with Reparixin, a CXCR1/2 receptor inhibitor; 3. Test the efficacy of treatment with RB40.34 (P-selectin inhibitor), alone and in combination with Ruxolitinib. In the first study, we demonstrated by immunohistochemistry(IHC) the presence in the BM of Gata1low mice of elevated levels of CXCL1, and its receptors CXCR1/2, and TGF-β1. Particularly, the cells with higher expression of these cytokines were the megakaryocytes. In the second study, we found that treatment with Reparixin in Gata1low mice showed dose-dependent efficacy in reducing bone marrow and splenic fibrosis. Furthermore, by IHC analysis we demonstrated that the treatment induced a decrease in the expression of TGF-β1. In the third study, we found that treatment with RB40.34 in combination with Ruxolitinib normalizes the phenotype of Gata1low mice, reducing fibrosis and the content of TGF-β and CXCL1 in the bone marrow, and restoring the architecture of hematopoiesis in the bone marrow and spleen. In summary, these data provide preclinical evidence that treatment with Reparixin and RB40.34 in combination with Ruxolitinib are effective on reversing the myelofibrotic trait in the Gata1low mouse model and encourage clinical trials to validate these compounds in human patients with PMF.

Pharmacological targeting of P-selectin to halt the progression of myelofibrosis in the Gata1low mouse model

Primary myelofibrosis is a clonal hematopoietic disorder characterized by marked degrees of systemic inflammation. The release of pro-inflammatory factors by clonal hematopoietic cell populations cause the remodeling of a specialized microenvironment, defined niche, in which the hematopoietic stem cells reside. The main source of pro-inflammatory cytokines is represented by malignant megakaryocytes. The bone marrow and spleen from myelofibrosis patients, as well as those from the Gata1low mouse model of the disease, contain increased number of abnormal megakaryocytes. These cells express on their surface high levels of the adhesion receptor P-selectin that, by triggering a pathological megakaryocyte-neutrophil emperipolesis, lead to increased bioavailability of TGF-β1 in the microenvironment and disease progression. Gata1low mice develop with age a phenotype similar to that of patients with myelofibrosis. We previously demonstrated that deletion of the P-selectin gene in Gata1low mice prevented the development of the myelofibrotic phenotype in these mice. In the current study, we tested the hypothesis that pharmacological inhibition of P-selectin may rescue the fibrotic phenotype of Gata1low mice. To test this hypothesis, we have investigated the phenotype expressed by old Gata1low mice treated with the anti-mouse monoclonal antibody against P-selectin RB40.34, alone or in combination with the JAK2 inhibitor Ruxolitinib. The results showed that the combined therapy normalized the phenotype of Gata1low mice with limited toxicity by reducing fibrosis, TGF-β1 and CXCL1 content in the BM and spleen and by restoring hematopoiesis in the bone marrow and the normal architecture of the spleen. In conclusion, pharmacological inhibition of P-selectin was effective in targeting malignant megakaryocytes and the microenvironmental abnormalities that affect the hematopoietic stem cell compartment in this model. These results suggest that P-selectin and JAK1/2 inhibitors in combination may represent a valid therapeutic option for patients with myelofibrosis.

Estudio de marcadores moleculares en neoplasias mieloproliferativas

Las neoplasias mieloproliferativas (NM) son un grupo de enfermedades clonales de la célula hematopoyética madre. Entre las NM clásicas se encuentran la policitemia vera (PV), la trombocitemia esencial (TE) y la mielofibrosis primaria (MFP). Durante muchos años el diagnóstico de estas patologías se hacía por exclusión utilizando biomarcadores de clonalidad poco específicos. En el año 2005, la descripción de la mutación JAK2V617F supuso un avance importante en el diagnóstico de estas patologías. Posteriormente, se han descrito mutaciones en otros genes como mutaciones en MPL, TET2, ASXL1, IDH1, IDH2, c-CBL, EZH2, IKZF1 y LNK, en distintas neoplasias mieloides y en porcentaje variable. Aun así, ninguno de estos genes son marcadores específicos de ninguna NM y todavía existe un porcentaje elevado de pacientes con TE y MFP sin un marcador de clonalidad conocido. Además, todos estos genes se han descrito como eventos genéticos implicados en la transformación de una NM a leucemia mieloide aguda. El objetivo de este proyecto fue estudiar varios marcadores moleculares en neoplasias mieloproliferativas Philadelphia negativas. En primer lugar, se estudió la modulación de la carga alélica JAK2V617F en pacientes con PV o TE que recibieron tratamiento citoreductor y a su vez se analizó la dinámica natural de la carga alélica en pacientes que no recibieron tratamiento. Posteriormente, se analizaron la presencia de alteraciones en los genes previamente mencionados, en distintos grupos de pacientes. En primer lugar, se analizó la presencia de mutaciones en TET2, ASXL1, IDH1, IDH2 y CBL en un grupo de pacientes JAK2 y MPL negativos, para determinar la frecuencia de alteraciones de estos genes en este grupo de pacientes y determinar su valor en el diagnóstico de estas patologías. En segundo lugar, se estudió la presencia de mutaciones en estos genes incluyendo EZH2, IKZF1 y LNK para estudiar la incidencia y el valor pronóstico de estas alteraciones en las NM que progresan a mielofibrosis.

