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MSMS identification of phospholipids and glycerol 3-phosphate using collision induced dissociation (CID) fragmentation.

Supplementary Figure 1 from Nm23-H1 Indirectly Promotes the Survival of Acute Myeloid Leukemia Blast Cells by Binding to More Mature Components of the Leukemic Clone

Mass spectrometry data: free fatty acids and phospholipids from cells that were drug treated in the presence of 13C D-glucose

Negative ion mass spectrometry (phospholipid) analysis of lipid extracts from HL60 and K562 cells after 24 hr BaP treatment (0.5 mM bezafibrate, BEZ, and 5 µM medroxyprogesterone acetate, MPA).

Mass spectrometry data: glycerol 3-phosphate and glycerolipids

B cell non-Hodgkin lymphomas (B-NHLs) are the most common adult hematological cancers and many remain incurable. Development of chemotherapy regimens is confounded by the prevalence of B-NHL in older, frailer patients and the chemo-protective tumor microenvironment. Although biological therapies such as rituximab have significantly improved outcomes and selective kinase inhibitors are showing promise, the rate of new drug discovery remains disappointing, the treatments expensive and long-term benefits uncertain. An alternative strategy is redeployment of available, inexpensive and non-toxic drugs. Here, we demonstrate the antiproliferative and mitochondrial superoxide (MSO) driven pro-apoptotic activities of bezafibrate (BEZ) and medroxyprogesterone acetate (MPA) against B-NHL cells, with a bias toward MZL, in the presence and absence of the microenvironmental signal CD40L. Our study is the first to confirm the presence of CD40L within the lymph node of B-NHL and its capacity to drive B-NHL proliferation. These findings implicate BEZ + MPA as a potential therapeutic strategy in B-NHL.

Background: We and others have identified the aldo-keto reductase AKR1C3 as a potential drug target in prostate cancer, breast cancer and leukaemia. As a consequence, significant effort is being invested in the development of AKR1C3-selective inhibitors. Methods: We report the screening of an in-house drug library to identify known drugs that selectively inhibit AKR1C3 over the closely related isoforms AKR1C1, 1C2 and 1C4. This screen initially identified tetracycline as a potential AKR1C3-selective inhibitor. However, mass spectrometry and nuclear magnetic resonance studies identified that the active agent was a novel breakdown product (4-methyl(de-dimethylamine)-tetracycline (4-MDDT)). Results: We demonstrate that, although 4-MDDT enters AML cells and inhibits their AKR1C3 activity, it does not recapitulate the anti-leukaemic actions of the pan-AKR1C inhibitor medroxyprogesterone acetate (MPA). Screens of the NCI diversity set and an independently curated small-molecule library identified several additional AKR1C3-selective inhibitors, none of which had the expected anti-leukaemic activity. However, a pan AKR1C, also identified in the NCI diversity set faithfully recapitulated the actions of MPA. Conclusions: In summary, we have identified a novel tetracycline-derived product that provides an excellent lead structure with proven drug-like qualities for the development of AKR1C3 inhibitors. However, our findings suggest that, at least in leukaemia, selective inhibition of AKR1C3 is insufficient to elicit an anticancer effect and that multiple AKR1C inhibition may be required.

Chronic lymphocytic leukemia (CLL) remains associated with low complete response rates and high relapse rates. This is in part due to poor understanding of CLL biology and thus inadequate targeting of therapy. For years CLL has been proposed as bi-compartmental: the quiescent tumor in the periphery and the proliferating cells within specific microenvironments. Historically the bone marrow was considered the major tissue of the CLL microenvironment. However, many recent innovative studies have categorically shown that peripheral CLL cells are derived from the lymph nodes (LN). Proliferation here is largely driven by helper T cells via CD40-CD40L engagement. Critically, in vitro studies have shown that such engagement additionally protects LN CLLs from apoptosis. Agents inducing apoptosis in non-CD40 engaged CLL cells are frequently ineffective against those continually engaged with CD40L. This emphasizes that, in order to improve responses and prevent relapse, novel therapies must be assessed against CD40L engaged CLL cells to show effective targeting against the LN. This review discusses the evidence supporting the superior involvement of the LN in CLL, how CD40L engaged CLL studies should be conducted, and the novel therapies studied in vitro and in vivo that have been proposed to be effective in this setting.

