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Hypomethylating agents (HMA) have become the backbone of nonintensive acute myeloid leukemia/myelodysplastic syndrome (AML/MDS) treatment, also by virtue of their activity in patients with adverse genetics, for example, monosomal karyotypes, often with losses on chromosome 7, 5, or 17. No comparable activity is observed with cytarabine, a cytidine analogue without DNA-hypomethylating properties. As evidence exists for compounding hypermethylation and gene silencing of hemizygous tumor suppressor genes (TSG), we thus hypothesized that this effect may preferentially be reversed by the HMAs decitabine and azacitidine. An unbiased RNA-sequencing approach was developed to interrogate decitabine-induced transcriptome changes in AML cell lines with or without a deletion of chromosomes 7q, 5q or 17p. HMA treatment preferentially upregulated several hemizygous TSG in this genomic region, significantly derepressing endogenous retrovirus (ERV)3–1, with promoter demethylation, enhanced chromatin accessibility, and increased H3K4me3 levels. Decitabine globally reactivated multiple transposable elements, with activation of the dsRNA sensor RIG-I and interferon regulatory factor (IRF)7. Induction of ERV3–1 and RIG-I mRNA was also observed during decitabine treatment in vivo in serially sorted peripheral blood AML blasts. In patient-derived monosomal karyotype AML murine xenografts, decitabine treatment resulted in superior survival rates compared with cytarabine. Collectively, these data demonstrate preferential gene derepression and ERV reactivation in AML with chromosomal deletions, providing a mechanistic explanation that supports the clinical observation of superiority of HMA over cytarabine in this difficult-to-treat patient group. Significance: These findings unravel the molecular mechanism underlying the intriguing clinical activity of HMAs in AML/MDS patients with chromosome 7 deletions and other monosomal karyotypes. See related commentary by O'Hagan et al., p. 813

Pancreatic cancer (PDAC) is a highly aggressive malignancy for which the identification of novel therapies is urgently needed. Here, we establish a human PDAC organoid biobank from 31 genetically distinct lines, covering a representative range of tumor subtypes, and demonstrate that these reflect the molecular and phenotypic heterogeneity of primary PDAC tissue. We use CRISPR-Cas9 genome editing and drug screening to characterize drug-gene interactions with ARID1A and BRCA2. We find that missense- but not frameshift mutations in the PDAC driver gene ARID1A are associated with increased sensitivity to the kinase inhibitors dasatinib (p < 0.0001) and VE-821 (p < 0.0001). We conduct an automated drug-repurposing screen with 1,172 FDA-approved compounds, identifying 26 compounds that effectively kill PDAC organoids, including 19 chemotherapy drugs currently approved for other cancer types. We validate the activity of these compounds in vitro and in vivo. The in vivo validated hits include emetine and ouabain, compounds which are approved for non-cancer indications and which perturb the ability of PDAC organoids to respond to hypoxia. Our study provides proof-of-concept for advancing precision oncology and identifying candidates for drug repurposing via genome editing and drug screening in tumor organoid biobanks.

Key Points mRNA decapping gene EDC4 is a novel fusion partner of MLL in AML. Genes functioning in mRNA decapping may compose a distinct group of MLL fusion partners that links MLL function with mRNA decapping in AML.

Colorectal cancer (CRC) accounts for about 10% of cancer deaths worldwide. Colon carcinogenesis is critically influenced by the tumor microenvironment. Cancer associated fibroblasts (CAFs) and tumor associated macrophages (TAMs) represent the major components of the tumor microenvironment. TAMs promote tumor progression, angiogenesis and tissue remodeling. However, the impact of the molecular crosstalk of tumor cells (TCs) with CAFs and macrophages on monocyte recruitment and their phenotypic conversion is not known in detail so far. In a 3D human organotypic CRC model, we show that CAFs and normal colonic fibroblasts are critically involved in monocyte recruitment and for the establishment of a macrophage phenotype, characterized by high CD163 expression. This is in line with the steady recruitment and differentiation of monocytes to immunosuppressive macrophages in the normal colon. Cytokine profiling revealed that CAFs produce M-CSF, and IL6, IL8, HGF and CCL2 secretion was specifically induced by CAFs in co-cultures with macrophages. Moreover, macrophage/CAF/TCs co-cultures increased TC invasion. We demonstrate that CAFs and macrophages are the major producers of CCL2 and, upon co-culture, increase their CCL2 production twofold and 40-fold, respectively. CAFs and macrophages expressing high CCL2 were also found in vivo in CRC, strongly supporting our findings. CCL2, CCR2, CSF1R and CD163 expression in macrophages was dependent on active MCSFR signaling as shown by M-CSFR inhibition. These results indicate that colon fibroblasts and not TCs are the major cellular component, recruiting and dictating the fate of infiltrated monocytes towards a specific macrophage population, characterized by high CD163 expression and CCL2 production.

(1) Background: A large body of research has examined the positive effects of physical activity on physical and mental health. However, for some, excessive exercise can develop into an addiction that is detrimental to their health. In the present study, we examine potential personal (self-control, self-concordance) and social (social support) resources that we assume to be related to exercise dependence. (2) Methods: One hundred and forty athletes from different endurance sports participated in an online survey. Exercise dependence, self-control, self-concordance, and social support were assessed using questionnaires that are well-established in health and sport psychology. Additionally, further sport-relevant and demographic variables were assessed. (3) Results: Correlational analyses supported our hypotheses that exercise dependence is negatively correlated with the personal resources trait, state self-control, and self-concordance. Social support, however, was not significantly correlated with exercise dependence. Furthermore, the results of a mediation analysis revealed that the relationship between both personal traits (self-control, self-concordance) and exercise dependence was mediated by state self-control. (4) Conclusions: Our results indicate that trait self-control and self-concordance might be important personal resources that protect against exercise dependence by making state self-control available.

BackgroundAlthough most breast cancer patients are treated these days with a curative intention, there are still many patients progressing to metastatic disease. The advent of anti-HER2 therapy has prominently prolonged the time of disease progression and survival for those patients, where a decent proportion is suffering from a HER2+ tumor. Beside classical approaches via antibodies against HER2, the use of Chimeric Antigen Receptor T (CAR-T) cells also in a solid cancer context is getting more and more attention.MethodsIn our study we evaluated the efficacy of Trastuzumab and HER2+ targeting CAR-T cells in a panel of human cancer cell lines (SK-OV3, Hs578T and JIMT-1) with different HER2 expression levels. The tumor cells were seeded in the presence or absence of different immune cells (PBMC, monocytes, NK cells or T cells) and cultured as 3D spheroids in a matrix-based system. The tumor growth and if applicable the invasion of the immune cells was measured via fluorescence-based live cell imaging. On the last experiment day, a metabolic read-out (CellTiter-Glo, CTG assay) was performed.ResultsTrastuzumab inhibited the tumor growth in a dose-dependent manner in the HER2+ cell line SK-OV3. The efficacy was increased specifically when NK cells were added to the culture. The HER2 positive cell line JIMT-1 was resistant to Trastuzumab treatment, which is in line with published data. The model was derived from a Trastuzumab-refractory patient. Interestingly, the addition of NK cells induced a marked increase of activity in this model as well. The HER2 targeted CART cells eradicated the 3D spheroids of SK-OV3 as well as JIMT-1 in a dose-dependent manner. The untransduced control T cells did not influence the tumor growth at all. The HER2- cell line Hs578T served as a negative control in the Trastuzumab as well as in the CAR-T cell experiments, and proved to be resistant to any treatment in this study. Taken together the 3D live cell imaging platform proved to be a feasible tool for efficacy testing of biologics as well as cellular therapies.ConclusionsOur in house developed HER2 CAR-T cells proved to be specific and effective in eradicating the targeted cancer cells. The mechanism behind the modulated sensitivity of the HER2+ JIMT-1 cells against HER2-targeted treatment will help to shed some light in possible resistance mechanism and hopefully have some translational value for patients suffering from this disease.

Solid tumors are often considered as abnormal organs composed of the cancerous cells and their surrounding tumor microenvironment (TME) containing fibroblasts, immune cells, blood and lymphatic vessels, and the extracellular matrix. The heterotypic interactions between this diversity of cell types within the TME are maintained through a wide variety of secreted proteins, resulting in a favorable milieu for the progression of the malignancy. The interactions between tumor cells and TME are complex and remain poorly understood. Here we investigated this by developing a unique nascent proteomic approach in tumor tissues.Precision cut cancer tissue slices (PCCTS) maintain tissue heterogeneity with different cell types and preserved TME. Cultivation of PCCTS provides an ex vivo model for tumor tissues. We developed an approach for PCCTS's nascent proteome analysis, using pulsed-SILAC (stable isotope labeling with amino acids in cell culture) labeling combined with click-chemistry to selectively isolate and quantify newly synthesized proteins in the TME upon applying a cellular perturbation. It is a powerful tool to selectively enrich secretory proteins from culture media even with presence of serum. Primary human ovarian tumors (phOVT) and patient derived xenografts (PDX) were used to produce the PCCTS with thickness of 150µm to 300µm. The different cell types and extracellular matrix of PCCTS make the depletion period of cells from the amino acids (methionine, lysine and arginine) prior the AHA-SILAC treatment difficult to define. The PCCTS need longer depletion periods than the 2D cell culture, the longer the depletion period the better depletion efficiency. Following, PCCTS were cultured in AHA-SILAC media and treated with cisplatin. PCCTS and culture media (containing secreted proteins) were harvested; newly synthesized proteins were enriched via click-chemistry and analyzed with mass spectrometry. The labeling time of 10 to 12h showed a good labeling efficiency of more than 60%, still further optimizations are needed. Different PCCTS showed various labeling efficiency indicating the patients heterogeneity. The nascent proteome analysis with cisplatin treatment demonstrated different protein regulations in patients suggesting different drug responses. STRING analysis can be applied to predict the protein-protein interactions. The immunohistochemical staining of the same PCCTS can be processed to further validate the results of the proteome analysis. In conclusion, we established a pulsed SILAC-AHA treatment approach for the PCCTS with the TME. This unique approach allows tracking the compositional and dynamic changes within the proteome and monitoring the direct proteome response at rapid time scale. It can be used to reveal a part of the proteome that has been poorly understood in the tumor tissues and contribute to studying cellular communication and finding new therapeutic targets. Citation Format: Meng Dong, Karim Aljakouch, Kathrin Böpple, Bernd Winkler, Julia Schüler, Frank Essmann, Hans-Georg Kopp, Jeroen Krijgsveld, Walter E. Aulitzky. Nascent proteome analysis of tumor cells and their microenvironment in cultured human tumor tissues [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 325.

Colorectal cancer (CRC) is one of the most frequent cancers accounting for 12% of all deaths from cancer in Europe (2016). Furthermore, CRC patients are prone to develop metastases and have a poor prognosis underpinning the need for new targeted and broadly applicable therapeutic strategies. Tumor cell secretion contributes to hallmarks of cancer e.g. hyperproliferation, evasion of growth suppression and resistance to cell death. Dysregulated secretion is thus a driver of cancer progression and therefore holds promise as a general therapeutic target for the treatment of cancers. The overall aim of this study is to gain a quantitative understanding of the cancer secretome on a systems level to elucidate the influence of the microenvironment on selected players and vice versa the influence of the secretome in vitro and later on in vivo. In a first attempt to elucidate the role of secreted factors in colon cancer progression, we cultured 20 CRC cell lines (five PDX derived and 15 commercial lines) in 3D spheroid cultures and compared the secreted cytokine profile with the 2D culture conditions of the same CRC cell line panel. We analyzed 45 cytokines in duplicates by using a human specific magnetic bead-based assay. Of the 45 cytokines, 41 were detected at relevant levels in at least one cell line in either 2D or 3D. Four cytokines were exclusively secreted in a 3D setting: IFN alpha (tumorigenic), IL-17A (pro-inflammatory), TNF alpha (pro-inflammatory and tumorigenic) and VEGF-D (pro-metastatic). Every cell line displayed a distinct cytokine profile depending on the culture conditions. 13 lines showed more cytokines upregulated in 3D, four lines displayed more cytokines upregulated in 2D and 3 lines exhibited an equal number of cytokines up-regulated in the two culture conditions. Eleven cytokines were specifically higher secreted when the cells were cultured in 3D across the majority of the 20 cell lines. Most of them with pro-inflammatory, pro-angiogenic and tumorigenic capabilities. A cluster analysis helped to identify four clusters, two comprising mainly 3D cultured cell line and two primarily consisting of 2D datasets. The main discriminators for these four clusters were the secretion levels of VEGF-A, IL-1Ralpha and HGF. The thorough characterization of the secretome in different in vitro conditions revealed the influence of the tumor microenvironment on basic tumor characteristics like proliferation and invasiveness. Based on this data set we gained a quantitative understanding of the cancer secretome on a systems level. From here on we will further elucidate the influence of the microenvironment on selected players of the secretome and vice versa the influence of the secretome on tumor biology in vitro and later on in vivo. Citation Format: Jacqueline Bersano, Katharina Schaich, Kanstantsin Lashuk, Julia Schüler. Culturing colorectal cancer cell lines in 3D spheroid cultures drives their cytokine profile towards a pro-inflammatory, tumorigenic and highly metastatic phenotype [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 2009.

