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Aberrant activation in fibroblast growth factor signaling has been implicated in the development of various cancers, including squamous cell lung cancer, squamous cell head and neck carcinoma, colorectal and bladder cancer. Thus, fibroblast growth factor receptors (FGFRs) present promising targets for novel cancer therapeutics. Here, we evaluated the activity of a novel pan‐FGFR inhibitor, rogaratinib, in biochemical, cellular and in vivo efficacy studies in a variety of preclinical cancer models. In vitro kinase activity assays demonstrate that rogaratinib potently and selectively inhibits the activity of FGFRs 1, 2, 3 and 4. In line with this, rogaratinib reduced proliferation in FGFR‐addicted cancer cell lines of various cancer types including lung, breast, colon and bladder cancer. FGFR and ERK phosphorylation interruption by rogaratinib treatment in several FGFR‐amplified cell lines suggests that the anti‐proliferative effects are mediated by FGFR/ERK pathway inhibition. Furthermore, rogaratinib exhibited strong in vivo efficacy in several cell line‐ and patient‐derived xenograft models characterized by FGFR overexpression. The observed efficacy of rogaratinib strongly correlated with FGFR mRNA expression levels. These promising results warrant further development of rogaratinib and clinical trials are currently ongoing (ClinicalTrials.gov Identifiers: NCT01976741, NCT03410693, NCT03473756)., What's new? Deregulated fibroblast growth factor receptor (FGFR) signaling is involved in tumorigenesis and cancer progression. Here, the authors report on a novel pan‐FGFR inhibitor, rogaratinib, that potently and highly selectively prevents the activity of FGFRs 1, 2, 3, and 4. Rogaratinib inhibits cell proliferation in various FGFR‐addicted cancers in vitro, including colon, lung, and bladder cancer. Rogaratinib also exhibits strong in vivo efficacy in several cell line‐ and patient‐derived xenograft models characterized by FGFR mRNA overexpression with good tolerability. Altogether, these data warrant the further development of rogaratinib for treatment of cancers with FGFR alterations, and clinical trials are currently ongoing.

Despite the recent advances in the treatment of ovarian cancer, it remains an area of high unmet medical need. Epithelial ovarian cancer is associated with high levels of mesothelin expression, and therefore, mesothelin is an attractive candidate target for the treatment of this disease. Herein, we investigated the antitumor efficacy of the mesothelin-targeting antibody-drug conjugate (ADC) anetumab ravtansine as a novel treatment option for ovarian cancer in monotherapy and in combination with the antitumor agents pegylated liposomal doxorubicin (PLD), carboplatin, copanlisib and bevacizumab. Anetumab ravtansine showed potent antitumor activity as a monotherapy in ovarian cancer models with high mesothelin expression. No activity was seen in mesothelin-negative models. The combination of anetumab ravtansine with PLD showed additive anti-proliferative activity in vitro, which translated into improved therapeutic in vivo efficacy in ovarian cancer cell line- and patient-derived xenograft (PDX) models compared to either agents as a monotherapy. The combination of anetumab ravtansine with the PI3Kα/δ inhibitor copanlisib was additive in the OVCAR-3 and OVCAR-8 cell lines in vitro, showing increased apoptosis in response to the combination treatment. In vivo, the combination of anetumab ravtansine with copanlisib resulted in more potent antitumor activity than either of the treatments alone. Likewise, the combination of anetumab ravtansine with carboplatin or bevacizumab showed improved in vivo efficacy in the ST081 and OVCAR-3 models, respectively. All combinations were well-tolerated. Taken together, these data support the development of anetumab ravtansine for ovarian cancer treatment and highlight its suitability for combination therapy with PLD, carboplatin, copanlisib, or bevacizumab.

Purpose: Radium-223 dichloride (radium-223, Xofigo), a targeted alpha therapy, is currently used for the treatment of patients with castration-resistant prostate cancer (CRPC) with bone metastases. This study examines the mode-of-action and antitumor efficacy of radium-223 in two prostate cancer xenograft models. Experimental Design: Mice bearing intratibial LNCaP or LuCaP 58 tumors were randomized into groups (n = 12–17) based on lesion grade and/or serum PSA level and administered radium-223 (300 kBq/kg) or vehicle, twice at 4-week intervals. X-rays and serum samples were obtained biweekly. Soft tissue tumors were observed macroscopically at sacrifice. Tibiae were analyzed by gamma counter, micro-CT, autoradiography and histology. Results: Radium-223 inhibited tumor-induced osteoblastic bone growth and protected normal bone architecture, leading to reduced bone volume in LNCaP and abiraterone-resistant LuCaP 58 models. Furthermore, radium-223 resulted in lower PSA values and reduced total tissue and tumor areas, indicating that treatment constrains prostate cancer growth in bone. In addition, radium-223 suppressed abnormal bone metabolic activity as evidenced by decreased number of osteoblasts and osteoclasts and reduced level of the bone formation marker PINP. Mode-of-action studies revealed that radium-223 was deposited in the intratumoral bone matrix. DNA double-strand breaks were induced in cancer cells within 24 hours after radium-223 treatment, and PSA levels were significantly lower 72 hours after treatment, providing further evidence of the antitumor effects. Conclusions: Taken together, radium-223 therapy exhibits a dual targeting mode-of-action that induces tumor cell death and suppresses tumor-induced pathologic bone formation in tumor microenvironment of osseous CRPC growth in mice. Clin Cancer Res; 23(15); 4335–46. ©2017 AACR.

The DNA damage response (DDR) secures the integrity of the genome of eukaryotic cells. DDR deficiencies can promote tumorigenesis but concurrently may increase dependence on alternative repair pathways. The ataxia telangiectasia and Rad3-related (ATR) kinase plays a central role in the DDR by activating essential signaling pathways of DNA damage repair. Here, we studied the effect of the novel selective ATR kinase inhibitor BAY 1895344 on tumor cell growth and viability. Potent antiproliferative activity was demonstrated in a broad spectrum of human tumor cell lines. BAY 1895344 exhibited strong monotherapy efficacy in cancer xenograft models that carry DNA damage repair deficiencies. The combination of BAY 1895344 with DNA damage–inducing chemotherapy or external beam radiotherapy (EBRT) showed synergistic antitumor activity. Combination treatment with BAY 1895344 and DDR inhibitors achieved strong synergistic antiproliferative activity in vitro, and combined inhibition of ATR and PARP signaling using olaparib demonstrated synergistic antitumor activity in vivo. Furthermore, the combination of BAY 1895344 with the novel, nonsteroidal androgen receptor antagonist darolutamide resulted in significantly improved antitumor efficacy compared with respective single-agent treatments in hormone-dependent prostate cancer, and addition of EBRT resulted in even further enhanced antitumor efficacy. Thus, the ATR inhibitor BAY 1895344 may provide new therapeutic options for the treatment of cancers with certain DDR deficiencies in monotherapy and in combination with DNA damage–inducing or DNA repair–compromising cancer therapies by improving their efficacy.