An abundant form of DNA damage caused by reactive oxygen species is 8-oxo-7,8-dihydroguanine for which the base excision repair protein 8-oxoguanine-DNA glycosylase 1 (OGG1) is a major repair enzyme. To assess the location and intracellular activity of the OGG1 protein in response to oxidative stress, we have utilised a fluorescence-quench molecular beacon switch containing a 8-oxo-dG:C base pair and a fluorescent and quencher molecule at opposite ends of a hairpin oligonucleotide. Oxidative stress was induced by treatment with potassium bromate. Flow cytometry demonstrated a concentration-dependent increase in the activity of OGG1 that was detected by the fluorescence produced when the oligonucleotide was cleaved in the cells treated with potassium bromate. This signal is highly specific and not detectable in OGG1 knock out cells. Induction of OGG1 activity is not a result of induction of OGG1 gene expression as assessed by qPCR suggesting a role for protein stabilisation or increased OGG1 catalytic activity. High resolution confocal microscopy pinpointed the location of the fluorescent molecular beacon in live cells to perinuclear regions that were identified as mitochondria by co-staining with mitotracker dye. There is no evidence of cut beacon within the nuclear compartment of the cell. Control experiments with a positive control beacon (G:C base pair and lacking the DAB quencher) did not result in mitochondrial localisation of fluorescence signal indicating that the dye does not accumulate in mitochondria independent of OGG1 activity. Furthermore, faint nuclear staining was apparent confirming that the beacon structure is able to enter the nucleus. In conclusion, these data indicate that the mitochondria are the major site for OGG1 repair activity under conditions of oxidative stress.

The aldo-keto reductase AKR1C3, has been shown to regulate myelopoiesis via its ability to metabolise prostaglandin D2 (PGD2). Other studies have demonstrated the oxidative activation of polycyclic aromatic hydrocarbon (PAH) procarcinogens by AKR1C3 in cell-free systems. This is the first study that addresses whether AKR1C3 mediates carcinogen activation within intact living cells following manipulation of AKR1C3 by molecular intervention. Quantitative RT-PCR identified AKR1C3 as the predominant AKR1C isoform expressed in acute myeloid leukemia (AML). Exposure of K562 and KG1a myeloid cell lines to the known AKR1C3 substrate 7,12-dimethylbenz(a)anthracene-3,4-dihydrodiol (7,12-DMBA-3,4-diol) resulted in both single strand DNA breaks and oxidative DNA damage as measured using conventional and FPG-modified comet assays respectively. PGD2-keto reductase activity was shown to be correlated with relative AKR1C3 expression and together with quantitative real time PCR was used to validate the RNAi-knockdown of AKR1C3 in K562 cells. Knockdown of AKR1C3 did not alter single strand DNA breaks following 7,12-DMBA-3,4-diol exposure but significantly decreased oxidative DNA damage. A similar interrelationship between AKR1C3 activity and 7,12-DMBA-3,4-diol mediated oxidative DNA damage but not single strand breaks was observed in KG1a cells. Finally, AKR1C3 knockdown also resulted in spontaneous erythroid differentiation of K562 cells. Since K562 cells are a model of AML blast crisis of chronic myeloid leukemia (CML) the data presented here identify AKR1C3 as a novel mediator of carcinogen-induced initiation of leukemia, as a novel regulator of erythroid differentiation and paradoxically as a potential new target in the treatment of CML.

