A novel Bruton's tyrosine kinase inhibitor CC-292 in combination with the proteasome inhibitor carfilzomib impacts the bone microenvironment in a multiple myeloma model with resultant antimyeloma activity.

Bruton's tyrosine kinase (Btk) modulates B-cell development and activation and has an important role in antibody production. Interestingly, Btk may also affect human osteoclast (OC) function; however, the mechanism was unknown. Here we studied a potent and specific Btk inhibitor, CC-292, in multiple myeloma (MM). In this report, we demonstrate that, although CC-292 increased OC differentiation, it inhibited OC function via inhibition of c-Src, Pyk2 and cortactin, all involved in OC-sealing zone formation. As CC-292 did not show potent in vitro anti-MM activity, we next evaluated it in combination with the proteasome inhibitor, carfilzomib. We first studied the effect of carfilzomib on OC. Carfilzomib did not have an impact on OC-sealing zone formation but significantly inhibited OC differentiation. CC-292 combined with carfilzomib inhibited both sealing zone formation and OC differentiation, resulting in more profound inhibition of OC function than carfilzomib alone. Moreover, the combination treatment in an in vivo MM mouse model inhibited tumor burden compared with CC-292 alone; it also increased bone volume compared with carfilzomib alone. These results suggest that CC-292 combined with carfilzomib augments the inhibitory effects against OC within the bone microenvironment and has promising therapeutic potential for the treatment of MM and related bone disease.

The epoxyketone-based proteasome inhibitors carfilzomib and orally bioavailable oprozomib have anti-resorptive and bone-anabolic activity in addition to anti-myeloma effects.

Proteasome inhibitors (PIs), namely bortezomib, have become a cornerstone therapy for multiple myeloma (MM), potently reducing tumor burden and inhibiting pathologic bone destruction. In clinical trials, carfilzomib, a next generation epoxyketone-based irreversible PI, has exhibited potent anti-myeloma efficacy and decreased side effects compared with bortezomib. Carfilzomib and its orally bioavailable analog oprozomib, effectively decreased MM cell viability following continual or transient treatment mimicking in vivo pharmacokinetics. Interactions between myeloma cells and the bone marrow (BM) microenvironment augment the number and activity of bone-resorbing osteoclasts (OCs) while inhibiting bone-forming osteoblasts (OBs), resulting in increased tumor growth and osteolytic lesions. At clinically relevant concentrations, carfilzomib and oprozomib directly inhibited OC formation and bone resorption in vitro, while enhancing osteogenic differentiation and matrix mineralization. Accordingly, carfilzomib and oprozomib increased trabecular bone volume, decreased bone resorption and enhanced bone formation in non-tumor bearing mice. Finally, in mouse models of disseminated MM, the epoxyketone-based PIs decreased murine 5TGM1 and human RPMI-8226 tumor burden and prevented bone loss. These data demonstrate that, in addition to anti-myeloma properties, carfilzomib and oprozomib effectively shift the bone microenvironment from a catabolic to an anabolic state and, similar to bortezomib, may decrease skeletal complications of MM.

Impaired bortezomib binding to mutant ß5 subunit of the proteasome is the underlying basis for bortezomib resistance in leukemia cells.

Proteasome inhibition is a novel treatment for several hematological malignancies. However, resistance to the proteasome inhibitor bortezomib (BTZ, Velcade) is an emerging clinical impediment. Mutations in the ß5 subunit of the proteasome, the primary target of BTZ, have been associated with drug resistance. However, the exact mechanism by which these mutations contribute to BTZ resistance, is still largely unknown. Toward this end, we here developed BTZ-resistant multiple myeloma (8226) and acute lymphoblastic leukemia (CCRF-CEM) cell line models by exposure to stepwise increasing concentrations of BTZ. Characterization of the various BTZ-resistant cells revealed upregulation of mutant ß5 subunit of the proteasome. These newly identified ß5-subunit mutations, along with previously described mutations, formed a mutation cluster region in the BTZ-binding pocket of the ß5 subunit, that of the S1 specificity pocket in particular. Moreover, we provide the first evidence that the mechanism underlying BTZ resistance in these tumor cells is impaired binding of BTZ to the mutant ß5 subunit of the proteasome. We propose that proteasome subunit overexpression is an essential compensatory mechanism for the impaired catalytic activity of these mutant proteasomes. Our findings further suggest that second-generation proteasome inhibitors that target the α7 subunit of the proteasome can overcome this drug resistance modality.

