PI3Kδ and PI3Kγ isoforms have distinct functions in regulating pro-tumoural signalling in the multiple myeloma microenvironment.

Phosphoinositide-3-kinase and protein kinase B (PI3K-AKT) is upregulated in multiple myeloma (MM). Using a combination of short hairpin RNA (shRNA) lentivirus-mediated knockdown and pharmacologic isoform-specific inhibition we investigated the role of the PI3K p110γ (PI3Kγ) subunit in regulating MM proliferation and bone marrow microenvironment-induced MM interactions. We compared this with inhibition of the PI3K p110δ (PI3kδ) subunit and with combined PI3kδ/γ dual inhibition. We found that MM cell adhesion and migration were PI3Kγ-specific functions, with PI3kδ inhibition having no effect in MM adhesion or migration assays. At concentration of the dual PI3Kδ/γ inhibitor duvelisib, which can be achieved in vivo we saw a decrease in AKT phosphorylation at s473 after tumour activation by bone marrow stromal cells (BMSC) and interleukin-6. Moreover, after drug treatment of BMSC/tumour co-culture activation assays only dual PI3kδ/γ inhibition was able to induce MM apoptosis. shRNA lentiviral-mediated targeting of either PI3Kδ or PI3Kγ alone, or both in combination, increased survival of NSG mice xeno-transplanted with MM cells. Moreover, treatment with duvelisib reduced MM tumour burden in vivo. We report that PI3Kδ and PI3Kγ isoforms have distinct functions in MM and that combined PI3kδ/γ isoform inhibition has anti-MM activity. Here we provide a scientific rationale for trials of dual PI3kδ/γ inhibition in patients with MM.

NRF2-driven miR-125B1 and miR-29B1 transcriptional regulation controls a novel anti-apoptotic miRNA regulatory network for AML survival.

Transcription factor NRF2 is an important regulator of oxidative stress. It is involved in cancer progression, and has abnormal constitutive expression in acute myeloid leukaemia (AML). Posttranscriptional regulation by microRNAs (miRNAs) can affect the malignant phenotype of AML cells. In this study, we identified and characterised NRF2-regulated miRNAs in AML. An miRNA array identified miRNA expression level changes in response to NRF2 knockdown in AML cells. Further analysis of miRNAs concomitantly regulated by knockdown of the NRF2 inhibitor KEAP1 revealed the major candidate NRF2-mediated miRNAs in AML. We identified miR-125B to be upregulated and miR-29B to be downregulated by NRF2 in AML. Subsequent bioinformatic analysis identified putative NRF2 binding sites upstream of the miR-125B1 coding region and downstream of the mir-29B1 coding region. Chromatin immunoprecipitation analyses showed that NRF2 binds to these antioxidant response elements (AREs) located in the 5' untranslated regions of miR-125B and miR-29B. Finally, primary AML samples transfected with anti-miR-125B antagomiR or miR-29B mimic showed increased cell death responsiveness either alone or co-treated with standard AML chemotherapy. In summary, we find that NRF2 regulation of miR-125B and miR-29B acts to promote leukaemic cell survival, and their manipulation enhances AML responsiveness towards cytotoxic chemotherapeutics.

Curcumin: potential for hepatic fibrosis therapy?

The beneficial antioxidative, anti-inflammatory and antitumorigenic effects of curcumin have been well documented in relation to cancer and other chronic diseases. Recent evidence suggests that it may be of therapeutic interest in chronic liver disease. Hepatic fibrosis (scarring) occurs in advanced liver disease, where normal hepatic tissue is replaced with collagen-rich extracellular matrix and, if left untreated, results in cirrhosis. Curcumin inhibits liver cirrhosis in a rodent model and exerts multiple biological effects in hepatic stellate cells (HSCs), which play a central role in the pathogenesis of hepatic fibrosis. In response to liver injury, these cells proliferate producing pro-inflammatory mediators and extracellular matrix. Curcumin induces apoptosis and suppresses proliferation in HSCs. In addition, it inhibits extracellular matrix formation by enhancing HSC matrix metalloproteinase expression via PPARgamma and suppressing connective tissue growth factor (CTGF) expression. In this issue, Chen and co-workers propose that curcumin suppresses CTGF expression in HSC by inhibiting ERK and NF-kappaB activation. These studies suggest that curcumin modulates several intracellular signalling pathways in HSC and may be of future interest in hepatic fibrosis therapy.

Haem oxygenase-1: a target for dietary antioxidants.

HO-1 (haem oxygenase-1) is a stress-response enzyme involved in the catabolism of haem. In animal models, it plays a key protective role in vascular disease. HO-1 has anti-inflammatory effects in macrophages and is induced by a range of stimuli, including antioxidants, in various cell types. As dietary antioxidants are considered to be beneficial in vascular disease, their protective effects may occur through induction of HO-1. Emerging evidence suggests that a range of dietary and other naturally occurring antioxidants stimulate HO-1 expression in various cell types, although regulation by these compounds has not been investigated in detail. These studies suggest that HO-1 may be a target for dietary therapy in vascular disease.

Haem oxygenase-1 in inflammation.

