Sphingosine kinase 2 inhibition synergises with bortezomib to target myeloma by enhancing endoplasmic reticulum stress

Published: April 14, 2017The proteasome inhibitor bortezomib has proven to be invaluable in the treatment of myeloma. By exploiting the inherent high immunoglobulin protein production of malignant plasma cells, bortezomib induces endoplasmic reticulum (ER) stress and the unfolded protein response (UPR), resulting in myeloma cell death. In most cases, however, the disease remains incurable highlighting the need for new therapeutic targets. Sphingosine kinase 2 (SK2) has been proposed as one such therapeutic target for myeloma. Our observations that bortezomib and SK2 inhibitors independently elicited induction of ER stress and the UPR prompted us to examine potential synergy between these agents in myeloma. Targeting SK2 synergistically contributed to ER stress and UPR activation induced by bortezomib, as evidenced by activation of the IRE1 pathway and stress kinases JNK and p38MAPK, thereby resulting in potent synergistic myeloma apoptosis in vitro. The combination of bortezomib and SK2 inhibition also exhibited strong in vivo synergy and favourable effects on bone disease. Therefore, our studies suggest that perturbations of sphingolipid signalling can synergistically enhance the effects seen with proteasome inhibition, highlighting the potential for the combination of these two modes of increasing ER stress to be formally evaluated in clinical trials for the treatment of myeloma patients.Craig T. Wallington-Beddoe, Melissa K. Bennett, Kate Vandyke, Lorena Davies, Julia R. Zebol, Paul A.B. Moretti, Melissa R. Pitman, Duncan R. Hewett, Andrew C.W. Zannettino and Stuart M. Pitso

Combination of RAD001 (everolimus) and docetaxel reduces prostate and breast cancer cell VEGF production and tumour vascularisation independently of sphingosine-kinase-1

Resistance to docetaxel is a key problem in current prostate and breast cancer management. We have recently discovered a new molecular mechanism of prostate cancer docetaxel chemoresistance mediated by the mammalian target of rapamycin (mTOR)/sphingosine-kinase-1 (SK1) pathway. Here we investigated the influence of this pathway on vascular endothelial growth factor (VEGF) production and tumour vascularisation in hormone resistant prostate and breast cancer models. Immunofluorescent staining of tumour sections from human oestrogen receptor (ER)-negative breast cancer patients showed a strong correlation between phosphorylated P70S6 kinase (mTOR downstream target), VEGF and SK1 protein expression. In hormone-insensitive prostate (PC3) and breast (MDA-MB-231 and BT-549) cancer cell lines the mTOR inhibitor RAD001 (everolimus) has significantly inhibited SK1 and VEGF expression, while low dose (5 nM) docetaxel had no significant effect. In these cell lines, SK1 overexpression slightly increased the basal levels of VEGF, but did not block the inhibitory effect of RAD001 on VEGF. In a human prostate xenograft model established in nude mice, RAD001 alone or in combination with docetaxel has suppressed tumour growth, VEGF expression and decreased tumour vasculature. Overall, our data demonstrate a new mechanism of an independent regulation of SK1 and VEGF by mTOR in hormone-insensitive prostate and breast cancers

Intracranial injection of dengue virus induces interferon stimulated genes and CD8(+) T cell infiltration by sphingosine kinase 1 independent pathways

We have previously reported that the absence of sphingosine kinase 1 (SK1) affects both dengue virus (DENV) infection and innate immune responses in vitro. Here we aimed to define SK1-dependancy of DENV-induced disease and the associated innate responses in vivo. The lack of a reliable mouse model with a fully competent interferon response for DENV infection is a challenge, and here we use an experimental model of DENV infection in the brain of immunocompetent mice. Intracranial injection of DENV-2 into C57BL/6 mice induced body weight loss and neurological symptoms which was associated with a high level of DENV RNA in the brain. Body weight loss and DENV RNA level tended to be greater in SK1-/- compared with wildtype (WT) mice. Brain infection with DENV-2 is associated with the induction of interferon-β (IFN-β) and IFN-stimulated gene (ISG) expression including viperin, Ifi27l2a, IRF7, and CXCL10 without any significant differences between WT and SK1-/- mice. The SK2 and sphingosine-1-phosphate (S1P) levels in the brain were unchanged by DENV infection or the lack of SK1. Histological analysis demonstrated the presence of a cellular infiltrate in DENV-infected brain with a significant increase in mRNA for CD8 but not CD4 suggesting this infiltrate is likely CD8+ but not CD4+ T-lymphocytes. This increase in T-cell infiltration was not affected by the lack of SK1. Overall, DENV-infection in the brain induces IFN and T-cell responses but does not influence the SK/S1P axis. In contrast to our observations in vitro, SK1 has no major influence on these responses following DENV-infection in the mouse brain.Wisam H. Al-Shujairi, Jennifer N. Clarke, Lorena T. Davies, Mohammed Alsharifi, Stuart M. Pitson, Jillian M. Car

