Combined inhibition of Aurora-A and ATR kinase results in regression of MYCN-amplified neuroblastoma

Amplification of MYCN is the driving oncogene in a subset of high-risk neuroblastoma. The MYCN protein and the Aurora-A kinase form a complex during S phase that stabilizes MYCN. Here we show that MYCN activates Aurora-A on chromatin, which phosphorylates histone H3 at serine 10 in S phase, promotes the deposition of histone H3.3 and suppresses R-loop formation. Inhibition of Aurora-A induces transcription-replication conflicts and activates the Ataxia telangiectasia and Rad3 related (ATR) kinase, which limits double-strand break accumulation upon Aurora-A inhibition. Combined inhibition of Aurora-A and ATR induces rampant tumor-specific apoptosis and tumor regression in mouse models of neuroblastoma, leading to permanent eradication in a subset of mice. The therapeutic efficacy is due to both tumor cell-intrinsic and immune cell-mediated mechanisms. We propose that targeting the ability of Aurora-A to resolve transcription-replication conflicts is an effective therapy for MYCN-driven neuroblastoma (141 words)

Combined inhibition of Aurora-A and ATR kinases results in regression of MYCN-amplified neuroblastoma

Amplification of MYCN is the driving oncogenic change in a subset of high-risk neuroblastomas. The MYCN protein and the Aurora-A kinase form a complex during the S phase that stabilizes MYCN. Here we show that MYCN activates Aurora-A on chromatin, which phosphorylates histone H3 at serine 10 in the S phase, promotes the deposition of histone H3.3 and suppresses R-loop formation. Inhibition of Aurora-A induces transcription–replication conflicts and activates ataxia telangiectasia and Rad3-related (ATR) kinase, which limits double-strand break accumulation upon Aurora-A inhibition. Combined inhibition of Aurora-A and ATR kinases induces rampant tumor-specific apoptosis and tumor regression in mouse models of neuroblastoma, leading to permanent eradication in a subset of mice. The therapeutic efficacy is due to both tumor cell-intrinsic and immune cell-mediated mechanisms. We propose that targeting the ability of Aurora-A to resolve transcription–replication conflicts is an effective therapy for MYCN-driven neuroblastoma

Pseudonocardia hispaniensis sp. nov., a novel actinomycete isolated from industrial wastewater activated sludge