Differential expression of apoptosis-related genes from death receptor pathway in chronic myeloproliferative diseases

Background Chronic myeloproliferative disorders (MPDs) are clonal haematopoietic stem cell malignancies characterised by an accumulation of mature myeloid cells in bone marrow and peripheral blood. Deregulation of the apoptotic machinery may be associated with MPD physiopathology. Aims To evaluate expression of death receptors` family members, mononuclear cell apoptosis resistance, and JAK2 allele burden. Subjects and Methods Bone marrow haematopoietic progenitor CD34 cells were separated using the Ficoll-hypaque protocol followed by the Miltenyi CD34 isolation kit, and peripheral blood leukocytes were separated by the Haes-Steril method. Total RNA was extracted by the Trizol method, the High Capacity Kit was used to synthesise cDNA, and real-time PCR was performed using SybrGreen in ABIPrism 7500 equipment. The results of gene expression quantification are given as 2(-Delta Delta Ct). The JAK2 V617F mutation was detected by real-time allelic discrimination PCR assay. Peripheral blood mononuclear cells (PBMCs) were isolated by the Ficoll-hypaque protocol and cultured in the presence of apoptosis inducers. Results In CD34 cells, there was mRNA overexpression for fas, faim and c-flip in polycythaemia vera (PV), essential thrombocythaemia (ET) and primary myelofibrosis (PMF), as well as fasl in PMF, and dr4 levels were increased in ET. In leukocytes, fas, c-flip and trail levels were increased in PV, and dr5 expression was decreased in ET. There was an association between dr5 and fasl expression and JAK2V617F mutation. PBMCs from patients with PV, ET or PMF showed resistance to apoptosis inducers. Conclusions The results indicate deregulation of apoptosis gene expression, which may be associated with MPD pathogenesis leading to accumulation of myeloid cells in MPDs.

Molecular diagnostics of myeloproliferative neoplasms

Since the discovery of the JAK2 V617F mutation in the majority of the myeloproliferative neoplasms (MPN) of polycythemia vera, essential thrombocythemia and primary myelofibrosis ten years ago, further MPN-specific mutational events, notably in JAK2 exon 12, MPL exon 10 and CALR exon 9 have been identified. These discoveries have been rapidly incorporated into evolving molecular diagnostic algorithms. Whilst many of these mutations appear to have prognostic implications, establishing MPN diagnosis is of immediate clinical importance with selection, implementation and the continual evaluation of the appropriate laboratory methodology to achieve this diagnosis similarly vital. The advantages and limitations of these approaches in identifying and quantitating the common MPN-associated mutations are considered herein with particular regard to their clinical utility. The evolution of molecular diagnostic applications and platforms has occurred in parallel with the discovery of MPN-associated mutations, and it therefore appears likely that emerging technologies such as next-generation sequencing and digital PCR will in the future play an increasing role in the molecular diagnosis of MPN. Accepted for publication 30 April 2015 doi:10.1111/ejh.12578

Expressão gênica de metaloproteinases e de seus reguladores em neoplasias mieloproliferativas: associação com biomarcadores de angiogênese e status mutacional

