PGC1α Driven Mitochondrial Biogenesis within the Bone Marrow Stromal Cells of the Acute Myeloid Leukemia Micro-Environment Is a Pre-Requisite for Mitochondrial Transfer to Leukemic Blasts

Background

The bone marrow (BM) microenvironment is essential for the growth and proliferation of acute myeloid leukemia (AML). Recently we have reported that mitochondria are transferred from BM stromal cells (BMSC) to AML blasts through tunnelling nanotubes (TNTs), a process which supports leukemic proliferation in-vitro and in-vivo. Moreover, we showed that tumor derived NOX2 generates superoxide which provides the stimulus to drive mitochondrial transfer in this way (Marlein at al. 2017 Blood). In the present study we specifically examine the effects of AML derived superoxide on BMSC mitochondrial biogenesis and subsequent mitochondrial transfer to AML.

Methods

Primary AML blasts and BMSC were obtained from patient bone marrow, with informed consent and under approval from the UK National Research Ethics Service. Animal experiments were performed following approval by the UK Home Office and University of East Anglia Animal Welfare and Ethical Review Board. Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) activity was analysed using Western blotting and lentiviral-mediated knockdown. Mitochondrial transfer was assessed and quantified in vitro using a MitoTracker green FM staining and flow cytometry. AML derived superoxide was quenched by glutathione and diphenyleneiodonium (DPI). Leukemic proliferation and tumor volume, in an NSG AML xenograft subcutaneous model, were monitored through bioluminescent live animal imaging.

Results

First we quantified levels of mitochondrial transfer with and without inhibition of superoxide activity. We found that the anti-oxidants glutathione and DPI significantly reduced levels of mitochondrial transfer from BMSC to AML. We next quantified mitochondrial biogenesis within BMSC, showing that BMSC had increased mitochondrial DNA and increased MitoTracker Green FM staining in response to co-culture with AML. Next we report that this increase in mitochondrial content also occurred in response to hydrogen peroxide, which mimics the effects of AML-derived superoxide. In addition, we show that PGC1α (master regulator of mitochondrial biogenesis) levels in the nucleus of BMSC increased when cultured with AML. Moreover, knockdown of PGC1α inhibited mitochondrial biogenesis in BMSC when cultured with AML or activated with hydrogen peroxide. Furthermore, transfer of mitochondria from BMSC to AML was substantially reduced when BMSC-derived PGC1α was silenced. Finally, we engrafted control or PGC1α knockdown BMSC together with OCI-AML3 cells (AML cell line) into the sub-cutaneous tissue of NSG mice. We found significantly reduced AML tumour volume when the AML was transplanted with the PGC1α knockdown BMSC compared to transplant of AML with the control knockdown stromal cells.

Conclusion

Here we report that the superoxide generated by AML drives BMSC mitochondrial biogenesis and subsequent mitochondrial transfer between BMSC and leukemic blasts. Targeting this pathway may provide an effective novel therapeutic approach in AML