Pharmacodynamic and pharmacogenomic study of the nanoparticle conjugate of camptothecin CRLX101 for the treatment of cancer.

CRLX101 is a nanopharmaceutical consisting of cyclodextrin-based polymer molecule and camptothecin. The CRLX101 nanoparticle is designed to concentrate and slowly release camptothecin in tumors over an extended period of time. Tumor biopsy and blood samples collected from patients with advanced solid malignancies before and after CRLX101 treatment are subjected to immunohistochemistry and pharmacogenomics. The expression of Topoisomerase-1, Ki-67, CaIX, CD31 and VEGF decreased after CRLX101 treatment. The expressions of these proteins are inversely proportional with survival duration of the patients. The Drug Metabolism Enzymes and Transporters (DMET) array shows an allele frequency in patients similar to global populations with none of the SNPs associated with toxicity. The results suggest that the observed lower toxicity is not likely to be due to different genotypes in SNPs. CRLX101 demonstrates a promising anti-tumor activity in heavily pre-treated or treatment-refractory solid tumor malignancies presumably by inhibition of proliferation and angiogenesis correlating with tumor growth inhibition. From the clinical editor: In this cancer treatment study clinical samples collected from patients were subjected to immunohistochemistry and pharmacogenomics. The expressions of key proteins that are inversely proportional with survival duration of the patients decreased after treatment with CRLX101, a camptothecin slow-release nanoparticle conjugate. This anti-tumor activity in heavily pre-treated and treatment resistant solid tumors, promises a novel therapeutic approach.

Combining histone deacetylase inhibitor vorinostat with aurora kinase inhibitors enhances lymphoma cell killing with repression of c-Myc, hTERT, and microRNA levels.

MK-0457 and MK-5108 are novel aurora kinase inhibitors (AKi) leading to G(2)-M cell-cycle arrest. Growth and survival of multiple lymphoma cell lines were studied with either drug alone or in combination with vorinostat, a histone deacetylase inhibitor (HDACi), using MTS and Annexin V assays, followed by molecular studies. Either of the AKi alone at 100 to 500 nmol/L resulted in approximately 50% reduced cell growth and 10% to 40% apoptosis. Addition of vorinostat reactivated proapoptotic genes and enhanced lymphoma cell death. Quantitative PCR and immunoblotting revealed that epigenetic and protein acetylation mechanisms were responsible for this activity. The prosurvival genes Bcl-X(L) and hTERT were downregulated 5-fold by combination drug treatment, whereas the proapoptotic BAD and BID genes were upregulated 3-fold. The p53 tumor suppressor was stabilized by an increased acetylation in response to vorinostat and a reduced Ser315 phosphorylation in response to aurora kinase A. Vorinostat or trichostatin A decreased MYC mRNA and protein as well as c-Myc-regulated microRNAs. MYC is a critical gene in these responses, as MYC knockdown combined with the expression of the c-Myc antagonist MXD1 raised cell sensitivity to the effects of either AKi. Thus, the HDACi vorinostat leads to both transcriptional and posttranscriptional changes to create a proapoptotic milieu, sensitizing cells to mitosis-specific agents such as AKis.

Human non-CG methylation: are human stem cells plant-like?

Non-CG methylation is well characterized in plants, where it appears to play a role in gene silencing and genomic imprinting. Although strong evidence for the presence of non-CG methylation in animals has been available for some time, both its origin and function remain elusive. In this review we discuss available evidence on non-CG methylation in animals in light of evidence suggesting that the human stem cell methylome contains significant levels of methylation outside the CG site.

Preclinical results of camptothecin-polymer conjugate (IT-101) in multiple human lymphoma xenograft models.

