Key Points• Bone marrow adipocytes support AML survival.• AML induces adipocyte lipolysis of triglyceride to free fatty acids and subsequent transport by FABP4. Abstract

Acute myeloid leukemia (AML) is an age-related disease that is highly dependent on the bone marrow (BM) microenvironment. With increasing age, tissues accumulate senescent cells, characterized by an irreversible arrest of cell proliferation and the secretion of a set of proinflammatory cytokines, chemokines, and growth factors, collectively known as the senescence-associated secretory phenotype (SASP). Here, we report that AML blasts induce a senescent phenotype in the stromal cells within the BM microenvironment and that the BM stromal cell senescence is driven by p16INK4a expression. The p16INK4a-expressing senescent stromal cells then feed back to promote AML blast survival and proliferation via the SASP. Importantly, selective elimination of p16INK4a+ senescent BM stromal cells in vivo improved the survival of mice with leukemia. Next, we find that the leukemia-driven senescent tumor microenvironment is caused by AML-induced NOX2-derived superoxide. Finally, using the p16-3MR mouse model, we show that by targeting NOX2 we reduced BM stromal cell senescence and consequently reduced AML proliferation. Together, these data identify leukemia-generated NOX2-derived superoxide as a driver of protumoral p16INK4a-dependent senescence in BM stromal cells. Our findings reveal the importance of a senescent microenvironment for the pathophysiology of leukemia. These data now open the door to investigate drugs that specifically target the “benign” senescent cells that surround and support AML.

Acute myeloid leukemia (AML) cells exhibit a high level of spontaneous apoptosis when cultured in vitro but have a prolonged survival time in vivo, indicating that tissue microenvironment plays a critical role in promoting AML cell survival. In vitro studies have shown that bone marrow mesenchymal stromal cells (BM-MSC) protect AML blasts from spontaneous and chemotherapy-induced apoptosis. Here, we report a novel interaction between AML blasts and BM-MSCs, which benefits AML proliferation and survival. We initially examined the cytokine profile in cultured human AML compared with AML cultured with BM-MSCs and found that macrophage migration inhibitory factor (MIF) was highly expressed by primary AML, and that IL8 was increased in AML/BM-MSC cocultures. Recombinant MIF increased IL8 expression in BMMSCs via its receptor CD74. Moreover, the MIF inhibitor ISO-1 inhibited AML-induced IL8 expression by BM-MSCs as well as BM-MSC-induced AML survival. Protein kinase C b (PKCb) regulated MIF-induced IL8 in BM-MSCs. Finally, targeted IL8 shRNA inhibited BM-MSC-induced AML survival. These results describe a novel, bidirectional, prosurvival mechanism between AML blasts and BM-MSCs. Furthermore, they provide biologic rationale for therapeutic strategies in AML targeting the microenvironment, specifically MIF and IL8.

SummmaryThe novel multi-kinase inhibitor TG02 has selectivity against cell cycle and transcriptional cyclin dependent kinases (CDKs) as well as fms-like tyrosine kinase receptor-3 (FLT3). Inhibition of transcriptional CDKs preferentially depletes short-lived proteins such as MCL1. We evaluated the in vitro toxicity of TG02 to primary acute myeloid leukaemia (AML) cells in the presence of survival signalling pathway activation by cytokines and fibronectin. One hundred nanomolar TG02 induced a median decrease of 40% in bulk cell survival and 43% in the CD34 + CD38 À CD123 + subset. A 90% inhibitory concentration of 500 nmol/l indicated that TG02 toxicity is not halted by protective cell cycle arrest. Samples with FLT3 internal tandem duplication were not preferentially targeted. By flow cytometry, TG02 treatment caused loss of RNA Polymerase II serine 2 phosphorylation in patient samples, which correlated strongly with BAX activation (R 2 =0Á89), suggesting these as potential biomarkers for clinical studies. MCL1 and XIAP expression also decreased. Repeated brief exposure to TG02 in MOLM-13 cells did not result in compensatory up-regulation of survival protein expression. In conclusion, TG02 is potently cytotoxic towards CD34 + CD38À CD123 + and bulk AML cells, despite protective signalling pathway activation. This antitumour activity is most likely mediated by dephosphorylation of RNA Polymerase II leading to depletion of survival molecules such as MCL1 and XIAP, with subsequent BAX activation and apoptosis.

