Simultaneous Sessions III: New molecular pathways and markers in MPN and mastocytosis
The BCR-ABL-negative myeloproliferative neoplasms (MPN) are characterised by constitutive activation of the JAK-STAT pathway in haematopoietic progenitor cells (HPC), typically resulting from somatic mutations in one of three genes; JAK2, MPL or CALR. It is known from murine models that activation of the JAK-STAT pathways results in hyperproliferation of HPCs in the bone marrow niche, and it has recently been shown that this leads to exhaustion and premature aging in a percentage of HPCs. The impact of the activation of TP53 in the context of cells harbouring MPN-driver mutations remains unclear however, and whether these ‘TP53-high HPCs' contribute to the mature cell populations in MPNs or are out-competed by ‘TP53-low', HPC remains unknown.
To determine the effect of JAK-STAT activation on DNA damage and TP53 pathways in mature myeloid cells of patients with myeloproliferative disorders.
Peripheral blood samples from patients with essential thrombocythaemia (ET, n = 10), polycythaemia vera (PV, N = 11) and myelofibrosis (MF, n = 7) as well as healthy controls (NC, n = 11) were fractioned by gradient separation and bead selection into granulocyte (PMN), mononuclear (MNC) and T-cell (TC) fraction. Transcriptomic profiling was performed by expression array and validated by RNA-seq in a subset of patients. Cell fractionation and purity was confirmed by computational decomposition and supervised differential expression, co-expression and gene set enrichment analysis was then performed to identify disease specific pathway mis-regulation.
Both PMN and MNC fractions displayed strong enrichment for hallmark TP53 pathway activation and DNA damage pathways. As expected, no TP53 pathway activation was seen in the T-cell fraction, confirming that this is restricted to the myeloid compartment. Interestingly, TP53 pathway activation directly correlated with JAK-STAT pathway activation, suggesting the p53 activation results directly from the effect of the MPN-driver mutations.
Given that TP53 is capable of activating a number of biological processes including cell cycle arrest, apoptosis and autophagy we looked at expression levels of a number of transcriptional targets of TP53. There was significantly increased expression of the cyclin-dependent kinase inhibitor p21 (CDKN1A) a well-known transcriptional target of p53, in the MNC fraction in all disease groups, and in the PMN fractions of PV and MF. Additionally, we noted upregulation of anti-apoptotic BCL-XL (BCL2L1) in a manner that mimicked CDKN1A upregulation.
These data show that mature myeloid lineages in patients with MPNs harbour activation of DNA damage and TP53 pathways with transcriptional activation of down-stream effectors in PMN and MNC compartments that correlate with JAK-STAT activation. Typically, engagement of TP53 pathways serves to limit proliferation of mutation harbouring cells, however the upregulation of anti-apoptotic genes may offer a potential mechanism for the persistent of these cells. These data suggest that MPN clones may develop mechanisms that permit escape from TP53 mediated death, and offers potential insight into novel therapeutic targets.