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Natural killer (NK) cells play a pivotal role in controlling cancer. Multiple extracellular receptors and internal signaling nodes tightly regulate NK activation. Cyclin-dependent kinases of the mediator complex (CDK8 and CDK19) were described as a signaling intermediates in NK cells. Here, we report for the first time the development and use of CDK8/19 inhibitors to suppress phosphorylation of STAT1S727 in NK cells and to augment the production of the cytolytic molecules perforin and granzyme B (GZMB). Functionally, this resulted in enhanced NK-cell–mediated lysis of primary leukemia cells. Treatment with the CDK8/19 inhibitor BI-1347 increased the response rate and survival of mice bearing melanoma and breast cancer xenografts. In addition, CDK8/19 inhibition augmented the antitumoral activity of anti–PD-1 antibody and SMAC mimetic therapy, both agents that promote T-cell–mediated antitumor immunity. Treatment with the SMAC mimetic compound BI-8382 resulted in an increased number of NK cells infiltrating EMT6 tumors. Combination of the CDK8/19 inhibitor BI-1347, which augments the amount of degranulation enzymes, with the SMAC mimetic BI-8382 resulted in increased survival of mice carrying the EMT6 breast cancer model. The observed survival benefit was dependent on an intermittent treatment schedule of BI-1347, suggesting the importance of circumventing a hyporesponsive state of NK cells. These results suggest that CDK8/19 inhibitors can be combined with modulators of the adaptive immune system to inhibit the growth of solid tumors, independent of their activity on cancer cells, but rather through promoting NK-cell function.

KRAS is the most frequently mutated driver of pancreatic, colorectal, and non–small cell lung cancers. Direct KRAS blockade has proved challenging, and inhibition of a key downstream effector pathway, the RAF–MEK–ERK cascade, has shown limited success because of activation of feedback networks that keep the pathway in check. We hypothesized that inhibiting SOS1, a KRAS activator and important feedback node, represents an effective approach to treat KRAS-driven cancers. We report the discovery of a highly potent, selective, and orally bioavailable small-molecule SOS1 inhibitor, BI-3406, that binds to the catalytic domain of SOS1, thereby preventing the interaction with KRAS. BI-3406 reduces formation of GTP-loaded RAS and limits cellular proliferation of a broad range of KRAS-driven cancers. Importantly, BI-3406 attenuates feedback reactivation induced by MEK inhibitors and thereby enhances sensitivity of KRAS-dependent cancers to MEK inhibition. Combined SOS1 and MEK inhibition represents a novel and effective therapeutic concept to address KRAS-driven tumors. Significance: To date, there are no effective targeted pan-KRAS therapies. In-depth characterization of BI-3406 activity and identification of MEK inhibitors as effective combination partners provide an attractive therapeutic concept for the majority of KRAS-mutant cancers, including those fueled by the most prevalent mutant KRAS oncoproteins, G12D, G12V, G12C, and G13D. See related commentary by Zhao et al., p. 17. This article is highlighted in the In This Issue feature, p. 1

KAT6A and KAT6B are histone acetyl transferase (HAT) that are controlling gene expression by transferring acetyl groups to histones. Yu et al (Oncogene, 2017 36, 2910-2918) have shown that KAT6A has a role in breast cancer development by activating the ERα promoter and was found frequently amplified in 11% and is overexpressed in 15% of breast cancers. Altogether, these data support KAT6A as an attractive target in estrogen receptor driven breast cancer. KAT6B is a close homolog that shares 91% identity in the acetyl coA binding site. While the prevalence of gene amplification for KAT6B in breast cancer is only 1-2%, it is often overexpressed. In this study, we are exploring how KAT6A and KAT6B are contributing to ERα expression and the growth of KAT6A amplified breast cancer cell lines. To decipher each protein's role, we have designed specific siRNA against KAT6A and KAT6B and we tested their action either individually or in combination in the KAT6A gene amplified breast cancer cell lines T-47D, CAM-1 and ZR-75. Following treatment of T-47D, CAMA-1 and ZR75 cell lines with KAT6A or B siRNA we observed a decrease of ERα gene and protein expression for all siRNAs. The effect was more pronounced when using KAT6A over KAT6B and the effect of both siRNA A/B were additive. Phenotypically, the impact on cell line proliferation by applying clonogenic and incucyte assays, mirrors what is observed on ERα gene and protein expression. The effect on cellular proliferation of KAT6A is more prominent than KAT6B silencing, while the combined KAT6A + KAT6B silencing being more impactful than KAT6A silencing alone. In conclusion, our data show that KAT6A and KAT6B share similar function in the control of ER gene and protein expression. This study suggests that KAT6A and KAT6B may be paralogs, one compensating for the loss of the other. This translates into a more pronounced antiproliferative effect when both genes are silenced concomitantly. Citation Format: Isabelle Meaux, Dimitri Gorge-Bernat, Angele Paz, Catherine Geslin, Laurent Debussche, Jürgen Moll, Christophe Henry. Roles of KAT6A and KAT6B in the biology of estrogen positive breast cancer [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 2125.

Over the past years, the HIPPO tumor suppressor pathway and its downstream effector Yes1-associated transcriptional regulator (YAP1) present increasing interest in cancer research. The YAP1-TEAD complex regulates transcription for cell proliferation, survival, plasticity, and migration. Aberrant activation of YAP1 has been reported for multiple different cancer types. To estimate the proportion of cases where YAP1/TEAD-dependent transcription may be responsible for tumor development or evolution and evaluate the pharmacodynamics of novel TEAD-inhibitors, we have identified a transcriptional signature of YAP1 activity. The signature comprises 500 genes, positive and negative downstream effectors at about equal proportions, selected by differential expression analyses from 10 published datasets reporting, in total, 19 YAP1-activation and 18 YAP1-inhibition experiments. YAP1/TEAD-dependent transcription was scored in single samples as the difference Rp - Rn, where Rp is the mean fractional rank of the positive YAP1-effectors, and Rn, that of the negative effectors. We so scored, and called, YAP1 activation in normal tissues (GTEx collection; N~9000), in primary, recurrent, or metastatic tumors (TCGA; N~10000), and in cell lines (CCLE; N~1000) for the selection of models. We further applied the signature to follow the degree of YAP1 inhibition in cell and tumor models either after genetic knockdown of HIPPO-YAP1 pathway components or by pharmacologic inhibition using small-molecule TEAD-inhibitors. Our data confirm the robustness of the YAP1-transcription signature as a biomarker for both, tumor indication selection and pharmacodynamics of YAP1-TEAD activity. Citation Format: Emmanuel Spanakis, Loreley Calvet, Odette Dos Santos, Colette Dib, Sukhvinder Sidhu, Jurgen Moll, Laurent Debussche, Jack Pollard, Iris Valtingojer. A transcriptomic signature for measuring YAP1 activity in patient samples and tumor models [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 2161.

Summary Misoriented division of neuroprogenitors, by loss-of-function studies of centrosome or spindle components, has been linked to the developmental brain defects microcephaly and lissencephaly. As these approaches also affect centrosome biogenesis, spindle assembly, or cell-cycle progression, the resulting pathologies cannot be attributed solely to spindle misorientation. To address this issue, we employed a truncation of the spindle-orienting protein RHAMM. This truncation of the RHAMM centrosome-targeting domain does not have an impact on centrosome biogenesis or on spindle assembly in vivo. The RHAMM mutants exhibit misorientation of the division plane of neuroprogenitors, without affecting the division rate of these cells, resulting against expectation in megalencephaly associated with cerebral cortex thickening, cerebellum enlargement, and premature cerebellum differentiation. We conclude that RHAMM associates with the spindle of neuroprogenitor cells via its centrosome-targeting domain, where it regulates differentiation in the developing brain by orienting the spindle., Graphical Abstract, Highlights • Cerebral cortex apical progenitors (APs) and cerebellar GCPs exhibit planar division • Spindle-associated RHAMM orients the division plane of these neuroprogenitors • Excessive apicobasal division of APs causes cerebral cortex thickening • Decreased planar division of GCPs causes cerebellum enlargement, Ploubidou and colleagues assess the impact of misorientation of the mitotic spindle of neuronal progenitor cells on brain development. They employ a spindle misorientation model expressing truncated RHAMM, exhibiting no centrosome biogenesis or spindle assembly defects in vivo. The data demonstrate that spindle misorientation in cerebellum and cerebrum neuroprogenitors results in megalencephaly and that RHAMM regulates differentiation in the developing brain.

Hypofertility is a risk factor for the development of testicular germ cell tumors (TGCT), but the initiating event linking these pathologies is unknown. We hypothesized that excessive planar division of undifferentiated germ cells promotes their self-renewal and TGCT development. However, our results obtained from mouse models and seminoma patients demonstrated the opposite. Defective planar divisions of undifferentiated germ cells caused their premature exit from the seminiferous tubule niche, resulting in germ cell depletion, hypofertility, intratubular germ cell neoplasias, and seminoma development. Oriented divisions of germ cells, which determine their fate, were regulated by spindle-associated RHAMM—a function we found to be abolished in 96% of human seminomas. Mechanistically, RHAMM expression is regulated by the testis-specific polyadenylation protein CFIm25, which is downregulated in the human seminomas. These results suggested that spindle misorientation is oncogenic, not by promoting self-renewing germ cell divisions within the niche, but by prematurely displacing proliferating cells from their normal epithelial milieu. Furthermore, they suggested RHAMM loss-of-function and spindle misorientation as an initiating event underlying both hypofertility and TGCT initiation. These findings identify spindle-associated RHAMM as an intrinsic regulator of male germ cell fate and as a gatekeeper preventing initiation of TGCTs. Cancer Res; 76(21); 6382–95. ©2016 AACR.

Breast cancer (BC) comprises multiple distinct subtypes that differ genetically, pathologically, and clinically. Here, we describe a robust protocol for long-term culturing of human mammary epithelial organoids. Using this protocol, >100 primary and metastatic BC organoid lines were generated, broadly recapitulating the diversity of the disease. BC organoid morphologies typically matched the histopathology, hormone receptor status, and HER2 status of the original tumor. DNA copy number variations as well as sequence changes were consistent within tumor-organoid pairs and largely retained even after extended passaging. BC organoids furthermore populated all major gene-expression-based classification groups and allowed in vitro drug screens that were consistent with in vivo xeno-transplantations and patient response. This study describes a representative collection of well-characterized BC organoids available for cancer research and drug development, as well as a strategy to assess in vitro drug response in a personalized fashion. The heterogeneity of breast cancer subtypes can be captured using organoid cultures that can facilitate drug screens that corroborate with patient responses.

Clinical studies of pharmacologic agents targeting the insulin-like growth factor (IGF) pathway in unselected cancer patients have so far demonstrated modest efficacy outcomes, with objective responses being rare. As such, the identification of selection biomarkers for enrichment of potential responders represents a high priority for future trials of these agents. Several reports have described high IGF2 expression in a subset of colorectal cancers, with focal IGF2 amplification being responsible for some of these cases. We defined a novel cut-off value for IGF2 overexpression based on differential expression between colorectal tumors and normal tissue samples. Analysis of two independent colorectal cancer datasets revealed IGF2 to be overexpressed at a frequency of 13% to 22%. An in vitro screen of 34 colorectal cancer cell lines revealed IGF2 expression to significantly correlate with sensitivity to the IGF1R/INSR inhibitor BI 885578. Furthermore, autocrine IGF2 constitutively activated IGF1R and Akt phosphorylation, which was inhibited by BI 885578 treatment. BI 885578 significantly delayed the growth of IGF2-high colorectal cancer xenograft tumors in mice, while combination with a VEGF-A antibody increased efficacy and induced tumor regression. Besides colorectal cancer, IGF2 overexpression was detected in more than 10% of bladder carcinoma, hepatocellular carcinoma and non-small cell lung cancer patient samples. Meanwhile, IGF2-high non-colorectal cancer cells lines displayed constitutive IGF1R phosphorylation and were sensitive to BI 885578. Our findings suggest that IGF2 may represent an attractive patient selection biomarker for IGF pathway inhibitors and that combination with VEGF-targeting agents may further improve clinical outcomes. Mol Cancer Ther; 16(10); 2223–33. ©2017 AACR.

MDM2-p53 antagonists block the interaction between the Tumor Protein p53 and MDM2, its key negative regulator, and represent a new therapeutic concept for cancer therapy. MDM2-p53 antagonists are designed to restore p53 activity in TP53 wild-type tumors. Several MDM2-p53 antagonists are currently being evaluated in early clinical development. BI-907828 is a novel and potent MDM2-p53 antagonist with optimized drug-like properties that has shown efficacy in human tumor xenograft models at daily low oral dose as well as intermittent high dose schedules. Recent preclinical studies in syngeneic mouse models of cancer have demonstrated that BI-907828, apart from its direct tumor-targeting activity, also has immunomodulatory activity shown to contribute to efficacy. Single-agent BI-907828 induced anti-tumor immunological memory that controlled tumor growth in a re-challenge study. Moreover, a dual combination with an anti-mouse PD-1 checkpoint inhibitor resulted in synergistic efficacy in a syngeneic mouse model of cancer (AACR 2018, abstract 4866). Here we present data for the triple combination of BI-907828 with two checkpoint inhibitors. In syngeneic mouse tumor models (Colon-26 and B16-F10), the combination of BI-907828 with tool antibodies against mouse PD-1 and mouse LAG-3 shows high response rates of 50-90% with tumor regressions observed for even very large tumors. Moreover, efficacy of the triple combination is superior to each single agent and all dual combinations. An antibody-mediated depletion study suggests a contribution of CD8+ T cells but not of CD4+ T cells to full efficacy of the triple combination. FACS analysis of tumors isolated from Colon-26 tumor-bearing mice indicates that treatment with the triple combination leads to expansion of tumor-infiltrating CD8+ T cells. In summary, BI-907828 is a novel, potent, orally bioavailable MDM2-p53 antagonist that shows synergistic efficacy in a triple combination with antibodies targeting the immune checkpoints PD-1 and LAG-3 in syngeneic mouse models of cancer. BI-907828 is currently under evaluation in a Phase I clinical study (NCT03449381). Citation Format: Dorothea Rudolph, Ulrike Weyer-Czernilofsky, Markus Reschke, Martina Sykora, Jörg Rinnenthal, Sophia Blake, Gabriela Gremel, Andreas Wernitznig, Andreas Gollner, Norbert Kraut, Jürgen Moll. BI-907828, a novel and potent MDM2-p53 antagonist, acts synergistically in a triple combination with anti-PD-1 and anti-LAG-3 antibodies in syngeneic mouse models of cancer [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 3197.

We have previously reported that over-expression of a panel of 119 genes correlates with the metastatic potential of pancreatic carcinoma cells. We sought to identify and functionally characterize candidate tumour metastasis promoting genes among this library using a secondary phenotype-assisted screen. Here we report the discovery of the metastasis-promoting function of a hitherto not characterized gene located on chromosome 14 (ORF138), which we have named 'novel metastasis-promoting gene 1' (NVM-1). The NVM-1 transcript is extensively alternatively spliced, is expressed endogenously in a number of different tissues, and is strongly over-expressed at the protein level in a variety of human tumour types. Importantly, NVM-1 expression stimulates the migratory and invasive behaviour of tumour cells and promotes metastasis formation in experimental animals in vivo. Up-regulation of FMNL2 and MT1E and down-regulation of TIMP4 and MHC-I is observed as a consequence of NVM-1 expression. Together these data identify NVM-1 as a gene that is functionally involved in tumour metastasis, and suggest that NVM-1 may constitute a promising therapeutic target for inhibition of tumour metastasis.

