The Sertoli cells are critical regulators of testis differentiation and development. In the adult, however, their known function is restricted largely to maintenance of spermatogenesis. To determine whether the Sertoli cells regulate other aspects of adult testis biology we have used a novel transgenic mouse model in which Amh-Cre induces expression of the receptor for Diphtheria toxin (iDTR) specifically within Sertoli cells. This causes controlled, cell-specific and acute ablation of the Sertoli cell population in the adult animal following Diphtheria toxin injection. Results show that Sertoli cell ablation leads to rapid loss of all germ cell populations. In addition, adult Leydig cell numbers decline by 75% with the remaining cells concentrated around the rete and in the sub-capsular region. In the absence of Sertoli cells, peritubular myoid cell activity is reduced but the cells retain an ability to exclude immune cells from the seminiferous tubules. These data demonstrate that, in addition to support of spermatogenesis, Sertoli cells are required in the adult testis both for retention of the normal adult Leydig cell population and for support of normal peritubular myoid cell function. This has implications for our understanding of male reproductive disorders and wider androgen-related conditions affecting male health. The Sertoli cells are essential regulators of testis differentiation and fetal masculinization through expression of SRY, secretion of AMH and induction of fetal Leydig cell development (reviewed in There is now increasing evidence to suggest that testosterone levels in adult and ageing men are important for maintaining wellbeing Use of controlled cell ablation to study development and function has a long history of utility in testis biology. Studies using cytotoxins such as busulfan (for germ cell ablation) Mice were housed and bred under standard conditions of care. Experiments passed local ethical review and were conducted with licenced permission under the UK Animal Scientific Procedures Act (1986), Home Office licence number PPL 60/4200.+/− (MacGreen) mice bred to Amh-Cre;iDTR mice were identified by genotyping from ear or tail DNA for the presence of Cre and iDTR using standard PCR with appropriate primers listed in +/+;iDTR+/+, Mac-Green and Stra8-Cre+/+;iDTR+/+. Adult animals  were treated with a single dose of DTX (100 ng) as previously described Mice were generated as previously described 2 asphyxiation and subsequent cervical dislocation. Blood was harvested by cardiac puncture for hormonal profile analysis. Body weight and reproductive organs  weights were recorded. Collected tissues were either fixed or frozen for RNA or protein analysis.Animals were culled at different times after DTX injection see  by CO2 aTestis RNA was extracted using Trizol  and levels of specific mRNA species were measured by real-time PCR following reverse transcription of the isolated RNA. Total RNA was reverse transcribed at 50°C using random hexamers and Moloney murine leukaemia virus reverse transcriptase  as described previously Tissues were fixed in Bouins for 6 hours, stored in ethanol (70%) and embeded in paraffin or in resin (see below). Tissue sections (5 µm) were dewaxed in xylene then rehydrated in graded ethanol solutions. For histology, slides were stained with Hemaetoxylin/Eosin and Picrosirius Red using standard protocols. For immunostaining, slides were subjected to antigen-retrieval in a pressure cooker for 5 minutes with 0.01 M citrate buffer (pH 6.0). To quench endogenous peroxidases activity, slides were incubated in 0.3% hydrogen peroxide (v/v) in Tris-buffered saline (TBS) for 30 minutes at room temperature (RT). Nonspecific activity was blocked using the appropriate normal blocking serum  and 5% BSA  in TBS for 30 minutes at RT followed by incubation overnight at 4°C with the primary antibody diluted in blocking serum. The primary antibodies used in this study are detailed in For stereological analysis, testes were embedded in Technovit 7100 resin, cut into sections (20 µm), and stained with Harris’ hematoxylin. The total testis volume was estimated using the Cavalieri principle After blood collection, sera were separated and stored at −20°C. Serum testosterone was measured using a commercial kit  while FSH and LH were measured by ELISA as previously described 2 and the testes were left on ice for 30 min. The contralateral testis was injected with vehicle (0.01 M MgCl2 in PBS) as a control. After incubation, the testes were fixed in Bouins for 6 hours, embedded in paraffin and processed for immunohistochemistry as above.Testes were collected from Amh-Cre;iDTR animals 7 days after treatment with DTX at pnd50. The interstitium of the testes was injected with 15 µl of 10 mg/ml EZ-Link Sulfo-NHS-LC-Biotin  freshly diluted in PBS containing 0.01 M MgClFigures were compiled using Adobe Photoshop CS6 and Adobe Illustrator CS6 .Data were analysed using Student T test, one-way ANOVA with the appropriate post hoc tests (Turkey’s multiple comparison or Dunnet’s tests), Bartlett’s test or Chi-square test. Specific details for each experiment are provided in figure legends. Graph Prism version 5  or Minitab  were used for analysis. When required, data were normalised by box-cox transformation. Values are expressed as means ± SEM.+/− line +/−, or iDTR+/− animals, but resulted in complete ablation of Sertoli cells in Amh-Cre+/−;iDTR+/− mice   Ablation of Sertoli cells at pnd50 led to a significant reduction in testis weight, 7, 30, 90 days and one year later . This waermatid) . Apoptotermatid) . To exclCnn1 and Myh11 were significantly reduced, however, for up to one year after Sertoli cell ablation  mice +/−;iDTR+/−;CSFR-GFP+/− mice. This permitted tracking of macrophages, following Sertoli cell ablation, through macrophage expression of GFP. Analysis confirmed that macrophage localization was largely restricted to peritubular and interstitial spaces with penetration of less than 1% of the total tubular sections observed despite the presence of cellular debris in all tubules  acts to regulate movement of substances into the seminiferous tubules and to prevent immune cell invasion of the tubules. In adult (pnd80) mice with Sertoli cell ablation, the PTMC layer remained intact in most tubules, which allowed us to determine the contribution of the PTMC and basement membrane to the BTB. Following biotin injection  tubules . This su tubules , arrows,response .Abundant Leydig cells remained in the interstitial tissue 7 days after DTX injection and the cells appeared normal. By 30 days after injection Leydig cells remained present singly or in groups throughout the interstitium but the nuclei of these cells often had an altered, elongated shape and the cytoplasm was vacuolated and/or reduced in size , which sDespite the significant reduction in Leydig cell number, average circulating testosterone concentrations were not significantly different from control animals at any time-point following Sertoli cell ablation , and semThe adult Leydig cells differentiate from progenitor cells in the peritubular region of the testis with a possible contribution also coming from perivascular cells There is no direct contact between Sertoli cells and Leydig cells and loss of adult Leydig cells following Sertoli cell ablation would suggest that there must be a Sertoli cell-derived factor(s) that is required to maintain the Leydig cells. This factor may act directly on the Leydig cell or may be mediated through the PTMC, which have been previously shown to regulate Leydig cell function in vitro systems, have reported the presence of Sertoli cell–derived factors, which inhibit Leydig cell function, although their nature is unknown The maintenance of basal circulating testosterone, despite loss of 75% of the Leydig cell population following Sertoli cell ablation, suggests that the remaining Leydig cells may have become hyperactive. This effect is also seen following neonatal Sertoli cell ablation Previous studies have shown that the adult Leydig cell population will regenerate from stem cells following ablation by the cytotoxic drug EDS As might be expected, Sertoli cell ablation in adult animals led to a rapid immune response in the testis with an increase in interstitial macrophages apparent by seven days and an increase in cytokine levels, which peaked at 30 days post-ablation. It is known that the resident testicular interstitial macrophages form a close physical and functional relationship with the Leydig cells The BTB is generated largely by the presence of specialised junctions between adjacent Sertoli cells near to the basement membrane In a previous study a gradual loss of Sertoli cells was shown over a one-year period in mice lacking BCL2L2 (BCLW) Data from this study should be read in context with previous results from our group showing developmental changes in the testis following Sertoli cell ablation in the fetus or in neonatal/prepubertal life The Sertoli cells are recognized as the primary regulators of testis development Figure S1Diphtheria toxin-mediated Sertoli cell-specific ablation. (A) Inheritance of iDTR and Cre Recombinase transgenes relative to testicular histology following DTX injection. Inheritance of both transgenes is required for Sertoli cell-specific ablation. 697 bp = iDTR-negative; 253 bp = iDTR-positive; 320 bp = Cre-negative (positive control for PCR amplification); 110 bp = Cre-positive; Marker (m); Water (w). .(TIF)Click here for additional data file.Figure S2Diphtheria-toxin does not cause increased apoptosis in other organs. (A) Histology of non-gonadal tissues  demonstrating no off-target effects 7 or 30 days post-DTX injection . (B) Immunostaining for CLEAVED CASPASE 3 protein in adrenal and kidney of adult Amh-Cre;iDTR vehicle or DTX-treated mice 7 days after ablation . DTX treatment to ablate Sertoli cells engenders no off-target apoptosis in other tissues. d,days; dtx, injected with toxin; veh: vehicle control.(TIF)Click here for additional data file.Table S1Primers used for Genotyping and qRT-PCR.(DOCX)Click here for additional data file.Table S2Details of Antibodies and detection methods used.(DOCX)Click here for additional data file.