Histone deacetylase inhibitors (HDIs) are a new class of drugs with significant antileukemic activity. To explore mechanisms of disease-specific HDI activity in acute myeloid leukaemia (AML), we have characterised expression of all 18 members of the histone deacetylase family in primary AML blasts and in four control cell types, namely CD34+ progenitors from umbilical cord, either quiescent or cycling (post-culture), cycling CD34+ progenitors from GCSF-stimulated adult donors and peripheral blood mononuclear cells. Only SIRT1 was consistently overexpressed (>2 fold) in AML samples compared with all controls, while HDAC6 was overexpressed relative to adult, but not neo-natal cells. HDAC5 and SIRT4 were consistently underexpressed. AML blasts and cell lines, exposed to HDIs in culture, showed both histone hyperacetylation and, unexpectedly, specific hypermethylation of H3 lysine 4. Such treatment also modulated the pattern of HDAC expression, with strong induction of HDAC11 in all myeloid cells tested and with all inhibitors (valproate, butyrate, TSA, SAHA), and lesser, more selective, induction of HDAC9 and SIRT4. The distinct pattern of HDAC expression in AML and its response to HDIs is of relevance to the development of HDI-based therapeutic strategies and may contribute to observed patterns of clinical response and development of drug resistance.

Ingenol 3-angelate (PEP005) is a selective small molecule activator of protein kinase C (PKC) extracted from the plant Euphorbia peplus, whose sap has been used as a traditional medicine for the treatment of skin conditions including warts and cancer. We report here that PEP005 also has potent antileukemic effects, inducing apoptosis in myeloid leukemia cell lines and primary acute myeloid leukemia (AML) cells at nanomolar concentrations. Of importance, PEP005 did not induce apoptosis in normal CD34+ cord blood myeloblasts at up to 2-log concentrations higher than those required to induce cell death in primary AML cells. The effects of PEP005 were PKC dependent, and PEP005 efficacy correlated with expression of PKC-delta. The delta isoform of PKC plays a key role in apoptosis and is therefore a rational potential target for antileukemic therapies. Transfection of KG1a leukemia cells, which did not express PKC-delta or respond to PEP005, with enhanced green fluorescent protein (EGFP)-PKC-delta restored sensitivity to induction of apoptosis by PEP005. Our data therefore suggest that activation of PKC-delta provides a novel approach for treatment of acute myeloid leukemia and that screening for PKC-delta expression may identify patients for potential responsiveness to PEP005. (Blood. 2005;106:1362-1368)

Treatment of MSCV-GFP-transduced HL60 promyelocytic cells with all-trans retinoic acid (ATRA) resulted in a significant increase in GFP expression. The increased GFP expression was observed by 16 hr and was dependent on de novo protein production. This effect was specific to ATRA and unrelated to cell differentiation because it was not induced by dimethyl sulfoxide. Furthermore, a similar increase in GFP expression was observed in MSCV-GFP-transfected K562 cells, which do not differentiate when exposed to ATRA. Significantly increased GFP expression was seen at doses as low as 0.5 nM ATRA and was abrogated by AGN193109, an antagonist of retinoid signaling. We therefore conclude that this increase in gene expression is mediated by retinoic acid receptors. The long terminal repeat (LTR) region of MSCV contains candidate retinoic acid response elements and response elements for the ATRA-inducible transcription factor C/EBPalpha. We suggest that the increase in GFP expression is driven by the action of ATRA-activated host cell transcription factors. These findings offer a method to increase the expression of retroviral transgenes either in vitro or in vivo by treatment with low doses of retinoic acid that are clinically achievable and well tolerated. This use of inducible host cell transcription factors offers an alternative to engineering novel LTR regulatory sequences in order to increase transgene expression.

