No abstract

Ponatinib is the only approved tyrosine kinase inhibitor (TKI) suppressing BCR-ABL1 T315I-mutated cells in chronic myeloid leukemia (CML). However, due to side effects and resistance, BCR-ABL1 T315I-mutated CML remains a clinical challenge. Hydroxyurea (HU) has been used for cytoreduction in CML for decades. We found that HU suppresses or even eliminates BCR-ABL1 T315I + sub-clones in heavily pretreated CML patients. Based on this observation, we investigated the effects of HU on TKI-resistant CML cells in vitro. Methods: Viability, apoptosis and proliferation of drug-exposed primary CML cells and BCR-ABL1+ cell lines were examined by flow cytometry and 3 H-thymidine-uptake. Expression of drug targets was analyzed by qPCR and Western blotting. Findings: HU was more effective in inhibiting the proliferation of leukemic cells harboring BCR-ABL1 T315I or T315I-including compound-mutations compared to cells expressing wildtype BCR-ABL1. Moreover, HU synergized with ponatinib and ABL001 in inducing growth inhibition in CML cells. Furthermore, HU blocked cell cycle progression in leukemic cells, which was accompanied by decreased expression of CDK4 and CDK6. Palbociclib, a more specific CDK4/CDK6-inhibitor, was also found to suppress proliferation in primary CML cells and to synergize with ponatinib in producing growth inhibition in BCR-ABL1 T315I + cells, suggesting that suppression of CDK4/CDK6 may be a promising concept to overcome BCR-ABL1 T315I-associated TKI resistance. Interpretation: HU and the CDK4/CDK6-blocker palbociclib inhibit growth of CML clones expressing BCR-ABL1 T315I or complex T315I-including compound-mutations. Clinical studies are required to confirm single drug effects and the efficacy of`ponatinib+HUandponatinib+palbociclibcombinations in advanced CML. Funding: This project was supported by the Austrian Science Funds (FWF) projects F4701-B20, F4704-B20 and P30625.

UDP-GalNAc:polypeptide GalNAc transferase (ppGalNAcT; EC 2.4.1.41) catalyzes the first step in mucin-type O-glycosylation. To date, several members of this large enzyme family have been analyzed in detail. In this study we present cloning, expression and characterization of the first representative of this type of glycosyltransferase from mollusk origin, namely from Biomphalaria glabrata. The full length sequence of the respective gene was obtained by screening of a cDNA library using homology-based PCR. The entire gene codes for a protein consisting of 600 amino acids comprising the features of a typical type II membrane protein containing a cytoplasmic tail at the N-terminus, a transmembrane and a catalytic domain as well as a ricin-like motif at the C-terminus. Sequence comparison with ppGalNAcTs from various species revealed high similarities in terms of structural architecture. The enzyme is O-glycosylated but does not have any putative N-glycosylation sites. All four tested acceptor peptides were functional substrates, with Muc2 being the best one. Further biochemical parameters tested, confirmed a close relationship to the family of yet known ppGalNAcTs.

It is well known that efficient functioning of photosynthetic (PET) and respiratory electron transport (RET) in cyanobacteria requires the presence of either cytochrome c(6) (Cytc(6)) or plastocyanin (PC). By contrast, the interaction of an additional redox carrier, cytochrome c(M) (Cytc(M)), with either PET or RET is still under discussion. Here, we focus on the (putative) role of Cytc(M) in cyanobacterial respiration. It is demonstrated that genes encoding the main terminal oxidase (cytochrome c oxidase, COX) and cytochrome c(M) are found in all 44 totally or partially sequenced cyanobacteria (except one strain). In order to check whether Cytc(M) can act as electron donor to COX, we investigated the intermolecular electron transfer kinetics between Cytc(M) and the soluble Cu(A) domain (i.e. the donor binding and electron entry site) of subunit II of COX. Both proteins from Synechocystis PCC6803 were expressed heterologously in E. coli. The forward and the reverse electron transfer reactions were studied yielding apparent bimolecular rate constants of (2.4+/-0.1)x10(5) M(-1) s(-1) and (9.6+/-0.4)x10(3) M(-1) s(-1) (5 mM phosphate buffer, pH 7, 50 mM KCl). A comparative analysis with Cytc(6) and PC demonstrates that Cytc(M) functions as electron donor to Cu(A) as efficiently as Cytc(6) but more efficient than PC. Furthermore, we demonstrate the association of Cytc(M) with the cytoplasmic and thylakoid membrane fractions by immunobloting and discuss the potential role of Cytc(M) as electron donor for COX under stress conditions.

