Inhibition of Enhancer of Zeste Homolog 2 Induces Blast Differentiation, Impairs Engraftment and Prolongs Survival in Murine Models of Acute Myeloid Leukemia.

BACKGROUND: Acute myeloid leukemia (AML) is the malignant proliferation of immature myeloid cells characterized by a block in differentiation. As such, novel therapeutic strategies to promote the differentiation of immature myeloid cells have been successful in AML, although these agents are targeted to a specific mutation that is only present in a subset of AML patients. In the current study, we show that targeting the epigenetic modifier enhancer of zeste homolog 2 (EZH2) can induce the differentiation of immature blast cells into a more mature myeloid phenotype and promote survival in AML murine models. METHODS: The EZH2 inhibitor EPZ011989 (EPZ) was studied in AML cell lines, primary in AML cells and normal CD34+ stem cells. A pharmacodynamic assessment of H3K27me3; studies of differentiation, cell growth, and colony formation; and in vivo therapeutic studies including the influence on primary AML cell engraftment were also conducted. RESULTS: EPZ inhibited H3K27me3 in AML cell lines and primary AML samples in vitro. EZH2 inhibition reduced colony formation in multiple AML cell lines and primary AML samples, while exhibiting no effect on colony formation in normal CD34+ stem cells. In AML cells, EPZ promoted phenotypic evidence of differentiation. Finally, the pretreatment of primary AML cells with EPZ significantly delayed engraftment and prolonged the overall survival when engrafted into immunodeficient mice. CONCLUSIONS: Despite evidence that EZH2 silencing in MDS/MPN can promote AML pathogenesis, our data demonstrate that the therapeutic inhibition of EZH2 in established AML has the potential to improve survival.

Mass Cytometry as a Tool for Investigating Senescence in Multiple Model Systems.

Cellular senescence is a durable cell cycle arrest as a result of the finite proliferative capacity of cells. Senescence responds to both intrinsic and extrinsic cellular stresses, such as aging, mitochondrial dysfunction, irradiation, and chemotherapy. Here, we report on the use of mass cytometry (MC) to analyze multiple model systems and demonstrate MC as a platform for senescence analysis at the single-cell level. We demonstrate changes to p16 expression, cell cycling fraction, and histone tail modifications in several established senescent model systems and using isolated human T cells. In bone marrow mesenchymal stromal cells (BMSCs), we show increased p16 expression with subsequent passage as well as a reduction in cycling cells and open chromatin marks. In WI-38 cells, we demonstrate increased p16 expression with both culture-induced senescence and oxidative stress-induced senescence (OSIS). We also use Wanderlust, a trajectory analysis tool, to demonstrate how p16 expression changes with histone tail modifications and cell cycle proteins. Finally, we demonstrate that repetitive stimulation of human T cells with CD3/CD28 beads induces an exhausted phenotype with increased p16 expression. This p16-expressing population exhibited higher expression of exhaustion markers such as EOMES and TOX. This work demonstrates that MC is a useful platform for studying senescence at a single-cell protein level, and is capable of measuring multiple markers of senescence at once with high confidence, thereby improving our understanding of senescent pathways.

Acute myeloid leukemia induces protumoral p16INK4a-driven senescence in the bone marrow microenvironment.

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.

Myeloma-derived macrophage inhibitory factor regulates bone marrow stromal cell-derived IL-6 via c-MYC.

Multiple myeloma (MM) remains an incurable malignancy despite the recent advancements in its treatment. The protective effects of the niche in which it develops has been well documented; however, little has been done to investigate the MM cell's ability to 're-program' cells within its environment to benefit disease progression. Here, we show that MM-derived macrophage migratory inhibitory factor (MIF) stimulates bone marrow stromal cells to produce the disease critical cytokines IL-6 and IL-8, prior to any cell-cell contact. Furthermore, we provide evidence that this IL-6/8 production is mediated by the transcription factor cMYC. Pharmacological inhibition of cMYC in vivo using JQ1 led to significantly decreased levels of serum IL-6-a highly positive prognostic marker in MM patients. CONCLUSIONS: Our presented findings show that MM-derived MIF causes BMSC secretion of IL-6 and IL-8 via BMSC cMYC. Furthermore, we show that the cMYC inhibitor JQ1 can reduce BMSC secreted IL-6 in vivo, irrespective of tumor burden. These data provide evidence for the clinical evaluation of both MIF and cMYC inhibitors in the treatment of MM.

HIF1α drives chemokine factor pro-tumoral signaling pathways in acute myeloid leukemia.

