Genetic Engineering in Combination with Semi-Synthesis Leads to a New Route for Gram-Scale Production of the Immunosuppressive Natural Product Brasilicardin†A.

Brasilicardin A (1) consists of an unusual anti/syn/anti-perhydrophenanthrene skeleton with a carbohydrate side chain and an amino acid moiety. It exhibits potent immunosuppressive activity, yet its mode of action differs from standard drugs that are currently in use. Further pre-clinical evaluation of this promising, biologically active natural product is hampered by restricted access to the ready material, as its synthesis requires both a low-yielding fermentation process using a pathogenic organism and an elaborate, multi-step total synthesis. Our semi-synthetic approach included a) the heterologous expression of the brasilicardin A gene cluster in different non-pathogenic bacterial strains producing brasilicardin A aglycone (5) in excellent yield and b) the chemical transformation of the aglycone 5 into the trifluoroacetic acid salt of brasilicardin A (1 a) via a short and straightforward five-steps synthetic route. Additionally, we report the first preclinical data for brasilicardin A.

A novel LysR-type regulator negatively affects biosynthesis of the immunosuppressant brasilicardin.

Brasilicardin A (BraA) is a promising immunosuppressive compound produced naturally by the pathogenic bacterium Nocardia terpenica IFM 0406. Heterologous host expression of brasilicardin gene cluster showed to be efficient to bypass the safety issues, low production levels and lack of genetic tools related with the use of native producer. Further improvement of production yields requires better understanding of gene expression regulation within the BraA biosynthetic gene cluster (Bra-BGC); however, the only so far known regulator of this gene cluster is Bra12. In this study, we discovered the protein LysRNt, a novel member of the LysR-type transcriptional regulator family, as a regulator of the Bra-BGC. Using in vitro approaches, we identified the gene promoters which are controlled by LysRNt within the Bra-BGC. Corresponding genes encode enzymes involved in BraA biosynthesis as well as the key Bra-BGC regulator Bra12. Importantly, we provide in vivo evidence that LysRNt negatively affects production of brasilicardin congeners in the heterologous host Amycolatopsis japonicum. Finally, we demonstrate that some of the pathway related metabolites, and their chemical analogs, can interact with LysRNt which in turn affects its DNA-binding activity.

An efficient system for stable markerless integration of large biosynthetic gene clusters into Streptomyces chromosomes.

The bacteria of the genus Streptomyces are among the most important producers of biologically active secondary metabolites. Moreover, recent genomic sequence data have shown their enormous genetic potential for new natural products, although many new biosynthetic gene clusters (BGCs) are silent. Therefore, efficient and stable genome modification techniques are needed to activate their production or to manipulate their biosynthesis towards increased production or improved properties. We have recently developed an efficient markerless genome modification system for streptomycetes based on positive blue/white selection of double crossovers using the bpsA gene from indigoidine biosynthesis, which has been successfully applied for markerless deletions of genes and BGCs. In the present study, we optimized this system for markerless insertion of large BGCs. In a pilot test experiment, we successfully inserted a part of the landomycin BGC (lanFABCDL) under the control of the ermEp* promoter in place of the actinorhodin BGC (act) of Streptomyces lividans TK24 and RedStrep 1.3. The resulting strains correctly produced UWM6 and rabelomycin in twice the yield compared to S. lividans strains with the same construct inserted using the PhiBT1 phage-based integration vector system. Moreover, the system was more stable. Subsequently, using the same strategy, we effectively inserted the entire BGC for mithramycin (MTM) in place of the calcium-dependent antibiotic BGC (cda) of S. lividans RedStrep 1.3 without antibiotic-resistant markers. The resulting strain produced similar levels of MTM when compared to the previously described S. lividans RedStrep 1.3 strain with the VWB phage-based integration plasmid pMTMF. The system was also more stable. KEY POINTS: • Optimized genome editing system for markerless insertion of BGCs into Streptomyces genomes • Efficient heterologous production of MTM in the stable engineered S. lividans strain.