The canonical WNT signaling pathway is crucial for intestinal stem cell renewal and aberrant WNT signaling is an early event in colorectal cancer (CRC) development. Here, we show for the first time that WNT2 is one of the most significantly induced genes in CRC stroma as compared to normal stroma. The impact of stromal WNT2 on carcinoma formation or progression was not addressed so far. Canonical WNT/β-catenin signaling was assessed using a 7TGP-reporter construct. Furthermore, effects of WNT2 on fibroblast migration and invasion were determined using siRNA-mediated gene silencing. Tumor cell invasion was studied using organotypic raft cultures and in vivo significance was assessed via a xenograft mouse model. We identified cancer-associated fibroblasts (CAFs) as the main source of WNT2. CAF-derived WNT2 activated canonical signaling in adenomatous polyposis coli/β-catenin wild-type colon cancer cells in a paracrine fashion, whereas no hyperactivation was detectable in cell lines harboring mutations in the adenomatous polyposis coli/β-catenin pathway. Furthermore, WNT2 activated autocrine canonical WNT signaling in primary fibroblasts, which was associated with a pro-migratory and pro-invasive phenotype. We identified FZD8 as the putative WNT2 receptor in CAFs. Three-dimensional organotypic co-culture assays revealed that WNT2-mediated fibroblast motility and extracellular matrix remodeling enhanced cancer cell invasion of cell lines even harboring mutations in the adenomatous polyposis coli/β-catenin pathway. Thus, suggesting a tumor-promoting influence on a broad range of CRC. In line, WNT2 also promotes tumor growth, invasion and metastasis in vivo. Moreover, high WNT2 expression is associated with poor prognosis in human CRC. The identification of the pro-malignant function of stromal derived WNT2 in CRC classifies WNT2 and its receptor as promising stromal targets to confine cancer progression in combination with conventional or targeted therapies.

// Dominik Schnerch 1 , Julia Schuler 2 , Marie Follo 1 , Julia Felthaus 1 , Dagmar Wider 1 , Kathrin Klingner 2 , Christine Greil 1 , Justus Duyster 1 , Monika Engelhardt 1 , Ralph Wasch 1 1 Department of Hematology, Oncology and Stem Cell Transplantation, Medical Center-University of Freiburg, Faculty of Medicine, University of Freiburg, Freiburg, Germany 2 Oncotest GmbH, Freiburg, Germany Correspondence to: Ralph Wasch, email: ralph.waesch@uniklinik-freiburg.de Keywords: AML, antimitotic therapy, APC/C, mitotic slippage, proteasome inhibition Received: December 22, 2016 Accepted: February 07, 2017 Published: February 18, 2017 ABSTRACT Elderly and frail patients, diagnosed with acute myeloid leukemia (AML) and ineligible to undergo intensive treatment, have a dismal prognosis. The small molecule inhibitor volasertib induces a mitotic block via inhibition of polo-like kinase 1 and has shown remarkable anti-leukemic activity when combined with low-dose cytarabine. We have demonstrated that AML cells are highly vulnerable to cell death in mitosis yet manage to escape a mitotic block through mitotic slippage by sustained proteasome-dependent slow degradation of cyclin B. Therefore, we tested whether interfering with mitotic slippage through proteasome inhibition arrests and kills AML cells more efficiently during mitosis. We show that therapeutic doses of bortezomib block the slow degradation of cyclin B during a volasertib-induced mitotic arrest in AML cell lines and patient-derived primary AML cells. In a xenotransplant mouse model of human AML, mice receiving volasertib in combination with bortezomib showed superior disease control compared to mice receiving volasertib alone, highlighting the potential therapeutic impact of this drug combination.

In order to exploit the complex tumour microenvironment, immunoreactivity in a multitude of cell-based assays is required to predict mode of action (MOA), pharmacodynamics, and efficacy downstream. We have developed an integrated approach starting with single cell and co-culture cell-based assays in 2D and 3D formats, followed by an in vivo set-up based on humanized mice to monitor an immunotherapeutic's interaction with specific cell types such as T cells including CAR-T cells, natural killer cells, macrophages, dendritic cells, neutrophils, and fibroblasts. Rituximab, a monoclonal antibody against CD20, is mainstay in the therapy for a broad variety of B-cell malignancies. Its MOA is not fully understood as direct signalling, complement dependent cellular cytotoxicity and antibody dependent cellular cytotoxicity (ADCC) all seem to have an impact. To elucidate the share and effect each of those mechanism has, we tested Rituximab in our immuno-oncology translational platform. Furthermore, our immuno-oncology platform was utilized to assess efficacy, bystander effects and off-target toxicity of a CD19 CAR-T cell therapy. In vitro assays for efficacy and bystander effect involved co-culture of the CAR-T cells with a mixture of non-target expressing cells and mid to high target antigen expressing cells, where T cell activation and cell death of both target cell types was quantified. Beyond efficacy the assay platforms were used to examine the influence of the therapy, antibodies or CAR-T cells, on normal human cells. These in vitro assays utilizing primary human cells from healthy tissue and/or differentiated iPCS-derived cells were used to assess cytotoxicity. The primary tissues were selected based on their potential safety risk by either expressing low levels of the target antigen or being major organs to de-risk for off-target effects. The results from our in vitro screen were validated in a xenograft in vivo system in the presence and absence of human immune cells. Four cell line derived xenografts were tested in vivo in immunocompromised mice subcutaneously and disseminated. The presence of human immune cells enhanced the antitumoral activity of Rituximab markedly, although the targeted effect of Rituximab induced already a significant prolongation in the overall survival (p< 0.005; Log-rank (Mantel-Cox) test).Over the last 30 years IO has progressed considerably with approvals for the use of various IO therapeutics including vaccines, cytokines, tumor-directed monoclonal antibodies, immune checkpoint inhibitors as well as Chimeric antigen receptor (CAR) and T cell receptor (TCR) engineered T cell therapies. Comprehensive characterization of novel IO compounds using a multitude of such in vitro and in vivo assays is essential to provide insights and understanding of the detailed mechanism of action and identify potential toxicity issues to ultimately improve translation to the clinic. Citation Format: Shilina Roman, Julia Schüler, Sanne Holt, Jeroen DeGroot, Omar Aziz, Ian Waddell. An integrated in vitro and in vivo approach providing insights in immune modulation [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 6705.

Three-dimensional (3D) cell culture platforms are increasingly being used in cancer research and drug development. Using patient derived xenograft (PDX) as tumor cell source the clinical relevance of those platforms increases significantly. In combination with live cell imaging capabilities a powerful tool for dynamic studies of drug-cell interactions arises. In the current study we determined the penetration of anti-EGFR antibodies cetuximab and panitumumab in NSCLC spheroids in different culture conditions (w/wo human dermal fibroblasts, HDF) and in combination with the permeabilization-enhancing peptide iRGD. Six different NSCLC PDX models were selected based on their EGFR protein expression level (ranging from high to low determined by immunohistochemistry on PDX tissue). Cetuximab and panitumumab, were labelled with an Alexa-Fluor488 dye and their real-time uptake kinetics were quantified in an IncuCyte S3 device (1 image/well/h, Sartorius). Apart from the absolute fluorescence within the spheroid, we developed a mathematical description, fitting intensity levels in separate regions of the spheroids to estimate spatial dependent penetration of the compound in a time-resolved manner. The accumulation of EGFR antibodies over time was positively correlated with the target expression on the corresponding PDX. In addition, the invasion of the antibodies was significantly influenced by the compactness of the spheroids. The compactness of the tumor spheroid was model immanent and dependent on the molecular make-up of the respective tumor line. Enhancing the compactness by adding HDF to the tumor spheroid reduced the infiltration of both tested antibodies markedly. The simultaneous treatment of anti-EGFR antibodies with iRGD enhanced the penetration capacity in a dose dependent manner. The data of the current study highlight the importance of the tumor microenvironment (TME) for the sensitivity of tumor cells towards anticancer treatment. The modulation of the TME will be an important approach to increase antitumoral activity by enhancing the compound concentration in the tumor tissue. In general, this screening approach can be applied to fluorescence-labelled large molecules as well as compounds with intrinsic fluorescence characteristics. The combination of life cell imaging and image analysis is a promising tool in preclinical drug development to optimize compounds with regard to tissue penetration and binding efficiency. Citation Format: Kanstantsin Lashuk, Hagen Klett, Agon Kolica, Claudia Göttlich, Julia Schüler. Co-administration of iRGD and therapeutic EGFR-antibodies Cetuximab and Panitumumab enhances drug penetration in NSCLC PDX based 3D multicellular spheroids [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 3041.

Purpose: 4-1BB (CD137) is a key costimulatory immunoreceptor and promising therapeutic target in cancer. To overcome limitations of current 4-1BB–targeting antibodies, we have developed PRS-343, a 4-1BB/HER2 bispecific molecule. PRS-343 is designed to facilitate T-cell costimulation by tumor-localized, HER2-dependent 4-1BB clustering and activation. Experimental Design: PRS-343 was generated by the genetic fusion of 4-1BB–specific Anticalin proteins to a variant of trastuzumab with an engineered IgG4 isotype. Its activity was characterized using a panel of in vitro assays and humanized mouse models. The safety was assessed using ex vivo human cell assays and a toxicity study in cynomolgus monkeys. Results: PRS-343 targets 4-1BB and HER2 with high affinity and binds both targets simultaneously. 4-1BB–expressing T cells are efficiently costimulated when incubated with PRS-343 in the presence of cancer cells expressing HER2, as evidenced by increased production of proinflammatory cytokines (IL2, GM-CSF, TNFα, and IFNγ). In a humanized mouse model engrafted with HER2-positive SK-OV-3 tumor cells and human peripheral blood mononuclear cells, PRS-343 leads to tumor growth inhibition and a dose-dependent increase of tumor-infiltrating lymphocytes. In IND-enabling studies, PRS-343 was found to be well tolerated, with no overt toxicity and no relevant drug-related toxicologic findings. Conclusions: PRS-343 facilitates tumor-localized targeting of T cells by bispecific engagement of HER2 and 4-1BB. This approach has the potential to provide a more localized activation of the immune system with higher efficacy and reduced peripheral toxicity compared with current monospecific approaches. The reported data led to initiation of a phase I clinical trial with this first-in-class molecule. See related commentary by Su et al., p. 5732

Therapy resistance is a major clinical problem in cancer medicine and crucial for disease relapse and progression. Therefore, the clinical need to overcome it, particularly for aggressive tumors such as pancreatic cancer, is very high. Aberrant activation of an epithelial–mesenchymal transition (EMT) and an associated cancer stem cell phenotype are considered a major cause of therapy resistance. Particularly, the EMT-activator ZEB1 was shown to confer stemness and resistance. We applied a systematic, stepwise strategy to interfere with ZEB1 function, aiming to overcome drug resistance. This led to the identification of both its target gene miR-203 as a major drug sensitizer and subsequently the class I HDAC inhibitor mocetinostat as epigenetic drug to interfere with ZEB1 function, restore miR-203 expression, repress stemness properties, and induce sensitivity against chemotherapy. Thereby, mocetinostat turned out to be more effective than other HDAC inhibitors, such as SAHA, indicating the relevance of the screening strategy. Our data encourage the application of mechanism-based combinations of selected epigenetic drugs with standard chemotherapy for the rational treatment of aggressive solid tumors, such as pancreatic cancer.

Digital pathology, driven by the increasing capabilities of modern computers, is an emerging field within medical research and diagnostics. A re-occurring task in pathology is the analysis of immunohistochemical (IHC) stains, i.e. stains in which a specific type of immune cell is highlighted using corresponding antibodies. Automatic quantification of these images is a challenge due to large image sizes of up to 10 gigapixels, but provides a more objective and reproducible evaluation than the exhaustive task of manual analysis. In this context, we compare counting measures against area-based measures in the case of cytoplasmic and membrane-bound IHC stains. Our evaluation indicates a superior performance of the area-based method which reaches a Jaccard index of approximately 80%, while cell nuclei count-based approaches can be severely affected by variance due to masking effects when the cytoplasmic chromogenic staining covers the blue nuclear counterstain

Objectives: Novel personalized therapeutic approaches are urgently needed for patients with metastatic clear cell renal cell carcinoma (ccRCC). Methods and materials: We combined the development of a primary patient-derived ccRCC cell line with a phenotypic drug screen consisting of 101 approved anticancer compounds. Results: We identified the MNNG HOS transforming gene (MET)-anaplastic lymphoma receptor tyrosine kinase (ALK) inhibitor crizotinib as the top hit of our drug screen, whereas compounds targeting the vascular endothelial growth factor (VEGF) or mammalian target of rapamycin (mTOR) pathway showed no or only minor in vitro activity. Among the known major crizotinib targets MET, ALK, and ROS-1, only MET was expressed in our ccRCC cell line. Subsequent sequence analysis revealed a heterozygous R988C mutation of the MET gene and a VHL deletion in both the primary tumor and the tumor-derived ccRCC cell line. However, we were unable to show an activation of MET and, further, MET knockdown did not result in increased apoptosis or cytotoxicity. Therefore, our results suggest that MET R988C does not function as a major oncogenic driver mutation but rather represents a sequence variant. However, we provide evidence that the cytotoxic effect of crizotinib in our cell line model correlates with its ability to inhibit P-glycoprotein (ABCB1)-associated transport functions. Conclusions: Our study shows that a phenotypic screen of a patient-derived tumor cell line can identify compounds with antitumor activity but with an unexpected mode of action. Our results underscore that target validation and phenotype-genotype correlations remain a major experimental challenge. The implications of our findings for a personalized management of patients with cancer are discussed. r 2014