Analysis of cell cycle progression by 5-bromo-2′-deoxyuridine (BrdU) incorporation is commonly used for evaluating the mode of action of anticancer drugs, but usually requires a high number of cells and large amounts of monoclonal antibodies. In addition, manual sample handling is not suitable for high throughput. To circumvent these limitations, we have developed a miniaturized method to measure BrdU incorporation into DNA directly in 96-wells plates. Adherent cells were grown in 96-well plates in the absence or presence of compounds of interest. After BrdU pulse labeling or pulse chase, cells were harvested, transferred to polymerase chain reaction (PCR) V-bottom plates, and fixed by adding methanol. DNA denaturation was performed directly in the plates by heat using a PCR thermocycler. BrdU incorporation was detected by indirect immunocytochemical staining, and cellular DNA was counterstained using propidium iodide. Samples were acquired by a BD FACSCalibur with BD Multiwells Auto sampler or BD HTS. We defined a dynamic range of the optimal cell number, for which cells maintained exponential growth up to 72 h. The assay was robust up to 30,000 cells per well. BrdU dot plots of cell cycle phases showed an excellent separation of cell populations, and DNA histograms showed a low coefficient of variation. Thermal denaturation was suitable for 96-well plates to detect BrdU incorporation with a good signal-to-noise ratio, and cluster analysis allowed fingerprint readouts for drug sensitivity and mechanism of action as exemplified for paclitaxel and doxorubicin. This method provided rapid high-throughput BrdU/DNA content analysis with high accuracy and reproducibility, accompanied by a reduction in reagent consumption. A critical step was identified as the standardization of DNA denaturation using a PCR thermocycler. Here,we show some applications of this method for cell cycle studies in drug discovery. © 2010 International Society for Advancement of Cytometry

MDM2 inhibitors block the interaction between the Tumor Protein p53 (TP53) and MDM2, its key negative regulator, and represent a new therapeutic concept in cancer therapy. MDM2 inhibitors are designed to restore p53 activity in TP53 wild-type tumors. Several MDM2 inhibitors are currently being evaluated in early clinical development. While the tumor targeting activity of MDM2 inhibitors has been well documented, recent preclinical data with the tool compound Nutlin-3 have shown that p53 activation by local, intra-tumoral injections induces anti-tumor immunity (Cancer Res 2017, 77(9) 2292-2305). Here we present data on BI 907828, a novel and potent MDM2 inhibitor with optimized drug-like properties that is suitable for intermittent dose schedules in syngeneic mouse models of cancer. Our data show that immune modulation contributes to efficacy of BI 907828 after oral administration in a Colon-26 syngeneic mouse model. Responding mice develop anti-tumor immunity that can control the tumor growth of a secondary tumor without any further treatment in a re-challenge study. A T cell depletion study shows a contribution of CD8+ T cells but not of CD4+ T cells to single-agent efficacy with BI 907828. Moreover, preclinical data in a Colon-26 syngeneic mouse model of cancer show synergistic efficacy for the combination of a PD-1 checkpoint inhibitor with BI 907828. In summary, BI 907828 is a novel, potent, orally bioavailable MDM2 inhibitor that shows synergistic efficacy in combination with a PD-1 checkpoint inhibitor in syngeneic mouse models of cancer. Citation Format: Dorothea Rudolph, Markus Reschke, Sophia Blake, Jörg Rinnenthal, Andreas Wernitznig, Ulrike Weyer-Czernilofsky, Andreas Gollner, Christian Haslinger, Pilar Garin-Chesa, Jens Quant, Darryl B. McConnell, Kraut Norbert, Jürgen Moll. BI 907828: A novel, potent MDM2 inhibitor that induces antitumor immunologic memory and acts synergistically with an anti-PD-1 antibody in syngeneic mouse models of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4866.

The programmed cell death-1 (PD-1) receptor provides inhibitory checkpoint signals to activated T cells upon binding to its ligands, PD-L1 and PD-L2, which are expressed on antigen-presenting cells and cancer cells leading to suppression of T-cell effector function and tumor immune evasion. Blockade of the PD-1 axis using either anti-PD-1 or anti-PD-L1 approved monoclonal antibodies (mAbs) results in improved T-cell effector function and anti-tumor immune responses. Durable tumor responses occur in 15-30% of cancer patients. BI 754091, a humanized IgG4 mAb with high affinity against hPD-1 blocks the interaction between PD-1 and PD-L1 or PD-L2. BI 754091 was characterized in a panel of binding, blocking and functional cell-based assays. In addition, efficacy and safety was assessed in mice and in cynomolgus monkeys, respectively. The ability of BI 754091 to stimulate cytokine production in exhausted human T cells in vitro was tested in an autologous assay system with antigen-specific memory CD4+ T cells being re-stimulated by antigen-pulsed dendritic cells in the presence of BI 754091 or isotype control. Under these assay conditions the majority of T cells co-expressed the exhaustion markers PD-1 and LAG-3 on their surface. Furthermore, PD-L1 and PD-L2 were expressed on the dendritic cells. At the end of the experiment supernatants were harvested and analyzed for IFNγ secretion as a measure for T-cell activation. BI 754091 showed a potent dose dependent T-cell activation. The average fold increase of IFNγ was 7.9 as compared to isotype control, with an average EC50 of 0.9 nM. The in vivo activity of BI 754091 was determined in MC-38 tumor-bearing mice, using a mouse strain where parts of the extracellular domain of murine PD-1 was replaced by the corresponding human PD-1 domain (C57BL/6NTac-PDCD1tm(PDCD1)Arte mice). A dose of 10 mg/kg BI 754091, given either as single treatment or in a twice weekly schedule, induced significant tumor growth inhibition (median TGI of 83% and 90%, respectively) and complete responses (CRs) in some tumors (3 CRs out of 10 and 2 CRs out of 10, respectively). BI 754091 binds to PD-1 from cynomolgus monkeys with comparable affinities as to human PD-1, thus allowing pharmacokinetic and toxicological assessment in this species. Repeated high doses of BI 754091 were well tolerated without adverse immune-related effects. BI 754091 is currently undergoing clinical investigations (NCT02952248). Citation Format: Markus Zettl, Melanie Wurm, Otmar Schaaf, Iñigo Tirapu, Sven Mostböck, Markus Reschke, Stephan-Michael Schmidbauer, Lee Frego, Ivo C. Lorenz, Michael Thibodeau, Diann Blanset, Elisa Oquendo Cifuentes, Jürgen Moll, Norbert Kraut, Eric Borges, Anne Vogt, Jonathon Sedgwick, Irene C. Waizenegger. In vitro and in vivo characterization of the PD-1 targeting antibody BI 754091 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4558.

MDM2 inhibitors block the interaction between the Tumor Protein p53 (TP53) and MDM2, its key negative regulator, and represent a new therapeutic concept for cancer therapy. MDM2 inhibitors are designed to restore p53 activity in TP53 wild-type tumors. Several MDM2 inhibitors are currently being evaluated in early clinical development with mainly daily dosing regimens. However, recent clinical data suggest myelosuppression as an on-target, dose-limiting toxicity for this class of inhibitors. Particularly, thrombocytopenia could limit the clinical utility of MDM2 inhibitors. Hence there is a need to mitigate these side effects and to improve the therapeutic window. One approach is less frequent dosing to allow bone marrow recovery while still maintaining efficacious exposure levels and clinical activity. Here we present data on BI 907828, a novel and potent MDM2 inhibitor with optimized drug-like properties including a reliable, dose-linear PK across species with good bioavailability after oral dosing that allows various dose schedules. Remarkably, a single oral dose of 2 mg/kg led to tumor regressions in a SJSA-1 xenograft model in all treated mice, as did treatment on a daily low dose schedule. Data on PK, PD and efficacy relationships in this xenograft model will be presented including dose-dependent induction of TP53 target genes and markers of apoptosis. Moreover, BI 907828 significantly prolonged the survival by more than 50 days with a daily oral dose of 2.5 mg/kg in a difficult to treat disseminated MOLM-13 AML model, as noted by profiling of a clinical-stage MDM2 inhibitor. In summary, BI 907828 is a novel, potent, orally bioavailable MDM2 inhibitor that shows excellent efficacy with daily low dose but also intermittent high dose schedules in preclinical models of cancer. Citation Format: Dorothea Rudolph, Andreas Gollner, Sophia Blake, Jörg Rinnenthal, Andreas Wernitznig, Ulrike Weyer-Czernilofsky, Christian Haslinger, Pilar Garin-Chesa, Jens Quant, Darryl B. McConnell, Jürgen Moll, Kraut Norbert. BI 907828: A novel, potent MDM2 inhibitor that is suitable for high-dose intermittent schedules [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4868.