It has long been known that methylated cytosines deaminate at higher rates than unmodified cytosines and constitute mutational hotspots in mammalian genomes. The repertoire of naturally occurring cytosine modifications, however, extends beyond 5-methylcytosine to include its oxidation derivatives, notably 5-hydroxymethylcytosine. The effects of these modifications on sequence evolution are unknown. Here, we combine base-resolution maps of methyl- and hydroxymethylcytosine in human and mouse with population genomic, divergence and somatic mutation data to show that hydroxymethylated and methylated cytosines show distinct patterns of variation and evolution. Surprisingly, hydroxymethylated sites are consistently associated with elevated C to G transversion rates at the level of segregating polymorphisms, fixed substitutions, and somatic mutations in tumors. Controlling for multiple potential confounders, we find derived C to G SNPs to be 1.43-fold (1.22-fold) more common at hydroxymethylated sites compared to methylated sites in human (mouse). Increased C to G rates are evident across diverse functional and sequence contexts and, in cancer genomes, correlate with the expression of Tet enzymes and specific components of the mismatch repair pathway . Based on these and other observations we suggest that hydroxymethylation is associated with a distinct mutational burden and that the mismatch repair pathway is implicated in causing elevated transversion rates at hydroxymethylated cytosines. Most cytosines that occur in a CpG context in mammalian genomes are methylated. Methylation has important functional consequences in the cell but also affects genome evolution. Notably, methylated cytosines are prone to deaminate and constitute mutational hotspots in mammalian genomes. Recently, a series of other modifications, derived from the oxidation of methylated cytosines, was shown to exist in various mammalian cell types including embryonic stem cells. The most abundant of these modifications is 5-hydroxymethylcytosine. In this work, we ask whether methylated and hydroxymethylated cytosines are subject to the same mutational biases or lead to distinct patterns of genome evolution. To do so, we examine differences between individuals, between species, and between normal and cancer tissues alongside high-resolution maps of DNA methylation and hydroxymethylation in the human and mouse genomes. Unexpectedly, we find that hydroxymethylated cytosines are associated with more cytosine to guanine changes in both human and mouse populations, in closely related species, and in the context of somatic evolution in tumors. Based on multiple lines of evidence, we suggest that the different patterns of sequence evolution at methylated and hydroxymethylated sites are owing to differences in how these sites are handled by the DNA repair machinery. In mammalian genomes, most cytosines that occur in a CpG context are methylated. 5-methylcytosines (5mCs) at CpG dinucleotides exhibit mutation rates an order of magnitude above that of unmodified cytosines, a consequence both of their greater propensity to deaminate and error-prone repair of the resulting thymine Recently, it has become clear that the repertoire of naturally occurring cytosine modifications in mammals extends beyond 5mC to include a series of modifications derived from successive rounds of 5mC oxidation: 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) In the context of the high mutational burden of 5mC and considering that 5hmC can be present as a stable epigenetic mark, we wondered whether methylated and hydroxymethylated sites might be associated with distinct patterns of sequence evolution, perhaps as a consequence of divergent mutational biases. For example, in mammalian systems, repair of 5hmU:G mismatches (derived from 5hmC deamination) by the glycosylases TDG and SMUG1 is less error-prone than dealing with 5mC-derived T:G mispairs Here, to elucidate the evolutionary repercussions of hydroxymethylation, we integrate population genomic, inter-species divergence and somatic mutation data from tumors with publicly available base-resolution maps of 5hmC and 5mC in human and mouse ES cells DNA methylation at single nucleotide resolution is commonly detected using bisulfite sequencing. While bisulfite sequencing provides a high-coverage view of methylation across the genome, it does not discriminate between 5hmC and 5mC modifications: any site identified as modified by bisulfite sequencing could be 5hmC or 5mC. However, alternative sequencing strategies, notably Tet-assisted bisulfite sequencing (TAB-Seq) We then focussed on residues located outside of repeat regions, covered by at least ten sequencing reads in the pertinent bisulfite experiment, and amenable to accurate SNP calling . FurtherM. spretus genome – relative to the M. musculus-M. spretus ancestral genome and M. musculus methylation state – echoes this result: C to G rates are higher at 5hmC sites than at 5mC sites , suggesting stronger average purifying selection at 5mC sites  and regional (±500 nt) GC content, chromatin state, biotype, the upstream neighbouring nucleotide and the methylation level of the focal cytosine , and GC content levels methylation patterns in ES cells are predictive of patterns in the germline and can therefore contribute mechanistically to a 5hmC-related mutation signature. To evaluate this assumption we first considered base resolution 5hmC maps for mouse neurons  ed pairs  and rateThe genomic incidence of hydroxymethylation has previously been examined for different stages of mouse spermatogenesis, using a chemical labelling method followed by enrichment and sequencing We reasoned that – if elevated C to G rates are mechanistically linked to hydroxymethylation – they might be higher at sites where the 5hmC mark is more prevalent. Hydroxymethylation is non-stoichiometric and sites classified as 5hmC are typically hydroxymethylated in a minority of cells in the population. We therefore tested whether cytosines with higher levels of hydroxymethylation exhibit higher SNP rates. This is indeed the case in human  and largely insensitive to upstream nucleotide context, suggesting that we are not dealing with a known, context-dependent mutational process.We then examined the relationship between C to G rates in tumors and the expression of Tet proteins. Tet proteins catalyse the oxidation of 5mC to 5hmC and therefore constitute a critical rate-limiting step for 5hmC generation, as evident in lower genome-wide levels of 5hmC in mouse ES cells where Tet1/2 protein levels are diminished following shRNA-mediated knock-down Considering correlations separately for 11 different types of cancer  complex MutSα, which recognizes post-replicative single-base mismatches The results above are consistent with a model where hydroxymethylation has a causal role in generating higher C to G rates at 5hmC sites. A mutational bias associated with hydroxymethylation might come as a surprise. Several That MMR might be implicated in 5hmC-related mutagenesis is intriguing. MMR operates immediately after replication when it needs to discriminate the newly replicated from the template strand, thus exhibiting an intrinsic requirement for asymmetry. In bacteria this requirement is catered for by transient asymmetric methylation Intriguingly, examining our cancer data, we discovered strong correlations between %C2G and three components of MMR: MSH2, MSH6, and MBD4 . MSH2 ande facto asymmetric sites. In contrast, the latter study used a synthetic probe that only contained fully symmetrically hydroxymethylated CpGs . It therefore seems possible, and consistent with in vitro replication studies, which normally only consider a single modified site, that MSH6 might preferentially associate with asymmetrically hydroxymethylated sites. This might explain why asymmetrically hydroxymethylated sites suffer from higher mutation rates, as suggested by It is further worth noting that MSH6 – the MutSα protein that makes direct contact with DNA Based on these observations we suggest the following model that links MMR, hydroxymethylation and elevated C to G transversion rates: 1.) 5hmC can be further oxidized by the Tet family of enzymes to 5fC and 5caC 2.) During DNA replication, 5caC:G pairing induces exonuclease activity of the replicating DNA polymerase δ and is targeted by MutSα Alternatively, one might consider a slightly more complex model where mutagenic effects derive from an interaction between the MMR and BER DNA repair pathways: After TDG glycosylase removes 5caC/5fC, the resulting abasic site is hijacked by an MMR-affiliated TLS polymerases, leading to elevated transversion rates. An analogous scenario has been suggested for the MSH2- and UNG2-dependent generation of C to G transversion by the TLS polymerase Rev1 in the context of somatic hypermutation ftp://neomorph.salk.edu/mc/h1_c_basecalls.tar.gz), principally to render results more comparable between species and allow detection of lowly (down to 10%) hydroxymethylated sites. We excluded residues that are part of repeats as annotated in UCSC (hg18) and added information on hydroxymethylation status, assayed at base-resolution for the same cell line Starting with all cytosine residues in the human reference genome, we confined analysis to cytosines covered by at least 10 reads in the genome-wide bisulfite sequencing of the H1 hESC cell line conducted by Lister et al ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/pilot_data/technical/reference/ancestral_alignments). We combined (hydroxy)methylation and polymorphism data after converting all coordinates to hg19 using the liftOver tool Data on single nucleotide polymorphisms in the human population from the 1000 Genomes Project ftp://ftp.ensembl.org/pub/mnt2/release-75/emf/ensembl-compara/epo_6_primate/) was