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Molecular mimicry is one of the leading mechanisms by which infectious agents can induce autoimmunity. Whether a similar mechanism triggers an antitumor immune response is unexplored, and the role of antiviral T cells infiltrating the tumor has remained anecdotal. To address these questions, we first developed a bioinformatic tool to identify tumor peptides with high similarity to viral epitopes. Using peptides identified by this tool, we demonstrated that, in mice, preexisting immunity toward specific viral epitopes enhanced the efficacy of cancer immunotherapy via molecular mimicry in different settings. To understand whether this mechanism could partly explain immunotherapy responsiveness in humans, we analyzed a cohort of patients with melanoma undergoing anti-PD1 treatment who had a high IgG titer for cytomegalovirus (CMV). In this cohort of patients, we showed that high levels of CMV-specific antibodies were associated with prolonged progression-free survival and found that, in some cases, peripheral blood mononuclear cells (PBMC) could cross-react with both melanoma and CMV homologous peptides. Finally, T-cell receptor sequencing revealed expansion of the same CD8+ T-cell clones when PBMCs were expanded with tumor or homologous viral peptides. In conclusion, we have demonstrated that preexisting immunity and molecular mimicry could influence the response to immunotherapies. In addition, we have developed a free online tool that can identify tumor antigens and neoantigens highly similar to pathogen antigens to exploit molecular mimicry and cross-reactive T cells in cancer vaccine development.

Supplementary Tables 1-3 and Figures 1-6

Supplementary Data File S1

The supplementary Figures 1 to 3 demonstrate the gating procedure post flow cytomety analysis.

Supplementary Figure S1. Dasatinib-induced rapid lymphocyte mobilization and tumor growth rate correlation Supplementary Figure S2. Immunohistochemical staining of CD8+ T cells in the tumor tissues of A) vehicle treated control group and B) dasatinib treated group Supplementary Figure 3. Confirmation of CD4+, CD8+ T cells and NK cell depletion by FACS Supplementary Figure 4. Immunophenotypic analysis of B16.OVA tumor infiltrating leukocytes Supplementary Figure 5. Immunophenotypic analysis of 1956 tumor infiltrating leukocytes Supplementary Figure 6. Immunophenotypic analysis of MC38 tumor infiltrating leukocytes Supplementary Figure 7. Immunophenotypic analysis of 4T1 tumor infiltrating leukocytes Supplementary Table S1. A summary of anti-mouse antibodies used for CyTOF mass cytometry analysis Supplementary Table S2. Frequencies of intratumoral T cell subsets analysed by CyTOF.

XLSX file 452K, DSRT Oncology drug collection

We generated ex vivo drug-response and multiomics profiling data for a prospective series of 252 samples from 186 patients with acute myeloid leukemia (AML). A functional precision medicine tumor board (FPMTB) integrated clinical, molecular, and functional data for application in clinical treatment decisions. Actionable drugs were found for 97% of patients with AML, and the recommendations were clinically implemented in 37 relapsed or refractory patients. We report a 59% objective response rate for the individually tailored therapies, including 13 complete responses, as well as bridging five patients with AML to allogeneic hematopoietic stem cell transplantation. Data integration across all cases enabled the identification of drug response biomarkers, such as the association of IL15 overexpression with resistance to FLT3 inhibitors. Integration of molecular profiling and large-scale drug response data across many patients will enable continuous improvement of the FPMTB recommendations, providing a paradigm for individualized implementation of functional precision cancer medicine.Significance:Oncogenomics data can guide clinical treatment decisions, but often such data are neither actionable nor predictive. Functional ex vivo drug testing contributes significant additional, clinically actionable therapeutic insights for individual patients with AML. Such data can be generated in four days, enabling rapid translation through FPMTB.See related commentary by Letai, p. 290.This article is highlighted in the In This Issue feature, p. 275

XLSX file 36K, Clinical characteristics of patients at the time of diagnosis and DSRT, and responses of previous therapies

Dasatinib, a broad-range tyrosine kinase inhibitor, induces rapid mobilization of lymphocytes and clonal expansion of cytotoxic cells in leukemia patients. Here, we investigated whether dasatinib could induce beneficial immunomodulatory effects in solid tumor models. The effects on tumor growth and on the immune system were studied in four different syngeneic mouse models (B16.OVA melanoma, 1956 sarcoma, MC38 colon, and 4T1 breast carcinoma). Both peripheral blood (PB) and tumor samples were immunophenotyped during treatment. Although in vitro dasatinib displayed no direct cytotoxicity to B16 melanoma cells, a significant decrease in tumor growth was observed in dasatinib-treated mice compared with vehicle-treated group. Further, dasatinib-treated melanoma-bearing mice had an increased proportion of CD8+ T cells in PB, together with a higher amount of tumor-infiltrating CD8+ T cells. Dasatinib-mediated antitumor efficacy was abolished when CD4+ and CD8+ T cells were depleted with antibodies. Results were confirmed in sarcoma, colon, and breast cancer models, and in all cases mice treated daily with dasatinib had a significant decrease in tumor growth. Detailed immunophenotyping of tumor tissues with CyTOF indicated that dasatinib had reduced the number of intratumoral regulatory T cells in all tumor types. To conclude, dasatinib is able to slow down the tumor growth of various solid tumor models, which is associated with the favorable blood/tumor T-cell immunomodulation. The assessment of synergistic combinatorial therapies with other immunomodulatory drugs or targeted small-molecule oncokinase inhibitors is warranted in future clinical trials. Cancer Immunol Res; 5(2); 157–69. ©2017 AACR.

11 supplement tables

Supplement text file including supplement figures, supplementary table legends and supplement methods

XLSX file 977K, Drug sensitivity profiles of AML and control samples

PDF file 400K, Supplementary Figures 1-9; Supplementary Tables 4-9 and Supplementary Methods

Purpose: Dasatinib is a short-acting dual ABL/SRC family tyrosine kinase inhibitor (TKI), which is frequently used to treat chronic myeloid leukemia. Although very effective, patients taking dasatinib often display severe adverse effects, including pleural effusions and increased risk of bleeding primarily in the gastrointestinal tract. The actual causes of these side effects are currently undetermined. We hypothesize that endothelial cells (ECs) that line the inner walls of blood vessels and control the traffic to the underlying tissues might be involved.Experimental Design: The effects of TKIs on ECs were studied by various assays, such as real-time cell impedance measurements, live-cell microscopy, wound healing, Western blot, and an in vivo model.Results: Dasatinib uniquely causes a profound, dose-dependent disorganization of the EC monolayers. Dasatinib promoted the disassembly of cell–cell contacts, altered cell–matrix contacts, and further altered the wound healing. A key observation is that this effect is fully reversible after drug washout. In line with these in vitro observations, intraperitoneal administration of dasatinib to mice caused significant vascular leakage in the intestine. The underlying molecular mechanism of dasatinib-induced reorganization of the actin involves ROCK activation, which increases the amount of the phosphorylation of myosin light chain and consequently activates the non-muscle myosin II.Conclusions: Our data are consistent with a scenario in which dasatinib triggers a transient increase in vascular leakage that probably contributes to adverse effects such as bleeding diathesis and pleural effusions. Clin Cancer Res; 23(21); 6697–707. ©2017 AACR.

MPFC gating strategy

Purpose: Leukemic stem cells (LSCs) may harbor important resistance to tyrosine kinase inhibitors in chronic myelogenous leukemia (CML). We identified Philadelphia chromosome (Ph)–positive CD34+CD38− bone marrow cells (here denoted LSCs) and addressed their response-predictive value in patients with CML (n = 48) subjected to nilotinib in the ENEST1st trial (NCT01061177).Experimental design: Two flow cytometry–based cell sorting methods were used with multiparameter-directed CD45- (MPFC) and BCR-ABL1 probe-linked (FISH) identification of Ph-positive cells, respectively.Results: We observed a positive correlation between the proportion of LSCs at diagnosis and established prognostic markers (blast count, spleen size, Sokal score, and hemoglobin). Conversely, a high LSC burden predicted for an inferior molecular response at 3 (MPFC and FISH), 6 (MPFC), 9 (FISH), and 15 months (FISH). During nilotinib therapy, the proportion of LSCs decreased rapidly. At 3 months, a median of only 0.3% LSCs remained among CD34+CD38− cells, and in 33% of the patients the LSC clone was not detectable anymore (FISH). The response kinetics was similar in LSC fractions as it was in the progenitor and unseparated bone marrow cell fractions.Conclusions: The proportion of LSCs at diagnosis, as analyzed by two independent methodologies, reflects the biology of the disease and appeared as a prognostic and response-predictive marker in patients with CML subjected to first-line nilotinib therapy. Clin Cancer Res; 22(16); 4030–8. ©2016 AACR.