Mithramycin A and Mithralog EC-8042 Inhibit SETDB1 Expression and Its Oncogenic Activity in Malignant Melanoma.

Malignant melanoma is the most deadly skin cancer, associated with rising incidence and mortality rates. Most of the patients with melanoma, treated with current targeted therapies, develop a drug resistance, causing tumor relapse. The attainment of a better understanding of novel cancer-promoting molecular mechanisms driving melanoma progression is essential for the development of more effective targeted therapeutic approaches. Recent studies, including the research previously conducted in our laboratory, reported that the histone methyltransferase SETDB1 contributes to melanoma pathogenesis. In this follow-up study, we further elucidated the role of SETDB1 in melanoma, showing that SETDB1 modulated relevant transcriptomic effects in melanoma, in particular, as activator of cancer-related secreted (CRS) factors and as repressor of melanocyte-lineage differentiation (MLD) and metabolic enzymes. Next, we investigated the effects of SETDB1 inhibition via compounds belonging to the mithramycin family, mithramycin A and mithramycin analog (mithralog) EC-8042: melanoma cells showed strong sensitivity to these drugs, which effectively suppressed the expression of SETDB1 and induced changes at the transcriptomic, morphological, and functional level. Moreover, SETDB1 inhibitors enhanced the efficacy of mitogen-activated protein kinase (MAPK) inhibitor-based therapies against melanoma. Taken together, this work highlights the key regulatory role of SETDB1 in melanoma and supports the development of SETDB1-targeting therapeutic strategies for the treatment of melanoma patients.

The Mithralog EC-7072 Induces Chronic Lymphocytic Leukemia Cell Death by Targeting Tonic B-Cell Receptor Signaling.

B-cell receptor (BCR)-dependent signaling is central for leukemia B-cell homeostasis, as underscored by the promising clinical results obtained in patients with chronic lymphocytic leukemia (CLL) treated with novel agents targeting components of this pathway. Herein, we demonstrate that the mithralog EC-7072 displays high ex vivo cytotoxic activity against leukemia cells from CLL patients independently from high-risk prognostic markers and IGHV mutational status. EC-7072 was significantly less toxic against T cells and NK cells and did not alter the production of the immune effector molecules IFN-γ and perforin. EC-7072 directly triggered caspase-3-dependent CLL cell apoptosis, which was not abrogated by microenvironment-derived factors that sustain leukemia cell survival. RNA-sequencing analyses revealed a dramatic EC-7072-driven reprograming of the transcriptome of CLL cells, including a wide downregulation of multiple components and targets of the BCR signaling pathway. Accordingly, we found decreased levels of phosphorylated signaling nodes downstream of the BCR. Crosslinking-mediated BCR activation antagonized CLL cell death triggered by EC-7072, increased the phosphorylation levels of the abovementioned signaling nodes and upregulated BCL2 expression, suggesting that the mithralog disrupts CLL cell viability by targeting the BCR signaling axis at multiple levels. EC-7072 exerted similar or higher antileukemic activity than that of several available CLL therapies and displayed additive or synergistic interaction with these drugs in killing CLL cells. Overall, our findings provide rationale for future investigation to test whether EC-7072 may be a potential therapeutic option for patients with CLL and other B-cell malignancies.

An efficient blue-white screening system for markerless deletions and stable integrations in Streptomyces chromosomes based on the blue pigment indigoidine biosynthetic gene bpsA.