Phytoestrogens and xenoestrogens: the contribution of diet and environment to endocrine disruption.

Some endocrine disrupting compounds such as phthalates and phenols act non-genomically by inhibiting the sulfotransferase (SULT 1E1 and SULT 1A1) isoforms which inactivate estrogens by sulfonation. A range of environmental phenolic contaminants and dietary flavonoids was tested for inhibition of the human SULT 1A1, 1E1 and 2A1 isoforms. In particular, the plasticisers 4-n-octyl- and 4-n-nonyl-phenol inhibit SULT 1E1 with IC(50) values of 0.16 microM vs. 10nM estradiol while the 2-substituted chlorophenols show similar values. Flavonoids are also SULT inhibitors; tricin is a competitive inhibitor of SULT 1E1 with a K(i) of 1.5+/-0.8 nM. In a small pilot study to determine whether ingestion of soy flavonoids would affect SULT1A1 activity in vivo as well as in vitro, sulfonation of daidzein was reduced in a group of women 'at risk' of breast cancer, as compared with controls, although the SULT 1A1*1/SULT 1A1*2 allele ratio was not different. Endocrine disrupting effects in man may be multifactorial when components from both the diet and the environment act at the same point in steroid metabolism.

Substrate characterisation of a recombinant sulfotransferase SULT1 and mRNA expression in chub (Leuciscus cephalus) tissues.

We have studied the role and regulation of sulfonation of xenobiotics and endogenous substrates in Leuciscus cephalus, an abundant and environmentally relevant freshwater fish. A sulfotransferase 1 (SULT1) cDNA and promoter region was cloned from chub liver and the cDNA expressed in Escherichia coli. The translated protein displayed 51% and 50% amino acid identity to mSULT1D1, hSULT1A1, respectively. We identified two conserved co-substrate binding motifs for 3'-phosphoadenosine 5'-phosphosulfate: RKGxxGDWKxxFT and YPKSGTxW. The recombinant SULT displayed a strong preference towards the isoflavones genistein (K(m)=1.7 microM) and daidzein (K(m)=4.4 microM) and lesser activity towards the endogenous substrate dopamine. Based on sequence identity and substrate preferences, the SULT was classified as SULT1,3. SULT1,3 mRNA expression was highest in the liver and kidney with low levels expressed in brain, gonad, and gill. Mature males displayed higher hepatic SULT1,3 mRNA expression compared to females. Analysis of the promoter region revealed several putative half palindromic estrogen response elements (ERE).

Non-genomic effects of endocrine disrupters: inhibition of estrogen sulfotransferase by phenols and chlorinated phenols.

Phenols are used world-wide and their presence in the environment is a cause of increasing concern. Despite evidence to suggest that, in general, they bind poorly to estrogen receptors, they are suspected of being endocrine disrupters. Here, we show that 2, x-substituted phenols are potent inhibitors of estrogen sulfotransferase with IC(50) values at low- or sub-micromolar levels. Our results demonstrate a potential non-genomic mechanism of action for these compounds and suggest that, where viable alternatives exist, both phenols substituted in the 2-position and their metabolic precursors should be avoided.

Phytoestrogens are potent inhibitors of estrogen sulfation: implications for breast cancer risk and treatment.

