Page 100 - Haematologica March 2020

A. Caulier et al.
YODA1 significantly repressed GPA induction (Figure 2C, D), and this effect was dose-dependent (Online Supplementary Figure S3A). Using the UT7/EPO cell line that proliferates in the presence of EPO and expresses a high level of GPA at baseline (Online Supplementary Figure S1A, right), we observed a similar dose-dependent GPA downregulation after exposure to YODA1 (Figure 2E and Online Supplementary Figure S3B, E). We ruled out any YODA1 off-target effect using a Sh-RNA lentiviral strate- gy that specifically knocks down PIEZO1. Transduction efficiency was >90%, as assessed by GFP expression. The Sh-RNA-mediated PIEZO1 decrease was 65% at the RNA level (Online Supplementary Figure S4A). At the protein level, the decrease was 50% as assessed by MFC (Online Supplementary Figure S4B) in comparison to the level in cells transduced with a Sh-scrambled control (Sh-SCR). The PIEZO1 decrease was confirmed using western blot- ting and immunofluorescence (Online Supplementary Figure S4C, D). Following exposure to YODA1, GPA expression decreased in Sh-SCR-transduced cells but not in cells transduced with Sh-PIEZO1 (Figure 2F). The effect was related to GFP intensity, being weaker in cells expressing Sh-PIEZO1 at a lower level (Online Supplementary Figure S4E). Sh-PIEZO1 had the opposite effect of YODA1 on GPA expression since it enhanced erythroid differentiation by increasing the percentage of GPA+ cells compared to the control Sh-SCR. (Figure 2F and Online Supplementary Figure S4E). Sh-PIEZO1 did not revert the decreased prolif- eration induced by YODA1 exposure, and induced a reduced proliferation rate by itself in comparison to Sh- SCR (Online Supplementary Figure S4F).
PIEZO1 activation delays erythroid differentiation in human CD34+-derived erythroid cells
We then investigated whether the erythroid blockage induced by YODA1 in leukemic cell lines was confirmed in human primary cells. Sorted CD34+ cells were driven into erythroid differentiation (Online Supplementary Figure S2A). A dose of 1 mM YODA1 was selected for drug expo- sure based on dose-escalation data (Online Supplementary Figure S5A). Erythroid differentiation was assessed on day 10 by MFC and cytology. YODA1 reduced cell amplifica- tion without increasing cell death or apoptosis (Online Supplementary Figure S5B-D). Importantly, it led to a dras- tic delay in GPA acquisition, with accumulation of imma- ture CD36+/CD117+ erythroblasts and a decrease in CD71+/GPAHigh mature cells (Figure 3A-D). These results were confirmed by cytology, which showed an increase in immature erythroid precursors after exposure to YODA1 (Figure 3E, F). To investigate the timing of the YODA1 effect more precisely, we synchronized the cell culture (Online Supplementary Figure S2B). CD36+/GPA- colony- forming unit-erythroid cells were sorted on day 7 and exposed sequentially to 1 mM YODA1 or DMSO from day 7, 10, or 13 to the end of the culture. Red cell terminal dif- ferentiation was evaluated as the mean percentage of enu- cleated cells on day 21. The YODA1 effect was maximal when cells were exposed early, from day 7 to 10, as shown by the significant decrease in the Hoechst negative fraction at day 21 (Online Supplementary Figure S6A, B). A decrease in the ratio of enucleated cells was also observed on cytological analyses after early exposure, with the effect being weaker when YODA1 was administered on day 13 (Online Supplementary Figure S6C). We showed here that erythroid differentiation was delayed but not blocked
after PIEZO1 activation, since precursors reached final enucleation, but at a slower rate. Taken together, these data argue for a YODA1 effect at the transition between the GPAlow and GPAhigh stages, corresponding to the begin- ning of the differentiation of erythroid precursors.26
PIEZO1 activation delays erythroid differentiation through transcriptional control
Using RT-qPCR we investigated whether the YODA1- induced delay in erythroid differentiation was driven by global transcriptional control. In human primary erythroid cells, EPO-mediated GPA mRNA induction was inhibited by YODA1, indicating that the GPA repression occurred at the transcriptional level. PIEZO1 activation also reduced EPO-mediated α- and β-globin mRNA induction (Figure 4A). Since GATA2 expression during erythropoiesis is associated with the early proliferative stage while GATA1 is the key protagonist of terminal differentiation, we eval- uated the GATA2/GATA1 mRNA ratio after exposure to YODA1. It increased significantly in comparision with the ratio after exposure to DMSO (Figure 4B). A broader analysis of erythroid gene expression confirmed that pri- mary erythroblasts exposed to YODA1 displayed an immature erythroid profile, with higher expression of BMI1 and STAT5A mRNA and lower ALAS2, EPOR, AHSP and SLC4A1 mRNA expression (Figure 4C).
YODA1-induced inhibition of erythroid differentiation is Ca2+-dependent and does not require activation
of the Gardos channel
PIEZO1-mediated red cell dehydration in HX is Ca2+- dependent; since PIEZO1 is a non-selective cation chan- nel, we next evaluated whether this was also the case dur- ing erythropoiesis. In UT7/GM cells, YODA1 caused a strong dose-dependent increase in cytosolic Ca2+ (Figure 5A). This effect was abolished in Ca2+-free medium, con- sistent with YODA1 causing a strong Ca2+ influx from the extracellular to the intracellular compartment (Figure 5A, B). Ca2+ chelation using 2 mM ethylene glycol tetra-acetic acid (EGTA) totally blocked the YODA1-mediated decrease of GPA; this was rescued by addition of 2 mM calcium chloride (Figure 5C). The same effect was observed after intracellular Ca2+ chelation using BAPTA- AM (data not shown). Since red cell dehydration in PIEZO1- HX involves a Ca2+-dependent secondary activation of the Gardos channel,20 we investigated whether the effect of PIEZO1 on erythroblasts was also Gardos-dependent. As shown in Figure 5D, in UT7/GM cells, Senicapoc, a selec- tive Gardos channel inhibitor, did not revert the YODA1- associated blockade of GPA, indicating that YODA1- induced inhibition of erythroid differentiation did not require secondary activation of the Gardos channel.
PIEZO1 effect on erythroid differentiation involves modulation of NFAT, ERK1/2, and STAT5 pathways in UT7 cells and human primary erythroblasts
We next investigated Ca2+-dependent signaling path- ways activated downstream PIEZO1 in erythroid cells. Since NFAT is a well-known target of Ca2+-signaling in many cell types,27 we evaluated whether it was involved in erythroid cells. Calcineurin inhibition using 5 mM tacrolimus completely blocked the YODA1-induced GPA repression in UT7/EPO cells (Figure 6A). Moreover, expo- sure to YODA1 led to substantial nuclear translocation of NFATc1, significantly increasing the similarity score value
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