featuring the Echo acoustic technology
TITLES and AUTHORS
The BCR-ABL1 fusion gene is a driver oncogene in chronic myeloid leukaemia and 30–50% of cases of adult acute lymphoblastic leukaemia1. Introduction of ABL1 kinase inhibitors (for example, imatinib) has markedly improved patient survival2, but acquired drug resistance remains a challenge3, 4, 5. Point mutations in the ABL1 kinase domain weaken inhibitor binding6 and represent the most common clinical resistance mechanism...
Drug combination testing in the pharmaceutical industry has typically been driven by late-stage opportunistic strategies rather than by early testing to identify drug combinations for clinical investigation that may deliver improved efficacy. A rationale for combinations exists across a number of diseases in which pathway redundancy or resistance to therapeutics are evident. However, early assays are complicated by the absence of both assay formats representative of disease biology and robust infrastructure to screen drug combinations in a medium-throughput capacity. When applying drug combination testing studies, it may be difficult to translate a study design into the required well contents for assay plates because of the number of compounds and concentrations involved. Dispensing these plates increases in difficulty as the number of compounds and concentration points increase and compounds are subsequently rolled onto additional labware. We describe the development of a software tool, in conjunction with the use of acoustic droplet technology, as part of a compound management platform, which allows the design of an assay incorporating combinations of compounds. These enhancements to infrastructure facilitate the design and ordering of assay-ready compound combination plates and the processing of combinations data from high-content organotypic assays.
The Malaria Box, assembled by the Medicines for Malaria Venture, is a set of 400 structurally diverse, commercially available compounds with demonstrated activity against blood-stage Plasmodium falciparum. The compounds are a representative subset of the 20,000 in vitro antimalarials identified from the high-throughput screening efforts of St. Jude Children's Research Hospital (TN, USA), Novartis and GlaxoSmithKline. In addition, a small set of active compounds from commercially available libraries was added to this group, but it has not previously been published. Elucidation of the biochemical pathways on which these compounds act is a major challenge; therefore, access to these compounds has been made available free of charge to the investigator community. Here, the Malaria Box compounds were tested for activity against the formation of b-hematin, a synthetic form of the heme detoxification biomineral, hemozoin. Further, the mechanism of action of these compounds within the malaria parasite was explored. Ten of the Malaria Box compounds demonstrated significant inhibition of b-hematin formation. In this assay, doseeresponse data revealed IC50 values ranging from 8.7 to 22.7 mM for these hits, each of which is more potent than chloroquine (a known inhibitor of hemozoin formation). The in vitro antimalarial activity of these ten hits was confirmed in cultures of the chloroquine sensitive D6 strain of the parasite resulting in IC50 values of 135e2165 nM, followed by testing in the multidrug resistant strain, C235. Cultures of P. falciparum (D6) were then examined for their heme distribution following treatment with nine of the commercially available confirmed compounds, seven of which disrupted the hemozoin pathway.
The High Throughput Biomedicine (HTB) unit at FIMM Technology Centre provides a wide range of biomedical high throughput assays. In collaboration with research groups and the Hospital District of Helsinki and Uusimaa, we have set up drug sensitivity and resistance testing (DSRT) platform with a set of 450 approved and investigational oncology drugs on patient samples. Ex vivo drug testing is currently performed at FIMM with primary cancer cells from patients with leukaemia and multiple myeloma as well as cancer cell lines and the drug sensitivity responses are integrated with molecular profiling such as exome sequencing, transcriptomics and phosphoproteomics. Currently, DSRT is run in 384-well plate format in 5 concentrations for each drug or in 1536-well plate format with 9 concentrations per drug and the assay plates are pre-drugged using acoustic droplet ejection with Labcyte® Echo® 550. The viability and cell death of cells is measured after 72 h and results are analysed using Dotmatics Studies – software and an in-house developed interface, Breeze. Application of the platform to AML patient samples has uncovered taxonomic drug-response subtypes and individualised therapy based on DSRT has resulted in several clinical responses. The DSRT platform enables drug repositioning, provides new combinatorial possibilities and allows for linking drug sensitivities to predictive biomarkers. We are developing the platform with additional readouts and increasing the number of drugs. Our Labcyte Access Workstation, with Echo 550 and Echo® 525 integration, allows development and set-up of miniaturised follow-up assays, such as reverse-phase protein array and qPCR, using non-contact acoustic dispensing.
In vitro studies show that estrogens acutely modulate synaptic function in both sexes. These acute effects may be mediated in vivo by estrogens synthesized within the brain, which could fluctuate more rapidly than circulating estrogens. For this to be the case, brain regions that respond acutely to estrogens should be capable of synthesizing them. To investigate this question, we used quantitative real-time PCR to measure expression of mRNA for the estrogen-synthesizing enzyme, aromatase, in different brain regions of male and female rats. Importantly, because brain aromatase exists in two forms, a long form with aromatase activity and a short form with unknown function, we targeted a sequence found exclusively in long-form aromatase. With this approach, we found highest expression of aromatase mRNA in the amygdala followed closely by the bed nucleus of the stria terminalis (BNST) and preoptic area (POA); we found moderate levels of aromatase mRNA in the dorsal hippocampus and cingulate cortex; and aromatase mRNA was detectable in brainstem and cerebellum, but levels were very low. In the amygdala, gonadal/hormonal status regulated aromatase expression in both sexes; in the BNST and POA, castration of males down-regulated aromatase, whereas there was no effect of estradiol in ovariectomized females. In the dorsal hippocampus and cingulate cortex, there were no differences in aromatase levels between males and females or effects of gonadal/hormonal status. These findings demonstrate that long-form aromatase is expressed in brain regions that respond acutely to estrogens, such as the dorsal hippocampus, and that gonadal/hormonal regulation of aromatase differs among different brain regions.
We developed a systematic algorithmic solution for quantitative drug sensitivity scoring (DSS), based on continuous modeling and integration of multiple dose-response relationships in high-throughput compound testing studies. Mathematical model estimation and continuous interpolation makes the scoring approach robust against sources of technical variability and widely applicable to various experimental settings, both in cancer cell line models and primary patient-derived cells. Here, we demonstrate its improved performance over other response parameters especially in a leukemia patient case study, where differential DSS between patient and control cells enabled identification of both cancer-selective drugs and drug-sensitive patient sub-groups, as well as dynamic monitoring of the response patterns and oncogenic driver signals during cancer progression and relapse in individual patient cells ex vivo. An open-source and easily extendable implementation of the DSS calculation is made freely available to support its tailored application to translating drug sensitivity testing results into clinically actionable treatment options.
The BCR-ABL translocation is found in chronic myeloid leukemia (CML) and in Ph+ acute lymphoblastic leukemia (ALL) patients. Although imatinib and its analogues have been used as front-line therapy to target this mutation and control the disease for over a decade, resistance to the therapy is still observed and most patients are not cured but need to continue the therapy indefinitely. It is therefore of great importance to find new therapies, possibly as drug combinations, which can overcome drug resistance. In this study, we identified eleven candidate anti-leukemic drugs that might be combined with imatinib, using three approaches: a kinase inhibitor library screen, a gene expression correlation analysis, and literature analysis. We then used an experimental search algorithm to efficiently explore the large space of possible drug and dose combinations and identified drug combinations that selectively kill a BCR-ABL+ leukemic cell line (K562) over a normal fibroblast cell line (IMR-90). Only six iterations of the algorithm were needed to identify very selective drug combinations.The efficacy of the top forty-nine combinations was further confirmed using Ph+ and Ph- ALL patient cells, including imatinib-resistant cells. Collectively, the drug combinations and methods we describe might be a first step towards more effective interventions for leukemia patients, especially those with the BCR-ABL translocation.
The High Throughput Biomedicine (HTB) unit at the Institute for Molecular Medicine Finland FIMM was established in 2010 to serve as a national and international academic screening unit providing access to state of the art instrumentation for chemical and RNAi-based high throughput screening. The initial focus of the unit was multiwell plate based chemical screening and high content microarray-based siRNA screening. However, over the first four years of operation, the unit has moved to a more flexible service platform where both chemical and siRNA screening is performed at different scales primarily in multiwell plate-based assays with a wide range of readout possibilities with a focus on ultraminiaturization to allow for affordable screening for the academic users. In addition to high throughput screening, the equipment of the unit is also used to support miniaturized, multiplexed and high throughput applications for other types of research such as genomics, sequencing and biobanking operations. Importantly, with the translational research goals at FIMM, an increasing part of the operations at the HTB unit is being focused on high throughput systems biological platforms for functional profiling of patient cells in personalized and precision medicine projects.
Single-cell genomics is a powerful tool for exploring the genetic makeup of environmental microorganisms, the vast majority of which are difficult, if not impossible, to cultivate with current approaches. Here we present a comprehensive protocol for obtaining genomes from uncultivated environmental microbes via high-throughput single-cell isolation by FACS. The protocol encompasses the preservation and pretreatment of differing environmental samples, followed by the physical separation, lysis, whole-genome amplification and 16S rRNA–based identification of individual bacterial and archaeal cells. The described procedure can be performed with standard molecular biology equipment and a FACS machine. It takes <12 h of bench time over a 4-d time period, and it generates up to 1 μg of genomic DNA from an individual microbial cell, which is suitable for downstream applications such as PCR amplification and shotgun sequencing. The completeness of the recovered genomes varies, with an average of ∼50%.
Inositol-requiring enzyme 1 alpha (IRE1α) is a transmembrane sensor protein with both kinase and ribonuclease activity, which plays a crucial role in the unfolded protein response (UPR). Protein misfolding in the endoplasmic reticulum (ER) lumen triggers dimerization and subsequent trans-autophosphorylation of IRE1α. This leads to the activation of its endoribonuclease (RNase) domain and splicing of the mRNA of the transcriptional activator XBP1, ultimately generating an active XBP1 (XBP1s) implicated in multiple myeloma survival. Previously, we have identified human IRE1α as a target for the development of kinase inhibitors that could modulate the UPR in human cells, which has particular relevance for multiple myeloma and other secretory malignancies. Here we describe the development and validation of a 384-well high-throughput screening assay using DELFIA technology that is specific for IRE1α autophosphorylation. Using this format, a focused library of 2312 potential kinase inhibitors was screened, and several novel IRE1α kinase inhibitor scaffolds were identified that could potentially be developed toward new therapies to treat multiple myeloma.