We investigated the ability of 37 flavonoids and flavonoid sulfoconjugates, including some abundant dietary constituents, to act as substrates and/or inhibitors of the sulfotransferase and sulfatase enzymes that interconvert active estrogens and inactive estrogen sulfates in human tissues. The enzymes studied include estrogen sulfotransferase, the thermostable phenolsulfotransferase that acts on a range of substrates including estrogens; steroid sulfatase; and two related enzymes, monoamine phenolsulfotransferase and arylsulfatase A. Several dietary flavonoids, including the soy isoflavones genistein and daidzein, were sulfated by these human sulfotransferases. Many flavonoids were potent inhibitors of thermostable phenolsulfotransferase. Genistein and equol were potent mixed inhibitors of hepatic estrogen sulfotransferase, with inhibitory constant values of 500 nM and 400 nM, respectively. Monoamine phenolsulfotransferase activity was relatively unaffected by flavonoids, but this enzyme was mainly responsible for the sulfation of flavonoids at concentrations greater than 1 micro M. Of the compounds tested, only daidzein 4,7-bisulfate, a trace metabolite in humans, significantly inhibited steroid sulfatase in the micromolar concentration range. Hence, dietary flavonoids may be able to influence the bioavailability of endogenous estrogens, and disrupt endocrine balance, by increasing the ratio of active estrogens to inactive estrogen sulfates in human tissues.

Immune evasion by muscle-specific gene expression in dystrophic muscle.

Muscle tissue from Duchenne muscular dystrophy patients and the Dmd(mdx/mdx) (hereafter referred to as mdx) mouse is characterized by an abundance of necrotic myofibers and infiltrating macrophages. Both features may provide additional stimulus to the immune response directed against novel antigens, such as those delivered by gene therapy vectors. It has previously been shown that the immune evasion achieved by adeno-associated virus in healthy muscle fails in one model of muscular dystrophy. Here, we examined the immune response to adenoviral vectors and their transgenes in normal and mdx mice. We found that mdx mouse muscles contain 20 times more macrophages and 7 times more dendritic cells than healthy muscles. This higher professional antigen-presenting cell content results in a stronger immune response to antigens that can be directly presented by those cells, including viral antigens and constitutively expressed transgene products. However, we did not detect a significant immune response to beta-galactosidase expressed specifically in muscle, even at high expression levels. This result suggests that cross-presentation is not more effective in mdx mouse muscle, and that targeted vectors and tissue-specific promoters may be useful tools for evasion of the immune response in dystrophic muscle.

The dynamics of the T-cell antitumor response: chemokine-secreting dendritic cells can prime tumor-reactive T cells extranodally.

Direct administration of dendritic cells (DCs) genetically modified to express secondary lymphoid tissue chemokine (SLC) into growing B16 melanoma could result in a substantial, sustained influx of T cells within the mass with only a transient increase in T-cell numbers in the draining lymph node (DLN). DCs were retained at the tumor site with only a very small percentage trafficking to the DLN. The T cells infiltrating the tumor mass expressed the activation marker CD25 within 24 h and developed IFN-gamma-secreting function within 7 days as tumor growth was inhibited. Similar results were obtained in lymphotoxin alpha-/- mice, which lacked peripheral lymph nodes. Our data demonstrate that effective T-cell priming can occur extranodally and result in measurable antitumor effects in vivo.

Do dietary phytoestrogens influence susceptibility to hormone-dependent cancer by disrupting the metabolism of endogenous oestrogens?

Phytoestrogens are natural constituents of our diets that have been suggested to protect against hormone-dependent breast cancer. Some of the diverse effects of these compounds may be attributed to ligand-dependent differences in their interaction with oestrogen receptor sub-classes. However, phytoestrogens can also inhibit enzymes that are involved in the generation and removal of endogenous steroid hormones. Among the most potent effects of dietary phytoestrogens is their ability to inhibit the sulphotransferases that sulphate both oestrogenic steroids and a variety of environmental chemicals, including dietary pro-carcinogens. Circulating steroid sulphates are thought to be the major source of oestradiol in post-menopausal breast tumours and sulphation is a key step in the activation of some dietary pro-carcinogens. Hence the inhibition of sulphotransferases by dietary phytoestrogens may have complex effects upon human susceptibility to breast cancer.

Up-regulation of steroid sulphatase activity in HL60 promyelocytic cells by retinoids and 1alpha,25-dihydroxyvitamin D3.