We previously developed an efficient deletion system for streptomycetes based on the positive selection of double-crossover events using bpsA, a gene for producing the blue pigment indigoidine. Using this system, we removed interfering secondary metabolite clusters from Streptomyces lividans TK24, resulting in RedStrep strains with dramatically increased heterologous production of mithramycin A (up to 3-g/l culture). This system, however, required a time-consuming step to remove the resistance marker genes. In order to simplify markerless deletions, we prepared a new system based on the plasmid pAMR18A. This plasmid contains a large polylinker with many unique restriction sites flanked by apramycin and kanamycin resistance genes and the bpsA gene for selecting a double-crossover event. The utility of this new markerless deletion system was demonstrated by its deletion of a 21-kb actinorhodin gene cluster from Streptomyces lividans TK24 with 30% efficiency. We used this system to efficiently remove the matA and matB genes in selected RedStrep strains, resulting in biotechnologically improved strains with a highly dispersed growth phenotype involving non-pelleting small and open mycelia. No further increase in mithramycin A production was observed in these new RedStrep strains, however. We also used this system for the markerless insertion of a heterologous mCherry gene, an improved variant of the monomeric red fluorescent protein, under the control of the strong secretory signal sequence of the subtilisin inhibitor protein, into the chromosome of S. lividans TK24. The resulting recombinant strains efficiently secreted mCherry into the growth medium in a yield of 30 mg/l.

Inhibition of FLT3 and PIM Kinases by EC-70124 Exerts Potent Activity in Preclinical Models of Acute Myeloid Leukemia.

Internal tandem duplication (ITD) or tyrosine kinase domain mutations of FLT3 is the most frequent genetic alteration in acute myelogenous leukemia (AML) and are associated with poor disease outcome. Despite considerable efforts to develop single-target FLT3 drugs, so far, the most promising clinical response has been achieved using the multikinase inhibitor midostaurin. Here, we explore the activity of the indolocarbazole EC-70124, from the same chemical space as midostaurin, in preclinical models of AML, focusing on those bearing FLT3-ITD mutations. EC-70124 potently inhibits wild-type and mutant FLT3, and also other important kinases such as PIM kinases. EC-70124 inhibits proliferation of AML cell lines, inducing cell-cycle arrest and apoptosis. EC-70124 is orally bioavailable and displays higher metabolic stability and lower human protein plasma binding compared with midostaurin. Both in vitro and in vivo pharmacodynamic analyses demonstrate inhibition of FLT3-STAT5, Akt-mTOR-S6, and PIM-BAD pathways. Oral administration of EC-70124 in FLT3-ITD xenograft models demonstrates high efficacy, reaching complete tumor regression. Ex vivo, EC-70124 impaired cell viability in leukemic blasts, especially from FLT3-ITD patients. Our results demonstrate the ability of EC-70124 to reduce proliferation and induce cell death in AML cell lines, patient-derived leukemic blast and xenograft animal models, reaching best results in FLT3 mutants that carry other molecular pathways' alterations. Thus, its unique inhibition profile warrants EC-70124 as a promising agent for AML treatment based on its ability to interfere the complex oncogenic events activated in AML at several levels. Mol Cancer Ther; 17(3); 614-24. ©2018 AACR.

Correction to: Increased heterologous production of the antitumoral polyketide mithramycin a by engineered Streptomyces lividans TK24 strains.

The original publication contains error error in the Materials and Methods section and in the acknowledgement section.

Increased heterologous production of the antitumoral polyketide mithramycin A by engineered Streptomyces lividans TK24 strains.

Mithramycin A is an antitumor compound used for treatment of several types of cancer including chronic and acute myeloid leukemia, testicular carcinoma, hypercalcemia and Paget's disease. Selective modifications of this molecule by combinatorial biosynthesis and biocatalysis opened the possibility to produce mithramycin analogues with improved properties that are currently under preclinical development. The mithramycin A biosynthetic gene cluster from Streptomyces argillaceus ATCC12956 was cloned by transformation assisted recombination in Saccharomyces cerevisiae and heterologous expression in Streptomyces lividans TK24 was evaluated. Mithramycin A was efficiently produced by S. lividans TK24 under standard fermentation conditions. To improve the yield of heterologously produced mithramycin A, a collection of derivative strains of S. lividans TK24 were constructed by sequential deletion of known potentially interfering secondary metabolite gene clusters using a protocol based on the positive selection of double crossover events with blue pigment indigoidine-producing gene. Mithramycin A production was evaluated in these S. lividans strains and substantially improved mithramycin A production was observed depending on the deleted gene clusters. A collection of S. lividans strains suitable for heterologous expression of actinomycetes secondary metabolites were generated and efficient production of mithramycin A with yields close to 3 g/L, under the tested fermentation conditions was achieved using these optimized collection of strains.