A novel actinomycete, designated PA3T, was isolated from an oil refinery wastewater treatment plant, located in Palos de la frontera, Huelva, Spain, and characterized taxonomically by using a polyphasic approach. Phylogenetic analysis based on 16S rRNA gene sequences showed that the isolate formed a distinct subclade in the Pseudonocardia tree together with Pseudonocardia asaccharolytica DSM 44247T. The chemotaxonomic properties of the isolate, for example, the presence of MK-8 (H4) as the predominant menaquinone and iso-C16:0 as the major fatty acid are consistent with its classification in the genus Pseudonocardia. DNA:DNA pairing experiments between the isolate and the type strain of P. asaccharolytica DSM 44247T showed that they belonged to separate genomic species. The two strains were readily distinguished using a combination of phenotypic properties. Consequently, it is proposed that isolate PA3T represents a novel species for which the name Pseudonocardia hispaniensis sp. nov. is proposed. The type strain is PA3T (= CCM 8391T = CECT 8030T).Cuesta Amat, G.; Soler Hernández, A.; Alonso Molina, JL.; Ruvira, M.; Lucena, T.; Arahal, D.; Goodfellow, M. (2013). Pseudonocardia hispaniensis sp. nov., a novel actinomycete isolated from industrial wastewater activated sludge. Antonie van Leeuwenhoek. 103(1):135-142. doi:10.1007/s10482-012-9792-1S1351421031Alonso JL, Cuesta G, Ramírez GW, Morenilla JJ, Bernácer I, Lloret RM (2009) Manual de técnicas avanzadas para la identificación y control de bacterias filamentosas. Epsar-Generalitat Valenciana, España, p 21–36Ara I, Tsetseg B, Daram D, Suto M, Ando K (2011) Pseudonocardia mongoliensis sp. nov. and Pseudonocardia khuvsgulensis sp. nov., isolated from soil. Int J Syst Evol Microbiol 61:747–756Arahal DR, Sánchez E, Macián MC, Garay E (2008) Value of recN sequences for species identification and as a phylogenetic marker within the family ‘‘Leuconostocaceae’’. Int Microbiol 11:33–39Auffret M, Labbé D, Thouand G, Greer CW, Fayolle-Guichard F (2009) Degradation of a mixture of hydrocarbons, gasoline, and diesel oil additives by Rhodococcus aetherivorans and Rhodococcus wratislaviensis. Appl Environ Microbiol 75:7774–7782Cashion P, Hodler-Franklin MA, McCully J, Franklin M (1977) A rapid method for base ratio determination of bacterial DNA. Anal Biochem 81:461–466Chen HH, Qin S, Li J, Zhang YQ, Xu LH, Jiang CL, Kim CJ, Li WJ (2009) Pseudonocardia endophytica sp. nov., isolated from pharmaceutical plant Lobelia clavata. Int J Syst Evol Microbiol 59:559–563De Ley J, Cattoir H, Reynaerts A (1970) The quantitative measurement of DNA hybridization from renaturation rates. Eur J Biochem 12:133–142Duangmal K, Thamchaipenet A, Matsumoto A, Takahashi Y (2009) Pseudonocardia acaciae sp. nov., isolated from roots of Acacia auriculiformis A. Cunn. ex Benth. Int J Syst Evol Microbiol 59:1487–1491Gordon RE, Barnett DA, Handerhan JE, Pang CH-N (1974) Nocardia coeliaca, Nocardia autotrophica, and the nocardin strain. Int J Syst Bacteriol 24:54–63Hamid ME, Minnikin DE, Goodfellow M, Ridell M (1993) Thin-layer chromatographic analysis of glycolipids and mycolic acids from Mycobacterium farcinogenes, Mycobacterium senegalense and related taxa. Zbl Bakt 279:354–367Hasegawa T, Takizawa M, Tanida S (1983) A rapid analysis for chemical grouping of aerobic actinomycetes. J Gen Microbiol 29:319–322Henssen A (1957) Beiträge zur Morphologie und Systematik der thermophilen Actinomyceten. Arch Mikrobiol 26:373–414Huang,Y, Goodfellow M (2012) Genus Pseudonocardia Hennsen 1957, 408VP emend. In: Goodfellow M, Kämpfer P, Busse H-J, Trujillo M, Suzuki KE, Ludwig W, Whitman WB (eds) Bergey’s manual of systematic bacteriology, 2nd edn, vol 5, part B. Springer, New YorkHuang Y, Wang L, Lu Z, Hong L, Liu Z, Tan GYA, Goodfellow M (2002) Proposal to combine the genera Actinobispora and Pseudonocardia in an emended genus Pseudonocardia, and description of Pseudonocardia zijingensis sp. nov. Int J Syst Evol Microbiol 52:977–982Huss VAR, Festl H, Schleifer KH (1983) Studies on the spectrophotometric determination of DNA hybridization from renaturation rates. Syst Appl Microbiol 4:184–192Kaewkla O, Franco CMM (2010) Pseudonocardia adelaidensis sp. nov., an endophytic actinobacterium isolated from the surface-sterilized stem of a grey box tree (Eucalyptus microcarpa). Int J Syst Evol Microbiol 60:2818–2822Kaewkla O, Franco CMM (2011) Pseudonocardia eucalypti sp. nov., an endophytic actinobacterium with a unique knobby spore surface, isolated from roots of a native Australian eucalyptus tree. Int J Syst Evol Microbiol 61:742–746Kämpfer P, Kohlweyer U, Thiemer B, Andreesen JR (2006) Pseudonocardia tetrahydrofuranoxydans sp. nov. Int J Syst Evol Microbiol 56:1535–1538Labeda DP, Goodfellow M, Chun J, Zhi XY, Li WJ (2011) Reassessment of the systematics of the suborder Pseudonocardineae: transfer of genera within the family Actinosynnemataceae Labeda and Kroppenstedt 2000 emend. Zhi et al. 2009 into an emended family Pseudonocardiaceae Embley et al. 1989 emend. Zhi et al. 2009. Int J Syst Evol Microbiol 61:1259–1264Lane DJ (1991) 16S/23S rRNA sequencing. In: Stackebrandt E, Goodfellow M (eds) Nucleic acid techniques in bacterial systematics. Wiley, Chichester, pp 115–148Lechevalier MP, Lechevalier H (1970) Chemical composition as a criterion in the classification of aerobic actinomycetes. Int J Syst Bacteriol 20:435–443Lechevalier MP, Stern AER, Lechevalier HA (1981) Phospholipids in the taxonomy of actinomycetes. Zbl Bakt Suppl 11:111–116Li