Wound healing in control conditions.

Wound healing in the presence of 100 nM dasatinib.

Evans Blue endothelial permeability assay shows leakage in the intestine of mice treated with dasatinib.

100nM dasatinib was added on a monolayer of HUVECs.

Recovery of HUVEC monolayer after washing out dasatinib.

The molecular pathways that drive cancer progression and treatment resistance are highly redundant and variable between individual patients with the same cancer type. To tackle this complex rewiring of pathway cross-talk, personalized combination treatments targeting multiple cancer growth and survival pathways are required. Here we implemented a computational–experimental drug combination prediction and testing (DCPT) platform for efficient in silico prioritization and ex vivo testing in patient-derived samples to identify customized synergistic combinations for individual cancer patients. DCPT used drug–target interaction networks to traverse the massive combinatorial search spaces among 218 compounds (a total of 23,653 pairwise combinations) and identified cancer-selective synergies by using differential single-compound sensitivity profiles between patient cells and healthy controls, hence reducing the likelihood of toxic combination effects. A polypharmacology-based machine learning modeling and network visualization made use of baseline genomic and molecular profiles to guide patient-specific combination testing and clinical translation phases. Using T-cell prolymphocytic leukemia (T-PLL) as a first case study, we show how the DCPT platform successfully predicted distinct synergistic combinations for each of the three T-PLL patients, each presenting with different resistance patterns and synergy mechanisms. In total, 10 of 24 (42%) of selective combination predictions were experimentally confirmed to show synergy in patient-derived samples ex vivo. The identified selective synergies among approved drugs, including tacrolimus and temsirolimus combined with BCL-2 inhibitor venetoclax, may offer novel drug repurposing opportunities for treating T-PLL.Significance: An integrated use of functional drug screening combined with genomic and molecular profiling enables patient-customized prediction and testing of drug combination synergies for T-PLL patients. Cancer Res; 78(9); 2407–18. ©2018 AACR.

Schematics of the computational steps of DCPT platform, along with detailed evaluation of the predicted combinations tested in the T-PLL and AML patient samples ex vivo.

Consort diagram

LSC analysis at 3 months time point by FISH

Globally, breast cancer is among the most diagnosed cancer types for women. Current and upcoming breast cancer therapies are being investigated in combination with compounds that stimulate an immune response, but whether the therapeutic agents themselves have unexpected immunomodulatory effects is often overlooked. Here, we have developed a method to grow 3D cultures of intact fragments of patient-derived tissue (Patient-Derived Explant Cultures; PDECs) to assess the preclinical potential of studying human tumor cells and immune cells simultaneously ex vivo Single cell sequencing, flow cytometry, gene expression profiling and cytokine profiling data show that the tumor immunocontexture is conserved in PDECs and that these resident immune cells respond to distinct immune stimulus We performed gene expression profiling, flow cytometry, and cytokine profiling of drug-treated human explants and found that metformin has antitumor potential through the activation of antigen presenting cells. We further validated in vitro that metformin-mediated APC activation is largely through mitochondrial respiration inhibition irrespective of the presence of tumor cells. Our PDEC platform highlights the preclinical potential of ex vivo explants by simultaneously offering information of tumor and immune cell toxicity and mechanism. Citation Format: Rita J. Turpin, Ruixian Liu, Pauliina Munne, Aino Peura, Jenna Rannikko, Gino Philips, Natasha Salmelin, Elina Hurskainen, Ilida Suleymanova, Minna Mutka, Tuomo Meretoja, Johanna Mattson, Satu Mustjoki, Päivi Saavalainen, Diether Lambrechts, Jeroen Pouwels, Maija Hollmén, Juha Klefström. TIL-containing patient-derived explant cultures reveal role of metformin on antigen presenting cell activation. [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 4122.

Background: Natural killer (NK) cells are emerging as a promising therapeutic option in cancer. To better understand how cancer cells evade NK cells, we studied interacting NK and blood cancer cells using single-cell and genome-scale functional genomics screens. Methods: We performed multiplexed single-cell RNA-seq (scRNA-seq) on co-cultures of NK cells and 26 cell lines representing diverse blood cancers. Using screens of pooled DNA-barcoded cell lines (PRISM), we quantified the sensitivity of over 60 blood cancer cell lines and integrated the results with CCLE multi-omics to uncover molecular correlates of tumor cell susceptibility to NK cells. We performed 12 genome-scale CRISPR loss-of-function and gain-of-function screens of cancer-cell intrinsic NK cell resistance mechanisms across 7 blood cancer cell lines. Finally, we investigated the mechanisms-of-action of 65 genome-scale screen hits using CRISPR screens with scRNA-seq readout in both tumor and NK cells. Results: At single-cell resolution, interaction of NK and cancer cells induced distinct activation states in both cell types depending on the cancer cell lineage and molecular phenotype. NK cells transitioned either into an activated state characterized by 4-1BB, GITR, TIM-3, and TIGIT or a state marked by type I interferon signature. Tumor cells responded to NK cell attack by activating interferon gamma (IFNy) signaling, inducing MHC class I. The activation states correlated with sensitivity to NK cells, ranging from more sensitive myeloid to more resistant B-lymphoid cancers. Molecular correlates of increased sensitivity included expression of activating receptor ligands NCR3LG1, PVR, and ULBP1, whereas TRAF3 mutations correlated with resistance. CRISPR screens uncovered cancer cell-intrinsic genes driving sensitivity and resistance, including antigen presentation and death receptor signaling mediators and adhesion molecules. The screens identified new ​​blood cancer-specific NK cell inhibitory regulators (SELPLG, SPN, and MYB ) and genes previously underappreciated in NK cell evasion, including protein fucosylation and transcriptional regulators (e.g. GFI1B). CRISPR screens with scRNA-seq readout identified MHC-I, IFNy, and NF-κB regulation as underlying mechanisms. Cancer cell knockout of positive regulators of NK cell response (CD58, NCR3LG1) induced an inactive NK cell state, providing experimental evidence how cancer cell-intrinsic genetic alterations can shape the molecular profile of attacking immune cells to promote immune evasion. Conclusions: By integrating diverse functional genomics screens and patient genomic profiles, we provide a comprehensive landscape of potential biomarkers and functionally validated genetic mechanisms which influence how NK cells recognize and kill malignant cells. The results offer a roadmap to facilitate development of NK-cell based immunotherapy for blood cancers and beyond. Citation Format: Olli Dufva, Sara Gandolfi, Jani Huuhtanen, Olga Dashevsky, Khalid Saeed, Jay Klievink, Petra Nygren, Jonas Bouhlal, Jenni Lahtela, Anna Näätänen, Bishwa Ghimire, Tiina Hannunen, Pekka Ellonen, Hanna Duàn, Jason Theodoropoulos, Essi Laajala, Jouni Härkönen, Petri Pölönen, Merja Heinäniemi, Shizuka Yamano, Ryosuke Shirasaki, David Barbie, Jennifer Roth, Rizwan Romee, Michal Sheffer, Harri Lähdesmäki, Dean A. Lee, Ricardo De Matos Simoes, Matti Kankainen, Constantine S. Mitsiades, Satu Mustjoki. Single-cell functional genomics of natural killer cell evasion by tumor cells [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 667.

Despite the success of immune checkpoint inhibitors in the clinic, they only benefit a fraction of patients. A theoretical strategy to increase efficacy would be to enhance such antibodies with Fc-mediated effector mechanisms. Current IgG1 antibodies are excellent activators of natural killer cells yet neglect a crucial effector population, neutrophils. Hence, we designed a cross-hybrid Fc-fusion peptide against PD-L1 able to elicit simultaneously effector mechanisms of an IgG1 but also IgA1, consequently activating neutrophils, in order to combine multiple effector mechanisms. Moreover, to prevent toxicities, these Fc-fusion peptides were cloned in oncolytic adenoviruses whose replication is restricted to the tumor. Our oncolytic adenoviruses were able to selectively infect tumor cells, secrete the cross-hybrid Fc-fusion peptides able to bind to PD-L1 and activate multiple immune components enhancing tumor cytotoxicity compared to FDA-approved immune checkpoint inhibitors. We validated this in various human and murine cancer cell lines and also renal cell carcinoma patient derived organoids from four patients. In conclusion, our cross-hybrid Fc-fusion peptides demonstrate that activating multiple immune effector populations increases tumor cytotoxicity potentially leading to improved clinical outcomes. Citation Format: Firas Hamdan, Erkko Ylösmäki, Jacopo Chiaro, Yvonne Giannoula, Maeve Long, Manlio Fusciello, Sara Feola, Beatriz Martins, Michaela Feodoroff, Gabriella Antignani, Otto Kari, Moon Hee Lee, Petrus Järvinen, Harry Nisen, Anna Kreutzman, Satu Mustjoki, Thomas G McWilliams, Mikaela Grönholm, Vincenzo Cerullo. Characterization in patient derived tumor organoids of novel oncolytic adenoviruses expressing enhanced cross-hybrid IgGA Fc PD-L1 inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1867.