Purpose: Targeted thorium-227 conjugates (TTC) represent a new class of molecules for targeted alpha therapy (TAT). Covalent attachment of a 3,2-HOPO chelator to an antibody enables specific complexation and delivery of the alpha particle emitter thorium-227 to tumor cells. Because of the high energy and short penetration range, TAT efficiently induces double-strand DNA breaks (DSB) preferentially in the tumor cell with limited damage to the surrounding tissue. We present herein the preclinical evaluation of a mesothelin (MSLN)-targeted thorium-227 conjugate, BAY 2287411. MSLN is a GPI-anchored membrane glycoprotein overexpressed in mesothelioma, ovarian, pancreatic, lung, and breast cancers with limited expression in healthy tissue. Experimental Design: The binding activity and radiostability of BAY 2287411 were confirmed bioanalytically. The mode-of-action and antitumor potency of BAY 2287411 were investigated in vitro and in vivo in cell line and patient-derived xenograft models of breast, colorectal, lung, ovarian, and pancreatic cancer. Results: BAY 2287411 induced DSBs, apoptotic markers, and oxidative stress, leading to reduced cellular viability. Furthermore, upregulation of immunogenic cell death markers was observed. BAY 2287411 was well-tolerated and demonstrated significant antitumor efficacy when administered via single or multiple dosing regimens in vivo. In addition, significant survival benefit was observed in a disseminated lung cancer model. Biodistribution studies showed specific uptake and retention of BAY 2287411 in tumors and enabled the development of a mechanistic pharmacokinetic/pharmacodynamic model to describe the preclinical data. Conclusions: These promising preclinical results supported the transition of BAY 2287411 into a clinical phase I program in mesothelioma and ovarian cancer patients (NCT03507452).

Mesothelin (MSLN)-targeted thorium conjugate (MSLN-TTC; BAY 2287411) is the first targeted alpha therapy in clinical development for the treatment of patients suffering from MSLN-positive mesothelioma and ovarian cancer (NCT03507452). It consists of the MSLN-targeting antibody anetumab, covalently attached to a 3,2-HOPO chelator complexing the alpha-emitter thorium-227. The main mode of action of MSLN-TTC is the induction of clustered DNA double-strand breaks upon alpha decay of thorium-227, resulting in cell death. The preclinical efficacy of MSLN-TTC has been demonstrated previously. The tumor control achieved by external beam radiation is partly driven by immune-stimulatory effects (Vatner et al; Frontiers in Oncology 2014). The essential mechanisms can be attributed to different pathways, including (a) induction of immunogenic cell death and (b) activation of the STING-pathway resulting in the secretion of pro-inflammatory cytokines (Vanpouille-Box C et al; NatComm 2017). We therefore investigated whether the targeted alpha therapy MSLN-TTC is also able to induce both of these pathways in vitro. The human ovarian cancer MSLN-expressing cell line OVCAR-3 was exposed to MSLN-TTC resulting in a dose dependent upregulation of markers of immunogenic cell death, e.g. calreticulin and HMGB-1. Further, exposure of OVCAR-3 cells to MSLN-TTC resulted in the secretion of pro-inflammatory cytokines, including IFN-β, IL-6 and IP-10. The efficacy of MSLN-TTC was evaluated in a syngeneic subcutaneous tumor model. As MSLN-TTC is not cross-reactive to murine MSLN, an MC38 cell line stably transfected with human MSLN was established (MC38-hMSLN). In vitro, MSLN-TTC induced specific reduction of MC38-hMSLN cell viability. Following single dose administration to MC38-hMSLN tumor-bearing mice, MSLN-TTC induced dose dependent antitumor activity with complete tumor eradication in 10 out of 36 treated animals. Interestingly, when tumor-free animals were re-inoculated with MC38-hMSLN cells 121 days post treatment, no tumor growth was observed. In contrast, tumors grew in animals inoculated with B16F10 cells, suggesting the development of an immune memory response against MC38-hMSLN cells. In summary, the data presented demonstrate that MSLN-TTC is able to induce immunogenic cell death and secretion of pro-inflammatory cytokines in vitro. Further, MSLN-TTC monotherapy evokes an immune-stimulatory effect in vivo. Citation Format: Urs B. Hagemann, Pascale LeJeune, Jenny Karlsson, Christoph A. Schatz, Alan S. Cuthbertson, Hartwig Hennekes, Karl Ziegelbauer, Dominik Mumberg. MSLN-TTC (BAY 2287411) induces immunogenic cell death and secretion of pro-inflammatory cytokines in vitro and triggers an immune memory effect against a mouse tumor model [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 3926.