Estrongiloidiasis en inmunosuprimidos

Strongyloides stercoralis es el único helminto con reproducción intracorpórea, en inmunosuprimidos acelera la producción larvaria y propicia desenlaces fatales. Objetivo: Comparar el comportamiento clínico y de laboratorio de la infección por S. stercoralis en inmunosuprimidos con y sin VIH. Métodos: Estudio restrospectivo (1984 a 2005). Se evaluaron 328 pacientes inmunosuprimidos con estrongiloidiasis, VIH+ (n=99) y VIH- con neoplasias, tratamiento esteroideo o desnutridos (n=229). Se realizó hematología con fórmula leucocitaria, cuantificación de inmunoglobulinas séricas y exámenes de heces seriados con métodos directo, Kato, Kinyoun y Baermann; se incluyó cultivo en agar modificado a partir del 2000. Resultados: Los hombres prevalecieron en ambos grupos, VIH+ 88 % y VIH- 63 %. La edad promedio fue 44 años en VIH- y 37 años en VIH+. Recuento normal de leucocitos se demostró en 50 % de los pacientes de ambos grupos; cifras menores de 5 000 leucocitos predominaron en VIH+ (34 %, 20 %) y por encima de 10 000 en VIH- (31 %, 14 %). Menos de 500 eosinófilos/mm3 se detectaron en 60 % de los VIH+ y 25 % de los VIH-. La diarrea, común en ambos grupos, afectó más a VIH+ (86%) que a VIH- (62 %), tendencia a la consistencia líquida (VIH+ 90 %, VIH- 77 %), cronicidad (VIH+ 76%, VIH- 65 %) y pérdida de peso (VIH+ 72 %, VIH- 48 %). Conclusiones: La infección por S. stercoralis ocasiona gastroenteropatías más severas en pacientes VIH+ que en otros inmunosuprimidos, contribuyendo al desgaste orgánico. La eosinofilia es un indicador confiable de infección pero su ausencia no la excluye. En inmunosuprimidos, su despistaje debe ser rutinario.

Strongyloides stercoralis is the only intestinal helminth with intracorporeal reproduction, in immunosuppressed patients accelerates larval production and promotes fatal outcomes. Objective: To identify the symptoms and lab hematological values during S. stercoralis infection in immunosuppressed patients with or without HIV. Methods: retrospective study (1984-2005). We evaluated 328 immunosuppressed patients with strongyloidiasis, infected with HIV (n=99) and not infected with HIV, with neoplasms, under steroid treatment and undernourished (n=229). Hematology exam with leukocyte formula, quantification of serum immunoglobulins and stool tests with the direct methods, Kato, Kinyoun and Baermann were performed. Modified agar culture was included since the year 2000. Results: Male sex prevailed in both groups, 88 % (HIV+) and 63 % (VIH-). The average age was 44 years in VIH- and 37 years in HIV+. Normal leukocyte count was demonstrated in 50 % of patients in both groups; figures below 5 000 leukocytes predominated in HIV+ (34 %, HIV- 20 %) and above 10 000 in VIH- (31 %, HIV+ 14 %). Less than 500 eosinophils were detected in 60 % of HIV+ and 25 % of VIH-. Diarrhea, common in both groups, affected HIV+ more than VIH- (HIV+ 86%, VIH- 62 %), with a tendency to liquid consistency (HIV+ 90 %, VIH- 77 %), chronicity (HIV+ 76%, VIH- 65 %) and greater weight loss (HIV+ 72 %, VIH- 48 %). Conclusions: The infection by S. stercoralis causes more severe gastroenteropathies in HIV+ patients than in other immunosuppressed patients, contributing to a greater weight loss. Eosinophilia is a reliable indicator of infection but its absence does not exclude it. In immunosuppressed patients, screening should be routine.