The redeployed drug combination of bezafibrate and medroxyprogesterone acetate (designated BaP) has potent in vivo anticancer activity in acute myelogenous leukemia (AML) and endemic Burkitt lymphoma (eBL) patients; however, its mechanism-of-action is unclear. Given that elevated fatty acid biosynthesis is a hallmark of many cancers and that these drugs can affect lipid metabolism, we hypothesized that BaP exerts anticancer effects by disrupting lipogenesis. We applied mass spectrometry–based lipidomics and gene and protein expression measurements of key lipogenic enzymes [acetyl CoA carboxylase 1 (ACC1), fatty acid synthase (FASN), and stearoyl CoA desaturase 1 (SCD1)] to AML and eBL cell lines treated with BaP. BaP treatment decreased fatty acid and phospholipid biosynthesis from 13C D-glucose. The proportion of phospholipid species with saturated and monounsaturated acyl chains was also decreased after treatment, whereas those with polyunsaturated chains increased. BaP decreased SCD1 protein levels in each cell line (0.46- to 0.62-fold; P < 0.023) and decreased FASN protein levels across all cell lines (0.87-fold decrease; P = 1.7 × 10−4). Changes to ACC1 protein levels were mostly insignificant. Supplementation with the SCD1 enzymatic product, oleate, rescued AML and e-BL cells from BaP cell killing and decreased levels of BaP-induced reactive oxygen species, whereas supplementation with the SCD1 substrate (and FASN product), palmitate, did not rescue cells. In conclusion, these data suggest that the critical anticancer actions of BaP are decreases in SCD1 levels and monounsaturated fatty acid synthesis. To our knowledge, this is the first time that clinically available antileukemic and antilymphoma drugs targeting SCD1 have been reported. Cancer Res; 75(12); 2530–40. ©2015 AACR.

Nm23-H1 plays complex roles in the development of diverse cancers including breast carcinoma, high-grade lymphomas, and acute myeloid leukemia (AML). In the case of AML and lymphomas, serum Nm23-H1 protein is elevated with the highest levels correlating with poorest prognosis. A recent study identified that this association is most likely causal in AML and that Nm23-H1 acts as an AML cell survival factor. In this study, we report heterogeneity in the ability of AML samples to bind and respond to Nm23-H1, and we offer evidence that binding is essential for improved survival. Further, we show that the subset of AMLs that bind Nm23-H1 do not do so through the putative Nm23-H1 receptor MUC1*. Although rNm23-H1 promoted the survival of the most primitive blasts within responding AMLs, it was not these cells that actually bound the protein. Instead, rNm23-H1 bound to more mature CD34lo/CD34− and CD11b+ cells, revealing an indirect survival benefit of Nm23-H1 on primitive blasts. In support of this finding, the survival of purified blast cells was enhanced by medium conditioned by more mature cells from the clone that had been stimulated by rNm23-H1. Levels of interleukin 1β (IL1β) and IL6 in rNm23-H1 conditioned medium mirrored the potency of the conditioned media to promote blast cell survival. Furthermore, Nm23-H1 expression was significantly associated with IL1β and IL6 expression in primary uncultured AML samples. These findings have implications for the role of Nm23-H1 in AML and its use as a prognostic marker. Additionally, they offer the first evidence of novel cross-talk between cell populations within the tumor clone. Cancer Res; 71(3); 1177–86. ©2010 AACR.

Background Tumor cells in chronic lymphocytic leukemia accumulate in the periphery through the proliferation of a minority of cells in lymph nodes. The proliferative and survival signals in these proliferation centers include interactions with T lymphocytes expressing CD40 ligand. We have demonstrated that the low toxicity combination of bezafibrate and medroxyprogesterone acetate induces mitochondrial superoxide-mediated apoptosis of non-CD40-liganded cells but not of cells exposed to CD40 ligand. Here, we assessed the ability of dasatinib and lycorine to restore bezafibrate- and medroxyprogesterone acetate- induced apoptosis in cells exposed to CD40 ligand. In parallel experiments we compared the ability of dasatinib to induce apoptosis of cells co-treated with fludarabine.Design and Methods Primary chronic lymphocytic leukemia and peripheral blood mononuclear cells were exposed to drug combinations for 72 hours on control and CD40 ligand-expressing fibroblast monolayers. Cells were harvested and analyzed for apoptosis and levels of mitochondrial superoxide using flow cytometry. In some experiments cells were removed from CD40 ligand at 48 hours, retreated and analyzed after a further 24 hours. The effect of CD40 ligand and drug treatments on mitochondrial superoxide levels were assessed.Results As previously described, dasatinib rendered cells sensitive to fludarabine but only when CD40 ligand was removed for the last 24 hours of culture. In contrast, lycorine restored the bezafibrate- and medroxyprogesterone acetate-induced apoptosis associated with mitochondrial superoxide even during continuous exposure to CD40 ligand. Furthermore, combined bezafibrate, medroxyprogesterone acetate and lycorine had little effect against normal peripheral blood mononuclear cells, whereas dasatinib with fludarabine induced high levels of apoptosis.Conclusions Our data indicate the potential of bezafibrate, medroxyprogesterone acetate and lycorine as novel therapy in chronic lymphocytic leukemia and have important implications for the reported potential of c-abl kinase inhibitors in this disease.

