The ruxolitinib effect: understanding how molecular pathogenesis and epigenetic dysregulation impact therapeutic efficacy in myeloproliferative neoplasms.

The myeloproliferative neoplasms (MPN), polycythaemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF) are linked by a propensity to thrombosis formation and a risk of leukaemic transformation. Activation of cytokine independent signalling through the JAK/STAT cascade is a feature of these disorders. A point mutation in exon 14 of the JAK2 gene resulting in the formation of the JAK2 V617F transcript occurs in 95% of PV patients and around 50% of ET and PMF patients driving constitutive activation of the JAK/STAT pathway. Mutations in CALR or MPL are present as driving mutations in the majority of remaining ET and PMF patients. Ruxolitinib is a tyrosine kinase inhibitor which inhibits JAK1 and JAK2. It is approved for use in intermediate and high risk PMF, and in PV patients who are resistant or intolerant to hydroxycarbamide. In randomised controlled trials it has demonstrated efficacy in spleen volume reduction and symptom burden reduction with a moderate improvement in overall survival in PMF. In PV, there is demonstrated benefit in haematocrit control and spleen volume. Despite these benefits, there is limited impact to induce complete haematological remission with normalisation of blood counts, reduce the mutant allele burden or reverse bone marrow fibrosis. Clonal evolution has been observed on ruxolitinib therapy and transformation to acute leukaemia can still occur. This review will concentrate on understanding the clinical and molecular effects of ruxolitinib in MPN. We will focus on understanding the limitations of JAK inhibition and the challenges to improving therapeutic efficacy in these disorders. We will explore the demonstrated benefits and disadvantages of ruxolitinib in the clinic, the role of genomic and clonal variability in pathogenesis and response to JAK inhibition, epigenetic changes which impact on response to therapy, the role of DNA damage and the role of inflammation in these disorders. Finally, we will summarise the future prospects for improving therapy in MPN in the JAK inhibition era.

Epigenetics in Myeloproliferative Neoplasms.

A decade on from the description of JAK2 V617F, the MPNs are circumscribed by an increasingly intricate landscape. There is now evidence that they are likely the result of combined genetic dysregulation, with several mutated genes involved in the regulation of epigenetic mechanisms. Epigenetic changes are not due to a change in the DNA sequence but are reversible modifications that dictate the way in which genes may be expressed (or silenced). Among the epigenetic mechanisms, DNA methylation is probably the best described. Currently known MPN-associated mutations now include JAK2, MPL, LNK, CBL, CALR, TET2, ASXL1, IDH1, IDH2, IKZF1 and EZH2. Enhancing our knowledge about the mutation profile of patients may allow them to be stratified into risk groups which would aid clinical decision making. Ongoing work will answer whether the use of epigenetic therapies as alterative pathway targets in combination with JAK inhibitors may be more effective than single agent treatment.

QUADrATiC: scalable gene expression connectivity mapping for repurposing FDA-approved therapeutics.

BACKGROUND: Gene expression connectivity mapping has proven to be a powerful and flexible tool for research. Its application has been shown in a broad range of research topics, most commonly as a means of identifying potential small molecule compounds, which may be further investigated as candidates for repurposing to treat diseases. The public release of voluminous data from the Library of Integrated Cellular Signatures (LINCS) programme further enhanced the utilities and potentials of gene expression connectivity mapping in biomedicine. RESULTS: We describe QUADrATiC ( http://go.qub.ac.uk/QUADrATiC ), a user-friendly tool for the exploration of gene expression connectivity on the subset of the LINCS data set corresponding to FDA-approved small molecule compounds. It enables the identification of compounds for repurposing therapeutic potentials. The software is designed to cope with the increased volume of data over existing tools, by taking advantage of multicore computing architectures to provide a scalable solution, which may be installed and operated on a range of computers, from laptops to servers. This scalability is provided by the use of the modern concurrent programming paradigm provided by the Akka framework. The QUADrATiC Graphical User Interface (GUI) has been developed using advanced Javascript frameworks, providing novel visualization capabilities for further analysis of connections. There is also a web services interface, allowing integration with other programs or scripts. CONCLUSIONS: QUADrATiC has been shown to provide an improvement over existing connectivity map software, in terms of scope (based on the LINCS data set), applicability (using FDA-approved compounds), usability and speed. It offers potential to biological researchers to analyze transcriptional data and generate potential therapeutics for focussed study in the lab. QUADrATiC represents a step change in the process of investigating gene expression connectivity and provides more biologically-relevant results than previous alternative solutions.

A multi-centre retrospective study of rituximab use in the treatment of relapsed or resistant warm autoimmune haemolytic anaemia.

