Mechanisms of disease transformation in myeloproliferative neoplasms

Myeloproliferative neoplasms are heterogeneous clonal haematological malignancies where disease initiation occurs by acquisition of a somatic mutation in a haematopoietic stem cell. This mutation most often occurs in a janus kinase-signal transducer and activator of transcription signalling gene which drives constitutive janus kinase-signal transducer and activator of transcription signalling, with ensuing clonal expansion of downstream progenitor cells. Importantly, intra-tumoural cellular and molecular heterogeneity underscores the phenotypic heterogeneity of myeloproliferative neoplasms. Various factors drive disease progression but additional somatic mutations contribute significantly. Current myeloproliferative neoplasm therapies show limited effects on modifying disease biology and treatment resistance is common, associated with disease progression. Understanding whether distinct mutational profiles are associated with treatment resistance and examining the molecular signatures of mutant disease- driving haematopoietic stem and progenitor cells may reveal novel therapeutic targets and most importantly, lead to development of new treatments for these patients. Single-cell genomics approaches are ideally placed to achieve this through multi-omic technologies that can isolate mutant and non-mutant disease-driving stem and progenitor cells.
My overarching aims were to firstly to understand whether distinct mutational profiles were associated with treatment-resistant/intolerant myeloproliferative neoplasms and increased risk of disease progression and secondly to gain insights into the mechanisms driving transformation within a molecularly-defined myeloproliferative neoplasm subgroup through studying the disease- driving haematopoietic stem and progenitor cells. Study of two treatment-intolerant/resistant myeloproliferative neoplasm cohorts uncovered distinct molecular profiles. In hydroxycarbamide- resistant/intolerant essential thrombocythaemia, I observed increased TP53 and SF3B1 mutations in patients with myelofibrotic progression which importantly was not alleviated with ruxolitinib treatment. Analysis of a second, more advanced myeloproliferative neoplasm cohort of ruxolitinib-resistant/intolerant myelofibrosis patients showed a novel finding of enhanced RAS- pathway mutations.
To understand how SF3B1 mutations collaborated with JAK2V617F mutations to accelerate myelofibrotic progression, I applied two single-cell multi-omic approaches. The first, 10X genomics, characterised the haematopoietic cellular landscape in JAK2V617F-SF3B1-mutated myeloproliferative neoplasms and the second, TARGET-seq, paired mutation and gene expression information in the same cell to enable gene and splicing signature analysis of the mutated disease-driving and also non-mutant haematopoietic stem and progenitor cells.
Through 10X genomics analysis, I identified an abnormal expansion in earlier erythroid, megakaryocyte and most prominently eosinophil-basophil-mast cell progenitors in JAK2V617F- SF3B1-mutated haematopoietic stem and progenitor cells. I resolved the clonal architecture of JAK2V617F-SF3B1-mutated myeloproliferative neoplasms, observing that in the majority of cases, double mutant cells were present and that SF3B1 was the founding event.
Uniquely, full-length transcriptome TARGET-seq made analysis of SF3B1-mutated aberrant splicing events possible. This uncovered novel biologically relevant aberrant splicing events in the STAT1 gene. These events were a reduction in intron retention which would have an expected consequence of increased STAT1 activation. This analysis couples abnormal splicing with enhanced janus kinase-signal transducer and activator of transcription signalling to support a mechanism for an accelerated myeloproliferative neoplasm phenotype in JAK2V617F-SF3B1- mutated myeloproliferative neoplasms.
In conclusion, this analysis established SF3B1 mutations in myeloproliferative neoplasms promote myelofibrotic disease progression and resolved the clonal architecture of JAK2V617F- SF3B1-mutated myeloproliferative neoplasms identifying abnormally expanded haematopoietic stem and progenitor cells populations. SF3B1-mutant specific splicing signatures were discovered that would be expected to augment janus kinase-signal transducer and activator of transcription signalling supporting mechanism by which SF3B1 cooperates with JAK2V617F to promote an accelerated myeloproliferative neoplasm phenotype. The overarching aim is translating this new knowledge into patient benefit. Incorporation of spliceosome mutation information into clinical tools may improve risk stratification and specifically for JAK2V617F- SF3B1-mutated myeloproliferative neoplasms, a move towards precision medicine may be achievable through development of therapies targeting STAT1.

Keywords: JAK2
myeloproliferative neoplasms
myeloid
splicing
Advisor: Mead, A
Harrison, C
Psaila, B