Oxidative stress as a result of elevated levels of reactive oxygen species (ROS) has been observed in almost all cancers, including leukaemia, where they contribute to disease development and progression. However, cancer cells also express increased levels of antioxidant proteins which detoxify ROS. This includes glutathione, the major antioxidant in human cells, which has recently been identified to have dysregulated metabolism in human leukaemia. This suggests that critical balance of intracellular ROS levels is required for cancer cell function, growth, and survival. Nuclear factor (erythroid-derived 2)-like 2 (NRF2) transcription factor plays a dual role in cancer. Primarily, NRF2 is a transcription factor functioning to protect nonmalignant cells from malignant transformation and oxidative stress through transcriptional activation of detoxifying and antioxidant enzymes. However, once malignant transformation has occurred within a cell, NRF2 functions to protect the tumour from oxidative stress and chemotherapy-induced cytotoxicity. Moreover, inhibition of the NRF2 oxidative stress pathway in leukaemia cells renders them more sensitive to cytotoxic chemotherapy. Our improved understanding of NRF2 biology in human leukaemia may permit mechanisms by which we could potentially improve future cancer therapies. This review highlights the mechanisms by which leukaemic cells exploit the NRF2/ROS response to promote their growth and survival.

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Direct co-operation between sensitiser molecules BAD and NOXA in mediating apoptosis suggests that therapeutic agents which sensitise to BAD may complement agents which sensitise to NOXA. Dynamic BH3 profiling is a novel methodology that we have applied to the measurement of complementarity between sensitiser BH3 peptide mimetics and therapeutic agents. Using dynamic BH3 profiling, we show that the agent TG02, which downregulates MCL-1, sensitises to the BCL-2-inhibitory BAD-BH3 peptide, whereas the BCL-2 antagonist ABT-199 sensitises to MCL-1 inhibitory NOXA-BH3 peptide in acute myeloid leukaemia (AML) cells. At the concentrations used, the peptides did not trigger mitochondrial outer membrane permeabilisation in their own right, but primed cells to release Cytochrome C in the presence of an appropriate trigger of a complementary pathway. In KG-1a cells TG02 and ABT-199 synergised to induce apoptosis. In heterogeneous AML patient samples we noted a range of sensitivities to the two agents. Although some individual samples markedly favoured one agent or the other, in the group as a whole the combination of TG02 + ABT-199 was significantly more cytotoxic than either agent individually. We conclude that dynamic NOXA and BAD BH3 profiling is a sensitive methodology for investigating molecular pathways of drug action and complementary mechanisms of chemoresponsiveness.

Phosphoinositide-3-kinase (PI3K) is an enzyme group, known to regulate key survival pathways in acute myeloid leukaemia (AML). It generates phosphatidylinositol-3,4,5-triphosphate, which provides a membrane docking site for protein kinaseB activation. PI3K catalytic p110 subunits are divided into 4 isoforms; α,β,δ and γ. The PI3Kδ isoform is always expressed in AML cells, whereas the frequency of PI3Kγ expression is highly variable. The functions of these individual catalytic enzymes have not been fully resolved in AML, therefore using the PI3K p110δ and p110γ-targeted inhibitor IPI-145 (duvelisib) and specific p110δ and p110γ shRNA, we analysed the role of these two p110 subunits in human AML blast survival. The results show that PI3Kδ and PI3Kγ inhibition with IPI-145 has anti-proliferative activity in primary AML cells by inhibiting the activity of AKT and MAPK. Pre-treatment of AML cells with IPI-145 inhibits both adhesion and migration of AML blasts to bone marrow stromal cells. Using shRNA targeted to the individual isoforms we demonstrated that p110δ-knockdown had a more significant anti-proliferative effect on AML cells, whereas targeting p110γ-knockdown significantly inhibited AML migration. The results demonstrate that targeting both PI3Kδ and PI3Kγ to inhibit AML-BMSC interactions provides a biologic rationale for the pre-clinical evaluation of IPI-145 in AML.