Approximately 80% of patients diagnosed with acute myeloid leukemia (AML) die as a consequence of failure to eradicate the tumor from the bone marrow microenvironment. We have recently shown that stroma-derived interleukin-8 (IL-8) promotes AML growth and survival in the bone marrow in response to AML-derived macrophage migration inhibitory factor (MIF). In the present study we show that high constitutive expression of MIF in AML blasts in the bone marrow is hypoxia-driven and, through knockdown of MIF, HIF1α and HIF2α, establish that hypoxia supports AML tumor proliferation through HIF1α signaling. In vivo targeting of leukemic cell HIF1α inhibits AML proliferation in the tumor microenvironment through transcriptional regulation of MIF, but inhibition of HIF2α had no measurable effect on AML blast survival. Functionally, targeted inhibition of MIF in vivo improves survival in models of AML. Here we present a mechanism linking HIF1α to a pro-tumoral chemokine factor signaling pathway and in doing so, we establish a potential strategy to target AML.

High NRF2 expression controls endoplasmic reticulum stress induced apoptosis in multiple myeloma.

Multiple myeloma (MM) is an incurable disease characterized by clonal plasma cell proliferation. The stress response transcription factor Nuclear factor erythroid 2 [NF-E2]-related factor 2 (NRF2) is known to be activated in MM in response to proteasome inhibitors (PI). Here, we hypothesize that the transcription factor NRF2 whose physiological role is to protect cells from reactive oxygen species via the regulation of drug metabolism and antioxidant gene plays an important role in MM cells survival and proliferation. We report for the first time that NRF2 is constitutively activated in circa 50% of MM primary samples and all MM cell lines. Moreover, genetic inhibition of constitutively expressed NRF2 reduced MM cell viability. We confirm that PI induced further expression of NRF2 in MM cell lines and primary MM. Furthermore, genetic inhibition of NRF2 of PI treated MM cells increased ER-stress through the regulation of CCAAT-enhancer-binding protein homologous protein (CHOP). Finally, inhibition of NRF2 in combination with PI treatment significantly increased apoptosis in MM cells. Here we identify NRF2 as a key regulator of MM survival in treatment naive and PI treated cells.

Inflammatory Differences in Plaque Erosion and Rupture in Patients With ST-Segment Elevation Myocardial Infarction.

BACKGROUND: Plaque erosion causes 30% of ST-segment elevation myocardial infarctions, but the underlying cause is unknown. Inflammatory infiltrates are less abundant in erosion compared with rupture in autopsy studies. We hypothesized that erosion and rupture are associated with significant differences in intracoronary cytokines in vivo. METHODS AND RESULTS: Forty ST-segment elevation myocardial infarction patients with <6 hours of chest pain were classified as ruptured fibrous cap (RFC) or intact fibrous cap (IFC) using optical coherence tomography. Plasma samples from the infarct-related artery and a peripheral artery were analyzed for expression of 102 cytokines using arrays; results were confirmed with ELISA. Thrombectomy samples were analyzed for differential mRNA expression using quantitative real-time polymerase chain reaction. Twenty-three lesions were classified as RFC (58%), 15 as IFC (38%), and 2 were undefined (4%). In addition, 12% (12 of 102) of cytokines were differentially expressed in both coronary and peripheral plasma. I-TAC was preferentially expressed in RFC (significance analysis of microarrays adjusted P<0.001; ELISA IFC 10.2 versus RFC 10.8 log2 pg/mL; P=0.042). IFC was associated with preferential expression of epidermal growth factor (significance analysis of microarrays adjusted P<0.001; ELISA IFC 7.42 versus RFC 6.63 log2 pg/mL, P=0.036) and thrombospondin 1 (significance analysis of microarrays adjusted P=0.03; ELISA IFC 10.4 versus RFC 8.65 log2 ng/mL, P=0.0041). Thrombectomy mRNA showed elevated I-TAC in RFC (P=0.0007) epidermal growth factor expression in IFC (P=0.0264) but no differences in expression of thrombospondin 1. CONCLUSIONS: These results demonstrate differential intracoronary cytokine expression in RFC and IFC. Elevated thrombospondin 1 and epidermal growth factor may play an etiological role in erosion.

Leukemic blasts program bone marrow adipocytes to generate a protumoral microenvironment.

Despite currently available therapies, most patients diagnosed with acute myeloid leukemia (AML) die of their disease. Tumor-host interactions are critical for the survival and proliferation of cancer cells; accordingly, we hypothesize that specific targeting of the tumor microenvironment may constitute an alternative or additional strategy to conventional tumor-directed chemotherapy. Because adipocytes have been shown to promote breast and prostate cancer proliferation, and because the bone marrow adipose tissue accounts for up to 70% of bone marrow volume in adult humans, we examined the adipocyte-leukemia cell interactions to determine if they are essential for the growth and survival of AML. Using in vivo and in vitro models of AML, we show that bone marrow adipocytes from the tumor microenvironment support the survival and proliferation of malignant cells from patients with AML. We show that AML blasts alter metabolic processes in adipocytes to induce phosphorylation of hormone-sensitive lipase and consequently activate lipolysis, which then enables the transfer of fatty acids from adipocytes to AML blasts. In addition, we report that fatty acid binding protein-4 (FABP4) messenger RNA is upregulated in adipocytes and AML when in coculture. FABP4 inhibition using FABP4 short hairpin RNA knockdown or a small molecule inhibitor prevents AML proliferation on adipocytes. Moreover, knockdown of FABP4 increases survival in Hoxa9/Meis1-driven AML model. Finally, knockdown of carnitine palmitoyltransferase IA in an AML patient-derived xenograft model improves survival. Here, we report the first description of AML programming bone marrow adipocytes to generate a protumoral microenvironment.

