Rachel E. Piddock

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.

NADPH oxidase-2 derived superoxide drives mitochondrial transfer from bone marrow stromal cells to leukemic blasts

Improvements in the understanding of the metabolic cross-talk between cancer and its microenvironment are expected to lead to novel therapeutic approaches. Acute myeloid leukemia (AML) cells have increased mitochondria compared with nonmalignant CD34+ hematopoietic progenitor cells. Furthermore, contrary to the Warburg hypothesis, AML relies on oxidative phosphorylation to generate adenosine triphosphate. Here we report that in human AML, NOX2 generates superoxide, which stimulates bone marrow stromal cells (BMSC) to AML blast transfer of mitochondria through AML-derived tunneling nanotubes. Moreover, inhibition of NOX2 was able to prevent mitochondrial transfer, increase AML apoptosis, and improve NSG AML mouse survival. Although mitochondrial transfer from BMSC to nonmalignant CD34+ cells occurs in response to oxidative stress, NOX2 inhibition had no detectable effect on nonmalignant CD34+ cell survival. Taken together, we identify tumor-specific dependence on NOX2-driven mitochondrial transfer as a novel therapeutic strategy 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 Brutons 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.

The Role of PI3K Isoforms in Regulating Bone Marrow Microenvironment Signaling Focusing on Acute Myeloid Leukemia and Multiple Myeloma

Despite the development of novel treatments in the past 15 years, many blood cancers still remain ultimately fatal and difficult to treat, particularly acute myeloid leukaemia (AML) and multiple myeloma (MM). While significant progress has been made characterising small-scale genetic mutations and larger-scale chromosomal translocations that contribute to the development of various blood cancers, less is understood about the complex microenvironment of the bone marrow (BM), which is known to be a key player in the pathogenesis of chronic lymphocytic leukaemia (CLL), AML and MM. This niche acts as a sanctuary for the cancerous cells, protecting them from chemotherapeutics and encouraging clonal cell survival. It does this by upregulating a plethora of signalling cascades within the malignant cell, with the phosphatidylinositol-3-kinase (PI3K) pathway taking a critical role. This review will focus on how the PI3K pathway influences disease progression and the individualised role of the PI3K subunits. We will also summarise the current clinical trials for PI3K inhibitors and how these trials impact the treatment of blood cancers.

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.

PGC-1α driven mitochondrial biogenesis in stromal cells underpins mitochondrial trafficking to leukemic blasts

Acute myeloid leukemia (AML) is a disease known to be heavily reliant on its bone microenvironment to survive and proliferate [1, 2]. We have previously shown that AML disease progression is enabled by the transfer of functional mitochondria to the malignant cell from bone marrow stromal cells (BMSC) [3, 4]. This process was shown to be stimulated by superoxide generated by NADPH oxidase-2 (NOX2) on the AML blast [3]. However, beyond the stimulation of reactive oxygen species in BMSC, the mechanisms controlling mitochondrial transfer in BMSC have yet to be elucidated. There are no apparent adverse effects on BMSC after donation of mitochondria to AML blasts, implying the presence of a mechanism whereby the BMSC can recover their metabolic potential. The master regulator of mitochondrial biogenesis [5], peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), has been implicated in cancer progression and metabolism [6, 7]. In these studies, PGC-1α is up-regulated and causes an increased accumulation of functional mitochondria. Here, we investigate the effect AML has on the mitochondrial mass, bio-energetic potential, and PGC-1α expression in BMSC. First, using MitoTracker Green staining and flow cytometry, we determined the mitochondrial levels in primary BMSC (n= 8) after co-culture with AML blasts. In Fig. 1a, we found significantly elevated mitochondrial levels in BMSC after co-culture, implying that AML blasts stimulate BMSC to produce more mitochondria. We next wanted to see if this increase in mitochondrial mass caused an increase in mitochondrial based metabolism. To do this we analyzed BMSC oxygen consumption rate using the Seahorse extracellular flux assay. Increased mitochondrial respiration was observed in BMSC (n= 4) after co-culture with AML blasts (Fig. 1b, c). Moreover, in Fig. 1d, we show that BMSC from patients with AML had increased mitochondrial respiration compared to BMSC from healthy individuals (n= 3). Together, these results show that BMSC from patients with AML have increased mitochondrial mass and functional bio-energetic consequence in BMSC metabolism. All primary patient BMSC samples used are highlighted in Supplementary Table 1. As the transcription factor PGC-1α is known to cause increased mitochondrial biogenesis [5], we examined PGC1α expression in BMSC after co-culture with AML blasts. RNA expression of PGC-1α in BMSC (n= 5) was increased in BMSC after co-culture with AML blasts compared to BMSC cultured alone (Fig. 1f). Next, we showed that total PGC-1α protein was elevated in BMSC after AML co-culture compared to control (Fig. 1f). Moreover, we show that BMSC have increased nuclear levels of PGC-1α after co-culture with AML compared to BMSC cultured alone (Fig. 1f), an effect which was reversed upon the addition of N-acetylcysteine to the coculture (Supplementary Figure 1). Previous studies have shown that AMPK can be stimulated by ROS [8] and in turn * Kristian M. Bowles k.bowles@uea.ac.uk