MDM2 inhibitors block the interaction between the Tumor Protein p53 (TP53) and MDM2, its key negative regulator, and represent a new therapeutic concept for cancer therapy. MDM2 inhibitors are designed to restore p53 activity in TP53 wild-type tumors. Several MDM2 inhibitors are currently being evaluated in early clinical development with mainly daily dosing regimens. However, recent clinical data suggest myelosuppression as an on-target, dose-limiting toxicity for this class of inhibitors. Particularly thrombocytopenia could limit the clinical utility of MDM2 inhibitors. Hence there is a need to mitigate these side effects and to improve the therapeutic window. One approach is less frequent dosing to allow bone marrow recovery while still maintaining efficacious exposure levels and clinical activity. Here we present pharmacokinetic data on BI 907828, a novel and potent MDM2 inhibitor with optimized drug-like properties. Due to its high permeability, good physiological solubility and low systemic clearance, the compound shows a high bioavailability in mice, rats, dogs and minipigs. BI 907828 is not absorption-limited, even at multiples of the efficacious dose, and shows a reliable, dose-linear PK with low variability in AUC and Cmax across species. Based on a good PK-PD correlation with efficacy in preclinical models and a robust human PK prediction based on in vivo PK data across species, a low human efficacious dose is predicted. The PK properties of BI 907828 enable a variety of different clinical dosing schedules with the potential to improve the therapeutic index with respect to on-target safety. In summary, BI 907828 is a novel, potent, orally available MDM2 inhibitor with excellent PK properties and low estimated human dose suitable for high-dose intermittent dose schedules in the clinic. Citation Format: Joerg Rinnenthal, Dorothea Rudolph, Sophia Blake, Andreas Gollner, Andreas Wernitznig, Ulrike Weyer-Czernilofsky, Christian Haslinger, Pilar Garin-Chesa, Jürgen Moll, Norbert Kraut, Darryl McConnell, Jens Quant. BI 907828: A highly potent MDM2 inhibitor with low human dose estimation, designed for high-dose intermittent schedules in the clinic [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4865.

Lymphocyte activation gene-3 (LAG-3) is an inhibitory receptor involved in maintaining immunological tolerance via regulation of T-cell activation, proliferation and response. Ligand-mediated activation of LAG-3 negatively regulates T-cell activity that is thought to actively contribute to tumor immune evasion beyond the established programmed cell death-1 (PD-1) pathway. BI 754111, a humanized IgG4 monoclonal antibody (mAb) with high affinity against LAG-3, blocks the interaction between LAG-3 and MHC II. BI 754111 was characterized in a panel of binding, blocking and functional cell-based assays; safety assessment was done in cynomolgus monkeys. BI 754111 is not mouse cross-reactive; therefore a surrogate mLAG-3 antibody was used for in vivo mouse efficacy studies. The ability of BI 754111 to stimulate cytokine production by exhausted human T cells in vitro was tested in an autologous assay system with antigen specific memory T cells being re-stimulated by antigen-pulsed dendritic cells in the presence of increasing amounts of BI 754111 or BI 754091 (anti-hPD-1 mAb) or a combination of increasing amounts of BI 754111 and a saturating dose of BI 754091. Under these assay conditions the majority of T cells co-expressed the exhaustion markers PD-1 and LAG-3 on the surface. At the end of the experiment supernatants were harvested and analyzed for IFNγ secretion as a measure of T-cell activation. Monotherapy of BI 754111 moderately increased IFNγ secretion (average of 1.8 fold) compared to BI 754091 monotherapy (6.9-fold average increase). Combining BI 754111 with BI 754091 was synergistic and led to a 13.2-fold increase in IFNγ secretion. MC-38 tumor-bearing mice (C57BL/6NTac-PDCD1tm(PDCD1)Arte mice expressing parts of the human instead of the murine extracellular domain of PD-1) were used to determine the in vivo activity of the anti-LAG3 and anti-PD-1 combination. A mouse tool antibody against mLAG-3 (IgG1 D265A) and BI 905725 (a mouse IgG1 D265A version of BI 754091, anti-hPD-1 mAb) were tested at a dose of 10 mg/kg in a q3or4d schedule as monotherapy or in combination. No anti-tumor activity was observed under mLAG-3 mAb treatment, whereas treatment with anti-PD-1 resulted in a median TGI of 100% and 4 out of 10 tumors showed a complete response. The combination of anti-PD-1 and anti-LAG-3 resulted in a median TGI of 103% and doubled the number of complete responses (8/10). BI 754111 binds to cynomolgus monkey LAG-3 with comparable affinity as to hLAG-3 thus allowing pharmacokinetic and toxicological assessment in this species. Repeated high doses of BI 754111 were well tolerated without adverse immune-related side effects. The above results describe synergistic effects upon combination of PD-1 and LAG-3 treatment thus supporting the ongoing clinical trial in which BI 754111 is being tested in combination with BI 754091 (NCT03156114). Citation Format: Markus Zettl, Melanie Wurm, Otmar Schaaf, Iñigo Tirapu, Sven Mostböck, Markus Reschke, Stephan-Michael Schmidbauer, Lee Frego, Ivo C. Lorenz, Michael Thibodeau, Diann Blanset, Elisa Oquendo Cifuentes, Jürgen Moll, Norbert Kraut, Eric Borges, Anne Vogt, Jonathon Sedgwick, Irene C. Waizenegger. Characterization of the LAG-3 targeting antibody BI 754111 in monotherapy and in combination with the anti-PD-1 antibody BI 754091 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4547.

Patients with advanced stages of hepatocellular carcinoma (HCC) face a poor prognosis. Although encouraging clinical results have been obtained with multikinase inhibitor sorafenib, the development of improved therapeutic strategies for HCC remains an urgent goal. Aurora kinases are key regulators of the cell cycle, and their uncontrolled expression promotes aneuploidy and tumor development. In tissue microarray analyses, we detected aurora-A kinase expression in all of the examined 93 human HCC samples, whereas aurora-B kinase expression levels significantly correlated with the proliferation index of HCCs. In addition, two human HCC cell lines (Huh-7 and HepG2) were tested positive for aurora-A and -B and revealed Ser10 phosphorylation of histone H3, indicating an increased aurora-B kinase activity. The antiproliferative features of a novel aurora kinase inhibitor, PHA-739358, currently under investigation in phase 2 clinical trials for other solid tumors, were examined in vitro and in vivo. At concentrations exceeding 50nM, PHA-739358 completely suppressed tumor cell proliferation in cell culture experiments and strongly decreased histone H3 phosphorylation. Cell cycle inhibition and endoreduplication were observed at 50 nM, whereas higher concentrations led to a complete G2/M-phase arrest. In vivo, administration of PHA-739358 resulted in significant tumor growth inhibition at a well-tolerated dose. In combination with sorafenib, additive effects were observed. Remarkably, when tumors restarted to grow under sorafenib monotherapy, subsequent treatment with PHA-739358 induced tumor shrinkage by up to 81%. Thus, targeting aurora kinases with PHA-739358 is a promising therapeutic strategy administered alone or in combination with sorafenib for patients with advanced stages of HCC.

The emergence of resistance to imatinib (IM) mediated by mutations in the BCR-ABL domain has become a major challenge in the treatment of chronic myeloid leukemia (CML). Here, we report on studies performed with a novel small molecule inhibitor, PHA-739358, which selectively targets Bcr-Abl and Aurora kinases A to C. PHA-739358 exhibits strong antiproliferative and proapoptotic activity against a broad panel of human BCR-ABL–positive and –negative cell lines and against murine BaF3 cells ectopically expressing wild-type (wt) or IM-resistant BCR-ABL mutants, including T315I. Pharmacologic synergism of IM and PHA-739358 was observed in leukemia cell lines with subtotal resistance to IM. Treatment with PHA-739358 significantly decreased phosphorylation of histone H3, a marker of Aurora B activity and of CrkL, a downstream target of Bcr-Abl, suggesting that PHA-739358 acts via combined inhibition of Bcr-Abl and Aurora kinases. Moreover, strong antiproliferative effects of PHA-739358 were observed in CD34+ cells derived from untreated CML patients and from IM-resistant individuals in chronic phase or blast crisis, including those harboring the T315I mutation. Thus, PHA-739358 represents a promising new strategy for treatment of IM-resistant BCR-ABL-positive leukemias, including those harboring the T315I mutation. Clinical trials investigating this compound in IM-resistant CML have recently been initiated.