Protein kinase activity of phosphoinositide 3-kinase regulates cytokine-dependent cell survival

Extent: 14 p.The dual specificity protein/lipid kinase, phosphoinositide 3-kinase (PI3K), promotes growth factor-mediated cell survival and is frequently deregulated in cancer. However, in contrast to canonical lipid-kinase functions, the role of PI3K protein kinase activity in regulating cell survival is unknown. We have employed a novel approach to purify and pharmacologically profile protein kinases from primary human acute myeloid leukemia (AML) cells that phosphorylate serine residues in the cytoplasmic portion of cytokine receptors to promote hemopoietic cell survival. We have isolated a kinase activity that is able to directly phosphorylate Ser585 in the cytoplasmic domain of the interleukin 3 (IL-3) and granulocyte macrophage colony stimulating factor (GM-CSF) receptors and shown it to be PI3K. Physiological concentrations of cytokine in the picomolar range were sufficient for activating the protein kinase activity of PI3K leading to Ser585 phosphorylation and hemopoietic cell survival but did not activate PI3K lipid kinase signaling or promote proliferation. Blockade of PI3K lipid signaling by expression of the pleckstrin homology of Akt1 had no significant impact on the ability of picomolar concentrations of cytokine to promote hemopoietic cell survival. Furthermore, inducible expression of a mutant form of PI3K that is defective in lipid kinase activity but retains protein kinase activity was able to promote Ser585 phosphorylation and hemopoietic cell survival in the absence of cytokine. Blockade of p110α by RNA interference or multiple independent PI3K inhibitors not only blocked Ser585 phosphorylation in cytokine-dependent cells and primary human AML blasts, but also resulted in a block in survival signaling and cell death. Our findings demonstrate a new role for the protein kinase activity of PI3K in phosphorylating the cytoplasmic tail of the GM-CSF and IL-3 receptors to selectively regulate cell survival highlighting the importance of targeting such pathways in cancer.Daniel Thomas, Jason A. Powell, Benjamin D. Green, Emma F. Barry, Yuefang Ma, Joanna Woodcock, Stephen Fitter, Andrew C.W. Zannettino, Stuart M. Pitson, Timothy P. Hughes, Angel F. Lopez, Peter R. Shepherd, Andrew H. Wei, Paul G. Ekert and Mark A. Guthridg

Outcomes of a randomized controlled trial assessing a smartphone Application to reduce unmet needs among people diagnosed with CancEr (ACE)

© 2019 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. Background: Smartphone technology represents an opportunity to deliver practical solutions for people affected by cancer at a scale that was previously unimaginable, such as information, appointment monitoring, and improved access to cancer support services. This study aimed to determine whether a smartphone application (app) reduced the unmet needs among people newly diagnosed with cancer. Methods: A single blind, multisite randomized controlled trial to determine the impact of an app-based, 4-month intervention. Newly diagnosed cancer patients were approached at three health service treatment clinics. Results: Eighty-two people were randomized (intervention; n = 43 and control; n = 39), average age was 59.5 years (SD: 12.9); 71% female; 67% married or in a de facto relationship. At baseline, there were no differences in participants’ characteristics between the groups. No significant effects, in reducing unmet needs, were demonstrated at the end of intervention (4-month) or 12-month follow-up. Overall, 94% used the app in weeks 1-4, which decreased to 41% in weeks 13-16. Mean app use time per participant: Cancer Information, 6.9 (SD: 18.9) minutes; Appointment Schedule, 5.1 (SD: 9.6) minutes; Cancer Services 1.5 minutes (SD: 6.8); Hospital Navigation, 1.4 (SD: 2.8) minutes. Conclusions: Despite consumer involvement in the design of this smartphone technology, the app did not reduce unmet needs. This may have been due to the study being underpowered. To contribute to a meaningful understanding and improved implementation of smartphone technology to support people affected by cancer, practical considerations, such as recruitment issues and access to, and confidence with, apps, need to be considered. Australian New Zealand Clinical Trials Registration (ACTRN) Trial Registration: 12616001251415; WEF 7/9/2016