Mesothelin (MSLN)-targeted thorium conjugate (MSLN-TTC; BAY 2287411) is a targeted alpha therapy consisting of the MSLN-targeting antibody anetumab, covalently attached to a 3,2-HOPO chelator complexing the alpha-emitter thorium-227. We previously demonstrated the potent in vivo activity of MSLN-TTC in cell line and patient-derived xenograft models, and further, demonstrated its combination potential with DNA damage repair inhibitors. MSLN-TTC is currently in clinical development for the treatment of patients with MSLN-positive mesothelioma and ovarian cancer (NCT03507452). Development of cellular resistance mechanisms are often co-evolutionary to the treatment regimen given to the respective individual. As such, it is well described that ovarian cancer patients have a decreased overall survival when tested positive for markers of acquired drug resistance (ADR), e.g. phospho-glycoprotein (P-gp) (Herzog TJ et al; Oncotarget 2016). However, MSLN-TTC should also be effective independent of P-gp expression, as the major mode of action of MSLN-TTC is the induction of irreparable DNA double-strand breaks upon alpha decay by thorium-227, resulting in apoptotic cell death. To confirm this hypothesis, we first determined the involvement of P-gp in cellular resistance mechanisms in ovarian cancer. To this end, immunohistochemical staining of ovarian cancer patient samples for P-gp was performed. Expression of P-gp was observed in 15% (7/48) primary and relapsing specimens. We then evaluated the potency of MSLN-TTC in MSLN-expressing cell line models in vitro using the isogenic cell line pair OVCAR-8 (P-gp negative) and NCI-RES-ADR (P-gp positive) in comparison to the standard of care (SOC) therapies cisplatin, paclitaxel, doxorubicin and vinorelbine. Whereas paclitaxel and doxorubicin showed a 100- and 10-fold decrease in in vitro potency on the P-gp positive cell line NCI-RES-ADR vs the P-gp negative cell line OVCAR-8, the potency of MSLN-TTC on the two isogenic cell lines remained unaffected. To further confirm and explore these observations, in vivo xenograft models were conducted. Similar to the in vitro results, the in vivo efficacy of MSLN-TTC was not affected by the P-gp status in the OVCAR-8 vs NCI-RES-ADR xenograft model. In contrast, limited efficacy of paclitaxel and doxorubicin was observed in the P-gp-positive MSLN-expressing NCI-RES-ADR and Hela-MATU-ADR models. Similar, vinorelbine showed no efficacy in the Hela-MATU-ADR xenograft. In summary, these data demonstrate that the efficacy of MSLN-TTC is independent of the P-gp status. Further, these data suggest that MSLN-TTC may be active in patients relapsing after standard therapy. Citation Format: Urs B. Hagemann, Sabine Zitzmann-Kolbe, Alexander Kristian, Carolyn Sperl, Christoph A. Schatz, Roger M. Bjerke, Alan S. Cuthbertson, Hartwig Hennekes, Karl Ziegelbauer, Dominik Mumberg. MSLN-TTC (BAY 2287411) demonstrates increased activity in comparison to standard of care chemotherapy in models of acquired drug resistance [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 3937.

Radium-223 dichloride (Ra-223, Xofigo®) is a targeted alpha therapy which prolongs the survival of castration-resistant prostate cancer (CRPC) patients with symptomatic bone metastases. As a calcium-mimetic, Ra-223 selectively binds to hydroxyapatite and targets areas of high bone turnover such as bone metastases. Here, we report the effects of Ra-223 on development and progression of osteolytic bone lesions and on survival in the syngeneic intratibial MBT-2 murine bladder cancer model in immunocompetent mice. Female 5-7-week-old C3H/HeNHsd mice were injected intratibially with 5x105 MBT-2 cells on day 0. The mice were stratified based on body weight (n=40 per group) on day 5 and Ra-223 (300 kBq/kg, i.v.) or vehicle control was administered on days 5, 33, and 61. The tumor-induced osteolytic lesion area was imaged using radiography on days 10, 18, 25, 76, and at sacrifice. The mice were sacrificed when they met the pre-defined sacrifice criteria (> 20% body weight loss, palpable tumor outgrowth from the bone or general deterioration of health status). The study was terminated on day 158. In this study, the first mice met the sacrifice criteria on study day 24 and the survival curves plateaued at 52% after 84 days in the control group and at 68% after 112 days in the Ra-223 treatment group. Survival on day 40 was significantly improved in the Ra-223 treatment group compared to the vehicle control (p< 0.001), but the effect was not statistically significant at the end of the study. Based on X-ray imaging 25 days after cancer cell inoculation, Ra-223 treatment also significantly decreased the bone lesion area compared to the vehicle treatment (p=0.0041). After initially sacrificing the mice with extensive bone lesions, tumor growth in bone was stabilized around day 25. The main reason for sacrifice at a later stage was breathing difficulties due to lung metastases, and in the Ra-223-treated mice only one mouse was sacrificed due to bone metastases. Ra-223 treatment was well-tolerated; no body weight loss was observed. In summary, radium-223 dichloride (Xofigo®) demonstrates moderate single agent in vivo efficacy in the intratibial MBT-2 murine bladder cancer model of osteolytic bone metastasis. Retaining a functional immune system in syngeneic mouse models is particularly relevant for the characterization of potential immune-stimulatory effects of Ra-223. The promising results obtained in this model warrant further investigation of Ra-223 in combination with immuno-oncological treatments like checkpoint inhibitors. Citation Format: Tiina E. Kähkönen, Mari I. Suominen, Jenni H. Mäki-Jouppila, Birgitta Sjöholm, Ilmari Ahonen, Dominik Mumberg, Karl Ziegelbauer, Jussi M. Halleen, Sanna-Maria Käkönen, Arne Scholz. Efficacy of single agent radium-223 in the syngeneic MBT-2 bladder cancer bone growth model in mice [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 3936.

C4.4A (LYPD3) has been identified as a cancer- and metastasis-associated internalizing cell surface protein that is expressed in non–small cell lung cancer (NSCLC), with particularly high prevalence in the squamous cell carcinoma (SCC) subtype. With the exception of skin keratinocytes and esophageal endothelial cells, C4.4A expression is scarce in normal tissues, presenting an opportunity to selectively treat cancers with a C4.4A-directed antibody–drug conjugate (ADC). We have generated BAY 1129980 (C4.4A-ADC), an ADC consisting of a fully human C4.4A-targeting mAb conjugated to a novel, highly potent derivative of the microtubule-disrupting cytotoxic drug auristatin via a noncleavable alkyl hydrazide linker. In vitro, C4.4A-ADC demonstrated potent antiproliferative efficacy in cell lines endogenously expressing C4.4A and inhibited proliferation of C4.4A-transfected A549 lung cancer cells showing selectivity compared with a nontargeted control ADC. In vivo, C4.4A-ADC was efficacious in human NSCLC cell line (NCI-H292 and NCI-H322) and patient-derived xenograft (PDX) models (Lu7064, Lu7126, Lu7433, and Lu7466). C4.4A expression level correlated with in vivo efficacy, the most responsive being the models with C4.4A expression in over 50% of the cells. In the NCI-H292 NSCLC model, C4.4A-ADC demonstrated equal or superior efficacy compared to cisplatin, paclitaxel, and vinorelbine. Furthermore, an additive antitumor efficacy in combination with cisplatin was observed. Finally, a repeated dosing with C4.4A-ADC was well tolerated without changing the sensitivity to the treatment. Taken together, C4.4A-ADC is a promising therapeutic candidate for the treatment of NSCLC and other cancers expressing C4.4A. A phase I study (NCT02134197) with the C4.4A-ADC BAY 1129980 is currently ongoing. Mol Cancer Ther; 16(5); 893–904. ©2017 AACR.

