Plasma cell-derived mtDAMPs activate the macrophage STING pathway, promoting myeloma progression.

Mitochondrial damage-associated molecular patterns (mtDAMPs) include proteins, lipids, metabolites, and DNA and have various context-specific immunoregulatory functions. Cell-free mitochondrial DNA (mtDNA) is recognized via pattern recognition receptors and is a potent activator of the innate immune system. Cell-free mtDNA is elevated in the circulation of trauma patients and patients with cancer; however, the functional consequences of elevated mtDNA are largely undefined. Multiple myeloma (MM) relies upon cellular interactions within the bone marrow (BM) microenvironment for survival and progression. Here, using in vivo models, we describe the role of MM cell-derived mtDAMPs in the protumoral BM microenvironment and the mechanism and functional consequence of mtDAMPs in myeloma disease progression. Initially, we identified elevated levels of mtDNA in the peripheral blood serum of patients with MM compared with those of healthy controls. Using the MM1S cells engrafted into nonobese diabetic severe combined immunodeficient gamma mice, we established that elevated mtDNA was derived from MM cells. We further show that BM macrophages sense and respond to mtDAMPs through the stimulator of interferon genes (STING) pathway, and inhibition of this pathway reduces MM tumor burden in the KaLwRij-5TGM1 mouse model. Moreover, we found that MM-derived mtDAMPs induced upregulation of chemokine signatures in BM macrophages, and inhibition of this signature resulted in egress of MM cells from the BM. Here, we demonstrate that malignant plasma cells release mtDNA, a form of mtDAMPs, into the myeloma BM microenvironment, which in turn activates macrophages via STING signaling. We establish the functional role of these mtDAMP-activated macrophages in promoting disease progression and retaining MM cells in the protumoral BM microenvironment.

Suramin: Effectiveness of analogues reveals structural features that are important for the potent trypanocidal activity of the drug.

Suramin was the first effective drug for the treatment of human African sleeping sickness. Structural analogues of the trypanocide have previously been shown to be potent inhibitors of several enzymes. Therefore, four suramin analogues lacking the methyl group on the intermediate rings and with different regiochemistry of the naphthalenetrisulphonic acid groups and the phenyl rings were tested to establish whether they exhibited improved antiproliferative activity against bloodstream forms of Trypanosomes brucei compared to the parent compound. The four analogues exhibited low trypanocidal activity and weak inhibition of the antitrypanosomal activity of suramin in competition experiments. This indicates that the strong trypanocidal activity of suramin is most likely due to the presence of methyl groups on its intermediate rings and to the specific regiochemistry of naphthalenetrisulphonic acid groups. These two structural features are also likely to be important for the inhibition mechanism of suramin because DNA distribution and nucleus/kinetoplast configuration analyses suggest that the analogues inhibit mitosis while suramin inhibits cytokinesis.

PGC-1α induced mitochondrial biogenesis in stromal cells underpins mitochondrial transfer to melanoma.

INTRODUCTION: Progress in the knowledge of metabolic interactions between cancer and its microenvironment is ongoing and may lead to novel therapeutic approaches. Until recently, melanoma was considered a glycolytic tumour due to mutations in mitochondrial-DNA, however, these malignant cells can regain OXPHOS capacity via the transfer of mitochondrial-DNA, a process that supports their proliferation in-vitro and in-vivo. Here we study how melanoma cells acquire mitochondria and how this process is facilitated from the tumour microenvironment. METHODS: Primary melanoma cells, and MSCs derived from patients were obtained. Genes' expression and DNA quantification was analysed using Real-time PCR. MSC migration, melanoma proliferation and tumour volume, in a xenograft subcutaneous mouse model, were monitored through bioluminescent live animal imaging. RESULTS: Human melanoma cells attract bone marrow-derived stromal cells (MSCs) to the primary tumour site where they stimulate mitochondrial biogenesis in the MSCs through upregulation of PGC1a. Mitochondria are transferred to the melanoma cells via direct contact with the MSCs. Moreover, inhibition of MSC-derived PGC1a was able to prevent mitochondrial transfer and improve NSG melanoma mouse tumour burden. CONCLUSION: MSC mitochondrial biogenesis stimulated by melanoma cells is prerequisite for mitochondrial transfer and subsequent tumour growth, where targeting this pathway may provide an effective novel therapeutic approach in melanoma.

ROS-mediated PI3K activation drives mitochondrial transfer from stromal cells to hematopoietic stem cells in response to infection.