used to reconstruct substitutions along the chimp lineage. We only considered residues that were cytosines in both human and orang-utan.The Ensembl 6-primate alignment  from the ENCODE project As done for human, we start from a list of all cytosines in the mouse genome (mm9) and subsequently remove nucleotides covered by fewer than 10 reads in the bisulfite sequencing study of the E14TG2a mESC line conducted by http://www.sanger.ac.uk/resources/mouse/genomes/), which are derived from three wild sub-species: Mus musculus domesticus, Mus musculus musculus, and Mus musculus castaneusMus spretus, a sister taxon to Mus musculus also included in the strain sequencing effort and rat as the outgroup to polarize polymorphisms. Specifically, based on the mouse-rat (mm10-rn5) pairwise alignment from UCSC we retrieved the corresponding Mus spretus variants and inferred the base ancestral to all Mus musculus strains by parsimony. We only considered sites where genotype calls were made across all strains and further confined analysis to sites where the genotype was congruent between the mouse reference genome and the 129P2/OlaHsd strain from which the mESC line was derived. Note that the bisulfite sequencing study of Stadler et al. In the absence of extensive genome-wide polymorphism data for wild mice populations, we considered polymorphisms across a collection of laboratory mouse strains sequenced by the Sanger Institute Local (±50 nt) and regional (±500 nt) GC content around each eligible cytosine as well as the upstream/downstream neighbouring nucleotides were computed from the reference and reconstructed ancestral sequence for mouse and human. Choice of either ancestral or reference sequence here has no significant impact on the results and we therefore only present data derived from the ancestral sequence.Chromatin context is strongly associated with mutation rates in cancer genomes bcNoise and spanDel filters . We excluded mutations in poorly mappable genomic regions  for cytosines in a CpG context in mouse (EPS)Click here for additional data file.Figure S2Derived allele frequencies. Derived allele frequencies (DAFs) in the human population for all eligible 5hmC, 5mC, and C sites and matched 5hmC and 5mC sites. When methylation status is assigned based on mapping bisulfite sequencing reads to the reference genomes (Top panel: Unmatched – reference genotype assumed), there is a striking excess of high frequency derived alleles for unmethylated cytosines. This excess disappears when considering only sites for which the H1 hESC genotype has been confirmed as a cytosine (Middle panel: Unmatched – H1 genotype). This suggests that the excess is caused by alleles where the reference carries the C allele but the cell line (and the majority of the human population) carry the T allele – so that mapping bisulfite reads to the reference would mistakenly indicate the presence of an unmethylated cytosine. The DAF spectra for unmatched but not matched 5mC and 5hmC sites differ significantly from each other (see main text). Note that a similar analysis of DAFs is not appropriate for mouse because the inbred laboratory strains considered here do not constitute a natural evolving population for which allele frequencies would provide a meaningful window into the evolutionary process.(EPS)Click here for additional data file.Figure S3Enrichment of mESC 5hmC residues in 5hmC-enriched regions in the male germline. Gan et al (EPS)Click here for additional data file.Table S1Matching criteria and ranges for matched pairs analyses.(DOCX)Click here for additional data file.Table S2List of cancer samples classified by cancer subtype.(TXT)Click here for additional data file.Table S3Correlations between the expression of mismatch repair, base excision repair and Tet genes and C to G transversion rates across 346 cancer genomes.(XLSX)Click here for additional data file.Table S4ENCODE data used to genotype H1 hESC.(DOCX)Click here for additional data file.


The LKB1 tumour suppressor is a serine/threonine kinase that functions as master regulator of cell growth, metabolism, survival and polarity. LKB1 is frequently mutated in human cancers and research spanning the last two decades have begun decoding the cellular pathways deregulated following LKB1 inactivation. This work has led to the identification of vulnerabilities present in LKB1-deficient tumour cells. Pre-clinical studies have now identified therapeutic strategies targeting this subset of tumours that promise to benefit this large patient population harbouring LKB1 mutations. Here, we review the current efforts that are underway to translate pre-clinical discovery of therapeutic strategies targeting LKB1 mutant cancers into clinical practice. Liver kinase B1  was first identified as the causal mutation in Peutz–Jeghers Syndrome (PJS), a rare inherited autosomal dominant disorder characterised by the development of benign gastrointestinal hamartomas and the early onset of cancer  Shackel. The pasThe predisposition of PJS patients to early-onset tumours predicted that LKB1 inactivation is an important event in carcinogenesis . Subsequex vivo  and two regulatory subunits . Adenosine monophosphate-activated protein kinase is activated upon the direct binding of ADP or AMP to a γ subunit where AMPK undergoes a conformational change leading to the phosphorylation of Thr172 on the activation loop of the α subunits  for intracellular transport  have not been uncovered  mouse models of lung cancer with the combination of the SRC inhibitor dasatinib, MEK inhibitor AZD6244, and the dual PIK3CA and mTOR inhibitor BEZ235 resulted in decreased primary and metastatic lung tumours  following LKB1 inactivation as having an integral role in metastasis. The link between deregulation of MARKs and metastasis is important because the inactivation of Lkb1 promotes metastasis in the Krasncovered . A recenncovered . Inactivs kinase . Lastly,lkb1 in mice have confirmed that LKB1 inactivation has an important role in tumourigenesis in a variety of tissues. Genetically engineered mouse models have proven to be a valuable tool to understand the molecular basis of disease following inactivation of LKB1. Biallelic loss of lkb1 is embryonic lethal in mice, yet, heterozygous lkb+/− mice develop gastrointestinal hamartomas that closely recapitulate the pathophysiology of the PJS patients  models will provide comprehensive nd colon  , KrasG12D/Lkb1−/− (KL) and KrasG12D/p53−/− (KP) GEMMs. Surprisingly, the authors discovered that K and KP lung tumours were responsive to docetaxol+selumetinib therapy, whereas KL tumours were unresponsive to therapy. Detailed molecular analysis revealed low activation of the MEK/ERK pathway in KL lung nodules, thus explaining the lack of therapeutic response. Interestingly, in a separate pre-clinical study comparing the response of K, KL and KP GEMMs to phenformin, it was discovered that KL tumours showed a significant response to phenformin, while K and KP lung tumours remained resistant, thus demonstrating that Lkb1 inactivation determined sensitivity to phenformin  to test novel therapeutics in vitro and in vivo studies. Novel combination therapies targeting LKB1 vulnerabilities in cancer will need to be rigorously tested in both cell-culture-based and animal models. Because most tumours eventually develop resistance to therapy, it will be important to choose pre-clinical in vivo models that readily develop resistance and accurately model the heterogeneity present in most tumours. Although xenografts remain a highly effective in vivo means to screen isogenic tumour lines, they should be used in combination with autochthonous models such as GEMMs or with PDXs. This will likely facilitate defined treatment modalities that selectively target this large subset of tumours. Finally, using imaging-based preclinical studies in animal models, such as GEMMs and PDXs, to rigorously test new therapeutic strategies hold great promise for efficiently translating novel drugs that target LKB1 vulnerabilities into clinical trials (As every single approach has its limitations, it is reasonable to assume that systematic and efficient translation of new therapies targeting LKB1 vulnerabilities will require the development and integration of both cell-based and mouse-based research platforms. The use of 2D and 3D culture systems as well as the development of organoid models hold promise for accurately bridging