As a result of the increasing cost pressure on healthcare systems, the depletion of easily addressable and well-validated target groups in drug development and the requirement of public research to contribute to innovative treatment paradigms, broad partnerships between industry and academia are becoming increasingly important. However, owing to different goals and drivers, hurdles have to be overcome to exploit the full potential of such alliances. The factors that need to be taken into account during set-up and management of such alliances and the result and impact all of this has on drug discovery have not been analyzed in a systematic manner until now. This will be the focus of this review, using the strategic alliance between the German Cancer Research Center and Bayer HealthCare as an example.

Despite remarkable results in treating certain cancers, immune checkpoint inhibitors (ICIs) are lack of activity in ‘cold' tumors and not always effective in inflamed ‘hot' tumors. In addition to aberrant activation in cancer cells, the PI3K pathway plays both positive and negative roles in immune response. Therefore, the overall outcome of PI3K inhibition on anti-tumor immunity and the combination strategy with ICIs should be carefully investigated. In this study, PI3K inhibition alone and in combination with anti-PD-1/L1 were evaluated in a set of 8 representative syngeneic tumor models non-responding or insensitive to ICIs. We first compared an oral pan-PI3K inhibitor BAY 1082439 dosed continuously (QD) versus intermittently (2On/5Off), and performed a comprehensive evaluation of an approved intravenously dosed pan-PI3K inhibitor copanlisib (2On/5Off). We found that intermittent treatment, regardless oral or iv, produced better anti-tumor efficacy, increased intratumoral CD8+/Treg ratio and a better safety profile, while continuous inhibition of PI3K led to a significant reduction in splenic B cells and lymphoid organ weight. In addition, intermittent treatment of PI3K inhibitor with subsequent combination of anti-PD-1 induced complete tumor regression in 50-100% of animals bearing TreghighA20 tumor compared to 0% response in the monotherapy groups. Synergistic combination was also demonstrated in M2-TAMhigh CT26 and MC38 CRC models. Of note, no tumor growth was observed in a re-challenge study conducted 3 months post complete tumor regression in the combination group of the CT26 model, indicating that tumor specific memory T cells were generated which prevented tumor recurrence. Analysis of tumor infiltrating leukocytes revealed significant reductions in Treg and/or M2-TAM but increases in CD8+ T cell, Granzyme B+ cell, M1-TAM and activated DC. The findings in mice were also confirmed in human PBMC derived Treg (induced by TGF-β) and M2 (induced by M-CSF/IL-4) differentiation assays. The pan-PI3K inhibitor copanlisib, but not a PI3Kδ selective inhibitor, effectively blocked TGF-β-induced Treg (CD25+/FoxP3+) differentiation, despite both inhibitors suppressing CD3-stimulated Treg proliferation. Furthermore, copanlisib not only blocked M2 differentiation, but also could redirect differentiated M2 to immune stimulating M1 even in the presence of M-CSF/IL-4. Taken together, pulsatile pan-PI3K inhibition could effectively convert an immune suppressive effect observed with continuous treatment to a favorable anti-tumor immune response. Combination of intermittently dosed PI3K inhibitor copanlisib with ICIs therefore might be a promising strategy to overcome the resistance induced by intratumoral oncogenic signaling and an immune suppressive tumor microenvironment. Citation Format: Sarah Glaeske, Franziska Huebner, Anna Anurin, Andreas Janzer, Sabine Zitzmann-Kolbe, Juliane Paul, Katja Glaeske, Sandra Berndt, Dominik Mumberg, Matyas Gorjanacz, Karl Ziegelbauer, Bertolt Kreft, Ningshu Liu. Pulsatile inhibition of PI3K converts immune suppression by Tregs and M2-TAM to anti-tumor immune response in animal models insensitive or resistant to the monotherapies of PI3K and checkpoint inhibitors [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 LB-123.

Targeted alpha therapy (TAT) agents deliver high linear energy transfer (LET) alpha-radiation selectively to tumors. The first TAT to be approved is radium-223 which prolongs overall survival in metastatic castration resistant prostate cancer (mCRPC) patients with symptomatic bone metastasis. Radium-223 shows a selective uptake in newly formed bone matrix such as bone metastasis and binds to hydroxyapatite. The PSMA targeted thorium-227 conjugate PSMA-TTC represents another TAT approach in mCRPC. It consists of a fully human PSMA IgG antibody covalently linked to the chelator moiety (3,2 HOPO). This antibody-chelator conjugate is radiolabeled with thorium-227, which decays with a half-life of 18.7 days to radium-223 via alpha-particle emission. Herein we describe tumor targeting and anti-tumor activity of PSMA-TTC in two PSMA positive patient derived xenograft (PDx) models of prostate cancer with different characteristics. In vivo biodistribution and anti-tumor efficacy were analyzed after i.v. injection of PSMA-TTC at radioactive doses from 75-500 kBq/kg and protein doses of either 0.14 or 0.43 mg/kg to tumor bearing mice. Initially, the PDx model KuCap1 (provided by Prof. O. Ogava, University of Kyoto, Japan), a prostate cancer model resistant to the second generation antiandrogen enzalutamide, was analyzed. In this model PSMA-TTC showed strong dose dependent tumor growth inhibition starting at a single dose of 75 kBq/kg. Moreover, after a single i.v. administration of PSMA-TTC at 300 kBq/kg 9 out of 10 mice (90%) showed either stable disease or tumor regression for at least 33 days after treatment. The observed activity was highly selective, as injection of a radiolabeled control conjugate at 300 kBq/kg showed only limited tumor growth inhibition. Next, PSMA-TTC was tested in the hormone- and enzalutamide-sensitive prostate cancer PDx model ST1273 (South Texas Accelerated Research Therapeutics, San Antonio, Texas). A single i.v. injection of PSMA-TTC resulted in significant tumor accumulation of thorium-227 for more than 3 weeks, whereas a radiolabeled isotype control conjugate did not show tumor uptake. In addition, single i.v. administration of PSMA-TTC showed strong dose dependent anti-tumor activity while limited tumor growth inhibition was observed for a radiolabeled isotype control conjugate. Single doses of either 250 or 500 kBq/kg resulted in a 100 % response rate 4 weeks after treatment, with all animals showing partial or even complete regression of tumor growth. No significant effects on body weight were detected compared to vehicle treated animals. In summary, PSMA-TTC shows strong anti-tumor activity in patient derived prostate cancer models which were either sensitive or resistant to standard of care drugs. These data warrant further clinical investigation of this targeted alpha pharmaceutical investigational agent. Citation Format: Stefanie Hammer, Urs B. Hagemann, Sabine Zitzmann-Kolbe, Aasmund Larsen, Christine Ellingsen, Oliver von Ahsen, Jenny Karlsson, Roger M. Bjerke, Olav B. Ryan, Pascale Lejeune, Hartwig Hennekes, Alan Cuthbertson, Karl Ziegelbauer, Dominik Mumberg. Preclinical activity of PSMA-TTC, a targeted alpha therapeutic in patient-derived prostate cancer models [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 844.

