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  • 1
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    High-Throughput Drug Sensitivity and Resistance Testing (DSRT) Platform Reveals Novel Candidate Drugs For Advanced Phase BCR-ABL1-Positive Leukemia
    American Society of Hematology ; 2013
    In:  Blood Vol. 122, No. 21 ( 2013-11-15), p. 2719-2719
    Details
    In: Blood, American Society of Hematology, Vol. 122, No. 21 ( 2013-11-15), p. 2719-2719
    Abstract: Advanced BCR-ABL1-positive leukemias (chronic myeloid leukemia in blast crisis and Ph+ALL) remain a therapy challenge despite advances in tyrosine kinase inhibitor (TKI) therapy. Emergence of primary and secondary resistance due to gatekeeper and compound mutations within the BCR-ABL1 kinase domain is common even with the novel 2nd and 3rd generation TKIs (dasatinib, nilotinib, ponatinib). We set out to identify novel candidate drugs for advanced BCR-ABL1-positive leukemias by using an unbiased high-throughput drug testing platform and utilizing both primary patient cells and cell lines. Methods As a study material we used 3 CML cell lines representing different types of CML blast phases. In addition to commonly used K562 cells, EM-2 and MOLM-1 cell lines were tested. AML cell lines (AML-193, AP-1060, HL60ATCC, HL60TB, Kasumi-1, KG-1, ME-1, MOLM-13, MONO-MAC-6, MUTZ-2, MV4-11, NOMO-1, SH-2, SHI-1, SIG-M5, SKM-1, THP-1) were used as cell line controls. To verify the results obtained from cell lines, primary bone marrow (BM) cells were derived from 2 TKI-resistant CML BC patients. Patient 1 had developed resistance to imatinib and dasatinib due to a T315I mutation, whereas patient 2 was resistant to nilotinib, dasatinib and ponatinib due to a V299L and a compound mutation. BM cells from 4 healthy individuals were used as controls. The functional profiling of drug responses was performed with a high-throughput drug sensitivity and resistance testing (DSRT) platform comprising of 306 anti-cancer agents (FDA/EMA approved, investigational and experimental compounds). Cells were dispensed to pre-drugged 386-well plates of 5 different concentrations and incubated in a humidified incubator with 5% CO2 at 37 °C for 72 hours. Cell viability was measured by using a luminescent cell viability assay (CellTiter-Glo). From plate reads a Drug Sensitivity Score (DSS) was calculated for each drug as a measure of cytotoxicity. In addition to DSRT, Human Phospho-Kinase Array Kit (R & D systems) was used to analyze the phosphokinase profile in patient samples. Results Based on initial comparisons between CML and AML cells lines, nonspecific cytotoxic drugs, which showed high activity in all cell lines, were omitted from further analysis. The DSS scores from different CML cells lines correlated relatively closely (EM-2 vs. K-562, r=0.89; EM-2 vs. MOLM-1, r=0.82; K-562 vs. MOLM-1, r=0.78; p 〈 0.0001 for all correlations). We next ranked the DSRT data according to the DSS values with most sensitive drugs showing the highest DSS scores. The primary cells from CML BC were further normalized against the median values from healthy controls, resulting in leukemia-specific sensitivity scores (sDSS). Ranked results from the DSRT analysis are shown in the Table. As expected, the cell lines were sensitive to TKIs, with the exception of the MOLM-1, which showed only modest sensitivity. The clinically TKI-resistant patient samples were also TKI-resistant ex vivo, further validating the DSRT assay data. Drugs which showed efficacy in both the cell lines and the TKI-resistant patients included HSP90 inhibitors (NVP-AUY922, BIIB021), a NAMPT inhibitor daporinad and the protein translation inhibitor omacetaxine (homoharringtonine). Phosphokinase antibody array results from the patient samples showed increased expression of the HSP27 protein as a putative biomarker for HSP90 inhibitor response. Conclusions DSRT is a powerful assay for identifying novel candidate molecules for refractory BCR-ABL1-positive leukemias. Our results indicate that HSP90 and NAMPT inhibitors in particular warrant further clinical evaluation both by analyzing a larger set of primary patient samples and by performing proof-of-concept clinical studies. The results also pave way for designing rational combination therapy strategies. Disclosures: Mustjoki: Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau. Porkka:BMS: Consultancy, Research Funding, Speakers Bureau; Novartis: Consultancy, Research Funding, Speakers Bureau.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V122.21.2719.2719
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    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2013
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  • 2
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    Exome Sequencing of Aggressive Natural Killer Cell Leukemia and Drug Profiling Highlight Candidate Driver Pathways in Malignant Natural Killer Cells
    American Society of Hematology ; 2015
    In:  Blood Vol. 126, No. 23 ( 2015-12-03), p. 700-700
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    In: Blood, American Society of Hematology, Vol. 126, No. 23 ( 2015-12-03), p. 700-700
    Abstract: Background Natural killer (NK) cell malignancies are rare lymphoid neoplasms characterized by aggressive clinical behavior and poor treatment outcomes. Clinically they are classified as extranodal NK/T-cell lymphoma, nasal type (NKTCL) and aggressive NK cell leukemia (ANKL). Both subtypes are almost invariably associated with Epstein-Barr virus (EBV). Recently, genomic studies in NKTCL have identified recurrent somatic mutations in JAK-STAT pathway molecules STAT3 and STAT5b as well as in the RNA helicase gene DDX3X in addition to previously detected chromosomal aberrations. Here, we identified somatic mutations in 4 cases of ANKL in order to understand whether these entities share common alterations at the molecular level. To further establish common patterns of deregulated oncogenic signaling pathways operating in malignant NK cells, we performed drug sensitivity profiling using NK cell lines representing ANKL, NKTCL and other malignant NK cell proliferations. We aimed to identify sensitivities to agents that selectively target components of pathways required for survival of malignant NK cells in an unbiased manner. Methods Exome sequencing was performed on peripheral blood or bone marrow of ANKL patients using the NK cell negative fraction or other healthy tissue as control. Profiling of drug responses was performed with a high-throughput drug sensitivity and resistance testing (DSRT) platform comprising 461 approved and investigational oncology drugs. The NK cell lines KAI3, KHYG-1, NKL, NK-YS, NK-92, SNK-6 and YT and IL-2-stimulated and resting NK cells from healthy donors were used as sample material. All drugs were tested on a 384-well format in 5 different concentrations over a 10,000-fold concentration range for 72 h and cell viability was measured. A Drug Sensitivity Score (DSS) was calculated for each drug using normalized dose response curve values. Results The ANKL patients displayed mutations in genes reported as recurrently mutated in NKTCL, such as FAS, TP53, NRAS, STAT3 and DDX3X. Additionally, novel alterations in genes previously implicated in the pathogenesis of NKTCL were detected. These included an inactivating mutation in INPP5D (SHIP), a negative regulator of the PI3K/mTOR pathway and a missense mutation in PTPRK, a negative regulator of STAT3 activation. Interestingly, the total number of nonsilent somatic mutations in 3 out of 4 ANKL patients (97, 82 and 45) was remarkably high compared to other hematological malignancies analyzed in our variant calling pipeline. Analysis of drug sensitivities in NK cell lines showed a close correlation between all cell lines and a markedly higher correlation with those of IL-2 stimulated than resting healthy NK cells, suggesting that malignant NK cells may share a common drug response pattern. Furthermore, in an unsupervised hierarchical clustering the NK cell lines formed a distinct group from other leukemia cell lines tested (Fig. A). Among pathway-selective compounds (namely, kinase inhibitors and rapalogs), the drugs most selective for malignant NK cells fell into two major categories: PI3K/mTOR inhibitors (e.g. temsirolimus, buparlisib) and inhibitors of aurora and polo-like kinases such as rigosertib and GSK-461364 (Fig. B). JAK inhibitors (e.g. ruxolitinib, gandotinib) and CDK inhibitors (e.g. dinaciclib) showed strong efficacy in both malignant NK cells and IL-2 activated healthy NK cells. Conclusions Our exome sequencing results suggest that candidate driver alterations affecting similar signaling pathways underlie the pathogenesis of ANKL as has been reported in NKTCL. Drug sensitivity profiling highlights the PI3K/mTOR pathway as a potential major driver of malignant NK cell proliferation, whereas JAK-STAT signaling appears to be essential in both healthy and malignant NK cells. Components of these pathways harbored mutations in our small cohort of ANKL patients and have been shown to be deregulated by mutations or other mechanisms in previous studies, underlining their importance as putative drivers. The systematic large-scale characterization of drug responses also identified these pathways as potential targets for novel therapy strategies in NK cell malignancies. Figure 1. (A) Unsupervised hierarchical clustering based on drug sensitivity scores (DSS) of NK, AML, CML and T-ALL cell lines. (B) Scatter plot comparing DSS of malignant NK cell lines (average) and healthy IL-2 stimulated NK cells. Figure 1. (A) Unsupervised hierarchical clustering based on drug sensitivity scores (DSS) of NK, AML, CML and T-ALL cell lines. (B) Scatter plot comparing DSS of malignant NK cell lines (average) and healthy IL-2 stimulated NK cells. Disclosures Mustjoki: Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V126.23.700.700
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    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2015
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  • 3
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    Identification and Clinical Exploration of Individualized Targeted Therapeutic Approaches in Acute Myeloid Leukemia Patients By Integrating Drug Response and Deep Molecular Profiles
    American Society of Hematology ; 2017
    In:  Blood Vol. 130, No. Suppl_1 ( 2017-12-07), p. 854-854
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    In: Blood, American Society of Hematology, Vol. 130, No. Suppl_1 ( 2017-12-07), p. 854-854
    Abstract: Introduction Most patients with acute myeloid leukemia (AML) are still missing effective options for targeted treatments. Here, we applied individualized systems medicine (ISM) concept1 by integrating deep molecular profiles (genomics, transcriptomics) and ex vivo drug response profiles with 521 oncology drugs in 154 AML patient samples. The aim was to identify new treatment opportunities for molecular subsets of AML patients. When feasible, ISM guided treatment opportunities were applied clinically for AML patient treatment. Serial samples were available to identify molecular alterations in response to targeted drug treatment and to monitor therapeutic success or failure. We also aimed at testing the impact of bone marrow stromal cell conditioned media on drug response profiles in AML patients2. Methods Samples from bone marrow or blood of 122 AML patients and 17 healthy donors were obtained with written consent and ethical approval (239/13/03/00/2010 and 303/13/03/01/2011) from the Hematology Clinic, Comprehensive Cancer Center, Helsinki University Hospital. The ex vivo drug sensitivity and resistance testing (DSRT) assay was performed with 521 approved oncology drugs and investigational oncology compounds as described earlier1. In this study, freshly isolated mononuclear cells were randomly resuspended either in standard mononuclear cell medium (MCM, PromoCell) or in human bone marrow stroma derived conditioned medium (CM) for drug testing. DNA samples from same mononuclear cells were subjected to whole exome and transcriptome sequencing and data were analyzed as described previsously2. Hierarchical clustering and non-parametric rank correlation were performed with drugs and samples. Wilcoxon sign ranked test was applied between wild type and mutated samples to identify significant mutation-drug associations. Results Hierarchical clustering was largely independent of clinical features such as disease status or risk class. A strong drug sub-cluster with a unique response profile was composed of that of the MDM2 antagonist idasanutlin along with BCL-2 inhibitors navitoclax and venetoclax (Figure). BET inhibitors (JQ1, I-BET151, birabresib) and MEK inhibitors (trametinib, selumetinib) were positively correlated with each other suggesting an association between bromodomain mediated epigenetic deregulation and up-regulation of the MEK pathway in a subset of patients. Comparison between patient samples profiled in CM (n=77) vs MCM medium (n=77) indicated higher efficacy of MDM2 modulator idasanutlin in MCM while BET inhibitors responded more strongly in CM. Other differences observed earlier by Karjalainen et al1 between the two media types were also validated. Furthermore, 16 chemorefractory and one diagnostic stage patients were treated with the targeted drugs suggested by this ISM approach. We observed complete remission or leukemia free state in 35% (6/17) of the AML patients given tailored treatment in an observational study. The targeted drugs used for clinical translation included ruxolitinib (in n=4 patients), temsirolimus (n=5), trametinib (n=4), sunitinib (n=7), dasatinib (n=7), sorafeninb (n=4), omacetaxine (n=3) and dexamethasone (n=5). Summary This study highlights the potential of individualized systems medicine (ISM) approach in the identification of effective treatment opportunities for individual patients with AML. Identifying molecular markers for ex vivo drug responses can help to assign treatments to the patient subgroups most likely to respond in clinical trials. Figure Figure. Disclosures Heckman: Orion Pharma: Research Funding; Novartis: Research Funding; IMI2 project HARMONY: Research Funding; Pfizer: Research Funding; Celgene: Research Funding. Porkka: Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V130.Suppl_1.854.854
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2017
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  • 4
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    AML Specific Targeted Drugs Identified By Drug Sensitivity and Resistance Testing: Comparison of Ex Vivo Patient Cells with in Vitro Cell Lines
    American Society of Hematology ; 2014
    In:  Blood Vol. 124, No. 21 ( 2014-12-06), p. 2163-2163
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    In: Blood, American Society of Hematology, Vol. 124, No. 21 ( 2014-12-06), p. 2163-2163
    Abstract: Introduction Many drug discovery efforts and pharmacogenomic studies are based on testing established cancer cell lines for their sensitivity to a given drug or a panel of drugs. This approach has been criticized due to high selectivity and fast proliferation rate of cancer cell lines. To explore new therapeutic avenues for acute myeloid leukemia (AML) and to compare experimental model systems, we applied high-throughput Drug Sensitivity and Resistance Testing (DSRT) platform with 305 approved and investigational drugs for 28 established AML cell lines and compared their drug responses with our earlier study of 28 ex vivo AML patient samples (Pemovska et al., 2013). We then correlated drug sensitivities with genomic and molecular profiles of the samples. Methods DSRT was carried out with 305 clinical, emerging and experimental drugs and small molecule chemical inhibitors. The drugs were tested at five different concentrations over a 10,000-fold concentration range. Cell viability was measured after 72 hours using Cell Titre Glow assay. IC50 values were calculated with Dotmatics software and drug sensitivity scores (DSS, a modified area under the curve metric) were derived for each drug (Yadav et al., 2014). Nimblegen's SeqCap EZ Designs Comprehensive Cancer Design kit was used to identify mutations from 578 oncogenes in cell lines. Results