Hematopoietic stem cells (HSCs) undergo rapid expansion in response to stress stimuli. Here we investigate the bioenergetic processes which facilitate the HSC expansion in response to infection. We find that infection by Gram-negative bacteria drives an increase in mitochondrial mass in mammalian HSCs, which results in a metabolic transition from glycolysis toward oxidative phosphorylation. The initial increase in mitochondrial mass occurs as a result of mitochondrial transfer from the bone marrow stromal cells (BMSCs) to HSCs through a reactive oxygen species (ROS)-dependent mechanism. Mechanistically, ROS-induced oxidative stress regulates the opening of connexin channels in a system mediated by phosphoinositide 3-kinase (PI3K) activation, which allows the mitochondria to transfer from BMSCs into HSCs. Moreover, mitochondria transfer from BMSCs into HSCs, in the response to bacterial infection, occurs before the HSCs activate their own transcriptional program for mitochondrial biogenesis. Our discovery demonstrates that mitochondrial transfer from the bone marrow microenvironment to HSCs is an early physiologic event in the mammalian response to acute bacterial infection and results in bioenergetic changes which underpin emergency granulopoiesis.

Mitochondrial oxidative phosphorylation in cutaneous melanoma.

The Warburg effect in tumour cells is associated with the upregulation of glycolysis to generate ATP, even under normoxic conditions and the presence of fully functioning mitochondria. However, scientific advances made over the past 15 years have reformed this perspective, demonstrating the importance of oxidative phosphorylation (OXPHOS) as well as glycolysis in malignant cells. The metabolic phenotypes in melanoma display heterogeneic dynamism (metabolic plasticity) between glycolysis and OXPHOS, conferring a survival advantage to adapt to harsh conditions and pathways of chemoresistance. Furthermore, the simultaneous upregulation of both OXPHOS and glycolysis (metabolic symbiosis) has been shown to be vital for melanoma progression. The tumour microenvironment (TME) has an essential supporting role in promoting progression, invasion and metastasis of melanoma. Mesenchymal stromal cells (MSCs) in the TME show a symbiotic relationship with melanoma, protecting tumour cells from apoptosis and conferring chemoresistance. With the significant role of OXPHOS in metabolic plasticity and symbiosis, our review outlines how mitochondrial transfer from MSCs to melanoma tumour cells plays a key role in melanoma progression and is the mechanism by which melanoma cells regain OXPHOS capacity even in the presence of mitochondrial mutations. The studies outlined in this review indicate that targeting mitochondrial trafficking is a potential novel therapeutic approach for this highly refractory disease.

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.

Mitochondria and the Tumour Microenvironment in Blood Cancer.

The bone marrow (BM) is a complex organ located within the cavities of bones. The main function of the BM is to produce all the blood cells required for a normal healthy blood system. As with any major organ, many diseases can arise from errors in bone marrow function, including non-malignant disorders such as anaemia and malignant disorders such as leukaemias. This article will explore the role of the bone marrow, in normal and diseased haematopoiesis, with an emphasis on the requirement for intercellular mitochondrial transfer in leukaemia.

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.

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.

Front-line glioblastoma chemotherapeutic temozolomide is toxic to Trypanosoma brucei and potently enhances melarsoprol and eflornithine.

Sleeping sickness is an infectious disease that is caused by the protozoan parasite Trypanosoma brucei. The second stage of the disease is characterised by the parasites entering the brain. It is therefore important that sleeping sickness therapies are able to cross the blood-brain barrier. At present, only three medications for chemotherapy of the second stage of the disease are available. As these trypanocides have serious side effects and are difficult to administer, new and safe anti-trypanosomal brain-penetrating drugs are needed. For these reasons, the anti-glioblastoma drug temozolomide was tested in vitro for activity against bloodstream forms of T. brucei. The concentration of the drug required to reduce the growth rate of the parasites by 50% was 29.1 µM and to kill all trypanosomes was 125 µM. Importantly, temozolomide did not affect the growth of human HL-60 cells up to a concentration of 300 µM. Cell cycle analysis revealed that temozolomide induced DNA damage and subsequent cell cycle arrest in trypanosomes exposed to the compound. As drug combination regimes often achieve greater therapeutic efficacy than monotherapies, the interactions of temozolomide with the trypanocides eflornithine and melarsoprol, respectively, was determined. Both combinations were found to produce an additive effect. In conclusion, these results together with well-established pharmacokinetic data provide the basis for in vivo studies and potentially for clinical trials of temozolomide in the treatment of T. brucei infections and a rationale for its use in combination therapy, particularly with eflornithine or melarsoprol.

Identification of Bruton's tyrosine kinase as a therapeutic target in acute myeloid leukemia.

