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S. von Palffy et al.
(C-C motif) ligand 28 (CCL28). To investigate whether the positive regulators identified also affected CD34+CD38low CML cells from BM, we performed a more focused screen using only the top regulating cytokines in a 96-well format. All cytokines identified as positive regulators of primitive CML PB cells also increased the number of primitive CML BM cells (Figure 1C). However, only IL-3, IL-1a/b, GM-CSF, IL-6, MSTNpp, and IFN-γ were able to expand cell numbers at least 2-fold, which was used as the cut-off in the original screen.
We next investigated whether the positive regulators identified also expanded normal CD34+CD38low BM cells, enriched for HSC (Figure 1D). Whereas IL-3 expanded the normal BM cells during a 7-day culture, none of the other cytokines increased the total cell number. However, MSTNpp promoted their survival, as indicated by a high- er cell number compared to the no cytokine control.
MSTNpp increases the progenitor potential of chronic myeloid leukemia and normal bone marrow cells
Of the top regulators identified, we selected MSTNpp for further studies given its strong effect on primary CML cells and its previously unexplored role in normal and malignant hematopoiesis. MSTNpp is produced by mus- cle cells and secreted into the bloodstream where its rec- ognized function is to bind and regulate the muscle inhibiting myokine myostatin (also known as GDF-8).22 To study the growth dynamics of CML cells in response to different concentrations of MSTNpp, a dose titration experiment was performed on CD34+ CML cells. In a 7- day culture, MSTNpp increased the cell number in all treated wells in a dose-dependent manner (Figure 2A), with similar results observed during three days of culture (Online Supplementary Figure S2A). Adding a polyclonal anti-MSTNpp antibody to the culture reduced the growth promoting effect of MSTNpp on CML cells, confirming the specificity of the observed response (Figure 2B).
To investigate whether MSTNpp had a positive effect also on primitive murine CML cells, Lin– Sca-1+ c-Kit+ (LSK) BM cells from transgenic Scl-tTA/BCR-ABL mice20 were cultured in vitro with or without MSTNpp. In a 7-day culture, MSTNpp greatly increased the cell number compared to untreated cells (Figure 2C). For wild-type murine LSK BM cells from B6.SJL mice, MSTNpp nearly maintained the cell number, hence promoting cell survival compared to no cytokine control (Figure 2D). These results are in agreement with our findings using human CD34+CD38low chronic phase CML PB and BM cells (Figure 1B and C), as well as for normal CD34+CD38low BM cells (Figure 1D).
Given the robust response of MSTNpp in chronic phase CML, we hypothesized that MSTNpp could have a simi- lar growth promoting effect on other myeloid malignan- cies. Therefore, we cultured primary blasts from five AML patients and three CD34+ blast crisis CML patients with and without MSTNpp, and evaluated the cell num- bers at day 3 or 4 and day 7 (Online Supplementary Figure S2B and C). Out of the five AML patients tested, two responded to MSTNpp with a slight increase in survival at day 3. At day 7, however, this effect was no longer seen. Another AML patient showed a response to MSTNpp only at day 7, but not at day 3. For the CD34+ blast crisis CML cells, one out of the three samples responded to MSTNpp stimulation with increased sur-
vival; the other two, including a sample with a T315I- mutation, did not respond. These results suggest that, unlike chronic phase CML cells, blast crisis CML cells and AML blasts are not consistent in their response to MSTNpp.
Next, we investigated the possible effects of MSTNpp on cellular differentiation of primary human CD34+ CD38low CML cells. Since CD34 expression, in contrast to CD38 expression, correlates with stem cell activity of in vitro cultured cells,23,24 we used CD34 as a marker to assess whether the identified cytokines would maintain the CML cells in a primitive state. Notably, whereas cells cul- tured in IL-3 and GM-CSF displayed reduced CD34 expression, IL-1a/b, IL-6, and MSTNpp stimulation resulted in retained CD34 expression, suggesting that these cytokines maintained the primitive state of the cells better (Figure 2E).
As CD34 expression of MSTNpp expanded cells was retained in liquid culture, we further investigated whether MSTNpp stimulation promotes the colony forming capacity of CML CD34+ cells. To this end, CD34+ or CD34+CD38low cells were cultured with or without MSTNpp for seven days in serum free media, prior to plating in methylcellulose media. MSTNpp significantly increased the number of CML colonies compared to con- trol cells without cytokine (Figure 2F). Moreover, pre- stimulation with MSTNpp increased the colony output upon replating (Figure 2F), suggesting that MSTNpp expands CML cells with self-renewal capacity.
We also investigated the effects of MSTNpp on the colony forming ability of normal CD34+CD38low BM cells after one week in culture. Although MSTNpp did not expand normal CD34+CD38low BM cells during a 7-day culture (Figure 1D), MSTNpp had a marked effect on the subsequent colony forming ability of the stimulated cells as compared to no cytokine control (Figure 2G and Online Supplementary Figure S2D). These findings show that MSTNpp promotes the colony forming capacity of both CML cells and normal hematopoietic stem and progenitor cells (HSPC).
Finally, to study the effect of MSTNpp on primitive CML cells in a setting that more closely resembles the BM microenvironment, we performed co-culture experiments where CD34+ CD38low CML cells were cultured on a monolayer of MSC established from primary CML BM (Figure 2H). When cultured on stroma, there were twice as many CML cells in the culture after three days, com- pared to the no stroma control. This suggests that the CML MSC produce growth factors important for the growth and survival of primitive CML cells. When adding MSTNpp to the co-culture, the cell numbers increased even further (approx. 2-fold), showing that primitive CML cells respond to MSTNpp also in a setting where other stimuli are present. These findings suggest that the effect of MSTNpp on CML cells is physiologically rele- vant.
MSTNpp affects chronic myeloid leukemia cells through mechanisms independent of myostatin
Myostatin is a transforming growth factor beta (TGF-b) superfamily member,25 synthesized as part of the myo- statin gene (MSTN) and produced primarily by muscle cells. The MSTN gene encodes a biologically-inactive pre- cursor protein consisting of a signaling peptide, the MSTNpp-domain and the myostatin-domain. Proteolytic
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haematologica | 2020; 105(8)