Molecular mimicry is known to be one of the leading mechanisms by which infectious agents may induce autoimmunity. However, whether a similar mechanism triggers anti-tumor immune response is unexplored, and the role of anti-viral T-cells infiltrating the tumor has remained anecdotal. To address this question, we first developed a bioinformatic tool to identify tumor peptides with high similarity to viral epitopes. Using peptides identified by this tool, we showed that, in mice, viral pre-existing immunity enhanced the efficacy of cancer immunotherapy via molecular mimicry. Specifically, when treated with a cancer vaccine consisting of peptides with a high degree of homology with specific viral peptides, the mice with induced pre-existing immunity to these viral peptides showed significantly better anti-tumor response.To understand whether this mechanism could partly explain immunotherapy-response in humans, we analyzed a cohort of melanoma patients undergoing PD1 treatment with high IgG titer for Cytomegalovirus (CMV). In this cohort of patients, we showed that high level of CMV-antibodies was associated with a prolonged progression free survival, and found that in some cases PBMCs could cross-react with both melanoma and CMV homologous peptides. Finally, T-cell TCR sequencing revealed expansion of the same CD8+ T-cell clones, when PBMCs were pulsed with tumor- or homologous viral peptides.In conclusion, we have demonstrated that pre-existing immunity and molecular mimicry could explain part of the response observed in immunotherapy. Most importantly, we have developed a tool able to identify tumor antigens and neoantigens based on their similarity to pathogen antigens, in order to exploit molecular mimicry and cross-reactive T-cells in cancer vaccine development. Citation Format: Jacopo Chiaro, Henna Kasanen, Thomas Whalley, Cristian Capasso, Sara Feola, Mikaela Grönholm, Firas Hamdan, Karita Peltonen, Micaela Hernberg, Siru Mäkelä, Hanna Karhapää, Paul Brown, Beatriz Martins, Manlio Fusciello, Erkko Ylösmäki, Anna Kreutzman, Satu Mustjoki, Barbara Szomolay, Vincenzo Cerullo. Viral molecular mimicry influences the antitumor immune response in murine and human melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1488.

Natural killer (NK) cells exhibit potent activity in pre-clinical models of diverse hematologic malignancies and solid tumors and infusion of high numbers of NK cells, either autologous or allogeneic, after their ex vivo expansion and activation, has been feasible and safe in clinical studies. To systematically define molecular features in human tumor cells which determine their degree of sensitivity to human allogeneic NK cells, we quantified the NK cell responsiveness of hundreds of molecularly-annotated “DNA-barcoded” solid tumor cell lines in multiplexed format (PRISM; Profiling Relative Inhibition Simultaneously in Mixtures approach), correlating cytotoxicity scores for each cell line with the CCLE transcriptional data (RNA-seq), to reveal genes that are associated with resistance or sensitivity to NK cells. In addition, we applied genome-scale CRISPR-based gene editing screens in several solid tumor cell lines to interrogate, at a functional level, which genes regulate tumor cell response to NK cells. Based on these orthogonal studies, NK sensitive tumor cells tend to exhibit high levels of the NK cell-activating ligand B7-H6 (NCR3LG1); low levels of the inhibitory ligand HLA-E; microsatellite instability (MSI) status; high transcriptional signature for chromatin remodeling complexes and low antigen presentation machinery genes. Treatment with an HDAC inhibitor reduced the sensitivity of SW620 colon cancer cells, increased antigen presentation machinery, including HLA-E, and reduced B7-H6. Importantly, we observe that transcriptional signatures of NK cell-sensitive tumor cells correlate with immune checkpoint inhibitor resistance in clinical samples. Strikingly, comprehensive analysis of the CCLE transcriptional signatures revealed that cell lines with mesenchymal-like program tend to be more sensitive to NK cells treatment, compared with cell lines of epithelial-like program. Indeed, mesenchymal tumors tend to have lower expression of antigen presentation machinery in both CCLE and TCGA, suggesting a link between these two machieneries. This study provides a comprehensive map of mechanisms regulating tumor cell responses to NK cells, with implications for future biomarker-driven applications of NK cell immunotherapies. Citation Format: Michal Sheffer, Emily Lowry, Nicky Beelen, Minasri Borah, Suha Naffar-Abu Amara, Chris C. Mader, Jennifer Roth, Aviad Tsherniak, Olga Dashevsky, Sara Gandolfi, Samantha Bender, Jordan Bryan, Cong Zhu, Li Wang, Ricardo De-Matos Simoes, Channing Yu, Yiguo Hu, Olli Dufva, Marios Giannakis, Todd Golub, Rizwan Romee, Satu Mustjoki, Aedin C. Culhane, Lotte Wieten, Constantine S. Mitsiades. Landscape of molecular events regulating tumor cell responses to natural killer cells [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO041.

STAT5 is a member of the signal transducer and activator of transcription family of proteins and is widely recognized as an oncogenic master regulator of hematological malignancies. To date, therapeutic approaches to attenuate aberrant activity have focused on upstream kinase targets such as JAK2, Bcr-Abl, Flt3, and Flt3-ITD. To date, there is no STAT5-selective inhibitor in clinical development. More recently, PROTAC approaches have been deployed successfully against the STAT3 isoform and demonstrated efficacy and safety in preclinical models. We herein report the identification of a potent and selective, non-PROTAC, small molecule degrader of STAT5 protein, JPX-0750. Upon binding, JPX-0750 covalently and selectively binds to a cysteine residue on STAT5 via an electrophilic warhead, which induces profound destabilization of STAT5. The unfolding/destabilization effect can be observed via Western blot, isothermal denaturation of purified protein and HDX exchange. In a range of AML and TPLL cell lines exposure to JPX-750 at nM concentrations leads to a rapid and dose-dependant degradation of both phospho-STAT5 and total STAT5 with resultant IC50's between 80-200 nM. Washout experiments determined that exposure to JPX-0750 at 1 µM for 2-4 h is sufficient to degrade STAT5, inhibit downstream targets, and induce apoptosis. Moreover, in the same washout experiments, 50% STAT5 recovery post-washout required >72 h suggesting a long pharmacodynamic effect. Against STAT3, degrading effects are observed later, 24 h post washout (6 h treatment), indicating that anti-STAT5 activity is more rapid in AML cells and in line with the inherent stability of each protein. No effect was observed on either STAT1 or pSTAT1 upon exposure to JPX-750. Importantly, compared to standard AML cell lines JPX-750 demonstrated similar low nM potency in 25/30 primary AML blasts and TPLL patient samples, including those with poor prognostic markers. JPX-750 exhibits a large therapeutic window for AML versus pooled human fibroblasts and hematopoietic stem cells (ca 100 fold). In a preclinical MV4;11 luciferase model, structurally related JPX-700, at 5 mg/kg (IP, qd) significantly reduced leukemic burden, suppressed tumour dissemination to both the lung and liver, and had no effect on body weight, organ histology or blood parameters. In summary, JPX-750 represents a new class of potent and selective small molecule degraders of STAT5 protein. Citation Format: Ji Sung Park, Gary Tin, Elvin D. de Araujo, Anna Orlova, Helena Sorger, Florian Grebien, Elizabeth Heyes, Mulu Geletu, Ruth Villalonga, Angel Sampedro, Abootaleb Sedighi, Marco Herling, Satu Mustjoki, Mohammad S. Eram, Siawash Ahmar, Richard Moriggl, Jeff A. O'Meara, John Proudfoot, Kay Noel, Dziyana Kraskouskaya, Roman Fleck, Patrick T. Gunning. A potent and selective small molecule degrader of STAT5 for the treatment of hematological malignancies [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 LB-108.

Despite the great promise of immunotherapy, only relatively few patients show long-term complete remission. In addition, immunotherapy is not effective against every cancer type. Cancer types with low mutational burden such as breast cancer (BCa), for example, are thought to not elicit an immune reaction and are therefore considered non-immunogenic. A major hurdle in the advancement of immunotherapy is the lack of preclinical models that faithfully preserve the tumor microenvironment and recapitulate human disease. We have developed a method to grow 3D cultures of intact fragments of patient-derived tissue (Patient-Derived Explant Cultures; PDECs), which has been used to assess the efficacy of novel treatment regimens on real human tumors (Haikala et al., Nature Comm. 2019; Tervonen et al., Oncogene 2016). PDECs offer many advantages over other cancer model systems; PDECs preserve the human tumor microenvironment, reflect inter- and intra-patient tumor heterogeneity and results are obtained within one week. Up to 16 experimental samples can be obtained from one clinical sample, allowing the same ‘patient' to be experimentally treated with different drug regimens and/or measurement of more than one endpoint.To assess whether PDECs are also a suitable model for IO studies we performed immunoprofiling experiments using FACS, RT-PCR, cytokine profiling and immunofluorescence, showing that the immune-contexture, i.e. the number and activation status of different immune cell types, is similar in PDECs than in the primary tumor they were derived from. Subsequently, we addressed whether the resident immune cells in PDECs were functional. Preliminary data showed that direct T cell activation in PDECs led to apoptosis of cancer cells and invoked a complex immune response, as assessed using cytokine profiling and FACS. Treatment with the immune checkpoint inhibitor anti-PD-1 had more moderate effects. These data reflect the clinical reality where very few BCa patients respond to immune checkpoint monotherapy, but also show that T cells in BCa can be activated to kill the cancer cells, suggesting that with the right treatment strategies, BCa can be targeted with immunotherapy. All in all, our data show that the tumor's immunocontexture is conserved in PDECs and that resident immune cells in PDECs can be activated to attack cancer cells, and thus support the use of PDECs as an ex vivo model system to study immuno-oncology in real human cancers for the development of the immunotherapies of tomorrow. Citation Format: Rita Turpin, Pauliina Munne, Ilida Suleymanova, Satu Mustjoki, Jeroen Pouwels, Juha Klefström. Patient-derived explant cultures (PDECs) as a model system for immuno-oncology studies [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 1115.

Immune escape mechanisms promote tumor progression and are hurdles of cancer immunotherapy. Removing immunosuppressive cells before treatment can enhance efficacy. Tyrosine kinase inhibitors (TKI) may be of interest to combine with immunotherapy, as it has been shown that the inhibitor sunitinib reduces myeloid suppressor cells in patients with renal cell carcinoma and dasatinib promotes expansion of natural killer–like lymphocytes in chronic myeloid leukemia (CML). In this study, the capacity of dasatinib and imatinib to reduce myeloid suppressor cells and to induce immunomodulation in vivo was investigated ex vivo. Samples from CML patients treated with imatinib (n = 18) or dasatinib (n = 14) within a Nordic clinical trial (clinicalTrials.gov identifier: NCT00852566) were investigated for the presence of CD11b+CD14−CD33+ myeloid cells and inhibitory molecules (arginase I, myeloperoxidase, IL10) as well as the presence of natural killer cells, T cells (naïve/memory), and stimulatory cytokines (IL12, IFNγ, MIG, IP10). Both imatinib and dasatinib decreased the presence of CD11b+CD14−CD33+ myeloid cells as well as the inhibitory molecules and the remaining myeloid suppressor cells had an increased CD40 expression. Monocytes also increased CD40 after therapy. Moreover, increased levels of CD40, IL12, natural killer cells, and experienced T cells were noted after TKI initiation. The presence of experienced T cells was correlated to a higher IFNγ and MIG plasma concentration. Taken together, the results demonstrate that both imatinib and dasatinib tilted the immunosuppressive CML tumor milieu towards promoting immune stimulation. Hence, imatinib and dasatinib may be of interest to combine with cancer immunotherapy. Mol Cancer Ther; 14(5); 1181–91. ©2015 AACR.

Introduction: Tumor microenvironment (TME) and limited immune surveillance impact lymphoma pathogenesis and survival. Immune checkpoint receptors, such as programmed cell death-1 (PD1), lymphocyte-activation gene-3 (LAG3) and T-cell immunoglobulin and mucin domain-3 (TIM3), mediate signals leading to T-cell exhaustion and immune escape. Here, we have characterized the immunological profiles of diffuse large B-cell lymphoma (DLBCL) and associated the findings with outcome. Methods: We utilized the NanoString nCounter platform with a 770-gene PanCancer Immune panel to profile gene expression from 81 RNA samples collected from high-risk DLBCL patients. Multiplex immunohistochemistry (mIHC) with digital image analysis was used to characterize the T-cell phenotypes (CD3, CD4, CD8, PD1, LAG3, and TIM3) in two DLCBL patient cohorts (n=52 and n=136). The findings from gene expression profiling and mIHC were correlated with clinical parameters and patient outcome (i.e. progression-free survival (PFS) and overall survival (OS)). Results: Gene expression profiling revealed a high degree of heterogeneity among DLCBL. Unsupervised hierarchical clustering identified gene clusters differentially expressed between the patients. The distinct clusters contained genes for cytolytic factors and immune checkpoint molecules (GZMB, PRF1, IFNG, TIM3, LAG3), T-cells (CD3, CD2, CD28), macrophages (CD68, CD163), B-cells (MS4A1, CD19, CD79A/B), and extracellular matrix (FN1, ITGA1/5/6, VEGFA). mIHC revealed that the proportion of CD3+ T-cells varied markedly between the patients (median 24.8%, range 0.5-65.1%). On average, 8.8% (0-34.3%) of the CD3+ T-cells were positive for TIM3, whereas 0.6% (0-5.4%) were positive for LAG3. High content of TIM3+ cells was associated with poor outcome (PFS: HR 1.06, 95%CI 1.02-1.10, P=0.003; OS: HR=1.12, 95%CI 1.04-1.20, P=0.003) in a cohort of 52 DLBCL patients independent of the international prognostic index (IPI). In addition, high proportion of TIM3+CD3+ cells associated with poor survival independently of IPI (PFS: HR 1.07, 95%CI 1.00-1.14, P=0.040; OS: HR 1.087, 95%CI 1.01-1.17, P=0.019). The prognostic impact of TIM3 on survival was validated in a separate mIHC cohort of 136 DLBCL patients (PFS: HR 1.07, 95%CI 1.02-1.14, P=0.014; OS: HR 1.10, 95%CI 1.03-1.16, P=0.002). Conclusions: Our data demonstrates that the molecular immunological profile of DLBCL is heterogenic and that the expression of T-cell exhaustion marker TIM3 correlates with poor prognosis in patients with DLBCL. A more detailed characterization of immune cell composition in the TME and its impact on survival is ongoing. Citation Format: Suvi-Katri Leivonen, Matias Autio, Oscar Bruck, Satu Mustjoki, Judit M. Joergensen, Marja-Liisa Karjalainen-Lindsberg, Klaus Beiske, Harald Holte, Sirpa Leppä. Clinical impact of T-cell exhaustion in patients with diffuse large B-cell lymphoma [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 980.

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.

Elevated MYC levels sensitize tumor cells to apoptosis but the therapeutic potential of this mechanism remains unclear. We find, in a model of MYC-driven breast cancer, that pharmacological activation of AMPK dramatically synergizes with BCL-2/BCL-XL inhibitors to activate MYC-dependent apoptosis. We demonstrate the translational potential of an AMPK and BCL- 2/BCL-XL co-targeting strategy in ex vivo and in vivo models of MYC-high breast cancer. Metformin combined with either navitoclax or venetoclax efficiently inhibits tumor growth, confers survival benefits and induces tumor infiltration by immune cells. However, withdrawal of the drugs allowed tumor re- growth with presentation of PD1+/CD8+ T cell infiltrates, suggesting immune escape. A two-step treatment regimen, beginning with neoadjuvant metformin+venetoclax to induce apoptosis and followed by tumor resection and adjuvant metformin+venetoclax+anti-PD1 treatment to overcome immune escape, led to durable antitumor responses even after drug withdrawal. We demonstrate that pharmacological reactivation of MYC-dependent apoptosis is a powerful antitumor strategy involving both tumor cell depletion and immunosurveillance. Citation Format: Heidi M. Haikala, Johanna M. Anttila, Mariel Savelius, Elsa Marques, Tiina Raatikainen, Mette Ilander, Henna Hakanen, Johanna Mattson, Paivi Heikkila, Marjut Leidenius, Heikki Joensuu, Satu Mustjoki, Panu Kovanen, Martin Eilers, Joel D. Leverson, Juha T. Klefström. Pharmacological reactivation of MYC-dependent apoptosis cooperates with anti-PD1 immunotherapy [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 LB-189.