HL60 promyeloid cells express both classes of oestrogen receptor (ERalpha and ERbeta). We show that hydrolysis of oestrone sulphate by steroid sulphatase is a major source of oestrone in HL60 cells, and that most of the released oestrone is not metabolized further to 17beta-oestradiol. Treatment of HL60 cells with retinoids or 1alpha,25-dihydroxyvitamin D3 increased steroid sulphatase mRNA and activity in parallel with the induction of CD11b, an early marker of myeloid differentiation that is expressed before the differentiating cells stop proliferating. Use of agonists and antagonists against retinoid receptor-alpha and retinoid receptor-X revealed that both classes of retinoid receptor can drive steroid sulphatase up-regulation. Steroid sulphatase activity fluctuates during the cell cycle, being highest around the transition from G1 to S phase. During the differentiation of HL60 cells induced by all-trans-retinoic acid or 1alpha,25-dihydroxyvitamin D3, there is increased conversion of 17beta-oestradiol into oestrone by an oxidative 17beta-hydroxysteroid dehydrogenase. Treatment of Caco-2 colon adenocarcinoma cells with all-trans-retinoic acid or 1alpha,25-dihydroxyvitamin D3 also increases 17beta-oestradiol oxidation to oestrone. An increase in local oestrone production therefore occurs in multiple cell types following treatment with retinoids and 1alpha,25-dihydroxyvitamin D3. The possible involvement of locally produced oestrogenic steroids in regulating the proliferation and differentiation of myeloid cells is discussed.

T cell-dependent antitumor immunity mediated by secondary lymphoid tissue chemokine: augmentation of dendritic cell-based immunotherapy.

Secondary lymphoid tissue chemokine (SLC) is a CC chemokine that is selective in its recruitment of naive T cells and dendritic cells (DCs). In the lymph node, SLC is believed to play an important role in the initiation of an immune response by colocalizing naive T cells with DC-presenting antigen. Here, we used SLC as a treatment for tumors established from the poorly immunogenic B16 melanoma. Intratumoral injections of SLC inhibited tumor growth in a CD8+, T cell-dependent manner. SLC elicited a substantial infiltration of DCs and T cells into the tumor, coincident with the antitumor response. We next used SLC gene-modified DCs as a treatment of established tumors. Intratumoral injections of SLC-expressing DCs resulted in tumor growth inhibition that was significantly better than either control DCs or SLC alone. Distal site immunization of tumor-bearing mice with SLC gene-modified DCs pulsed with tumor lysate elicited an antitumor response whereas control DCs did not. We also found that s.c. injection of lysate-pulsed DCs expressing SLC promoted the migration of T cells to the immunization site. This report demonstrates that SLC can both induce antitumor responses and enhance the antitumor immunity elicited by DCs.

Estrogenic alkylphenols induce cell death by inhibiting testis endoplasmic reticulum Ca(2+) pumps.

Industrial alkylphenols in the environment may act as "xenoestrogens" to disrupt testicular development and decrease male fertility. Amongst possible targets for these compounds are testicular Sertoli cells, which nurture the developing sperm cells. We demonstrate that SERCA 2 and 3 Ca(2+) pumps are relatively abundant in rat testis microsomal membranes, and also in Sertoli, myoid, and TM4 cells (a Sertoli cell line). A number of estrogenic alkylphenols such as nonylphenol, octylphenol, bisphenol A, and butylated hydroxytoluene all inhibit testicular Ca(2+) ATPase in the low micromolar concentration range. These agents also mobilize intracellular Ca(2+) in intact TM4 cells in a manner consistent with the inhibition of ER Ca(2+) pumps. Alkylphenols dramatically decrease the viability of TM4 cells, an effect that is reversed by either a caspase inhibitor or by BAPTA, and is therefore consistent with Ca(2+)-dependent cell death via apoptosis. We postulate that alkylphenols disrupt testicular development by inhibiting ER Ca(2+) pumps, thus disturbing testicular Ca(2+) homeostasis.

Analysis of muscle creatine kinase regulatory elements in recombinant adenoviral vectors.