PURPOSE: Camptothecin (CPT) has potent broad-spectrum antitumor activity by inhibiting type I DNA topoisomerase (DNA topo I). It has not been used clinically because it is water-insoluble and highly toxic. As a result, irinotecan (CPT-11), a water-soluble analogue of CPT, has been developed and used as salvage chemotherapy in patients with relapsed/refractory lymphoma, but with only modest activity. Recently, we have developed a cyclodextrin-based polymer conjugate of 20-(S)-CPT (IT-101). In this study, we evaluated the preclinical antilymphoma efficacy of IT-101 as compared with CPT-11. EXPERIMENTAL DESIGN: We determined an in vitro cytotoxicity of IT-101, CPT-11, and their metabolites against multiple human lymphoma cell lines. In human lymphoma xenografts, the pharmacokinetics, inhibitions of tumor DNA topo I catalytic activity, and antilymphoma activities of these compounds were evaluated. RESULTS: IT-101 and CPT had very high in vitro cytotoxicity against all lymphoma cell lines tested. As compared with CPT-11 and SN-38, IT-101 and CPT had longer release kinetics and significantly inhibit higher tumor DNA topo I catalytic activities. Furthermore, IT-101 showed significantly prolonged the survival of animals bearing s.c. and disseminated human xenografts when compared with CPT-11 at its maximum tolerated dose in mice. CONCLUSIONS: The promising present results provide the basis for a phase I clinical trial in patients with relapsed/refractory lymphoma.

The novel histone deacetylase inhibitor, LBH589, induces expression of DNA damage response genes and apoptosis in Ph- acute lymphoblastic leukemia cells.

We investigated the mechanism of action of LBH589, a novel broad-spectrum HDAC inhibitor belonging to the hydroxamate class, in Philadelphia chromosome-negative (Ph(-)) acute lymphoblastic leukemia (ALL). Two model human Ph(-) ALL cell lines (T-cell MOLT-4 and pre-B-cell Reh) were treated with LBH589 and evaluated for biologic and gene expression responses. Low nanomolar concentrations (IC(50): 5-20 nM) of LBH589 induced cell-cycle arrest, apoptosis, and histone (H3K9 and H4K8) hyperacetylation. LBH589 treatment increased mRNA levels of proapoptosis, growth arrest, and DNA damage repair genes including FANCG, FOXO3A, GADD45A, GADD45B, and GADD45G. The most dramatically expressed gene (up to 45-fold induction) observed after treatment with LBH589 is GADD45G. LBH589 treatment was associated with increased histone acetylation at the GADD45G promoter and phosphorylation of histone H2A.X. Furthermore, treatment with LBH589 was active against cultured primary Ph(-) ALL cells, including those from a relapsed patient, inducing loss of cell viability (up to 70%) and induction of GADD45G mRNA expression (up to 35-fold). Thus, LBH589 possesses potent growth inhibitory activity against including Ph(-) ALL cells associated with up-regulation of genes critical for DNA damage response and growth arrest. These findings provide a rationale for exploring the clinical activity of LBH589 in the treatment of patients with Ph(-) ALL.

Transgene-induced CCWGG methylation does not alter CG methylation patterning in human kidney cells.

Several reports suggest that C(m)CWGG methylation tends not to co-exist with (m)CG methylation in human cells. We have asked whether or not methylation at CCWGG sites can influence CG methylation. DNA from cells expressing an M.EcoRII-GFP fusion was actively methylated at CCWGG sites. CG methylation as measured by R.HpaII/R.MspI ratios was unchanged in cells expressing the transgene. Cloned representatives of C(m)CWGG methylated DNA often contained, or were adjacent to an ALU repeat, suggesting that M.EcoRII-GFP actively methylated gene-rich R-band DNA. The transgenic methyltransferase applied C(m)CWGG methylation to a representative human promoter that was heavily methylated at CG dinucleotides (the SERPINB5 promoter) and to a representative promoter that was essentially unmethylated at CG dinucleotides (the APC promoter). In each case, the CG methylation pattern remained in its original state, unchanged by the presence of neighboring C(m)CWGG sites. Q-PCR measurements showed that RNA expression from the APC gene was not significantly altered by the presence of C(m)CWGG in its promoter. Kinetic studies suggested that an adjacent C(m)CWGG methylation site influences neither the maintenance nor the de novo methylation activities of purified human Dnmt1. We conclude that C(m)CWGG methylation does not exert a significant effect on CG methylation in human kidney cells.

Prostate cancer molecular markers GSTP1 and hTERT in expressed prostatic secretions as predictors of biopsy results.