J, Zhao GZ, Huang HY, Zhu WY, Lee JC, Kim CJ, Xu LH, Zhang LX, Li WJ (2010) Pseudonocardia rhizophila sp. nov., a novel actinomycete isolated from a rhizosphere soil. Antonie Van Leeuwenhoek 98:77–83Liu ZP, Wu JF, Liu ZH, Liu SJ (2006) Pseudonocardia ammonioxydans sp. nov., isolated from coastal sediment. Int J Syst Evol Microbiol 56:555–558Lucena T, Pascual J, Garay E, Arahal DR, Macián MC, Pujalte MJ (2010) Haliea mediterranea sp. nov., a new marine gammaproteobacterium. Int J Syst Evol Microbiol 60:1844–1848Ludwig W et al (2004) ARB: a software environment for sequence data. Nucleic Acids Res 32:1363–1371Mahendra S, Alvarez-Cohen L (2005) Pseudonocardia dioxanivorans sp. nov., a novel actinomycete that grows on 1,4-dioxane. Int J Syst Evol Microbiol 55:593–598Mesbah M, Premachandran U, Whitman WB (1989) Precise measurement of the G+C content of deoxyribonucleic acid by high-performance liquid chromatography. Int J Syst Bacteriol 39:159–167MIDI (2008) Sherlock microbial identification system operating manual, version 6.1. MIDI Inc., NewarkMinnikin DE, O’Donnell AG, Goodfellow M, Alderson G, Athalye M, Schaal A, Parlett JH (1984) An integrated procedure for the extraction of isoprenoid quinones and polar lipids. J Microbiol Methods 2:233–241Nam S-W, Chun J, Kim S, Kim W, Zakrzewska-Czerwinska J, Goodfellow M (2003) Tsukamurella spumae sp. nov., a novel actinomycete associated with foaming in activated sludge plants. Syst Appl Microbiol 26:367–375Okoh A, Ajisebutu S, Babalola G, Trejo-Hernandez MR (2001) Potential of Burkholderia cepacia RQ1 in the biodegradation of heavy crude oil. Int Microbiol 4:83–87Park SW, Park ST, Lee JE, Kim YM (2008) Pseudonocardia carboxydivorans sp. nov., a carbon monoxide-oxidizing actinomycete, and an emended description of the genus Pseudonocardia. Int J Syst Evol Microbiol 58:2475–2478Pruesse E, Quast C, Knittel K, Fuchs B, Ludwig W, Peplies J, Glöckner FO (2007) SILVA: a comprehensive online resource for quality checked and aligned ribosomal RNA sequence data compatible with ARB. Nucleic Acids Res 35:7188–7196Qin S, Su YY, Zhang YQ, Wang HB, Jiang CL, Xu LH, Li WJ (2008) Pseudonocardia ailaonensis sp. nov., isolated from soil in China. Int J Syst Evol Microbiol 58:2086–2089Qin S, Zhu WY, Jiang JH, Klenk HP, Li J, Zhao GZ, Xu LH, Li WJ (2010) Pseudonocardia tropica sp. nov., an endophytic actinomycete isolated from the stem of Maytenus austroyunnanensis. Int J Syst Evol Microbiol 60:2524–2528Qin S, Xing K, Fei SM, Lin Q, Chen XM, Li WJ, Jiang JH (2011) Pseudonocardia sichuanensis sp. nov., a novel endophytic actinomycete isolated from the root of Jatropha curcus L. Antonie Van Leeuwenhoek 99:395–401Rehfuss M, Urban J (2005) Rhodococcus phenolicus sp. nov., a novel bioprocessor isolated actinomycete with the ability to degrade chlorobenzene, dichlorobenzene and phenol as sole carbon sources. Syst Appl Microbiol 28:695–701Reichert K, Lipski A, Pradella S, Stackebrandt E, Altendorf K (1998) Pseudonocardia asaccharolitica sp. nov. and Pseudonocardia sulfidoxidans sp. nov., two new dimethyl disulfide-degrading actinomycetes and emended description of the genus Pseudonocardia. Int J Syst Bacteriol 48:441–449Sakiyama Y, Thao NKN, Vinh HV, Giang NM, Miyadoh S, Hop DV, Ando K (2010) Pseudonocardia babensis sp. nov., isolated from plant litter. Int J Syst Evol Microbiol 60:2336–2340Sasser M (1990) Identification of bacteria by gas chromatography of cellular fatty acids. MIDI Tech Note 101:1–7Schäfer J, Busse HJ, Kämpfer P (2009) Pseudonocardia parietis sp. nov., from the indoor environment. Int J Syst Evol Microbiol 59:2449–2452Seviour RJ, Kragelund C, Kong Y, Eales K, Nielsen JL, Nielsen PH (2008) Ecophysiology of the actinobacteria in activated sludge systems. Antonie Van Leeuwenhoek 94:21–33Shirling EB, Gottlieb D (1966) Methods for characterization of Streptomyces species. Int J Syst Bacteriol 16:313–340Staneck JL, Roberts GD (1974) Simplified approach to identification of aerobic actinomycetes by thin-layer chromatography. Appl Microbiol 28:226–231Warwick S, Bowen T, McVeigh HP, Embley TM (1994) A phylogenetic analysis of the family Pseudonocardiaceae and the genera Actinokineospora and Saccharothrix with 16S rRNA sequences and a proposal to combine the genera Amycolata and Pseudonocardia in an emended genus Pseudonocardia. Int J Syst Bacteriol 44:293–299Wayne LG, Brenner DJ, Colwell RR, Grimont PAD, Kandler O, Krichevsky MI, Moore LH, Moore WEC, Murray RGE, Stackebrandt E, Starr MP, Trüper HG (1987) International committee on systematic bacteriology report on the ad hoc committee on the reconciliation of approaches to bacterial systematics. Int J Syst Bacteriol 37:463–464Yarza P, Ludwig W, Euzeby J, Amann R, Schleifer KH, Glöckner FO, Rossello-Mora R (2010) Update of the all-species living tree project based on 16S and 23S rRNA sequence analyses. Syst Appl Microbiol 33:291–299Zhao GZ, Li J, Zhu WY, Li XP, Tian SZ, Zhao LX, Xu LH, Li WJ (2011a) Pseudonocadia bannaensis sp. nov., a novel actinomycete isolated from the surface-sterilized roots of Artemisiae annua L. Antonie Van Leeuwenhoek 100:35–42Zhao GZ, Li J, Huang HY, Zhu WY, Zhao LX, Tang SK, Xu LH, Li WJ (2011b) Pseudonocardia artemisiae sp. nov., isolated from surface-sterilized Artemisia annua L. Int J Syst Evol Microbiol 61:1061–1065Zhao GZ, Li J, Huang HY, Zhu WY, Park DJ, Kim CJ, Xu LH, Li WJ (2011c) Pseudonocardia kunmingensis sp. nov., an actinobacterium isolated from surface-sterilized roots of Artemisia annua L. Int J Syst Evol Microbiol 61:2292–229