No abstract

In the last decade, pieces of evidence for TDP-43-mediated mitochondrial dysfunction in neurodegenerative diseases have accumulated. In patient samples, in vitro and in vivo models have shown mitochondrial accumulation of TDP-43, concomitantly with hallmarks of mitochondrial destabilization, such as increased production of reactive oxygen species (ROS), reduced level of oxidative phosphorylation (OXPHOS), and mitochondrial membrane permeabilization. Incidences of TDP-43-dependent cell death, which depends on mitochondrial DNA (mtDNA) content, is increased upon ageing. However, the molecular pathways behind mitochondrion-dependent cell death in TDP-43 proteinopathies remained unclear. In this review, we discuss the role of TDP-43 in mitochondria, as well as in mitochondrion-dependent cell death. This review includes the recent discovery of the TDP-43-dependent activation of the innate immunity cyclic GMP-AMP synthase/stimulator of interferon genes (cGAS/STING) pathway. Unravelling cell death mechanisms upon TDP-43 accumulation in mitochondria may open up new opportunities in TDP-43 proteinopathy research.

Point mutations in the ABL1 kinase domain are an important mechanism of resistance to tyrosine kinase inhibitors (TKI) in BCR-ABL1-positive and, as recently shown, BCR-ABL1-like leukemias. The cell line Ba/F3 lentivirally transduced with mutant BCR-ABL1 constructs is widely used for in vitro sensitivity testing and response prediction to tyrosine kinase inhibitors. The transposon-based Sleeping Beauty system presented offers several advantages over lentiviral transduction including the absence of biosafety issues, faster generation of transgenic cell lines, and greater efficacy in introducing large gene constructs. Nevertheless, both methods can mediate multiple insertions in the genome. Here we show that multiple BCR-ABL1 insertions result in elevated IC50 levels for individual TKIs, thus overestimating the actual resistance of mutant subclones. We have therefore established flow-sorting-based fractionation of BCR-ABL1-transformed Ba/F3 cells facilitating efficient enrichment of cells carrying single-site insertions, as demonstrated by FISH-analysis. Fractions of unselected Ba/F3 cells not only showed a greater number of BCR-ABL1 hybridization signals, but also revealed higher IC50 values for the TKIs tested. The data presented highlight the need to carefully select transfected cells by flow-sorting, and to control the insertion numbers by FISH and real-time PCR to permit unbiased in vitro testing of drug resistance.