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.ConclusionsOur 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.

Abstract 4327: Bone marrow adipocytes drive transcriptional changes in leukemic blasts to enhance their capacity to derive energy from free fatty acid metabolism

Introduction: Most patients diagnosed with acute myeloid leukaemia [AML] die of their disease and the average age of patients at diagnosis is 72 years. For this reason, new therapeutic strategies with tolerability in the fragile, less fit population is necessary for reducing the mortality rate associated with this disease. The tumor microenvironment is an emerging target in the search for less cytotoxic therapies. We have previously shown that the adipocyte component of the bone marrow (BM) is a key player in blast survival, proliferation and chemotherapy evasion. In this study, we draw focus onto the status of an adipocyte rich environment in the context of leukemia and highlight the key players in regulating leukemic cell metabolism through transport and metabolism of fatty acids. Objective: We hypothesize that the presence of adipocytes within the proximity of AML blasts creates a FA rich environment for increased β - oxidation within the blasts. Methods: We used primary AML blasts and normal CD34+ hematopoietic stem cells (HSC) following informed consent (LRCEref07/H0310/146). Adipocytes were derived from bone marrow stromal cells (BMSC). Differential expression analysis of RNA sequencing data (GEO ID: GSE49642, GSE48846) was used to compare respiratory gene signatures of BM AML blasts, peripheral blood obtained AML and normal CD34+ HSC. Fatty acid binding protein 4 (FABP4) and carnitine palmitoyltransferase 1A (CPT1A) were identified as one of the key genes involved in the lipolysis and oxidation signature differential expression. Oxygen consumption rate (OCR) of adipocyte co-cultured blasts and normal CD34+ were analyzed using Seahorse technology. Lentiviral-knockdown of FABP4 and CPT1A were performed on blasts prior to in vivo xenograft mouse model injection. Results: Leukemic blasts showed increased adipocyte lipolysis stimulation compared with normal CD34+ cells. Moreover, adipocytes increased transcriptional activation of FABP4 and CPT1A in malignant blasts compared to CD34+ HSC. FABP4 inhibitor reduced AML blast survival when cells were cultured with adipocytes which is in contrast to normal CD34+ HSC. Moreover, AML had increased oxygen consumption rate when grown on adipocytes which was inhibited by etomoxir (β - oxidation inhibitor). Finally in-vivo lentiviral mediated knockdown of FABP4 and CPT1A reveled an increased survival of AML xenografts. Conclusion: Our results show that the stimulation of lipolysis and fatty acid oxidative genes that contribute to the genetic signatures of these processes are a malignant blast exclusive profile. Interventions at a molecular level reveal survival favorable outcomes in xenograft models suggesting the need for enhancing strategies which include targeting the FABP4 and CPT1A axis. Our data provide a biologic rationale for exploring future therapies that target the adipocyte/AML interactions. Citation Format: Manar Shafat, Thomas Oellerich, Sebastian Mohr, Stephen Robinson, Dylan Edwards, Rachel Piddock, Amina Abdul-Aziz, Christopher Marlein, Matthew Fenech, Jeremy Turner, Matthew Lawes, Lyubov Zaitseva, Johnathan Watkins, Kristian Bowles, Stuart Rushworth. Bone marrow adipocytes drive transcriptional changes in leukemic blasts to enhance their capacity to derive energy from free fatty acid metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4327. doi:10.1158/1538-7445.AM2017-4327