Furthermore, there is currently no viable method for prospectively assessing drug synergy directly in human patients in order to potentially tailor therapies. To address these issues we have employed the previously described CIVO platform and developed a quantitative approach for investigating multiple combination hypotheses simultaneously in single living tumors. This platform provides a rapid, quantitative and cost effective approach to compare and prioritize drug combinations based on evidence of synergistic tumor cell killing in the live tumor context. Using a gemcitabine resistant model of pancreatic cancer, we efficiently investigated nine rationally selected Abraxane-based combinations employing only 19 xenografted mice. Among the drugs tested, the BCL2/BCLxL inhibitor ABT-263 was identified as the one agent that synergized with Abraxane® to enhance acute induction of localized apoptosis in this model of human pancreatic cancer. Importantly, results obtained with CIVO accurately predicted the outcome of systemic dosing studies in the same model where superior tumor regression induced by the Abraxane/ABT-263 combination was observed compared to that induced by either single agent. This supports expanded use of CIVO as an in vivo platform for expedited in vivo drug combination validation and sets the stage for performing toxicity-sparing drug combination studies directly in cancer patients with solid malignancies.While advances in high-throughput screening have resulted in increased ability to identify synergistic anti-cancer drug combinations, validation of drug synergy in the  While the past several decades of cancer research have led to a vastly increased understanding of the complex mechanisms that underlie cancer cell survival, particularly in the face of stressors such as chemotherapy, the failure rate of new oncology drugs in clinical trials compared to other disease areas is still exceedingly high . This rin vivo analysis in the preclinical setting. Statistically thorough quantification of synergy using intact in vivo tumor models is resource intensive and time consuming, particularly when an investigator desires to compare multiple drug combinations of interest simultaneously. Barriers to in vivo anti-cancer drug combination analysis include, but are not limited to requirements for scale-up of sufficient quantities of compound for in vivo dosing, establishment of proper dosing regimens to avoid toxicity and legitimately evaluate combination effects, and large numbers of tumor-bearing animals to achieve study significance. Furthermore, statistically rigorous assessment of cancer drug synergy, even for a simple two-compound drug combination, in the context of the human clinic is not currently feasible. Therefore, a need exists for a methodology that incorporates a multiplexed framework for statistically valid drug combination analysis, but importantly is adapted to a relevant in vivo system that recapitulates the dynamic genotypic and phenotypic heterogeneity of a tumor in its microenvironment.Discovery, validation, and prioritization of effective synergistic drug combinations for pursuit in the clinic is a factorial problem that is not efficiently addressed by current methods of in vivo cancer drug combination analysis that is reproducible, quantitative and statistically rigorous, feasible for incorporation into existing drug development undertakings including human clinical trials and most notably, predictive of long term systemic outcomes. Towards this goal, we adapted the previously described CIVO microinjection platform, which enables assessment of multiple drugs simultaneously in single living solid tumors, to in vivo, in-tumor investigation of drug combinations. First, we optimized CIVO to perform head-to-head comparisons of a panel of possible combination therapies to select the best candidate(s) for further investigation. Second, we combined CIVO with a statistically rigorous model of combination effects to test specific combination therapies for synergistic anti-tumor effects.We therefore sought to develop an approach to ® (nab paclitaxel) and thus ultimately provide a rational alternative to gemcitabine in patients who are resistant to treatment with this drug. Of the nine combinations analyzed with our method, the combination of Abraxane® with the BCL2/BCLxL inhibitor ABT-263 was found to induce synergistic pancreatic tumor cell apoptosis, a result which was verified upon systemic administration of these agents in an independent pre-clinical combination study.In this study, we demonstrate the potential of these methods through a focused study in a preclinical model of gemcitabine-refractory pancreatic cancer. Specifically, the CIVO platform was used to identify agents that enhance the efficacy of Abraxane® was manufactured by Celgene Corporation .Compounds used in the CIVO and systemic studies were purchased from Selleck Chemicals , Chemietek  and Medkoo Biosciences (Mitomycin C). AbraxaneMia PaCa2 pancreatic cancer cell line (ATCC) were cultured in DMEM (ThermoFisher) plus 10% fetal bovine serum. Cultures were grown at 37°C, 5% CO2.6 million cells per mouse as per protocol described in [3. At this size, tumors exhibited multiple aspects of a heterogeneous microenvironment including regions of vascularization and hypoxia, collagenous extracellular matrix, vascular endothelial cells, and infiltrating macrophages  using the MiaPaCa2 cells inoculated at 5x10rophages . Microin® was formulated in 0.9% saline and administered intravenously at 10, 20 or 30 mpk; ABT-263 was formulated in 60% Phosal 50, 30% PEG400 and 10% ethanol and administered by oral gavage at 100 mpk. Mice were enrolled for study when tumors reached a volume of 200 mm3. Tumor volume was calculated as V = length x width x height, all three dimensions measured using digital calipers [For systemic drug efficacy studies, AbraxaneFollowing tumor resections, 2 mm thick sections perpendicular to the injection columns, were fixed in 10% buffered formalin for 48 hours, scanned and processed as previously described . Rabbit f(r). To mitigate the influence of pre-existing necrosis on these measurements, injection sites that fall within largely acellular tumor regions are excluded prior to quantitative analysis.Quantitative analysis of IHC was performed using custom software   with the lme4 package for linear mixed models.In this investigation, Abraxanes models . In the 1(r) is the radial response induced by drug 1, β2(r) is the radial response induced by drug 2, βs(r) is the additional synergistic effect of the combination of both drugs, and α(r) is the response due to the act of injection. A value of βs(r) greater than zero indicates synergy.Once a combination of two compounds is selected, CIVO can subsequently be employed to test for synergy of those compounds. For this purpose, we model the radial response to the combination asAn important consideration regarding this response model is that pharmacologically, combined responses are not characterized by such algebraic sums . Typical1(r), β2(r), and βs(r), and α(r) modeled as a tumor-dependent random effect. Fitting this model requires the use of four needles per tumor: one with both drugs in combination, one with drug 1 alone, one with drug 2 alone, and one with vehicle alone. Positive values of βs(r) with p<0.05 are taken to indicate statistically significant synergy.The model parameters are estimated via a linear mixed model with fixed effects βUnder certain restrictive assumptions, the model parameters estimated from the combination model above can be used to determine the combination index (CI) of Chou and Talalay . The CI D1 of agent 1 and D2 of agent 2 have a combined fractional effect x, and (Dx) 1 and (Dx) 2, respectively, are the doses of agents 1 and 2 alone required to achieve the same fractional effect. A value of x equal to 50% (the “median” effect) is often targeted. A CI less than 1 indicates synergy.Where doses By applying the median effect relationship  that relSee ® to induce mitotic arrest, localized tumor exposure to Abraxane® induced a substantial increase in the fraction of pHH3-postive cells surrounding the site of microinjection [® exposure was minimal and limited to the immediate region around the site of injection where drug exposures tend to be super-physiological. Similar to Abraxane, most of the other single agents tested induced no increase or modest increases in apoptotic tumor cell death ..® to indll death . One not® revealed that the addition of Abraxane® induced a modest increase in apoptosis when combined with each of the single agents tested  was significantly higher than 0 at a wide range of radial distances extending out to at least 1200 μm for the combination of Abraxane® and ABT-263  was not significantly different from 0 for the combination of Abraxane® and ABT-199 at any of the radial distances tested  were systemically treated with Abraxane, ABT-263 or a combination thereof, based on dosing regimens previously described for both drugs [® and ABT-263, while Abraxane® showed prominent single agent efficacy compared to vehicle, combining it with ABT-263 even at the lowest Abraxane® dose yielded a significantly superior growth inhibitory effect and durable tumor remission (® single agent arm and none (0/8) in the ABT-263 single agent arm. Lower doses of Abraxane® (10 and 20 mpk) combined with ABT-263 100 mpk also led to 25% (2/8) complete remission  in live tumors. This led us to select ABT-263 as a target of interest for combination with Abraxane® in tumors derived from the MiaPaCa2 pancreatic cancer cell line. Second, we demonstrated the ability to identify and statistically validate the presence of synergy in response to a specific combination. This allowed us to confirm that the combination response of ABT-263 and Abraxane® exhibits synergy in the same sense as this term is used to describe drug interactions in cell-based assays and with similar precision, bringing this level of rigor for the first time into the in vivo realm. We also demonstrated the absence of drug synergy using ABT-199, a closely related derivative of ABT-263, in combination with Abraxane®. The responses correlated with the expression of the respective target proteins thereby demonstrating the capability of the CIVO platform to identify context-specific drug combinations. Finally, key to demonstrating the utility of our approach, the synergistic interaction between Abraxane® and ABT-263 observed upon co-localized microinjection, was borne out in a traditional 4-arm preclinical drug combination study where tumor growth inhibition was measured over time following systemic drug treatment. This suggests that the localized anti-tumor drug combination responses, measured within days of drug microinjection by histological biomarker analysis, are meaningful in terms of accurately predicting the results of longer term studies where effects of systemically administered drugs can be assessed by more traditional measures of tumor size and progression. Our findings are consistent with previously published data that demonstrate enhanced efficacy in other cancer models when ABT-263 is paired with a taxane [in vivo, enabling efficient prioritization of drug combinations