A selective ATP-competitive sphingosine kinase inhibitor demonstrates anti-cancer properties

The dynamic balance of cellular sphingolipids, the sphingolipid rheostat, is an important determinant of cell fate, and is commonly deregulated in cancer. Sphingosine 1-phosphate is a signaling molecule with anti-apoptotic, pro-proliferative and pro-angiogenic effects, while conversely, ceramide and sphingosine are pro-apoptotic. The sphingosine kinases (SKs) are key regulators of this sphingolipid rheostat, and are attractive targets for anti-cancer therapy. Here we report a first-in-class ATP-binding site-directed small molecule SK inhibitor, MP-A08, discovered using an approach of structural homology modelling of the ATP-binding site of SK1 and in silico docking with small molecule libraries. MP-A08 is a highly selective ATP competitive SK inhibitor that targets both SK1 and SK2. MP-A08 blocks pro-proliferative signalling pathways, induces mitochondrial-associated apoptosis in a SK-dependent manner, and reduces the growth of human lung adenocarcinoma tumours in a mouse xenograft model by both inducing tumour cell apoptosis and inhibiting tumour angiogenesis. Thus, this selective ATP competitive SK inhibitor provides a promising candidate for potential development as an anti-cancer therapy, and also, due to its different mode of inhibition to other known SK inhibitors, both validates the SKs as targets for anti-cancer therapy, and represents an important experimental tool to study these enzymes.Melissa R. Pitman, Jason A. Powell, Carl Coolen, Paul A.B. Moretti, Julia R. Zebol, Duyen H. Pham, John W. Finnie, Anthony S. Don, Lisa M. Ebert, Claudine S. Bonder, Briony L. Gliddon, Stuart M. Pitso

Sphingosine Kinase-1 Is Required for Toll Mediated β-Defensin 2 Induction in Human Oral Keratinocytes

Host defense against invading pathogens is triggered by various receptors including toll-like receptors (TLRs). Activation of TLRs is a pivotal step in the initiation of innate, inflammatory, and antimicrobial defense mechanisms. Human beta-defensin 2 (HBD-2) is a cationic antimicrobial peptide secreted upon gram-negative bacterial perturbation in many cells. Stimulation of various TLRs has been shown to induce HBD-2 in oral keratinocytes, yet the underlying cellular mechanisms of this induction are poorly understood.Here we demonstrate that HBD-2 induction is mediated by the Sphingosine kinase-1 (Sphk-1) and augmented by the inhibition of Glycogen Synthase Kinase-3beta (GSK-3beta) via the Phosphoinositide 3-kinase (PI3K) dependent pathway. HBD-2 secretion was dose dependently inhibited by a pharmacological inhibitor of Sphk-1. Interestingly, inhibition of GSK-3beta by SB 216763 or by RNA interference, augmented HBD-2 induction. Overexpression of Sphk-1 with concomitant inhibition of GSK-3beta enhanced the induction of beta-defensin-2 in oral keratinocytes. Ectopic expression of constitutively active GSK-3beta (S9A) abrogated HBD-2 whereas kinase inactive GSK-3beta (R85A) induced higher amounts of HBD-2.These data implicate Sphk-1 in HBD-2 regulation in oral keratinocytes which also involves the activation of PI3K, AKT, GSK-3beta and ERK 1/2. Thus we reveal the intricate relationship and pathways of toll-signaling molecules regulating HBD-2 which may have therapeutic potential

S1P lyase regulates DNA damage responses through a novel sphingolipid feedback mechanism

The injurious consequences of ionizing radiation (IR) to normal human cells and the acquired radioresistance of cancer cells represent limitations to cancer radiotherapy. IR induces DNA damage response pathways that orchestrate cell cycle arrest, DNA repair or apoptosis such that irradiated cells are either repaired or eliminated. Concomitantly and independent of DNA damage, IR activates acid sphingomyelinase (ASMase), which generates ceramide, thereby promoting radiation-induced apoptosis. However, ceramide can also be metabolized to sphingosine-1-phosphate (S1P), which acts paradoxically as a radioprotectant. Thus, sphingolipid metabolism represents a radiosensitivity pivot point, a notion supported by genetic evidence in IR-resistant cancer cells. S1P lyase (SPL) catalyzes the irreversible degradation of S1P in the final step of sphingolipid metabolism. We show that SPL modulates the kinetics of DNA repair, speed of recovery from G2 cell cycle arrest and the extent of apoptosis after IR. SPL acts through a novel feedback mechanism that amplifies stress-induced ceramide accumulation, and downregulation/inhibition of either SPL or ASMase prevents premature cell cycle progression and mitotic death. Further, oral administration of an SPL inhibitor to mice prolonged their survival after exposure to a lethal dose of total body IR. Our findings reveal SPL to be a regulator of ASMase, the G2 checkpoint and DNA repair and a novel target for radioprotection

