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Basal-like breast cancers (BLBC) are characterized by defects in homologous recombination (HR), deficient mitotic checkpoint, and high-proliferation activity. Here, we discover CIP2A as a candidate driver of BLBC. CIP2A was essential for DNA damage–induced initiation of mouse BLBC-like mammary tumors and for survival of HR–defective BLBC cells. CIP2A was dispensable for normal mammary gland development and for unperturbed mitosis, but selectively essential for mitotic progression of DNA damaged cells. A direct interaction between CIP2A and a DNA repair scaffold protein TopBP1 was identified, and CIP2A inhibition resulted in enhanced DNA damage–induced TopBP1 and RAD51 recruitment to chromatin in mammary epithelial cells. In addition to its role in tumor initiation, and survival of BRCA-deficient cells, CIP2A also drove proliferative MYC and E2F1 signaling in basal-like triple-negative breast cancer (BL-TNBC) cells. Clinically, high CIP2A expression was associated with poor patient prognosis in BL-TNBCs but not in other breast cancer subtypes. Small-molecule reactivators of PP2A (SMAP) inhibited CIP2A transcription, phenocopied the CIP2A-deficient DNA damage response (DDR), and inhibited growth of patient-derived BLBC xenograft. In summary, these results demonstrate that CIP2A directly interacts with TopBP1 and coordinates DNA damage–induced mitotic checkpoint and proliferation, thereby driving BLBC initiation and progression. SMAPs could serve as a surrogate therapeutic strategy to inhibit the oncogenic activity of CIP2A in BLBCs. Significance: These results identify CIP2A as a nongenetic driver and therapeutic target in basal-like breast cancer that regulates DNA damage–induced G2–M checkpoint and proliferative signaling.

High-grade serous ovarian cancer (HGSC) is the most prevalent and lethal ovarian cancer type. While HGSC usually responds well to primary treatment, most cases eventually relapse. Functional precision medicine - tailoring individualized treatments based on functional in vitro assays on patient-derived cells - has been recently employed in cancer clinical trials. Cancer organoids - three-dimensional, self-organizing, self-renewing cell cultures that recapitulate original tissue structure and function - have been applied as cellular models in these trials. However, in case of HGSC, organoid derivation has proven time consuming and inefficient, hindering their application in functional precision medicine due to a short time window, in which therapy for each patient needs to be selected. To address this problem, we aimed to establish whether drug vulnerabilities at HGSC relapse could be predicted using organoids derived from the primary disease cells. We derived sequential organoid models from material sampled during primary treatment and at relapse. Then, for organoid pairs (primary-relapse), we performed large-scale drug response profiling of a library of 370 compounds (approved drugs or drugs in clinical development), in 384-well microplate format, alone or in combination with a standard HGSC chemotherapeutic agent carboplatin. First, we found that HGSC organoid responses to standard chemotherapeutics retrospectively correlated to observed clinical treatment outcomes. But further, for each patient we identified compounds with pronounced cytotoxicity both in the primary and in the relapsed model, amounting to 66% of all hits (7% were primary-specific and 27% relapse-specific). We then focused on identifying patient-specific hits rather than compounds displaying general toxicity in all patient models. Based on a potential clinical applicability, for three patients we selected compounds for validation in organoid outgrowth assay, with prolonged (>1 month) drug-free period post-treatment. In two patients, AZD4573, a selective CDK9 inhibitor in clinical development for hematological malignancies, at nanomolar concentrations caused eradication of organoids when combined with carboplatin. Organoids from the third patient were vulnerable to nitazoxanide, an approved anti-helminthic agent and an inhibitor of mitochondrial oxidative phosphorylation. Importantly, the selected final hits were identified solely based on screening in organoid models from primary disease. In summary, we here demonstrate that HGSC organoids derived from primary disease material predict a majority of patient-specific drug vulnerabilities of organoids derived from the relapsed HGSC lesions. This indicates that patient stratification in functional precision medicine for treatment of HGSC relapse could be prospectively performed at the primary disease stage. Citation Format: Wojciech Senkowski, Laura Gall-Mas, Matias M. Falco, Yilin Li, Kari Lavikka, Mette C. Kriegbaum, Jaana Oikkonen, Daria Bulanova, Elin J. Pietras, Karolin Voßgröne, Yan-Jun Chen, Erdogan P. Erkan, Mia K. Høg, Ida M. Larsen, Tarja Lamminen, Katja Kaipio, Jutta Huvila, Anni Virtanen, Lars H. Engelholm, Pernille Christiansen, Eric Santoni Rugiu, Kaisa Huhtinen, Olli Carpén, Johanna Hynninen, Sampsa Hautaniemi, Anna Vähärautio, Krister Wennerberg. A platform utilizing high-grade serous ovarian cancer organoids for prospective patient stratification in functional precision medicine. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5779.

Triple negative breast cancer (TNBC) is a highly aggressive type of breast cancer with poor prognosis that accounts approximately for 15% of breast cancer cases. The lack of targetable hormonal receptors or HER2/ErbB2 makes TNBC therapeutically challenging and existing drug treatments are often ineffective. TNBC is also a highly heterogeneous class of breast cancer, and different genetic approaches have not been so far successful in identifying clinically useful patient stratification strategies to date. In previous studies, we have discovered a correlation between targeted phospho-proteomics and mitotic inhibitor sensitivities in TNBC cells, suggesting the potential of using phospho-proteome profiles to identifying markers predictive of actionable therapeutic strategies. In the current study, we further explored the potential of phospho-proteomics for TNBC stratification and drug chemo-sensitivity prediction. We screened 25 TNBC cell lines against 517 oncology compounds, and in parallel ran mass spectrometry-based global phospho-proteome profiling of the same cell lines, resulting in 4283 confident phospho-peptide quantifications. Using multitask multiple kernel learning, we integrated the phospho-peptide data with other publicly available omics data for the 25 TNBC cell lines (genomics, transcriptomics, metabolomics, and methylomics), and developed a multi-omics machine learning model to quantify the predictive contribution of each omics profile to drug response prediction. When considering all the 517 compounds, we found that the phospho-proteomic and gene expression data were most informative for the prediction of compound’s chemo-sensitivity. This integrated approach has the potential to both identify panels of multi-omics markers for individual drug classes, as well as to stratify the heterogeneous TNBC cells based on their genomic, molecular and functional profiles. Citation Format: Prson Gautam, Xiangju Qin, Julia Vainonen, Srikar Nagelli, Johanna Lampe, Juha Klefström, Jukka Westermarck, Krister Wennerberg, Tero Aittokallio. Therapeutic stratification of triple negative breast cancer by integrating chemosensitivity & phospho-proteome profiles [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-10-28.

Identifying effective oncogenic targets is challenged by the complexity of genetic alterations in cancer and their poorly understood relation to cell function and survival. This, combined with the fact that the majority of the proteome is predicted to be undruggable, may explain why genomic approaches have had only moderate success in identifying and translating oncogenic drug targets. There is a need for methods that rapidly and accurately identify “pharmacologically effective” targets without the requirement for priori knowledge of complex signaling networks. We developed an approach that uses machine learning to relate results from unbiased phenotypic screening of kinase inhibitors to their biochemical activity data. This process, which we call idTRAX (Identification of Drug TaRgets and Anti-targets by Cellular and Molecular Cross-referencing), identifies targets that are pharmacologically responsive and readily druggable. Additionally, the identified targets are not typically overcome by the robustness of signaling networks, because only targets that effectively induce a phenotype upon pharmacological engagement are selected. We applied this methodology to triple negative breast cancer, which still lacks targeted therapy. We screened 19 breast cancer cell lines with ˜500 small-molecule kinase inhibitors with annotated kinase activity data and identified cell-line specific kinase targets, i.e. kinases whose inhibition causes cell line-specific cytotoxicity or cytostaticity. We were able to identify various unique cell line selective kinase addictions along with well-known driver kinase addictions, for example, dependence on FGFR2 in the MFM-223 cell line and AKT kinases in MFM-223, CAL-148, and others. We also found that triple negative breast cancer cell lines exhibit heterogeneous target patterns, indicating the need of personalized medicine approach to tackle them. We further compared candidate targets identified by idTRAX with those reported from RNAi- and CRISPR-based screens. Strikingly, the correlation between targets identified by the pharmacological approach and those identified by knockdown/knockout-based screens was low [average rank correlation (ρ) = 0.0273- 0.1074], suggesting that gene-silencing approaches may not be the most efficient at identifying targets for small-molecule drug discovery or repurposing. Our approach provides a platform for rapidly identifying sample-specific drug targets and potentially guiding personalised therapy regimens. Citation Format: Gautam P, Jaiswal A, Aittokallio T, Hassan A-A, Wennerberg K. A phenotypic screening and machine learning platform efficiently identifies triple negative breast cancer-selective and readily druggable targets [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-06-25.

Extensive utilization of organoids from high-grade serous ovarian carcinoma (HGSOC), the most common and lethal ovarian cancer, has been hampered by low success rates of long-term culture and scarcity of fresh tumor material. Here we present the development of a novel method for efficient generation, expansion and use of HGSOC organoids from cryopreserved tumor material. First, we assessed commonly used organoid media components and found that supplements such as FGF-2, R-Spondin1, Wnt or Noggin had negative impact on the HGSOC organoid derivation. But further, we found that supplementation with FGF-4, which has not been used in cancer organoid culture before, is beneficial for HGSOC organoid growth. Through extensive testing of various supplements and their combinations, we designed two novel HGSOC organoid media formulations - Medium 1 (M1) and Medium 2 (M2). Using M1 and M2 enabled generation and long-term expansion of living HGSOC organoid biobank with markedly improved success rate than in previous reports (55% vs. 23-38%). The organoids were established from cryopreserved tumor material, demonstrating the feasibility of using frozen tissue biobanks for HGSOC organoid derivation. Overall, we generated a collection of 18 expandable HGSOC organoid lines from 11 patients, encompassing samples from different tissue sites and disease progression stages. We validated the organoids using whole-genome sequencing, immunohistochemistry and single-cell RNA sequencing and demonstrated that they are genetically and phenotypically representative of original patient samples over long-term culture. Based on available patient consents, we deposited 3 organoid lines in a publicly accessible biobank. Finally, we investigated whether organoid drug responses correlate to those observed earlier in the clinic in the corresponding patients. Organoid-based drug-response profiling of clinically used HGSOC drug collection was performed in 384-well microplate format. To explore whether growth conditions impact correlation between organoid drug responses and clinical response, we compared the organoid drug responses in the nutrient-rich M1/M2 growth media to the ones observed in human plasma-like medium (HPLM), supplemented with relevant niche factors from M1/M2. Organoid drug responses correlated with clinical treatment outcomes, but only for organoids maintained in HPLM (Spearman r = 0.987, p=0.007 in HPLM vs 0.607, p=0.167 in growth medium, n=7), highlighting the importance of culture conditions in organoid-based functional assays. Taken together, we introduce a resource for efficient development and use of HGSOC organoids from cryopreserved material in ovarian cancer research. Citation Format: Wojciech Senkowski, Laura Gall Mas, Matias M. Falco, Yilin Li, Kari Lavikka, Mette C. Kriegbaum, Jaana Oikkonen, Daria Bulanova, Elin J. Pietras, Karolin Voßgröne, Erdogan P. Erkan, Terese K. Høj, Mia K. Høg, Tarja Lamminen, Katja Kaipio, Anni Virtanen, Lars H. Engelholm, Pernille Christiansen, Eric Santoni-Rugiu, Kaisa Huhtinen, Olli Carpén, Johanna Hynninen, Sampsa Hautaniemi, Anna Vähärautio, Krister Wennerberg. Efficient establishment and utilization of a high-grade serous ovarian cancer organoid biobank [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3069.

Purpose: Ovarian cancer is the most lethal gynecological cancer. Histologically, about 90% of the ovarian cancers have epithelial origin and more than 75% are characterized as high-grade serous ovarian cancer (HGSOC). Despite having a good primary response to the standard platinum-taxane based chemotherapy, almost all HGSOC patients experience a relapsed disease. The 5-year survival rate of ovarian cancer is only 38% globally, indicating need for the development of novel efficient treatments to fight the resistance. Here we have established a personalized drug screening and evaluation platform using HGSOC patient samples and medicines approved for treatment of different types of cancers. Methods: Tissues collected from ovarian cancer patients were grown as 3-dimensional (3D) ex vivo long-term tumor organoid cultures. Seven HGSOC cell lines were additionally grown as spheroids or as xenograft tumors to establish them as 3D tumor organoid cultures for drug efficiency evaluation. Cancer stemness and further classification of samples were determined via immunofluorescent staining with relevant antibodies (pax8, Ki67, acetylated tubulin, p53, cytokeratin-8) and consequent high-throughput imaging using the ImageXpress Micro Confocal microscope (Molecular Devices). The images were analyzed and quantified using MetaXpress software. Organoid growth and response to tested treatments was validated via invasive growth and survival assays. Both assays were used to monitor resistance of organoids towards cisplatin and carboplatin treatment and towards suggested potential new treatments. Cancer drugs were selected according to suggestions based on RNA seq, DNA and/or drug screenings done in cancer cell lines. A preclinical mouse model (in immunodeficient NOD/Shi-scid/IL-2Rγnull (NOG) mice) was set up to be used for in vivo validation by monitoring the tumor growth in response to the treatment. Results: Currently, 20 patient tumor samples received from Turku University Hospital, Finland (KH, JH) have been cultured in 4 different culture media to obtain highest possible survival rates for each sample. Organoids from ovarian cancer cell lines are characterized for their resistance to increasing concentrations of platinum treatment. Notably, organoid analysis protocols and screening conditions have been setup for the high-throughput microscopy. These include organoid growth, survival, and invasiveness/aggressiveness. We are currently set up for screening organoids with the first drugs suggested by bioinformatics-based genetic pathway analysis and drug screening studies in cancer cell lines. The results of the screen will be presented. This study is a part of a large EU-funded ovarian cancer project DECIDER (https://www.deciderproject.eu/). Citation Format: Aikaterini Skorda, Kaisa Huhtinen, Wojciech Senkowski, Krister Wennerberg, Sampsa Hautaniemi, Johanna Hynninen, Tuula Kallunki. Ex vivo screening and analysis of novel effective treatments for ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 192.

KRAS mutations are found in 95% of pancreatic ductal adenocarcinoma (PDAC), where they are key cancer drivers. Effective anti-KRAS therapeutics are thus anticipated to make a significant improvement in PDAC treatment. Despite the promise of inhibiting KRAS effector signaling, such inhibitors have not demonstrated significant activity as monotherapy, and there are concerns regarding potential normal tissue toxicity. Inhibitor combinations may overcome limitations of both efficacy and toxicity. We performed a 525-drug chemical biology screen to identify combinations that enhanced the cytotoxic activity of inhibitors of the RAF-MEK-ERK and the PI3K-AKT-mTOR effector pathways. Many such combinations were identified for RAF effector pathway inhibitors, while few were identified for PI3K pathway inhibitors. Generally, the same classes of inhibitors were identified for inhibitors of the RAF, MEK, and ERK nodes of the MAP kinase (MAPK) cascade. We found that inhibitors of the PI3K-AKT-mTOR pathway, microtubules, HDAC, and HSP90 each synergistically enhanced the cytotoxicity of RAF, MEK, and ERK inhibitors, in both conventional and PDX-derived PDAC cell lines. Mechanistically, we found that a shared basis for this synergy was enhanced loss of MYC protein, a key ERK substrate. Our studies identify promising combinations of KRAS effector inhibitors for PDAC treatment. Citation Format: Adrienne D. Cox, Craig M. Goodwin, Kirsten L. Bryant, Irem Dagliyan, Samuel D. George, Kelly E. Lucas, Prson Gautam, Krister Wennerberg, Channing J. Der. Inhibitor combinations targeting KRAS effector signaling in KRAS-mutant pancreatic cancer [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 SY20-02.

Cell surface mono-ADP ribosyltransferases (ARTs) transfer the ADP-ribose moiety from NAD to amino acid residues on target proteins and post-translationally regulate their function. Most mammalian ARTs are extracellular with glycosylphosphatidylinositol (GPI) anchors. ARTs can target cell surface proteins on bystander cells or soluble proteins in the tumor microenvironment, particularly P2RX7 on T cells1. We hypothesized that an evolutionarily conserved parallel protective role may be provided by ART1 expression in lung cancer cells, particularly during times of cellular stress2. A mouse KP1 lung tumor cell line with ART1 expression was established from KRASG12D/P53-/- mice and transduced with shRNA targeting ART1 (KP1-sh) or an empty vector (KP1-ev). For in vitro studies, KP1 WT cells were treated with an ER stress inducer thapsigargin, with radiation, or with chemotherapy. Using human materials, we evaluated ART1 expression in NSCLC tumors by whole tumor RT-PCR/RNAseq, immunofluorescence (IF), and immunohistochemistry (IHC). For evaluation of tumor growth in immune competent models, KP1 cells were injected into syngeneic C57BL/6 mice into the flank or intravenously to generate orthotopic lung tumors. Following in vitro treatment with thapsigargin, expression of ART1 increased 11.8-fold in KP1 WT cells (24 hrs). Similarly, following radiation (8Gy x 3,) ART1 expression increased 1.93-fold by RT-PCR and surface protein expression increased by 1.87 fold (p 1. PMID: 7930612 2. PMID: 25292337 Citation Format: Sumit Mukherjee, Erik Wennerberg, Clarey Hung, Najla Saadallah, Shashi Kariyawasam, Christopher Agrusa, Amanda Valeta, Nasser Altorki, Timothy McGraw, Sandra Demaria, Brendon Stiles. Art1, an extracellular mono-ADP-ribosyltransferase, is upregulated in response to cellular stress and promotes lung cancer growth [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1820.

Extracellular NAD+ (eNAD) is released from stressed and dying cells where it acts as a proinflammatory mediator. However, in the presence of mono-ADP-ribosyltransferases (ARTs) eNAD serves as a substrate for ADP-ribosylation of the P2 × 7 receptor (P2RX7), resulting in NAD-induced cell death (NICD). P2RX7 is expressed on several immune cells including CD103+ conventional type I dendritic cells (cDC1s) which are essential for initiation of tumor-specific immune responses as well as on tissue resident memory T cells (TRMs), of which high tumor infiltration has been associated with improved survival in non-small cell lung cancer (NSCLC) [1]. NICD of P2 × 7R+ TRMs was recently identified as a regulator of TRM tissue homeostasis [2]. CD38 is expressed on activated immune cells and can reduce eNAD levels by converting eNAD into ADPR. We tested the hypothesis that ART1 expression in NSCLC constitutes a mechanism of immune resistance by mediating NICD of P2 × 7R+ TRMs and P2 × 7R+ cDC1s. ART1 expression in human NSCLC samples was determined by immunohistochemistry scoring of NSCLC tumors using a tissue microarray (TMA) (n=493). In addition, matched tumor and adjacent normal lung tissue samples were analyzed for ART1 expression by qPCR (n=40) and for infiltration of TRM and cDC1s by flow cytometry. A mouse KP1 lung tumor cell line with high ART1 expression was established from KRASG12D/P53−/− mice and transduced with a doxycycline-inducible shRNA targeting ART1 (ART1KD). KP1 cells and their derivatives were intravenously injected into syngeneic C57BL/6 mice to generate orthotopic lung tumors. Lung tumor nodules were enumerated two weeks after tumor inoculation using H&E-stained FFPE lung sections and lung-derived cell suspensions were analyzed by flow cytometry for infiltration of T cell and DC subsets. Increased expression of ART1 in tumor compared to normal lung was observed in 55% of NSCLC samples by TMA analysis, and confirmed by qPCR. The frequency of P2RX7+ CD8 TRMs (p In conclusion, we show that ART1 is overexpressed in NSCLC and identify ART1-mediated NICD of P2 × 7R+ TRMs and cDC1s as a possible novel immune escape mechanism in NSCLC. Furthermore, we describe a novel role of CD38 expression on immune cells in its protection against NICD. 1. Nizard, M., et al., Induction of resident memory T cells enhances the efficacy of cancer vaccine. Nat Commun, 2017. 8: p. 15221. 2. Stark, R., et al., T RM maintenance is regulated by tissue damage via P2RX7. Sci Immunol, 2018. 3(30). Citation Format: Erik Wennerberg, Clarey Hung, Amanda Valeta, Timothy McGraw, Sandra Demaria, Brendon Stiles. ART1 tumor expression mediates immune resistance in non-small cell lung cancer by elimination of P2 × 7R+ CD8 tissue resident memory T cells and conventional type I dendritic cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2158.

RAS pathway mutations are found in nearly 75% of high-risk embryonal rhabdomyosarcoma (ERMS). While RAS oncoproteins are well-established therapeutic targets for many adult human cancers, still very little is known about the role of RAS mutations in the development and maintenance of ERMS. By sequencing, we identified cell lines and PDX tumors harboring activating mutations in H- or NRAS. Further, we showed that mutant H- or NRAS was critical for the growth of all RAS-mutant ERMS cell lines and that RAF/MEK/ERK signaling pathway, but not PI3K/AKT, was mediator of RAS dependency in these cells. However, in vivo treatment of RAS-mutant ERMS xenografts with the MEK inhibitor trametinib showed modest response as compared to BRAF-mutant astrocytoma xenografts. We reasoned that similarly to other RAS-driven cancers, ERMS cells and tumors are able to acquire resistance to inhibitors of the RAF/MEK/ERK pathway. We performed drug-sensitizing pooled CRISPR library screen and identified that inhibition of ERK2 potentiated trametinib treatment. We show that combining trametinib with ERK1/2 inhibitor leads to potent synergistic ERK inhibition and ERMS tumor growth suppression. Citation Format: Angelina V. Vaseva, Abhik Bandyopadhyay, Vanessa Del Pozo, Craig M. Goodwin, Prson Gautam, Krister Wennerberg, Kris C. Wood, Yidong Chen, Channing J. Der, Peter J. Houghton. Parallel targeting of RAF/MEK/ERK pathway in RAS-mutant embryonal rhabdomyosarcoma [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A54.

Introduction/Purpose: Cancer therapy is increasingly moving towards individualized care and therapy, but there are still gaps between what is known and described on the molecular level about cancers and what is applied in the clinic. In an attempt to bridge the knowledge gap, we at the Institute for Molecular Medicine Finland (FIMM) have set up an Individualized Systems Medicine program that integrates clinical information, molecular profiling and functional information about individual patients’ cancers (Pemovska et al, Cancer Discov, 2013). Central to this program is the Drug Sensitivity and Resistance Testing (DSRT) where we functionally profile the responses of primary cancer cells to a comprehensive clinical oncology and signal transduction inhibitor drug collection of 528 compounds. Methods: Acoustic dispensing platforms are integral to the success of this profiling activity. We have to date produced approximately 3000 drug sets as dose response assay ready plates. The acoustic dispensing allows for making pre-drugged single drug plate sets and/or drug combination plates within hours after sampling of the cells. The plates are also readily sent to researchers anywhere in the world for running comparable assays at other sites. The drugging reproducibility is excellent generating results with correlations of 0.98 or higher in replicate assays. We have developed in-house software solutions to aid these processes: a script for quick creation of transfer list for combination plates and automated analysis pipelines with web-based software interfaces to enable the screening biologists to analyze the screening results effectively. Results: The results of these assays are used to explore and understand cancer biology in terms of druggability, functional heterogeneity and mechanism of drug response and resistance. The profiling data can be used to stratify and position the relevance of specific drugs in different diseases and has been used to identify novel clinically relevant activities of existing and investigational drugs (see e.g. Pemovska et al, Nature, 2015). This information is further utilized to establish hypotheses on drug combinations selectively targeting individual cancers and their predictive biomarkers, which can be explored in the clinic by our clinical collaborators to guide the treatment of the individual patient. Conclusions: In summary, we describe our platform for a functional drug sensitivity testing within our individualized cancer systems medicine program, which generates consistent biological and clinically relevant data. Citation Format: Sergey G. Kuznetsov, Alexander Ianevski, Evgeny Kulessky, Karoliina Laamanen, Elina Lehtinen, Maria Nurmi, Swapnil Potdar, Jani Saarela, Katja Suomi, Laura Turunen, Krister Wennerberg, Päivi Tammela. Ex vivo drug sensitivity testing of primary cells for precision cancer medicine [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 2153.

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.

Tumors can suppress the host immune system by employing a variety of cellular immune modulators, such as regulatory T cells, tumor-associated macrophages, and myeloid-derived suppressor cells (MDSC). In the peripheral blood of patients with advanced stage melanoma, there is an accumulation of CD14+HLA-DRlo/− MDSC that suppress autologous T cells ex vivo in a STAT-3–dependent manner. However, a precise mechanistic basis underlying this effect is unclear, particularly with regard to whether the MDSC induction mechanism relies on cell–cell contact of melanoma cells with CD14+ cells. Here, we show that early-passage human melanoma cells induce phenotypic changes in CD14+ monocytes, leading them to resemble MDSCs characterized in patients with advanced stage melanoma. These MDSC-like cells potently suppress autologous T-cell proliferation and IFN-γ production. Notably, induction of myeloid-suppressive functions requires contact or close proximity between monocytes and tumor cells. Further, this induction is largely dependent on production of cyclooxygenase-2 (COX-2) because its inhibition in these MDSC-like cells limits their ability to suppress T-cell function. We confirmed our findings with CD14+ cells isolated from patients with advanced stage melanoma, which inhibited autologous T cells in a manner relying up prostaglandin E2 (PGE2), STAT-3, and superoxide. Indeed, PGE2 was sufficient to confer to monocytes the ability to suppress proliferation and IFN-γ production by autologous T cells ex vivo. In summary, our results reveal how immune suppression by MDSC can be initiated in the tumor microenvironment of human melanoma. Cancer Res; 73(13); 3877–87. ©2013 AACR.

The STAT3 transcription factor is hyperactivated in many cancers where it induces cancer progression by driving drug resistance, metastasis and pro-cancer inflammation. We therefore sought new strategies to target the activation of STAT3. Many kinases and signalling pathways can cause STAT3 activation, and it is not well understood how this is regulated in cancer cells. In some rare solid and hematological cancers, such as NK-/T-cell large granular lymphocytic leukemia and inflammatory hepatocellular adenomas, STAT3 has also been found to be mutated, resulting in a constitutively active STAT3 protein. In testing mock transfected vs. STAT3(Y640F)-transfected cells, as a means to introduce activated STAT3 in already transformed cells, in a screen against 525 anticancer agents, we identified that activated STAT3 protects the cells from cytotoxic effects of several classes of anticancer compounds. Therefore, we performed RNAi screens to identify kinases and phosphatases that regulate STAT3 activity and therefore may act as drug-sensitizing targets. Using STAT3 transcription-driven luciferase reporter cell lines expressing active wild type (WT) or mutant (Y640F) STAT3, we identified several candidate genes (CDC7, CDK8, CSK, CSNK2A1, PI4KII, PTPRH, DDR2) whose downregulation led to differential STAT3 transcriptional activity. Strikingly, none of the hits caused highly selective effect on either mutant or wild-type STAT3 expressing cells, suggesting that the phosphoregulation of hyperactive mutant and IL6 induced wild-type STAT3 are mechanistically similar. Using small molecule inhibitors targeting CDK8, CSNK2A1, DDR2 and CDC7, we confirmed that STAT3 transcriptional activity was inhibited in a dose and time dependent fashion without severely affecting cell viability. Small molecules targeting of CSNK2A1, DDR2 and CDC7 kinase activity caused a slow inhibition of STAT3 transcriptional activity where the strongest effects were only seen after 72 h. CDK8 kinase inhibition, on the other hand, reduced STAT3 activity effectively already after 4 h. This suggests that CDK8 regulates STAT3 activity more directly than CSNK2A1, DDR2 and CDC7. Inhibition of CDK8 and CSNK2A1 resulted in decrease of STAT3 phosphorylation in the STAT3(Y640F)-transfected cells, and knockdown of CSNK2A1 inhibited the nuclear localization of STAT3. In conclusion, we have found that hyperactive STAT3(Y640F) can protect cancer cells from several drug classes, mimicking the case when hyperactivated wild type STAT3 is acting as malignancy promoting protein in cancers. In a functional genetics screen, we found six kinases and a phosphatase that regulate transcriptional activity of STAT3. Using small molecule inhibitors, we could confirm that targeting the hit kinases regulate STAT3 transcriptional activity. Together, our data suggest that there are multiple kinases that may be targeted to counteract STAT3 mediated drug resistance in cancers. Citation Format: Elina Parri, Heikki Kuusanmäki, Arjan van Adrichem, Meri Kaustio, Laura Turunen, Krister Wennerberg. Identifying kinases and phosphatases that regulate STAT3 activity [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 3343.

Objective: High-grade serous ovarian cancer (HGSOC) remains the most lethal gynecologic malignancy. More than 50% of high-grade serous ovarian cancer (HGSOC) patients die due to recurrent disease within 5 years. Recurrent tumor clones are selected by chemotherapy treatment from heterogeneous subpopulations of cancer cells, and differ phenotypically. The objective of current study is to develop a high-throughput chemical biology approach to analyze phenotypic heterogeneity in HGSOC following progression of the disease. Methods: We utilize the Drug Sensitivity and Resistance Testing (DSRT) platform to assess the effects of 528 approved and investigational anti-cancer agents on primary HGSOC isolates. Samples are collected from ascites and/or solid high-grade ovarian tumors from the patients at different disease stages. DSRT profiling of the samples integrates viability and toxicity readouts with high-content imaging in order to assess the drug responses of phenotypically different cell subpopulations of epithelial cancer cells and stromal component. Results: Drug sensitivity profiling of ten primary and six relapse patient samples revealed heterogeneous drug sensitivity profiles characterized by common responsiveness to HDAC inhibitors, CDK inhibitors, and PI3K/mTOR inhibitors (mean drug sensitivity score (DSS) > 10). Relapse HGSOC samples demonstrated general trend towards resistance to a broad spectrum of chemotherapeutics and targeted drugs. Imaging-based DSRT profiling revealed that in both primary and relapse ascites-derived cancer cells the response to platinum drugs and DNA damaging agents associated with upregulation of transcriptional regulators of stemness, including BMI1, SOX2 and NANOG. The ascites-derived CK18-expressing epithelial cells collected from the same patient a) before chemotherapy, b) after 6 cycles of Carboplatin treatment and c) at the relapse demonstrated an increasing sensitivity to aldehyde dehydrogenase inhibitor disulfiram. These preliminary findings suggest that increase in a subpopulation of cancer cells with stem-like characteristics may accompany recurrence in HGSOC. Conclusions: Compound sensitivity testing combined with high-content imaging appears an informative tool to explore therapeutic vulnerabilities in chemoresistant subpopulations of ovarian cancer cells, and improves the accuracy of drug sensitivity profiling. Chemical biology screening of prospectively collected primary HGSOC samples can also help identifying phenotypic changes associated with chemoresistance development. Citation Format: Daria R. Bulanova, Katja Kaipio, Tarja Lamminen, Aleksandr Ianevski, Tero Aittokallio, Olli Carpen, Krister Wennerberg. Chemical biology approach to phenotypic intra-tumor heterogeneity in high-grade serous ovarian cancer [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 875.