Coinfection with Hymenolepis nana, Hymenolepis diminuta, Giardia intestinalis, and Human Immunodeficiency Virus: A Case Report with Complex Immunologic Interactions.

AbstractWe describe the case of a 43-year-old human immunodeficiency virus-infected man receiving combined antiretroviral therapy and coinfected with Hymenolepis nana, Hymenolepis diminuta, and Giardia intestinalis, presenting as chronic diarrhea and critical weight loss. Immunological aspects of these interactions are reviewed.

Improved Detection of Strongyloides stercoralis in Modified Agar Plate Cultures.

AbstractA modification of Koga agar plate culture was performed, consisting of a 2 × 2-cm cellophane paper centered on the agar plate to prevent bacterial contamination of the agar and daily dish examinations (days 2-5). Between January 2000 and July 2005, we examined 1,708 infection-suspected patients, of which 147 (8.6%) harbored S. stercoralis. Single modified agar plate cultures exhibited superior sensitivity (93.2%), compared with different three-sample screening methods (sensitivity-Baermann: 76.6%, formalin-ethyl acetate: 22%, and direct smear: 15.3%). Agar plate cultures stand out as helpful alternatives for improved detection and therapy monitoring in poor countries and endemic areas. Combined with Baermann methods, they provide increased probability for S. stercoralis detection.

High-Quality Draft Genome Sequence of the Actinobacterium Nocardia terpenica IFM 0406, Producer of the Immunosuppressant Brasilicardins, Using Illumina and PacBio Technologies.

The bacterium Nocardia terpenica IFM 0406 is known as the producer of the immunosuppressant brasilicardin A. Here, we report the completely sequenced genome of strain IFM 0406, which facilitates the heterologous expression of the brasilicardin biosynthetic gene cluster but also unveils the intriguing biosynthetic capacity of the strain to produce secondary metabolites.

Producción de anticuerpos IgA contra componentes proteicos del huevo de Ascaris lumbricoides en el suero de niños infectados / Production of IgA antobodies against protein components of eggs from Ascaris lumbricoides in serum of infected children

La ascariasis es una enfermedad causada por el nematodo A. lumbricoides afectando a 1 billón de personas en todo el mundo. En este trabajo se analiza la respuesta de anticuerpos IgA específicos frente a extractos de huevos de A. lumbricoides en el suero de niños de comunidades rurales del estado Miranda. Se evaluaron 100 niños entre 3 y 14 años de edad y se tomaron muestras de suero y el diagnóstico coproparasitológico. Según lo reportado fueron clasificados en: infectados solo por A. lumbricoides (Asc+), no infectados (NSOP), infectados con A. lumbricoides y otros parásitos (Asc+/OP+) e individuos no infectados por A. lumbricoides pero con otros parásitos intestinales (Asc-/OP+). El extracto fue probado por ELISA, Western Blot e inmunocitoquímica. El perfil electroforético del extracto mostró 7 bandas proteicas de 102, 92, 85, 65, 50, 45 y 30kDa. Por la ELISA, el 26% de los pacientes infectados por A. lumbricoides fueron seropositivos, mientras que el 56% en el grupo NSOP fueron seronegativos. El ensayo de inhibición con extracto de A. lumbricoides permitió identificar 3 bandas proteicas especificas reconocidas por la inmunoglobulina IgA de 92, 85 y 65 kDa diferentes a las reconocidas por el extracto de verme adulto. El nivel de anticuerpos fue significativamente mayor en los infectados que en los no infectados. La superficie de los huevos fue reconocida por la IgA en el suero de los individuos infectados. Estos resultados, indican que el huevo de Ascaris induce anticuerpos específicos contra proteínas que podrían ser utilizados como antígenos en pruebas serológicas que complementan el diagnóstico de Ascariasis.