The strong link between gene expression of mitotic Aurora kinases and cancer has stimulated a very high interest in developing Aurora kinase inhibitors for cancer therapy. Validation of Aurora kinases as targets, and development of pharmacodynamic biomarkers for inhibitors of Aurora kinases, provides an example of how target validation can help the drug discovery process, and also of how to interpret results depending on the technology used. In this review, we outline the principal tools, concepts, and strategies of target and biomarker validation for Aurora kinases, with emphasis on validation results derived from RNA-interference experiments. These data were essential for the decision to enter the next steps in drug development and for the selection of the appropriate biomarkers for clinical trials.

Mutations in the kinase domain of Bcr-Abl are the most common cause of resistance to therapy with imatinib in patients with chronic myelogenous leukemia (CML). Second-generation Bcr-Abl inhibitors are able to overcome most imatinib-resistant mutants, with the exception of the frequent T315I substitution, which is emerging as a major cause of resistance to these drugs in CML patients. Structural studies could be used to support the drug design process for the development of inhibitors able to target the T315I substitution, but until now no crystal structure of the T315I Abl mutant has been solved. We show here the first crystal structure of the kinase domain of Abl T315I in complex with PHA-739358, an Aurora kinase inhibitor currently in clinical development for solid and hematologic malignancies. This compound inhibits in vitro the kinase activity of wild-type Abl and of several mutants, including T315I. The cocrystal structure of T315I Abl kinase domain provides the structural basis for this activity: the inhibitor associates with an active conformation of the kinase domain in the ATP-binding pocket and lacks the steric hindrance imposed by the substitution of threonine by isoleucine. [Cancer Res 2007;67(17):7987–90]

The postnatal mammalian ovary contains the primary follicles, each comprising an immature oocyte surrounded by a layer of somatic granulosa cells. Oocytes reach meiotic and developmental competence via folliculogenesis. During this process, the granulosa cells proliferate massively around the oocyte, form an extensive extracellular matrix (ECM) and differentiate into cumulus cells. As the ECM component hyaluronic acid (HA) is thought to form the backbone of the oocyte-granulosa cell complex, we deleted the relevant domain of the Receptor for HA Mediated Motility (RHAMM) gene in the mouse. This resulted in folliculogenesis defects and female hypofertility, although HA-induced signalling was not affected. We report that wild-type RHAMM localises at the mitotic spindle of granulosa cells, surrounding the oocyte. Deletion of the RHAMM C-terminus in vivo abolishes its spindle association, resulting in impaired spindle orientation in the dividing granulosa cells, folliculogenesis defects and subsequent female hypofertility. These data reveal the first identified physiological function for RHAMM, during oogenesis, and the importance of this spindle-associated function for female fertility.

Polo-like kinase 1 (Plk1), a member of the Polo-like kinase family of serine/threonine kinases, is a key regulator of multiple steps in mitosis. Here we report on the pharmacological profile of volasertib, a potent and selective Plk inhibitor, in multiple preclinical models of acute myeloid leukemia (AML) including established cell lines, bone marrow samples from AML patients in short-term culture, and subcutaneous as well as disseminated in vivo models in immune-deficient mice. Our results indicate that volasertib is highly efficacious as a single agent and in combination with established and emerging AML drugs, including the antimetabolite cytarabine, hypomethylating agents (decitabine, azacitidine), and quizartinib, a signal transduction inhibitor targeting FLT3. Collectively, these preclinical data support the use of volasertib as a new therapeutic approach for the treatment of AML patients, and provide a foundation for combination approaches that may further improve and prolong clinical responses.

Aurora protein kinases are key players in different steps during mitosis and their activity is required for correct cell division. The inhibition of Aurora kinases is regarded as a promising approach for the development of novel anticancer agents. In the last few years several templates for ATP-competitive kinase inhibition have been successfully developed towards the inhibition of Aurora kinases, and the first compounds have recently entered clinical studies. This review summarises the patent literature up until March 2005 and the characteristics of the low molecular weight Aurora inhibitors described so far.

Cell motility is an essential element of tumor dissemination, allowing organ infiltration by cancer cells. Using mouse LB lymphoma cells transfected with standard CD44 (CD44s) cDNA (LB-TRs cells) or with the alternatively spliced CD44 variant CD44v4-v10 (CD44v) cDNA (LB-TRv cells), we explored their CD44-dependent cell migration. LB-TRv cells, but not LB-TRs or parental LB cells, bound soluble hyaluronic acid (HA) and other glycosaminoglycans (GAGs), and exclusively formed, under physiological shear force, rolling attachments on HA substrate. Furthermore, LB-TRv cells, but not LB-TRs cells or their parental LB cells, displayed accelerated local tumor formation and enhanced accumulation in the peripheral lymph nodes after s.c. inoculation. The aggressive metastatic behavior of i.v.-injected LB-TRV cells, when compared with that of other LB-transfectants, is attributed to more efficient migration to the lymph nodes, rather than to local growth in the lymph node. Injection of anti-CD44 monoclonal antibody or of the enzyme hyaluronidase also prevented tumor growth in lymph nodes of BALB/c mice inoculated with LB-TRv cells. The enhanced in vitro rolling and enhanced in vivo local tumor growth and lymph node invasion disappeared in LB cells transfected with CD44v cDNA bearing a point mutation at the HA binding site, located at the distal end of the molecule constant region. These findings show that the interaction of cell surface CD44v with HA promotes cell migration both in vitro and in vivo, and they contribute to our understanding of the mechanism of cell trafficking, including tumor spread.

The CD44v6 epitope has been widely reported to be expressed in human mammary carcinomas, yet its prognostic significance is controversial and its function in mammary tumors and mammary glands is unknown. To begin to resolve these issues, we analysed in detail the normal postnatal expression patterns and regulation of the CD44v6 epitope in murine mammary glands. We demonstrate that significant CD44v6 epitope expression is first seen during puberty, and that after puberty CD44v6 epitope expression follows the estrous cycle. CD44v6 epitope expression is observed in the myoepithelium and also less widely in luminal epithelial cells. During lactation, CD44v6 epitope expression is turned off and reappears during involution. The CD44 variant isoform bearing the v6 epitope is CD44v1-v10. Using HC11, a mammary epithelial cell line with stem cell characteristics, and facilitated by the cloning of the murine CD44 promoter, we show that growth factors and hormones which regulate ductal growth and differentiation modulate CD44 transcription. Together our data suggest that the CD44v6 epitope is expressed in mammary epithelial stems cells and in lineages derived from these cells, and that CD44v6 expression is regulated in part by hormones and growth factors such as IGF-1 and EGF which regulate the growth and differentiation of the mammary epithelium. The function of these same growth factors and hormones is often perturbed in mammary carcinomas, and we suggest that CD44v6 expression in tumors reflects this perturbation. We conclude that the expression of the CD44v6 epitope observed in some mammary tumors reflects the stem cell origin of breast tumors, and that whether or not the CD44v6 epitope is expressed in a mammary tumor is determined by the differentiation status of the tumor cells.

The glycosaminoglycan hyaluronate (HA) is part of the extracellular environment in bone marrow. We show here that HA activates signal transduction cascades important for hemopoiesis. In myeloid and lymphoid long-term bone marrow cultures (LTBMC), treatment with hyaluronidase (HA’ase) results in reduced production of both progenitor and mature cells. Exogeneous HA added to LTBMC had the opposite effect: it enhanced hematopoiesis. The effect of HA is mediated through two different HA receptors on bone marrow macrophage-like cells, one of which is CD44 while the other is unknown. HA induces bone marrow macrophages to secrete IL-1β (CD44-dependent) and IL-6 (CD44-independent). The two receptors address different signal transduction pathways: CD44 links to a pathway activating p38 protein kinase while the other yet unknown receptor induces Erk activity. There was no difference of the effect of HA and HA’ase on hematopoiesis in LTBMC and on cytokine production by macrophages in CD44-deficient mice compared with wild-type mice, indicating that the CD44 hyaluronate receptor and its signal transduction can be compensated for. Our data suggest a regulatory role for the extracellular matrix component HA in hematopoiesis and show the induction of signal transduction by HA receptors.