Bruton's tyrosine kinase (BTK) is a cytoplasmic protein found in all hematopoietic cell lineages except for T cells. BTK mediates signaling downstream of a number of receptors. Pharmacologic targeting of BTK using ibrutinib (previously PCI-32765) has recently shown encouraging clinical activity in a range of lymphoid malignancies. This study reports for the first time that ibrutinib inhibits blast proliferation from human acute myeloid leukemia (AML) and that treatment with ibrutinib significantly augmented cytotoxic activities of standard AML chemotherapy cytarabine or daunorubicin. Here we describe that BTK is constitutively phosphorylated in the majority of AML samples tested, with BTK phosphorylation correlating highly with the cell's cytotoxic sensitivity toward ibrutinib. BTK-targeted RNAi knockdown reduced colony-forming capacity of primary AML blasts and proliferation of AML cell lines. We showed that ibrutinib binds at nanomolar range to BTK. Furthermore, we showed ibrutinib's antiproliferative effects in AML are mediated via an inhibitory effect on downstream nuclear factor-κB survival pathways. Moreover, ibrutinib inhibited AML cell adhesion to bone marrow stroma. Furthermore, these effects of ibrutinib in AML were seen at comparable concentrations efficacious in chronic lymphocytic leukemia. These results provide a biological rationale for clinical evaluation of BTK inhibition in AML patients.

Trypanocidal and cysteine protease inhibitory activity of isopentyl caffeate is not linked in Trypanosoma brucei.

Previously, it was reported that caffeic acid esters inhibit the growth of bloodstream forms of Trypanosoma brucei and the activity of its major lysosomal cathepsin L-like cysteine protease, TbCATL. However, whether this trypanocidal activity is due to inactivation of TbCATL has not so far been demonstrated. Caffeic acid isopentyl ester (isopentyl caffeate) displayed antitrypanosomal activity against T. brucei bloodstream forms with minimum inhibitory concentration (MIC) and 50 % growth inhibition (GI50) values of 1 and 0.31 µg/ml, respectively. The ester also inhibited the activity of purified TbCATL but with a 27-fold higher half maximal inhibitory concentration (IC50) value of 8.5 µg/ml compared to its GI50 value. In contrast to previous suggestion, isopentyl caffeate did not interact with the active site of TbCATL but inhibited the enzyme in a non-competitive way. In addition, the ester was ineffective in blocking the proteolysis in the lysosome of the parasite, which, however, is a hallmark for inhibitors whose trypanocidal action is through inactivation of TbCATL. These results suggest that the antitrypanosomal activity of isopentyl caffeate (and probably of other caffeic acid esters) cannot be attributed to inhibition of TbCATL. Nevertheless, caffeic acid esters are interesting compounds with promising antitrypanosomal activity. This is supported by a more than 100 times less sensitivity of human HL-60 cells to isopentyl caffeate indicating that the ester has a favourable selectivity profile.

Understanding life and death decisions in human leukaemias.

Human leukaemia cells have an often unique ability to either undergo apoptotic cell death mechanisms or, at other times, undergo proliferative expansion, sometimes to the same stimulus such as the pluripotent cytokine TNFα (tumour necrosis factor α). This potential for life/death switching helps us to understand the molecular signalling machinery that underlies these cellular processes. Furthermore, looking at the involvement of these switching signalling pathways that may be aberrant in leukaemia informs us of their importance in cancer tumorigenesis and how they may be targeted pharmacologically to treat various types of human leukaemias. Furthermore, these important pathways may play a crucial role in acquired chemotherapy resistance and should be studied further to overcome in the clinic many drug-resistant forms of blood cancers. In the present article, we uncover the relationship that exists in human leukaemia life/death switching between the anti-apoptotic pro-inflammatory transcription factor NF-κB (nuclear factor κB) and the cytoprotective antioxidant-responsive transcription factor Nrf2 (nuclear factor-erythroid 2-related factor 2). We also discuss recent findings that reveal a major role for Btk (Bruton's tyrosine kinase) in both lymphocytic and myeloid forms of human leukaemias and lymphomas.

The high Nrf2 expression in human acute myeloid leukemia is driven by NF-κB and underlies its chemo-resistance.

NF-E2-related factor 2 (Nrf2) transcription factor regulates a range of cytoprotective transcriptional responses, preventing further cellular injury by removing biochemical damage and renewing tissue. Here we show that acute myeloid leukemia (AML) cells possess greater constitutive nuclear levels of Nrf2 than normal control CD34(+) cells because of an imbalance between mRNA expression levels of Nrf2 and its inhibitor Keap1 but not through their somatic mutation. Elevated Nrf2 was reduced by NF-κB inhibitors. Using promoter assays, ChIP and siRNA knockdown, we demonstrated NF-κB subunits p50 and p65 induce transcription of Nrf2 in AML cells at a specific promoter κB-site and that long-term lentiviral miRNA-knockdown of Nrf2 significantly reduced clonogenicity of AML patient cells and improved their chemotherapeutic responsiveness. Normal physiologic Nrf2 protects cells from damage, but here we have exposed aberrant continuous nuclear activation of Nrf2 in AML that allows cell survival, even against cytotoxic chemotherapeutics. We show for the first time that Nrf2, an important regulator of several biologic processes involved in the progression of cancer, has abnormal NF-κB-driven constitutive expression in AML. Such a mechanism allows for a greater cytoprotective response in human AML cells and encourages their evasion of chemotherapy-induced cytotoxicity, which is necessary for improved clinical outcomes.

