Page 116 - 2020_08-Haematologica-web

F. Kramer et al.
fibrotic bone marrow. In overt fibrotic bone marrow, Ptpn2 and Ptpn11 gene expression was enhanced. Since TC-PTP (Ptpn2) has previously been ascribed an essential role in normal hematopoietic function,23,24 we further focused on PDGFRb regulation by TC-PTP. Given the con- clusive increase in Ptpn2 gene expression in overt fibrotic bone marrow, we next analyzed TC-PTP protein expres- sion in the different fibrotic stages in Gata-1low mice. Quantitative analysis of TC-PTP protein expression by PLA confirmed an increased expression in early and overt myelofibrosis (Figure 6A, original PLA images are shown in Online Supplementary Figure S5). In addition, in situ imag- ing showed ubiquitous expression of TC-PTP in the bone marrow of Gata-1low mice (Figure 6B). TC-PTP staining was positive in a wide variety of hematopoietic cells, megakaryocytes and in spindle-shaped cell structures, raising the question if TC-PTP directly regulates PDGFRb in bone marrow stromal cells. To determine the interac- tion of PDGFRb and TC-PTP, we again applied the sensi- tive proximity ligation technique (Figure 6C) and detected increased interaction of PDGFRb and TC-PTP in early and overt fibrotic bone marrow of Gata-1low mice (Figure 6D, original PLA images are shown in Online Supplementary Figure S5).
early and overt myelofibrosis, potentially counteracting PDGFRb phosphorylation. Likewise, Ptpn2 KD increased PDGFRb tyrosine phosphorylation at Y751 and Y1021 and resulted in enhanced downstream AKT and PLCγ1 signal- ing in fibroblasts. Furthermore, Ptpn2 KD cells showed a growth condition-dependent increase in expansion rate. Thus, while PDGF signaling is differentially regulated dur- ing PMF, PTP seem novel and so far unrecognized compo- nents in disease development. Previously not applied in bone marrow, PLA might add to diagnostics as a novel technique.
Intense efforts have been made to understand the mech- anisms leading to PMF, focusing on genetic analysis. However, the PMF-associated driver mutations leading to aberrant activation of JAK-STAT signaling are not unique to PMF but also occur in other MPN, namely essential thrombocythemia and polycythemia vera. Although one of the driver mutations is sufficient to induce PMF in patients, they are often accompanied by other mutations and epigenetic changes.25,26 However, there is still no dis- tinct molecular marker available for the respective MPN, emphasizing that the underlying mechanisms directing the different MPN are not yet understood. The discovery of JAK-STAT-associated mutations led to the development of JAK inhibitors and since its US Food and Drug Administration approval in 2011, the JAK1/2 inhibitor ruxolitinib has become part of combined standard therapy for PMF patients. Long-term treatment with ruxolitinib reduces spleen size and prolongs the overall survival of PMF patients.27 However, there is no improvement or reversal of bone marrow fibrosis. Furthermore, efficacy of ruxolitinib is limited by drug resistance,28 and JAK inhibi- tion does not abrogate clonal proliferation.29 Recently, PDGFRa signaling was shown to remain active despite JAK2 inhibition in vivo and is a cell-intrinsic bypass for maintaining downstream ERK signaling upon ruxolitinib treatment.30 To date, allogeneic stem cell transplantation is the only curative treatment for PMF; however, transplan- tation is only suitable for a subset of high-risk patients and limited by comorbidities and donor availability.31,32
TC-PTP in PDGFRb signaling and proliferation in fibroblasts in vitro
Our findings of increased PDGFRb and TC-PTP expres- sion and interaction led us to further analyze the regula- tion of PDGFRb by TC-PTP in fibroblasts in vitro. We knocked down Ptpn2 in NIH-3T3 fibroblasts and stimulat- ed cells with PDGF-BB (Figure 7A). Transfection with Ptpn2-targeting siRNA resulted in a moderate but signifi- cant knockdown (KD) compared to cells transfected with non-targeting (NT) siRNA (Figure 7B). We observed a con- secutive increase in PDGFRb phosphorylation at Y751 and downstream AKT signaling in Ptpn2 KD cells (Figure 7C and F). However, there was no substantial effect on down- stream ERK signaling (Figure 7G). PDGFRb phosphoryla- tion at Y1021, as well as downstream PLCγ activation, was increased in Ptpn2 KD cells (Figure 7D and E). We further monitored proliferation of Ptpn2 KD cells (Figure 7H) and did not find any evident differences in proliferation in cells cultured in complete growth medium containing 10% fetal bovine serum (FBS). However, when cells were exposed to serum-reduced medium (1% FBS), Ptpn2 KD cells showed increased growth rates compared to NT con- trol cells.
Gata-1low mice were characterized by a marked throm- bocytopenia, splenomegaly and progressive anemia start- ing before 5 months of age. Enrichment analyses of RNAseq data from early fibrotic bone marrow of Gata-1low mice revealed that genes implicated in PDGF binding are most over-represented within the up-regulated genes. We further observed enhanced PDGFRb and PDGF-B protein expression at 15 months of age, along with an increase in PDGFRb–PDGF-B interaction, analyzed by PLA.
Discussion
In this study, we provide detailed analyses of the expression patterns of PDGFRb signaling in the bone mar- row of Gata-1low mice at different fibrotic disease stages using RNAseq, qPCR, in situ protein expression analyses, multiplex staining and PLA. Early and overt fibrotic bone marrow was characterized by increased expression of PDGF signaling components and overt fibrosis by an increase in PDGFRb–PDGF-B interaction. Since PDGFRb tyrosine phosphorylation levels were not increased, the regulation of PDGFRb by PTP was investigated. Ptpn2 gene as well as TC-PTP protein expression was increased in fibrotic bone marrow of Gata-1low mice. Furthermore, enhanced PDGFRb–TC-PTP interaction was observed in
However, we did not detect an increase in PDGFRb tyrosine phosphorylation in the bone marrow of Gata-1low mice. This observation raised the question as to whether other mechanisms are involved in the regulation of the receptor in fibrotic bone marrow. Since a number of PTP have been identified, which site-selectively dephosphory- late PDGFRb, namely PTP1B, TC-PTP, SHP-1, SHP-2, PTP- PEST and DEP-1,19-22 an increased expression of these PTP might be responsible for the absence of an increased PDGFRb phosphorylation. Indeed, our data showed dis- tinct dynamics in gene expression of these PTP.
We observed decreased Ptpn1 and Ptprj gene expression in pre-fibrotic bone marrow in Gata-1low mice. The rele- vance of these PTP as potential diagnostic markers could be validated in a translational clinical approach. In con- trast, Ptpn2, Ptpn11 and Ptpn12 showed an increased gene
2092
haematologica | 2020; 105(8)