that merit further investigation in more resource intensive studies, thereby potentially greatly reducing the timeframe from effective drug combination identification to clinical trials.In this study we present a novel application of the CIVO microinjection platform that enables rapid identification and quantitative evaluation of drug combinations a taxane , 24, 25.in vivo have been proposed, notably using the popular Chou Talalay approach for combination analysis [in vivo drug screening capability [Alternative strategies for assessing anti-cancer drug combinations analysis . To use analysis . In contpability  suggests® versus the combination of Abraxane® and ABT-263. The data is supportive of a previously described model proposing a mechanism for the synergistic anti-tumor activity induced by ABT-263 and taxanes [in vivo demonstration in support of this model of synergy [® where clear anti-tumor synergy is observed. This may allow for lower, more tolerable doses of ABT-263 to be administered to patients so that anti-tumor effects are maintained under a reduced requirement for high tumor drug concentrations, thereby decreasing unintended impact on normal cell compartments.The combination of Abraxane and ABT-263 may represent a novel therapy, with a demonstrated mechanistic basis for anti-tumor synergy, for pancreatic cancer patients who have failed gemcitabine-based treatments. An interesting observation, as revealed by the CIVO study, was the reciprocal relationship between localized CC3 and pHH3 positive cell subpopulations in response to Abraxane taxanes . Three p synergy . ConsistWhile the studies presented here represent critical steps forward, ultimately the value of CIVO technology as applied to drug combinations lies in direct investigation of combination efficacy in the human cancer clinic. It is reasonable to expect that localized responses to direct drug microinjection into solid tumors, while the tumor mass is still in the patient, will capture elements of tumor biology and tumor drug interactions not captured by advanced preclinical models. For instance, patient-derived xenografts preserve some of the complex biology observed in human tumors, and a recent study demonstrated the feasibility of using such models in a high throughput screen format . HoweverIn summary, we have presented a novel method for diagnostic assessment of drug combinations in living tumors using the CIVO technology. This approach can currently be applied to medium throughput drug combination screens that should significantly reduce the preclinical costs and time to validation for translation of drug combinations into the clinic. More importantly, CIVO technology holds the potential for use in toxicity-sparing studies directly in human patients to segregate responders from non-responders enabling improved clinical trial stratification.D is any dose, β is the fractional effect at that dose, and D50 is the dose for which β = 50%. More generally, we can write this in terms of a dose Dx that produces a fractional response x asThe median effect relationship for a first-order dose response relationship  isD is not included in this equation, because it captures effects unrelated to the drug). Thus, plugging this in for x and writing the complete equation as a function of radial distance gives, blood vessel endothelial cells (CD31), α-smooth muscle actin (αSMA) and infiltrating macrophages (S100A9) in the tumor stroma.(TIF)Click here for additional data file.S2 FigRadial effect curves show fraction pHH3+ and CC3+ cells as a function of radial distance from the site of injection. Data are averaged across four tumors. Error bars denote SEM.(TIF)Click here for additional data file.S3 Fig® (ABX) 10 or 20 mpk IV Days 1–3 or combination of ABT 263 and Abraxane® using the same dosing regimen as the single agents. Drug efficacy with respect to vehicle was assessed in all arms via tumor volume measurements when tumors met censoring criteria (See ex vivo images of tumors from each treatment arm on Day 24 and Day 35.(A-B) MiaPaCa2 xenografted mice (n = 8 per treatment cohort) were treated systemically with vehicle (control), ABT 263 100 mpk PO Days 1–3, Abraxaneeria See . Data ar(TIF)Click here for additional data file.S4 FigPlots represent body weight of mice recorded over time during treatment (Days 1–3) and 12 days after cessation of treatment across all treatment groups. Data are averaged across all tumors in the respective cohorts. Error bars represent SEM.(TIF)Click here for additional data file.


The FANCJ DNA helicase is mutated in hereditary breast and ovarian cancer as well as the progressive bone marrow failure disorder Fanconi anemia (FA). FANCJ is linked to cancer suppression and DNA double strand break repair through its direct interaction with the hereditary breast cancer associated gene product, BRCA1. FANCJ also operates in the FA pathway of interstrand cross-link repair and contributes to homologous recombination. FANCJ collaborates with a number of DNA metabolizing proteins implicated in DNA damage detection and repair, and plays an important role in cell cycle checkpoint control. In addition to its role in the classical FA pathway, FANCJ is believed to have other functions that are centered on alleviating replication stress. FANCJ resolves G-quadruplex (G4) DNA structures that are known to affect cellular replication and transcription, and potentially play a role in the preservation and functionality of chromosomal structures such as telomeres. Recent studies suggest that FANCJ helps to maintain chromatin structure and preserve epigenetic stability by facilitating smooth progression of the replication fork when it encounters DNA damage or an alternate DNA structure such as a G4. Ongoing studies suggest a prominent but still not well-understood role of FANCJ in transcriptional regulation, chromosomal structure and function, and DNA damage repair to maintain genomic stability. This review will synthesize our current understanding of the molecular and cellular functions of FANCJ that are critical for chromosomal integrity. FANCJ  or BRCA1 interacting helicase (BRIP1) was first discovered by its physical interaction with BRCA1, a known tumor suppressor and mediator of double strand break (DSB) repair . ConsistFigure 1). The reader is referred to several recently published excellent reviews on the functions of the FA gene products and the overall workings of the FA pathway and its importance in chromosomal stability . In addition, it is believed that FANCJ functions in a broader role to suppress replication stress (see subsequent sections).In accord with its role as a tumor suppressor, FANCJ was identified as the gene mutated in the J complementation group of Fanconi anemia (FA), a rare disorder characterized by progressive bone marrow failure, skeletal abnormalities, and cancer . Currenttability . One nottability . FANCJ lFigure 2). A signature motif in this FANCJ family of DNA helicases is an Iron–Sulfur (Fe–S) cluster, characterized by four conserved cysteine residues, residing within the helicase core domain , I, Ia, II, III, IV, V, VI] found in DEAH superfamily two helicases . A 5′ tail of 15 nucleotides (nt) is required and 35 nt is optimal for FANCJ to catalyze appreciable unwinding of the simple 5′ tailed duplex , consistent with its translocation directionality. Backbone continuity in the pre-existing 5′ single-stranded DNA tail, but not the 3′ single-stranded tail, of the forked duplex substrate within six nt of the single-stranded DNA-double stranded DNA junction is required for FANCJ to initiate unwinding of the adjacent duplex. However, disruption of the sugar phosphate backbone by a polyglycol modification in either the translocating or non-translocating strand within the duplex region inhibits FANCJ helicase activity . However, FANCJ fails to unwind a four-stranded Holliday Junction structure, another key intermediate of HR repair that can lead to cross-over or non-crossover recombinant products  DNA substrate that represents a key early intermediate of HR repair . Followiproducts . Thus, FFigure 3). For both triplexes and G4s, FANCJ requires a 5′ single-stranded DNA tail, consistent with its 5′–3′ directionality of translocation. For unwinding triplexes this 5′ tail must reside on the pyrimidine motif third strand that invades the major groove of the underlying DNA double helix. FANCJ has the capacity to resolve intermolecular (two-stranded or four-stranded) as well as unimolecular G4 substrates , and therefore inhibit DNA strand exchange activity of RAD51 . The ability of RPA to stimulate FANCJ displacement of TRF1 or TRF2 from forked duplex substrates harboring telomeric repeats may be important for remodeling chromosome ends during DNA replication or repair. Indeed, FANCJ was localized to telomeres of living cells that operate according to the alternative lengthening of telomere (ALT) pathway . However, this inhibition is overcome by the presence of RPA in the FANCJ reaction mixtures under conditions that RPA alone had little effect . The interface between FANCJ and the interacting BRCA1 BRCT repeats was further defined in structural studies , raising the possibility that the protein interaction of FANCJ with BLM is affected by phosphoSer-990. More recently, a second FANCJ phosphorylation dependent interaction was identified at Thr-1133 . Phosphorylated FANCJ Thr-1133 interacts with the BRCT repeats of Topoisomerase IIb binding protein 1 (TopBP1) to promote an ATR-dependent checkpoint in response to replication stress , affects the DNA damage response similar to Ser-990 phosphorylation. When acetylation is prevented, cellular ICL resistance is achieved by a reduced need for Rad54-mediated HR repair and enhanced dependence on the translesion polymerase η. This modulation of repair pathway mechanism could stem