UBASH3B knockout Downregulates STAT1 Mediated Interferon Signalling and Megakaryocyte Related Genes in a JAK2 V617F Positive MPN Model

Abstract

Interferon (IFN) signalling via STAT1 plays an important role in the pathogenesis and treatment of myeloproliferative neoplasms (MPN). Single cell sequencing approaches have implicated megakaryocyte (MKC) biased stem cells with enhanced intrinsic activation of IFN signalling pathways in MPN patients as critical mediators of disease initiation and maintenance. UBASH3B is a histidine phosphatase and has been identified as a potential regulator of malignant haematopoiesis and as a mediator of JAK/STAT1 IFN signalling in lymphoid cells. RNA sequencing (RNA-seq) data in JAK2 V617F positive UKE-1, HEL (both own lab) and SET2 (GSE91062) cells demonstrate that UBASH3B is significantly downregulated following administration of the JAK inhibitor ruxolitinib (RUX). We observed that UBASH3B expression in CD34+ cells in patients with primary myelofibrosis (GSE53482) is significantly enhanced but reduced in patients with essential thrombocythaemia(ET) and polycythaemia (GSE103237) and hypothesized that UBASH3B may contribute to MPN pathogenesis. UKE-1 is a JAK2 V617F positive primitive haematopoietic cell line derived from an ET patient. It shows features of intrinsic IFN signalling and expresses genes related to MKC development and differentiation providing a useful model of MPN stem cell behaviour. CRISPR-Cas9 mediated KO of UBASH3B was performed in UKE-1 cells. A ribonucleoprotein complex of guide RNA and Cas9 protein was nucleofected into cells. Individual colonies were assessed for UBASH3B status using western blotting and Sanger sequencing. Four distinct, healthy KO clones were established for the UKE-1 cell line and compared to the original wildtype cell population. RNA-seq and phospho-proteomic analysis were undertaken in two KO clones. Extracted RNA was sequenced on Illumina platforms with a minimum of 45 million individual paired reads per sample. Differential gene expression was analysed using DESEQ2. Whilst, matched cell lysates underwent tandem mass tagging, high pH fractionation, and phospho-peptide enrichment was undertaken prior to liquid chromatography-mass spectrometry for total and phospho-proteomic analysis. RNA-seq and total proteomic analysis were consistent in demonstrating a significant downregulation of STAT1 (Log2FC-0.79, FDR 9.03E-49) and STAT2 (Log2FC -0.70, FDR 9.05E-20) with over-enrichment analysis identifying downregulation of type 1 IFN signalling pathways and pathways related to MKC and platelet function as shown in Figure 1. The significant downregulation of genes associated with platelet function, MKC development and differentiation included GATA1, GP1BA, ITGA2B, SELP, VWF and genes identified as mediators of fibrosis in MPN as demonstrated in Figure 2. Targeted real time PCR for STAT1, GP1BA and LTPB1 confirmed the same transcriptional patterns were present in both additional knockout clones. Knockout clones show a diminished response to exogenous IFN alpha administration consistent with downregulation of these pathways. Consistently reduced proliferation is observed in the KO clones by a variety of methodologies. Comparison to the transcriptional response in RUX treated UKE-1 cells demonstrates that at 24 hours post RUX treatment significant STAT1 downregulation is observed but not accompanied by the same pattern of down-regulation of MKC and fibrosis associated genes. The same was seen in cells persistently treated with high dose RUX for greater than 5 weeks. These results show a significant downregulation of IFN signalling in UBASH3B KO cells linked to a significant downregulation of MKC associated genes further highlighting the link observed in MPN stem cells. They implicate UBASH3B as a potential mediator of IFN signalling in a JAK2 V617F context and support a hypothesis that downregulating IFN signalling in MPN stem cells may have disease modifying potential by altering differentiation and pro-fibrotic potential. RUX treatment downregulates STAT1 without activating the same transcriptional programme associated with MKC gene downregulation and this may help explain the limited disease modifying effect of RUX observed in patients. This work has generated an important KO cell line model which will permit the molecular characterisation of differential STAT1/IFN pathway activation and allow further study to understand the regulation of MKC differentiation and pro-fibrotic gene transcription observed in MPN stem cells. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal