Laura Turunen

Precision Cancer Medicine in the Acoustic Dispensing Era Ex Vivo Primary Cell Drug Sensitivity Testing

Cancer therapy is increasingly becoming individualized, but there are also big gaps between the molecular knowledge of individual cancers we can generate today and what can be applied in the clinic. In an attempt to bridge this knowledge gap between cancer genetic and molecular profiling and clinically useful information, an individualized systems medicine program has been established at the Institute for Molecular Medicine Finland (FIMM), University of Helsinki, and the Helsinki University Hospital. Central to this program is drug sensitivity and resistance testing (DSRT), in which responses of primary cancer cells to a comprehensive clinical oncology and signal transduction drug collection are monitored. The drug sensitivity information is used with molecular profiling to establish hypotheses on individual cancer-selective targeting drug combinations and their predictive biomarkers, which can be explored in the clinic. Here, we describe how acoustic droplet ejection is enabling DSRT in our cancer individualized systems medicine program to (1) generate consistent but configurable assay-ready plates and determine how this affects data quality, (2) flexibly prepare drug combination testing plates, (3) dispense reagents and cells to the assay plates, and (4) perform ultra-miniaturized follow-up assays on the cells from DSRT plates.

Methods for High-throughput Drug Combination Screening and Synergy Scoring

Gene products or pathways that are aberrantly activated in cancer but not in normal tissue hold great promises for being effective and safe anticancer therapeutic targets. Many targeted drugs have entered clinical trials but so far showed limited efficacy mostly due to variability in treatment responses and often rapidly emerging resistance. Toward more effective treatment options, we will need multi-targeted drugs or drug combinations, which selectively inhibit the viability and growth of cancer cells and block distinct escape mechanisms for the cells to become resistant. Functional profiling of drug combinations requires careful experimental design and robust data analysis approaches. At the Institute for Molecular Medicine Finland (FIMM), we have developed an experimental-computational pipeline for high-throughput screening of drug combination effects in cancer cells. The integration of automated screening techniques with advanced synergy scoring tools allows for efficient and reliable detection of synergistic drug interactions within a specific window of concentrations, hence accelerating the identification of potential drug combinations for further confirmatory studies.

Antiviral Properties of Chemical Inhibitors of Cellular Anti-Apoptotic Bcl-2 Proteins

Viral diseases remain serious threats to public health because of the shortage of effective means of control. To combat the surge of viral diseases, new treatments are urgently needed. Here we show that small-molecules, which inhibit cellular anti-apoptotic Bcl-2 proteins (Bcl-2i), induced the premature death of cells infected with different RNA or DNA viruses, whereas, at the same concentrations, no toxicity was observed in mock-infected cells. Moreover, these compounds limited viral replication and spread. Surprisingly, Bcl-2i also induced the premature apoptosis of cells transfected with viral RNA or plasmid DNA but not of mock-transfected cells. These results suggest that Bcl-2i sensitizes cells containing foreign RNA or DNA to apoptosis. A comparison of the toxicity, antiviral activity, and side effects of six Bcl-2i allowed us to select A-1155463 as an antiviral lead candidate. Thus, our results pave the way for the further development of Bcl-2i for the prevention and treatment of viral diseases.

The high throughput biomedicine unit at the institute for molecular medicine Finland: high throughput screening meets precision medicine.

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.