The 28 established AML cell lines were in general more sensitive to the drugs as compared to the 28 ex vivo patient samples, with some important exceptions. Sensitivity towards many targeted drugs was observed in both AML cell lines and in patient samples. These included inhibitors of MEK (e.g. trametinib in 56% of cell lines and 36% of ex vivo samples), mTOR (e.g. temsirolimus in 42% and 32%) and FLT3 (quizartinib in 28% and 18%). Overall, drug responses between cell lines and ex vivo patient cells in AML showed an overall correlation coefficient of r=0.81. BCL2 inhibitors (venetoclax and navitoclax) showed more sensitivity in ex vivo patient cells than in AML cancer cell lines, whereas responses to anti-mitotic agents (docetaxel, camptothecin, vincristine) showed stronger responses in cell lines (Figure). Only 7% of AML cell lines exhibited responses to a broad-spectrum tyrosine kinase inhibitor dasatinib, in contrast to 36% patient samples. AML cell lines that carried FLT3 mutations showed high sensitivity to FLT3 inhibitors. Similarly, cell lines harbouring mutations in RAS or RAF were strongly sensitive to MEK inhibitors. MEK and FLT3 inhibitor responses were mutually exclusive, indicating alternative pathway dependencies in cell lines. However, these pharmacogenomics correlations were not as clearly seen in the clinical samples. Summary These data revealed a few important differences as well as many similarities between established AML cell lines and primary AML patient samples in terms of their response to a panel of cancer drugs. The hope is that patient-derived primary cells in ex vivo testing predict clinical response better as compared to the established cancer cell lines, which indeed seem to overestimate the likelihood of responses to many drugs. On the other hand, cancer cell line studies may also underestimate the potential of dasatinib and BCL2 inhibitors as emerging AML therapeutics. References 1. Pemovska T, Kontro M, Yadav B, Edgren H, Eldfors S, Szwajda A, et al. Individualized systems medicine strategy to tailor treatments for patients with chemorefractory acute myeloid leukemia. Cancer Discovery. 2013 Dec;3(12):1416-29 2. Yadav B, Pemovska T, Szwajda A, Kulesskiy E, Kontro M, Karjalainen R, et al. Quantitative scoring of differential drug sensitivity for individually optimized anticancer therapies. Scientific reports. 2014;4:5193. Figure: Correlation of average drug responses (n=305) between 28 AML cell lines and 28 AML ex vivo patient samples Figure:. Correlation of average drug responses (n=305) between 28 AML cell lines and 28 AML ex vivo patient samples Disclosures Heckman: Celgene: Research Funding. Porkka:BMS: Honoraria; BMS: Research Funding; Novartis: Honoraria; Novartis: Research Funding; Pfizer: Research Funding. Kallioniemi:Medisapiens: Consultancy, Membership on an entity's Board of Directors or advisory committees.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V124.21.2163.2163
    RVK:
    XA 33000
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    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2014
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  • 5
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    Development of a Cancer Pharmacopeia-Wide Ex-Vivo Drug Sensitivity and Resistance Testing (DSRT) Platform: Identification of MEK and mTOR As Patient-Specific Molecular Drivers of Adult AML and Potent Therapeutic Combinations with Dasatinib
    American Society of Hematology ; 2011
    In:  Blood Vol. 118, No. 21 ( 2011-11-18), p. 2487-2487
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    In: Blood, American Society of Hematology, Vol. 118, No. 21 ( 2011-11-18), p. 2487-2487
    Abstract: Abstract 2487 Introduction: The molecular drivers of adult AML as well as the determinants of drug response are poorly understood. While AML genomes have recently been sequenced, many cases do not harbor druggable mutations. Treatment options are particularly limited for relapsed and refractory AML. Due to the molecular heterogeneity of the disease, optimal therapy would likely consist of individualized combinations of targeted and non-targeted drugs, which poses significant challenges for the conventional paradigm of clinical drug testing. In order to better understand the molecular driver signals, identify individual variability of drug response, and to discover clinically actionable therapeutic combinations and future opportunities with emerging drugs, we established a diagnostic ex-vivo drug sensitivity and resistance testing (DSRT) platform for adult AML covering the entire cancer pharmacopeia as well as many emerging anti-cancer compounds. Methods: DSRT was implemented for primary cells from adult AML patients, focusing on relapsed and refractory cases. Fresh mononuclear cells from bone marrow aspirates ( 〉 50% blast count) were screened in a robotic high-throughput screening system using 384-well plates. The primary screening panel consisted of a comprehensive collection of FDA/EMA-approved small molecule and conventional cytotoxic drugs (n=120), as well as emerging, investigational and pre-clinical oncology compounds (currently n=90), such as major 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). The drugs are tested over a 10,000-fold concentration range resulting in a dose-response curve for each compound and with combinations of effective drugs explored in follow-up screens. The same samples also