Adenoviral gene transfer holds promise for gene therapy, but effective transduction of a large and distributed tissue such as muscle will almost certainly require systemic delivery. In this context, the use of muscle-specific regulatory elements such as the muscle creatine kinase (MCK) promoter and enhancer will avoid potentially harmful ectopic expression of transgenes. We describe here the development and testing of adenoviral vectors containing small, striated muscle-specific, highly active MCK expression cassettes. One of these regulatory elements (CK6) is less than 600 bp in length and is 12% as active as the CMV promoter/enhancer in muscle. A recombinant adenoviral vector containing this regulatory element retains very high muscle specificity, expressing 600-fold higher levels of transgene in muscle than in liver. Muscle-specific regulatory elements may also increase persistence of transduced muscle cells. Adenoviral transduction of dendritic cells has been shown to stimulate cytotoxic T-lymphocyte (CTL) responses directed against transgene epitopes. We show that human dendritic cells infected in vitro with MCK-containing adenoviruses do not express significant levels of transgene. Furthermore, while adenoviral vectors containing nonspecific promoters are normally cleared from muscle tissue within 1 month, we show that MCK-containing vectors express significant levels of transgene 4 months after intramuscular injection.

Gene-modified dendritic cells for use in tumor vaccines.

Dendritic cells (DCs) are potent antigen-presenting cells capable of priming activation of naive T cells. Because of their immunostimulatory capacity, immunization with DCs presenting tumor antigens has been proposed as a treatment regimen for cancer. The results from translational research studies and early clinical trials point to the need for improvement of DC-based tumor vaccines before they become a more broadly applicable treatment modality. In this regard, studies suggest that genetic modification of DCs to express tumor antigens and/or immunomodulatory proteins may improve their capacity to promote an antitumor response. Because the DC phenotype is relatively unstable, nonperturbing methods of gene transfer must be employed that do not compromise viability or immunostimulatory capacity. DCs expressing transgenes encoding tumor antigens have been shown to be more potent primers of antitumor immunity both in vitro and in animal models of disease; in some measures of immune priming, gene-modified DCs exceeded their soluble antigen-pulsed counterparts. Cytokine gene modification of DCs has improved their capacity to prime tumor antigen-specific T cell responses and promote antitumor immunity in vivo. Here, we review the current status of gene-modified DCs in both human and murine studies. Although successful results have been obtained to date in experimental systems, we discuss potential problems that have already arisen and may yet be encountered before gene-modified DCs are more widely applicable for use in human clinical trials.

rhGGF2 protects against cisplatin-induced neuropathy in the rat.

In many patients treated with cisplatin a peripheral sensory neuropathy develops. This side-effect is considered dose-limiting, and therefore restricts the total dose of cisplatin that can be administered. Recent in vitro and in vivo studies suggest that recombinant human Glial Growth Factor 2 (rhGGF2) has neuroprotective effects. This prompted us to investigate in a rat model whether rhGGF2 ameliorates cisplatin neuropathy. A total of 48 rats were randomly divided into four groups of 12 rats each. Three groups received cisplatin and were treated with either 0.1 mg/kg rhGGF2, 0.3 mg/kg rhGGF2 or placebo. The fourth group (saline/placebo) served as age-matched controls. In the cisplatin/placebo treated rats a neuropathy developed, as determined by measurements of the nerve conduction velocity (NCV). Treatment with rhGGF2 dose-dependently protected against the neuropathy. Histological examination and morphometric analysis revealed that rhGGF2 also protects against cisplatin-induced changes in the morphology and size of DRG satellite cell nuclei. In a control study rhGGF2 did not affect normal NCV development. We conclude that rhGGF2 treatment is of benefit in the treatment of cisplatin neuropathy in the rat.

Sulfation of "estrogenic" alkylphenols and 17beta-estradiol by human platelet phenol sulfotransferases.

