In VitroandIn VivoInteractions between Stromal and Leukemic Cells

Understanding the mechanisms underlying changes in the microenvironment caused by pathological cells is essential to develop new therapeutic strategies. Such modifications allow leukemia cells to modify their microenvironment in order to promote their growth.

Here, we studiedin vivoandin vitrointeractions between Primary Myelofibrosis and the microenvironment. This clonal malignant disorder is caused by three major driver mutations: JAK2V617F, MPLW515L and CALR exon 9 mutations.

We performed 72h co-cultures of the mesenchymal stromal cell line MS5 and the MO7e human hematopoietic cell line transduced with either the MPLW515L mutation (MO7eMPLW515L) or GFP as a control. As Mo7e cells are dependent on the human growth factor h-GM-CSF, co-cultures were performed with and without it

Flow cytometry confirmed that leukemic cells expressing the MPLW515L mutation proliferated actively in the absence of hGM-CSF, unlike the control MO7e GFP cells. In addition, the presence of MS5 stromal cells did not have a major impact on the proliferation of MO7eMPLW515L but promoted MO7e GFP cell survival.

Thus the MPLW515L mutation activates signaling pathways that make the cells independent of cytokines and stroma.

On the other hand, the number of stromal cells increased upon co-culture with either MO7e GFP or MO7e W515L and in the presence of h-GM-CSF. Thus, either growth factor addition or the MPLW515L mutation allows MO7e cells to enhance the proliferation of stromal cells.

To test our observations in primary cells, we also performed 72h co-cultures using isolated murine cKIT+ hematopoietic progenitor cells from JAK2V617F knock-in or wild type mice. The impact of stromal cells was evaluated in the presence or absence of thrombopoietin (TPO). Cells were analyzed by flow cytometry.

The results showed that MS5 stromal cells induced proliferation of both primary wild type and JAK2V617F cKIT+ hematopoietic cells independently of TPO. Moreover, the proliferation of MS5 stromal cells was also significantly induced by both wild type and JAK2617F cKIT + primary cells.

In vivointeractions were studied using transgenic mice expressing CXCL12HT-dsRED crossed with JAK2V617F mice (CXCL2-HT-JAK2V617F). We followed different parameters, namely the weight and the size of the spleen, the different blood parameters, the number of hematopoietic progenitors in bone marrow, spleen and blood, and the cellularity and the number of fibroblast colonies (CFU-F).

Results showed that CXCL12 HT JAK2KI mice developed significant splenomegaly, associated with changes in blood parameters characteristic of JAK2KI mice, i.e. increases in white blood cells, red blood cells, hematocrit, and hemoglobin. There was also an increase inblood, spleenand bone marrow progenitors compared to the CXCL12HT GFP control mice.

The number of fibroblast colonies (CFU-F), reflecting the activity of mesenchymal stem cells (MSC) in the microenvironment, was also greatly increased, especially in the marrow of CXCL12HT JAK2KI mice, indicating that the malignant clone modified the microenvironment of the mice.

Taken together, our findings show that hematopoietic cells, either normal or leukemic, can modify their own microenvironmentin vitroandin vivo. This offers a new avenue for therapeutic targeting in the treatment of this type of hemopathologies.