Acute myeloid leukaemia (AML) causes life-threatening deficits of functional blood cells that require management using red cell and platelet transfusion and aggressive treatment of neutropenic infections. Current cytotoxic chemotherapy further worsens the problem of reduced haemopoiesis and two-thirds of patients are too frail to tolerate intensive chemotherapy at all. Median survival amongst these patients remains at3 months emphasizing the urgent need for anti-AML therapies that do not suppress haemopoiesis. Our laboratory studies showed combined Bezafibrate and Medroxyprogesterone acetate (BaP) had activity against AML without toxicity to normal stem cells. Here we report the safety and efficacy of BaP in 20 patients (19 AML, 1 high-risk myelodysplasia) for whom intensive chemotherapy was not an option. No patient exhibited haematological toxicity from BaP. Eleven patients took BaP alone for4 weeks. One reverted from high risk myelodysplasia and remains transfusion independent after 201 weeks of therapy. Three AML patients gained major haematological improvements for 22-30 weeks; in one, marrow was available to document a partial AML response. Thus, this trial indicates that BaP therapy has potential for treatment of elderly and relapsed AML.

BackgroundThe majority of acute myeloid leukaemia (AML) patients are over sixty years of age. With current treatment regimens, survival rates amongst these, and also those younger patients who relapse, remain dismal and novel therapies are urgently required. In particular, therapies that have anti-leukaemic activity but that, unlike conventional chemotherapy, do not impair normal haemopoiesis.Principal findingsHere we demonstrate the potent anti-leukaemic activity of the combination of the lipid-regulating drug bezafibrate (BEZ) and the sex hormone medroxyprogesterone acetate (MPA) against AML cell lines and primary AML cells. The combined activity of BEZ and MPA (B/M) converged upon the increased synthesis and reduced metabolism of prostaglandin D(2) (PGD(2)) resulting in elevated levels of the downstream highly bioactive, anti-neoplastic prostaglandin 15-deoxy Delta(12,14) PGJ(2) (15d-PGJ(2)). BEZ increased PGD(2) synthesis via the generation of reactive oxygen species (ROS) and activation of the lipid peroxidation pathway. MPA directed prostaglandin synthesis towards 15d-PGJ(2) by inhibiting the PGD(2) 11beta -ketoreductase activity of the aldo-keto reductase AKR1C3, which metabolises PGD(2) to 9alpha11beta-PGF(2alpha). B/M treatment resulted in growth arrest, apoptosis and cell differentiation in both AML cell lines and primary AML cells and these actions were recapitulated by treatment with 15d-PGJ(2). Importantly, the actions of B/M had little effect on the survival of normal adult myeloid progenitors.SignificanceCollectively our data demonstrate that B/M treatment of AML cells elevated ROS and delivered the anti-neoplastic actions of 15d-PGJ(2). These observations provide the mechanistic rationale for the redeployment of B/M in elderly and relapsed AML.

Members of the aldo-keto reductase (AKR) superfamily, particularly the AKR1C subfamily, are emerging as important mediators of the pathology of cancer. Agents that inhibit these enzymes may provide novel agents for either the chemoprevention or treatment of diverse malignancies. Recently, jasmonates, a family of plant stress hormones that bear a structural resemblance to prostaglandins, have been shown to elicit anticancer activities both in vitro and in vivo. In this study, we show that jasmonic acid (JA) and methyl jasmonate (MeJ) are capable of inhibiting all four human AKR1C isoforms. Although JA is the more potent inhibitor of recombinant AKR1C proteins, including the in vitro prostaglandin F synthase activity of AKR1C3, MeJ displayed greater potency in cellular systems that was, at least in part, due to increased cellular uptake of MeJ. Moreover, using the acute myelogenous leukemia cell lines HL-60 and KG1a, we found that although both jasmonates were able to induce high levels of reactive oxygen species in a dose-dependent fashion, only MeJ was able to induce high levels of mitochondrial superoxide (MSO), possibly as an epiphenomenon of mitochondrial damage. There was a strong correlation observed between MSO formation at 24 hours and reduced cellularity at day 5. In conclusion, we have identified AKR1C isoforms as a novel target of jasmonates in cancer cells and provide further evidence of the promise of these compounds, or derivatives thereof, as adjunctive therapies in the treatment of cancer. [Cancer Res 2009;69(11):4769–75]