During inflammation, activated monocytes (Mo) migrate into tissues where they interact with extracellular matrix components such as hyaluronate (HA), produced in high amounts at inflammatory sites. We determined whether Mo that had invaded sites of cutaneous inflammation bind HA and express the putative HA receptors CD44 isoforms, ICAM-1, or receptor for hyaluronate-mediated motility (RHAMM). In cutaneous inflammation, activated infiltrating Mo displayed high HA avidity and expressed epitopes encoded by CD44s, CD44 variant exons v3, v4, v5, v6, v7, and v9, and ICAM-1, but not RHAMM. We further investigated how activation affects the avidity of Mo for HA and which receptors were responsible for such binding. Mo freshly purified from human peripheral blood bound little HA and expressed CD44s but no epitopes encoded by CD44v exons, ICAM-1, or RHAMM. During short-term tissue culture, Mo upregulated their HA avidity and expression of ICAM-1, CD44s, and epitopes encoded by CD44v, all of which were further augmented by IFN-γ or lipopolysaccharide, whereas RHAMM was not detectable. Thus in vitro activated Mo resembled Mo that had migrated to inflammatory sites in vivo . Lipolysaccharide or IFN-γ-induced HA binding was inhibited by more than 90% with monoclonal antibodies directed against N- terminal HA binding domains of CD44s, but not by monoclonal antibodies against CD44v epitopes or ICAM-1. In conclusion, we show that upon in vitro or in vivo activation, Mo enhance their capacity to bind HA. This is critically dependent upon the expression of CD44s epitopes. Regulated CD44–HA interactions may be important for the ability of Mo to migrate into and within sites of inflammation and for Mo effector functions.

Constitutive expression of a rat CD44 variant isoform, rCD44v4-v7, on murine T cells accelerates immune responsiveness. Because prolonged immunodeficiency can be a major drawback in allogeneic bone marrow transplantation, we considered it of special interest to see whether repopulation of lethally irradiated syngeneic and allogeneic mice may be influenced by constitutive expression of the rCD44v4-v7 transgene. When lethally irradiated syngeneic and allogeneic mice were reconstituted with bone marrow cells (BMC) from rCD44v4-v7 transgenic (TG) or nontransgenic (NTG) mice, the former had a clear repopulation advantage: thymocytes expanded earlier after reconstitution and, as a consequence, higher numbers of lymphocytes were recovered from spleen and lymph nodes. Lymphocytes also displayed functional activity in advance to those from mice reconstituted with BMC from NTG mice. Most importantly, after the transfer of BMC from TG mice into an allogeneic host, the frequency of host-reactive T cells decreased rapidly. Apparently, this was due to accelerated induction of tolerance. Because these effects were counterregulated by an rCD44v6-specific antibody, it is likely that they could be attributed to the rCD44v4-v7 TG product. Thus, expression of a CD44 variant isoform at high levels facilitated reconstitution with allogeneic BMC by accelerated establishment of tolerance and the regaining of immunocompetence.

Two key features of myeloma cells are the deregulation of the cell cycle and the dependency on the expression of the BCL2 family of anti-apoptotic proteins. The cell division cycle 7 (CDC7) is an essential S-phase kinase and emerging CDC7 inhibitors are effective in a variety of preclinical cancer models. These compounds also inhibit CDK9 which is relevant for MCL-1 expression. The activity and mechanism of action of the dual CDC7/CDK9 inhibitor PHA-767491 was assessed in a panel of multiple myeloma cell lines, in primary samples from patients, in the presence of stromal cells and in combination with drugs used in current chemotherapeutic regimens. We report that in all conditions myeloma cells undergo cell death upon PHA-767491 treatment and we report an overall additive effect with melphalan, bortezomib and doxorubicin, thus supporting further assessment of targeting CDC7 and CDK9 in multiple myeloma.

Expression of CD44 and of specific splice-variants of CD44 has been causally related to metastatic behaviour in a variety of carcinomas and lymphomas. To elucidate whether, in principle, similar splice-variants could be involved in glioma cell invasion we examined the expression of CD44 and its splice-variants in a series of 38 primary human brain tumors (28 astrocytomas, WHO grade I-III and 10 glioblastomas, WHO grade IV) and in cell lines derived from 9 glioblastomas. All brain tumors examined showed strong immunoreactivity for an N-terminal epitope present on all CD44 isoforms known. Using a polyclonal antiserum raised against the complete sequence encoded by variant exons v3 to v10, CD44 splice-variants could be detected irrespective of the grade of malingnancy in many of the tumor samples at a low level and often restricted to only a few clustered tumor cells. Thus, the N-terminal epitope probably indicates the presence of the smallest and most ubiquitous isoform CD44s. Interestingly, all glioblastomas expressed CD44 variants whereas expression in astrocytomas WHO grade I, II, and III could only be detected in about half of the tumor samples. Using reverse transcriptase-PCR we were able to detect different CD44 splice-variants in the glioblastoma cell lines and in cultured primary astrocytic cells. Glioblastoma cells analyzed by flow cytometry showed the expected binding capacity for hyaluronic acid which could be increased twofold after pretreatment with hyaluronidase. The results presented show that there is low expression of CD44 variants in human tumors of astrocytic origin. Expression of CD44 and its splice-variants could contribute to the migration capacity of neoplastic astrocytes, and may be considered as a target for new diagnostic and therapeutic approaches in the clinical management of brain tumors.

Members of the CD44 family of cell surface hyaluronate-binding proteins have been implicated in cell migration, cell-matrix interactions and tumor progression. To determine whether these proteins might play a role in the normal functions of Schwann cells and in their tumorigenesis, we examined the patterns of CD44 expression in Schwann cells from rat peripheral nerve, rat Schwann cell tumor lines, and human schwannomas. Normal rat spinal nerves and primary Schwann cell cultures expressed standard CD44 (CD44s) but not alternatively spliced variant isoforms. In contrast, rat Schwann cell tumor lines expressed both CD44s and a number of variants, including proteins containing sequences encoded by exon v6. Furthermore, we found that these cell lines bind hyaluronate, and that their cell surface hyaluronate binding correlates with CD44 expression. All of the human schwannomas also expressed CD44 variants, especially epitopes encoded by exon v5, the border between v7 and v8, and v9-10. These data indicate that Schwann cells normally express CD44s, that Schwann cell tumors express both CD44s and particular variants of CD44, and that CD44s and possibly variants of CD44 are involved in hyaluronate recognition by Schwann cell tumors.

Biomarkers that indicate biological activity and/or efficacy are a potentially useful tool in the development of molecularly targeted therapeutics. Most pharmacodynamic assays measure the response to drugs in tissues, a procedure requiring tissue biopsies, which is often not available. In this study, we identify GDF15 as a potential plasma biomarker for Danusertib (formerly PHA-739358), an Aurora kinase inhibitor, currently in phase I/II clinical studies. Cell lysates and cell-free culture supernatants from human tumor isogenic cell lines were assessed for GDF15 levels following treatment with Danusertib, by using Western Blotting and Elisa. GDF15 levels were also measured in plasma from xenografted mice as well as in plasma from patients before and after Danusertib treatment. At the protein level an increased level of GDF15 was found in tissue culture cells after Danusertib treatment and GDF15 expression was also detected in plasma of xenografted mice treated with the compound. Hence, both cellular and xenograft models of human cancer showed a clear correlation with p53 pathway activation. Danusertib treatment also resulted in a significantly increased expression of GDF15 protein levels in the plasma of examined patients, underlining technical feasibility of translating this putative biomarker to clinical studies. Determination of GDF15 levels in plasma is feasible and enabled us to follow the kinetic of Danusertib activity in plasma from cancer xenografted mice and from patients. These findings should allow us to evaluate systematically the kinetics of p53 activation by Danusertib in future clinical studies.

