Novel cell intrinsic factors in the pathogenesis of JAK2 V617F positive myeloproliferative neoplasms

Abstract

The BCR-ABL negative myeloproliferative neoplasms (MPN) comprise a heterogeneous group of chronic blood cancers arising from the myeloid haematopoietic stem cells. The majority of cases are characterised by the presence of the JAK2 V617F driver mutation. Most remaining cases of MPN are characterised by the presence of mutations in CALR or MPL. All three driver mutations drive constitutive activation of the JAK-STAT signalling pathway as a hallmark of MPN pathogenesis. Despite this molecular similarity, there is a wide spectrum of disease phenotype observed across MPN. Broadly patients are classified into polycythaemia vera (PV), characterised by overproduction of red blood cells, essential thrombocythaemia (ET), characterised by overproduction of platelets, and myelofibrosis (MF), characterised by extensive bone marrow scarring. A small number of individuals progress on to a leukaemic blast phase (BP-MPN) of disease. All patients have an elevated rate of thrombosis which is currently effectively managed by antiplatelet therapy and cytoreduction for selected patients at highest risk. Prognosis is hugely variable and is dependent on multiple factors including disease phenotype, driver mutation status, the presence of additional pathogenic mutations, epigenetic dysregulation and patient factors. Whilst, in general, patients with ET and PV generally have a life expectancy of long years this is still reduced in comparison to age matched controls. In addition, these individuals are frequently symptomatic. In contrast, the prognosis for individuals with MF or BP-MPN is much worse and has a significant impact on quality of life.

Whilst haematopoietic stem cell transplant is potentially curative it is not suitable for the vast majority of patients due to associated toxicity and risk of mortality. Hydroxycarbamide is a frequently utilised cytoreductive therapy but has no disease modifying activity. Ruxolitinb, a JAK1/2 inhibitor, is effective at cytoreduction and provides symptom improvement but does not offer any significant degree of disease modification despite directly targeting the activated JAK-STAT pathway. Interferon-alpha (IFN-alpha) therapy is effective and can induce gradual molecular responses in a subset of patients. These responses are slow and take many years to develop and this treatment is often poorly tolerated in individuals with more aggressive forms of disease. Combination therapy which enhances the efficacy of either ruxolitinib or IFN-alpha has the potential to build on the benefits of these therapies for patients.

Cell intrinsic factors which modulate the activity of intracellular signalling cascades or the epigenetic regulation of the transcriptional response of the cell have provided some novel therapeutic targets in early phase development. Initially I aimed to use ruxolitinib as a means of JAK inhibition to identify novel cell intrinsic factors facilitating the pathogenesis of MPN.

Initially, I undertook a combination of bioinformatic approaches to determine the common transcriptional response to ruxolitinib across three JAK2 V617F positive MPN cell line models and the gene expression changes occurring at the point of megakaryocytic differentiation in CD34+ cells in MPN patients and healthy controls. UBASH3B expression was very significantly downregulated by ruxolitinib in all three cell line models, differentially expressed in MPN patients, upregulated at the point of megakaryocytic differentiation in MPN and has previously been implicated in the regulation of malignant myeloid haematopoiesis. In addition, ruxolitinib was observed to consistently downregulate genes associated with the unfolded protein response which is upregulated at the point of dysregulated megakaryopoiesis in MPN.

Using CRISPR-Cas9 I generated a UBASH3B KO model in a JAK2 V617F UKE1 cell line. The resulting phenotype was characterised by a significant downregulation of genes related to megakaryocyte development, genes related to IFN signalling and a loss of response to the anti-proliferative effects of exogenous IFN-alpha. Re-expression of UBASH3B did not rescue the original phenotype and shRNA mediated knockdown was inconsistent with the CRISPR-Cas9 KO phenotype. Ultimately this phenotype was determined not to be a gene specific event but rather could be replicated by single cell dilutional colony selection in UKE1 WT cells.

These “low IFN, low megakaryocytic” cells were investigated further given the importance of these two cellular processes in MPN biology. Loss of GATA1 expression was identified as the critical factor underlying loss of megakaryocytic gene expression. ATAC sequencing demonstrated loss of chromatin accessibility in enhancer regions identified as GATA1 binding sites highlighting the role of GATA1 in the regulation of chromatin accessibility. GATA1 was identified to directly regulate expression of TBL1X, a core component of the NCoR/SMRT epigenetic complex which was demonstrated to be upregulated during early megakaryocytic priming of CD34+ cells. Co-localisation of NCoR/SMRT components was significantly over-enriched in genomic regions showing loss of chromatin accessibility and GATA1 binding peaks suggesting an important role in the co-regulation of GATA1 target genes for further investigation.

The exact mechanism of IFN-alpha in MPN is not fully elucidated and therefore the differential response to exogenous IFN-alpha in the “low IFN, low megakaryocytic” cells was also investigated. I identified the upregulation of the unfolded protein response and in particular PERK/ATF4/CHOP arm driving apoptosis in the responsive cell populations. Co-treatment with bortezomib was synergistic in enhancing cell apoptosis and restoring responsiveness to IFN-alpha therapy and offers a novel combination therapy for further investigation in MPN.

Thesis is embargoed until 31 July 2025.

Date of Award	Jul 2024
Original language	English
Awarding Institution	Queen's University Belfast
Sponsors	Irish Clinical Academic Training (ICAT) Programme & Wellcome Trust
Supervisor	Ken Mills (Supervisor), Mary Frances McMullin (Supervisor) & Jaine Blayney (Supervisor)