Paediatric Strategy Forum for medicinal product development of DNA damage response pathway inhibitors in children and adolescents with cancer: ACCELERATE in collaboration with the European Medicines Agency with participation of the Food and Drug Administration

DNA damage response inhibitors have a potentially important therapeutic role in paediatric cancers; however, their optimal use, including patient selection and combination strategy, remains unknown. Moreover, there is an imbalance between the number of drugs with diverse mechanisms of action and the limited number of paediatric patients available to be enrolled in early-phase trials, so prioritisation and a strategy are essential. While PARP inhibitors targeting homologous recombination-deficient tumours have been used primarily in the treatment of adult cancers with BRCA1/2 mutations, BRCA1/2 mutations occur infrequently in childhood tumours, and therefore, a specific response hypothesis is required. Combinations with targeted radiotherapy, ATR inhibitors, or antibody drug conjugates with DNA topoisomerase I inhibitor-related warheads warrant evaluation. Additional monotherapy trials of PARP inhibitors with the same mechanism of action are not recommended. PARP1-specific inhibitors and PARP inhibitors with very good central nervous system penetration also deserve evaluation. ATR, ATM, DNA-PK, CHK1, WEE1, DNA polymerase theta and PKMYT1 inhibitors are early in paediatric development. There should be an overall coordinated strategy for their development. Therefore, an academia/industry consensus of the relevant biomarkers will be established and a focused meeting on ATR inhibitors (as proof of principle) held. CHK1 inhibitors have demonstrated activity in desmoplastic small round cell tumours and have a potential role in the treatment of other paediatric malignancies, such as neuroblastoma and Ewing sarcoma. Access to CHK1 inhibitors for paediatric clinical trials is a high priority. The three key elements in evaluating these inhibitors in children are (1) innovative trial design (design driven by a clear hypothesis with the intent to further investigate responders and non-responders with detailed retrospective molecular analyses to generate a revised or new hypothesis); (2) biomarker selection and (3) rational combination therapy, which is limited by overlapping toxicity. To maximally benefit children with cancer, investigators should work collaboratively to learn the lessons from the past and apply them to future studies. Plans should be based on the relevant biology, with a focus on simultaneous and parallel research in preclinical and clinical settings, and an overall integrated and collaborative strategy