Despite the impressive impact of CTLA4 and PD1-targeted immuno-oncologic (IO) therapies, a large proportion of patients fail to respond. The observed variance in treatment efficacy has been linked to heterogeneity in the immune cell distribution of individual patients. Lymphocyte activation gene 3 (LAG3, CD223) is one of the newest IO targets entering the clinic. Triggering of LAG3 on T-cells by HLA-DR -ligands has a well-established role in the negative regulation of T-cell function. Although for example on activated regulatory T-cells (Treg) LAG3 is widely expressed, the detailed effects of LAG3 on various cell types are still unknown. We profiled over 30,000 CD45+ lymphocytes cells using a novel paired single-cell RNA and T-cell receptor (TCR) αβ chain (10x Genomics) sequencing method for peripheral blood samples from two patients receiving anti-LAG3 and anti-PD1 combination treatment in multicentre phase I trial. The sequenced cells were from IO-treatment naïve metastatic melanoma patients from before, after 4 weeks and after 12 weeks of the start of therapy. For validation, we performed TCRβ-sequencing and flow cytometry analysis of a larger cohort of melanoma patients (n = 12) enrolled in the same study. To gain in-depth understanding of the immune cell subsets, we used a recently described method to seek matching mutual neighbors between patients to normalize the interpatient variation to enable a systematic comparison across patients. To identify phenotypic clusters, we used a graph theory-based clustering method SNN-Cliq and built predictive machine learning classifiers to assess the reproducibility of learnt clusters. After optimizing our choice of input genes and parameters, we identified 16 distinct clusters that define the T-cell roadmap of anti-LAG3 and anti-PD1 treatment that includes 6 CD8+, 8 CD4+ (including Treg cluster), and 2 other clusters. We identified 4 CD8+ T-cell clusters of increasing cytotoxicity profile using a cytotoxicity score and the pseudotime algorithm Monocle3. The most highly cytotoxic clusters increased and a cluster of lower cytotoxicity score decreased during the treatment. In addition, we defined 2 CD8+ exhaustion clusters. During treatment, we observed a decrease in the exhausted T-cell cluster defined by LAG3 and PDCD1 expression, but an increase in TIGIT+ exhaustion cluster. Furthermore, ordering the cluster of FOXP3+ Treg cells along pseudotime trajectory revealed two different fates for Treg cells, where the other fate showed significantly decreased expression of LAG3 and PDCD1, adding evidence of the effect of the treatment on Treg cells. TCRαβ analysis revealed 19 individual expanded clonotypes of size of 100 sequenced individual cells. The true transcriptomic heterogeneity of identical clonotypes was revealed as most clonotypes spanned several of the 16 different clusters, challenging our view of clonal expansion. Furthermore, we were able to assess the temporal phenotypic changes in individual clones during the course of immunotherapy. Most clonotypes, including cytotoxic and exhausted clones, had more homogenous transcriptomes before the start of the treatment, but diversified during the therapy, suggesting a release of immunologic break in these clones. In summary, we defined the evolution of immunological response to anti-LAG3 and anti-PD1 therapy cell by cell, and described an increase in the heterogeneity of cytotoxicity in CD8+ cells, distinct fates of Treg cells and the release of transcriptomic profiles of individual T-cell clonotypes during IO treatment. Citation Format: Jani Huuhtanen, Henna H.E. Hakanen, Tapio Lönnberg, Olli Dufva, Katriina Peltola, Siru Mäkelä, Micaela Hernberg, Petri Bono, Kreutzman Anna, Satu Mustjoki. Single-cell roadmap of the evolution of T-cell response during anti-LAG3 and anti-PD1 combination treatment in metastatic melanoma patients [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A134.

Anti-PD1 therapy has proven to be effective in various cancer types, but not all patients benefit from the therapy. Further, no comprehensive immunologic monitoring during anti-PD1 treatment has yet been published. In this study, we aimed to discover the effects of anti-PD1 therapy on the immune system, especially on NK and NKT-cells, which are less studied, but known to be involved in antitumor immune events. Peripheral blood samples from immuno-oncology (IO) naive metastatic melanoma patients (n=20) were obtained before the first infusion of pembrolizumab or nivolumab (D0), then 1 and 3 months after the initiation of treatment. From each time-point, complete blood counts (CBC) were obtained, and comprehensive immunophenotyping of NK, NKT, and T-cells was performed with multicolor flow cytometry. Moreover, 92 different serum cytokines were measured using the Olink inflammation panel. The protein levels are presented as arbitrary units of normalized protein expression, NPX, on Log2 scale. The CBC revealed that the proportion of lymphocytes (mean D0 30.6% vs. 3mo 24.9%, p=0.02), decreased during the treatment but no changes were observed in absolute numbers or in other leukocytes. Immunophenotyping of lymphocyte subpopulations revealed that the frequency of NKT brighT-cells was increased (D0 1.8% vs. 1mo 2.3%, p=0.01) and that cytotoxic NK CD56dim cells expressed more CD25 (D0 19.5% vs. 1mo 23.7%, p=0.02) and CD45RO (D0 14.7% vs. 1mo 21.1%, p=0.03) surface markers after 1 month of therapy. The cytokine assay indicated that during anti-PD1 treatment the levels of CXC family cytokines were increased in the serum; CXCL9 (D0 470 vs. 1mo 1070, p=0.0003, D0 470 vs. 3mo 1227, p=0.0007), CXCL11 (D0 49.8 vs. 1mo 81.8, p=0.005), CXCL10 (D0 1000 vs. 1mo 2078, p=0.0003). Also, an increase in IL-12B (D0 31.6 vs. 1mo 41.2, p=0.003, D0 31.6 vs. 3mo 32.4, p=0.04) and TNFRSF9 (D0 126.4 vs. 1mo 181.0, p=0.01, D0 126.4 vs. 3mo 149.0, p=0.04) levels was observed.To further examine these results, patients were categorized into two cohorts: responders (R, n=6, PFS=17.0 months) and patients with progressive disease (PD, n=9, PFS=5.0 months) in terms of the duration of progression-free survival (PFS) and decrease in tumor burden. 5 patients were excluded due to challenging clinical evaluation of response. When examining the differences between these cohorts, CBC indicated a significant decrease in the mean frequency of lymphocytes in PD (D0 26.9% vs. 3mo 19.2%, p=0.04), but not in the R cohort. The responders had also higher frequency of lymphocytes at 1- and 3-month time-points (R 34.5% vs. PD 25.4%, p=0.02, R 32.4% vs. PD 19.2%, p=0.01, respectively) and lower frequency of neutrophils before initiation and after 1 and 3 months of treatment (R 50.8% vs. PD 58.6%, p=0.04, R 45.3% vs. PD 59.4%, p=0.01, R 49.8% vs. PD 64.5%, p=0.04, respectively). The CBC absolute counts revealed that the responders had less neutrophils (R 2.8 109/L vs. PD 4.9 109/L, p=0.04) and monocytes (R 0.4 109/L vs. PD 0.7 109/L, p=0.04) after 3 months of treatment when compared to PD. The immunophenotyping showed that responders had more NKT dim cells before (R 10.1% vs. PD 3.5%, p=0.03) and after 3 months of therapy (R 15.7% vs. PD 3.7%, p=0.03) and the increase in NKT bright frequency was observed only in R (D0 2.5% vs. 1mo 3.4%, p=0.04) and not in PD cohort. The cytokine assay indicated that the CXCL9 was increased in R cohort (D0 476.8 vs. 1mo 1480.2, p=0.01), but not in PD, making the cytokine levels greater in R (R 1480.2 vs. PD 639.3, p=0.01) after 1 month of therapy. Based on our preliminary results, we believe that high frequency of NKT-cells in blood is related to positive treatment response, and in addition to T-cells, also NK and NKT-cells may play a key role in the antitumor response induced by PD-1 inhibition. Hence, further research on the effect of anti-PD1 therapy on NK and NKT-cells is needed to better understand their role in positive therapy response. Citation Format: Henna H.E. Hakanen, Micaela Hernberg, Siru Mäkelä, Bhagwan Yadav, Oscar Brück, Susanna Juteau, Laura Kohtamäki, Mette Ilander, Satu Mustjoki, Kreutzman Anna. Metastatic melanoma patients responding to PD1 therapy have higher proportion of peripheral blood NKT-cells [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A130.