from the role of FANCJ and its acetylation upregulating DNA end resection required for HR. Dynamic acetylation of FANCJ was also found to maintain checkpoint signaling following DNA damage , especially because FANCJ status dramatically influences BLM protein stability by a proteasomal degradation pathway  identified in individuals with early breast cancer and normal genotypes for BRCA1 or BRCA2  affected catalytic activity in a distinct manner. The P47A substitution in the highly conserved Walker A box (motif I) inactivated the ATPase and helicase functions of FANCJ, whereas the M299I mutation located in the Fe-S cluster upregulated its ATPase activity . The alanine to proline substitution is adjacent to one of the highly conserved cysteine residues important for chelation of Fe atoms. Inheritance of the A349P mutation and a second mutation encoding a prematurely truncated FANCJ protein resulted in intrauterine growth failure and death as a stillborn fetus with a gestational age of 22 weeks . Biophysical analysis of the purified recombinant FANCJ-Q25A mutant protein disrupted in the Q motif revealed that its assembly state was dramatically altered compared to the recombinant WT FANCJ protein. FANCJ-Q25A protein was only a monomer, whereas FANCJ-WT protein was nearly equally monomer and dimer  found in both RNA and DNA helicases that is predicted to coordinate ATP binding and hydrolysis to catalytic DNA strand separation . Although expression of either R251C or Q255H mutant could not rescue the cisplatin sensitivity of a fancj null cell line, the two mutations exerted markedly different effects on the biochemical functions of FANCJ in vitro. Both FANCJ motif Ia mutants abolished DNA helicase activity. The R251C mutation strongly interfered with FANCJ DNA binding and consequently its DNA-dependent ATPase activity. Instead, the Q255H mutant displayed elevated FANCJ DNA binding, a normal ATPase function, and ability to translocate on single-stranded DNA. In this regard, the Q255H mutant behaved similarly to the FANCJ-A349P mutant which was also able to translocate on single-stranded DNA in an ATP-dependent manner but failed to unwind even short 20 bp duplex substrates  that impair DNA helicase (but not necessarily ATPase activity) demonstrate that catalytic DNA unwinding is vital for FANCJ function and suggests that FANCJ heterozygosity may contribute to tumorigenicity or disease-associated phenotypes. For a further discussion of helicase-inactivating mutations as a basis for dominant negative phenotypes, see . Consistent with this observation, CtIP is also delayed in its recruitment to DSBs in cells that are deficient in MRE11 exonuclease, that have reduced FANCJ recruitment to DSBs . FANCJ localization to UV light induced DNA crosslinks also requires the MLH1 interaction as well as the upstream MMR protein MSH2  protein MLH1 . While MPso-ICLs . This anein MSH2 . This isein MSH2 . Localizein MSH2 . CoordinFigure 1), FANCD2 recruitment to Pso-ICLs, not but laser-induced DSBs, was dependent on FANCJ . Consistent with these observations, FANCJ is recruited to Pso-ICLs much earlier than FANCD2. These findings correlate with those of the Andreassen lab that reported that FANCJ foci formed normally in FANCD2-deficient cells after exposure to the DNA cross-linker MMC . The precise role of FANCJ in BLM recruitment to laser-induced DSBs or its function at DNA ends is being investigated, and a potential partnership between FANCJ and BLM in processive strand resection is a possibility . MMR proteins initially recruit FANCJ. However, the further accumulation of FANCJ requires dual incision by the NER endonucleases XPF and XPG  and MMR proteins promote the localization of the FANCJ to sites of UV-induced lesions  often display transcriptional deficiencies which are likely to culminate in pleiotropic symptoms including developmental abnormalities and features of premature aging . For exReplication stress can be a source of genomic instability. Cellular data have implicated FANCJ in helping to cope with replication stress induced exogenously by chemical exposure. Human cells mutated in FANCJ or acutely depleted of FANCJ by RNA interference are sensitive to DNA cross-linking agents that block fork progression , or HU tAside from its proposed role in DNA end processing to initiate HR repair (previous section), FANCJ may help to process DNA structures associated with stalled replication forks or regulate such processing events. The interaction between FANCJ and MLH1 appears to be critical for cells to recover from replication stress induced by ICLs or the DNA polymerase inhibitor, aphidicolin. As mentioned above, deletion of MSH2 suppresses replication restart defects in cells lacking the FANCJ–MLH1 interaction . Thus, tin vivo and exert biological effects on DNA replication and transcription, and play unique structural roles in telomere capping . A recent study using Xenopus egg extract and single-stranded plasmid DNA template with a predicted G4-forming sequence validated the role of FANCJ to promote DNA synthesis through G4 structures . In subsequent work, it was shown that FANCJ, together with the RecQ helicases WRN and BLM, preserve epigenetic stability by helping to efficiently couple histone recycling with replication fork progression through their ability to resolve G4s that impede smooth DNA synthesis ; however, this hypothesis remains to be formally tested in human cells. It is well known that G-rich sequences predicted to form G4 are enriched in promoter regions especially at transcriptional start sites  of FANCJ in the FA pathway of ICL repair is still not well understood. In addition, cellular and biochemical studies have demonstrated an important role of FANCJ to resolve G4 DNA structures that potentially interfere with normal chromosome packaging, replication, and transcription. FANCJ interacts with a number of DNA damage response proteins and appears to be a key player in maintaining chromosomal stability through its involvement in DNA repair and checkpoint signaling. With FANCJ’s emergence as a uniquely important genome caretaker, future studies may explore FANCJ as a potential target for clinical and therapeutic strategies. In spite of the tremendous progress, much still remains to be learned about FANCJ and avenues for biomedical advances.In this review, we have attempted to provide a comprehensive view of what now appears to be multi-faceted roles of FANCJ in cellular DNA metabolism. Discovered 13 years ago as a BRCA1-interacting protein, FANCJ helicase is now regarded as a The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Forty-two participants with cerebral palsy were recruited for a study examining traditional and novel indicators of cardiovascular risk (McPhee et al., 2015  Specifications tableValue of the data•There is a paucity of literature reporting the prevalence of cardiovascular disease risk in adults with cerebral palsy; a population believed to be at elevated risk due to their mobility impairments.•These data are included for comparative purposes of the existing literature, and may inform systematic and/or meta-analytic reviews.•These data may inform future studies in non-ambulatory adults with cerebral palsy as they presented greater absolute risk for cardiovascular disease.In the present data, we highlight the prevalence of cardiovascular and metabolic risk in adults with cerebral palsy (CP) who are either ambulatory or non-ambulatory. We provide cardiovascular disease (CVD) risk factor information that is consistent with predictors from the Framingham Heart Study Study participants’ characteristics are described elsewhere Measurements of supine brachial artery systolic blood pressure, diastolic blood pressure, and mean arterial pressure were obtained using an automated sphygmomanometer  in all 42 participants. Hypertension was defined as per the Eighth Joint National Committee 2). Waist circumference was measured while in a supine position using an adapted technique as per the National Institutes of Health (NIH) Supine height without shoes was measured to the nearest 0.5 cm using an anthropometric tape measure. Body mass was measured to the nearest 0.1 kg using a digital wheelchair scale . Body mass index (BMI) was determined by dividing the participant׳s weight (kg) by their height squared , high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), TC/HDL-C, and triglycerides. Participant labeled Eppendorf tubes were sent to Hamilton Regional Laboratory Medicine Program – McMaster Medical Centre Campus for analysis of the previously identified lipid markers. Glucose was examined using the Glucose Hexokinase Reagent Set . All coefficients of variation between duplicates were <10% for each metabolic marker.2 was defined as being obese as per the NIH Descriptive results of lifestyle-related and metabolic risk factors are shown in