As neoplasias mieloproliferativas (NMPs) BCR-ABL1 negativas compreendem a mielofibrose primária (PMF), trombocitemia essencial (TE) e a policitemia vera (PV). A patogênese e progressão dessas NMPs não estão completamente elucidadas. As metaloproteinases de matriz (MMPs) degradam a matriz extracelular, ativando citocinas e fatores de crescimento que, por sua vez, participam da tumorigênese e angiogênese. O objetivo deste estudo foi avaliar a relação da expressão gênica das MMPs, TIMPs, HIF1-α e SPARC com os marcadores angiogênicos bFGF e VEGFA em pacientes com MF e TE, considerando o status mutacional; bem como avaliar a regulação desses genes em camundongos submetidos à hipóxia, e em modelos HIF1-α(-/-) e VHL(-/-). Foram incluídos 21 pacientes com MF, 21 com MF pós-TE, 6 com MF pós-PV, 23 com TE e 78 indivíduos controle. As análises realizadas foram: dosagem sérica e expressão de RNAm de MMP2, MMP9, TIMP1, TIMP2 e SPARC, hemograma, determinação da proteína C reativa ultrassensível, determinação das concentrações de VEGFA e bFGF e avaliação das mutações nos genes JAK2, cMPL e CALR. A avaliação da densidade microvascular da medula óssea foi feita em 30 dos pacientes incluídos. Os pacientes com MFP, MFPTE e TE apresentaram maior expressão de MMP2, SPARC, TIMP1, TIMP2 e bFGF quando comparados aos seus controles (P<0,05), enquanto MMP9 foi mais expressa nos pacientes com MFPTE e TE (P= 0,011 e P=0,047, respectivamente). Os pacientes com TE apresentaram maior expressão de HIF1-α e VEGFA em relação ao grupo controle (P<0,05). Pacientes com MF JAK2V617F positivos apresentaram maiores concentrações de MMP9, TIMP2, bFGF e VEGFA quando comparados aos pacientes portadores de mutações na CALR (P<0,05). Os pacientes com TE JAK2V617F positivos apresentaram maiores concentrações de MMP2 e TIMP2 (P=0,049 e P=0,020, respectivamente). As concentrações das proteínas estudadas não apresentaram correlação com a carga alélica de JAK2V617F e nem com a densidade microvascular da medula óssea. Células de medula óssea de camundongos submetidos à hipóxia apresentaram maior expressão de MMP2 e TIMP1 comparados aos camundongos em normóxia. Camundongos VHL(-/-) apresentaram aumento na expressão dos genes MMP2, MMP9, TIMP1, TIMP2 e VEGFA. Diferentemente, embriões HIF1-α(-/-) não foram considerados um bom modelo para este estudo devido ao envolvimento das MMPs na embriogênese/organogênese. Frente aos resultados encontrados, pode-se sugerir que a maior expressão de MMP2, SPARC e de bFGF estão associadas às NMPs. A mutação JAK2V617F foi associada a maiores concentrações de MMPs, TIMP2 VEGFA e bFGF. HIF1-α foi mais expresso na PV e na TE, sugerindo uma possível regulação da expressão das MMPs e TIMPs nessas doenças.

Unraveling the molecular underpinnings of Philadelphia-negative myeloproliferative neoplasms: implications for future therapeutic strategies

The present thesis encompasses the two researches projects I conducted during my PhD program in Molecular Biology and Pathology. The common thread is represented by the analysis of the signaling pathways implicated in the pathophysiology of the two most aggressive Philadelphia-negative myeloproliferative neoplasms, namely, atypical chronic myeloid leukemia (aCML) and primary myelofibrosis (PMF). In the last decade, since the description of the JAK2V617F mutation in 2005, the field of the molecular characterization of Philadelphia-negative myeloproliferative neoplasms has experienced an astonishing implementation that led to the discovery of 16 new mutations involving signal transduction, epigenetic modifiers, cell cycle regulators. Nevertheless, their pathogenetic relevance and whether they could represent good “druggable” candidates have to be proved yet. In the first section I provide the first report of the signaling cascade down-stream the rare cytogenetic lesion t(8;9)(p22;p24)/PCM1-JAK2 associated with aCML, finding that it selectively activates the ERK1/2 signaling without affecting JAK/STAT phosphorylation. In the second part, I investigated the implication of the ε isoform of novel Protein kinase Cs (PKCs) in the pathophysiology of the aberrant megakaryocytopoiesis in PMF, concluding that the over-expression of PKCε detains a crucial relevance in the aberrant behavior of PMF megakaryocytes and its inhibition is capable to restore their normal differentiation and abrogate the anti-apoptotic signaling. Both results are discussed in the view of their therapeutic implications. In case PCM1/JAK2-related hematologic neoplasms, ERK-inhibitors rather than JAK-inhibitors (i.e. ruxolitinib) should be considered as a “tailored” drugs. In case of PMF, PKCε-inhibitors (i.e. εV1-2 peptide) configure as an appealing strategy to re-direct the megakaryocytic neoplastic clone.

Patient perspectives of a diagnosis of myeloproliferative neoplasm in a case control study

Background: Myeloproliferative neoplasms (MPNs) including the classic entities; polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis are rare diseases with unknown aetiology. The MOSAICC study, is an exploratory case–control study in which information was collected through telephone questionnaires and medical records. Methods: As part of the study, 106 patients with MPN were asked about their perceived diagnosis and replies correlated with their haematologist’s diagnosis. For the first time, a patient perspective on their MPN diagnosis and classification was obtained. Logistic regression analyses were utilised to evaluate the role of variables in whether or not a patient reported their diagnosis during interview with co-adjustment for these variables. Chi square tests were used to investigate the association between MPN subtype and patient reported categorisation of MPN. Results: Overall, 77.4 % of patients reported a diagnosis of MPN. Of those, 39.6 % recognised MPN as a ‘blood condition’,23.6 % recognised MPN as a ‘cancer’ and 13.2 % acknowledged MPN as an ‘other medical condition’. There was minimal overlap between the categories. Patients with PV were more likely than those with ET to report their disease as a ‘blood condition’. ET patients were significantly more likely than PV patients not to report their condition at all.Patients from a single centre were more likely to report their diagnosis as MPN while age, educational status, and WHO re-classification had no effect. Conclusions: The discrepancy between concepts of MPN in patients could result from differing patient interest in their condition, varying information conveyed by treating hematologists, concealment due to denial or financial concerns. Explanations for the differences in patient perception of the nature of their disease, requires further, larger scale investigation.