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.

Introduction: Ex vivo drug sensitivity studies of samples derived from acute myeloid leukemia (AML) patients have been shown to be predictive of in vivo response. These findings are based on a limited number of well-characterized agents for which in vivo patient response data and ex vivo drug sensitivity data—on that same patient—are available. To show the feasibility of scaling such ex vivo studies to large drug screens, we characterized the reproducibility of expression-based models of drug response across two independent data sets—one generated at the Oregon Health and Science University (OHSU) and the second at the Institute for Molecular Medicine Finland (FIMM). Methods: We harmonized two large-scale AML ex vivo studies screened for drug response and profiled transcriptomically—OHSU (303 AML patient samples and 160 drugs) and FIMM (48 AML samples and 480 drugs). The two panels have 94 drugs in common. Log-logistic curves were fit to the dose-response data and area under the dose-response curves (AUCs) were calculated. Predictive modeling using Ridge regression or an integrative Bayesian approach was performed for each drug AUC independently using 202 highly-variable and/or cancer-associated genes as features. Results: For each of the 94 drugs in common between the two data sets, we trained a Ridge regression model on the OHSU data set, used the model to predict response in the FIMM data set, and calculated the Pearson correlation between the predicted and observed FIMM responses. 41 of the 94 drug models had a positive and statistically significant correlation [false discovery rate (FDR) < 20%; mean ρ = 0.43; 95% CI = 0.29 – 0.77]. Drugs corresponding to the top decile of these significant models (mean ρ = 0.54; 95% CI = 0.48 – 0.77) clustered into four primary classes: MEK inhibitors (PD184352, Selumetinib, and Trametinib), EGFR/VEGFR inhibitors (Cabozantinib, Erlotinib, Foretinib, and Sorafenib), and singletons Venetoclax and Sirolimus. To confirm these results, we applied a second modeling approach—an integrative Bayesian machine learning method—that allows systematic combination of both data sets. Training and evaluation of this approach using 10-fold cross validation yielded 82 positive and statistically significant correlations (FDR < 20%; mean ρ = 0.35; 95% CI = 0.13 – 0.58). Five of nine drugs (Cabozantinib, Selumetinib, Sirolimus, Sorafenib, and Trametinib) corresponding to the top decile of these significant models (mean ρ = 0.54; 95% CI = 0.49 – 0.60) overlapped with drugs from the top decile of Ridge results (one-sided Fisher p = 2.5 x 10-4) Conclusions: Our results using independent data sets and two statistical approaches suggest that certain drugs (including MEK and EGFR/VEGFR inhibitors) are amenable to expression-based predictive modeling in AML. Future work will focus on inferring individual biomarkers of response. Citation Format: Brian S. White, Suleiman A. Khan, Muhammad Ammad-ud-din, Swapnil Potdar, Mike J. Mason, Cristina E. Tognon, Brian J. Druker, Caroline A. Heckman, Olli P. Kallioniemi, Stephen E. Kurtz, Kimmo Porkka, Jeffrey W. Tyner, Tero Aittokallio, Krister Wennerberg, Justin Guinney. Gene expression predicts ex vivo drug sensitivity in acute myeloid leukemia [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 3883.

Despite the pivotal role of phosphatases in cancer cell signalling, systemic understanding of phosphatase targets is still at infancy. Protein phosphatase 2A (PP2A) is a human tumor suppressor complex. PP2A inhibition is a requirement for human cell transformation and PP2A regulates many cancer critical signalling pathways. Importantly, emerging data indicates that reactivation of PP2A tumor suppressor activity could provide entirely novel approach for cancer therapy. Here, we present first systemic analysis of phosphoprotein targets (dephosphorylome) regulated by PP2A in cancer cells. Based on data, PP2A regulates cancer critical signalling pathways, including entire EGFR-RAS-RAF-MEK-ERK cascade, and functions as a master regulator of MYC function. At network level, PP2A targets critical cellular processes such as chromosome organization, RNA splicing, and nuclear envelope assembly. Surprisingly, soft clustering of PP2A dephosphorylome revealed that most phospho-target residues are subject to only unidirectional regulation in cancer cells. Moreover, targets show intracellular gradient where phosphatase inhibition dominates nuclear phosphorylation balance. Since phosphoregulation is critical for cancer drug responses, dephosphorylome was correlated with cancer cell responses to over 300 drugs. Importantly, cancer therapies could be broadly classified based on their dephosphorylome, both at quantitative and qualitative manner. Finally, we demonstrate the utility of this large dataset by validating the role of PP2A in MEK inhibitor resistance in KRAS mutant cancer cells via regulation of RAF, mTor and MYC. The study presents first systemic resource to understand potential of PP2A manipulation in cancer cell signalling and drug responses. The study also uncovers generally important insights to phosphoregulation. Citation Format: Otto Kauko, Susumu Imanishi, Evgeny Kulesskiy, Teemu D. Laajala, Laxmana Yetukuri, Artur Padzik, Mikael Jumppanen, Pekka Haapaniemi, Bhagwan Yadaw, Veronika Suni, Taru Varila, Garry Corthals, Wennerberg Krister, Tero Aittokallio, Jukka Westermarck. Systemic map of protein phosphatase 2A (PP2A)-regulated phosphotargets and drug responses in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5560. doi:10.1158/1538-7445.AM2017-5560

As part of our individualized systems medicine (ISM) program, personalized treatment options are provided to clinicians based on in-depth genomic and molecular profiling as well as ex vivo drug sensitivity and resistance testing (DSRT) of leukemia patients (Pemovska et al. Cancer Discovery, 2013). In chemorefractory AML patients (n = 17), the ISM strategy has resulted in up to 35% response rate when individually selected targeted drugs have been applied in patient treatment. The responses achieved have, however, been transient and patients have typically relapsed quickly. Here, we aimed to understand the molecular basis of such treatment failures by quantitating the kinetics of individual cancer subclones before, during and after targeted treatments, as well as at the time of relapse and disease progression. Longitudinal serial samples from 13 AML patients were studied at multiple steps during leukemia progression and drug response. Clonal evolution of leukemic subclones was studied by both exome sequencing to get genome-wide overviews of disease progression, as well as by ultra-deep (>10,000x) amplicon resequencing with unique molecular identifiers to identify rare clones carrying specific cancer-relevant mutations. Nine of the 13 patients (69%) had multiple clones by exome sequencing and displayed branching evolution. In five patients who received treatment with targeted inhibitors we observed a significant differential therapeutic response of the individual AML subclones during therapy. In some cases, this could be directly attributed to the molecular mechanisms of drug response and resistance, such as the loss of NF1 in a subclone leading to cytarabine resistance or the loss of the FLT3-positive subclone in a patient responding to sunitinib treatment. Despite a prominent drug response at the level of the subclone carrying the driver mutations, in all these patients a new subclone emerged that led to progression of the disease. In three of the patients, the dominant clone appearing at relapse was already detected as a minor subclone in the diagnostic sample by amplicon resequencing. Amplicon sequencing enabled us to detect these minor subclones (down to 0,5% frequency) that were missed by exome sequencing. The results suggest that relapses in AML may arise because the drug-resistant subclone exists already before the onset of therapy. Overall, it is necessary to quantify tumor evolution and drug responses at the level of cancer subclones. Ultra-deep resequencing can be used to monitor drug responses at the subclone level, even at very low frequencies. This could facilitate early detection of small subclones with important prognostic implications, as well as the design of intelligent combinations of targeted drugs that could block such subclones. Citation Format: Poojitha N Ojamies, Mika Kontro, Henrik Edgren, Pekka Ellonen, Sonja Lagstrom, Henrikki Almusa, Timo Miettinen, Samuli Eldfors, David Tamborero, Krister Wennerberg, Caroline Heckman, Kimmo Porkka, Maija Wolf, Olli Kallioniemi. Responses of AML patients to tailored drug regimens: monitoring cancer subclones by ultra-deep resequencing. [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 2378.

Culture of human prostate cancer (PCa) progenitor cells would facilitate the discovery and testing of new, potentially curative therapies for PCa. Here, we established and characterized patient-derived conditionally reprogrammed cells (CRCs) from seven patients to determine their biological properties (genomic, transcriptomic, protein expression) and to apply these cells to test efficacies of 306 emerging and clinically approved drugs. The patient samples came from both primary prostate cancer as well as from advanced Castration-Resistant Prostate Cancer (CRPC). Patients with primary prostate cancer generated six benign CRC cultures which all had an androgen receptor (AR)-negative, basal/transit-amplifying phenotype with few CNAs. In 3D cell culture, these cells could re-express AR. The CRCs from a CRPC patient (HUB.5) displayed multiple CNAs, many of which were shared with the parental tumor. We carried out high-throughput drug-response studies with 306 emerging and clinical cancer drugs. Bcl-2 family inhibitor navitoclax emerged as the most potent cancer-specific drug for the CRCs from CRPC patient. Other drug efficacies observed included taxanes, mepacrine and retinoids, thus covering both existing (and clinically validated) as well as new drug efficacies. In conclusion, comprehensive cancer-pharmacopeia-wide drug testing of CRCs from a CRPC patient highlighted both known and novel drug sensitivities in PCa, including navitoclax, which is currently tested in clinical trials of CRPC. Citation Format: Khalid Saeed, Vesa Rahkama, Samuli Eldfors, Dmitrii Bychkov, John Patrick Mpindi, Bhagwan Yadav, Tero Aittokallio, Peter Horvath, Donna Peehl, Lassi Paavolainen, Caroline Heckman, Krister Wennerberg, Tuomas Mirtti, Antti Rannikko, Olli Kallioniemia, Päivi Östling, Taija af Hällström. Comprehensive drug testing of patient-derived conditionally reprogrammed cells from castration-resistant prostate cancer. [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 608.

Radiation-induced immunogenic cell death (ICD) is a key mechanism whereby local radiation therapy (RT) can elicit anti-tumor immune responses and synergize with immune checkpoint inhibitors in enhancing tumor responses. ATP, which is an essential signal of ICD, activates tumor-resident dendritic cells (DCs) promoting their ability to cross-present tumor-derived antigens to T cells. Interestingly, while release of ATP by RT is dose-dependent (Golden et al., OncoImmunology 2014), a large RT dose of 20 Gy was not effective in inducing anti-tumor T cells and synergize with anti-CTLA-4 (Dewan et al., Clin Cancer Res 2009). Extracellular ATP is rapidly catabolized to adenosine (ADO) by ectonucleotidases CD39 and CD73, which are expressed on tumor cells and immune cells. ADO has immunosuppressive effects, inhibiting DC- and effector T cell-activation, while promoting regulatory T cells (Tregs). Here, we tested the hypothesis that conversion of ATP to ADO hinders generation of effective anti-tumor immunity by high dose RT. Mice were inoculated s.c. with TSA breast cancer cells or MCA38 colon cancer cells on day 0 and assigned to treatment with: (1) control mAb; (2) anti-CD73 (TY/23); (3) RT (4) RT+TY/23. TY/23 (200 μg) was administered i.p. on day 11, 14, 17 and 20. RT was given locally as single 20 Gy dose on day 12. On day 18, some tumors were harvested for flow cytometry analysis of DC and T cells. Mice were monitored for tumor progression. HPLC was used to measure ADO levels in supernatants from 24 h co-cultures of bone marrow-derived DCs and irradiated TSA cells.In vitro, antibody blockade of CD73, the rate-limiting enzyme in the generation of ADO, reduced the levels of ADO in the supernatant and restored the activation of DCs cultured with irradiated TSA cells. In irradiated tumors, anti-CD73 reduced the percentage of Tregs within the tumor-infiltrating CD4+ T cell population (7.9±2.5% in RT+TY/23 vs 20±0.8% in RT, p Our data show that adenosinergic signaling regulates the ability of RT to induce anti-tumor immunity, affecting activation of both DCs and effector T cells. ADO blockade may represent a promising strategy to enhance the immunogenicity of irradiated tumors by improving the ability of RT to induce in situ tumor vaccination. Citation Format: Erik Wennerberg, Aranzazu Mediero, Tuere Wilder, Silvia Formenti, Bruce Cronstein, Sandra Demaria. Adenosine regulates radiation therapy-induced antitumor immunity. [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 4033.

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.

The T315I gatekeeper mutation confers resistance to majority of approved ABL1 inhibitors, with only ponatinib demonstrating efficacy in BCR-ABL1(T315I)-driven disease. However, vascular adverse events and selection of resistant compound mutations limit its clinical utility. Hence, there is an unmet need for novel therapies for patients with gatekeeper-mutated Ph+ leukemia. In this study we integrated comprehensive drug sensitivity testing with structural analysis to characterize the tyrosine kinase inhibitor axitinib as a putative novel therapy for BCR-ABL1(T315I)-driven leukemias. To address this we profiled BCR-ABL1(T315I)-driven CML/Ph+ ALL patient samples against 300 anti-cancer compounds (approved and investigational drugs). Ex vivo drug sensitivity testing of primary cells derived from a Ph+ ALL patient revealed a marked and cancer-selective response to the VEGFR inhibitor axitinib. Strikingly, axitinib exhibited higher sensitivity in T315I positive Ph+ patient samples in comparison to T315I negative CML and ALL patient samples. In line with the ex vivo drug response data, axitinib inhibited the kinase activity of ABL1(T315I) with similar potency as its primary target VEGFR2, while the potency to non-mutated ABL1 was 30-fold lower. Analogously, in engineered Ba/F3 cells, axitinib showed a 10-fold higher inhibition of T315I than non-mutated ABL1-driven autophosphorylation and cell growth. To better understand the molecular mechanisms of the BCR-ABL1(T315I) selectivity, we solved the crystal structure of axitinib in complex with ABL1(T315). The structure revealed that axitinib bound to a mutation induced active conformation of ABL1(T315I), different than the binding mode in non-mutated ABL1, likely explaining the increased potency towards ABL1(T315I). Moreover, axitinib occupied a distinct binding space than all approved ABL1 inhibitors, signifying that axitinib will have a unique mutation vulnerability profile. Compassionate two week treatment of a CML patient harboring the T315I mutation with axitinib, resulted in a 5-fold reduction of T315I transcript levels in the bone marrow, further suggesting that axitinib can produce specific and effective responses in patients with BCR-ABL1(T315I)-driven leukemia. In conclusion, we demonstrate that axitinib potently inhibits BCR-ABL1(T315I) via a gatekeeper mutant-selective mechanism. Since axitinib is in clinical use for treatment of refractory renal cell carcinoma with a manageable safety profile, our data provide a sound basis for readily repurposing axitinib for BCR-ABL1(T315I)-driven leukemia. Finally, the distinct mechanism of inhibition by axitinib serves as an exemplar for development of even more effective gatekeeper-mutant selective inhibitors targeting ABL1 as well as other clinically important kinases, such as EGFR and KIT. Citation Format: Tea Pemovska, Eric Johnson, Mika Kontro, Gretchen A. Repasky, Jeffrey Chen, Peter Wells, Ciarán N. Cronin, Michele McTigue, Olli Kallioniemi, Kimmo Porkka, Brion W. Murray, Krister Wennerberg. Axitinib targets gatekeeper-mutant BCR-ABL1(T315I)-driven leukemia in a distinct and selective fashion. [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 676. doi:10.1158/1538-7445.AM2015-676