undergo deep molecular profiling including exome- and transcriptome sequencing, as well as phosphoproteomic analysis. Results: DSRT data from 11 clinical AML samples and 2 normal bone marrow controls were bioinformatically processed and resulted in several exciting observations. First, overall drug response profiles of the AML samples and the controls were distinctly different suggesting multiple leukemia-selective inhibitory effects. Second, the MEK and mTOR signaling pathways emerged as potential key molecular drivers of AML cells when analyzing targets of leukemia-specific active drugs. Third, potent new ex-vivo combinations of approved targeted drugs were uncovered, such as mTOR pathway inhibitors with dasatinib. Fourth, data from ex-vivo DSRT profiles showed excellent agreement with clinical response when serial samples were analyzed from leukemia patients developing clinical resistance to targeted agents. Summary: The rapid and comprehensive DSRT platform covering the entire cancer pharmacopeia and many emerging agents has already generated powerful insights into the molecular events underlying adult AML, with significant potential to facilitate individually optimized combinatorial therapies, particularly for recurrent leukemias. DSRT will also serve as a powerful hypothesis-generator for clinical trials, particularly for emerging drugs and drug combinations. The ability to correlate response profiles of hundreds of drugs in clinical ex vivo samples with deep molecular profiling data will yield exciting new translational and pharmacogenomic opportunities for clinical hematology. Disclosures: Mustjoki: Novartis: Honoraria; Bristol-Myers Squibb: Honoraria. Porkka:Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding. Kallioniemi:Abbot/Vysis: Patents & Royalties; Medisapiens: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Bayer Schering Pharma: Research Funding; Roche: Research Funding.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V118.21.2487.2487
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    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2011
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  • 6
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    Mutational Landscape of Aggressive Natural Killer Cell Leukemia and Drug Sensitivity Profiling Reveal Therapeutic Options in Natural Killer Cell Malignancies
    American Society of Hematology ; 2016
    In:  Blood Vol. 128, No. 22 ( 2016-12-02), p. 2921-2921
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    In: Blood, American Society of Hematology, Vol. 128, No. 22 ( 2016-12-02), p. 2921-2921
    Abstract: INTRODUCTION Natural killer (NK) cell malignancies are rare aggressive neoplasms that are classified by the WHO as extranodal NK/T-cell lymphoma, nasal type (NKTCL) and aggressive NK-cell leukemia (ANKL). Recently, genome and exome level studies in NKTCL have shed light on the mutational spectrum of the disease. However, somatic mutations in ANKL have not been characterized. Here, we identified somatic mutations in 14 cases of ANKL to further clarify the genetic landscape underlying malignant NK cell proliferation. We compared the discovered variants to those detected in NKTCL to understand whether the two diseases harbor common molecular alterations. Moreover, we used high-throughput drug screening and RNA sequencing on NK cell lines derived from ANKL, NKTCL and other malignant NK cell proliferations to identify therapeutically actionable drivers of malignant NK cell growth. METHODS We performed whole-exome sequencing on genomic DNA extracted from peripheral blood or bone marrow samples of 14 ANKL patients. To compare the mutational profiles in ANKL and NTKCL, we re-analyzed the published whole-exome NKTCL data from Jiang et al. (Nat Genet 2015) using our somatic variant calling pipeline. For profiling of drug responses, we used a high-throughput drug sensitivity and resistance testing (DSRT) platform comprising 461 approved and investigational oncology drugs to screen the ANKL cell lines IMC-1, KHYG-1 and NK-92, NKTCL cell lines NK-YS and SNK-6 as well as DERL-7, KAI3, NKL, YT and IL-2-stimulated NK cells from healthy donors. All drugs were tested in 384-well format in 5 concentrations over a 10,000-fold concentration range for 72 h, cell viability was measured and normalized dose response curve values were used to calculate a drug sensitivity score (DSS) for each drug. Finally, we performed amplicon sequencing of known cancer driver genes and RNA sequencing on the cell lines and healthy NK cells to identify driver mutations and integrate gene expression profiles with drug sensitivity patterns. RESULTS We identified recurrent somatic activating mutations in STAT3 in 21% (3 of 14) of ANKL patients. Other mutated genes included RAS-MAPK pathway molecules (BRAF, NRAS, KRAS), protein phosphatases regulating JAK-STAT and PI3K-AKT-mTOR pathways (PTPRT, PTPRK, INPP5D) as well as several epigenetic modifiers (TET2, ARID2, KDM2B, SETD7, SETD2) and the tumor suppressor TP53. Interestingly, we detected mutations in genes recurrently mutated in NKTCL, such as the RNA helicase DDX3X and the cell surface receptor FAS. Re-analysis of the published NKTCL data revealed a high frequency of missense mutations in receptor type and non-receptor type protein phosphatases (e.g. PTPRC, PTPRR, PTPRT, PTPN1, PTPN2, PTPN3), many of which have established roles as negative regulators of JAK-STAT