Discovery of MINC1, a GTPase-Activating Protein Small Molecule Inhibitor, Targeting MgcRacGAP

The Rho family of Ras superfamily small GTPases regulates a broad range of biological processes such as migration, differentiation, cell growth and cell survival. Therefore, the availability of small molecule modulators as tool compounds could greatly enhance research on these proteins and their biological function. To this end, we designed a biochemical, high throughput screening assay with complementary follow-up assays to identify small molecule compounds inhibiting MgcRacGAP, a Rho family GTPase activating protein involved in cytokinesis and transcriptionally upregulated in many cancers. We first performed an in-house screen of 20,480 compounds, and later we tested the assay against 342,046 compounds from the NIH Molecular Libraries Small Molecule Repository. Primary screening hit rates were about 1% with the majority of those affecting the primary readout, an enzyme-coupled GDP detection assay. After orthogonal and counter screens, we identified two hits with high selectivity towards MgcRacGAP, compared with other RhoGAPs, and potencies in the low micromolar range. The most promising hit, termed MINC1, was then examined with cell-based testing where it was observed to induce an increased rate of cytokinetic failure and multinucleation in addition to other cell division defects, suggesting that it may act as an MgcRacGAP inhibitor also in cells.

Abstract 3746: Novel therapeutic possibilities for chemorefractory ovarian cancer patients identified by functional ex vivo drug sensitivity testing of primary cells from ascites

Ovarian cancer (OvCa) is the sixth most common cancer in women and a leading cause of death from gynecologic diseases. Poor prognosis in OvCa is due to late diagnosis and acquired resistance to conventional therapy. A significant setback for OvCa treatment is the lack of reliable biomarkers and the lack of effective targeted therapies. Our aim is to discover novel therapeutic opportunities with approved and emerging drugs for OvCa and then translate actionable drug efficacies and combinations as personalized therapy suggestions for the clinic. We have previously pioneered drug sensitivity and resistance testing (DSRT) of patient cells from leukemias for personalized medicine and for drug repositioning (Pemovska et al., Cancer Discovery, 2013; Pemovska et al. Nature, in press, 2014). Here, we tested primary cell cultures from the ascites fluid of relapsed chemorefractory OvCa patient samples using the DSRT platform for 305 drugs, consisting of both emerging and established cancer drugs. All drugs were tested in 5 concentrations to achieve a dose-response over a 10,000-fold concentration range. Both viability and cell toxicity assays were applied and results compared to a variety of normal cells and a database of >700 previously DSRT-tested cancer samples and cell lines of various types, including 30 OvCa cell lines. Genomic and transcriptomic profiling by next-gen sequencing was carried out in parallel. Most samples represent high-grade serous OvCa. Results from 5 primary ascites cultures tested so far showed a distinct drug sensitivity profile as compared to the 30 OvCa cell lines. The results on patient cells led to the discovery of previously unanticipated therapeutic possibilities. For example, in a 51-year old chemorefractory serous OvCa patient, genomic and transcriptomic analyses revealed a fusion gene of NRG-1, a target that was recently reported to involve the NRG1/ERBB3 activation loop in OvCa (Sheng et al. Cancer Cell, 2010). We found high expression of ERBB2 and ERBB3 by RNA seq and phospho-ERBB3, phospho-ERBB2 and phospho-EGFR by immunohistochemistry. In agreement with the molecular mechanism, DSRT analysis identified significant sensitivity of patient tumor cells to EGFR inhibitors, such as erlotinib and afatinib. Furthermore, drug combination testing identified highest combinatorial potential of dasatinib with erlotinib and afatinib in both viability and cell killing assays. In conclusion, DSRT testing together with genomic and transcriptomic profiling can identify and mechanistically validate tumor driver signals and pinpoint clinically actionable inhibitors and their combinations. Thus, this type of systems medicine profiling can significantly expand the power of current exclusively genomics-oriented personalized medicine approaches and help in drug repositioning to new indications. Citation Format: Astrid Murumagi, Akira Hirasawa, Mariliina Arjama, Katja Valimaki, Bhagwan Yadav, Jing Tang, Agnieszka Szwajda, Laura Turunen, John Patrick Mpindi, Teijo Pellinen, Krister Wennerberg, Ralf Butzow, Tero Aittokallio, Olli Kallioniemi. Novel therapeutic possibilities for chemorefractory ovarian cancer patients identified by functional ex vivo drug sensitivity testing of primary cells from ascites. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3746. doi:10.1158/1538-7445.AM2015-3746

Abstract 5588: Functional drug sensitivity and resistance profiling of AML patient cells defines a disease-specific combination of druggable signal addictions.