We have investigated the ability of alkylphenols to act as substrates and/or inhibitors of phenol sulfotransferase enzymes in human platelet cytosolic fractions. Our results indicate: (i) straight chain alkylphenols do not interact with the monoamine-sulfating phenol sulfotransferase (SULT1A3); (ii) short chain 4-n-alkylphenols (C < 8) are substrates for the phenol-sulfating enzymes (SULT1A1/2), which exhibit two activity maxima against substrates with alkyl chain lengths of C1-2 and C4-5; (iii) long chain 4-n-substituted alkylphenols (C >/= 8) are poor substrates and act as inhibitors of SULT1A1/2; (iv) human platelets contain two activities, of low and high affinity, capable of sulfating 17beta-estradiol, and 4-n-nonylphenol is a partial mixed inhibitor of the low affinity form of this activity. We conclude that by acting either as substrates or inhibitors of SULT1A1/2, alkylphenols may influence the sulfation, and hence the excretion, of estrogens and other phenol sulfotransferase substrates in humans.

Raf-1 protein kinase activity in T cells from aged mice.

Most of our models of signal transduction through the T-cell receptor (TCR) involve components and pathways first described in T-cell clones and T-cell lymphomas such as the Jurkat cell line (1). These studies, while providing valuable insights, are not always reliable guides to the analogous biochemical events in cells freshly isolated from live donors (2). Thus, studies of the effects of aging on T-cell activation must frequently begin with a detailed study of the responses of cells from young individuals. Studies of aging effects involve additional challenges, including the difficulty of purifying sufficient numbers of cells from specific subsets, and allowing for the inherent variability among donors of any age.

Age-related decline in activation of JNK by TCR- and CD28-mediated signals in murine T-lymphocytes.

c-Jun N-terminal kinase (JNK) is activated when T-lymphocytes are stimulated jointly through the T-cell receptor (TCR) and CD28, and it contributes to T-cell activation and IL-2 production through phosphorylation of transcription factors, including c-Jun. We performed in vitro kinase assays on JNK in CD4(+) T-cells, from young and old mice, activated by antibodies to CD3, CD4, and CD28, and found a approximately 2-fold decline in JNK activity at the peak of activation, but no significant change in the kinetics of stimulation or in the steady-state expression of JNK. We found a similar decline in c-Jun phosphorylation in stimulated CD4(+) T-cells from old mice, suggesting that JNK activation also declined with age in intact cells. Aging does not, however, alter the level of Ras activation by anti-CD3/CD4 +/- anti-CD28 or change the level of Ras protein in CD4(+) cells, suggesting that the JNK defect is due to changes in the regulation of other upstream regulators. Our results suggest that a decline with age in JNK responses may contribute to the decline in proliferation and IL-2 production seen in CD4(+) T-cells from old mice.

Age-sensitive and -insensitive pathways leading to JNK activation in mouse CD4(+) T-cells.

The c-Jun N-terminal kinase (JNK) can be activated in T-cells either by the combination of TCR and CD28 costimulation or by a variety of stress-related stimuli including UV light, H(2)O(2), and hyperosmolar sorbitol solutions. In T-lymphocytes, TCR/CD28 stimulation of JNK leads to induction of new gene expression via c-Jun, ATF-2, and Elk-1. Phosphorylation of c-Jun in CD4(+) T-cells stimulated by CD3/CD4/CD28 cross-linking declines with age, due to diminished activation of JNK. Here we show that the age-related decline in TCR/CD28 activation of JNK reflects two effects of age: the accumulation of memory cells (in which JNK stimulation is poor regardless of donor age) and age-dependent declines in JNK activation within the naive subset. Cyclosporin A inhibits induction of JNK function by TCR/CD28, PMA/ionomycin, ceramide, or H(2)O(2), but not induction by UV light or hyperosmolar sorbitol. Although aging impairs JNK induction by UV light, it has no effect on JNK activation by ceramide, H(2)O(2), or sorbitol. The data as a whole indicate that there are at least four pathways that activate JNK in CD4(+) T-cells, of which two are age-sensitive and two others unaffected by aging. Two of the pathways (UV and hyperosmolar sorbitol) are insensitive to cyclosporin inhibition. Finally, we show that the alterations in JNK function are not due to changes in the expression of MKK4, an upstream activator of JNK, and that another JNK kinase, MKK7, is not expressed in splenic T-cells.