Page 190 - 2021_12-Haematologica-web

Letters to the Editor
Figure 3. Validation of interferon-stimulated genes in immortalized megakaryocyte cell line (imMKCL). (A) Immunoblot analysis of total SRC expression on day 0, 2 and 4 megakaryocytes (MK) (left). GAPDH was used as loading control. Quantification of SRC expression in triplicate immunoblots and statistical analysis using one-way ANOVA with multiple comparisons (right). (B) Quantitative real-time polymerase chain reaction (qRT-PCR) validation showing relative expression of SRC, IFIT1, MX1, OAS2, IFIT3 and ISG15 on day 0, 2 and 4. Statistical analysis was performed using one-way ANOVA with multiple comparisons. (C) qRT-PCR validation showing relative expression of SRC, IFIT1, MX1, OAS2, IFIT3 and ISG15 on day 2 MK transfected with empty vector, WT-SRC or E527K-SRC in pSecTag- Hygro expression vector (triplicated transfection experiment). Statistical analysis was performed using one-way ANOVA with multiple comparisons. (D) qRT-PCR validation showing relative expression of SRC, RUNX1, IFIT1, MX1, OAS2, IFIT3 and ISG15 in MK from a control and the patient carrying the E527K SRC variant (3 technical repeats). Statistical analysis was performed using one-way ANOVA with multiple comparisons.
in the RNAseq dataset could not be explained by the lower SRC expression in E527K-SRC compared to WT- SRC MK. Though the difference between WT-SRC and E527K-SRC MK in the transfection experiment was only significantly different for IFIT3 expression, the difference for the other ISG was always more pronounced between E527K-SRC and the condition with the empty vector than when comparing WT-SRC with the empty vector. This difference could be due to the fact that these expres- sion studies were performed on day 2 immature imMKCL cells while the omics was performed on day 12 mature MK. Finally, expression studies were validated in HSC-derived day 12 mature MK from a patient carrying E527K-SRC3 and a control. No significant difference in SRC and RUNX1 expression could be detected while patient MK showed significantly decreased expression of all ISG except OAS2 (Figure 3D). The downregulation was most pronounced for IFIT1 and MX1, the two genes that were the most significantly downregulated in the RNAseq dataset (Figure 2C).
The combined omics approach detected IFNα/b signal- ing as most significantly downregulated pathway associ- ated with E527K-SRC hyperactivity. This was an unex- pected finding because interferons are generally consid- ered to be negative regulators of cellular proliferation and maturation.5 As qRT-PCR data showed no evidence for an altered response towards IFNα, the difference in ISG expression in E527K-SRC is due to a downstream effect. The proteomics (but not the RNAseq) data showed that STAT1 and STAT2 were significantly downregulated in E527K-SRC MK (Online Supplementary Table S2). Further studies must be undertaken to pinpoint the exact place of SRC in this pathway. In chronic myeloid leukemia, the fusion gene BCR-ABL is the result from the translocation between chromosomes 9 and 22. Similar to E527K-SRC, BCR-ABL is an overactive tyrosine kinase. Studies showed that BCR-ABL in hematopoietic cells caused the transcriptional suppression of ISG such as ISG15, IRF1, IRF9 and IFIT1,7 which resulted in impaired IFNα-medi- ated protection against viral infection and reversal of IFNα-dependent growth suppression, thereby promoting malignant transformation.7 As downregulated ISG were also present in E527K-SRC MK, this points to a clear sim- ilarity between both overactive kinases, SRC and BCR-ABL. The suppression of ISG in BCR-ABL cells is reflected by the suppression of JAK-STAT pathway com- ponents, including STAT1.7 It is also interesting that pre- vious proteomic studies using inducible pluripotent stem cell-derived MK from a patient with the GFI1B Q287* variant showed low expression levels of STAT1, MX1, IFIT1, IFIT3 and OAS2, similar to our findings.8 This study hypothesizes that the underlying pathway would be a failure of IFNg to activate its target genes via STAT1 although this was not experimentally studied. Of note, similar to SRC deficiency, GFI1B defects result in throm- bocytopenia, α granule deficiency and myelofibrosis.
In conclusion, we here describe that interferon signal- ing plays a role during megakaryopoiesis by acting down- stream of SRC signaling. However, the exact mechanisms
of how SRC can change ISG to influence megakary- opoiesis still remain unknown.
Lore De Kock,1 Fabienne Ver Donck,1 Chantal Thys,1 Anouck Wijgaerts,1 Koji Eto,2,3 Chris Van Geet1 and Kathleen Freson1
1Center for Molecular and Vascular Biology, Department of Cardiovascular Sciences, University of Leuven, Leuven, Belgium; 2Department of Clinical Application, Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan and 3Department of Regenerative Medicine, Chiba University Graduate School of Medicine, Chiba, Japan
Correspondence:
KATHLEEN FRESON - kathleen.freson@kuleuven.be doi:10.3324/haematol.2021.279248
Received: May 17, 2021.
Accepted: July 27, 2021.
Pre-published: August 5, 2021.
Disclosures: no conflicts of interest to disclose.
Contributions: LDK analyzed the results, performed the experiments, and wrote the manuscript; LDK and FVD performed statistical analyses; AW contributed to the sample preparation for RNAseq; FVD analyzed the RNA sequencing and shotgun proteomics data and performed the pathway analysis; CT performed imMKCL experiments; KE provided differentiation protocol and imMKCL cells; CVG studied E527K defective patients; KF designed the study and analysis plan and co-wrote the manuscript.
Funding: this work was supported by KULeuven BOF grant C14/19/096, FWO grant G072921N and research grants from Novo Nordisk and Swedish Orphan Biovitrum AB (SOBI).
Data sharing statement: RNA sequencing data will be released in European Genome-phenome Archive (EGA).
References
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