Ascariasis is a disease caused by the nematode A. lumbricoides affecting 1 billion people worldwide. In this study the specific response for IgA antibodies against extracts of A. lumbricoides eggs in the serum of children in rural areas of Miranda state. One hundred children were evaluated at ages 3 and 14 and were taken serum samples and diagnosis of stools. According to the reported, were classified into four groups: infected by A. lumbricoides (Asc +), uninfected (NSOP), infected A. lumbricoides and other parasite (Asc +/OP +); and individuals with other parasitic infections without A. lumbricoides (ASC/ OP+). The extract was tested by ELISA, Western blotting and immunocytochemestry. The electrophoretic profile showed 7 bands of 102, 92, 85, 65, 50, 45 and 30kDa. By ELISA, the 26% of patients in group A. lumbricoides were positive, while the 56% in the NSOP group were seronegative. Immune assay by inhibition with A. lumbricoides extract allowed identify three bands for the IgA antibodies of 92, 85 and 65 kDa different to the bands recognized by adult extracts of A. lumbricoiedes. The levels of antibodies were significantly higher in infected than non-infected children. In addition, the protein component on the surface of eggs was recognized by sera of infected individuals. These results indicate that eggs of Ascaris induce specific antibodies against proteins that could be used as antigens in serological diagnosis for complementing the diagnosis for Ascariasis.

Larvas rabditoides de Strongyloides stercoralis en orina en paciente con riñón trasplantado y estrongiloidiasis diseminada / Urinary rhabditiform larvae of Strongyloides stercoralis in disseminated disease affecting a kidney-transplanted patient

Strongyloides stercoralis, an intestinal nematode prevalent in tropical and subtropical zones, remains clinically silent or mildly symptomatic in immunecompetent individuals. In contrast, clinical dissemination and/or hyperinfection may induce severe widespread damage and fatal outcomes in immunecompromised hosts. This report describes a 53-year-old male renal transplant recipient, in whom standard immunosuppressive therapy did not prevent development of acute nephritis also coinciding with appearance of larvae in fecal smears. During a 3-days sequential copro-parasitological testing S. stercoralis larvae were identified in Baermann and agar culture tests simultaneously with rhabditiform larvae of S. stercoralis in his urinary sediment. Significant improvement of renal dysfunction with ivermectin therapy highlights the importance of incorporating S. stercoralis screening in pre-transplant protocols and post-transplant complications.

Strongyloides stercoralis, nemátode intestinal de alta prevalencia en zonas tropicales y subtropicales, se presenta clínicamente de forma asintomática u oligosintomática en personas inmunocompetentes. No obstante, la diseminación o hiperinfección por este parásito, puede producir severas complicaciones, a veces fatales en pacientes inmunosuprimidos. Este reporte describe a un paciente masculino de 53 años, trasplantado renal, cuyas complicaciones coinciden con la detección de larvas en las heces. La terapia inmunosupresora usual no impidió el desarrollo de nefritis aguda en este paciente. Los métodos de Baermann y cultivo en agar resultaron positivos en tres muestras consecutivas. Adicionalmente, en el sedimento urinario, se encontraron larvas rabditoides. Posterior al tratamiento con ivermectina la función renal evidenció significativa mejoría. El caso presentado enfatiza la necesidad de investigar la infección por S. stercoralis en los protocolos pre-trasplante en donantes y receptores, y en las complicaciones post-trasplante asociadas con eosinofilia, especialmente cuando proceden del trópico.

Inhibition of SP1 by the mithramycin analog EC-8042 efficiently targets tumor initiating cells in sarcoma.