Targeted Thorium-227 Conjugates (TTCs) represent a new class of targeted alpha therapy. In this compound family a 3,2-HOPO chelator, which binds thorium-227 with high affinity, is covalently attached to an antibody. This enables the specific delivery of the alpha particle emitter thorium-227 to tumor cells. Thorium-227 has a half-life of 18.7 days and decays via emission of an alpha particle to radium-223 (half-life of 11.4 days), a calcium-mimetic used in the treatment of CRPC [Henriksen et al. J Nucl Med, 2003]. The high linear energy transfer from the alpha-emitter thorium-227 induces clustered DNA double-strand breaks. Its short penetration range of 2-10 cell diameters limits the damage to the normal tissue surrounding the tumor. We present the preclinical evaluation of a mesothelin targeted thorium-227 conjugate (MSLN-TTC), the first TTC that will enter clinical development in MSLN-positive solid tumor indications, based on the fully human anti-MSLN monoclonal antibody anetumab. MSLN is a 40 kDa membrane-anchored glycoprotein with prominent overexpression in mesothelioma, ovarian, pancreatic, lung and breast cancer. In normal tissue, MSLN is confined mainly to the mesothelial cells of pleura, peritoneum and pericardium. In vitro, the mode of action of MSLN-TTC in cellular assays was demonstrated to induce DNA double strand breaks, leading to cell cycle arrest and subsequent reduced cell viability. In vivo, MSLN-TTC demonstrated potent tumor growth inhibition administered as a single-dose in cell- and patient-derived xenograft tumor models. Similar anti-tumor activity to single dose application was observed when the MSLN-TTC was applied at fractionated doses. A trend for dependence of anti-tumor activity on MSLN expression levels in preclinical tumor models was observed. Biodistribution studies evaluated the tumor accumulation of MSLN-TTC in xenograft models. These studies served to develop a mechanistic PK/PD model, which was used to predict the efficacious dose in humans. The initiation of clinical investigation of the MSLN-TTC in mesothelin positive cancers' is planned for 2018. Citation Format: Urs B. Hagemann, Alexander Kristian, Christine Ellingsen, Katrine Wickstroem, Anne Mobergslien, Jenny Karlsson, Roger M. Bjerke, Christoph Schatz, Christoph Kneip, Joachim Schuhmacher, Liv-Ingrid Oedegaardstuen, Hartwig Hennekes, Anna Tafuri, Dominik Mumberg, Hanno Wild, Karl Ziegelbauer, Alan S. Cuthbertson. Mesothelin targeted thorium-227 conjugate (MSLN-TTC): Preclinical evaluation of a new targeted alpha therapeutic in mesothelin-positive cancers [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 850.

Ovarian cancer remains an area of high unmet medical need, with 239,000 patients newly diagnosed per year. Here we describe the mesothelin-targeting antibody drug-conjugate anetumab ravtansine as a novel treatment option for ovarian cancer. Internalization of anetumab ravtansine and co-localization with lysosomal markers in ovarian cancer cells was accompanied by rapid resynthesis of mesothelin, which may allow consecutive anetumab ravtansine treatment cycles. The strong antitumor activity of anetumab ravtansine was preserved during repeated treatment cycles in the OVCAR-3 ovarian cancer in vivo model. Mechanistically, treatment with anetumab ravtansine caused mitotic arrest characterized by increased pHH3 signal and monopolar spindle structures. Interestingly, anetumab ravtansine monotherapy also induced DNA damage as indicated by focal γH2AX signals. Ultimately, the cells engaged in apoptotic cell death. Strong in vitro monotherapy activity was demonstrated in a set of 11 ovarian cancer cell lines with IC50s between 3 and 90nM. The in vivo efficacy of anetumab ravtansine dosed at 2.5 mg/kg Q3Dx3 i.v. anetumab ravtansine was evaluated in 9 human ovarian cancer models with varying mesothelin expression levels. Strong in vivo efficacy at this moderate dose with T/C Citation Format: Christoph A. Schatz, Maria Quanz, Urs B. Hagemann, Sabine Zitzmann-Kolbe, Beatrix Stelte-Ludwig, Sven Golfier, Charlotte Kopitz, Cem Elbi, Karl Ziegelbauer, Dominik Mumberg. Anetumab ravtansine has single-agent activity in mesothelin-expressing human ovarian cancer models and potentiates the activity of chemotherapeutics and targeted agents [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 1780.

Declining productivity with decreasing new molecular entity output combined with increased R&D spending is one of the key challenges for the entire pharmaceutical industry. In order to offset decreasing new molecular entity output, life-cycle management activities for established drugs become more and more important to maintain or even expand clinical indication and market opportunities. Life-cycle management covers a whole range of activities from strategic pricing to a next generation product launch. In this communication, we review how research organizations can contribute to successful life-cycle management strategies using phosphodiesterase 5 inhibitors as an example.