Malaria invasion of red blood cells involves multiple parasite-specific targets that are easily accessible to inhibitory compounds, making it an attractive target for antimalarial development. However, no current antimalarial agents act against host cell invasion.Plasmodium falciparum) and rodent (P. berghei) malarias. Multiple lines of evidence demonstrate that the action of azithromycin in inhibiting parasite invasion of red blood cells is independent of its inhibition of protein synthesis in the parasite apicoplast, opening up a new strategy to develop a single drug with multiple parasite targets. We identified derivatives of azithromycin and erythromycin that are better invasion inhibitors than parent compounds, offering promise for development of this novel antimalarial strategy.Here, we demonstrate that the clinically used macrolide antibiotic azithromycin, which is known to kill human malaria asexual blood-stage parasites by blocking protein synthesis in their apicoplast, is also a rapid inhibitor of red blood cell invasion in human  contains supplementary material, which is available to authorized users. Plasmodium falciparum. Current disease control measures rely on reducing exposure to the mosquito vector or treatment of clinical malaria cases with antimalarial drugs that target the disease causing blood-stage trophozoite; most of these act against the parasite’s food vacuole or interfere with pyrimidine synthesis of maturing trophozoites [Malaria is a mosquito-borne parasitic disease, which causes nearly 600,000 deaths every year, mainly in children under 5 years of age in sub-Saharan Africa . Most dehozoites –6. Of mahozoites –9. The phozoites . This deInvasion of blood-stage merozoites into host cells has been proposed as a potential target for antimalarial chemotherapy –14. InvaP. falciparum merozoite invasion, partly through lack of tractable invasion inhibition assays. The ability to assess the invasion inhibitory potential of compounds and their biological activity in vitro has been greatly enhanced with the development of a robust and efficient merozoite purification protocol and techniques to directly measure invasion inhibition [P. falciparum and P. berghei merozoite invasion in vitro. This represents the first time that a drug or drug class that has clinical applications against malaria has been demonstrated to inhibit parasite invasion of the red blood cell.Few compounds have been directly tested for ability to inhibit hibition , 18–20, hibition , 22, hephibition  and calchibition , 24) havhibition . In thisin vivo [In vitro experiments at clinically relevant concentrations show that these antibiotics have a ‘delayed death’ drug response. Treated parasites grow normally during the first lifecycle under treatment. Only during the second post-treatment cycle (after replication and reinvasion) are drug effects observed. Parasite death at this stage is thought to result from the inheritance of a defective apicoplast that is unable to synthesize isoprenoid precursors required for development [Antibiotics such as azithromycin and clindamycin (lincosamide antibiotic) have been proposed as potential partner drugs for artemisinin combination therapies owing to their extremely long half-life and good safety profile in vivo –29. Bothin vivo –32. In velopment –33. Thiselopment .50 of invasion inhibitory activity could be lowered through modification. These results provide the basis for a novel strategy of antimalarial drug development by advancing compounds that have dual mechanisms of action. Given the established safety and low cost of macrolides in widespread clinical use, development of macrolides as antimalarials with dual modalities against merozoite invasion and protein translation in the apicoplast is a promising strategy to counter the rapid emergence of drug resistance.In this study, we aimed to identify existing, and novel, compounds with invasion inhibitory activity that have the potential for development as effective antimalarials. We demonstrate that the antibiotic azithromycin can specifically inhibit merozoite invasion. Related macrolide antibiotics were also found to inhibit invasion, and the ICP. falciparum asexual blood-stage merozoite invasion of the host erythrocyte  or DMSO (38 μM), suggesting that choice of vehicle can impact azithromycin potency in vitro. Comparative screens of azithromycin potency over different treatment times demonstrated that the IC50 of merozoite invasion assays (1 hour exposure to drug) were surprisingly similar to the in cycle  invasion 10 μm; in cycle 6 μM; 1 cycle 7 μM, −(drug prepared in DMSO) invasion 38 μM (DMSO); in cycle 12 μM; 1 cycle assay 7 μM, Fig. in vitro.Application of the merozoite purification method of Boyle et al.  identifi ethanol 0 μM or D80mero DMSO 151 μM) and the invasion inhibitory control compound, heparin, was significantly more inhibitory to parasite growth than the same treatment of early ring stages at the time-point of 20 minutes post-invasion (T = 20 (exposed for 40 minutes); azithromycin P <0.001; heparin P <0.01). Additionally, pretreatment of erythrocytes, followed by washout of drug, had little or no inhibitory effect on invasion . This indicated that exposure of merozoites, but not very early ring stages or uninfected erythrocytes, was inhibitory to parasite growth.To confirm that azithromycin was inhibiting merozoite invasion directly in our assays and was not acting downstream of invasion during intracellular parasite growth, the effect of exposure of merozoites or early ring-stage parasites to the drug was examined; azithromycin was washed out of the cultures within 1 hour and parasitaemia assessed by flow cytometry 40 hours post-assay setup for all treatments (prior to the next round of parasite rupture and invasion of new host cells). Treatment of merozoites (10 minute incubation) and early ring stages for less than 1 hour (T = 0) with azithromycin (1 × IC80mero (380 μM (drug prepared in ethanol to limit nonspecific effects of vehicle)) prior to washing and addition of merozoites. There was minimal loss of invasion into erythrocytes pretreated with a 10 × IC80mero concentration of azithromycin for 1 hour relative to control , supporting the conclusion that it is the merozoite and its interaction with the erythrocyte, and not the erythrocyte itself, that is the target of inhibition.In order to confirm that the inhibitory activity was against purified merozoites and not the uninfected erythrocyte prior to merozoite invasion, erythrocytes were pretreated with azithromycin at a 10 × ICrol Fig. . In cont80mero), confirming that azithromycin is indeed inhibitory to merozoite invasion  and spiramycin (123 μM), while erythromycin (420 μM) was 42-fold less potent than azithromycin. Of interest was the very poor invasion inhibitory activity of dirithromycin (521 μM), which was 52-fold less potent than azithromycin, even though the IC50 values determined using 1 cycle growth assays were very similar  further confirmed specific inhibition of merozoite invasion .After identifying that azithromycin was inhibitory to merozoite invasion, we examined whether related drugs with a history of clinical use as antibiotics also had this property. Macrolides with a 14-membered macrolactone ring  and 16-membered ring (spiramycin) were tested using the same methods as azithromycin  than seen for azithromycin (10 μM). However, pre-treatment of erythrocytes with 2,972 μM of clindamycin (IC80mero) resulted in a 65 % reduction in merozoite invasion relative to untreated controls and there was evidence of erythrocyte lysis using these high levels of clindamycin  selected for reduced sensitivity to both azithromycin and erythromycin in 2 cycle (delayed death) assays. Sequencing of apicoplast ribosome genes revealed a G91D mutation in the rpl4 gene product, corresponding to the G112D mutation in Chlamydomonas reinhardtii, known to confer resistance to erythromycin [50120hr D10-AZRr, 9 μM; D10 parental, 0.16 μM; P <0.0001; Fig. 50mero , erythromycin  or clindamycin . Together, these data suggest that the invasion inhibitory activity of azithromycin is largely independent of apicoplast ribosomal protein synthesis, consistent with the current view that the apicoplast does not play a role in invasion.We tested further whether apicoplast ribosomal protein translation was the target of the inhibitory activity at the merozoite stage by comparison of the invasion inhibitory activity of azithromycin, erythromycin and clindamycin between the D10-PfPHG (azithromycin-sensitive) parasite used throughout this study and an azithromycin-resistant D10 derivative  that has a lower IC50 in 1 cycle growth inhibition assays [50 of merozoite invasion inhibition (13 μM) 32-fold compared to the parent compound  almost 3-fold compared to erythromycin A. The increased potency of Meg-erythromycin and erythromycin oxime compared to the parent drug indicates that various modifications to macrolides can lead to more potent invasion inhibitory activity.We tested a panel of macrolide analogues to determine whether the ICn assays  was test50 values lower than azithromycin in a 72 hour growth inhibition assay  had a substantially lower IC50 for merozoite invasion inhibition than azithromycin (38 μM (when prepared in DMSO); Fig. in vitro.Next we obtained a small panel of azithromycin analogues (in DMSO) from GlaxoSmithKline  –44 with 50 of the modified analogues was between 10- and 14-fold lower than that of azithromycin  and 90 hours  the IC80) reduced invasion to only 8 % of total contact events, reduced contacts leading to erythrocyte deformation to 27 % of contact events and increased the number of merozoites releasing without invading or deforming the erythrocyte to 65 % of all contact events, resulting in failure to invade for 92 % of all contact events. These results support the live filming observations for azithromycin in ethanol . As expected, at the same concentration the less potent parent azithromycin was less inhibitory to invasion (25 % contact events invaded) and as a result had fewer failed invasion events (75 %). Together, these findings establish a proof-of-concept that macrolides with more potent invasion inhibitory activity can be developed through chemical modification and that the dual activities of invasion inhibition and other inhibitory activities can be developed in single compounds and are not mutually exclusive.In order to assess whether the invasion inhibitory phenotype of a more potent analogue was similar to our observations for azithromycin in ethanol see Fig. , live finol Fig.  and agai50 for both in cycle  and 1 cycle  drug assays . Comparison of these compounds indicates that the cladinosyl group has a role in lowering the IC50 of invasion inhibition for azithromycin, but the presence of