Tumor initiating cells (TICs), responsible for tumor initiation, and cancer stem cells (CSCs), responsible for tumor expansion and propagation, are often resistant to chemotherapeutic agents. To find therapeutic targets against sarcoma initiating and propagating cells we used models of myxoid liposarcoma (MLS) and undifferentiated pleomorphic sarcoma (UPS) developed from human mesenchymal stromal/stem cells (hMSCs), which constitute the most likely cell-of-origin for sarcoma. We found that SP1-mediated transcription was among the most significantly altered signaling. To inhibit SP1 activity, we used EC-8042, a mithramycin (MTM) analog (mithralog) with enhanced anti-tumor activity and highly improved safety. EC-8042 inhibited the growth of TIC cultures, induced cell cycle arrest and apoptosis and upregulated the adipogenic factor CEBPα. SP1 knockdown was able to mimic the anti-proliferative effects induced by EC-8042. Importantly, EC-8042 was not recognized as a substrate by several ABC efflux pumps involved in drug resistance, and, opposite to the chemotherapeutic drug doxorubicin, repressed the expression of many genes responsible for the TIC/CSC phenotype, including SOX2, C-MYC, NOTCH1 and NFκB1. Accordingly, EC-8042, but not doxorubicin, efficiently reduced the survival of CSC-enriched tumorsphere sarcoma cultures. In vivo, EC-8042 induced a profound inhibition of tumor growth associated to a strong reduction of the mitotic index and the induction of adipogenic differentiation and senescence. Finally, EC-8042 reduced the ability of tumor cells to reinitiate tumor growth. These data suggest that EC-8042 could constitute an effective treatment against both TIC and CSC subpopulations in sarcoma.

Identification of Mithramycin Analogues with Improved Targeting of the EWS-FLI1 Transcription Factor.

PURPOSE: The goal of this study was to identify second-generation mithramycin analogues that better target the EWS-FLI1 transcription factor for Ewing sarcoma. We previously established mithramycin as an EWS-FLI1 inhibitor, but the compound's toxicity prevented its use at effective concentrations in patients. EXPERIMENTAL DESIGN: We screened a panel of mithralogs to establish their ability to inhibit EWS-FLI1 in Ewing sarcoma. We compared the IC50 with the MTD established in mice to determine the relationship between efficacy and toxicity. We confirmed the suppression of EWS-FLI1 at the promoter, mRNA, gene signature, and protein levels. We established an improved therapeutic window by using time-lapse microscopy to model the effects on cellular proliferation in Ewing sarcoma cells relative to HepG2 control cells. Finally, we established an improved therapeutic window using a xenograft model of Ewing sarcoma. RESULTS: EC-8105 was found to be the most potent analogue and was able to suppress EWS-FLI1 activity at concentrations nontoxic to other cell types. EC-8042 was substantially less toxic than mithramycin in multiple species but maintained suppression of EWS-FLI1 at similar concentrations. Both compounds markedly suppressed Ewing sarcoma xenograft growth and inhibited EWS-FLI1 in vivo CONCLUSIONS: These results provide a basis for the continued development of EC-8042 and EC-8105 as EWS-FLI1 inhibitors for the clinic. Clin Cancer Res; 22(16); 4105-18. ©2016 AACR.

EC-70124, a Novel Glycosylated Indolocarbazole Multikinase Inhibitor, Reverts Tumorigenic and Stem Cell Properties in Prostate Cancer by Inhibiting STAT3 and NF-κB.

Cancer stem cells (CSC) contribute to disease progression and treatment failure in prostate cancer because of their intrinsic resistance to current therapies. The transcription factors NF-κB and STAT3 are frequently activated in advanced prostate cancer and sustain expansion of prostate CSCs. EC-70124 is a novel chimeric indolocarbazole compound generated by metabolic engineering of the biosynthetic pathways of glycosylated indolocarbazoles, such as staurosporine and rebeccamycin. In vitro kinome analyses revealed that EC-70124 acted as a multikinase inhibitor with potent activity against IKKß and JAK2. In this study, we show that EC-70124 blocked concomitantly NF-κB and STAT3 in prostate cancer cells and particularly prostate CSCs, which exhibited overactivation of these transcription factors. Phosphorylation of IkB and STAT3 (Tyr705), the immediate targets of IKKß and JAK2, respectively, was rapidly inhibited in vitro by EC-70124 at concentrations that were well below plasma levels in mice. Furthermore, the drug blocked activation of NF-κB and STAT3 reporters and suppressed transcription of their target genes. Treatment with EC-70124 impaired proliferation and colony formation in vitro and delayed development of prostate tumor xenografts. Notably, EC-70124 had profound effects on the prostate CSC subpopulation both in vitro and in vivo Thus, EC-70124 is a potent inhibitor of the NF-κB and STAT3 signaling pathways and blocked tumor growth and maintenance of prostate CSCs. EC-70124 may provide the basis for developing new therapeutic strategies that combine agents directed to the CSC component and the bulk tumor cell population for treatment of advanced prostate cancer. Mol Cancer Ther; 15(5); 806-18. ©2016 AACR.

