Publisher Correction: Non-invasive prenatal sequencing for multiple Mendelian monogenic disorders using circulating cell-free fetal DNA.

In the version of this article originally published, some cases that were presented in Fig. 3 should have been underlined but were not. The appropriate cases have now been underlined. The error has been corrected in the print, PDF and HTML versions of the article.

Non-invasive prenatal sequencing for multiple Mendelian monogenic disorders using circulating cell-free fetal DNA.

Current non-invasive prenatal screening is targeted toward the detection of chromosomal abnormalities in the fetus1,2. However, screening for many dominant monogenic disorders associated with de novo mutations is not available, despite their relatively high incidence3. Here we report on the development and validation of, and early clinical experience with, a new approach for non-invasive prenatal sequencing for a panel of causative genes for frequent dominant monogenic diseases. Cell-free DNA (cfDNA) extracted from maternal plasma was barcoded, enriched, and then analyzed by next-generation sequencing (NGS) for targeted regions. Low-level fetal variants were identified by a statistical analysis adjusted for NGS read count and fetal fraction. Pathogenic or likely pathogenic variants were confirmed by a secondary amplicon-based test on cfDNA. Clinical tests were performed on 422 pregnancies with or without abnormal ultrasound findings or family history. Follow-up studies on cases with available outcome results confirmed 20 true-positive, 127 true-negative, zero false-positive, and zero-false negative results. The initial clinical study demonstrated that this non-invasive test can provide valuable molecular information for the detection of a wide spectrum of dominant monogenic diseases, complementing current screening for aneuploidies or carrier screening for recessive disorders.

MLF1 is a proapoptotic antagonist of HOP complex-mediated survival.

In the HAX1/HtrA2-OMI/PARL (HOP) mitochondrial protein complex, anti-apoptotic signals are generated by cleavage and activation of the serine protease HtrA2/OMI by the rhomboid protease PARL upon recruitment of both proteases to inner mitochondrial membrane protein HAX1 (HS1-associated protein X-1). Here we report the negative regulation of the HOP complex by human leukemia-associated myeloid leukemia factor 1 (MLF1). We demonstrate that MLF1 physically and functionally associates with HAX1 and HtrA2. Increased interaction of MLF1 with HAX1 and HtrA2 displaces HtrA2 from the HOP complex and inhibits HtrA2 cleavage and activation, resulting in the apoptotic cell death. Conversely, over-expressed HAX1 neutralizes MLF1's effect and inhibits MLF1-induced apoptosis. Importantly, Mlf1 deletion reverses B- and T-cell lymphopenia and significantly ameliorates the progressive striatal and cerebellar neurodegeneration observed in Hax1-/- mice, with a doubling of the lifespan of Mlf1-/-/Hax1-/- animals compared to Hax1-/- animals. Collectively, these data indicate that MLF1 serves as a proapoptotic antagonist that interacts with the HOP mitochondrial complex to modulate cell survival.

Design, synthesis and initial biological evaluation of a novel pladienolide analog scaffold.

A novel and simplified synthetic scaffold based on pladienolide was designed using a consensus pharmacophore hypothesis. An initial target was synthesized and evaluated to examine the role of the 3-hydroxy group and the methyl groups present at positions 10, 16, 20, 22 in 1, on biological activity. We report the first totally synthetic analog of this macrolide that shows biological activity. Our novel synthetic strategy enables the rapid synthesis of other new analogs of pladienolide in order to develop selective anticancer lead compounds.

Synthetic mRNA splicing modulator compounds with in vivo antitumor activity.

We report our progress on the development of new synthetic anticancer lead compounds that modulate the splicing of mRNA. We also report the synthesis and evaluation of new biologically active ester and carbamate analogues. Further, we describe initial animal studies demonstrating the antitumor efficacy of compound 5 in vivo. Additionally, we report the enantioselective and diastereospecific synthesis of a new 1,3-dioxane series of active analogues. We confirm that compound 5 inhibits the splicing of mRNA in cell-free nuclear extracts and in a cell-based dual-reporter mRNA splicing assay. In summary, we have developed totally synthetic novel spliceosome modulators as therapeutic lead compounds for a number of highly aggressive cancers. Future efforts will be directed toward the more complete optimization of these compounds as potential human therapeutics.