Model selection is one critical step in anti-cancer drug development. In the advent of immune modulatory compounds being part of most of the oncology drug development pipelines, the available preclinical models have to be characterized with respect to those assets. In the current study we analyzed 26 PDX models covering a broad range of solid cancer entities with regard to the phenotypic characteristics in a humanized environment. Well established PDX models from six different entities (breast, colon, renal, pancreatic, NSCL cancer and glioblastoma) were implanted subcutaneously into immune-compromised mice substituted with CD34+ hematopoietic stem cells (hu-mice). Tumor characterization included: tumor growth curves, take rates, human immune cell infiltrates of tumor (=TILs), peripheral blood, spleen and bone marrow (determined by flow cytometry, FC, and IHC) and secretion of human cytokines in murine serum (determined by cytokine array & Luminex based technology). Those data were compared to equivalent data-sets in non-humanized mice. The analysis of TILs revealed a large bandwidth of immune cell infiltration in the respective model ranging from 94% huCD45+ cells (renal cancer RXF 2773) to 3.54% huCD45+ (NSCLC LXFA 1041). Based on the percentage and of huCD4+ cell engraftment and the ratio of their subtypes (memory vs naïve) in untreated tumors the models were grouped into high (=hot), medium and low (=cold) infiltrated tumor models. The infiltration rate was stable across different experiments and specific for one distinct model. IHC analyses confirmed infiltration rates in tumor tissue determined by FC for CD45+, CD4+ and CD8+ cells. Furthermore, the models previously defined as “hot” or “cold” expressed distinct cytokine profiles in serum of tumor bearing mice. Amongst other cytokines, hot tumors secreted high levels of IL-17A, IL-18 BPa, MMP-9, Osteopontin, CD31, TIM-3 and YKL-40. In contrast, levels of GDF-15 and Lipocalin-2 were down regulated in those models compared to their cold counterpart. Of note, neither the amount nor the composition of human immune cell populations in peripheral blood of those mice was suitable to conclude on the amount of TILs in the respective model. Further validation of the cytokine profiles using immune modulating compounds on hot as well as cold PDX models are currently underway. Taken together, the characterization of PDX models with respect to their TIL composition facilitates model selection for innovative drugs in the immune-oncology field. The possibility to discriminate hot vs cold tumors by a minimal-invasive method like serum analysis of cytokine expression profile as potential biomarker will help to characterize the complete PDX collection of more than 500 models but will as well serve as additional read-out in preclinical I-O studies. Citation Format: Eva Oswald, Dorothee Lenhard, Stefanie Schmitt, Anna Edinger, Anne Löhr, Volker Knauff, Anke Behnke, Anne-Lise Peille, Julia Schüler. PDX models of solid cancer express distinct cytokine profiles in humanized mice relating to their tumor infiltrating lymphocyte landscape [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 1057.

The insulin-like growth factor (IGF)2/IGF1 receptor (IGF1R) signaling axis has an important role in intestinal carcinogenesis and overexpression of IGF2 is an accepted risk factor for colorectal cancer (CRC) development. Genetic amplifications and loss of imprinting contribute to the upregulation of IGF2, but insufficiently explain the extent of IGF2 expression in a subset of patients. Here, we show that IGF2 was specifically induced in the tumor stroma of CRC and identified cancer-associated fibroblasts (CAFs) as the major source. Further, we provide functional evidence that stromal IGF2, via the paracrine IGF1R/insulin receptor axis, activated pro-survival AKT signaling in CRC cell lines. In addition to its effects on malignant cells, autocrine IGF2/IGF1R signaling in CAFs induced myofibroblast differentiation in terms of alpha-smooth muscle actin expression and contractility in floating collagen gels. This was further augmented in concert with transforming growth factor-β (TGFβ) signaling suggesting a cooperative mechanism. However, we demonstrated that IGF2 neither induced TGFβ/smooth muscle actin/mothers against decapentaplegic (SMAD) signaling nor synergized with TGFβ to hyperactivate this pathway in two dimensional and three dimensional cultures. IGF2-mediated physical matrix remodeling by CAFs, but not changes in extracellular matrix-modifying proteases or other secreted factors acting in a paracrine manner on/in cancer cells, facilitated subsequent tumor cell invasion in organotypic co-cultures. Consistently, colon cancer cells co-inoculated with CAFs expressing endogenous IGF2 in mouse xenograft models exhibited elevated invasiveness and dissemination capacity, as well as increased local tumor regrowth after primary tumor resection compared with conditions with IGF2-deficient CAFs. In line, expression of IGF2 correlated with elevated relapse rates and poor survival in CRC patients. In agreement with our results, high-level coexpression of IGF2 and TGFβ was predicting adverse outcome with higher accuracy than increased expression of the individual genes alone. Taken together, we demonstrate that stroma-induced IGF2 promotes colon cancer progression in a paracrine and autocrine manner and propose IGF2 as potential target for tumor stroma cotargeting strategies.

Abstract. The aim of this study was to examine whether athletes differ from nonathletes regarding their mental rotation performance. Furthermore, it investigated whether athletes doing sports requiring distinguishable levels of mental rotation (orienteering, gymnastics, running), as well as varying with respect to having an egocentric (gymnastics) or an allocentric perspective (orienteering), differ from each other. Therefore, the Mental Rotations Test (MRT) was carried out with 20 orienteers, 20 gymnasts, 20 runners, and 20 nonathletes. The results indicate large differences in mental rotation performance, with those actively doing sports outperforming the nonathletes. Analyses for the specific groups showed that orienteers and gymnasts differed from the nonathletes, whereas endurance runners did not. Contrary to expectations, the mental rotation performance of gymnasts did not differ from that of orienteers. This study also revealed gender differences in favor of men. Implications regarding a differentiated view of the connection between specific sports and mental rotation performance are discussed.

Three studies investigated the relationship between the achievement motive and sport participation. It was expected that both the implicit and the explicit achievement motives are positively associated with how frequently people engage in sport activities. The implicit achievement motive was assessed with indirect motive measures; the explicit achievement motive was either inferred from participants’ personal goals or measured with self-reports. Two hundred five athletes participated including college students enrolled in leisure sport programs offered at their university (Study 1), amateur athletes registered in sports clubs (Study 2), and elite tennis athletes (Study 3). The implicit achievement motive consistently predicted sport participation in all three studies. In contrast, the explicit achievement motive was uncorrelated with sport participation. The interaction between the two motives did not yield an effect on sport participation. The results indicate that the implicit, unconscious need to achieve facilitates regular engagement in sport activities, but the explicit, conscious orientation toward achievement does not. The enrichment of sports environments with incentives for the implicit achievement motive may thus attract more people to participate in sport activities.

Receptor-targeted therapies have become standard in the treatment of various lymphomas. In view of its unparalleled specificity for the malignant B-cell clone, the B-cell receptor (BCR) on B cell lymphoma cells is a potential therapeutic target. We have used two BCR epitope mimicking peptides binding to the Burkitt's lymphoma cell lines CA46 and SUP-B8. We proved their functionality by demonstrating calcium flux and BCR-mediated endocytosis upon peptide receptor binding. Toxicity experiments in vitro via cross-linking of the BCR with tetramerized epitope mimics lead to apoptosis in both cell lines but was far more effective in SUP-B8 cells. We established a SUP-B8-based disseminated Burkitt's lymphoma model in NOD/SCID mice. Treatment of tumor-bearing mice with tetramerized epitope mimics had significant anti-tumor effects in vivo. We conclude that peptide-mediated, BCR-targeted therapy is a promising approach which may be explored and further developed for application in highly aggressive lymphoma.

Invasion and metastasis of carcinomas is promoted by the activation of the embryonic 'epithelial to mesenchymal transition' (EMT) program, which triggers cellular mobility and subsequent dissemination of tumour cells. We recently showed that the EMT-activator ZEB1 (zinc finger E-box binding homeobox 1) is a crucial promoter of metastasis and demonstrated that ZEB1 inhibits expression of the microRNA-200 (miR-200) family, whose members are strong inducers of epithelial differentiation. Here, we report that ZEB1 not only promotes tumour cell dissemination, but is also necessary for the tumour-initiating capacity of pancreatic and colorectal cancer cells. We show that ZEB1 represses expression of stemness-inhibiting miR-203 and that candidate targets of miR-200 family members are also stem cell factors, such as Sox2 and Klf4. Moreover, miR-200c, miR-203 and miR-183 cooperate to suppress expression of stem cell factors in cancer cells and mouse embryonic stem (ES) cells, as demonstrated for the polycomb repressor Bmi1. We propose that ZEB1 links EMT-activation and stemness-maintenance by suppressing stemness-inhibiting microRNAs (miRNAs) and thereby is a promoter of mobile, migrating cancer stem cells. Thus, targeting the ZEB1-miR-200 feedback loop might form the basis of a promising treatment for fatal tumours, such as pancreatic cancer.

Background With the aim of remaining viable, bacteria must deal with changes in environmental conditions, including increases in external osmolarity. While studies concerning bacterial response to this stress condition have focused on soil, marine and enteric species, this report is about Caulobacter crescentus, a species inhabiting freshwater oligotrophic habitats. Results A genomic analysis reported in this study shows that most of the classical genes known to be involved in intracellular solute accumulation under osmotic adaptation are missing in C. crescentus. Consistent with this observation, growth assays revealed a restricted capability of the bacterium to propagate under hyperosmotic stress, and addition of the compatible solute glycine betaine did not improve bacterial resistance. A combination of transcriptomic and proteomic analyses indicated quite similar changes triggered by the presence of either salt or sucrose, including down-regulation of many housekeeping processes and up-regulation of functions related to environmental adaptation. Furthermore, a GC-MS analysis revealed some metabolites at slightly increased levels in stressed cells, but none of them corresponding to well-established compatible solutes. Conclusion Despite a clear response to hyperosmotic stress, it seems that the restricted capability of C. crescentus to tolerate this unfavorable condition is probably a consequence of the inability to accumulate intracellular solutes. This finding is consistent with the ecology of the bacterium, which inhabits aquatic environments with low nutrient concentration. Electronic supplementary material The online version of this article (doi:10.1186/s12866-015-0404-x) contains supplementary material, which is available to authorized users.

Experimental tumors raised in rodents represent an important preclinical tool to develop innovative anticancer compounds before clinical testing. Amongst others such models include solid tumors raised in syngeneic fully immunocompetent hosts and tumors spontaneously growing in genetically engineered mice (GEM) and derivate thereof. These model platforms have gained additional value since the manipulation of the immune system to fight cancer has led to tangible benefits for cancer patients. In the current study, we analyzed somatic mutation profiles from whole-exome sequencing (WES) data in a panel of 14 different mouse models covering 6 major cancer types. 4 models were GEM-derived, all other lines were developed by injection of established cell lines into the corresponding mouse strain. In parallel, these models were evaluated for their sensitivity towards checkpoint inhibitors (α-CTLA-4, α-PD-1 or α-PDL-1) in mono- or combined therapy with cytostatic and/or targeted agents.WES achieved an average-of-coverage of 165X in tumor models and normal DNA. A median mutation rate of 34 somatic mutations (m)/MB was detected, ranging from 7 m/MB (GEM derived NSCLC model KP) to 328 m/MB (syngeneic NSCLC line Lewis Lung) in exons. Mutation rates were markedly lower in GEM-derived models as in syngeneic lines (median of 9 vs 43 m/MB). This reflects very well the different underlying carcinogenic mechanism of these two types of models. The cross-comparison of tissue-transplants vs cell lines from GEM-derived model KP revealed that 75% of the mutations found in the primary KP could also be detected in the corresponding cell lines KP1 and KP4. Of note, the mutation count increased 1.3- (KP4) and 2.9-fold (KP1) during cell line establishment. Every model depicted a distinct profile against modulators of the immune system dividing the panel in responders and non-responders. In our hands no significant correlation could be determined between mutational load and sensitivity towards checkpoint inhibition in vivo. This might be related to the fact that the dataset was not broad enough and the number of models per entity was too small, rendering the subtype analysis within the panel not feasible. However, a strong tendency was observed when investigating the colon lines Colon26, CT26 and MC38 showing best response to the combination of PD-1+CTLA-4 inhibitors and in parallel the highest mutation rates (52, 64 and 59 m/MB, respectively) compared to non-responders B16-F10, CloudmanS91, 4T1 and KP1 (23 m/MB on average). Mouse models of cancer are a relevant tool for preclinical studies specifically for immuno-oncology. The molecular characterization of these models will help to optimize their use in drug discovery. They will support the development of innovative drugs and indentification of biomarkers to classify the patient cohort profiting the most from these new compounds. Citation Format: Bruno Zeitouni, Cordula Tschuch, Jason M. Davis, Anne-Lise Peille, Yana Raeva, Manuel Landesfeind, Sheri Barnes, Julia B. Schüler. Whole-exome somatic mutation analysis of mouse cancer models and implications for preclinical immunomodulatory drug development [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 1840. doi:10.1158/1538-7445.AM2017-1840

Cancer stem cells (CSC) drive tumorigenesis and contribute to genotoxic therapy resistance, diffuse infiltrative invasion, and immunosuppression, which are key factors for the incurability of glioblastoma multiforme (GBM). The AC133 epitope of CD133 is an important CSC marker for GBM and other tumor entities. Here, we report the development and preclinical evaluation of a recombinant AC133×CD3 bispecific antibody (bsAb) that redirects human polyclonal T cells to AC133+ GBM stem cells (GBM-SC), inducing their strong targeted lysis. This novel bsAb prevented the outgrowth of AC133-positive subcutaneous GBM xenografts. Moreover, upon intracerebral infusion along with the local application of human CD8+ T cells, it exhibited potent activity in prophylactic and treatment models of orthotopic GBM-SC–derived invasive brain tumors. In contrast, normal hematopoietic stem cells, some of which are AC133-positive, were virtually unaffected at bsAb concentrations effective against GBM-SCs and retained their colony-forming abilities. In conclusion, our data demonstrate the high activity of this new bsAb against patient-derived AC133-positive GBM-SCs in models of local therapy of highly invasive GBM. Cancer Res; 75(11); 2166–76. ©2015 AACR.

