Acute Myeloid Leukaemia Induces p16 Driven Senescence in the Bone Marrow Microenvironment to Support Their Proliferation and Survival

Background

Acute myeloid leukaemia (AML) occurs most commonly in older people and depends on the bone marrow microenvironment. The aging bone marrow accumulates senescent cells, characterized by an irreversible arrest of cell proliferation and the secretion of numerous pro-inflammatory cytokines, chemokines and growth factors, termed the secretary associated senescence profile (SASP). Previously we shown that AML is capable of reprogramming bone marrow stromal cells (BMSC) to support blast survival and proliferation. Here, we hypothesis that AML programs a senescent aging phenotype in the BMSC, generating a SASP in the BMSC which then feedback to promote AML blast proliferation in the bone marrow niche.

Methods

We isolated primary AML cells and BMSC from the bone marrow, with informed consent and under approval from the UK National Research Ethics Service (LRCEref07/H0310/146). We characterized BMSC by flow cytometry for expression of CD90+, CD73+, CD105+ and CD45, and assayed cytokines and chemokines secreted by primary AML cells, primary BMSC and AML/BMSC co-cultures using a proteomic array. To assess senescence of BMSC, we measured p16 mRNA and protein levels and senescence associated β-Galactosidase activity. We also transplanted syngeneic AML blasts (generated from lineage negative cells transduced with the oncogene MN1) into p16-3MR mice (Demaria, M., et al. 2014 Cell Development), in which the herpes simplex virus thymidine kinase (HSV-TK) moiety of the p16-3MR transgene allows selective killing of senescent p16-expressing cells by the pro-drug ganciclovir (GCV). We also used the NSG xenograft model in which we subcutaneously transplanted primary human AML cells and BMSC (either control and p16-kockdown).

Results

We report that human AML cells induce BMSC to secrete a cytokine profile (including interleukin-6, interleukin-8, MIP1a and MIP2a). To examine whether AML cells can induce senescence in BMSC in culture, we co-cultured primary AML-derived BMSC with primary AML blasts and assayed for the senescent phenotype. We found that primary AML cells can induce p16 mRNA and protein and increase senescence associated β-Galactosidase activity in the BMSC after 6 days in culture. Furthermore, primary AML cells cultured on p16 knockdown BMSC had reduced survival compared to control wild type BMSC. In vivo we show that MN1 blasts engraft p16-3MR mice and the depletion of p16 positive stromal cells through treating animals with GVC compared to vehicle control significantly increased survival of MN1 engrafted mice. Finally, subcutaneous injection of human AML cells with human p16-KD BMSC significantly decreased tumour volume compared to injection of human AML cells with control-KD BMSC in mouse xenograft model.

Conclusion

Here we show that the AML blast induces a senescent phenotype in the bone marrow microenvironment which then feeds back to enhance the growth capacity of the AML blast. Targeting the senescent microenvironment may provide a novel therapeutic approach for the treatment of this disease.