OBJECTIVES: To develop noninvasive diagnostic tools for the early detection of prostate cancer (PCa). Current screening for PCa lacks sensitivity and specificity. Two molecular markers, telomerase activity and aberrant methylation of the glutathione S-transferase P1 (GSTP1) promoter, are found in more than 90% of PCa specimens. Additionally, these markers can be detected in bodily fluids such as urine and postprostatic massage urethral washes. METHODS: Expressed prostatic secretions (EPS) from men being evaluated for PCa were analyzed for human telomerase reverse transcriptase (hTERT) expression (the critical factor for telomerase activity) and GSTP1 methylation status. The results were compared with the prostate needle biopsy findings. RESULTS: EPS could be obtained from 86% of all subjects, and 90% of these samples yielded sufficient RNA and/or DNA for assaying. hTERT expression from EPS (n = 49) had 36% sensitivity and 66% specificity, and GSTP1 methylation from EPS (n = 58) had 46% sensitivity and 56% specificity for the detection of PCa. The combined analysis (n = 32) of hTERT and GSTP1 had 73% sensitivity and 43% specificity, giving a positive predictive value of 40% and a negative predictive value of 75%. CONCLUSIONS: These results demonstrate that EPS can be successfully obtained and easily tested for hTERT expression and GSTP1 methylation. Tests with a high negative predictive value, such as our combination assay results, could be useful in augmenting current PCa diagnostic procedures. For example, the examination of EPS for hTERT and GSTP1 methylation in patients with an elevated prostate-specific antigen level might be used in predicting which patients will have negative biopsies. The use of this assay could potentially eliminate up to 30% of costly and invasive needle biopsies.

Autorepression of c-myc requires both initiator and E2F-binding site elements and cooperation with the p107 gene product.

Myc proteins are transcriptional activators, but also repress transcription through initiator (Inr) elements. Repression requires the conserved Myc Box II, but the cis-acting element(s) required for c-myc autorepression have eluded definition. Since the gene has a candidate Inr at the P2 promoter, we tested whether Myc autorepression operates through the Inr/BoxII mechanism. Overexpression of c-Myc but not a Box II deletion mutation represses both c-myc P2 reporter genes and endogenous c-myc, as does Mxi1 expression. Only 45 nucleotides surrounding the P2 start suffice to mediate autorepression, but Myc and Mxi1 also downregulate P2 Inr mutations, suggesting other core promoter sequence requirements for autorepression. We tested the importance of conserved E2F sites, based on known Myc interaction with the pRb-related p107 and on the transrepressive effects of Rb family proteins. Myc, Mxi1, and p107 repress c-myc somewhat less well in the absence of E2F binding sites, while an E2F+Inr double mutation is not repressed at all by these gene products. Further, Myc repression at the c-myc P2 core promoter is augmented by p107, but not by pRb or p130, nor by p107 lacking the conserved pocket domain. Our data suggest that Myc autorepression requires both the c-myc Inr and E2F sites in cis, as well as p107 in trans. Consistent with this, we found that retrovirally transduced c-Myc cannot downregulate endogenous c-myc in p107-null fibroblasts, and show evidence that both Myc and p107 are present in a complex assembled at the c-myc P2 core promoter.

The growth-inhibitory Ndrg1 gene is a Myc negative target in human neuroblastomas and other cell types with overexpressed N- or c-myc.

A major prognostic marker for neuroblastoma (Nb) is N-myc gene amplification, which predicts a poor clinical outcome. We sought genes differentially expressed on a consistent basis between multiple human Nb cell lines bearing normal versus amplified N-myc, in hopes of finding target genes that might clarify how N-myc overexpression translates into poor clinical prognosis. Using differential display, we find the previously described growth-inhibitory gene Ndrg1 is strongly repressed in all tested Nb cell lines bearing N-myc amplification, as well as in a neuroepithelioma line with amplified c-myc. Overexpression of N-myc in non-amplified Nb cells leads to repression of Ndrg1, as does activation of an inducible c-myc transgene in fibroblasts. Conversely, N-myc downregulation in N-myc-amplified Nb cells results in re-expression of the Ndrg1, and stimuli known to induce Ndrg1 do so in Nb cells while simultaneously down-regulating N-myc. Relevant to these results, we demonstrate an in vitro interaction of Myc protein with the Ndrg1 core promoter. We also find that Ndrg1 levels increase dramatically during in vitro differentiation of two cell lines modeling neural and glial development, while c- and N-myc levels decline. Our results combined with previous information on the Ndrg1 gene product suggest that downregulation of this gene is an important component of N-Myc effects in neuroblastomas with poor clinical outcome. In support of this notion, we find that re-expression of Ndrg1 in high-Myc Nb cells results in smaller cells with reduced colony size in soft-agar assays, further underscoring the functional significance of this gene in human neuroblastoma cells.