Here, we report unbiased screens for genes expressed in metastatic tumor cells that are associated with cell motility. These screens identified Ier2, an immediate early gene of unknown function, as potentially having a role in tumor cell motility and metastasis. Knockdown of Ier2 in 3T3 fibroblasts inhibited their motility upon relief of contact inhibition in monolayer wounding assays. Furthermore, ectopic Ier2 expression promoted the motility and invasiveness of poorly metastatic 1AS pancreatic tumor cells in vitro. Relief of contact inhibition was associated with translocation of the Ier2 protein from the cytoplasm to the nucleus in both 3T3 fibroblasts and 1AS tumor cells. Importantly, ectopic Ier2 expression in 1AS cells stimulated metastasis formation when cells were implanted into experimental animals. Furthermore, we found elevated Ier2 expression in a wide variety of human tumor types. This correlated with poor metastasis-free and overall survival in patients with colorectal adenocarcinomas. Together, these data reveal Ier2 as a new player in the regulation of tumor progression and metastasis, and suggest that Ier2 may be useful prognostically and therapeutically in the management of cancer.

Most cancers are characterized by some degree of aneuploidy, although its relevance for tumor initiation or progression and the nature of the initial trigger are still not well understood. It was Theodor Boveri who first suggested a link between aneuploidy and cancer at the beginning of the last century, but it is only recently that the molecular mechanisms involved have started to be uncovered.The molecular mechanisms that are at the origin of aneuploidy and their cellular consequences. Based on these new findings molecular targets have emerged which could lead to a specific treatment of at least some types of aneuploid tumors.Therapeutic intervention specifically for aneuploid cells is a very promising approach, however, although new promising targets have been spotted they still need to be tested for proof of concept. Targeting the spindle checkpoint could be an interesting approach for cancer therapy, however, as for other mitotic targets, the open question of the therapeutic window and sensitivity of normal hemopoietic cells has to be considered carefully. Future challenges will not only include identifying and validating druggable targets related to the relevant pathways, but also finding predictive biomarkers to define the responding patient population(s).

PLoS one 6(4), e19164 (2011). doi:10.1371/journal.pone.0019164, Published by PLoS, Lawrence, Kan.

Aneuploidy is one of the major hallmarks of cancer cells and several paths towards aneuploidy have been described. However, the relevance for tumor initiation or progression and how tumors deal with the initial aneuploidy related stress response is still unclear and recent results suggest that aneuploidy can even have tumor suppressive effects under certain conditions. The molecular mechanisms leading to and sustaining growth of aneuploid cells are just at the beginning to be understood and might provide new targets for cancer drug development. We will discuss some of the ideas to specifically kill aneuploid cells by targeting key regulators of mitosis.

To dissect mechanisms that co-ordinate specific events in thymopoiesis we have characterized alterations in thymic structure and function caused by expression of a transgene. This gene encodes SV40Tag and is specifically expressed in a subset of thymic epithelial (TE) cells around birth. As a result the number of immortal TE cells increases, thymic mass increases (up to 3 g), and thymopoiesis is expanded. The latter is reflected by a approximately 100-fold increase of the major thymocyte subsets and increased peripheral T cell counts. Grossly hyperplastic thymi retain many but not all morphological features of a normal thymus. Also in grafts, SV40Tag+ TE cells steer expansion (up to 8 g) and organize a tissue with mainly cortex-like features that includes mainly SV40Tag+ TE cells, thymocytes, and macrophages. To investigate expression of specialized gene functions in the immortal TE cells, a cell line was derived. The Epi-A1 cell line expresses the genes for major histocompatibility complex class I and II, Thy-1, interleukin (IL)-6, IL-7, macrophage-colony-stimulating factor, and transforming growth factor-beta 3. Most importantly, Epi-A1 cells also express the IL-4 receptor and the c-kit ligand (KL), a factor that, in concert with commitment factors, channels progenitors into hemopoietic lineages. The expression of low constitutive levels of KL mRNA does not require IL-4, but KL mRNA levels are increased dramatically in response to IL-4. Since constitutive expression of KL mRNA in vivo is restricted to a small subset of TE cells in the thymus, our findings reveal a novel specific interaction between thymocytes and a specialized subset of TE cells.

Purpose: This study was conducted to assess the safety, tolerability, pharmacokinetics, and pharmacodynamics of the i.v. pan-aurora kinase inhibitor PHA-739358, danusertib, in patients with advanced solid tumors. Experimental Design: In part 1, patients received escalating doses of danusertib (24-hour infusion every 14 days) without filgrastim (granulocyte colony-stimulating factor, G-CSF). Febrile neutropenia was the dose-limiting toxicity without G-CSF. Further dose escalation was done in part 2 with G-CSF. Blood samples were collected for danusertib pharmacokinetics and pharmacodynamics. Skin biopsies were collected to assess histone H3 phosphorylation (pH3). Results: Fifty-six patients were treated, 40 in part 1 and 16 in part 2. Febrile neutropenia was the dose-limiting toxicity in part 1 without G-CSF. Most other adverse events were grade 1 to 2, occurring at doses ≥360 mg/m2 with similar incidence in parts 1 and 2. The maximum tolerated dose without G-CSF is 500 mg/m2. The recommended phase 2 dose in part 2 with G-CSF is 750 mg/m2. Danusertib showed dose-proportional pharmacokinetics in parts 1 and 2 with a median half-life of 18 to 26 hours. pH3 modulation in skin biopsies was observed at ≥500 mg/m2. One patient with refractory small cell lung cancer (1,000 mg/m2 with G-CSF) had an objective response lasting 23 weeks. One patient with refractory ovarian cancer had 27% tumor regression and 30% CA125 decline. Conclusions: Danusertib was well tolerated with target inhibition in skin at ≥500 mg/m2. Preliminary evidence of antitumor activity, including a partial response and several occurrences of prolonged stable disease, was seen across a variety of advanced refractory cancers. Phase II studies are ongoing. (Clin Cancer Res 2009;15(21):6694–701)

Determination of incorporation of the thymidine analog 5-bromo-2'-deoxyuridine (BrdU) into DNA is a widely used method to analyze the cell cycle (see UNIT 7.7). However, DNA denaturation is required for BrdU detection with the consequence that most protein epitopes are destroyed and their immunocytochemical detection for multiplex analysis is not possible. A novel assay is presented for identifying cells in active S-phase that does not require the DNA denaturation step but nevertheless detects BrdU. For this purpose, cells were pulsed for a short time by an alkenyl deoxyuridine (5-ethynyl-2'-deoxyuridine, EdU), which is incorporated into DNA. The nucleotide exposed ethynyl residue was then derivatized by a copper-catalyzed cycloaddition reaction ("click chemistry" coupling) using a BrdU azide probe. The resulting DNA-bound bromouracil moieties were then detected by commercial anti-BrdU monoclonal antibodies without the need for a denaturation step. This method has been tested using several cell lines and is preferred over traditional BrdU detection since it is more sensitive and allows multicolor and multiplex analysis in FCM and imaging.

Aurora kinases are key regulators of mitosis and inhibitors being developed by a wide range of pharmaceutical and biotechnology companies for the treatment of cancer. Tumor cells respond differentially on inhibition of different Aurora kinase family members and these differences have to be considered in the clinical development of small-molecule inhibitors with respect to the chosen indications, the schedules or the selection of appropriate end points and they should also guide the development of biomarkers. Preclinical validation of potential biomarkers for Aurora kinase inhibitors led to a first application in clinical trials, as exemplified for the phosphorylation of histone H3 to follow Aurora-B inhibition. This review discusses the criteria for translation into the clinic and the value of pharmacodynamic biomarkers and their potential, but also their limitations to be used as surrogate markers for clinical end points.

The large family of CD44 splice variants are likely to serve multiple functions in the embryo and in the adult organism. This is reflected in their complex patterns of expression. In molecular terms these functions are largely unknown. Certain splice variants (CD44v) can promote the metastatic behaviour of cancer cells. In human colon and breast cancer the presence of epitopes encoded by exon v6 on primary resected tumour material indicates poor prognosis. Metastasis-promoting splice variants differ from those that seem not to have a role in the induction of metastasis by the formation of homomultimeric complexes in the plasma membrane of cells. This may increase their affinity to ligands such as hyaluronate. The affinity can be further regulated over a range from low to very high by cell-specific modification. The fact that CD44v epitopes are found on normal epithelial cells such as skin, cervical epithelium and bladder enforces cautious evaluation of the significance of CD44v expression in human cancer. Nevertheless, certain epitopes can serve as tools in early diagnosis of certain cancers and will facilitate the development of specific targeted therapy.