MIF-Induced Stromal PKCß/IL8 Is Essential in Human Acute Myeloid Leukemia.

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 BM-MSCs 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 ß (PKCß) 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. Cancer Res; 77(2); 303-11. ©2016 AACR.

Complementary dynamic BH3 profiles predict co-operativity between the multi-kinase inhibitor TG02 and the BH3 mimetic ABT-199 in acute myeloid leukaemia cells.

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.

Targeting PI3Kδ and PI3Kγ signalling disrupts human AML survival and bone marrow stromal cell mediated protection.

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,ß,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.

Activity of Bruton's tyrosine-kinase inhibitor ibrutinib in patients with CD117-positive acute myeloid leukaemia: a mechanistic study using patient-derived blast cells.

BACKGROUND: Roughly 80% of patients with acute myeloid leukaemia have high activity of Bruton's tyrosine-kinase (BTK) in their blast cells compared with normal haemopoietic cells, rendering the cells sensitive to the oral BTK inhibitor ibrutinib in vitro. We aimed to develop the biological understanding of the BTK pathway in acute myeloid leukaemia to identify clinically relevant diagnostic information that might define a subset of patients that should respond to ibrutinib treatment. METHODS: We obtained acute myeloid leukaemia blast cells from unselected patients attending our UK hospital between Feb 19, 2010, and Jan 20, 2014. We isolated primary acute myeloid leukaemia blast cells from heparinised blood and human peripheral blood mononuclear cells to establish the activity of BTK in response to CD117 activation. Furthermore, we investigated the effects of ibrutinib on CD117-induced BTK activation, downstream signalling, adhesion to primary bone-marrow mesenchymal stromal cells, and proliferation of primary acute myeloid leukaemia blast cells. We used the Mann-Whitney U test to compare results between groups. FINDINGS: We obtained acute myeloid leukaemia blast cells from 29 patients. Ibrutinib significantly inhibited CD117-mediated proliferation of primary acute myeloid leukaemia blast cells (p=0·028). CD117 activation increased BTK activity by inducing phosphorylated BTK in patients with CD117-positive acute myeloid leukaemia. Furthermore, ibrutinib inhibited CD117-induced activity of BTK and downstream kinases at a concentration of 100 nM or more. CD117-mediated adhesion of CD117-expressing blast cells to bone-marrow stromal cells was significantly inhibited by Ibrutinib at 500 nM (p=0·028) INTERPRETATION: As first-in-man clinical trials of ibrutinib in patients with acute myeloid leukaemia commence, the data suggest not all patients will respond. Our findings show that BTK has specific pro-tumoural biological actions downstream of surface CD117 activation, which are inhibited by ibrutinib. Accordingly, we propose that patients with acute myeloid leukaemia whose blast cells express CD117 should be considered for forthcoming clinical trials of ibrutinib. FUNDING: Worldwide Cancer Research, The Big C, UK National Institutes for Health Research, the Humane Research Trust, the Department of Higher Education and Research of the Libyan Government, and Norwich Research Park.

Targeting BTK for the treatment of FLT3-ITD mutated acute myeloid leukemia.

Approximately 20% of patients with acute myeloid leukaemia (AML) have a mutation in FMS-like-tyrosine-kinase-3 (FLT3). FLT3 is a trans-membrane receptor with a tyrosine kinase domain which, when activated, initiates a cascade of phosphorylated proteins including the SRC family of kinases. Recently our group and others have shown that pharmacologic inhibition and genetic knockdown of Bruton's tyrosine kinase (BTK) blocks AML blast proliferation, leukaemic cell adhesion to bone marrow stromal cells as well as migration of AML blasts. The anti-proliferative effects of BTK inhibition in human AML are mediated via inhibition of downstream NF-κB pro-survival signalling however the upstream drivers of BTK activation in human AML have yet to be fully characterised. Here we place the FLT3-ITD upstream of BTK in AML and show that the BTK inhibitor ibrutinib inhibits the survival and proliferation of FLT3-ITD primary AML blasts and AML cell lines. Furthermore ibrutinib inhibits the activation of downstream kinases including MAPK, AKT and STAT5. In addition we show that BTK RNAi inhibits proliferation of FLT3-ITD AML cells. Finally we report that ibrutinib reverses the cyto-protective role of BMSC on FLT3-ITD AML survival. These results argue for the evaluation of ibrutinib in patients with FLT3-ITD mutated AML.

Oxidative stress responses and NRF2 in human leukaemia.

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.

The multi-kinase inhibitor TG02 overcomes signalling activation by survival factors to deplete MCL1 and XIAP and induce cell death in primary acute myeloid leukaemia cells.

The 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(-) 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) =0·89), 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(+) CD38(-) 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.