signaling. These findings potentially expand the subset of NK cell tumors where the JAK-STAT pathway is somatically activated and implicate deregulated JAK-STAT signaling as a major driver in these diseases. The malignant NK cell lines used in drug sensitivity profiling frequently harbored mutations in same genes and pathways, including STAT3 (N=3), STAT5B (N=1), DDX3X (N=2), KRAS (N=1), FAS (N=2) and several epigenetic modifiers, thus validating these cell lines as relevant disease models. The drug sensitivities in NK cell lines showed a high correlation and the cell lines formed a distinct group from other lymphoid and myeloid leukemia cell lines in unsupervised hierarchical clustering, suggesting an NK-cell specific drug response pattern. The most effective targeted drugs across all NK cell lines included HDAC inhibitors, inhibitors of Aurora and Polo-like kinases, JAK inhibitors, HSP inhibitors and CDK inhibitors as well as the Bcl-2 inhibitor navitoclax. Compared to other leukemia and lymphoma cell lines, JAK inhibitors, navitoclax and methotrexate emerged as the most NK-cell specific compounds. CONCLUSIONS Our genetic data demonstrate extensive heterogeneity in the mutational spectrum of ANKL and implicate JAK-STAT and RAS-MAPK signaling as well as disruption of epigenetic modifiers in the disease pathogenesis. Integrated drug sensitivity and gene expression profiling corroborates the JAK-STAT pathway as a major therapeutically actionable driver of malignant NK cell proliferation and identifies other potential novel targeted therapy options such as Bcl-2 inhibition in NK cell malignancies. Disclosures Suzumiya: Chugai: Honoraria, Research Funding; Toyama Chemical: Research Funding; Kyowa Hakko kirin: Research Funding; Astellas: Research Funding; Eisai: Honoraria, Research Funding; Takeda: Honoraria. Ohshima:Chugai: Research Funding, Speakers Bureau; Kyowa Kirin: Research Funding, Speakers Bureau. Mustjoki:Pfizer: Honoraria, Research Funding; Ariad: Research Funding; Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V128.22.2921.2921
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    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2016
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  • 7
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    Abstract 4679: Acquisition of cytarabine resistance leads to increased glucocorticoid sensitivity in AML
    American Association for Cancer Research (AACR) ; 2016
    In:  Cancer Research Vol. 76, No. 14_Supplement ( 2016-07-15), p. 4679-4679
    Details
    In: Cancer Research, American Association for Cancer Research (AACR), Vol. 76, No. 14_Supplement ( 2016-07-15), p. 4679-4679
    Abstract: Acquired resistance to standard chemotherapeutic agents, such as cytarabine, is a major challenge in the treatment of acute myeloid leukemia (AML). Here, we hypothesized that development of resistance to one chemotherapeutic agent may lead to increased sensitivity to other drugs. Hence, we sought to identify novel drug vulnerabilities that arise during the development of cytarabine resistance using both cytarabine resistant AML cell lines and samples from AML patients who had relapsed during cytarabine containing chemotherapy. We developed resistant variants of AML cell lines MOLM-13 and SHI-1 by long-term drug treatment with increasing doses of cytarabine. Profiling data from the in vitro generated cytarabine resistant cell line variants were systematically compared with corresponding data from 31 chemorefractory AML patient samples. All samples were subjected to genomic and transcriptomic profiling and high-throughput drug sensitivity and resistance testing with a panel of 250 chemical compounds (each in five doses). Cytarabine resistant AML cell line variants and patient samples showed co-resistance to other nucleoside analogues, such as cladribine, clofarabine and gemcitabine. Genomic profiling showed deletion of the deoxycytidine kinase gene DCK, a well-known genetic lesion related to cytarabine resistance, in both MOLM-13 and SHI-1 cytarabine resistant cell lines and in one chemorefractory AML patient. Importantly, comprehensive drug testing revealed that cytarabine resistant SHI-1 cell variants developed increased sensitivity to glucocorticoids, such as dexamethasone, methylprednisolone and prednisolone when compared to parental cells. This was accompanied by up-regulation of the glucocorticoid receptor NR3C1. We also observed acquisition of glucocorticoid sensitivity in paired samples from two AML patient cases who had relapsed after cytarabine containing chemotherapy. Systematic ex vivo drug testing of 31 relapsed and chemorefractory AML patient samples showed high sensitivity to dexamethasone in five (20%) and to prednisolone and methylprednisolone in four (13%) patient samples. In conclusion, our results from both cytarabine resistant AML cell lines and chemorefractory patient samples indicate that a subset of AML samples develop sensitivity to glucocorticoids. This novel finding indicates the need of detailed investigation of glucocorticoid efficacy in the clinic. Citation Format: Disha Malani, Astrid Murumägi, Bhagwan Yadav, Mika Kontro, Samuli Eldfors, Ashwini Kumar, Krister Wennerberg, Caroline Heckman, Kimmo Porkka, Maija Wolf, Tero Aittokallio, Olli Kallioniemi. Acquisition of cytarabine resistance leads to increased glucocorticoid sensitivity in AML. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4679.