Summary Histone deacetylase inhibitors (HDIs) are emerging as valuable new agents in the treatment of acute myeloid leukaemia (AML). However, since response rates to these agents alone are low, we sought to identify markers associated with responsiveness. In a trial of 20 patients treated with the HDI sodium valproate (VPA) in combination with all trans retinoic acid and theophylline, three patients responded clinically with one complete remission (CR) and two partial remissions. The in vivo response of the CR patient was mirrored by high in vitro sensitivity of their blasts to VPA, indicating that similar factors determine both in vivo and in vitro sensitivity. Microarray analysis of the primary AMLs and a panel of haemato-lymphoid cell lines, with a similar range of VPA sensitivities as the primary leukaemic blasts, identified elevated FOSB-expression as a potential marker of VPA sensitivity. Quantitative polymerase chain reaction confirmed overexpression of FOSB in the CR patient blasts compared to patients failing to achieve CR, and in a subset of a larger panel of AML samples. Overexpression of FOSB in K562 myeloid cells significantly increased in vitro sensitivity to VPA. Thus, we propose that FOSB is a novel, potential marker of VPA sensitivity in AML.

B-cell chronic lymphocytic leukemia (CLL), the most common leukemia in older adults, remains largely incurable and novel treatments are urgently required. We previously reported powerful pro-apoptotic actions of bezafibrate (BEZ) and medroxyprogesterone acetate (MPA) against Burkitts lymphoma cells. Here, we demonstrate that BEZ and MPA individually, and more potently when combined (BEZ+MPA), induce apoptosis of unsorted and CD19(+ve)-selected CLL cells and abrogate the pro-proliferative activity of CD40(L). This action was tumor cell specific, as the drugs had little impact on normal donor cells. The antiproliferative actions of BEZ+MPA were associated with the generation of reactive oxygen species (ROS), and the proapoptotic actions were associated with the generation of both ROS and mitochondrial superoxide (MSO). BEZ increased prostaglandin D(2) (PGD(2)) synthesis by CLL cells, and treatment with PGD(2) and its antineoplastic derivative 15dDelta(12,14,)PGJ(2) recapitulated BEZ-induced antiproliferative and proapoptotic actions. The PGD(2) receptor antagonist, BW868C, did not block BEZ or PGD(2) activity against CLL cells. The potency of BEZ+MPA against CLL cells mirrored that of chlorambucil, and BEZ+MPA combined with chlorambucil was more potent than either treatment alone. Given the known safety profiles of BEZ and MPA, our data warrant further investigation of their potential as novel therapy for CLL.

The supramolecular iron cylinder, [Fe(2)L(3)]Cl(4) (L = C(25)H(20)N(4)), shows unprecedented DNA binding in vitro, inducing intramolecular DNA coiling and also targeting Y-shaped DNA junctions. We investigated its effects on proliferation and survival in both tumor and normal cell lines. Iron cylinder reduced mitochondrial activity of cultures with potency similar to cisplatin, inhibited the cell cycle, and increased cell death by apoptosis. Associated with this, we observed a lowering of the association of propidium iodide with cellular DNA consistent with an observed competitive displacement of PI from naked DNA by cylinders. Importantly, and in contrast to existing anticancer drugs such as cisplatin, the iron cylinder [Fe(2)L(3)](4+) was not genotoxic. In summary, the design of metal complexes such as [Fe(2)L(3)](4+) with potential anticancer properties in the absence of genotoxicity may represent a significant step toward therapeutic advancement.