Pleiotropic Anti-Angiogenic and Anti-Oncogenic Activities of the Novel Mithralog Demycarosyl-3D-ß-D-Digitoxosyl-Mithramycin SK (EC-8042).

Demycarosyl-3D-ß-D-digitoxosyl-mithramycin SK (DIG-MSK) is a recently isolated analogue of mithramycin A (MTA) that showed differences with MTA in the DNA binding strength and selectivity. These differences correlated with a better therapeutic index and less toxicity in animal studies. Herein, we show that DIG-MSK displays a potent anti-tumor activity against different types of cancer cell lines, ovarian tumor cells being particularly sensitive to this drug. Of relevance, DIG-MSK exerts low toxicity on fibroblasts and peripheral blood mononuclear cells, this toxicity being significantly lower than that of MTA. In correlation with its antitumor activity, DIG-MSK strongly inhibited Sp1-mediated transcription and endogenous Sp1 mRNA expression, which correlated with the inhibition of the expression of key Sp1-regulated genes involved in tumorigenesis, including VEGFA, BCL2L1 (Bcl-XL), hTERT, BRCA2, MYC and SRC in several ovarian cells. Significantly, DIG-MSK was a stronger inhibitor of VEGFA expression than MTA. Accordingly, DIG-MSK also exhibited potent anti-angiogenic activity on microvascular endothelial cells. Likewise, it significantly inhibited the gene expression of VEGFR1, VEGFR2, FGFR, PDGFB and PDGFRA and, additionally, it induced the expression of the anti-angiogenic factors angiostatin and tunstatin. These effects correlated with a pro-apoptotic effect on proliferating microvascular endothelial cells and the inhibition of the formation of endothelial capillary structures. Overall, the pleiotropic activity of DIG-MSK in inhibiting key oncogenic and angiogenic pathways, together with its low toxicity profile, highlight the therapeutic potential of this new drug.

Antitumoral activity of the mithralog EC-8042 in triple negative breast cancer linked to cell cycle arrest in G2.

Triple negative breast cancer (TNBC) is an aggressive form of breast cancer. Despite response to chemotherapy, relapses are frequent and resistance to available treatments is often observed in the metastatic setting. Therefore, identification of new therapeutic strategies is required. Here we have investigated the effect of the mithramycin analog EC-8042 (demycarosil-3D-ß-D-digitoxosyl mithramycin SK) on TNBC. The drug caused a dose-dependent inhibition of proliferation of a set of TNBC cell lines in vitro, and decreased tumor growth in mice xenografted with TNBC cells. Mechanistically, EC-8042 caused an arrest in the G2 phase of the cell cycle, coincident with an increase in pCDK1 and Wee1 levels in cells treated with the drug. In addition, prolonged treatment with the drug also causes apoptosis, mainly through caspase-independent routes. Importantly, EC-8042 synergized with drugs commonly used in the therapy of TNBC in vitro, and potentiated the antitumoral effect of docetaxel in vivo. Together, these data suggest that the mithralog EC-8042 exerts an antitumoral action on TNBC cells and reinforces the action of standard of care drugs used in the therapy of this disease. These characteristics, together with a better toxicology profile of EC-8042 with respect to mithramycin, open the possibility of its clinical evaluation.