Numerous underlying molecular events have been described in acute myeloid leukemia (AML), but still, the fundamental disease mechanisms are poorly understood. Several targeted therapies have been investigated for improved AML therapy but have not succeeded to date, likely due to the inability to identify the patient subgroups that are most likely to benefit. Here, we describe functional profiling of AML patient cells ex vivo with a drug sensitivity and resistance (DSRT) platform in order to distinguish disease- and patient-specific molecular vulnerabilities and individualized therapeutic strategies. The oncology drug collection covers 306 anti-cancer agents including 131 approved, 107 investigational and 68 experimental compounds. Each compound is tested in a dose response series allowing for calculation of drug sensitivity scores. The functional exploration of AML patient samples was accompanied with comprehensive molecular profiling and clinical background data to link drug sensitivities to molecular aberrations and predictive biomarkers. Comparison of the drug sensitivity profiles of 24 AML patient and 5 control samples revealed that targeted inhibitors often exhibit no to little effect in the controls and the majority of AML patient samples with only a subset of patients showing very selective responses, indicating that cancer specific vulnerabilities can be detected with the DSRT platform. Clustering of the drug responses among patient and control samples identified distinct subgroups of patients and drugs. Each of these groups could be defined by a separate drug class, implying that the linked samples were addicted to the corresponding signaling networks. Specifically, we saw select addictions to drug classes such as dasatinib/VEGFR TKIs, MEK inhibitors, rapalogs, JAK inhibitors, mTOR inhibitors in 6 (25 %), 5 (21%), 5 (21%), 5 (21%), and 6 (25%) of the patients, respectively. Together, these sensitivities highlight a set of disease-specific and individualized signaling nodes that can serve as starting points for personalized combination therapy strategies in AML. Further, integration of DSRT results with molecular and clinical profiling link some of the selective drug responses to clinically actionable markers. For example, dasatinib sensitivity was found to be predominant in AML M5 patients, suggesting that this drug is a promising therapeutic candidate for relapsed and refractory AML M5 patients. In conclusion, the DSRT platform can be used as a functional interrogation of patient cells leading to the identification of druggable vulnerabilities that can be used for personalized medicine strategies. Furthermore, insight into disease-specific signal addictions and establishment of how these are linked can aid in deconvoluting the complex molecular disease mechanisms of cancers and generate strategies for novel drug development. Citation Format: Tea Pemovska, Bhagwan Yadav, Riikka Karjalainen, Evgeny Kulesskiy, Mika Kontro, Muntasir Mamun Majumder, Laura Turunen, Ida Lindenschmidt, Anna Lehto, Jonathan Knowles, Caroline Heckman, Kimmo Porkka, Tero Aittokallio, Olli Kallioniemi, Krister Wennerberg. Functional drug sensitivity and resistance profiling of AML patient cells defines a disease-specific combination of druggable signal addictions. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5588. doi:10.1158/1538-7445.AM2013-5588

Abstract 65: Comprehensive ex vivo drug sensitivity testing combined with in depth molecular profiling of AML patients cells provides individualized treatment strategies and reveals mechanisms of drug resistance.