Synthesis, characterization, and anti-melanoma activity of tetra-O-substituted analogs of nordihydroguaiaretic acid.

Synthesis of seven semi-synthetic analogs of NDGA is described. An approach to NDGA derivatization is described in which the ortho-phenolic groups are tethered together by one atom, forming a 5-membered heterocyclic ring. The analogs were evaluated for cytotoxicity in four cancer cell lines and compared to NDGA and tetra-O-methyl-NDGA (M4N) (1a). NDGA bis-cyclic sulfate (2a), NDGA bis-cyclic carbonate (2b), and methylenedioxyphenyl-NDGA (2d) and NDGA tetra acetate (1b) showed anti-cancer activity in vitro. Two compounds, (1b) and (2b), were evaluated for anticancer activity in a mouse xenograft model of human melanoma and showed dose-dependent activity.

Antitumor compounds based on a natural product consensus pharmacophore.

We report the design and highly enantioselective synthesis of a potent analogue of the spliceosome inhibitor FR901464, based on a non-natural product scaffold. The design of this compound was facilitated by a pharmacophore hypothesis that assumed key interaction types that are common to FR901464 and an otherwise unrelated natural product (pladienolide). The synthesis allows for the preparation of numerous novel analogues. We present results on the in vitro activity for this compound against several tumor cell lines.

Proteasomal inhibition stabilizes topoisomerase IIalpha protein and reverses resistance to the topoisomerase II poison ethonafide (AMP-53, 6-ethoxyazonafide).

Multiple myeloma (MM) is an incurable malignancy of plasma cells. Although multiple myeloma patients often respond to initial therapy, the majority of patients will relapse with disease that is refractory to further drug treatment. Thus, new therapeutic strategies are needed. One common mechanism of acquired drug resistance involves a reduction in the expression or function of the drug target. We hypothesized that the cytotoxic activity of topoisomerase II (topo II) poisons could be enhanced, and drug resistance overcome, by increasing the expression and activity of the drug target, topo II in myeloma cells. To test this hypothesis, we evaluated the cytotoxicity of the anthracene-containing topo II poison, ethonafide (AMP-53/6-ethoxyazonafide), in combination with the proteasome inhibitor bortezomib (PS-341/Velcade). Combination drug activity studies were done in 8226/S myeloma cells and its drug resistant subclone, 8226/Dox1V. We found that a 24-h treatment of cells with bortezomib maximally increased topo IIalpha protein expression and activity, and consistently increased the cytotoxicity of ethonafide in the 8226/S and 8226/Dox1V cell lines. This increase in cytotoxicity corresponded to an increase in DNA double-strand breaks, as measured by the neutral comet assay. Therefore, increasing topo IIalpha expression through inhibition of proteasomal degradation increased DNA double-strand breaks and enhanced the cytotoxicity of the topo II poison ethonafide. These data suggest that bortezomib-mediated stabilization of topo IIalpha expression may potentiate the cytotoxic activity of topo II poisons and thereby, provide a strategy to circumvent drug resistance.

Formation of pseudosymmetrical G-quadruplex and i-motif structures in the proximal promoter region of the RET oncogene.

A polypurine (guanine)/polypyrimidine (cytosine)-rich sequence within the proximal promoter region of the human RET oncogene has been shown to be essential for RET basal transcription. Specifically, the G-rich strand within this region consists of five consecutive runs of guanines, which is consistent with the general motif capable of forming intramolecular G-quadruplexes. Here we demonstrate that, in the presence of 100 mM K+, this G-rich strand has the ability to adopt two intramolecular G-quadruplex structures in vitro. Moreover, comparative circular dichroism (CD) and DMS footprinting studies have revealed that the 3'-G-quadruplex structure is a parallel-type intramolecular structure containing three G-tetrads. The G-quadruplex-interactive agents TMPyP4 and telomestatin further stabilize this G-quadruplex structure. In addition, we demonstrate that the complementary C-rich strand forms an i-motif structure in vitro, as shown by CD spectroscopy and chemical footprinting. This 19-mer duplex sequence is predicted to form stable intramolecular G-quadruplex and i-motif species having minimum symmetrical loop sizes of 1:3:1 and 2:3:2, respectively. Together, our results indicate that stable G-quadruplex and i-motif structures can form within the proximal promoter region of the human RET oncogene, suggesting that these secondary structures play an important role in transcriptional regulation of this gene.