    Type of Medium: Online Resource
    ISSN: 0008-5472 , 1538-7445
    URL: Article
    DOI: 10.1158/1538-7445.AM2016-4679
    RVK:
    XA 36000
    RVK:
    XA 10000
    Language: English
    Publisher: American Association for Cancer Research (AACR)
    Publication Date: 2016
    detail.hit.zdb_id: 2036785-5
    detail.hit.zdb_id: 1432-1
    detail.hit.zdb_id: 410466-3
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  • 8
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    Online Resource
    Abstract 3899: Discovery and clinical implementation of individualized therapies in acute myeloid leukemia based on ex vivo drug sensitivity testing and multi-omics profiling
    American Association for Cancer Research (AACR) ; 2018
    In:  Cancer Research Vol. 78, No. 13_Supplement ( 2018-07-01), p. 3899-3899
    Details
    In: Cancer Research, American Association for Cancer Research (AACR), Vol. 78, No. 13_Supplement ( 2018-07-01), p. 3899-3899
    Abstract: Acute myeloid leukemia (AML) is a heterogeneous disease characterized by multiple molecular subtypes and lack of effective targeted therapies. Here, we performed extensive molecular profiling and ex vivo drug testing with 515 approved and emerging cancer drugs on 164 AML patient samples. The aim was to i) assign individualized treatment options to advanced AML patients in real time, ii) explore drug response patterns across the molecular subtypes of AML and iii) identify opportunities to repurpose existing and emerging cancer drugs. Bone marrow samples (n=164) from 129 consecutive AML patients and 17 healthy donors were studied from the Helsinki University Hospital and the Haukeland University Hospital, Bergen. Mononuclear cells were resuspended either in mononuclear cell medium (MCM) or stroma conditioned medium (CM) and tested for drug sensitivity and resistance as previously described (PMID: 24056683) and studied by exome and transcriptome sequencing. The study protocol allowed us to return data to the clinician for consideration of novel treatment options. For the meta-analysis of associations between drug responses and molecular and clinical parameters, Wilcoxon signed ranked test and logistic regression were applied. Clustering of all patient samples based on ex vivo drug response patterns in both media types identified 7 distinct functional groups of AML. For example, a subgroup of samples was highly resistant to chemotherapeutics and all targeted drugs except BCL-2 inhibitors. The differences in drug responses in the two media types highlighted the importance of assay conditions for ex vivo drug testing. Strong clustering of several drugs in the same drug classes was often observed as well as clustering across different classes, for example between BET (JQ1, I-BET151, birabresib) and MEK (trametinib, cobimetinib) inhibitors. About 24 percent of the FLT3 negative AML patients manifested strong ex vivo sensitivity to glucocorticoids, highlighting a potential drug repositioning opportunity in this subset of AML patients. Overall, we identified 320 significant associations between drugs and mutated driver genes including association between NPM1 mutation and sensitivity to JAK inhibitors. Altogether, targeted treatment opportunities were clinically tested in 25 occasions in chemorefractory AML patients. The tailored clinical therapy led to transient complete remission or leukemia free state in 36% (9/25) of these cases. In conclusion, we discovered and clinically implemented individualized therapeutic options for AML patients, which resulted in a 36% clinical responses in a non-randomized proof-of-concept study. The associations identified between ex-vivo drug response and driver mutations provided novel drug repositioning opportunities in specific molecular subtypes. Citation Format: Disha Malani, Ashwini Kumar, Bhagwan Yadav, Mika Kontro, Swapnil Potdar, Oscar Brück, Sari Kytölä, Jani Saarela, Samuli Eldfors, Poojitha Ojamies, Karjalainen Riikka, Muntasir Mamun Majumder, Imre Västrik, Pekka Ellonen, Matti Kankainen, Minna Suvela, Siv Knappila, Alun Parson, Aino Palva, Pirkko Mattila, Evgeny Kulesskiy1, Laura Turunen, Karoliina Laamanen, Elina Lehtinen, Piia Mikkonen, Maria Nurmi, Sanna Timonen, Astrid Murumägi, Bjorn Tore Gjersten, Satu Mustjoki, Tero Aittokallio, Krister Wennerberg, Simon Anders, Maija Wolf, Caroline Heckman, Kimmo Porkka, Olli Kallioniemi. Discovery and clinical implementation of individualized therapies in acute myeloid leukemia based on ex vivo drug sensitivity testing and multi-omics profiling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3899.