We and others have demonstrated expression of the aldo-keto reductase AKR1C3 in myeloid leukemia cell lines and that inhibitors of the enzyme, including nonsteroidal anti-inflammatory drugs (NSAIDs), promote HL-60 differentiation in response to all-trans retinoic acid (ATRA) and 1alpha,25-dihydroxyvitamin D3 (D3). Here, we demonstrate that overexpression of AKR1C3 reciprocally desensitizes HL-60 cells to ATRA and D3, thus confirming the enzyme as a novel regulator of cell differentiation. AKR1C3 possesses marked 11-ketoreductase activity converting prostaglandin (PG) D2 to PGF2alpha. Supplementing HL-60 cultures with PGD2 mimicked treatment with AKR1C3-inhibitors by enhancing the differentiation of the cells in response to ATRA. However, PGD2 is chemically unstable, being converted first to PGJ2 and then stepwise to 15-deoxy-Delta(12,14)-prostaglandin J2(15Delta-PGJ2), a natural ligand for the peroxisome proliferator-activated receptor-gamma (PPARgamma). Consistent with this, PGD2 was rapidly converted to PGJ2 under normal tissue culture conditions but not in the presence of recombinant AKR1C3 when PGF2alpha was predominantly formed. In addition, PGJ2 but not PGF2alpha recapitulated the potentiation of HL-60 differentiation by PGD2 and AKR1C3 inhibitors. Furthermore, the capacity of all of these treatments to potentiate HL-60 cell differentiation was significantly reduced in the presence of the PPARgamma-antagonist GW 9662. We conclude that AKRIC3 protects HL-60 cells against ATRA and D3-induced cell differentiation by limiting the production of natural PPARgamma ligands via the diversion of PGD2 toward PGF2alpha and away from PGJ2. In addition, these observations identify AKR1C3 as plausible target for the non-cyclooxygenase-dependent antineoplastic actions of NSAIDs.

Background The COP9/signalosome (CSN) is a highly conserved eight subunit complex that, by deneddylating cullins in cullin-based E3 ubiquitin ligases, regulates protein degradation. Although studied in model human cell lines such as HeLa, very little is known about the role of the CSN in haemopoietic cells. Results Greater than 95% knockdown of the non-catalytic subunit CSN2 and the deneddylating subunit CSN5 of the CSN was achieved in the human myeloid progenitor cell line K562. CSN2 knockdown led to a reduction of both CSN5 protein and mRNA whilst CSN5 knockdown had little effect on CSN2. Both knockdowns inhibited CSN deneddylase function as demonstrated by accumulation of neddylated Cul1. Furthermore, both knockdowns resulted in the sequential loss of Skp2, Cdc4 and β-TrCP F-box proteins. These proteins were rescued by the proteasome inhibitor MG132, indicating the autocatalytic degradation of F-box proteins upon loss of CSN2 or CSN5. Interestingly, altered F-box protein gene expression was also observed in CSN2 and CSN5 knockdowns, suggesting a potential role of the CSN in regulating F-box protein transcription. Loss of either CSN subunit dramatically reduced cell growth but resulted in distinct patterns of cell death. CSN5 knockdown caused mitotic defects, G2/M arrest and apoptotic cell death. CSN2 knockdown resulted in non-apoptotic cell death associated with accumulation of both the autophagy marker LC3-II and autophagic vacuoles. Treatment of vector control K562 cells with the autophagy inhibitors 3-methyladenine and bafilomycin A1 recapitulated the growth kinetics, vacuolar morphology and LC3-II accumulation of CSN2 knockdown cells indicating that the cellular phenotype of CSN2 cells arises from autophagy inhibition. Finally, loss of CSN2 was associated with the formation of a CSN5 containing subcomplex. Conclusion We conclude that CSN2 is required for CSN integrity and the stability of individual CSN subunits, and postulate that CSN2 loss results in a phenotype distinct from that of cells lacking CSN5 possibly as a consequence of altered CSN5 activity within a resultant CSN subcomplex. Our data present the first evidence for the sequential loss of F-box proteins upon CSN manipulation and are the first to identify a potential link between CSN function and autophagy.