The NFκB transcription factor family facilitates the activation of dendritic cells (DCs) and CD4(+) T helper (Th) cells, which are important for protective adaptive immunity. Inappropriate activation of these immune cells may cause inflammatory disease, and NFκB inhibitors are promising anti-inflammatory drug candidates. Here, we investigated whether inhibiting the NFκB-inducing kinase IKK2 can attenuate crescentic GN, a severe DC- and Th cell-dependent kidney inflammatory disease. Prophylactic pharmacologic IKK2 inhibition reduced DC and Th cell activation and ameliorated nephrotoxic serum-induced GN in mice. However, therapeutic IKK2 inhibition during ongoing disease aggravated the nephritogenic immune response and disease symptoms. This effect resulted from the renal loss of regulatory T cells, which have been shown to protect against crescentic GN and which require IKK2. In conclusion, although IKK2 inhibition can suppress the induction of nephritogenic immune responses in vivo, it may aggravate such responses in clinically relevant situations, because it also impairs regulatory T cells and thereby, unleashes preexisting nephritogenic responses. Our findings argue against using IKK2 inhibitors in chronic GN and perhaps, other immune-mediated diseases.

For decades, the entire pharmaceutical industry has focused on a limited number of drug targets. Owing to advances in molecular biology and genome technology at the beginning of the 1990s, discovery and isolation of a large number of genes from the human genome became feasible. This triggered a multi billion US dollars investment by both biotechnology and pharmaceutical companies to gain access to and patent as many potential drug targets as possible. Although the combined effort of publicly funded projects and private investments resulted in rapid identification of essentially all genes of the human genome, harnessing this information to enable drug discovery has turned out to be more challenging and time consuming than initially anticipated.

BAY 1163877 is an orally available, highly potent and selective pan fibroblast growth factor receptor (FGFR) inhibitor. In an ongoing Phase 1 clinical trial (NCT01976741) BAY 1163877 showed clinical responses at exceptional tolerability in patients suffering from different tumor types including urothelial bladder carcinoma or lung tumors, which were selected based on elevated FGFR1-3 mRNA expression. In the preclinical phase, the compound demonstrated significant single agent anti-tumor activity in various tumor models with different FGFR alterations leading to FGFR overexpression (e.g. FGFR gene amplifications or mutations). Genetic alterations of FGFRs can also be found in breast cancer with 7.5 - 17% of all tumors harboring a FGFR1 gene amplification. Elevated FGFR1 mRNA levels can be found in up to 22% of breast cancer cell lines as well as clinical samples. Other FGFR alterations include FGFR2 or FGFR4 gene amplifications as well as elevated FGFR mRNA levels, which were reported in all breast cancer subtypes. We therefore investigated BAY1163877 monotherapy in various breast cancer models. Due to the favorable clinical safety profile of BAY1163877, we also examined a combination treatment with early line antihormonal therapies in hormone receptor positive breast cancer. In vitro profiling of BAY 1163877 in a number of breast cancer cell lines showed a clear association of efficacy with expression levels of different FGFR isoforms. The efficacy was further investigated in several patient- or cell line-derived breast cancer in vivo models. For instance, BAY 1163877 alone dosed 38mg/kg twice daily induced tumor growth inhibition of greater than 90% in a subcutaneous mouse syngeneic 4T1 breast cancer model expressing elevated levels of FGFR2. Resistance to endocrine therapy appears associated with FGFR1 gene amplification and may explain the poor prognosis of FGFR1 overexpressing tumors treated with adjuvant tamoxifen. We therefore investigated the combination of the panFGFR-inhibitor BAY 1163877 with the clinically used antihormonal compound fulvestrant in selected luminal breast cancer PDX models. Some of these models showed resistance to antihormonal treatment in monotherapy but improved in vivo efficacy in combined treatment using BAY 1163877 and fulvestrant. These data may warrant further clinical investigation of BAY1163877alone or in combination with antihormonal therapy in patients with FGFR overexpressing breast cancer. Citation Format: Oliver Politz, Peter Ellinghaus, Sebastian Bender, Sylvia Gruenewald, Franziska Siegel, Marie-Pierre Collin, Sabine Zitzmann-Kolbe, Dominik Mumberg, Karl Ziegelbauer. Preclinical activity of the FGFRinhibitor BAY 1163877 alone or in combination with antihormonal therapy in breast cancer [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 1079. doi:10.1158/1538-7445.AM2017-1079

PTEFb/CDK9 mediated transcription of short-lived anti-apoptotic survival proteins like Mcl-1 and Myc plays a critical role in cancer cell growth and survival in various tumor entities including AML. In addition, these survival proteins play important roles in the development of resistance to chemotherapy. We previously disclosed the preclinical profile of BAY 1143572, the first selective, orally available PTEFb/CDK9 inhibitor that entered clinical development [1-3]. BAY 1143572 had low nanomolar activity against PTEFb/CDK9, an at least 50-fold selectivity against other CDKs in enzymatic assays and broad anti-proliferative activity against a panel of tumour cell lines with sub-micromolar IC50 values. BAY 1143572 also showed single agent in vivo efficacy at tolerated doses in various xenograft tumour models in mice and rats upon once daily oral administration. To fully explore future treatment options using selective PTEFb/CDK9 inhibitors we initiated a follow-up program to identify novel PTEFb/CDK9 inhibitors for treatment of cancer with increased potency enabling i.v. treatment of patients. Extensive lead optimisation efforts, including various scaffold hops, led to the identification of BAY 1251152. In comparison to oral BAY 1143572, BAY 1251152 shows significantly increased biochemical (IC50 CDK9 = 3 nM) and cellular potency (IC50 MOLM13 = 29 nM), increased selectivity against CDK2 as well as high permeability and no efflux. The significantly reduced therapeutic dose and high solubility of BAY 1251152 enable the desired i.v. application. BAY 1251152 demonstrated excellent efficacy upon i.v. treatment in xenograft models (e.g. MOLM13) in mice and rats. BAY 1251152 is currently being evaluated in Phase I studies (NCT02635672; NCT02745743) to determine the safety, tolerability, pharmacokinetics and initial pharmacodynamic biomarker response in patients with advanced cancer. This presentation will highlight the key learnings from our PTEFb/CDK9 i.v. lead optimization program and disclose the structure of BAY 1251152 for the first time. [1]: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3022. [2]: 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 DDT02-02. doi:10.1158/1538-7445.AM2015-DDT02-02. [3]: 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 2828. doi:10.1158/1538-7445.AM2015-2828 Citation Format: Ulrich T. Luecking, Arne Scholz, Dirk Kosemund, Rolf Bohlmann, Hans Briem, Philip Lienau, Gerhard Siemeister, Ildiko Terebesi, Kirstin Meyer, Katja Prelle, Ray Valencia, Stuart Ince, Franz von Nussbaum, Dominik Mumberg, Karl Ziegelbauer, Michael Brands. Identification of potent and highly selective PTEFb inhibitor BAY 1251152 for the treatment of cancer: from p.o. to i.v. application via scaffold hops [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 984. doi:10.1158/1538-7445.AM2017-984