HO-1 (haem oxygenase-1) is a stress-inducible enzyme that plays a protective role in inflammation. Pro-inflammatory mediators, including lipopolysaccharide and cytokines, induce HO-1 expression. The 5'-flanking region of the HO-1 gene contains binding sites for the transcription factors that regulate inflammation, including nuclear factor-kappaB and activator protein 1. However, these do not appear to mediate lipopolysaccharide-induced HO-1 gene expression. In response to haem and antioxidants, murine HO-1 is regulated by the transcription factor Nrf2 (NF-E2-related factor 2). This transcription factor may also be important in the regulation of HO-1 by pro-inflammatory stimuli.

Inhibition of CD40-mediated endothelial cell activation with antisense oligonucleotides.

BACKGROUND: CD40-CD154 interactions play a pivotal role in the amplification of immune responses and, as such, represent an attractive target for immune intervention in a number of disease indications. We have previously shown that binding of human CD154 expressed on the Jurkat D1.1 cell line to porcine CD40 on pig aortic endothelial cells (PAECs) can lead to up-regulation of vascular cell adhesion molecule (VCAM)-1 and MHC class II. This activation can be completely inhibited by the addition of a monoclonal antibody (mAb) to human CD154. In this study, we explore an alternative approach to blocking this pathway with antisense oligonucleotides (ASOs). METHODS: Ten ASOs were generated on the basis of the porcine CD40 cDNA sequence. The ASOs that were found to reduce CD40 expression on PAECs were analyzed for their ability to reduce CD40-mediated PAEC activation. RESULTS: Four ASOs were found to significantly lower surface expression of porcine CD40 on PAECs 48 hr after transfection. Eight of the ASOs were seen to lead to mRNA cleavage products by ribonuclease protection assay. Of the four ASOs tested in the PAEC activation assay, one (ASO-9) showed a dramatic inhibition of PAEC activation (IC50 approximately 1 nM) results comparable to the use of a blocking mAb. Furthermore, we compared the effect of CD40 ASO on tumor necrosis factor alpha receptor signaling, in which we observed no effect, which confirmed ASO specificity. CONCLUSIONS: These results indicate that a CD40-dependent activation pathway can be inhibited with an ASO with high potency and specificity. ASO could be an attractive alternative therapy to the use of mAbs.

Human CD154 induces activation of porcine endothelial cells and up-regulation of MHC class II expression.

BACKGROUND: CD40 is expressed on a number of antigen-presenting cells and also on vascular endothelium. It has been shown that engagement of CD40 on vascular endothelium by CD154 on platelets and CD154-bearing cell lines leads to the induction of adhesion molecule expression. Having cloned porcine CD40, and shown that it is capable of binding human CD154, we investigate whether human CD154 can activate porcine endothelial cells (EC) through CD40 ligation. METHODS: Human Jurkat clone D1.1 (CD154+), or clone E6.1 (CD154-), were co-cultured with EC from pig aorta and human aorta and umbilical vein for various times in the presence or absence of blocking antibody to CD154. RESULTS: Human and pig EC were shown to express CD40 by flow cytometry by using soluble human CD154 (CD154Ckappa). Co-culture of pig EC with CD154-expressing Jurkat D1.1 cells led to the induction of E-selectin by 6 hr (peak 24 hr) and vascular cell adhesion molecule-1 (VCAM-1) by 6 hr (peak 48 hr). Similar results were also observed with human EC. Porcine EC were induced to up-regulate major histocompatibility complex class II at 24 hr by co-culture with Jurkat D1.1 cells through a CD40-dependent mechanism. In contrast, no up-regulation was observed on human EC. CONCLUSIONS: A number of cells can express CD154, including T cells, natural killer cells, and platelets, and these could signal graft EC through the CD40 pathway. These results demonstrate a possible role for the CD40 pathway in the activation of vascular endothelium in the rejection of porcine xenografts.

High sequence homology between human and pig CD40 with conserved binding to human CD154.

BACKGROUND: Understanding the molecular interactions between pig tissues and human immune cells is fundamental to achieving long-term pig to human xenograft survival. CD40 has been shown to be central in the interaction of T cells with many antigen-presenting cells including B cells, and dendritic cells. It has been clearly shown in vitro that human T cells can effectively recognize pig major histocompatibility complex proteins, and that various accessory molecule interactions are compatible between these species, including human CD28 with pig B7 family members (CD80/CD86). The importance of CD40 in transplantation has been established using blocking antibodies to its ligand, CD154, which prolong allograft survival in mouse and primate models. METHODS: Pig CD40 was cloned from a porcine spleen cDNA library and subsequently sequenced. Expression of pig CD40 was detected by flow cytometry using soluble human CD154 (hCD154-Ig). Results. Comparison of the derived amino acid sequence of pig with human shows 74% identity. Significantly, there is conservation between pig and human at 5 residues shown by mutagenesis studies to be essential for binding of human CD40 to CD154. hCD154Ckappa was shown to bind pig B cell lines and a proportion of human and pig lymphocytes and further confirmed by staining of COS cells transfected with pig CD40. Conclusions. Recipient human cells bearing CD154 will, therefore, be able to bind donor pig CD40, and these interactions might modulate effector functions and hence influence xenograft survival. Further investigation is necessary to ascertain the exact nature of these interactions and their implications for xenograft survival.