A technology that visualizes tumor stem cells with clinically relevant tracers could have a broad impact on cancer diagnosis and treatment. The AC133 epitope of CD133 currently is one of the best-characterized tumor stem cell markers for many intra- and extracranial tumor entities. Here we demonstrate the successful noninvasive detection of AC133(+) tumor stem cells by PET and near-infrared fluorescence molecular tomography in subcutaneous and orthotopic glioma xenografts using antibody-based tracers. Particularly, microPET with (64)Cu-NOTA-AC133 mAb yielded high-quality images with outstanding tumor-to-background contrast, clearly delineating subcutaneous tumor stem cell-derived xenografts from surrounding tissues. Intracerebral tumors as small as 2-3 mm also were clearly discernible, and the microPET images reflected the invasive growth pattern of orthotopic cancer stem cell-derived tumors with low density of AC133(+) cells. These data provide a basis for further preclinical and clinical use of the developed tracers for high-sensitivity and high-resolution monitoring of AC133(+) tumor stem cells.

The receptor tyrosine kinase (RTK) insulin-like growth factor-1 receptor (IGF1R) is implicated in various tumor entities including chronic lymphocytic leukemia (CLL), but its functional significance in this disease remains poorly characterized. Here, we show that the IGF1R protein is overexpressed in various CLL subsets, suggesting a contribution to CLL pathology. Indeed, we show that IGF1R knockdown in primary human CLL cells compromised their viability. Likewise, IGF1R inhibition with 3 structurally distinct compounds induced apoptosis, even in the presence of protective stroma components. Furthermore, IGF1R inhibition effectively limited CLL development in Eμ-TCL1 transgenic mice and of primary human CLL xenografts. In agreement with its prosurvival function, IGF1R inhibition affected the phosphorylation and/or expression of multiple signaling proteins. The multikinase inhibitor sorafenib yielded similar effects on these signaling elements as IGF1R inhibitors. Indeed, IGF1R appears to be a direct sorafenib target because sorafenib decreased IGF1R expression and phosphorylation, counteracted insulin-like growth factor-1 (IGF-1) binding to CLL cells, and lowered the in vitro kinase activity of recombinant, purified IGF1R. Thus, we demonstrate that blockade of IGF1R-mediated signaling represents a novel mechanism of action for sorafenib in CLL. Importantly, IGF1R inhibitors compromise CLL viability in their microenvironment context, implicating this RTK as a promising therapeutic target.

Background: Translocations involving the MLL gene located on chromosome 11q23 are usually found in de novo acute myeloid leukemia (AML) and generally confer a dismal prognosis unless the AF9 gene is involved (Döhner et al., Blood 2010;115:453-74). MLL can be fused to multiple different genes, resulting in the large and growing "MLL recombinome" (Meyer et al., Leukemia 2013;27:2165-76). Thus far, only two genes encoding proteins that are part of the mRNA decapping protein complex (i.e., DCP1A, DCPS) have been described as rarely being fused to MLL. Here, we describe an AML with an indolent disease course arising from myelodysplastic syndrome (MDS) that disclosed a unique MLL fusion with another component of the mRNA decapping complex, i.e., EDC4. Patient and Methods: Briefly, a 55 year-old female patient presented with an MDS [timepoint (t) -1] that within 10 months progressed to an AML with 2% blood and 40% bone marrow myeloblasts (t0). The patient refused treatment beyond supportive care. Six months later, a marked blast expansion of 80% was detectable in the blood (t1). The patient received 5 cycles of decitabine (t2, cycle 5), followed by 3 months of hydroxyurea (t3). Samples were depleted from CD3+ cells via MACS; CD3+ cells served as germline control. RNA sequencing libraries were prepared using the NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs), and the SureSelect Human All Exon v5 kit (Agilent Technologies) was used for exome capturing from gDNA. Next generation sequencing was performed on an Illumina Hiseq 2500. The analyses were performed as previously described (Kataoka et al. Nature Genetics 2015;47:1304-15, Becker et al. Blood 2014;123:1883-6). NOD scid gamma mice were used as hosts for patient derived tumor xenografts (PDX). Results: Standard metaphase cytogenetics at the diagnosis of AML (t0) revealed a previously undescribed translocation involving the MLL gene, i.e., t(11;16)(q23;q22), as the sole cytogenetic abnormality. The unknown fusion partner on chromosome 16 was identified by RNA-sequencing as the EDC4 gene (a.k.a. Ge-1), which encodes a key scaffold protein of the mRNA decapping complex; the fusion was confirmed by PCR on cDNA. The translocation led to the in-frame fusion of MLL exon 13 to EDC4 exon 6 which was linked by 19 nucleotides from EDC4 intron 5. The predicted amino acid sequence of the linker was ALNTLLR. Further analyses including exome sequencing on the samples collected over the disease course demonstrated STAG2 as a potential founder mutation that was already present during the MDS (t-1) and persisted throughout the disease course at variant allele frequencies (VAFs) of approximately 45-50%. At the time of transformation to AML (t0), the MLL-EDC4 and two RAS mutations (KRAS p.G13D, NRAS p.G12C) were detectable. Towards the terminal phase (t3), the RAS mutations disappeared and a clone that acquired a mutation in the FLT3 tyrosine kinase domain (TKD; p.D835V; VAF 43%) expanded. Primary blasts from the patient engrafted in NOD scid gamma mice and established a stable PDX serial transplantation mouse model used for drug testing. Conclusions: This report provides the first demonstration of an MLL-EDC4 in-frame fusion, with potential cooperativity with a founder mutation in the STAG2 splicing factor gene during the transformation of MDS to AML and the additional acquisition of a FLT3-TKD mutation during disease progression. RNA sequencing proved to be a very feasible approach to identify novel fusion partners of known oncogenes such as MLL. Disclosures Becker: BMS: Honoraria; Novartis: Honoraria. Kataoka:Yakult: Honoraria; Boehringer Ingelheim: Honoraria; Kyowa Hakko Kirin: Honoraria. Schüler:Oncotest GmbH: Employment. Ogawa:Kan research institute: Consultancy, Research Funding; Sumitomo Dainippon Pharma: Research Funding; Takeda Pharmaceuticals: Consultancy, Research Funding. Lübbert:Janssen-Cilag: Other: Travel Funding, Research Funding; Celgene: Other: Travel Funding; Ratiopharm: Other: Study drug valproic acid.

Background: CD137 (4-1BB) is a key costimulatory immunoreceptor and a member of the TNF-receptor (TNFR) superfamily. While multiple lines of evidence show that CD137 is a highly promising therapeutic target in cancer, current mAb-based approaches are not designed to achieve a tumor-target driven activation and may display toxicity and a limited therapeutic window due to peripheral T cell and NK cell activation. To overcome this limitation, we generated PRS-343, a CD137/HER2 bispecific that is designed to promote CD137 clustering by bridging CD137-positive T cells with HER2-positive tumor cells, thereby providing a potent costimulatory signal to tumor antigen-specific T cells. Methods: Anticalin® proteins are 18 kD protein therapeutics derived from human lipocalins. We utilized phage display to generate an Anticalin protein binding to CD137 with high affinity and specificity. PRS-343 was obtained by genetic fusion of the CD137-specific Anticalin protein to a variant of the HER2-targeting monoclonal antibody trastuzumab with an engineered IgG4 backbone. We have shown previously that the bispecific fusion PRS-343 targets CD137 and HER2 in a bispecific manner and efficiently activates T cells ex vivo in the presence of HER2-positive cells. Here, in vivo proof of concept data is presented utilizing a humanized mouse model in immunocompromised mice and the SK-OV-3 cell line as a HER2-positive xenograft. When tumors had reached a predefined size, mice received human PBMC via an intravenous route and weekly intraperitoneal injections of PRS-343 for three weeks. An IgG4 isotype antibody served as the negative control, while a CD137-targeting benchmark antibody and trastuzumab with an engineered IgG4 backbone (“tras-IgG4”) served as controls for monospecific targeting of CD137 and HER2, respectively. Results: PRS-343 activity was investigated at four different weekly doses of PRS-343 (4μg, 20μg, 100μg and 200μg). We found that PRS-343 dose-dependently led to strong tumor growth inhibition compared to treatment with the isotype control, and that the tumor response was accompanied by a significantly higher tumor infiltration with human lymphocytes (hCD45+). Interestingly, the anti-CD137 benchmark neither displayed tumor growth inhibition nor enhanced lymphocyte infiltration into tumors compared to isotype. The tras-IgG4 control was also devoid of lymphocyte infiltration into the tumor, but displayed a tumor growth inhibition comparable to PRS-343. Taken together, these data show that PRS-343 provided dual activity by both increasing the frequency of tumor-infiltrating lymphocytes by bispecific targeting of CD137 and HER2 as well as mediating direct tumor growth inhibition by the direct, monospecific targeting of HER2. Notably, the tumor growth inhibition provided by targeting HER2 did not require any antibody directed cellular cytotoxicity (ADCC) as both PRS-343 and the tras-IgG4 control lack the ability to interact with Fc-gamma receptors on NK cells that ADCC would require. The animal model also allowed investigating the potential safety of PRS-343: While the anti-CD137 benchmark accelerated the onset of graft-versus-host-disease and led to stronger expansion of CD8+ T cells in the peripheral blood compared to the isotype control group, both of these effects were absent for PRS-343. The data therefore support the envisaged mode of action of selective, tumor-localized costimulatory T cell activation, as well as the concept that such an approach may lead to higher efficacy and reduced systemic toxicity compared to conventional anti-CD137 mAbs. Conclusion: We report potent costimulatory T-cell engagement of the immunoreceptor CD137 in a HER2-dependent manner, utilizing the CD137/HER2 bispecific PRS-343. In a humanized mouse model, PRS-343 displays dual activity based on monospecific HER2-targeting and bispecific, tumor-localized costimulation of CD137. Compared to known CD137-targeting antibodies in clinical development, this approach has the potential to provide a more localized activation of the immune system with higher efficacy and reduced peripheral toxicity. The direct, monospecific HER2-targeting activity may provide an additional therapeutic benefit and work in synergy with local CD137 costimulation. The positive functional ex vivo and in vivo data of PRS-343 as well as the excellent developability profile support investigation of its anti-cancer activity in clinical trials. Citation Format: Marlon J. Hinner, Rachida-Siham Bel Aiba, Corinna Schlosser, Thomas Jaquin, Andrea Allersdorfer, Sven Berger, Alexander Wiedenmann, Gabriele Matschiner, Julia Schüler, Ulrich Moebius, Christine Rothe, Shane A. Olwill. Costimulatory T-cell engagement by PRS-343, a CD137 (4-1BB)/HER2 bispecific, leads to tumor growth inhibition and TIL expansion in humanized mouse model [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 B016.

Despite considerable advances, multiple myeloma (MM) remains incurable and the development of novel therapies targeting the interplay between plasma cells (PCs) and their bone marrow (BM) microenvironment remains essential. We investigated the effect of various agents in vitro on the proliferation, phenotype, morphology, actin polymerization and migration of MM cells and, in vivo, the tumour growth of L363-bearing non-obese diabetic severe combined immunodeficient mice with a deficient interleukin-2 receptor gamma chain (NSG). In vitro, we observed a dose-dependent cytotoxicity with bortezomib and sorafenib. Using RPMI8226 cells co-expressing histone 2B-mCherry and cytochrome c-GFP, bortezomib- and sorafenib-induced apoptosis was confirmed, and both agents combined showed synergism. Sorafenib induced CD138-downregulation and abolished CXCL12-induced actin polymerization. L363 cells expressed CCR4 and CCR5 and migrated to their common ligand CCL5. Chemotaxis to BM stroma cells was notable and significantly reduced by sorafenib. Downregulation of phospho-ERK appeared relevant for the inhibition of actin polymerization and chemotaxis. Sorafenib alone, and combined with bortezomib, showed substantial antitumour activity in L363-bearing NSG. Correspondingly, sorafenib induced clinical responses in MM-/AL-amyloidosis patients. We conclude that, in addition to the cytotoxic and anti-angiogenic effects of sorafenib, blocking of MM cell migration and homing represent promising mechanisms to interrupt the interplay between PCs and their supportive microenvironment.

