Inhibiting Matrix Metalloproteinases Hinders Acute Myeloid Leukaemia and Prevents Healthy Stem Cell Loss

Haematopoietic stem cells (HSCs), despite being very rare (<0.015% of bone marrow haematopoietic cells), maintain the turnover of all blood cells through a balance of quiescence, self-renewal and differentiation. Disruption of HSCs function and of the bone marrow (BM) microenvironment are key aspects of Acute Myeloid Leukaemia (AML). AML develops in adults and symptoms arise due to the loss of healthy haematopoietic cells. It is unknown exactly what factors contribute to this although it is clear that there is a progressive loss of BM HSCs in this disease. One hypothesis is that HSCs are pushed out of the BM niche by competing leukaemic blasts. To explore this, we used intravital 2-photon confocal microscopy in a live mouse model of leukaemia which allows us to visualise the dynamics of healthy haematopoietc cells at various stages of the disease. We monitored the development of extramedullary haematopoiesis (EMH) during AML growth and tested the function of HSCs found in these alternative sites to determine whether EMH acts as an alternative mechanism of blood cell maintenance in response to AML. Furthermore, we investigated the influence of an extracellular matrix metalloproteinase inhibitor, Prinomastat, on the loss of HSCs in this model. Prinomastat has been studied extensively in solid organ cancers as it has been shown capable of inhibiting cancer metastasis. In this study, we examined whether it might have a function in preventing the loss of HSCs from the bone during leukaemia infiltration.

C57BL/6 mice were injected with 100k of YFP-AML blasts and peripheral blood (PB) checked every four days for AML progression. Cellular dynamics were assessed by intravital microscopy (IVM) of the mouse calvarium and spleen at early (10%), medium (25%) and late (>25%) PB infiltration. To calculate the number of circulating HSCs and progenitors (HSPCs) blood was taken by cardiac puncture and analysed by flow cytometry for HSCPs absolute number. The same was done for BM, spleen and liver HSPCs. HSC functionality was determined by transplanting sorted Lin^- c-Kit⁺ Sca-1⁺ CD48- CD150+ (LKS Slam) cells from CD45.1 BM, spleen and liver of AML-burdened mice into lethally irradiated C57BL/6 mice. BM reconstitution was then analysed every four weeks. To analyse the role of extracellular matrix remodelling, C57BL/6 mice were transplanted with 100k AML cells tagged with yellow-fluorescent-protein (YFP) and then administered intravenous prinomastat daily. These mice were imaged and had bone marrow analysed using flow cytometry together with a control group at early, medium and late AML based on PB infiltration.

AML progression leads to a dramatic and progressive loss HSCPs in the BM. Intravital imaging showed an enhanced egress of healthy cells from the BM into the circulation. Conversely, we found a clear association between the extent of infiltration of the marrow and the number of HSCs found in the spleen and liver. Our transplantation experiments show that the extramedullary HSCs are functional and able to reconstitute the BM of lethally irradiated mice irrespective of the organ from which they were sorted. Treatment with prinomastat significantly reduced the number of HSCs and progenitors leaving the bone marrow (P=0.0001). In the treated mice, the number of BM HSCs was consistently higher at every infiltration time point when compared to untreated mice. In addition, prinomastat caused a reduction in the extent of extramedullary haematopoesis in both the spleen and liver.

This study provides a unique insight into the effect of AML on the dynamics of HSCs as disease progresses. Contrary to expectations, HSCs are not lost, but rather a majority appear to migrate from the bone marrow to sites of extramedullary haematopoiesis. These cells remain functional and are capable of regenerating haematopoiesis when transplanted into a recipient mouse. Further to this, we have demonstrated that by inhibiting the function of metalloproteinases using Prinomastat it is possible to prevent this loss of HSCs thus retain these cells within the bone marrow. These findings highlight the importance of the extracellular matrix in acute myeloid leukaemia and suggest that metalloproteinase inhibitors could potentially have a significant role in resisting the perturbations caused by AML on haematopoiesis.