The impact of transposable element activity on therapeutically relevant human stem cells

Human stem cells harbor significant potential for basic and clinical translational research as well as regenerative medicine. Currently ~ 3000 adult and ~ 30 pluripotent stem cell-based, interventional clinical trials are ongoing worldwide, and numbers are increasing continuously. Although stem cells are promising cell sources to treat a wide range of human diseases, there are also concerns regarding potential risks associated with their clinical use, including genomic instability and tumorigenesis concerns. Thus, a deeper understanding of the factors and molecular mechanisms contributing to stem cell genome stability are a prerequisite to harnessing their therapeutic potential for degenerative diseases. Chemical and physical factors are known to influence the stability of stem cell genomes, together with random mutations and Copy Number Variants (CNVs) that accumulated in cultured human stem cells. Here we review the activity of endogenous transposable elements (TEs) in human multipotent and pluripotent stem cells, and the consequences of their mobility for genomic integrity and host gene expression. We describe transcriptional and post-transcriptional mechanisms antagonizing the spread of TEs in the human genome, and highlight those that are more prevalent in multipotent and pluripotent stem cells. Notably, TEs do not only represent a source of mutations/CNVs in genomes, but are also often harnessed as tools to engineer the stem cell genome; thus, we also describe and discuss the most widely applied transposon-based tools and highlight the most relevant areas of their biomedical applications in stem cells. Taken together, this review will contribute to the assessment of the risk that endogenous TE activity and the application of genetically engineered TEs constitute for the biosafety of stem cells to be used for substitutive and regenerative cell therapiesS.R.H. and P.T.R. are funded by the Government of Spain (MINECO, RYC-2016- 21395 and SAF2015–71589-P [S.R.H.]; PEJ-2014-A-31985 and SAF2015–71589- P [P.T.R.]). GGS is supported by a grant from the Ministry of Health of the Federal Republic of Germany (FKZ2518FSB403)