Background Isolation and characterization of tumourigenic colon cancer initiating cells may help to develop novel diagnostic and therapeutic procedures. Methods We characterized a panel of fourteen human colon carcinoma cell lines and their corresponding xenografts for the surface expression of potential stem cell markers CD133, CD24, CD44, CDCP1 and CXCR4. In five cell lines and nine xenografts, mRNA expression of these markers was determined. Tumour growth behaviour of CD133+, CD133- and unsorted SW620 cells was evaluated in vivo. Results All five putative stem cell markers showed distinct expression patterns in the tumours examined. Two patient-derived cell lines highly expressed CD133 (> 85% of positive cells) and three other cell lines had an expression level of about 50% whereas in long-term culture based models CD133 expression ranged only from 0 to 20%. In 8/14 cell lines, more than 80% of the cells were positive for CD24 and 11/14 were over 70% positive for CD44. 10/14 cell lines expressed CDCP1 on ≥ 83% of cells. CXCR4 expression was determined solely on 94 L and SW480. Analyses of the corresponding xenografts revealed a significant reduction of cell numbers expressing the investigated surface markers and showed single cell fractions expressing up to three markers simultaneously. Statistical analysis revealed that the CXCR4 mRNA level correlates negatively with the protein expression of CD133, CD44, CD24 and CDCP1 in cell lines and xenografts. A lower differentiation grade of donor material correlated with a higher CDCP1 mRNA expression level in the respective tumour model. In vivo growth behaviour studies of SW620 revealed significantly higher take rates and shorter doubling times in the tumour growth of CD133 positive subclones in comparison to the unsorted cell line or CD133 negative subclones. Conclusions Our data revealed correlations in the expression of surface markers CD44 and CD24 as well as CD44 and CDCP1 and strongly suggest that CD133 is a stem cell marker within our colon carcinoma panel. Further studies will elucidate its role as a potential therapeutic target.

The farnesoid X receptor (FXR) is expressed predominantly in tissues exposed to high levels of bile acids and controls bile acid and lipid homeostasis. FXR(-/-) mice develop hepatocellular carcinoma (HCC) and show an increased prevalence for intestinal malignancies, suggesting a role of FXR as a tumor suppressor in enterohepatic tissues. The N-myc downstream-regulated gene 2 (NDRG2) has been recognized as a tumor suppressor gene, which is downregulated in human hepatocellular carcinoma, colorectal carcinoma and many other malignancies.We show reduced NDRG2 mRNA in livers of FXR(-/-) mice compared to wild type mice and both, FXR and NDRG2 mRNAs, are reduced in human HCC compared to normal liver. Gene reporter assays and Chromatin Immunoprecipitation data support that FXR directly controls NDRG2 transcription via IR1-type element(s) identified in the first introns of the human, mouse and rat NDRG2 genes. NDRG2 mRNA was induced by non-steroidal FXR agonists in livers of mice and the magnitude of induction of NDRG2 mRNA in three different human hepatoma cell lines was increased when ectopically expressing human FXR. Growth and metastasis of SK-Hep-1 cells was strongly reduced by non-steroidal FXR agonists in an orthotopic liver xenograft tumor model. Ectopic expression of FXR in SK-Hep1 cells reduced tumor growth and metastasis potential of corresponding cells and increased the anti-tumor efficacy of FXR agonists, which may be partly mediated via increased NDRG2 expression. FXR agonists may show a potential in the prevention and/or treatment of human hepatocellular carcinoma, a devastating malignancy with increasing prevalence and limited therapeutic options.

Cetuximab (Erbitux®) targets the epidermal growth factor receptor (EGFR) and is approved for treatment of colorectal and head and neck cancer. Despite wide expression of EGFR, only a subgroup of cancer patients responds to cetuximab therapy. In the present study we assessed the cetuximab response in vivo of 79 human patient-derived xenografts originating from five tumour histotypes. We analysed basic tumour characteristics including EGFR expression and activation, mutational status of KRAS, BRAF and NRAS, the expression of EGFR ligands and the activation of HER3 (ErbB3) and the hepatocyte growth factor receptor MET. Based on these results, a cetuximab response score including positive and negative factors affecting therapeutic response is proposed. Positive factors are high expression and activation of EGFR and its ligands epiregulin or amphiregulin, negative factors are markers for downstream pathway activation independent of EGFR. In cetuximab resistant NSCL adenocarcinoma LXFA 526 and LXFA 1647, overexpression due to gene amplification and strong activation of MET was identified. Knock-down of MET by siRNA in the corresponding cell lines showed that anchorage-independent growth and migration are dependent on MET. MET knock down sensitized LXFA 526L and LXFA 1647L to EGF. Combined treatments of a MET inhibitor and cetuximab were additive. Therefore, combination therapy of cetuximab and a MET inhibitor in selected lung cancer patients could be of high clinical significance.

ObjectivesThis research aimed to shed light on the relationship between flow experience and performance in sports using a marathon race as an example. We hypothesized that flow influences the marathon race performance by an indirect rewarding effect.We assumed that the positive quality of flow experience rewards the pre-race running activity and thereby enhances training behavior which again leads to high race performance. A methodological issue of the this was to compare the retrospective with the experience-sampling measure of flow.DesignThree studies with marathon runners (Ns ¼ 109, 112, 65 for Studies 1, 2, and 3, respectively) were conducted.MethodThey measured flow experience four times during a marathon race either retrospectively (Studies 1 and 2) or using an experience-sampling method during the race (Study 3). Additionally race performance and future running motivation (Studies 1, 2, and 3), pre-race training behavior (Studies 2 and 3) and flow experience in training (Study 3) were measured.ResultsThe results confirmed the hypothesis showing that flow during a marathon race is related to future running motivation, but is not directly linked to race performance. Instead, race performance was predicted by pre-race training behavior (Studies 2 and 3) which again was fostered by flow during the training (Study 3). The descriptive flow courses of the retrospective and the experience-sampling flow measures were comparable but also showed important differences.ConclusionsWe critically discuss the practical implications of the rewarding effect of flow on performance and the advantages of the retrospective and experience-sampling measure of flow. published

Introduction: Novel substances such as the next generation IMiD pomalidomide or the recently approved next generation proteasome inhibitor carfilzomib (Cfz) have considerably expanded our treatment options in MM, both of them influencing multiple myeloma (MM) interaction with bone marrow stroma cells (BMSCs), that provides an interesting target for anti MM therapy. More compounds directed at this disease critical crosstalk are currently under investigation, however the development of novel drugs remains inefficient, displayed by a substantial drop out rate of the 376 preclinical single agents tested since 1961 (Rongvaux, Annu Rev Immunol. 2013; Schüler, Expert Opin Biol Ther. 2013; Kortüm, CLML 2014). Our focus in the projected presented here was to develop a novel bone-derived in vitro 3D co-culture model specifically adapted to mimic the BM niche to more closely study the role of bone and BM bystander cells and to perform more reliable ex vivo compound screening in MM. Methods: Previous 2D models were compared to a novel 3D co-culture model (agarose matrix interlayer, 100 microwells/cm², 1.5mm in depth, permeable for oxygen+cytokines, but not for BMSCs utilizing U266, RPMI-8226, OPM-2 and primary BM patient (pt) cells, with and without HS-5 vs. M210B4 stroma support (Fig. A + Fig. B.a. for pt characteristics). Analyses covered Trypan Blue, Annexin/PI, MTT, FACS, cell cycle analyses and H2B-mCherry/cytochrome c-GFP assays (Udi, Br J Hematol. 2013). In a next step, primary bone-derived stroma cells were acquired from bones of C57BL/6 J mice. Bones were flushed, digested and FACS sorted in order to acquire single BM and bone bystander cell subtypes (MSPCs [mesenchymal stem and progenitor cells], endothelial cells, osteoblasts, PAS [PDFGRalphaSca1] and CaRs [CXCL12-abundant reticular cells]) which were then compared to HS-5 and M210B4 with regard to growth support, cytokine secretion and protection from anti-MM substances. Results: MMCLs and pt specimens were cultured at different concentrations (10 vs. 100 cells per microwell) with and without M210B4 demonstrating a growth advantage with vs. without M210B4 (Fig. B.b). Liquid overlay technique allowed cluster formation of pt specimens leading to more reliable propagation of pt material for up to 20d of culture. Apoptotic changes were assessed by confocal microscopy of RPMI8226 co-expressing fluorescently labelled histone 2B-mCherry (red) and cytochrome c-GFP (green) as indicators of late and early apoptosis. Comparing BMSCLs with regard to their MM growth support capacities, human HS-5 proved even more beneficent than M210B4 stroma (Fig. B.b). Phenotypic analyses of pt specimens showed decreased CXCR4 expression with vs. without BMSCs suggesting a dynamic regulation of homing molecules. The model was then used as an ex-vivo platform allowing both cytotoxicity and cell cycle analyses for the combination of bortezomib (Btz) vs. Cfz with ARRY-520 (kinesin spindle protein inhibitor). Btz (10nM) and Cfz (20nM) proved significantly cytotoxic compared to the control (U266 and pt specimens, respectively) after 48h of single agent treatment Fig.B.c). Compared to Btz (10nM, B10) and Cfz (20nM, C20) as single agents, the additional combination with ARRY-520 showed stronger additive cytotoxicity for Btz (A5+B10, median: 37.5% vs. 13.1%) than for Cfz (A5+C20, 38.6% vs. 34.1%). To note, the model could also be utilized for more profound analyses as depicted for G2/M cell cycle studies in Fig.B.d. 5nM ARRY-520 (A5) led to significant accumulation of OPM-2 cells in G2/M arrest after 48h of treatment confirming prior analyses (Hernández-Garcia Blood Suppl 4710,2014; Fig. B.d). Co-culture studies with different subsets of BM and bone-derived bystander cells are currently ongoing and will be presented at the meeting (Fig. B.e). Conclusions: More complex, 3D bone-derived high-throughput in vitro models are urgently needed to better predict the potency of preclinically tested agents and to better estimate the likelihood of their later clinical adoption into phase I-II trials. With this work, we provide an innovative model which reflects the BM microenvironment as a crucial predictor for in-vivo sensitivity as shown for ARRY-5200. This ex-vivo approach helps to better incorporate MM growth support by bone and BM-derived bystander cells and thus depicts a valid tool to better characterize the role of the BM niche in myeloma. Figure 1. Figure 1. Disclosures Engelhardt: Deutsch Krebshilfe: Other: grant. Wäsch:German Cancer Aid: Research Funding; Comprehensiv Cancer Center Freiburg: Research Funding; Janssen-Cilag: Research Funding; MSD: Research Funding.

Introduction: Hematopoietic stem and progenitor cells (HSPCs) represent the lifelong source of all blood cells and continuously renew the hematopoietic system by differentiation into mature blood cells. The process of differentiation is predominantly initiated in G1 phase of the cell cycle when stem cells leave their quiescent state. During G1 the anaphase-promoting complex or cyclosome (APC/C) associated with the coactivator Cdh1 is highly active and marks proteins for proteasomal degradation to regulate proliferation. In addition, Cdh1 has been shown to control terminal differentiation in neurons, muscle cells or osteoblasts. Here we show that Cdh1 is also a critical regulator of human HSPC differentiation and self-renewal. Methods: Human CD34+ cells were collected from peripheral blood (PB) of G-CSF mobilized donors and cultured in the presence of different cytokine combinations. To analyze cell division and self-renewal versus differentiation, CFSE staining was used in combination with flow cytometric detection of CD34 expression. The knockdown and overexpression of Cdh1 was achieved by lentiviral delivery of suitable vectors into target cells. After cell sorting transduced (GFP+) CD34+ cells were used for in vitro differentiation in liquid culture or CFU assay. For in vivo experiments purified cells were transplanted into NSG mice. Results: G-CSF mobilized CD34+ cells showed effective differentiation into granulocytes (SCF, G-CSF), erythrocytes (SCF, EPO) or extended self-renewal (SCF, TPO, Flt3-L) when stimulated in vitro. The differentiation was characterized by a fast downregulation of Cdh1 on protein level, while Cdh1 remained expressed under self-renewal conditions. A detailed analysis of different subsets, both in vitro and in vivo, showed high Cdh1 level in CD34+ cells and low expression in myeloid cells. Analysis of proliferation revealed lowest division rates during self-renewal, accompanied by higher frequency of CD34+ cells. The fastest proliferation was found after induction of erythropoiesis. These experiments also showed a more rapid decrease of HSPCs' colony-forming ability and of CD34+ cells during granulopoiesis after 2-3 cell divisions in contrast to a moderate decline under self-renewal conditions. The depletion of Cdh1 (Cdh1-kd) had no effect on total cell numbers or proliferation detected by CFSE during differentiation and self-renewal, but showed an increase in S phase cells. These results were confirmed at the single cell level by measuring the cell cycle length of individual cells. Independent of cell cycle regulation, Cdh1-kd cells showed a significant maintenance of CD34+ cells under self-renewal conditions and during erythropoiesis with lower frequency of Glycophorin A+ cells. In CFU assays, the Cdh1-kd resulted in less primary colony formation, notably CFU-GM and BFU-E, but significantly more secondary colonies compared to control cells. These results suggest that the majority of cells reside in a more undifferentiated state due to Cdh1-kd. The overexpression of Cdh1 showed reversed results with less S phase cells and tendency to increased differentiation in liquid culture and CFU assays. To further validate our results in vivo, we have established a NSG xenotransplant mouse model. Human CD34+ cells depleted of Cdh1 engrafted to a much higher degree in the murine BM 8 and 12 weeks after injection as shown by higher frequencies of human CD45+ cells. Moreover, we also found an increased frequency of human CD19+ B cells after transplantation of CD34+ Cdh1-kd cells. These results suggest an enhanced in vivo repopulation capacity of human CD34+ HSCs in NSG mice when Cdh1 is depleted. Preliminary data in murine hematopoiesis support our hypothesis showing enhanced PB chimerism upon Cdh1-kd. Looking for a mediator of these effects, we found the Cdh1 target protein TRRAP, a cofactor of many HAT complexes, increased upon Cdh1-kd under self-renewal conditions. We use currently RT-qPCR to determine, if this is caused by a transcriptional or post-translational mechanism. Conclusions: Loss of the APC/C coactivator Cdh1 supports self-renewal of CD34+ cells, represses erythropoiesis in vitro and facilitates engraftment capacity and B cell development of human HSPCs in vivo. This work was supported by Josè Carreras Leukemia Foundation grant DCJLS R10/14 (to ME+RW) Disclosures Ewerth: Josè Carreras Leukemia Foundation: Research Funding. Wäsch:German Cancer Aid: Research Funding; Comprehensiv Cancer Center Freiburg: Research Funding; Janssen-Cilag: Research Funding; MSD: Research Funding.