Radium-223 dichloride (radium-223, Xofigo®), a targeted alpha-therapy, improves overall survival in prostate cancer patients with bone metastases. It inhibits disease progression by reducing tumor growth and tumor-induced pathological bone reaction in breast and prostate cancer mouse models. Radium-223 is actively incorporated into the bone matrix by osteoblasts. Multiple myeloma (MM) is characterized by increased osteoclast and reduced or no osteoblast activity. Bortezomib (Velcade®), a treatment for MM, restores the impaired osteoblast activity in MM. Here, we report the effects of radium-223, bortezomib and their combination on myeloma cell proliferation in vitro and on myeloma bone disease model in mice. Proliferation assays were performed with human plasma cell leukemia (JJN-3, L-363), human MM (LP-1, MOLP-8, RPMI-8226 and OPM-2), and mouse MM (5TGM1) cells. Corresponding in vivo effects were studied in a syngeneic 5TGM1 mouse MM model. Female C57BL/KaLwRij mice (7 weeks old, n=15/group) were inoculated with 5TGM1 cells via tail vein and 26 days later, radium-223 (300 kBq/kg, single iv injection) and/or bortezomib (1 mg/kg ip, twice a week; total of 3 doses) or vehicle control were administered. The development of osteolytic lesions was detected by radiography. Hind limbs were used for histological analyses and total activity measurement was performed by a gamma-counter. TRAP-stained osteoclasts were counted at tumor-bone interface. Bortezomib inhibited proliferation of all cancer cell lines tested at 25 nM (JJN3 and OPM-2 at 2.5 nM) and radium-223 at 0.8 kBq/ml (L-363 and MOLP-8 at 0.2 kBq/ml) concentrations. Additive effects were observed with combination treatment in vitro. The 5TGM1 in vivo model demonstrated that both bortezomib and radium-223 decreased osteolytic lesion area as monotherapy (p In conclusion, radium-223 dichloride (Xofigo®) therapy in combination with bortezomib decreased osteolytic lesion area and almost completely eradicated tumor-associated osteoclasts in a mouse model of myeloma bone disease. Incorporation of radium-223 to bone matrix was improved, possibly via induction of osteoblast activity by bortezomib. These data suggest that combination of radium-223 and bortezomib could be a new effective therapy in MM, which is currently being investigated in a Phase Ib/II trial in patients with early relapsed MM (NCT02928029). Citation Format: Mari I. Suominen, Jukka P. Rissanen, Anniina Luostarinen, Katja M. Fagerlund, Birgitta Sjöholm, Esa Alhoniemi, Sanna-Maria Käkönen, Dominik Mumberg, Jussi M. Halleen, Karl Ziegelbauer, Arne Scholz. Additive benefits of radium-223 dichloride and bortezomib combination in a syngeneic 5TGM1 multiple myeloma mouse model [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 5202. doi:10.1158/1538-7445.AM2017-5202

The integrity of the genome of eukaryotic cells is secured by complex signaling pathways, known as DNA damage response (DDR). Recognition of DNA damage activates DDR pathways resulting in cell cycle arrest, induction of DNA repair, or cell death. Proteins that directly recognize aberrant DNA structures recruit and activate kinases of the DDR pathway, such as ATR (ataxia telangiectasia and Rad3-related). ATR responds to a broad spectrum of DNA damage, including double-strand breaks (DSB) and lesions derived from interference with DNA replication as well as increased replication stress. Therefore, inhibition of ATR kinase activity could be the basis for a novel anti-cancer therapy in tumors with increased DNA damage, deficiency in DNA damage repair or replication stress. Radium-223 dichloride (Xofigo®) is the first and only approved targeted alpha therapy so far. It is indicated for the treatment of patients with castration-resistant prostate cancer (CRPC), symptomatic bone metastases and no known visceral metastatic disease, based on improvement of overall survival. It exhibits strong cytotoxic effects on adjacent cells via the induction of DNA DSB. Here, we disclose for the first time the structure and functional characterization of the novel ATR kinase inhibitor BAY 1895344. In vitro, BAY 1895344 is a selective low-nanomolar inhibitor of ATR kinase activity, potently inhibiting proliferation of a broad spectrum of human tumor cell lines (median IC50 of 78 nM). A clear separation between highly sensitive (IC50 Our findings validate the concept of synthetic lethality of genetically determined DNA repair deficiency and ATR blockade by demonstrating strong monotherapy efficacy of the highly potent ATR inhibitor BAY 1895344 in a variety of tumor indications. Furthermore, the mechanism-based combination potential of DNA damage induction by Radium-223 with BAY 1895344 creates a powerful new treatment option for CRPC patients with bone metastases. The start of clinical investigation of BAY 1895344 is planned early 2017. Citation Format: Antje Margret Wengner, Gerhard Siemeister, Ulrich Luecking, Julien Lefranc, Philip Lienau, Gesa Deeg, Eleni Lagkadinou, Li Liu, Sven Golfier, Christoph Schatz, Arne Scholz, Franz von Nussbaum, Michael Brands, Dominik Mumberg, Karl Ziegelbauer. ATR inhibitor BAY 1895344 shows potent anti-tumor efficacy in monotherapy and strong combination potential with the targeted alpha therapy Radium-223 dichloride in preclinical tumor models [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 836. doi:10.1158/1538-7445.AM2017-836