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 Murumägi, Akira Hirasawa, Mariliina Arjama, Katja Välimäki, Bhagwan Yadav, Jing Tang, Agnieszka Szwajda, Laura Turunen, John Patrick Mpindi, Teijo Pellinen, Krister Wennerberg, Ralf Bützow, 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

Triple negative breast cancer (TNBC) is characterized by the lack of of estrogen, progesterone and HER2/ErbB2 receptors. It is a highly heterogeneous class of breast cancer and transcriptomics has recently been used to define 6 major subtypes of TNBC. We studied a panel of 15 TNBC cell lines using a chemical biology approach where we measure the responses to 306 approved and investigational oncology drugs. Clustering of cell lines based on their overall drug responses resulted in a strikingly different grouping compared to the gene expression derived one, highlighting that the current TNBC subtyping is not easily converted to differential sensitivities to drugs. To further evaluate the nature of the drug responses and to differentiate between their cell growth and cytotoxic effects, we multiplexed the standard cell viability readout in the cell line screening with detection of cytotoxicity. This simple multiplexed readout identified several drug classes that previously had been assumed to be cytotoxic based on strong effects on cell viability (cell numbers) while they in fact showed no or a very heterogeneous effect on cytotoxicity. Drug classes exhibiting this type of response included mTOR inhibitors, cyclin-dependent kinase inhibitors (eg. alvociclib), mitotic inhibitors (eg. paclitaxel) as well as proteasome inhibitors (eg. bortezomib) and RNA synthesis inhibitors (eg. dactinomycin). Further investigation of these drug classes showed that their static effects were reversible and in some cases the cells even overcame the inhibitory effect in the presence of the drug in a matter of a few days. Given the non-toxic responses to major classes of anticancer compounds such as mTOR inhibitors, we performed combination screens with these compounds to identify other drugs with which they may synergize to promote cancer cell specific killing. Surprisingly, we instead found that mTOR inhibitors had an antagonistic effect on the activity of many other cancer drugs such as different cytotoxic and antimitotic drugs, tyrosine kinase inhibitors, HDAC inhibitors and PARP inhibitors, suggesting that combining these classes of drugs may be counterproductive also in the clinic. We also found out that accessing a cytotoxic readout allowed us to identify effective synergistic drug combination concentrations that were not seen in cell viability readouts. For example, these synergistic toxic combination responses were seen in DU4475 cells when the MEK inhibitor trametinib was combined either with the PARP inhibitor iniparib or with the broad spectrum tyrosine kinase inhibitor ponatinib. In conclusion, multiplexed cell viability cell death readouts in drug sensitivity testing yields novel critical information on single drug and drug combination activities and liabilities. With this we were able to conclude that antimitotic, mTOR, CDK, proteasome and metabolic inhibitors have a heterogeneous cytotoxic effect across the panel of TNBC cell lines in contrast to their homogenous effect on metabolic inactivation. Citation Format: Prson Gautam, Leena Karhinen, Agnieszka Szwajda, Sawan Kumar Jha, Bhagwan Yadav, Tero Aittokallio, Krister Wennerberg. Identification of subgroups of triple negative breast cancer cells with selective responses to mTOR, CDK, mitotic and proteasome inhibitors [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-02-01.

Patients with Fanconi anemia (FA) are prone to develop malignancies at an early age. Besides hematologic malignancies, squamous cell carcinomas in the anogenital region and head and neck are also frequently found in these patients. The aim of this study was to generate a panel of head and neck squamous cell carcinoma (HNSCC) cell lines and xenografts of FA HNSCC, and to characterize these cell lines in comparison with a panel of seven cell lines from patients with sporadic HNSCC. Analyses have been done on sensitivity to DNA cross-linking agents, loss of heterozygosity profile, TP53 mutations, TP53 polymorphisms and the presence of human papillomavirus. Four FA HNSCC cell lines were established. Sensitivity to DNA cross-linking agents (cisplatin) in the FA HNSCC cell lines was on average 10 times higher as compared with the sporadic HNSCC cell lines. Human papillomavirus was not detected in any of the FA or sporadic cell lines. No differences were found in loss of heterozygosity pattern, TP53 mutation frequency and TP53 polymorphism between FA and sporadic HNSCC cell lines. This is the first report on the generation of squamous cell lines of FA patients. The FA HNSCC cell lines we have generated may be utilized for future studies and might aid in the development of new preventive therapies for FA patients. The genetic characteristics of these cell lines suggest that FA HNSCC are not very different from sporadic HNSCC, except for the sensitivity to cisplatin which is consistent with the known cellular FA phenotype.

Acute myeloid leukemia (AML) is a genetically heterogeneous disease characterized by significant clonal evolution. It is critical to understand clonal diversity and evolution during cancer progression and drug resistance in order to tailor curative therapies that block the growth of the multiple AML subclones in each patient. In our individualized systems medicine (ISM) initiative, we performed ex-vivo drug sensitivity and resistance testing (DSRT) with a comprehensive set of 306 cancer drugs on primary cells from 22 AML patients (Pemovska et al, Cancer Discovery, 2013). Objective clinical responses were seen in 3/6 chemorefractory patients treated based on DSRT results. Here, we applied next-generation genome and transcriptome sequencing in order to a) follow clonal progression of adult AML in patients during treatment with novel targeted drugs, b) identify putative cancer predisposing mutations in patients who later develop aggressive relapsed AML. Serial samples were obtained from each patient during diagnosis, treatment, remission and relapse. The data indicated significant clonal evolution and emergence of new subclones after relapse and drug resistance. In one such case, treatment of an AML patient with a combination of dasatinib-sunitinib-temsirolimus led to the selection of an already existing low-frequency subclone carrying ETV6-NTRK3 fusion. DSRT of the relapsed sample indicated addiction to IGF1R signalling, compatible with the NTRK3-fusion acting as a driver gene for drug resistance after this treatment. Similar trend where a low-frequency subclone was selected for in the resistance sample was seen in 3 other cases analyzed. Thereby, in depth analysis of clonal evolution from paired AML samples should facilitate the design of new combinatorial regimens to block relapse from arising. Altogether, we analyzed the genomic profile of 20 recurrent, chemorefractory AML cases. The profile was different between the individual patients and showed an overrepresentation of FLT3 and WT1 mutations, whereas previously reported recurrent mutations in primary AML such as DNMT3A, IDH1/2 and RUNX1 mutations were seen only in individual cases. We also analyzed the germline variants in the 20 cases, of which two had a positive family history. These patients were analyzed to identify putative variants predisposing these patients to refractory AMLs. The finnish population data comprising of exome sequencing data of 3700 individuals was used to filter the germline variants and assess their potential significance. This analysis indicated recurrent germline changes in multidrug resistance superfamily of genes, which suggests potential germline clues to the etiology to the chemorefractory, recurrent AML cases. Citation Format: Naga Poojitha Ojamies, Mika Kontro, Henrik Edgren, Samuli Eldfors, Pekka Ellonen, Tea Pemovska, Langstrom Sonja, Henrikki Almusa, Maija Lepisto, Tero Aittokallio, Krister Wennerberg, Caroline Heckman, Kimmo Porkka, Olli Kallioniemi. Analysis of clonal evolution of leukemia in vivo following novel targeted treatments. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 982. doi:10.1158/1538-7445.AM2014-982