Non-small cell lung cancer (NSCLC) is the largest subgroup of lung cancer, occurring at a frequency of over 80% of lung cancer cases. In up to 30% of NSCLC patients the oncogenic driver of tumor growth is a constitutively activated EGF receptor (EGFR), which plays a critical role in regulating multiple cellular processes, including proliferation, survival and apoptosis. Although these patients gain great benefit from treatment with EGFR tyrosine kinase inhibitors (TKI, e.g. erlotinib or gefitinib), development of resistance is inevitable. To model the emergence of drug resistance, an EGFR driven, gefitinib sensitive, patient-derived xenograft (PDX) NSCLC model was treated continuously with gefitinib in immunocompromised mice. The dose of daily treatment was adjusted according to tumor growth over a period of up to 91 days. In a first phase, dosing was high (40-50 mg/kg) to eradicate EGFR TKI sensitive cells. At the time point of maximal antitumoral activity dosing was reduced to 20-30 mg/kg ( = low dose) to preserve selection pressure. Between 69 and 91 days after dosing was initiated, drug-resistant tumors emerged in 4 out of 10 mice under high dose treatment. Resistant tumor fragments, which were re-implanted into a new cohort of mice and continuously treated with gefitinib, kept resistance also under high dose treatment. A comprehensive analysis using Western blot (WB), qPCR and sequencing was performed to identify the reason for resistance. In WB analysis we could show that signalling through EGFR was completely abrogated in all four resistant tumor sublines. Neither secondary mutations in EGFR (ex19-21) or KRAS (ex 1+2) could be detected, nor was the expression of cMET, AXL, HGF, PTEN or HER3 significantly increased in resistant tumors as shown by sequencing and qPCR respectively. However a more comprehensive WB analysis revealed several genes being activated in resistant compared to primary tumors. Depending on the subline, phospho-(p)-cMET, p-AKT, AXL & p-AXL, p-cRAF, p-MEK, p-ERK as well as HER3, IGF-R and ALK were up-regulated in resistant tumors. Based on these data we determined that signalling pathways such as the (RAS)-cRAF-MEK-ERK signalling cascade or enhanced cMET/AKT signalling were activated in drug-resistant tumors. These signalling pathways are known to be alternative pathways for EGFR signalling also in patients with acquired resistance indicating the clinical relevance of these models. To shed more light into the mechanism of resistance, whole exome sequencing analyses of the four resistant sublines as well as the original tumor model are underway. In summary, we have developed four NSCLC tumor sublines each harbouring a different mechanism of resistance to EGFR TKI treatment, modelling the emergence of drug-resistant NSCLC in patients. Herewith a strong preclinical tool for the development of innovative compounds targeting acquired EGFR resistance is now available. Citation Format: Cordula Tschuch, Kerstin Klingner, Dorothee Lehnhard, Anne Löhr, Yana Raeva, Anne-Lise Peille, Eva Oswald, Julia B. Schüler. Establishment and characterization of a patient-derived non-small cell lung cancer mouse model of acquired resistance towards anti-EGFR treatment. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A10.

Patient-derived tumor xenografts (PDX) have played a major role in the development of new cancer therapies and their strengths and weaknesses have gradually been elucidated. One major drawback of PDX is the lack of an immunological competent host. To overcome this hurdle we supplemented NSG/NOG mice with human hematopoietic stem cells (HSC) and subsequently examined growth characteristics of the non-small cell lung cancer (NSCLC) PDX model LXFA 923 in these mice. In parallel we monitored the presence of human and murine immune cells in different organs of the mouse. HSC (2×106) cells were isolated from healthy donors and injected intravenously into sub-lethally irradiated NSG or NOG mice (n = 43 mice in 3 ind. exp.). After 8 weeks LXFA 923 was transplanted subcutaneously (s.c.) into the pretreated mice. Murine peripheral blood was examined by flow cytometry for common murine and human markers expressed on immune cells (hCD14, mCD14, hCD3, mCD3, hCD56, mCD56, hCD19) once weekly. At the end of the experiment tumors and organs were analyzed for human cancer (CD44, CD133, CDCP1, CD166, CD24) and immune cell markers (hCD14, hCD3, hCD56, hCD19) by flow cytometry. Tumors and organs were additionally histologically and immunohistochemically examined. Growth of the implanted tumors was monitored by caliper measurement. Mice bearing only the subcutaneous PDX or the HSC served as control groups. Stable engraftment of human immune cells in immune-compromized mice was successfully achieved. Human immune cells expressing T-, B-, NK- and stem cell markers could be detected in different compartments (bone marrow, peripheral blood and spleen) of the tumor-bearing as well as non-tumor bearing mice. Furthermore, infiltrates of human monocytes (CD14+) as well as T cells (CD3+) could be detected in s.c. implanted tumor tissue. Implantation of LXFA 923 did not influence the proliferation of human immune cells in recipient mice. Growth behavior of the s.c. implanted PDX was not affected by the engraftment of HSC in the murine host. The histological architecture of LXFA 923 was similar when implanted s.c. in humanized or immunodeficient mice and it still closely resembles the patient donor material. In conclusion, our investigations validate the analysis of PDX in mice engrafted with human immune cells, as it enables the interaction of tumor cells with human immune cells as well as with murine stroma to be investigated. This preclinical PDX based in vivo platform provides a further step to support the development of new drugs targeting the host immune response. Citation Format: Eva Oswald, Kerstin Klingner, Dorothee Lenhard, Gabriele Niedermann, Julia B. Schüler. NSCLC PDX model for the evaluation of immuno-oncological treatment strategies. [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 5023. doi:10.1158/1538-7445.AM2015-5023

Recently, unsupervised gene expression-based signatures with prognostic and potential predictive implications were proposed for colorectal cancer (CRC) classification. The challenge is now to characterize the genomic alterations of CRC subtypes and their sensitivity to therapies. Patient derived xenograft models (PDX) may be a valuable tool for this purpose, as they retain the molecular features and drug response patterns of their parental patient tumors. In this study, we classified our collection of colon PDX into transcriptomic subtypes and investigated the associations with genomic alterations and in vivo responses to cetuximab (CTX), oxaliplatin (OXT) and irinotecan (IR). We determined expression profiles of 67 PDX models using Affymetrix HGU133 Plus2.0 arrays and applied the CRC assigner-786 gene expression signature reported by Sadanandam et al. to classify our PDX into 5 subtypes. We identified 34 (51%) transit-amplifying (TA), 15 (22%) goblet-like (GL), 10 (15%) inflammatory (IF), 7 (10%) enterocyte (ET) and 1 (1%) stem-like (SL) PDX models. TA showed a gene signature of WNT pathway activation whereas GL, ET and IF PDX subtypes harbored gene signatures of KRAS pathway activation. As analyzed by whole exome sequencing and Affymetrix SNP6.0 array, the GL, ET and IF subtypes were found to display different signatures of mutational processes, mutations and chromosomal rearrangement patterns than TA and SL. Of particular note, mutations in BRAF, PIK3CA/PTEN, TGFBR2/SMAD4 or NOTCH1 were mainly found in GL, ET or IF while mutations in APC, TP53 and KRAS were not associated with a given subtype. Regarding PDX drug sensitivity, 10/11 TA/SL PDX with unaltered KRAS/PIK3CA/PTEN/HER2 status were sensitive to CTX whereas 9/12 models with alterations were resistant. TA and SL PDX were also frequently sensitive to IR and OXT (6/10 and 4/10, respectively). In the IF subtype, 6/6 PDX showing KRAS, PIK3CA or PTEN alterations were resistant to CTX. Of interest, 5/5 IF PDX were sensitive to IR treatment. As in IF PDX, GL and ET often had alterations in KRAS/PIK3CA/PTEN/HER2 and were resistant to CTX. Moreover, these subtypes were resistant to both IR and OXT. Furthermore, the frequent KRAS pathway activation and the presence of actionable mutations in GL, ET and IF would advocate the testing of inhibitors (as monotherapy or in combination) of BRAF, PI3K or MEK in tumors of these subtypes while WNT inhibitors should be tested in TA models.Molecular classification of our colon PDX collection confirmed their similarities with patient tumors. Investigation of drug sensitivity resulted in the identification of PDX subtypes which respond poorly to standard therapies and which require different treatment options. Testing alternative and combined therapies in PDX may be a valuable approach to optimize personalized medicine. Citation Format: Anne-Lise Peille, Christina Gredy, Bruno Zeitouni, Armin Maier, Kerstin Klingner, Tim Kees, Julia Schüler, Thomas Metz, Heinz. Herbert Fiebig, Vincent Vuaroqueaux. Classification of colorectal PDX into transcriptomic subtypes associated with distinct genomic alteration profiles and in vivo response patterns to therapies. [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 1705. doi:10.1158/1538-7445.AM2015-1705

Introduction: In the past decade, substantial progress has been made in the understanding of multiple myeloma (MM) cell biology and its interaction with the bone marrow microenvironment (BMM). Binding of MM cells to BM stroma cells (BMSCs) alters the expression of SDF-1α and its receptor CXCR4, leading to the secretion of anti-apoptotic cytokines, promoting tumor growth, drug resistance and migration. MM cancer stem cells migrate to endosteal BM niches, where they escape therapies in a quiescent state causing relapse in the course of the disease. The development of novel agents that aim to target the MM and BMM interaction includes drugs as promising as 2nd and 3rdgeneration IMIDs or proteasome inhibitors. Despite these profound advances, the failure rate of preclinically proven cytotoxic single substances is sizeable, as preclinical models often lack the biological, genetic, etiological and immunological properties of the disease (Schüler, Expert. Opin. Biol. Ther. 2013; Kortüm. CLML 2014; Rongvaux. Annu Rev Immunol 2013). Methods & Results: We have previously demonstrated that BM interaction and homing to niches, mediated by the adhesion molecules CXCR4, CD49d and CD44, protect MM cell lines (MMCL) and primary plasma cells (PC) from the cytotoxic effect of anti-MM agents, such as bortezomib (Bor), vorinostat (Vor) and pomalidomide (Pom). Our in vitro and in vivo observed cytotoxic effects from Bor, Vor and Pom confirmed their potent cytotoxicity, whereas cocultivation with M2-10B4 substantially reduced apoptosis and induced tumor protective effects. Additional treatment with the CXCR4 inhibitor AMD3100 blocked CXCR4 in coculture, but left CD49d, CD44 and CD11a widely unchanged. Toxic or therapeutic effects from AMD3100 monotherapy were excluded for the doses used. Comparison of the CXCR4 antibody (ab)-clones 12G5, 44717 and 4G10 revealed that AMD3100 treatment of U266 cells reduced CXCR4 expression with use of 12G5 and 44717, whereas binding of both FITC- and PE-coupled 4G10 was not influenced, making the latter the most reliable for CXCR4 analysis. Use of image cytometry (IC) allowed accurate visualization of co-localisation of CXCR4 expression both on the cell surface and within the cytoplasm of MM cells. IC correlated with flow cytometry-determined CXCR4 expression and allowed the detailed assessment of treatment studies with and without anti-MM agents and AMD3100. Of note, AMD3100 resensitized MM cells to Bor, Vor and Pom (Waldschmidt. Blood 2012:2450), whereas carfilzomib (Cfz) reduced CXCR4 expression in MMCL and could not be antagonized by stroma coculture. Cfz sensitivity was not increased by adding AMD3100 (Simon. Blood 2013:3851). These preclinical studies need additional adaptation to the clinical setting in order to surpass prior drug failure rates, and there is a need to develop more broadly available and better predictive preclinical systems. Therefore, we are currently assessing a 3D co-culture MM model composed of agarose matrix interlayers, based on a novel liquid overlay technique. This model has been specifically adapted to MM cell and BM component interactions as described (Udi. BJH 2013; Zlei. Exp Hematol 2007; Schüler. EOBT 2013). MM cells are cultivated in conical microwells of a non-adherent agarose matrix after BMSCs were plated on the bottom of each plate, allowing the diffusion of soluble cytokines but no direct contact between BMSC and MMCL. Therein, we are presently testing novel anti-MM substances in comparison to our standard-coculture system. Conclusion: Targeting microenvironmental mediators, like SDF-1α and CXCR4, is a promising approach to expand the choice of antimyeloma agents and amplify the effects of established antimyeloma drugs, as previously shown by us and others for the combination of AMD3100 and Bor or Pom. However, as our knowledge on MM and its BMM has dramatically increased a great effort has been made in the preclinical testing of promising new anti-MM agents, and more complex high-throughput in vitro models are urgently needed to better predict the potency of these substances in order to reduce dropouts in clinical trials. We hereby provide a novel approach which better reflects the spatial growth of human MM samples in BMSC coculture, and more closely mimics the growth and proliferation of human MM clones in vivo. Disclosures No relevant conflicts of interest to declare.