In vivo and in silico determination of essential genes of Campylobacter jejuni

<p>Abstract</p> <p>Background</p> <p>In the United Kingdom, the thermophilic <it>Campylobacter </it>species <it>C. jejuni </it>and <it>C. coli </it>are the most frequent causes of food-borne gastroenteritis in humans. While campylobacteriosis is usually a relatively mild infection, it has a significant public health and economic impact, and possible complications include reactive arthritis and the autoimmune diseases Guillain-Barré syndrome. The rapid developments in "omics" technologies have resulted in the availability of diverse datasets allowing predictions of metabolism and physiology of pathogenic micro-organisms. When combined, these datasets may allow for the identification of potential weaknesses that can be used for development of new antimicrobials to reduce or eliminate <it>C. jejuni </it>and <it>C. coli </it>from the food chain.</p> <p>Results</p> <p>A metabolic model of <it>C. jejuni </it>was constructed using the annotation of the NCTC 11168 genome sequence, a published model of the related bacterium <it>Helicobacter pylori</it>, and extensive literature mining. Using this model, we have used <it>in silico </it>Flux Balance Analysis (FBA) to determine key metabolic routes that are essential for generating energy and biomass, thus creating a list of genes potentially essential for growth under laboratory conditions. To complement this <it>in silico </it>approach, candidate essential genes have been determined using a whole genome transposon mutagenesis method. FBA and transposon mutagenesis (both this study and a published study) predict a similar number of essential genes (around 200). The analysis of the intersection between the three approaches highlights the shikimate pathway where genes are predicted to be essential by one or more method, and tend to be network hubs, based on a previously published <it>Campylobacter </it>protein-protein interaction network, and could therefore be targets for novel antimicrobial therapy.</p> <p>Conclusions</p> <p>We have constructed the first curated metabolic model for the food-borne pathogen <it>Campylobacter jejuni </it>and have presented the resulting metabolic insights. We have shown that the combination of <it>in silico </it>and <it>in vivo </it>approaches could point to non-redundant, indispensable genes associated with the well characterised shikimate pathway, and also genes of unknown function specific to <it>C. jejuni</it>, which are all potential novel <it>Campylobacter </it>intervention targets.</p

Germline mutations in mitochondrial complex I reveal genetic and targetable vulnerability in IDH1-mutant acute myeloid leukaemia

The interaction of germline variation and somatic cancer driver mutations is underinvestigated. Here we describe the genomic mitochondrial landscape in adult acute myeloid leukaemia (AML) and show that rare variants affecting the nuclear- and mitochondriallyencoded complex I genes show near-mutual exclusivity with somatic driver mutations affecting isocitrate dehydrogenase 1 (IDH1), but not IDH2 suggesting a unique epistatic relationship. Whereas AML cells with rare complex I variants or mutations in IDH1 or IDH2 all display attenuated mitochondrial respiration, heightened sensitivity to complex I inhibitors including the clinical-grade inhibitor, IACS-010759, is observed only for IDH1-mutant AML. Furthermore, IDH1 mutant blasts that are resistant to the IDH1-mutant inhibitor, ivosidenib, retain sensitivity to complex I inhibition. We propose that the IDH1 mutation limits the flexibility for citrate utilization in the presence of impaired complex I activity to a degree that is not apparent in IDH2 mutant cells, exposing a mutation-specific metabolic vulnerability. This reduced metabolic plasticity explains the epistatic relationship between the germline complex I variants and oncogenic IDH1 mutation underscoring the utility of genomic data in revealing metabolic vulnerabilities with implications for therapy.Mahmoud A. Bassal, Saumya E. Samaraweera, Kelly Lim, Brooks A. Bernard, Sheree Bailey, Satinder Kaur, Paul Leo, John Toubia, Chloe Thompson-Peach, Tran Nguyen, Kyaw Ze Ya Maung, Debora A. Casolari, Diana G. Iarossi, Ilaria S. Pagani, Jason Powell, Stuart Pitson, Siria Natera, Ute Roessner, Ian D. Lewis, Anna L. Brown, Daniel G. Tenen, Nirmal Robinson, David M. Ross, Ravindra Majeti, Thomas J. Gonda, Daniel Thomas, Richard J. D, Andre