Acute myeloid leukemia (AML) is an aggressive, heterogeneous disease with few options for targeted therapy. Here, we describe a novel translational strategy termed Individualized Systems Medicine (ISM), in which we profile primary AML patient cells functionally, molecularly and clinically to identify novel treatment strategies for patients, monitor and predict disease progression and follow-up therapies, and elucidate drug response and resistance mechanisms. We developed a comprehensive ex vivo drug sensitivity and resistance testing (DSRT) strategy to screen AML patient blast cells ex vivo against a set of 202 conventional chemotherapeutic and targeted approved (n=119) and investigational (n=83) drugs. Quantitative leukemia-selective drug sensitivity scores for each drug were determined by comparing the area under the dose response curve from the patient cells to that of healthy control mononuclear cells. Analysis of consecutive samples from the same patients with DSRT and next-generation sequencing was applied to infer clonal evolution and potential mechanisms of drug response and resistance. Twenty-four samples from 16 recurrent and refractory AML patients were profiled by DSRT, sequencing and proteomic approaches. Several approved and late stage clinical investigated targeted drugs including multi-kinase inhibitors (e.g. dasatinib, sunitinib), TORC1 inhibitors (e.g. temsirolimus), JAK inhibitors (e.g. ruxolitinib) and MEK inhibitors (e.g. trametinib, selumetinib) showed selective leukemic-specific responses in 10-30% of AML samples from patients with recurrent disease. In two refractory AML cases where dasatinib, sunitinib and temsirolimus showed selective responses, the clinical administration of these compounds resulted in complete and partial remission, but was followed by resistance to the applied drugs. Re-sampling and DSRT retesting of cells confirmed diminished sensitivities to the administered drugs, but also indicated new acquired drug sensitivities. Exome and RNA sequencing of the serial samples from both patients revealed diverse subclonal populations characterized by multiple somatic mutations, which were either lost or gained during disease progression and represented drug sensitive or resistant subclones. In conclusion, our results suggest that an ISM strategy based on consecutive cancer sampling, ex vivo DSRT and analysis of clonal evolution could facilitate the rapid design of improved combinatorial therapies for AML. This strategy can also help tailor optimized therapies for patients, and prioritize introduction of new drugs for clinical testing. Citation Format: Krister Wennerberg, Tea Pemovska, Mika Kontro, Bhagwan Yadav, Evgeny Kulesskiy, Henrik Edgren, Samuli Eldfors, Riikka Karjalainen, Naga Poojitha Kota Venkata, Anna Lehto, Muntasir Mamun Majumder, Disha Malani, Astrid Murumagi, Laura Turunen, Jonathan Knowles, Tero Aittokallio, Caroline Heckman, Kimmo Porkka, Olli Kallioniemi. Comprehensive ex vivo drug sensitivity testing combined with in depth molecular profiling of AML patients cells provides individualized treatment strategies and reveals mechanisms of drug resistance. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 65. doi:10.1158/1538-7445.AM2013-65

Abstract 3188: Development of a cancer pharmacopeia-wide ex-vivo drug sensitivity and resistance testing (DSRT) platform for AML: Towards individually optimized therapy and improved understanding of drug resistance patterns

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL In order to discover unexpected anti-cancer efficacies of approved and emerging drugs, we established a diagnostic ex vivo drug sensitivity and resistance testing (DSRT) platform covering the entire cancer pharmacopeia as well as emerging anti-cancer compounds. Here, the platform was applied to analyze bone marrow (BM) mononuclear cells from 17 adult acute myeloid leukemia (AML) patients, 3 healthy donors as well as 7 AML cell lines. The DSRT panel covered FDA-approved small molecule oncology drugs (n=120), as well as emerging, investigational and pre-clinical oncology compounds (n=120), such as kinase (e.g. RTKs, checkpoint and mitotic kinases, Raf, MEK, JAKs, mTOR, PI3K), and non-kinase inhibitors (e.g. HSP, Bcl, activin, HDAC, PARP, Hh). To generate dose-response curves, each of the drugs was applied over a 10,000-fold concentration range. In addition, the samples underwent deep molecular profiling including exome- and transcriptome sequencing, as well as phosphoproteomic analysis. DSRT provided consistent and reliable data from ex vivo samples with a high correlation between data from individual healthy BM samples (r=0.93). Bioinformatic processing of the data from AML resulted in several key observations. First, overall drug response profiles of AML blast cells were distinctly different from healthy BM controls suggesting several potential leukemia-selective effects, such as multi-kinase (dasatinib), MEK, and mTOR inhibitors. Second, the overall drug responses from AML cell lines and the patient ex vivo samples showed differences, suggesting that ex vivo testing may reveal cancer-selective effects not previously seen in established cancer cell line panels. Third, the response data from patient samples clustered many drugs consistently into the expected functional classes (such as topoisomerase II inhibitors, MEK inhibitors and rapalogs), whereas other drug classes were more dispersed (such as FLT3 inhibitors with quizartinib clustering away from all other tyrosine kinase inhibitors), suggesting secondary targets playing a key role in drug efficacy. Fourth, analysis of serial samples from patients developing clinical resistance to targeted agents showed striking agreement between the ex-vivo DSRT profiles and clinical responses. In conclusion, comprehensive DSRT platform generated powerful novel insights on AML drug response and may enable individual optimization of therapies, particularly for recurrent leukemias. DSRT will also serve as a powerful hypothesis-generator for clinical trials, particularly for emerging drugs. The ability to correlate ex vivo response profiles for hundreds of drugs in clinical samples with deep molecular profiling data will yield exciting new translational and pharmacogenomic opportunities for cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3188. doi:1538-7445.AM2012-3188