This retrospective analysis assessed the response, safety and duration of response to standard dose rituximab 375 mg/m(2) weekly for four weeks as therapy for patients with primary or secondary warm autoimmune haemolytic anaemia (WAIHA), who had failed initial treatment. Thirty-four patients received rituximab for WAIHA in seven centres in the Republic of Ireland. The overall response rate was 70·6% (24/34) with 26·5% (9/34) achieving a complete response (CR). The time to response was 1 month post-initiation of rituximab in 87·5% (21/24) and 3 months in 12·5% (3/24) of patients. The median duration of follow-up was 36 months (range 6-90 months). Of the patients who responded, 50% (12/24) relapsed during follow up with a median time to next treatment of 16·5 months (range 6-60 months). Three patients were re-treated with rituximab 375 mg/m2 weekly for four weeks at relapse and responded. There was a single episode of neutropenic sepsis. Rituximab is an effective and safe treatment for WAIHA but a significant number of patients will relapse in the first two years post treatment. Re-treatment was effective in a small number of patients, suggesting that intermittent pulse treatment or maintenance treatment may improve long-term response.

Modification of the Histone Landscape with JAK Inhibition in Myeloproliferative Neoplasms.

Dysregulation of epigenetic processes is increasingly understood to play a role in the pathogenesis of myeloproliferative neoplasms (MPNs). Ruxolitinib, a JAK/STAT inhibitor, has proved a useful addition to the therapeutic arsenal for these disorders, but has limited disease modifying activity. We determined the effect of JAK inhibition on the histone landscape of MPN cells in cell line models of MPNs and validated using samples from the MAJIC randomised clinical trial of ruxolitinib in polycythaemia vera and essential thrombocythaemia. We demonstrated an epigenetic modifying effect of ruxolitinib using a histone modification assay. The majority of 21 histone H3 modifications were upregulated, with H3K27me3 and H3K36me2 significant in the combined cell line results. Chromatin immunoprecipitation and sequencing (CHIP-seq) for three marks of interest, H3K4me1, H3K4me3 and H3K27ac, was consistent with the histone modification assay showing a significant increase in H3K4me3 and H3K27ac peaks at promoter regions, both marks of active transcription. In contrast, RNA sequencing demonstrates a coordinated reduction in gene expression in a number of cell pathways including PI3K-AKT signalling, transcriptional misregulation in cancer and JAK-STAT signalling in spite of these histone changes. This highlights the complex mechanisms of transcriptional control within the cells which was reflected in analysis of the histone landscape in patient samples following ruxolitinib treatment.

Methylation age as a correlate for allele burden, disease status, and clinical response in myeloproliferative neoplasm patients treated with vorinostat.

The myeloproliferative neoplasms (MPNs) are a heterogeneous group of clonal neoplastic disorders. Driver mutations in JAK2, CALR, and MPL genes have been identified in the majority of cases. Alongside these, an increasing number of genes are repeatedly identified as mutated in MPN. These, including ASXL1, TET2, DMNT3A, and EZH2, have key roles in epigenetic regulation. Dysregulation of epigenetic processes is therefore a key feature of MPN. Vorinostat is a pan histone deacetylase inhibitor (HDACi) that has been investigated in MPN. DNA methylation (DNAm) is a well-defined epigenetic mechanism of transcription modification. It is known to be affected by ageing, lifestyle, and disease. Epigenetic ageing signatures have been previously described allowing calculation of a methylation age (MA). In this study we examined the effect of vorinostat on MA in MPN cell lines and in patients with polycythaemia vera (PV) and essential thrombocythaemia (ET) treated with vorinostat as part of a clinical trial. An older MA was observed in patients with a higher JAK2 V617F allele burden and those with a longer duration of disease. PV patients had a MA older than that predicted whilst MA was younger than predicted in ET. Treatment with vorinostat resulted in a younger MA in PV patients and older MA in ET patients, in both cases a trend towards the normal chronological age. When MA change was compared against response, nonresponse was associated with a younger than predicted MA in ET patients and a higher than predicted MA in PV patients. The link between MA and JAK2 mutant allele burden implies that allele burden has a role not only in clinical phenotype and disease evolution in MPN patients, but also in the overall methylation landscape of the mutated cells.

Chronic Myeloid Leukemia with e19a2 BCR-ABL1 Transcripts and Marked Thrombocytosis: The Role of Molecular Monitoring.

While most patients with chronic myeloid leukemia (CML) express either e13a2 or e14a2 BCR-ABL1 transcripts, a significant minority expresses variant transcripts, of which e19a2 is the most common. Although considered to have a relatively favourable outcome, reported responses to tyrosine kinase inhibitor (TKI) therapy are variable with molecular monitoring in CML patients with e19a2 BCR-ABL1 transcripts rarely reported. A case of e19a2 BCR-ABL1 CML with marked thrombocytosis is described in which the value of molecular monitoring is emphasised during treatment interruptions, dose reductions, and changes. This case serves to demonstrate the requirement for prospective real-time quantitative PCR (RQ-PCR) assays for patients with variant BCR-ABL1 transcript types and standardisation of such assays to enable modern patient management.