both the cladinosyl and desosamine groups is critical for azithromycin’s invasion inhibitory activity. While these data suggest that the desosamine group plays the critical role during invasion inhibition, technical limitations prevented the selective removal of the desosamine group.We were further able to remove both the cladinosyl and desosamine groups from azithromycin  [P. berghei sporozoites to enter liver cells in vitro using an established assay. GFP-expressing sporozoites were allowed to settle on and invade HepG2 cells. After free sporozoites were removed by washing, cells were fixed and then antibody-labeled without cell permeabilization, thereby preventing labeling of sporozoites that are inside host cells. We then compared the number of labeled cells  to the total number of cells  to determine invasion efficiency. There was a statistically significant, but modest, effect indicating that azithromycin may inhibit sporozoite invasion of hepatocytes at concentrations up to 100 μM azithromycin ; Fig. P. berghei sporozoites  ; the efftes Fig. .Toxoplasma gondii, which has orthologues of many Plasmodium invasion proteins and a similar invasion process [80 of azithromycin against P. falciparum merozoite , clearly indicating that azithromycin is a less potent inhibitor of T. gondii invasion in vitro. Importantly, there was no evidence of tachyzoite invasion inhibition in the presence of 500 μM erythromycin A, reflecting the results of the P. falciparum studies which showed that erythromycin A is a poor inhibitor of merozoite invasion compared to azithromycin. We next explored whether the azithromycin analogue 12e, which was found to be a more potent inhibitor of P. falciparum merozoite invasion than azithromycin (DMSO as vehicle), had improved tachyzoite invasion inhibitory activity compared to azithromycin. Analogue 12e (tested at 42 μM due to limited compound availability) was found to be a consistently more potent inhibitor of T. gondii tachyzoite invasion (48 % inhibition compared to non-inhibitory control) than much higher concentrations of azithromycin  to the low nanomolar range [In addition to its ‘delayed death’ effect on parasite growth, azithromycin has been reported to kill parasites within the first cycle of trations , 32. It trations . Given gar range –44. Yet Several lines of evidence indicate that azithromycin has a secondary mechanism and target(s) of action to inhibit invasion, in addition to binding to the 50S apicoplast ribosomal subunit as described for the ‘delayed death’ phenotype , 32. Cli50 values seen for both inhibitory phenotypes raises the intriguing possibility that this secondary mechanism of action could be targeted throughout the disease causing blood-stage of P. falciparum malaria.The near instantaneous inhibition of merozoite invasion caused by azithromycin also suggests that this drug can rapidly kill parasites through a secondary mechanism of action. Typical ‘delayed death’ inhibition by macrolide antibiotics results in the loss of apicoplast functionality , 32, witThe ability of azithromycin to rapidly inhibit merozoite invasion, in addition to the apicoplast-targeting ‘delayed death’ mechanism of action, is an exciting prospect for the development of macrolide-based drugs in combination therapies. This dual modality could see azithromycin assist in rapid clearance of parasites as well as offering longer-term treatment of the remaining low-level blood and liver-stage parasites through its activity against the apicoplast. Such a drug treatment strategy has the potential to increase drug efficacy, while reducing the chances for the development of resistance.in vitro merozoite invasion inhibitory activity of azithromycin required drug concentrations at the upper end of what is achieved clinically [in vivo fermentation process [50 by up to 5-fold. The azithromycin derivatives were tested only when solubilized in DMSO due to limitations in the amount of available material. Importantly, the use of DMSO as vehicle for solubilization reduced the potency of the parent compound azithromycin 4-fold relative to ethanol as vehicle. If this observation were to hold true for the derivatives, the invasion inhibitory IC50 of the compound 1j (5) [Although the inically , 32, we  process  produced process –44 lower50 of azithromycin (10 μM) and analogues  is comparable to that achieved for inhibitors that specifically target the function of essential merozoite proteins. Two recent proof-of-concept studies identified small molecule invasion inhibitory compounds targeting interactions between essential invasion ligands RON4-AMA1 (inhibitory range 30–6 μM) and MSP1-19 (replication inhibition 21.7 μM) [50 values in a very similar range to azithromycin and analogues. Another highly selective invasion inhibitor, the 3D7-AMA1-specific peptide inhibitor R1 [50 of 2.5 μM in assays using purified merozoites [The invasion inhibitory IC21.7 μM) , and repbitor R1 , has an rozoites . AzithroRemoval of azithromycin’s glycan groups provided insight into the structural requirements for invasion inhibitory activity. Removal of the cladinosyl sugar resulted in a 5-fold reduction in invasion inhibitory activity. Strikingly, removal of both sugars resulted in complete loss of invasion inhibitory activity, strongly suggesting that the desosamine sugar, rather than the cladinosyl, is critical for invasion inhibitory activity. Interestingly, the desosamine sugar is also considered the critical glycosylated group in binding of macrolides to microbial ribosomes  with cliPlasmodium merozoites  and T. gondii tachyzoites parasites suggests that the target of inhibition is shared amongst some apicomplexan invasive stages. The fact that the potency of azithromycin against P. falciparum (human) and P. berghei (rodent) malarias was very similar raises the possibility that targeting this pathway could be an effective strategy to treat other malaria species. The results of the T. gondii invasion assays suggest that host cell invasion of this more distantly related apicomplexan parasite can also be targeted by azithromycin, albeit at higher concentrations. Importantly, the analogue 12e had equal or better invasion inhibitory potency than azithromycin  when tested at a 6-fold lower concentration. This strongly suggests that analogues of azithromycin could be developed with superior inhibitory activity against tachyzoite invasion and confirms that the invasion inhibitory activity of azithromycin and analogues is shared across distantly related apicomplexan parasites.The fact that azithromycin inhibits host invasion by 2+ and MAPK through an as yet undefined mechanism . Intraciewed in ) so inteiewed in , 56. IntPlasmodium spp. merozoite invasion into erythrocytes in vitro. Invasion inhibition assays and live filming experiments show that azithromycin acts rapidly to stop merozoite entry very early during the invasion process. The mechanism of invasion inhibition appears to be independent of apicoplast ribosomal translation inhibition, which is the currently described mechanism of action of these drugs. Modification of both azithromycin and erythromycin A resulted in promising reductions in the invasion inhibitory IC50 to levels nearing those achieved clinically for the parent compounds, providing a proof-of-concept for the potential development of potent invasion inhibitors. Elucidation of the mechanism of action and subsequent improvements in compound potency could pave the way for development of macrolide antibiotics with dual modality as novel and potent antimalarials to complement frontline treatments for malaria. Furthermore, the use of drugs that inhibit merozoite invasion in combination with drugs that inhibit intra-erythrocytic parasite development may be an effective strategy to facilitate rapid parasite clearance to help improve clinical outcomes, reduce transmission, and minimize the development of resistance.The results of this study have identified for the first time that macrolide antibiotics, a diverse group of well-tolerated antibiotic drugs with antimalarial properties, inhibit + erythrocytes  according to established protocols [D10 parental and GFP fluorescent D10-PfPHG  parasiterotocols , 58. Synrotocols , 23, 57.rotocols , 18.P. falciparum growth and invasion inhibition is available in Fig. A diagram outlining the different drug inhibition assays used in this study to measure P. falciparum and P. berghei merozoites from E64-treated schizonts and merozoite invasion inhibition assays have been previously described [The protocols for filtration of viable escribed , 18, 45.P. berghei ANKA sporozoites dissected from infected Anopheles stephensi mosquitoes. Two-color invasion assays followed the protocol of Sinnis et al. [P. berghei constitutively expressing GFP [P. berghei merozoite surface proteins [.Liver-stage assays used s et al.  with thesing GFP  were addproteins  (500:1) proteins .Toxoplasma gondii invasion assays were performed using the Rh parasite strain according to existing protocols [rotocols  with the50 and IC80 concentrations were determined using GraphPad Prism  as previously described [90hr, 120hr and mero where appropriate.The ICescribed . The inh2, 5 % O2 and 5 % CO2), selected schizonts were observed until they ruptured and released their merozoites. Time-lapse videos of the invading merozoites were recorded with a high-resolution AxioCam MRm camera (Zeiss). ImageJ was used to perform image analysis and GraphPad Prism used to perform statistical analyses using a Chi-squared test.Live cell microscopy experiments were undertaken according to the method of Weiss et al. . BrieflyA summary of the chemical structures of the compounds used in this study is available in Fig. A tabular summary of data used to generate graphs and perform statistical tests presented in this manuscript Figs. ,e,f is a