There is an urgent need to develop better understanding of the drug responsivenes in ovarian cancer (OC), particularly in the clear cell and mucinous subtypes, which tend to be resistant to the commonly used platinum-based regimens. This would help to improve survival and facilitate precision medicine approaches to therapy. In order to discover previously unsuspected anti-cancer effects with approved and emerging drugs, we applied Drug Sensitivity and Resistance Testing (DSRT) technology (Pemovska et al., Cancer Discovery, 2013) to determine detailed dose-response effects of 300 drugs in 25 established OC cell lines (10 serous, 10 clear cell and 5 mucinous ovarian cancer cell lines). The panel covered 160 approved drugs as well as 162 emerging, investigational and pre-clinical oncology compounds such as kinase and non-kinase inhibitors. All drugs were tested in 5 different concentrations covering a 10,000-fold concentration range using Labcyte nano-dispensing technology, in order to generate detailed dose-response curves for each drug in each cell line. The area under the curve was estimated from the dose-response curve to obtain the drug sensitivity score. Bioinformatic processing of the drug response data from OC cell lines resulted in several key observations. First, the DSRT data made it possible to classify OC cell lines into four functional taxonomic subtypes based on comprehensive drug responses. Interestingly, these clusters did not depend on the histological origin of the OC cell lines. Second, the 322 drugs were clustered into functional subsets based on the response data across the 25 OC cell lines. Most of this clustering followed the expected chemical similarity and target space of the drugs, such as topoisomerase II inhibitors, MEK inhibitors, mTOR inhibitors and Taxanes each clustering in their own subgroups. Third, we found that many emerging, currently not-yet-approved drugs were active in subgroups of OC cell lines, including both kinase as well as non-kinase drugs, such as HDAC inhibitors Entinostat and Belinostat. DSRT technology provides a powerful strategy for assessing drug response in OC cell models, and it could also help to asses drug responses in patient-derived ex vivo model systems of OC. Analysis of correlations of DSRT data with genomic data of the cell lines is underway and will yield new translational and pharmacogenomic opportunities for OC. Citation Format: Akira Hirasawa, Astrid Murumägi, Mariliina Arjama, Bhagwan Yadav, John Patrick Mpindi, Krister Wennerberg, Tero Aittokallio, Daisuke Aoki, Olli Kallioniemi. Systematic high-throughput drug sensitivity and resistance testing (DSRT) of ovarian cancer cell lines indicates novel therapeutic possibilities with existing and emerging drugs. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5384. doi:10.1158/1538-7445.AM2014-5384

Most drug-testing approaches published so far focus on identifying a single drug that shows favorable response and is associated with a known cancer biomarker such as the drug Imatinib in BCR-ABL gene fusion positive cells. We developed and applied drug set enrichment analysis (DSEA) to find enriched patterns or statistically significant similarities (overlaps) between the drug responses of a test sample against a cohort of 182 previously screened cancer samples. The samples studied included established (ATCC) cancer cell lines, drug-resistant cancer cell models, ex-vivo patient cancer cells in primary cultures, including conditionally reprogrammed cancer cells from patients. DSEA is adopting Gene Set Enrichment Analysis statistics commonly used for gene expression analysis to high throughput drug testing data. Our drug screening (Pemovska et al., Cancer Discovery, 2013) was done with a panel of 306 established (FDA-approved) and emerging targeted cancer drugs such as tyrosine-kinase inhibitors (e.g. EGFR, PDGFR, BRAF, MET), S/T-type inhibitors, (e.g. MEK, Plk1, Akt, Aurora, Chk1), and inhibitors of other pathways (HDACs, Hh, BCL2, PI3K, PARP) and many others. The readout was based on viability of cells after a 72 hour culture. The DSEA approach is based on taking the top most sensitive drugs (above a defined sensitivity score cut-off) in an individual cancer sample and then identifying overlapping drug response profiles in previously screened reference samples. Our hypothesis is that the most sensitive drug sets in any given sample tend to show similar response profiles in a cohort of similar samples. We convey the correlations and drug set enrichment analysis results as dendrogram trees, plots and tables with enrichment and significance scores. Interestingly, our results show that clustering of drug sensitivity testing data does not place all cancer cell line samples within well-established subgroups based on biological features or histological origin. We find a similar tendency in ex vivo patient samples. Therefore, comprehensive drug response profiles seen may reveal novel biological data that reflect pharmacologically-relevant, phenotypic cancer cell states. DSEA could also provide novel means to subtype previously poorly characterized cancer samples based on their drug response profiles and thereby in the future facilitate the choice of therapies to patients whose cancers repond in an atypical way as compared to the expectations based on anatomical origin or genomic composition. Citation Format: John Patrick Mpindi, Dimitry Bychkov, Yadav Bhagwan, Disha Malani, Hirasawa Akira, Khalid Saeed, Susanne Hultsch, Sara Kangaspeska, Astrid Murumägi, Caroline A Heckman, Kimmo Porkka, Tero Aittokallio, Krister Wennerberg, Päivi Östling, Olli Kallioniemi. Drug set enrichment analysis : A computational approach to identify functional drug sets. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4184. doi:10.1158/1538-7445.AM2014-4184

The use of ABL1 kinase inhibitors has dramatically improved the outcome for chronic myeloid leukemia (CML) and Philadelphia-positive (Ph+) acute lymphoblastic leukemia (ALL) patients. However, resistance after treatment still poses a major clinical challenge. The most common resistance mechanism following treatment with first or second line therapy is the occurrence of a T315I mutation in the kinase domain of BCR-ABL1. Only one clinically available ABL1 inhibitor, ponatinib, has been shown to target this gatekeeper mutation, but has recently been associated with significant adverse effects. Hence, there is an unmet need for new and improved therapies for patients with T315I BCR-ABL1 leukemias. In this study we set out to functionally and molecularly profile BCR-ABL1 T315I-driven CML/Ph+ALL patient samples to understand the disease pathogenesis and identify novel therapies with a drug sensitivity and resistance testing (DSRT) platform covering 306 approved and investigational oncology compounds. Mononuclear cells isolated from patient bone marrow were plated with drugs on 384-well plates. Each compound was tested for its effect on cell growth and survival in a 10,000-fold concentration range enabling the generation of dose response curves and by comparing to healthy donor mononuclear cells, selective drug sensitivity scores (sDSS). Ex vivo DSRT results of one CML and two Ph+ALL patient samples with the T315I mutation revealed a marked and specific sensitivity (IC50 30-40nM) to the tyrosine kinase inhibitor axitinib, originally developed as a VEGFR inhibitor. Strikingly, sensitivity to axitinib was higher in these T315I positive patient samples than in T315I negative CML or ALL patient samples or any other leukemic samples. Supporting the notion that axitinib is a direct T315I BCR-ABL1 inhibitor we observed that Ba/F3 cells transformed with T315I BCR-ABL1 were sensitive to axitinib while the same cells transformed with wild type BCR-ABL1 were not. Finally, we discovered that axitinib has been described to have selective binding towards T315I ABL1 compared to the wild type kinase (Kd 1.5 nM vs. 36 nM, respectively, Davis et al. 2011, Nat. Biotechnol. 29:1046-1051). Based on this information, the T315I CML patient mentioned above was compassionately treated with axitinib for 2 weeks resulting in a rapid 4-fold reduction of the mutated transcript levels in blood suggesting targeted in vivo activity of the drug. In summary, we demonstrated that axitinib is a potent BCR-ABL1 T315I inhibitor both in vitro and in vivo. In light to the fact that axitinib is currently approved as a second line therapy for renal cell carcinoma and is well tolerated in patients, there is an opportunity to repurpose axitinib for Ph+ leukemia patients with T315I mutations with significantly shorter clinical development time. Citation Format: Tea Pemovska, Mika Kontro, Gretchen A. Repasky, Kimmo Porkka, Krister Wennerberg. The tyrosine kinase inhibitor axitinib targets T315I gatekeeper-mutant Philadelphia chromosome-positive leukemias in vitro and in vivo. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4763. doi:10.1158/1538-7445.AM2014-4763