Abstract 895: Quantitative drug sensitivity and resistance testing (DSRT) of primary ex vivo AML blasts highlights mTOR and MEK as potential key molecular driver signals of therapeutic significance

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Identification of signaling pathways that are required for the growth and differentiation block of cells from adult acute myeloid leukemia (AML) is urgently required to facilitate development of novel therapies. Here, we describe an approach to functionally determine molecular drivers of AML by quantitative drug sensitivity and resistance testing (DSRT) of AML blast cells in primary culture ex vivo. The selection of drugs covered the entire cancer pharmacopeia and much of the pipeline of drugs under development in the industry: 120 FDA approved small molecular cancer drugs and 120 emerging drugs, investigational compounds and signal transduction inhibitors. All compounds were tested over a 10,000-fold concentration range to generate quantitative and reliable dose-response data. In addition, whole exome and transcriptome sequencing and phophoproteomic profiling were also performed to derive a comprehensive understanding of the molecular AML-related aberrations on an individual basis. Comparison of 17 AML patient samples and 3 healthy bone marrow control samples based on ex vivo drug responses identified several classes of approved and investigational drugs that showed selective anti-AML activities: mTOR inhibitors (e.g. temsirolomus, everolimus, sirolimus), MEK inhibitors (e.g. AS703026, GSK1120212, RDEA119, selumetinib), tyrosine kinase inhibitors (e.g. dasatinib, ponatinib, sunitinib), Bcl-2 inhibitors (navitoclax) and HSP90 inhibitors (e.g. BIIB021, NVP-AUY922, tanespimycin). In particular, the rapamycin class of mTOR inhibitors and allosteric MEK inhibitors stood out as effective and selective inhibitors in 8/17 (47%) and 9/17 (52%) of the patients, respectively. Simultaneous data from other targeted inhibitors made it possible to dissect the critical steps in signaling and therapeutic efficacy. For example, PI3K and Akt inhibitors were not effective in these patients, suggesting that the mTOR dependency is mediated through a PI3K-Akt-independent pathway. Similarly, the dependency of MEK signaling appears to be through a Ras-Raf-independent pathway since Raf inhibitors were not effective. In conclusion, the DSRT platform allows us to derive quantitative data on the ex vivo drug response profiles of AML cells from individual patients. This information could be used as a diagnostic tool to optimize personalized therapies in the future. Our data demonstrate that mTOR and MEK signaling and the associated inhibitors are the most promising leads for improved AML therapeutics. This analysis also demonstrates gaps in our current understanding of the redundancy of key cancer cell signaling pathways and proves the significant value of data from experimental drug response testing ex vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 895. doi:1538-7445.AM2012-895