In chronic myeloid leukemia (CML), the occurrence of BCR-ABL1 T315I is associated with resistance against first- and second-generation BCR-ABL1 tyrosine kinase inhibitors (TKI). Ponatinib is a third generation TKI that exerts strong anti-neoplastic effects in advanced CML and is capable of suppressing the kinase activity of BCR-ABL1 T315I. However, therapy with ponatinib is associated with potentially severe side effects. In addition, resistance against ponatinib may develop in sub-clones carrying multiple (compound) mutations in BCR-ABL1. In addition, BCR-ABL1-independent oncogenic pathways contribute to drug resistance. For these patients, alternative therapies such as stem cell transplantation (SCT) or various drug combinations are often considered.Hydroxyurea (HU) is used for initial or palliative cytoreduction in CML. However, the effects of HU on TKI-resistant mutant sub-clones have not been examined so far. The aims of this study were to explore the effects of HU on CML clones carrying BCR-ABL1 T315I as individual mutation or in compound-context, and to investigate anti-leukemic effects of the drug combination ponatinib+HU. In in vitro studies, primary patient-derived cells, human CML cell lines (K562, KU812, KCL-22), and Ba/F3 cells expressing wild type (wt) BCR-ABL1, BCR-ABL1 T315I, or BCR-ABL1 compound mutants involving T315 were examined. Cell proliferation was quantified by measuring 3H-thymidine uptake. Drug effects on competitive clonal growth were analyzed by mixing two Ba/F3 clones, one expressing BCR-ABL1 T315I with GFP and one BCR-ABL1 T315I/E255V labeled by tdTomato, at a 1:1 ratio. Then, cells were exposed to HU, pontinib, or HU+ponatinib for 72 hours, and the percentage of viable cells in each clone was analyzed by flow cytometry. The in vivo response of primary CML cells carrying BCR-ABL1 T315I to HU was examined in 4 TKI-resistant CML patients who were treated with HU (1-3 g/day) for up to 18 months. In these patients, we measured white blood counts (WBC), differential counts, and BCR-ABL1 transcript levels in peripheral blood (PB) by qPCR. The percentage of BCR-ABL1 T315I compared to total BCR-ABL1 was determined by ligation-dependent PCR. In all 4 patients treated with HU, WBC and total BCR-ABL1 mRNA levels remained stable for 3-12 months. Surprisingly, in 3 of 4 patients, the leukemic sub-clone expressing BCR-ABL1 T315Iwas no longer detectable after HU-treatment. After 3 months, 2/4 patients received allogenic SCT. In the other 2 patients, the disease remained stable for 6 and 12 months, respectively. In our in vitro studies, HU was found to inhibit the growth of all BCR-ABL1+ cell lines, including K562 (IC50: 1120±89 µM), KU812 (IC50: 216±32 µM), and KCL-22 (IC50: 196±23 µM) as well as Ba/F3 cells harboring BCR-ABL1 T315I as single mutation (IC50: 74±25 µM) or as compound together with E255V (IC50: 86±2 µM), F311L (IC50: 76±20 µM), F359V (IC50: 69±10 µM), or G250E (IC50: 89±4 µM). Interestingly, Ba/F3 cells exhibiting BCR-ABL1 T315I alone or in compound configuration were more sensitive to HU compared to Ba/F3 cells expressing wt BCR-ABL1 (IC50: 236±49 µM). As expected, HU was also found to inhibit growth of primary CML cells. In subsequent experiments, HU and ponatinib were found to synergize with each other in inhibiting growth of K562, KU812, and KCL-22 cells as well as Ba/F3 cells carrying BCR-ABL1 T315I (Figure) or BCR-ABL1-T315I/F359V. In cell mix experiments, ponatinib exerted strong growth-inhibitory effects on Ba/F3-T315I cells but not on Ba/F3-T315I/E255V cells, whereas HU was found to produce stronger effects on Ba/F3-T315I/E255V cells, and only the combination of both drugs resulted in complete suppression of both cell lines. In conclusion, HU exerts strong sub-clone-specific anti-neoplastic effects in TKI-resistant CML cells, both in patients with BCR-ABL1 T315I+CML and in various cell line models, including sub-clones harboring BCR-ABL1 T315I as single mutation or in compound configuration. In addition, we show that HU and ponatinib produce strong synergistic anti-neoplastic effects on TKI resistant CML cells, including sub-clones carrying T315I. These observations may have clinical implications and may pave the way for more effective sub-clone-eradicating but also palliative or bridging-to-SCT concepts in advanced CML. Clinical studies are now warranted to define the exact value of the drug combination ponatinib+HU in TKI-resistant CML. Figure 1 Figure 1. Disclosures Sperr: Novartis: Honoraria; Amgen: Honoraria, Research Funding. Lion:Ariad: Honoraria; Amgen: Honoraria; BMS: Honoraria; Pfizer: Honoraria; Novartis: Honoraria, Research Funding. Hoermann:Ariad: Honoraria; Novartis: Honoraria; Gilead: Research Funding; Amgen: Honoraria. Deininger:CTI BioPharma Corp.: Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Research Funding; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gilead: Research Funding; Celgene: Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; Ariad: Consultancy, Membership on an entity's Board of Directors or advisory committees. Valent:Amgen: Honoraria; Novartis: Honoraria, Research Funding; Deciphera Pharmaceuticals: Research Funding; Celgene: Honoraria, Research Funding; Ariad: Honoraria, Research Funding.