Harnessing natural killer (NK) cells to attack tumors could improve immune-based cancer treatment strategies. However, mechanisms regulating sensitivity or resistance of cancer cells to the effector function of NK cells are incompletely understood. Here, we performed genome-scale CRISPR-Cas9 loss-of-function screens in human cancer cells to discover genes that influence susceptibility to primary human NK cells. To screen for genes essential for the interaction between NK cells and cancer cells, we infected human cancer cells expressing Cas9 with a genome-scale lentiviral guide RNA library. The resulting pool of knockout cells was exposed to NK cells expanded from peripheral blood of healthy donors. Enriched and depleted knockout clones were detected by next-generation sequencing of the integrated sgRNA cassettes, enabling identification of genes conferring resistance or susceptibility to NK cell-mediated lysis. The screens were performed in cell lines from diverse cancer types, including chronic myeloid leukemia (CML), B cell acute lymphoblastic leukemia, diffuse large B-cell lymphoma (DLBCL), and multiple myeloma. We recovered several known mechanisms of NK cell/cancer cell interactions, demonstrating feasibility of the screening approach. Loss of genes encoding components of the MHC class I complex (B2M, HLA-A, HLA-C, HLA-E) sensitized multiple cancer cell lines to NK cell-mediated lysis. This is consistent with missing-self recognition as a fundamental mechanism of NK cell activation. Furthermore, knockout of IFN-JAK-STAT signaling mediators led to increased tumor cell lysis, suggesting that MHC class I induction in response to NK cell-derived IFN gamma enables NK cell evasion by tumor cells. We also identified genes essential for effective NK cell-mediated lysis. NCR3LG1, encoding the B7-H6 ligand for the NKp30 activating NK cell receptor, was essential for NK cell lysis of CML cells. In contrast, knockout of apoptotic mediators and TRAIL pathway components conferred resistance to NK cell cytotoxicity in DLBCL cells, indicating heterogeneity in NK cell/cancer cell interactions between cancer types. Our data support a view that distinct mechanisms regulate sensitivity to NK cell cytotoxicity in different cancers. Importantly, our results indicate that loss-of-function mutations in the antigen-presenting machinery and the IFN-JAK-STAT pathway sensitize tumors to NK cell effector function. As alterations in these genes are associated with resistance to T-cell immunotherapies such as PD-1 blockade, NK cell-based therapies could be employed to overcome resistance in these patients. In summary, we suggest that systematic identification of mechanisms governing tumor immune susceptibility has the potential to uncover novel immunotherapy targets. Citation Format: Olli Dufva, Jay Klievink, Khalid Saeed, Matti Kankainen, Mette Ilander, Tiiina Hannunen, Sonja Lagström, Pekka Ellonen, Dean A Lee, Satu Mustjoki. Genome-scale CRISPR screens identify essential genes for tumor sensitivity to NK cells [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A065.

Background: Renal cell carcinoma (RCC) is considered one of the most immunogenic cancers with the highest number of indel mutations, frequent infiltration of T-cells, presence of many antigen-specific T-cell clones, and high immuno-oncologic (IO) sensitivity. Since less is known about natural killer (NK) cells in RCC, our primary aim was to investigate the intratumoral phenotype of NK cells as well as further assess the overall immune landscape of the tumors, peripheral blood (PB) and adjacent healthy kidney tissue, which may be critical for patient prognosis and predictions to targeted immunotherapies. Methods: We used multi-parameter flow cytometry together with a comprehensive immunostaining panel containing a total of 56 fundamental markers to cancer immunology to immunophenotype the tumor, adjacent healthy kidney tissue and presurgical peripheral blood (PB) samples from 31 RCC patients who underwent partial or radical nephrectomies. To study the intratumoral T-cell clonalities, T-cell receptor beta (TCRβ) deep sequencing was carried out with eight tumors. Results: Using hierarchical clustering (Spearman correlation distance and Ward linkage) and correlation analyses (Spearman correlation), we discovered that our patient cohort clustered into two distinct subgroups defined by a high (NKhigh, n=11; mean 29.7%) and low (NKlow, n=20; mean 9.4%) percentage of NK cells among the intratumoral lymphocyte population. Accordingly, the NKhigh subgroup had a lower percentage of T-cells (mean 36.9%) than the NKlow group (mean 65.7%), and overall, a significant negative correlation between T and NK cells was discovered. Our TCRβ sequencing results revealed a positive correlation between T-cell clonality and the intratumoral T-cell percentage, whereas the higher proportion of tumor NK cells associated with low T-cell clonality, possibly due to a polyclonal T-cell population. When we compared the expressions of the most clinically relevant IO markers (LAG-3 and PD-1) on the NK cells, LAG-3 was more expressed in the NKhigh group than in the NKlow group (21.3% vs 9.8%; p=0.08). In contrast, no differences were observed with PD-1. Clinical parameters such as tumor grade (Fuhrman), weight, size (diameter), the presence of necrosis, gender, or age of the patients did not differ between the two subgroups. To examine the overall immune landscape of RCC, we compared the cells from the tumor, PB, and healthy kidney tissue of seven patients. Our results showed that tumors have more NK cells compared to their corresponding T-cell-rich PB and healthy tissue counterparts, supporting our findings that some tumors accumulate NK cells. Compared to the adjacent healthy tissue, PD-1 and LAG-3 expressions were higher in the intratumoral CD8 cells. The expressions of PD-1 and LAG-3 on PB CD8+ T-cells or NK cells did not correlate with their intratumoral counterparts, whereas a positive correlation was found between the PB and tumor CD4+ T-cells for both LAG-3 and PD-1. Conclusions: Our study has led to the discovery of two distinct RCC tumor subgroups with differential expressions in the clinically leading molecules. These results suggest that immunophenotyping RCC patients may effectively aid in selecting those who will benefit the most from immune checkpoint inhibition therapies such as anti-PD1 and -LAG3. Prospective analyses on spatial immunoprofiling by multiplexed immunohistochemistry and mutational load by exome sequencing will be assessed next in order to find answers to why some tumors are NK-dominant, with expectations of understanding further the biologic differences between the two tumor types. Citation Format: Moon Hee Lee, Petrus Järvinen, Harry Nisén, Oscar Brück, Mette Ilander, Satu Mustjoki, Kreutzman Anna. High infiltration of NK cells expressing elevated LAG-3 in a subgroup of renal cell carcinoma patients [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A085.

We present an individualized systems medicine (ISM) approach to optimize cancer drug therapies one patient at a time. ISM is based on (i) molecular profiling and ex vivo drug sensitivity and resistance testing (DSRT) of patients' cancer cells to 187 oncology drugs, (ii) clinical implementation of therapies predicted to be effective, and (iii) studying consecutive samples from the treated patients to understand the basis of resistance. Here, application of ISM to 28 samples from patients with acute myeloid leukemia (AML) uncovered five major taxonomic drug-response subtypes based on DSRT profiles, some with distinct genomic features (e.g., MLL gene fusions in subgroup IV and FLT3-ITD mutations in subgroup V). Therapy based on DSRT resulted in several clinical responses. After progression under DSRT-guided therapies, AML cells displayed significant clonal evolution and novel genomic changes potentially explaining resistance, whereas ex vivo DSRT data showed resistance to the clinically applied drugs and new vulnerabilities to previously ineffective drugs. Significance: Here, we demonstrate an ISM strategy to optimize safe and effective personalized cancer therapies for individual patients as well as to understand and predict disease evolution and the next line of therapy. This approach could facilitate systematic drug repositioning of approved targeted drugs as well as help to prioritize and de-risk emerging drugs for clinical testing. Cancer Discov; 3(12); 1416–29. ©2013 AACR. See related commentary by Hourigan and Karp, p. 1336 This article is highlighted in the In This Issue feature, p. 1317

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.