BCL-2 inhibition in haematological malignancies: Clinical application and complications.

B-cell lymphoma-2 (BCL-2) family proteins are fundamental regulators of the intrinsic apoptotic pathway which modulate cellular fate. In many haematological malignancies, overexpression of anti-apoptotic factors (BCL-2, BCL-XL and MCL-1) circumvent apoptosis. To address this cancer hallmark, a concerted effort has been made to induce apoptosis by inhibiting BCL-2 family proteins. A series of highly selective BCL-2 homology 3 (BH3) domain mimetics are in clinical use and in ongoing clinical trials for acute myeloid leukaemia (AML), chronic myeloid leukaemia (CML), chronic lymphocytic leukaemia (CLL), and multiple myeloma (MM). These inhibitors serve as promising candidates, both as single agents or in combination therapy to improve patient outcomes. In other diseases such as follicular lymphoma, efficacy has been notably limited. There are also clinical problems with BCL-2 family inhibition, including drug resistance, disease relapse, tumour lysis syndrome, and clinically relevant cytopenias. Here, we provide a balanced view on both the clinical benefits of BCL-2 inhibition as well as the associated challenges.

Perlecan (HSPG2) promotes structural, contractile, and metabolic development of human cardiomyocytes.

Perlecan (HSPG2), a heparan sulfate proteoglycan similar to agrin, is key for extracellular matrix (ECM) maturation and stabilization. Although crucial for cardiac development, its role remains elusive. We show that perlecan expression increases as cardiomyocytes mature in vivo and during human pluripotent stem cell differentiation to cardiomyocytes (hPSC-CMs). Perlecan-haploinsuffient hPSCs (HSPG2+/-) differentiate efficiently, but late-stage CMs have structural, contractile, metabolic, and ECM gene dysregulation. In keeping with this, late-stage HSPG2+/- hPSC-CMs have immature features, including reduced ⍺-actinin expression and increased glycolytic metabolism and proliferation. Moreover, perlecan-haploinsuffient engineered heart tissues have reduced tissue thickness and force generation. Conversely, hPSC-CMs grown on a perlecan-peptide substrate are enlarged and display increased nucleation, typical of hypertrophic growth. Together, perlecan appears to play the opposite role of agrin, promoting cellular maturation rather than hyperplasia and proliferation. Perlecan signaling is likely mediated via its binding to the dystroglycan complex. Targeting perlecan-dependent signaling may help reverse the phenotypic switch common to heart failure.

TNF mediates the sustained activation of Nrf2 in human monocytes.

Modulation of monocyte function is a critical factor in the resolution of inflammatory responses. This role is mediated mainly by the production of TNF-α. Investigations of the actions of TNF have mostly focused on acute activation of other cell types such as fibroblasts and endothelial cells. Less is known about the effects of TNF on monocytes themselves, and little is known about the regulation of cell responses to TNF beyond the activation of NF-κB. In this study, we investigated the regulation of NF-E2-related factor 2 (Nrf2) cyctoprotective responses to TNF in human monocytes. We found that in monocytes TNF induces sustained Nrf2 activation and Nrf2 cytoprotective gene induction in a TNFR1-dependent manner. Under TNF activation, monocytes increased their expression of Nrf2-dependent genes, including NAD(P)H:quinone oxidoreductase 1 and glutamyl cysteine ligase modulatory, but not heme oxygenase-1. We also showed that autocrine TNF secretion was responsible for this sustained Nrf2 response and that Nrf2 activation by TNF was mediated by the generation of reactive oxygen species. Moreover, we showed that Nrf2-mediated gene induction can modulate TNF-induced NF-κB activation. These results show for the first time, to our knowledge, that TNF modulates prolonged Nrf2-induced gene expression, which in turn regulates TNF-induced inflammatory responses.

In Vivo Imaging of Bone Marrow Long-Chain Fatty Acid Uptake.

In vivo imaging enables the detection and visualization of many different processes occurring within the body. Fatty acid uptake is a fundamental cellular process which is essential for the use of free fatty acids (FFAs) as a fuel source for metabolism. Detection and visualization of in vivo FFA uptake in the bone marrow has been relatively unknown. Here, we describe the process of non-invasive bioluminescent imaging of in vivo FFA uptake within the bone marrow.