Hematological malignancies account for about 10% of newly diagnosed cancers in the US, their heterogeneity and diverse biological characteristics present unique therapeutic challenges. In order to develop more promising therapeutic strategies, the establishment of functional and reproducible in vivo models is widely pursued. A panel of 18 hematological cell lines comprising different entities including leukemia (acute lymphoblastic AML; chronic myeloid CML; chronic lymphatic leukemia CLL), diffuse large B cell lymphoma (DLBCL), as well as multiple myeloma (MM) was evaluated for in vivo engraftment using different mouse strains and application routes. 18 different cell lines were inoculated into 4 different immuncompromized mouse strains (NOG; NSG; NOD/SCID; SCID/beige nude) in 35 independent experiments. Cells were injected intravenously (i.v.), intratibialy (i.t.) or intraperitonealy (i.p.). Tumor growth was monitored via a) determination of overall survival, b) fluorescence-based in vivo imaging (IVI, Bruker FX, using CF750 labeled anti-hu CD33, CD19, CD45) c) flow cytometry and d) histological and IHC examination. Engraftment of hematological cell lines was clearly influenced by tumor entity, mouse strain and application route. The investigated AML cell lines engrafted in all three applied mouse strains (NOG; NSG; NOD/SCID) and injection routes. MOLM-13 infiltrated the bone marrow (BM) and the spleen (>90% infiltration, 21 days after tumor cell injection (=d21)), whereas MV4-11 cells disseminated mainly to the spleen (5% BM and 15% spleen infiltration, d21). In contrast THP-1 (80% BM and 2% spleen infiltration, d21) as well as NOMO-1 cells (30% BM and 3% spleen infiltration, d21) grew predominantly in the murine BM. MM cell lines (L363, RPMI8826, MM1R, MM1S) as well as B-CLL cell lines (Mec-1) failed to engraft reliably in NOD/SCID mice, irrespective of the application route. MM cells engrafted predominantly in the BM (20 - 60% depending on the cell line) of NSG mice when injected i.t.. i.v. injected MM cells did engraft in the BM but to a significantly lower extent than when injected i.t. (e.g. 20% vs. 2% BM infiltration of RPMI8226 cells, d35). Mec-1 cells infiltrated the BM and spleen of NSG and NOG mice reliably when injected i.v. and i.p. in the same individual (60% BM and 15% spleen infiltration, d28), whereas the same approach did not result in any engraftment when using SCID/beige nude mice. 3 out of 4 investigated DLBCL cell lines engrafted in vivo. Disseminated growth could be observed in NOG mice when injecting the cells i.v. or i.t. In all cases, tumors grew faster and infiltrated the BM more aggressively when injected I.t.. In summary, the engraftment of hematological cell lines strongly depends on the tumor microenvironment. The direct contact to the BM niche is a major survival benefit for the tumor cells as is the lack of NK cells in NOG and NSG mice. Citation Format: Eva Oswald, Kerstin Klingner, Ralph Waesch, Katja Zirlik, Monika Engelhardt, Julia B. Schüler. Influence of tumor microenvironment on engraftment capacity of hematological cell lines in immunocompromized mice. [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 3926. doi:10.1158/1538-7445.AM2014-3926

Introduction: Genetically engineered mouse models (GEMM) represent an attractive system for preclinical research since GEMM tumors develop in the presence of a competent immune system, thereby closely resembling the tumor microenvironment in patients. One example is the KP model (DuPage et al., 2009), which reflects the tumorigenesis of NSCLC in humans. KP tumors carry Kras and Trp53 mutations, comparable to subtypes of human NSCLC. Typical drawbacks of GEMM tumors include differences in their genetic make-up to human tumors and the very slow and heterogeneous tumor development, making the use of these models challenging for routine in vivo efficacy studies. The latter can be improved by grafting primary tumors on a genetically dissimilar member of the same strain, so-called allografting. Thus, the advantages of xenograft and GEMM models can be combined. Here, we described the establishment and characterization of allografts from the KP GEMM. Material & Methods: KP tumors were excised from the lungs of KP mice and implanted subcutaneously (s.c.) into C57BL/6N female mice. When tumor growth was detected, passaging steps from animal to animal were performed. Furthermore, two cell lines derived from KP tumors, KP1 and KP4 (provided by Müller et al.), were injected s.c., the developing tumors excised and directly re-implanted as described above. The passages were analyzed molecularly, histologically, and via IHC (e.g. Ki67 proliferation marker) , and compared to the original in-situ tumors. A therapeutic study with Erlotinib and BEZ235 was performed on KP1 allografted mice. Results: All investigated allografts showed similar histological patterns. Ki67 scores reflected the average time from implantation to passaging. The average passage times ranged from 19±3 days (KP4 cell line) to 26±6 days (KP1 cell line). Allografts from the original KP model showed a time to passaging of 23±5 days. The mRNA expression of EpCAM (CD326) was on average 10-fold higher in KP and KP1 allografted tumors compared to normal C57BL/6 lung tissue, which could be confirmed by flow cytometry and cell-binding assay, determining >99% EPCAM positive cells in KP1 tumors. All allografts carried the Kras mutation and loss of function point mutations in Trp53. The therapy study revealed no effect of Erlotinib on tumor growth, but a significant (p=0.0003) tumor load reduction in mice treated with BEZ235 (519 ± 244 mm³, n=5) compared to the control group (1678 ± 275 mm³, n=4) on day 28, respectively. Conclusion: The generation of s.c. transplantable allografts from the KP GEMM was successfully conducted. The allograft models enable the use of immunocompetent mice in a feasible time frame and still resemble key aspects of human disease. Further studies will elucidate the interaction of the allografts with the host and compare sensitivity characteristics between GEMM and allograft of the same model. Citation Format: Damaris Kukuk, Philipp Müller, Kerstin Klingner, Anne-Lise Peille, Alfred Zipelius, Julia B. Schüler. Establishment and characterization of allografts derived from a genetically engineered mouse model of non-small cell lung cancer (NSCLC). [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 100. doi:10.1158/1538-7445.AM2014-100

The stromal compartment of the bone marrow is composed of various cell types that provide trophic and instructive signals for hematopoiesis. The mesenchymal stem cell is believed to give rise to all major cellular components of the bone marrow microenvironment. Nemo-like kinase, Nlk, is a serine-threonine kinase that connects MAP kinase and Wnt signaling pathways; its in vivo function in mouse is unknown. We have generated mice with a targeted disruption of Nlk and find that the complex phenotype significantly varies with the genetic background. Whereas C57BL/6 mice lacking Nlk die during the third trimester of pregnancy, the 129/Sv background supports survival into adolescence; such mice are growth retarded and suffer from various neurological abnormalities. We show here that the Nlk deficiency syndrome includes aberrant differentiation of bone marrow stromal cells. Varying degrees of morphological abnormality, such as increased numbers of adipocytes, large blood sinuses and absence of bone-lining cells are observed in the bone marrow of mutant mice. Nlk deficient mice thus provide a novel model to study the genetic requirements for bone marrow stromal differentiation.

The in vitro antiproliferative activity of an aqueous mistletoe extract (AME) with a defined content of bioactive mistletoe lectin (ML) was tested in 25 human tumor cell lines, including 20 solid and 5 hematological malignancies and 47 human tumor xenografts. The antiproliferative activity of AME was compared to that of the standard cytotoxic agent doxorubicin (CAS 23214-92-8, adriamycin, ADR) using the sulforhodamin B, propidium iodide and soft agar colony forming assays, respectively. AME was highly cytotoxic in solid human tumors with mean IC70 values in the range of 0.17-1 ng ML/ml (2.8-17 pmol bioactive ML). On a molar basis, AME was 3 to 4 logs more potent than ADR and showed differential cytotoxicity towards tumors of the breast, small cell and non-small cell lung, prostate and renal cell cancers. AME was also highly active in hematological malignancies with steep dose response curves resulting in mean IC70 values of 0.12 ng ML/ml (2 pmol). The acute lymphoblastic leukemia cell line HL-60 was the most sensitive, the histiocytic lymphoma cell line U937 the most resistant hematological malignancy. It is important to stress that AME did not induce a biologically relevant increase of cell proliferation in any of the tumor cell lines tested. Our data suggest that AME has in vitro antitumor profiles similar to those of classical anticancer agents. Clear dose-response relationships were found in all of the performed experiments and interesting differential cytotoxicity patterns were observed. Experiments with sensitive tumor types identified in these in vitro studies are currently ongoing in order to demonstrate the anticancer activity of AME in different animal tumor models.

Introduction Hematopoietic stem cells (HSCs) and multipotent progenitor cells continuously maintain hematopoiesis by self-renewal and differentiation. The stem cell fate is tightly connected with the cell cycle, where the major regulator anaphase-promoting complex or cyclosome (APC/C) with its co-activators Cdc20 and Cdh1 marks cell cycle regulatory proteins, such as cyclin A and B, for proteasomal degradation and thus controls their activity. Known targets of Cdh1 are involved in regulation of self-renewal and granulopoiesis. This raises the hypothesis that Cdh1 may be a critical mediator of HSC proliferation, self-renewal and differentiation. Methods CD34+ cells were collected from peripheral blood (PB) of G-CSF mobilized donors and cultured in the presence of different cytokines. To analyze cell division and self-renewal versus differentiation, CFSE staining was used in combination with CD34 detection. The knockdown (kd) of Cdh1 was achieved by lentiviral delivery of specific shRNA into target cells. Results In vitro cultivation of CD34+ cells under conditions resulting in either self-renewal (SCF, TPO, Flt3-l) or differentiation/granulopoiesis (SCF, G-CSF) showed impressive downregulation of Cdh1 during culture. A high Cdh1 expression in CD34+ cells and lower expression in myeloid cells (CD41a+, CD15+, Glycophorin A+) reflects the situation we found in vivo in bone marrow (BM) subsets. Western blotting also revealed inactivation of Cdh1 by its specific inhibitor Emi1 which stabilized the ubiquitin ligase Skp2 and promoted cell cycle entry and proliferation by degrading the Cdk inhibitor p27. In addition, the APC/CCdh1 target cyclin B was upregulated. These data indicate that initial Cdh1 downregulation is required to promote cell cycle entry and proliferation of CD34+ HSCs under conditions mediating both self-renewal as well as differentiation. When cultured under self-renewal conditions, CD34+ cells showed diminished proliferation with cells residing in lower generations, whereas during granulopoiesis, cells accumulated within higher generations. These experiments also revealed a more rapid decrease of CD34+ cells in granulopoiesis after three cell divisions in contrast to a moderate decline under self-renewal conditions. We also found a decreased colony-forming ability in cells divided more than twice during granulopoiesis, which correlates with their lower CD34 expression. This is consistent with more symmetric divisions into CD34+ daughter cells under self-renewal conditions and gradual commitment during granulopoiesis. Our current experiments extent these analyses to immunofluorescence of Numb distribution in individual cells to elucidate the impact of Cdh1 on symmetric/asymmetric cell division. We could already show that Cdh1-kd led to expansion of CD34+ HSCs in vitro. To further validate our results in vivo, we have established a NOD/SCID/IL-2Rγ chain-/- (NSG) xenotransplant mouse model. Human CD34+ cells depleted of Cdh1 engrafted to a much higher degree in the murine BM 8 and 12 weeks after injection as shown by higher frequencies of engrafted human CD45+ cells. Moreover, we also found an increased frequency of human CD19+ B cells after transplantation of CD34+ Cdh1-kd cells. Further analyses of the contributing subsets to the pool of CD45+ human cells are ongoing. These results suggest an enhanced in vivo repopulation capacity of human CD34+ HSCs in NSG mice when Cdh1 is depleted. Conclusions APC/CCdh1 mediates cell cycle entry and proliferation during self-renewal and differentiation in CD34+ HSCs in vitro and improves engraftment capacity in vivo. Disclosures: No relevant conflicts of interest to declare.