T-cell prolymphocytic leukemia (T-PLL) is a mature T-cell neoplasm usually presenting at an aggressive phase and a marked resistance to conventional cytostatics, i.e. alkylators. The monoclonal antibody alemtuzumab induces high response rates, but virtually all patients relapse and the mostly elderly individuals are often ineligible for allogeneic stem cell transplantation. Overall, the treatment options for T-PLL are scarce and its general prognosis with average survival times of Note: This abstract was not presented at the meeting. Citation Format: Sabine Puetzer, Emma Andersson, Alexandra Schrader, Lesley Varghese, M Hülsemann, Petra Mayer, Marc-Henri Stern, Sebastian Newrzela, L Frenzel, Satu Mustjoki, Marco Herling. Towards novel strategies of targeting specific vulnerabilities of T-PLL 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 1372. doi:10.1158/1538-7445.AM2017-1372

The potential of immune checkpoint inhibition in many hematological malignancies such as chronic myeloid leukemia (CML) has not been characterized in detail. CML is a hematopoietic stem cell disease driven by the BCR-ABL fusion gene. Tyrosine kinase inhibitor (TKI) therapy is able to induce remissions in the majority of patients. Despite treatment responses, leukemic stem cells persist in the bone marrow (BM). Enhancing antitumor immune responses has been suggested as a potential strategy of enabling treatment discontinuation and disease control in CML. We hypothesized that the bone marrow may represent a distinct immunological milieu in CML patients. Furthermore, TKI therapy has been shown to induce immunological changes. Therefore, we evaluated the expression of immune checkpoint molecules in paired samples from BM and peripheral blood (PB) in chronic phase CML patients at diagnosis and during treatment with TKIs. In addition, we monitored changes in the BM T cell receptor (TCR) repertoire induced by TKI therapy and disease resolution. We performed multicolor flow cytometry on frozen PB and BM samples from chronic phase CML patients at diagnosis and during treatment with the tyrosine kinase inhibitors imatinib, dasatinib and nilotinib as well as healthy controls. We analyzed the expression of PD-1, CTLA-4, LAG-3, ICOS and TIM-3 on T cells and PD-L1, PD-L2, CD80 and CD86 on antigen-presenting cells and CD34+ leukemic progenitor cells. TCR repertoire was assayed by deep sequencing of the CDR3 region using the immunoSEQ assay. At diagnosis, CD8+ T cells in the bone marrow exhibited a more exhausted phenotype compared to peripheral blood as measured by PD-1 positivity (26.1 vs. 12.7%, p = 0.001). This difference was recapitulated in all T cell subsets, including effector memory (TEM), terminally differentiated (TEMRA), central memory (TCM) and naïve, with the highest PD-1 positivity in TEM and TEMRA cells. In addition, dendritic cells as well as leukemic CD34+ progenitor cells expressed higher levels of PD-L1 and CD86 in BM compared to PB at diagnosis. Interestingly, TKI therapy led to a reduction of PD-1-positive CD8+ T cells in the BM after 1 and 6 months of treatment. This was accompanied by a concomitant decrease in PD-L1 and CD86 positivity on dendritic cells. Finally, treatment with dasatinib led to a reduction in TCR repertoire diversity and increased clonality at 6 months, whereas imatinib or nilotinib did not alter the repertoire diversity. In conclusion, BM and PB seem to exhibit different immunological milieus in terms of expression of immune checkpoint molecules in CML. During TKI therapy both PD-1 and PD-L1 levels decrease, suggesting at least partial reversal of immune cell exhaustion. Treatment with the TKI dasatinib is able to drive the TCR repertoire towards a more clonal composition, which has been associated with good responses to checkpoint blockade. Thus, targeted therapies such as TKIs are able to modulate immune checkpoints and T cell activity in unexpected ways, providing a potential strategy for enhancing immunotherapies in combination treatment regimens. Citation Format: Olli Dufva, Tiina Kasanen, Mohamed El Missiry, Judith Klievink, Hanna Lähteenmäki, Satu Mustjoki. Tyrosine kinase inhibitor therapy modulates immune checkpoints and TCR repertoire diversity in chronic myeloid leukemia [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A115.

Somatic mutations in exons 1 and 2 of the Mediator complex subunit 12 (MED12) have been identified in 70% of uterine leiomyomas, 7-20% of uterine leiomyosarcomas, 59% of breast fibroadenomas and 67% of breast phyllodes tumors. In addition to female hormone-dependent tumors we have recently found the same specific missense and small in-frame insertion and deletion mutations in approximately 5% of chronic lymphocytic leukemias (CLL). In CLL the presence of MED12 mutations was also associated with markers of poor prognosis (IgVH (-), Zap70 (+), and Zap70-methylation). The surprising finding of the same mutations in a completely different tumor type prompted us to further screen other hematological malignancies for MED12 mutations. We have thus far collected samples of 107 T-cell acute lymphoblastic leukemias (T-ALL), 21 large granular lymphocyte leukemias (LGL), 33 acute myeloid leukemias (AML), 154 diffuse large B cell lymphomas (DLBCL), and 6 six follicular lymphomas (FL). Also a set of 30 additional CLLs was collected. The MED12 mutation status was determined by direct Sanger sequencing of exons 1 and 2 of the gene. A c.107T>G, p.L36R mutation was found in a single DLBCL case and c.100-8T>A, p.E33_D34insPQ in one AML sample. A novel variant of uncertain significance, c.-3A>G, was detected on 5’UTR of one FL sample. One T-ALL patient harbored a nonsense mutation affecting the last codon of exon 1 c.97G>T, p.E33X. No mutations were identified in the LGL or CLL samples. We are also analyzing approximately 100 new AML samples and 100 multiple myeloma samples. MED12 mutations are present, in addition to CLL, also in other hematological malignancies. The frequency, however, seems to be low and, as the number of samples in the preliminary analysis is limited, screening of larger sample sets is needed. The c.107T>G, p.L36R and c.100-8T>A, p.E33_D34insPQ mutations have previously been detected recurrently in uterine leiomyomas and in CLL. Further studies are required for evaluation of the effects of c.-3A>G and c.97G>T, p.E33X mutations. The latter mutation affects the last codon of exon 1, which is also a mutational hotspot in CLL, with recurrent missense mutations. MED12 exon 1 and 2 missense mutations disrupt the interactions between the Mediator complex and Cyclin C and cause loss of CDK8 kinase activity. Studies to determine the impact of the c.97G>T, p.E33X mutation on MED12 function are ongoing. Citation Format: Tuomas Heikkinen, Kati Kämpjärvi, Sirpa Leppä, Peter Hokland, Heikki Kuusanmäki, Satu Mustjoki, Marjatta Sinisalo, Caroline Heckman, Mika Kontro, Pia Vahteristo. Somatic MED12 mutations in hematological malignancies. [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 1169.

Introduction T-cell large granular lymphocyte (T-LGL) leukemia is a rare, clonal disease characterized by the expansion of CD8+ cytotoxic T-cells. We recently discovered that 40% of T-LGL leukemia patients have somatic mutations in the SH2-domain of the STAT3 gene (Koskela et al. NEJM 2012). As aberrant STAT3 activation can be observed in all patients with LGL-leukemia, we now aimed to discover whether patients without mutations in the STAT3 hotspot area harbor mutations in the other parts of the STAT3 gene. Methods Targeted STAT3 sequencing covering all 23 coding exons was done with in-house developed deep amplicon sequencing panel using the Illumina Miseq platform. The data was analyzed with a bioinformatics pipeline, which is based on calling of variants with specific counts/frequencies and filtering out false positives using the estimated error rate and quality data of amplicon reads. All samples with a frequency ratio ≥0.9 were considered to be true mutations after filtering of SNPs and low coverage variants. 111 LGL-leukemia patients with no known STAT3-mutations in the SH2 domain were analyzed. To explore the functional effects of mutations, expression constructs were generated with the identified variants and wild-type STAT3. The variants were expressed in HEK-293 cells carrying a STAT3-responsive SIE-reporter driven luciferase expression sequence to establish increased basal and IL6-stimulated STAT3 activity. Results With targeted amplicon sequencing, 3 patients were discovered to have STAT3 missense mutations in the DNA-binding domain. Two patients presented with the same H410R mutation with a variant allele frequency (VAF) of 49% and 8.8% respectively while another patient had a S381Y mutation (VAF 7%). The mutation H410R occurs in the DNA-binding domain in a highly conserved position, and results in conversion of histidine to arginine, which would predict for a slight increase in hydrophilicity. In addition to STAT3 DNA-binding domain mutations, one T-LGL patient had a novel F174S mutation in the coiled-coil domain of STAT3 (VAF 43%). The coiled-coil domain of STAT3 has previously been shown to be essential in SH2-domain mediated receptor binding and subsequent activation. Luciferase measurements of SIE-reporter HEK-293 cells transfected with constructs expressing either wild-type, variant F174S, H410R or Y640F STAT3 (the most common activating mutation in LGL leukemia) revealed the F174S and H410R variants to be as activating as the Y640F mutation in both unstimulated and IL-6 stimulated conditions. Conclusions T-LGL leukemia patients without STAT3 SH2-domain mutations harbor novel activating mutations in the DNA-binding and coiled-coil domain of STAT3. The frequency of mutations was 3.6% (4 of 111 patients). These findings further highlight the importance of screening the whole STAT3 gene in the diagnostic workup of LGL-leukemia and the central role of STAT3 in the pathogenesis of the disease. Citation Format: Emma I. Andersson, Hanna Rajala, Heikki Kuusanmäki, Arjan van Adrichem, Samuli Eldfors, Sonja Lagström, Thomas Olson, Michael Clemente, Pekka Ellonen, Caroline Heckman, Thomas P. Loughran, Jaroslaw P. Maciejewski, Satu Mustjoki. Novel somatic mutations in the DNA-binding and coiled-coil domain of the STAT3 gene in LGL-leukemia. [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 606. doi:10.1158/1538-7445.AM2015-606