Background: Polo-like kinase 1 (Plk1), a key regulator of cell cycle progression and accurate spindle assembly, is an attractive target for cancer drug discovery. We have previously shown that volasertib (BI 6727), a potent and selective small-molecule inhibitor of Plk, induces a distinct mitotic arrest phenotype in prometaphase (“polo-arrest”) with subsequent apoptosis in a variety of different cancer cell lines, irrespective of their mutational status. When used in vivo, volasertib administered intravenously shows potent anti-tumor activity in xenograft models of human epithelial cancers at well-tolerated doses. The present study was designed to extend the analysis of volasertib to additional preclinical models of human AML, including bone marrow samples from AML patients. Volasertib is the most advanced Plk inhibitor in clinical development and has demonstrated encouraging results in phase II clinical trials. It is currently being investigated in a phase III clinical trial in patients with previously untreated AML, who are ineligible for intensive remission induction therapy. Methods: A panel of human AML cell lines was used to evaluate pharmacodynamic biomarker modulation and anti-tumor effects of volasertib in vitro using FACS analysis, Western blot analysis and proliferation assays. This in vitro analysis of established AML cell lines was extended to proliferation assays using bone marrow samples from AML patients. In vivo anti-tumor activity of volasertib was tested in subcutaneous xenograft models as well as in multiple disseminated xenograft models of AML. Single-agent efficacy of volasertib and combination therapies were evaluated with existing and emerging AML drugs, including an approved cytotoxic drug (cytarabine), hypomethylating agents (decitabine, azacitidine) and a signal transduction inhibitor targeting FLT3 (quizartinib). Results: Volasertib potently inhibited proliferation of established AML cell lines in vitro with EC50 values of 16-169 nM. Proliferation assays with 15 ex vivo bone marrow samples from AML patients showed EC50 values of 8-8800 nM with a median EC50 of 37 nM. Volasertib showed potent anti-tumor activity at well tolerated doses in 3 subcutaneous xenograft models of AML (MV4-11, Molm-13 and a patient-derived AML model AML-6252). While single-agent volasertib at medium dose level (20 mg/kg q7d i.v. for 2 cycles) and single-agent cytarabine (100 mg/kg q3-4d i.p. for 2 cycles) showed moderate efficacy in the AML-6252 AML model, the combination showed improved efficacy. Moreover, efficacy of single-agent volasertib at high dose level (40 mg/kg q7d i.v. for 2 cycles) could be further improved by adding cytarabine to the treatment regime (Figure 1). A combination of volasertib with decitabine or azacitidine was tested in the MV4-11 subcutaneous AML xenograft model. Either combination therapy showed improved efficacy compared to the respective single-agent treatment groups. Volasertib showed also improved anti-tumor activity when tested in combination with the Flt-3 inhibitor quizartinib (5 or 10 mg/kg qd po for 2 cycles) in the MV4-11 AML model. While tumors in the quizartinib single agent treatment groups started to regrow around day 60 post treatment start, a combination with volasertib could control tumor growth long term until the study was terminated (day 87 post treatment start). Efficacy of volasertib was also tested in 3 disseminated xenograft models of AML (MV4-11, Molm-13 and THP-1). Efficacy read out in these disseminated models was based on tumor load measurements as detected by bioluminescence imaging and increased lifespan. Volasertib prolonged survival compared to vehicle treated animals in all three disseminated models of AML. Conclusions: These results indicate that volasertib is highly efficacious as a single agent in preclinical models of AML and shows potential for improved efficacy and good tolerability in combination with existing and emerging AML drugs. Figure 1: Efficacy of volasertib in combination with cytarabine in a patient-derived AML model (AML-6252) Figure 1:. Efficacy of volasertib in combination with cytarabine in a patient-derived AML model (AML-6252) Disclosures Rudolph: Boehringer Ingelheim RCV: Employment. Off Label Use: Volasertib is an investigational agent. Albrecht:Boehringer Ingelheim RCV GmbH & Co KG: Employment. Geiselmann:Boehringer Ingelheim RCV GmbH & Co KG: Employment. Impagnatiello:Boehringer Ingelheim RCV GmbH & Co KG: Employment. Garin-Chesa:Boehringer Ingelheim RCV: Employment. Wernitznig:Boehringer-Ingelheim: Employment. Moll:Boehringer-Ingelheim: Employment. Kraut:Boehringer Ingelheim RCV: Employment.

During the past decade, considerable advances have been made in our under­standing of the cellular and molecular biology of carcinogenesis. The analysis of the metastatic process, however, which is the chief contributor to cancer patient morbidity, has met with various obstacles due to its complexity. As a result, we still know very little about how cancer cells spread. It is generally assumed that cancer cells randomly diversify during tumor progression and that clones of such cells with metastatic properties are selected. The process by which these cells then metastasize is believed to occur via several steps, each of which can be linked to the aquisition of new cellular functions (see, e.g., reviews by Hart et al. 1989; Kerbel 1990). For example, a stationary epithelial cell must first be con­verted into a mobile, nonadherent cancer cell which can invade subepithelial tissue. The cell must then move towards lymphatic and blood vessels, enter these vessels, and survive during their transport within the fluid medium. It must also colonize draining lymphatic tissue and further disseminate through efferent lymphatics into the blood stream, extravasate from blood vessels and, finally, preferentially settle in various distant locations.

Antiprogestins of the 11β-aryl-substituted 19-norsteroid family are effectively used in inhibiting nidation and in terminating pregnancies. They are potentially useful in the treatment of progesterone-related diseases such as meningiomas and endometriosis and in inhibiting the growth of mammary tumors. However their long-term use is limited because of their inherent antiglucocorticoid activity. Here we have used molecular biological techniques to examine the antiglucocorticoid activity of a series of antiprogestins. The compounds we have analyzed contain different substitutents at the C-17 position and a change from the trans to cis configuration of the C-D steroid rings. Our results show that minor changes at the C-17 position but not in the configuration of the C and D rings produced antiprogestins with reduced antiglucocorticoid activity. Thus only subtle changes in the structure of classical antiprogestins are needed for the reduction of their antiglucocorticoid activities.

Poly (ADP-ribose) polymerase 1 and 2 (Parp-1 and Parp-2) are nuclear enzymes responsible for signaling the presence of DNA damages by catalyzing the addition of ADP-ribose units to DNA, histones and various DNA repair enzymes, thus facilitating DNA repair. Parp-1 has been gaining increasing interest as a therapeutic target for cancer in combination with DNA damaging agents but also as single agent in particular tumor settings, such as BRCA mutated tumors. While Parp-1 knock out mice are viable and display only defects in DNA repair, double Parp-1 and Parp-2 knock-out are embryonic lethal indicating that the absence of Parp-1 and Parp-2 is less tolerated in normal cells. We confirmed this concept in vitro by RNAi of Parp-1 and Parp-2, showing that the inhibition of Parp-1 alone is enough to kill tumor cells but spares normal cells. Hence a selective Parp-1 inhibitor might have a better safety profile, particularly in view of a long treatment. Here we report the discovery and the in vitro and in vivo characterization of a novel class of potent, orally available Parp-1 selective small molecule inhibitors with no activity on other Parp isoforms such as Parp-2, −3 and −5a (tankyrase), which differentiates it from current clinical Parp inhibitors. The most potent compounds were further evaluated in vitro and in vivo. These compounds are highly potent in inhibiting DNA damage-induced Parp-1 activity in cells and exhibit selective anti-proliferative and pro-apoptotic activity in tumor cell lines harbouring defects in DNA repair. In vivo, the compounds show an excellent pharmacokinetic profile with almost complete oral bioavailability and demonstrate efficacy as a single agent in xenograft tumor models with DNA repair deficiencies superior to reference Parp inhibitors. These compounds are very well tolerated with no overt toxicity even after prolonged exposure and their mechanism of action is confirmed both in tumors and peripheral blood cells of treated mice. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 691.