Page 186 - 2021_12-Haematologica-web

Letters to the Editor
Combined transcriptome and proteome profiling of SRC kinase activity in healthy and E527K defective megakaryocytes
Megakaryocytes (MK) and platelets express high levels of different SRC family kinases including the proto-onco- gene SRC, a tyrosine kinase that has been extensively studied in platelets.1 The germline heterozygous mis- sense variant E527K in SRC was detected in patients from three unrelated families with thrombocytopenia accompanied with bleeding symptoms, a paucity of α granules, and a variety of other variable symptoms including myelofibrosis, osteoporosis, facial dysmor- phism and behavioral problems.2-4 The E527K-SRC vari- ant resulted in increased kinase activity.2 These observa- tions suggested an important role of SRC in megakary- opoiesis, but defects in downstream pathways because of increased kinase activity remain unknown. In this study, we further explored the effect of SRC kinase signaling in megakaryopoiesis using omics approaches.
For RNA sequencing (RNAseq), CD34+ hematopoietic stem cells (HSC) were isolated from healthy controls before transduction with wild-type SRC (WT-SRC) and E527K-SRC lentiviral vectors in triplicate and differentia- tion to MK as described2 (Figure 1A). Day 12 MK were analyzed by flow cytometry for expression markers CD41 (ITGA2B) and CD42 (GP9) (Figure 1B). Though E527K-SRC cultures contained less CD41/CD42 double- positive MK, there was no difference in CD41 positive immature MK when compared to the WT condition. RNA was extracted from the complete cell population as the aim was to detect differences in genes regulating MK maturation and differentiation. A total of six WT-SRC and six E527K-SRC RNA samples were used for RNAseq to identify differentially expressed genes (DEG). Principal component analysis performed on the RNAseq datasets showed obvious clustering of WT-SRC and E527K-SRC MK samples according to condition (Figure 1C). The SRC gene for WT-SRC samples was covered by 8,661±901 read counts while only 2,988±410 read counts were detected for E527K-SRC MK samples but 80,83±8,06% of these reads contained the E527K variant. Relative expression levels of all significant DEG in E527K-SRC versus WT-SRC samples were visualized in a heatmap (Figure 1D). A total of 852 significant (false discovery rate [FDR]<0.05 and |log2FC|>1) DEG were detected (see the Online Supplementary Table S1 for the full dataset), of which 369 upregulated genes and 483 downregulated genes as visualized in the volcano plot (Figure 1E). Reactome pathway analysis showed that the downregu- lated DEG were enriched in pathways like platelet activa- tion, signaling and aggregation, interferon type I or α/β signaling, platelet degranulation, response to elevated platelet cytosolic Ca2+, and RUNX1 regulates genes involved in megakaryocytic differentiation and platelet function as top five (Figure 1F). On the other hand, the pathway analysis for upregulated DEG in E527K-SRC MK included interleukin-10 signaling, degradation of the extracellular matrix, chemokine receptors bind chemokines, collagen degradation and signaling by inter- leukins as top five pathways (Figure 1F). Interestingly, Barozzi et al. recently described that overactive SRC kinase may affect proplatelet formation by affecting the interaction of MK with the extracellular matrix.4
Shotgun proteomics was next used to analyze protein expression in transduced WT-SRC and E527K-SRC differ- entiated MK at day 12 (Figure 1A). Principal component analysis performed on the proteomics dataset showed a
clear separation between WT-SRC and E527K-SRC MK samples, with 66% of total variance explained by the first principal component and 13% explained by the second principal component (Figure 2A). Statistical analysis of WT-SRC and E527K-SRC MK proteomes detected 142 significant (FDR<0.01) differentially expressed proteins (DEP) (see the Online Supplementary Table S2 for the full dataset), of which 43 were upregulated and 99 downreg- ulated as shown in the volcano plot (Figure 2B). Similar to the RNAseq results, SRC was significantly downregu- lated in E527K-SRC MK (Figure 2B). Interestingly, the Reactome pathway analysis again identified an enrich- ment of downregulated DEP in the interferon pathways, but no platelet-related pathways were detected.
By comparing the set of DEG and DEP, 20 upregulated and 24 downregulated joined DEG/DEP sets were detect- ed in E527K-SRC MK. Reactome pathway analysis of these joined DEG/DEP identified an enrichment of inter- feron α/b signaling, interferon signaling and cytokine sig- naling in the immune system as significant (Figure 2C). This unbiased approach to identify pathways related to hyperactive SRC signaling in MK proposed decreased interferon α/b signaling as an unexpected player. Interferon signaling is neither well studied in megakary- opoiesis nor in SRC kinase signaling, encouraging our fur- ther studies in the immortalized megakaryocyte cell line (imMKCL) and in MK derived from a patient with the E527K-SRC variant. The top five downregulated interfer- on-stimulated genes (ISG) IFIT1, MX1, OAS2, IFIT3 and ISG15 (labeled in Figures 1E, 2B and 2C) were selected for these validation studies.
Interestingly, SRC protein expression was significantly increased from day 0 (non-differentiated) to days 2 and 4 differentiated imMKCL (Figure 3A; Online Supplementary Figure S1A and B). This increase was confirmed by quan- titative real-time ploymerase chain reaction (qRT-PCR) analysis (Figure 3B). The expression of ISG showed increased levels of IFIT1, MX1, and OAS2 during MK dif- ferentiation while no significant differences were detect- ed for IFIT3 and ISG15 (Figure 3B). The expression of RUNX1 in contrast to that of ITGA2B remained unchanged during megakaryopoiesis (Figure 3B).
Genetically modified imMKCL transduced with WT- SRC and E527K-SRC lentiviral vectors showed similar results as detected in the RNAseq study. A significant downregulation of all ISG was present in E527K-SRC MK compared to WT-SRC MK (Online Supplementary Figure S1C). Adding interferon α (IFNα) to the medium resulted in higher expression of ISG but no difference in reactivity was detected between E527K-SRC and WT-SRC MK (Online Supplementary Figure S1C). In order to exclude the possibility that the difference in ISG expression between WT-SRC and E527K-SRC would be due to the difference in SRC expression between those conditions or the lentiviral transduction, transfection experiments were performed. In addition, a comparison with MK transfect- ed with an empty vector was missing from previous experiments. Therefore, imMKCL were transfected with pSecTag2 empty, WT-SRC and E527K-SRC expression vectors and the expression of SRC and the different ISG was quantified in day 2 MK (Figure 3C). Compared to the condition with the empty vector, MK with WT-SRC and E527K-SRC now expressed similarly elevated SRC levels. Interestingly, both WT-SRC and E527K-SRC MK expressed significantly lower ISG levels compared to MK transfected with the empty vector (Figure 3C). Overexpression of SRC in the undifferentiated imMKCL affected ISG expression during megakaryopoiesis but this experiment also pointed out that the detection of the ISG
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haematologica | 2021; 106(12)