Mediator complex subunit 12 (MED12) is part of a Mediator, which is a large, highly conserved protein complex that participates in the regulation of general as well as gene-specific transcription. First somatic MED12 mutations were identified when exome sequencing revealed highly specific mutations in exon 2 in 70% of uterine leiomyomas studied. Additional mutations have subsequently been observed in exon 1, which further highlight the role of MED12 in these tumors. Functional analyses have indicated that exon 1 and exon 2 mutations lead to a diminished interaction between MED12 and other components of the Mediator kinase module, reduced Mediator-associated CDK kinase activity, and a unique global gene expression pattern. Recently, we observed similar mutations in chronic lymphocytic leukemia (CLL). Mutations were observed in 5% of the patients and the positive mutation status was associated with well-known poor prognosis markers in CLL. All the observed mutations both in uterine leiomyomas and CLL have been missense mutations or small insertions or deletions leading to an in-frame transcript. Interestingly, the first MED12 mutation in a patient with T-cell acute lymphoblastic leukemia (T-ALL) was recently identified through exome sequencing. This is a nonsense mutation affecting the mutation hotspot observed in CLL. It is also the first nonsense mutation identified in the MED12 N-terminal mutation hotspot area in any tumor type. The patient has received bone marrow transplantation and is now in complete remission, but small amounts of cancer cells were collected before the treatment. RNA was extracted from the cells, and cDNA sequencing revealed that only the mutant allele is expressed. We have now generated a construct with the MED12 specific mutation and transfected it into HEK293 Flp-In cells. Wild-type MED12 is being used as a control. Western blotting with N- and C-terminal MED12 antibodies indicates that the mutant protein is being produced but that the N-terminal part is lacking. We now aim to further characterize the function of this highly unusual mutation. Mutant and wild type protein will be produced for proteome-wide mass spectrometry analyses to detect possible effects on protein-protein interactions and also to gain more information on the translation start site. Also transcriptome-wide expression data will be produced to study the effect of the mutation on global gene expression profile. Overall, a mutation affecting the 5′ terminus of MED12 has now been identified in yet another cancer type, T-ALL. Our preliminary analyses indicate that the observed nonsense mutation escapes nonsense-mediated RNA decay and leads to a truncated protein product. Besides providing additional knowledge on the role of MED12 mutations in cancer, this exceptional mutation has potential to produce novel information on the normal MED12 function. Citation Format: Tuomas Heikkinen, Kati Kämpjärvi, Salla Keskitalo, Mikko Turunen, Heikki Kuusanmäki, Netta Mäkinen, Satu Mustjoki, Caroline Heckman, Markku Varjosalo, Pia Vahteristo. Nonsense MED12 mutation in a patient with T-cell acute lymphoblastic leukemia. [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 4964. doi:10.1158/1538-7445.AM2015-4964

Dasatinib, a tyrosine kinase inhibitor used in the treatment of chronic myeloid leukemia and acute lymphoblastic leukemia, has been shown to exert immunomodulatory effects in addition to direct oncokinase inhibition. Recently, we observed that dasatinib induces a rapid (within one hour after oral administration) and marked mobilization of lymphocytes (up to 8-fold increase in lymphocyte count), which closely follows the drug plasma concentration. In addition, in a subgroup of patients a clonal expansion of large granular lymphocytes occurs, which is correlated with good therapy response. As dasatinib-induced immunomodulatory effects are not leukemia specific, we aimed to exploit this activity to slow down or inhibit the growth of a syngeneic murine melanoma model and to characterize in detail the anti-tumor immune responses. Ovalbumin expressing murine melanoma cell line B16.OVA was grown under the skin of immunocompetent mice. The mice (n=6/group) were treated daily i.g. either with 30 mg/kg dasatinib or vehicle only. Blood was collected before tumor transplantation, before treatment, and on treatment days 4, 7 and 10. Tumor volumes were measured manually and specific growth rate was calculated based on the first and the last day of the treatment. In addition to white blood cell differential counts, immunophenotyping of blood and tumor homogenate was done by flow cytometry using antibodies against CD45.1, CD3, CD4, CD8b and NK1.1, and SIINFEKL-pentamer to detect B16.OVA-specific cells. To assess the functional properties of lymphocytes, we used antibodies against immune checkpoint inhibitors CTLA-4 and PD-1, and a degranulation marker CD107. On the 10th day of treatment, the tumor volumes were smaller in dasatinib group compared to control group, and there was a significant decrease in the tumor growth rate (0.06 vs. 0.18, p=0.01). Furthermore, dasatinib treated mice had increased proportion of CD8+ cells in the circulation (21% vs. 14%, p=0.04) and the CD4/CD8 ratio was significantly decreased (1.39 vs. 1.52, p= 0.04). During the tumor growth the mean CTLA-4 expression on CD8+ cells increased from 1.2% to 9% in the control group, whereas, in dasatinib group the increase was more modest (1.2% to 5.7%). Interestingly, 80% of tumor infiltrating CD8+ cells expressed PD1 antigen compared to To conclude, dasatinib treatment slowed down the tumor growth in B16.OVA mouse model, which could be associated with the immunomodulatory effects of dasatinib. However, combinatorial treatment regimens may be needed as dasatinib is not able to fully break the immune cell anergy induced by tumor cells. Citation Format: Can Hekim, Mette Ilander, Markus Vähä-Koskela, Paula Savola, Siri Tähtinen, Akseli Hemminki, Kimmo Porkka, Satu Mustjoki. Dasatinib-induced reduction of tumor growth is accompanied by the changes in the immune profile in melanoma B16.OVA mouse model. [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 2587. doi:10.1158/1538-7445.AM2014-2587

Exome-sequencing of matched tumor-normal sample pairs enables the identification of the full set of somatically acquired mutations that directly affect protein function in a given tumor. Inferring the functional significance of coding region mutations is relatively straightforward compared to mutations occurring in non-coding regions. Furthermore, exome sequencing enables sufficient sampling depth required for sensitive detection of somatic mutations in tumor samples. However, identification of novel driver mutations remains challenging due to the high levels of sequencing errors observed with current next generation sequencing technologies and due to passenger mutations which vastly out number driver mutations in any given tumor. Identification of novel driver mutations is especially challenging in the absence of cohort data. To identify driving somatic events in exome sequence data, we developed an analytical pipeline utilizing a combination of 3rd party tools for sequence read processing and mutation calling. This was coupled to an annotation framework that aids prioritization of driver mutation candidates for downstream validation experiments. The annotation framework integrates prior knowledge from reference databases on known cancer genes, sequence level evolutionary conservation and functional consequence predictions. Gene expression data from RNA-seq of the tumor sample can also be integrated in the annotations. To test this pipeline, we performed exome sequencing of a tumor-normal sample pair from an index patient diagnosed with T-cell derived large granular lymphocytic leukemia (T-LGL), which is a rare lymphoproliferative disease of previously unknown pathogenesis. The analysis pipeline identified a high-ranking candidate mutation in STAT3. Structural analysis indicated that the mutation resulted in a hydrophobic substitution at the SH2 dimerization interface, suggesting that the mutation stabilizes the active dimer form through increased hydrophobic interaction between monomers. Further validation experiments showed STAT3 SH2 hydrophobic substitutions to be recurrent in T-LGL and to result in constitutive STAT3 activation [1]. Application of the pipeline enabled identification of a novel class of oncogenic STAT3 SH2 domain mutations and established STAT3 as a key driver oncogene in T-LGL. Citation Format: Samuli Eldfors, Hanna LM Rajala, Pekka Ellonen, Emma I. Andersson, Sonja Lagström, Henrikki Almusa, Henrik Edgren, Maija Lepistö, Pirkko Mattila, Jonathan Knowles, Janna Saarela, Kimmo Porkka, Olli Kallioniemi, Satu Mustjoki, Caroline A. Heckman. Somatic mutation analysis pipeline for exome-sequencing data identifies oncogenic STAT3 mutations in T-LGL leukemia. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3164. doi:10.1158/1538-7445.AM2013-3164

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