Investigation of the role of marrow adipose tissue in the acute myeloid leukaemia bone marrow microenvironment.

Acute myeloid leukaemia (AML) is a cancer of the blood forming cells of the bone marrow (BM) and has a high mortality rate in the elderly population. New, tolerable therapeutic strategies are necessary for reducing the mortality rate associated with AML in this fragile, less fit population. The tumour microenvironment is an evolving target in the search for lower chemotherapy-induced toxicity and strategies that can encompass a larger sector of the population including the elderly. Adipocytes in the BM were long considered to be mere occupants however, emerging research has identified these cells to be more than such and have an active role in metabolism regulation and an endocrine organ in its own right. In the context of cancers, adipocytes now pose an attractive target for novel cancer treatments due to their ability to confer chemoresistance in the tumour microenvironment. Here, I show the active participation of adipocytes that enhance the survival and proliferation of the AMLs within the BM microenvironment (BMM) though the support of several cellular functions in the AMLs. I also show that adipocytes support metabolism of AML by providing them with energy substrates in the form of fatty acid which are then used to support the proliferation of the AMLs. Additionally, I provide preliminary results implicating genes that may be responsible for the homing and free fatty acquisition in the BMM. These findings therefore provide important pre-clinical evidence for targeting factors in the BMM that support the tumour survival which may be less toxic than current therapies that do not encompass the wider AML population

The bone marrow microenvironment – Home of the leukemic blasts

Acute Myeloid Leukaemia (AML) is a genetically, biologically and clinically heterogeneous set of diseases, which are characterised by an increased growth of abnormal myeloid progenitor cells within the bone marrow (BM). Ex-vivo AML exhibits a high level of spontaneous apoptosis. Furthermore, relapse for patients achieving remission occurs from minimal residual disease harboured within the BM microenvironment. Taken together, these observations illustrate the importance of the BM microenvironment in sustaining AML. While significant progress has been made elaborating the small-scale genetic mutations and larger-scale chromosomal translocations that contribute to the development of AML and its prognosis in response to treatment, less is understood about the complex microenvironment of the BM, which is known to be a key player in the pathogenesis of the disease. As we look towards future therapies, the consideration that the BM microenvironment is uniquely important as a niche for AML - coupled with the idea that leukaemic blasts are more likely to be genetically unstable and therefore evolve resistance to conventional chemotherapies - make the functions of the non-malignant cells of the BM attractive targets for therapy. In this review, we discuss the microanatomy of the BM and provide an overview of the evidence supporting the role of the BM microenvironment in creating conditions conducive to the survival and proliferation of AML blasts. Ultimately, we examine the therapeutic potential of uncoupling AML from the BM microenvironment

Ibrutinib inhibits SDF1/CXCR4 mediated migration in AML

Pharmacological targeting of BTK using ibrutinib has recently shown encouraging clinical activity in a range of lymphoid malignancies. Recently we reported that ibrutinib inhibits human acute myeloid leukemia (AML) blast proliferation and leukemic cell adhesion to the surrounding bone marrow stroma cells. Here we report that in human AML ibrutinib, in addition, functions to inhibit SDF1/CXCR4-mediated AML migration at concentrations achievable in vivo. It has previously been shown that SDF1/CXCR4-induced migration is dependent on activation of downstream BTK in chronic lymphocytic leukaemia (CLL) and multiple myeloma. Here we show that SDF-1 induces BTK phosphorylation and downstream MAPK signalling in primary AML blast. Furthermore, we show that ibrutinib can inhibit SDF1-induced AKT and MAPK activation. These results reported here provide a molecular mechanistic rationale for clinically evaluating BTK inhibition in AML patients and suggests that in some AML patients the blasts count may initially rise in response to ibrutinib therapy, analgous to similar clinical observations in CLL

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

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

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 micro-environment are expected to lead to novel therapeutic approaches. Acute myeloid leukemia (AML) cells have increased mitochondria compared to non-malignant CD34+ hematopoietic progenitor cells. Furthermore, contrary to the Warburg hypothesis, (AML) relies on oxidative phosphorylation to generate ATP. 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 tunnelling 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 non-malignant CD34+ cells occurs in response to oxidative stress, NOX2 inhibition had no detectable effect on non-malignant CD34+ cell survival. Taken together we identify tumor-specific dependence on NOX2 driven mitochondrial transfer as a novel therapeutic strategy in AML

AKT inhibition generates potent polyfunctional clinical grade AUTO1 CAR T-cells, enhancing function and survival

BACKGROUND: AUTO1 is a fast off-rate CD19-targeting chimeric antigen receptor (CAR), which has been successfully tested in adult lymphoblastic leukemia. Tscm/Tcm-enriched CAR-T populations confer the best expansion and persistence, but Tscm/Tcm numbers are poor in heavily pretreated adult patients. To improve this, we evaluate the use of AKT inhibitor (VIII) with the aim of uncoupling T-cell expansion from differentiation, to enrich Tscm/Tcm subsets. METHODS: VIII was incorporated into the AUTO1 manufacturing process based on the semiautomated the CliniMACS Prodigy platform at both small and cGMP scale. RESULTS: AUTO1 manufactured with VIII showed Tscm/Tcm enrichment, improved expansion and cytotoxicity in vitro and superior antitumor activity in vivo. Further, VIII induced AUTO1 Th1/Th17 skewing, increased polyfunctionality, and conferred a unique metabolic profile and a novel signature for autophagy to support enhanced expansion and cytotoxicity. We show that VIII-cultured AUTO1 products from B-ALL patients on the ALLCAR19 study possess superior phenotype, metabolism, and function than parallel control products and that VIII-based manufacture is scalable to cGMP. CONCLUSION: Ultimately, AUTO1 generated with VIII may begin to overcome the product specific factors contributing to CD19+relapse

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 hypothesise that specific targeting of the tumor microenvironment may constitute an alternative or additional strategy to conventional tumor-directed chemotherapy. Since adipocytes have been shown to promote breast and prostate cancer proliferation, and because the bone marrow adipose tissue (MAT) accounts for up to 70% of bone marrow volume in adult humans, we examined the adipocyte-leukaemia 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) mRNA is up-regulated in adipocytes and AML when in co-culture. FABP4 inhibition using FABP4 shRNA 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 (CPT1A) in an AML patient-derived xenograft model improves survival. Here we report the first description of AML programming bone marrow adipocytes to generate a pro-tumoral microenvironment

MIF-induced stromal PKCβ/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-MSC which benefits AML proliferation and survival. We initially examined the cytokine profile in cultured human AML compared to AML cultured with BMMSC and found that macrophage-migration inhibitory factor (MIF) was highly expressed by primary AML, and that interleukin-8 (IL-8) was increased in AML/BM-MSC co-cultures. Recombinant MIF increased IL-8 expression in BM-MSC via its receptor CD74. Moreover, the MIF inhibitor ISO-1 inhibited AML-induced IL-8 expression by BM-MSC as well as BMMSC- induced AML survival. Protein kinase C β (PKCβ) regulated MIF-induced IL-8 in BMMSC. Finally, targeted IL-8 shRNA inhibited BM-MSC-induced AML survival. These results describe a novel, bidirectional, pro-survival mechanism between AML blasts and BM-MSC. Furthermore, they provide biologic rationale for therapeutic strategies in AML targeting the microenvironment, specifically MIF and IL-8

Acute myeloid leukemia induces pro-tumoral 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 1 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