    Type of Medium: Online Resource
    ISSN: 0008-5472 , 1538-7445
    URL: Article
    DOI: 10.1158/1538-7445.AM2018-3899
    RVK:
    XA 36000
    RVK:
    XA 10000
    Language: English
    Publisher: American Association for Cancer Research (AACR)
    Publication Date: 2018
    detail.hit.zdb_id: 2036785-5
    detail.hit.zdb_id: 1432-1
    detail.hit.zdb_id: 410466-3
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  • 9
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    Elevated expression of S100A8 and S100A9 correlates with resistance to the BCL-2 inhibitor venetoclax in AML
    Springer Science and Business Media LLC ; 2019
    In:  Leukemia Vol. 33, No. 10 ( 2019-10), p. 2548-2553
    Details
    In: Leukemia, Springer Science and Business Media LLC, Vol. 33, No. 10 ( 2019-10), p. 2548-2553
    Type of Medium: Online Resource
    ISSN: 0887-6924 , 1476-5551
    URL: Article
    DOI: 10.1038/s41375-019-0504-y
    RVK:
    WA 15000
    Language: English
    Publisher: Springer Science and Business Media LLC
    Publication Date: 2019
    detail.hit.zdb_id: 2008023-2
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  • 10
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    Online Resource
    Abstract 458: Precision systems medicine in acute myeloid leukemia: real-time translation of tailored therapeutic opportunities arising from ex-vivo drug sensitivity testing and molecular profiling
    American Association for Cancer Research (AACR) ; 2019
    In:  Cancer Research Vol. 79, No. 13_Supplement ( 2019-07-01), p. 458-458
    Details
    In: Cancer Research, American Association for Cancer Research (AACR), Vol. 79, No. 13_Supplement ( 2019-07-01), p. 458-458
    Abstract: Acute myeloid leukemia (AML) is an aggressive disease of clonal hematopoietic progenitor cells. Here, we applied ex-vivo drug sensitivity and resistance testing on AML patient cells with 362 emerging and 153 approved cancer drugs together with genomic and transcriptomic profiling to identify and tailor therapies for patients with advanced disease. Ex-vivo testing with freshly isolated patient cells revealed cancer-specific efficacies of approved drugs in 97% of the 164 patient cases, including 47% of the cases with no actionable driver mutations. We identified 142 statistically significant associations between drug responses and somatic mutations, including increased sensitivity to JAK inhibitors in patients with NPM1 mutations. Transcriptomic profiles predicted drug responses better than genomics and helped to identify additional response markers, especially beyond mutations. For example, overexpression of HOX family genes was associated with sensitivity to JAK inhibitors in patients with NPM1 mutation. In a prospective study, we translated the functional drug response and molecular profile data to the clinic and suggested tailored therapy with targeted drugs for 26 relapsed or refractory AML patients. In an observational intervention study, acting on these recommendations resulted in a temporary complete clinical remission or leukemia-free state in 39% of the cases. In summary, we conclude that ex-vivo testing of drugs on patient AML cells i) revealed clinically actionable drug efficacies in almost all AML patients, including patients with no actionable mutations, ii) predicted cases with actionable driver mutations with no pharmacological dependency on the target, and iii) enabled real-time tailoring of therapy with 39% clinical response rate in chemorefractory advanced AML. Taken together, we believe this real-time systems medicine approach could become a powerful strategy for tailoring therapies for individual patients in the future. Citation Format: Disha Malani, Ashwni Kumar, Bhagwan Yadav, Mika Kontro, Swapnil Potdar, Oscar Bruck, Säri Kytölä, Jani Saarela, Samuli Eldfors, Riikka Karjalainen, Muntasir M. Majumder, Imre Västrik, Pekka Ellonen, Matti Kankainen, Minna Suvela, Siv Knappila, Alun Parson, Aino Palva, Pirkko Mattila, Evgeny Kulesskiy, Laura Turunen, Karoliina Laamanen, Elina Lehtinen, Maria Nurmi, Katja Suomi, Astrid Muruimägi, Bjorn T. Gjertsen, Satu Mustjoki, Simon Anders, Maija Wolf, Tero Aittokallio, Krister Wennerberg, Caroline Heckman, Kimmo Porkka, Olli Kallioniemi. Precision systems medicine in acute myeloid leukemia: real-time translation of tailored therapeutic opportunities arising from ex-vivo drug sensitivity testing and molecular profiling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 458.
    Type of Medium: Online Resource
    ISSN: 0008-5472 , 1538-7445
    URL: Article
    DOI: 10.1158/1538-7445.AM2019-458
    RVK:
    XA 36000
    RVK:
    XA 10000
    Language: English
    Publisher: American Association for Cancer Research (AACR)
    Publication Date: 2019
    detail.hit.zdb_id: 2036785-5
    detail.hit.zdb_id: 1432-1
    detail.hit.zdb_id: 410466-3
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