Ethonafide-induced cytotoxicity is mediated by topoisomerase II inhibition in prostate cancer cells.

Ethonafide is an anthracene-containing derivative of amonafide that belongs to the azonafide series of anticancer agents. The lack of cross-resistance in multidrug-resistant cancer cell lines and the absence of a quinone and hydroquinone moiety make ethonafide a potentially less cardiotoxic replacement for existing anthracene-containing anticancer agents. For this study, we investigated the anticancer activity and mechanism of ethonafide in human prostate cancer cell lines. Ethonafide was cytotoxic against three human prostate cancer cell lines at nanomolar concentrations. Ethonafide was found to be better tolerated and more effective at inhibiting tumor growth compared with mitoxantrone in a human xenograft tumor regression mouse model. Mechanistically, we found that ethonafide inhibited topoisomerase II activity by stabilizing the enzyme-DNA complex, involving both topoisomerase IIalpha and -beta. In addition, ethonafide induced a potent G(2) cell cycle arrest in the DU 145 human prostate cancer cell line. By creating stable cell lines with decreased expression of topoisomerase IIalpha or -beta, we found that a decrease in topoisomerase IIalpha protein expression renders the cell line resistant to ethonafide. The decrease in sensitivity to ethonafide was associated with a decrease in DNA damage and an increase in DNA repair as measured by the neutral comet assay. These data demonstrate that ethonafide is a topoisomerase II poison and that it is topoisomerase IIalpha-specific in the DU 145 human prostate cancer cell line.

Cytotoxic activity of Apomine is due to a novel membrane-mediated cytolytic mechanism independent of apoptosis in the A375 human melanoma cell line.

Apomine, a novel bisphosphonate ester, has demonstrated anticancer activity in a variety of cancer cell lines; however, its mechanism of cytotoxicity is not well understood. Previous work has demonstrated that Apomine induces cell death by activation of caspase-3 in several cancer cell types. However, we have demonstrated that Apomine induces cell death in the A375 human melanoma cell line through a novel membrane-mediated mechanism that is independent of caspase-3 activation. This mechanism of membrane lysis may apply to other bisphosphonates and may be an important mechanism for overcoming resistance to apoptosis. Interestingly, Apomine-mediated cell death in the A375 and UACC 3093 human melanoma cell lines is also independent of N-Ras farnesylation, which was a previously described mechanism of action for Apomine in other cancer cell types. These data suggest that Apomine induces cell death through a novel plasma membrane-mediated cytolytic pathway, independent of caspase-3 activation and N-Ras farnesylation.

Preclinical antitumor activity, pharmacokinetics and pharmacodynamics of imexon in mice.

Imexon, a novel pro-oxidant, thiol-binding agent, is currently in phase I/II clinical trials in patients with advanced solid tumors. The aim of this study was to characterize the preclinical pharmacology of imexon in vivo. We investigated the anticancer activity of imexon in several cancer cell lines grown as xenografts in severe combined immunodeficient mice. Imexon was active against both hematologic and solid tumor types. The maximally tolerated dose, at the selected dosing schedule, was 150 mg/kg. Using the maximally tolerated dose of imexon, we sought to identify a potential pharmacodynamic biomarker to monitor the mechanistic effect systemically. As imexon binds cellular thiols in vitro, thiol depletion by imexon in vivo was evaluated as a potential biomarker. Following a single 150 mg/kg dose of imexon by intraperitoneal injection, glutathione levels decreased by 40% at 3 h in mouse erythrocytes. In mouse plasma, imexon treatment led to a significant decrease in cystine levels 2-4 h after drug administration. Notably, by this time, free imexon plasma levels were nondetectable. By investigating the pharmacokinetics of imexon, we also found that imexon undergoes rapid clearance from plasma in a dose-independent fashion with a half-life of 12-15 min. In summary, imexon is active against several cancer types in vivo. Imexon also decreases circulating thiols and exhibits dose-independent pharmacokinetics in mice. Plasma cystine levels may represent a biomarker of imexon activity in vivo.