There is growing evidence that histone deacetylase inhibitors (HDAC-I) may have benefit in the treatment of multiple myeloma (MM). We assessed the in vitro responses of the MM cell lines H929, JJN3, KMS11 and U266 to the HDAC-Is trichostatin A (TSA), suberoylanilide hydroxamic acid (SAHA) and sodium valproate (SV). All three HDAC-Is induced dose dependent cell killing and the ranked sensitivity of the lines was remarkably constant with U266 cells being most resistant to all three inhibitors. Real time RT-PCR based analyses of the HDAC expression profiles, identified that the greater resistence of U266 cells was associated with 5–10 fold increased expression of a single HDAC from the sirtuin family of deacetylases (SIRT5). Each HDAC-I displayed different potencies against the cell lines; TSA killed in the range 0.1–1μM, SAHA in the range 1–10μM and SV at the higher doses of 1–10mM. Despite being less potent, the doses of SV are commensurate with those that can be achieved in vivo. Furthermore, we have identified synergy/cooperation between suboptimal SV concentrations and the proteasome inhibitor Velcade (Bortezomib). MTT assays using JJN3 cells, demonstrated that 1mM SV had little effect reducing survival to 89% whereas 0.6 nM velcade reduced cell survival to 42±5.5%. Killing by velcade was not significantly enhanced by the additional presence of 1μM dexamethasone (Dex)(36±3.4%). However, the combination of SV, Dex and Velcade resulted in near total cell killing (4.7±2.6% survival). Collectively, these observations suggested that SV may have value in MM therapy. However, we wished to also investigate the potency of SV against primary MM cells. We have developed an in vitro culture system in which primary MM plasma cells survive for up to 30 days. In this culture system we have observed efficient and selective killing of MM cells by 1mM SV that has been comparable or indeed greater than that observed in response to 1μM SAHA. Thus it appears that primary MM cells are more SV sensitive than MM cell lines. Together our findings indicate that SV may have value in MM therapy and in particular when combined with proteasome inhibitors. Our data also indicate that SIRT5 overexpression may be a resistance factor against such therapies.

Post-translational modifications of chromatin structure are recognised as having a potential role in the pathogenesis of acute myeloid leukaemia (AML). Histone deacetylases play a central role in determining the acetylation status of histones and are emerging as novel targets in AML therapy. Histone deacetylase inhibitors (HDIs) inhibit growth of primary AML blasts in vitro and demonstrate clinical activity in patients with relapsed AML. To date, three major classes of HDACs have been characterised which differ in their susceptibility to HDIs. However, little is known of the pattern of HDAC expression in AML and this limits the rational use of HDIs in this disease. We have analysed the pattern of class I, II and III HDAC expression in AML cell lines, primary AML blasts and CD34+ selected progenitors from cord blood and normal donors by real time quantitative PCR(RT-PCR). RT-PCR analyses demonstrated consistently increased expression in AML blasts of two HDACs compared with proliferating CD34+ve cells from cord blood (n=5) . HDAC2 (class I HDAC) was more highly expressed in 3/3 myeloid cell lines and 20/24 primary AML samples, compared to cord blood CD 34+ve cells. SIRT1 (class III HDAC) was also more highly expressed in 3/3 myeloid cell lines and 24/24 primary AMLs. In contrast, no marked differences were detected in expression of HDAC1, 3, 4, 5, 6, 7, 9, 10, 11 and SIRT 2–6. We therefore studied the impact of sodium valproate (SV), an HDI with reported activity in AML, on HDAC activity and expression. SV treatment resulted in time and dose dependent increases in histone acetylation and specific methylation at H3K4, in both AML cell lines and primary AML cells. We have shown that the mechanism of the increased methylation at H3K4 is partly a result of the preference of the methyltransferase enzyme for acetylated histones. Using quantitative RT-PCR we found that SV treatment of HL-60 cells resulted in increased expression of the gene for MLL, an enzyme known to be capable of methylating H3K4. These changes in chromatin were associated with dose dependent cell killing. Significant (p