Introduction: Asian and Caucasian populations are distinct regarding the prevalence of different tumor types due to differences in lifestyle and genetics. The molecular makeup of tumors of the same type can also vary. When developing drugs for the treatment of stomach cancer it is important to study tumors in both Asian and Caucasian backgrounds. In East Asia stomach cancer is the 2nd most common malignancy. The development of patient derived xenografts (PDX) from patients of East Asian origin is therefore critical. Oncotest established xenografts from stomach cancer patients who underwent surgery for the treatment of their cancer at the Seoul National University, South Korea. These Asian gastric cancer xenografts (GXA) were utilized for the investigation of molecular characteristics and sensitivity to anticancer agents. Material and Methods: 78 gastric tumor samples from SNU were implanted subcutaneously in nude mice and sequentially passaged. Tumor material was collected for molecular profiling, including mutational analysis by Sanger sequencing and OncoCarta panels I, II, III. The efficacy of standard of care drugs 5-fluoruracil, cisplatin and taxol was tested in vivo. Results: From the 78 implantations we were able to establish 21 Asian patient-derived gastric cancer xenografts, representing a take rate of 27%. The majority of tumors from which GXA xenografts were derived were poorly or moderately differentiated adenocarcinomas. The panel also includes mucinous adenocarcinomas and squamous cell carcinomas. The morphological features of the tumors (degree of differentiation) were preserved throughout serial passages. Mutational analysis for 18 established models revealed that TP53 (13/18, 72%), PTEN (6/9, as well as one with PTEN RNA levels too low for RT-PCR), KRAS (5/18, 28%) and PIK3CA (4/18, 22%) were frequently mutated in these gastric cancers. Mutations in EGFR, MLH1 and APC were also detected, but no mutations in NRAS (0/18) or BRAF (0/17) were found. Regarding sensitivity to anticancer agents, the response of the tumors varied strongly. Tumors were considered sensitive if the optimal treatment / control (T/C [%]) value was lower than 30%. 6 of 18 (33%) tumors were sensitive to 5-fluoruracil, 5 of 18 (28%) to paclitaxel and 3/18 (17 %) to cisplatin. Some gastric PDX were responsive to more than one chemotherapy, whereas 7 of 18 (39%) were not sensitive to any of these standard of care therapies. These results demonstrate (i) that standard of care therapies are not the therapy of choice for a large proportion of these tumors and (ii) great heterogeneity within the 18 analyzed gastric Asian patient- derived xenografts. Conclusion: In this study we developed a panel of Asian patient-derived gastric cancer xenografts with diverse molecular characteristics and responses to standard of care drugs. Citation Format: Rebekka Krumbach, Seong-Ho Kong, Woo Ho Kim, Julia Schüler, Andreas Ackermann, Vincent Vuaroqueaux, Gregory P. Donoho, Amit Aggarwal, Christoph Reinhard, Han-Kwang Yang, Thomas Beckers, Heiner Fiebig. Functional and molecular characteristics of 20 novel patient-derived xenografts of Asian gastric cancer. [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 2789. doi:10.1158/1538-7445.AM2013-2789

Abstract 1236 Hematopoietic stem cells (HSCs) and multipotent progenitor cells continuously maintain hematopoiesis by self-renewal and differentiation to all types of blood lineages. These unique processes are regulated by intrinsic and extrinsic signals (e.g. cytokines, cell-cell contacts) and strongly connects stem cell fate with the cell cycle. The ubiquitin-proteasome system regulates spatial and temporal abundance of proteins in the cell. During cell cycle, the anaphase-promoting complex or cyclosome (APC/C) with its co-activators Cdc20 and Cdh1 marks proteins for proteasomal degradation and thus controls their activity. Known targets of Cdh1, namely Skp2 and Id2, are involved in regulation of self-renewal and granulopoiesis (Wang et al., Blood 2011; Buitenhuis et al., Blood 2005). This raises the hypothesis that Cdh1 may be a critical upstream regulator of HSC differentiation. The analysis of human bone marrow cell subsets (CD34+, lymphoid and myeloid cells) revealed highest protein level of Cdh1 in CD34+ cells, lower expression in more mature lymphoid subsets (CD3+, CD19+) and only marginal expression in mature myeloid cells (CD41a+, CD11b+). These data suggest that Cdh1 is important to induce differentiation, but dispensable for maintaining the differentiated state. In vitro cultivation of G-CSF mobilized peripheral blood CD34+ cells under conditions resulting in either self-renewal (SCF, TPO, Flt3-l) or differentiation/granulopoiesis (SCF, G-CSF) showed downregulation of Cdh1 during culture compared to d0. Western blots did not only reveal decreasing levels of Cdh1, but also its inactivation by its specific inhibitor Emi1 which stabilized the ubiquitin ligase Skp2 and promoted cell cycle entry and proliferation by degrading the cyclin-dependent-kinase inhibitor p27. In addition, the APC/CCdh1 target cyclin B was upregulated. These data indicate that initial Cdh1 downregulation is required to promote cell cycle entry and proliferation of CD34+ HSCs under conditions mediating both self-renewal as well as differentiation. To analyze cell division/proliferation and self-renewal versus differentiation more closely, we used the fluorescent dye CFSE as an indicator of cell division in combination with CD34 to indicate the differentiation status. When cultured under self-renewal conditions using SCF, TPO and Flt3-l, CD34+cells showed enhanced proliferation with increased cells in higher generations, whereas using SCF and G-CSF to induce granulopoiesis, cells within lower generations were more prominent. These experiments also revealed a rapid decrease of CD34 expression in granulopoiesis after 3 cell divisions in contrast to a moderate decline under self-renewal conditions. This is consistent with more symmetric divisions into CD34+ daughter cells under self-renewal conditions and gradual cell cycle exit and differentiation under conditions that induce granulopoiesis. To further elucidate the role of Cdh1 for stem/progenitor cell fate, we used a lentiviral knockdown of Cdh1 in CD34+ cells. After 4 days of transduction and cell sorting, the cells were cultivated for 1 week in medium containing SCF, TPO and Flt3-l. Cdh1 depleted cells showed enhanced proliferation compared to the empty vector control and a higher expression of CD34. In colony forming unit (CFU) assays, we observed that CD34+ cells with Cdh1-knockdown were less efficient to differentiate to CFU-G, CFU-M and BFU-E. A higher potential to self-renew was validated by replating of these colonies, where the number with Cdh1-knockdown increased during serial replating. To validate our results in vivo, we have established a NOD/SCID/IL-2Rγ chain−/− (NSG) xenotransplant mouse model. The evaluation of engraftment capacity and differentiation potential of human Cdh1 depleted CD34+ cells in this model is ongoing. Our data establish the central cell cycle regulator APC/CCdh1 as a novel regulator of self-renewal and differentiation in CD34+ HSCs. Disclosures: No relevant conflicts of interest to declare.

Abstract 4995 Introduction: Leukemia and lymphoma account for a notable proportion of cancers worldwide. The heterogeneity and biological characteristics of hematological malignancies induce unique therapeutic challenges. It is well known that pluripotent as compared to differentiated cells possess the potential for anchorage independent growth in semisolid medium. This can be monitored via clonogenic or colony formation assays, in which cells grow in vitro in a three-dimensional (3D) manner without adherence to plastic culture material support. These assays can be utilized to evaluate growth and drug sensitivity of tumor stem and progenitor cells (Fiebig HH et al. Eur J Cancer 40:802, 2004). In addition, these 3D cell culture assays often mimic the in vivo scenario better than 2D cell culture assays with adherent tumor cells. Material and methods: For our ex vivo anti-tumor efficacy profiling using clonogenic assays, we established a panel of 20 hematological cell lines comprising different entities like acute lymphoblastic leukemia (ALL, 4 cell lines), acute myeloid leukemia (AML, 6 cell lines), chronic myeloid leukemia (CML, 5 cell lines), Hodgkin- (1 cell line) and non-Hodgkin-lymphoma (NHL, 3 cell lines), as well as multiple myeloma (MM, 3 cell lines). Tumor cells were injected into the flanks of NOD/SCID mice in order to obtain subcutaneous tumor xenografts, which were kept at low passages (n Results: Twenty-four standard of care agents were tested in terms of their ex vivo chemosensitivity (e.g. cytarabine, cyclophosphamide, dexamethasone, doxorubicin, etoposide, melphalan, prednisolone, vincristine), including selected targeted drugs also (e.g. bortezomib, imatinib, nilotinib, sorafenib). The drugs showed diverse patterns of selectivity and potency: vincristine, doxorubicin and cytarabine, but also the proteasome inhibitor bortezomib exhibited pronounced activity with IC50 values in the nanomolar range (mean IC50 = 1 – 100nM), not only in their respective clinical application, but also in various other tumor entities, such as in ALL and AML with use of bortezomib. Differential activity was determined e.g. for prednisolone and dexamethasone, which were active in a micromolar range (mean IC50 = 22 – 58μM) in the ALL cell lines CCRF-CEM and MOLT-4, AML cell lines NOMO-1, NHL DAUDI and U-937, as well as the MM cell line IM-9. All-trans-retinoic acid (mean IC50 = 1.3μM) as well as interferon-gamma-1b (mean IC50 = 0.43 μM) showed specific activity patterns with pronounced growth inhibition in AML (3/6 tested AML cell lines: KG-1, NOMO-1, OCI-AML2), but also in CML (1/5 tested CML cell lines: EM-2) and MM (1/3 tested MM cell lines: L-363). The strong correlation of both tyrosine kinase inhibitors imatinib and nilotinib (spearman coefficient: 0.73, p Conclusions: The presented panel screen using clonogenic assays is of great value for time and cost effective profiling of traditional cytotoxic as well as new targeted anti-cancer agents which can be confirmed in tumor models of hematological malignancies and can thereby guide to more effectively designed in vivo experiments. Diverse activity and resistance patterns ex vivo and in vivo also contribute to create clinical development strategies of standard and novel compounds. Disclosures: No relevant conflicts of interest to declare.

Background: PTEN alterations are major determinants of resistance to several new targeted therapies involving EGFR/PI3K/PTEN pathways. Recent findings also suggested implication of its associated PTENP1 pseudogene. PTEN can be altered at the gene or transcriptional level. PTEN expression is often higher in stroma than in tumor cells complicating readouts of assayed tumor samples. Taking advantage of the stroma of xenografted human tumors in nude mice being produced by the host, we characterized specific PTEN/PTENP1 tumor cell transcript expression levels and mutational status in a large panel of tumor models. Materials and Methods: A total of 192 patient-derived xenografts of 23 different histotypes were investigated. Human and murine PTEN, PTENP1 mRNA levels as well as PTEN gene copy numbers were quantified by quantitative polymerase chain reaction using species specific assays; PTEN transcript was analyzed by sequencing. Results were analyzed by tumor type and compared to sensitivity to cetuximab. Results: PTEN transcript alterations were observed in 32 of the 192 tumor xenografts (17%) and were shown to be due to loss of PTEN gene for 9, to loss of transcript expression for 4 and to frameshift or substitution-missense mutations for 14 and 7 samples, respectively. PTEN expression levels and alteration frequency were both associated with specific histotypes. Melanoma and breast cancer showed most PTEN alterations (6/11 and 6/16 respectively). PTENP1 pseudogene was shown to be well expressed in only 61% of tumor xenografts, whereby expression levels depended on the tumor type. No or low PTENP1 expression was found in tumor models having a PTEN alterations. Finally, in a tumor panel consisting of colon, non-small cell lung, gastric and head and neck cancer we found that none of the PTEN altered tumors analyzed were sensitive to cetuximab in vivo. Conclusion: This study confirmed the multiplicity of PTEN alterations occurring in cancer. PTEN expression levels and alterations were associated with PTENP1 expression levels and depended on tumor types. PTEN alterations were shown to be associated with tumor resistance to cetuximab and their impact on other targeted therapies involving EGFR/PI3K/PTEN pathways should be evaluated. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A52.

Abstract 3924 Targeted therapies in terms of monoclonal antibodies have become standard in the treatment of various lymphomas. Albeit being more specific than conventional therapy, the used antibodies target surface receptors both present on polyclonal and monoclonal hematopoietic cells. Due to its specificity for the malignant B-cell clone the B-cell receptor (BCR) is an ideal therapeutic target in lymphoma therapy. Moreover, using peptides has several advantages over whole antibodies: reduced immunogenicity, better epitope mimicry and tissue penetration, easier synthesis and more favourable pharmacokinetics (no uptake into the reticulo-endothelial system). Peptides mimicking the epitope recognized by lymphoma BCRs have therefore been praised as promising therapeutic tools for years (Lam, West J Med., 1993) but a proof-of-concept has only been published recently in mice bearing subcutaneous A20 lymphoma (Palmieri et al., Blood, 2010). In the current study, we have established a human cell line-derived disseminated Burkitt′s lymphoma model (SUP-B8) in NOD/SCID mice by intravenous injection. Our active principle was the tetramerized BCR binding peptide YSFEDLYRRGGK-biotin (termed T-peptide, Renschler et al., PNAS, 1994) which was applied intravenously on day (d) 12, 14, 16 and 19 after injection of the tumor cells, respectively. The therapeutic efficacy was evaluated in comparison to several control groups (tetramerized control peptide (termed C-peptide, RDYSYERLFGGK-biotin), vehicle (0.8% ACN in water, 200μl/d) and untreated animals). Tumor cell engraftment was monitored via daily surveillance of disease symptoms, FACS (anti-human lambda, CD19, anti-murine CD45) and fluorescence-based in vivo imaging system (FI, Kodak FX, Alexa750 labeled anti-human CD45) on days 12 and 21. SUP-B8 engrafted predominantly in the bone marrow (BM, take rate = 100%) and marrow infiltration increased in untreated mice between start and end of therapy from 1 ± 0.4% (d 12) to 39.8 ± 9.4% (d 21). Other sites of engraftment were subcutis (38%) and spleen (8%). The examined compounds were well tolerated in tumor-bearing mice, no acute toxicity could be observed and maximal body weight loss was below 15%. Treatment of xenograft mice with the tetramerized BCR-binding peptide significantly reduced bone marrow infiltration compared to controls (T-peptide 8.1 ± 4.6%, C-peptide: 32.8 ± 8%, p=0.037, vehicle: 30.5 ± 7.9%, p=0.029). Considering the short half-life of uncoupled peptides and the injection schedule every second day, this is a remarkable reduction. For further optimization of this promising therapeutic approach we plan to couple peptides to effector molecules via acid labile linkers; this is based on the evidence that confocal imaging of Burkitt lymphoma cell lines showed the processing of specific BCR binding peptides in acidic organelles of the cell. In summary, we conclude that BCR targeted peptide-based therapy is a feasible method with remarkable therapeutic results in vivo and future studies will focus on coupling specific peptides to appropriate effector molecules or combinational therapeutic approaches using conventional chemotherapeutics. Disclosures: No relevant conflicts of interest to declare.