BACKGROUND Bone metastases are associated with increased morbidity and poor prognosis in breast cancer patients. Radium-223 dichloride is a calcium mimetic that localizes to bone, providing targeted therapy for skeletal metastasis. METHODS We investigated the mode of action of radium-223 dichloride using breast cancer cell, osteoclast, and osteoblast cultures as well as a mouse model of breast cancer bone metastasis. A single dose of radium-223 dichloride was used in three different settings mimicking the prevention or treatment of bone metastasis. Disease progression was monitored using fluorescence and radiographic imaging and histological analyses. The effect of radium-223 dichloride alone and in combination with doxorubicin or zoledronic acid on survival of mice was analyzed by Kaplan-Meier methods. All statistical tests used were two-sided. RESULTS Radium-223 dichloride incorporated into bone matrix and inhibited proliferation of breast cancer cells and differentiation of osteoblasts and osteoclasts (all P values < .001) in vitro. In an established bone metastasis setting, radium-223 dichloride prevented tumor-induced cachexia (0/14 vs 7/14 control mice) and decreased osteolysis by 56% and tumor growth by 43% (all P values < .05). Radium-223 dichloride induced double-strand DNA breaks in cancer cells in vivo. Finally, radium-223 dichloride extended survival as a monotherapy (29.2 days, 95% confidence interval [CI] = 26.6 to 31.8 days, P = .039) and in combination with zoledronic acid (31.4 days, 95% CI = 28.8 to 34.0 days, P = .004) or doxorubicin (31.5 days, 95% CI = 29.5 to 33.5 days, P < .001) compared to the vehicle group (24.9 days, 95% CI = 23.4 to 26.4 days). Similar but even more pronounced effects were observed when radium-223 dichloride was administered in a preventive or micrometastatic setting. CONCLUSIONS Our findings strongly support the development of radium-223 dichloride for the treatment of breast cancer patients with or at high risk of developing bone metastases.

Introduction: ABC-DLBCL is a subtype of DLBCL with less favorable clinical outcomes to the standard of care (SoC) therapies. Constitutive activation of NF-κB by various genetic alterations in ABC-DLBCL has been identified as one of the key mechanisms driving chemotherapy resistance. Inhibition of B cell receptor (BCR) signaling with BTK (Bruton's tyrosine kinase) inhibitor ibrutinib demonstrated encouraging clinical responses in ABC-DLBCL. However, patients with CD79wt/MyD88mut, or CARD11mut did not respond to ibrutinib, indicating the need for new therapies targeting ibrutinib refractory ABC-DLBCL. Recent approval of PI3Kδ selective inhibitor idelalisib for the treatment of indolent NHL (iNHL) as monotherapy highlighted selective inhibition of PI3Kδ as an effective therapeutic strategy. However, idelalisib did not show clinical activity in DLBCL in a Phase I expansion cohort study. Here we report immunohistochemical (IHC) analysis of clinical tumor samples from follicular lymphoma (FL) and DLBCL patients and a series of in vitro and in vivo mechanistic and functional studies to explore the importance of PI3K isoforms in regulating key signaling pathways in ABC-DLBCL. Methods: Expression of PI3K isoforms and PTEN was assessed by IHC and western blot from a panel of 45 FL and 45 DLBCL primary tumors. Effects of PI3K inhibitors (PI3Kδ-selective inhibitor idelalisib, PI3Kα-selective inhibitor BLY-719, PI3Kβ-selective inhibitor TGX-221, predominant PI3Kα/δ inhibitor copanlisib and BTK inhibitor ibrutinib) on nuclear NF-κB activation were determined using stable cell lines expressing NF-κB-luciferase reporter (obtained by lentiviral infection), IHC staining of p65 NF-κB, expression of CCL4, IL-6, and IL-10 by RT-PCR and protein production by ELISA assays. In vitro and in vivo mechanisms of action were addressed by assessing the activities of the key survival signaling pathways. In vitro and in vivo anti-tumor activities were investigated using cell lines and patient derived xenograft ABC-DLBCL models representing the key molecular features of BCR-dependent and independent ABC-DLBCL. Results: Although PI3Kδ was predominantly expressed in both FL and DLBCL, high PI3Kα expression was more prevalent in DLBCL (60% vs 18%), a patient population resistant to PI3Kδ-selective inhibition in the clinic. Simultaneous inhibition of PI3Kα and PI3Kδ with BYL-719 plus idelalisib or copanlisib alone dramatically enhanced anti-tumor profile in ABC-DLBCL models compared to selective inhibition of PI3Kδ, PI3Kα or BTK. The anti-tumor activity was associated with suppression of p-AKT and a novel mechanism of blocking NF-κB activation driven by CD79mut, CARD11mut, A20mut or MyD88mutin vitro and in vivo. Suppression of NF-kB activation by PI3K inhibition is independent from AKT, but involves a novel mechanism of modulating c-IAP expression. Inhibition of PI3Kα/δ resulted in complete tumor regression in an ibrutinib-resistant MyD88mut-LY0257 patient-derived (PDx) ABC-DLBCL model. Furthermore, rebound activation of BTK and AKT was identified as a mechanism limiting CD79mut ABC-DLBCL to show robust response to PI3K and BTK inhibitors, respectively, as single agents in vivo. Combination of ibrutinib with PI3Kα/δ inhibitor copanlisib dosed intermittently iv was well tolerated and produced complete tumor regression in CD79Bmut TMD-8 cell line and Ly2298 PDx ABC-DLBCL models. Conclusions: High expression of PI3Kα in addition to PI3Kδ in ABC-DLBCL is associated with intrinsic resistance to PI3Kδ selective inhibition. Simultaneous inhibition of PI3Kα/δ by copanlisib modulates not only the PI3K/AKT pathway but also BCR-dependent and independent activation of nuclear NF-κB via a novel AKT-independent mechanism, indicating a promising utility for the treatment of clinically relevant ibrutinib-resistant ABC-DLBCL patients with CD79wt/MyD88mut, A20mut, or CARD11mut tumor genotypes. Combination of PI3Kα/δ and BTK inhibitors demonstrated promising potential for ibrutinib-responsive ABC-DLBCL to achieve complete tumor regression by blocking rebound activation of BTK and AKT. Thus, our findings presented here provide additional insights on intrinsic and acquired resistance mechanisms to selective PI3Kδ and BTK inhibitors and provide rationale for clinical development of PI3K inhibitors with specific isoform profile in combination for the treatment of ABC-DLBCL. Disclosures Paul: Bayer AG: Employment. Sojoun:Bayer AG: Employment. Wengner:Bayer AG: Employment. Zitzmann-Kolbe:Bayer AG: Employment. Sturz:Bayer AG: Employment. Haike:Bayer AG: Employment. Martin:Bayer AG: Employment. Mumberg:Bayer AG: Employment. Ziegelbauer:Bayer AG: Employment. Liu:Bayer AG: Employment.

Background and objective: Bone is the most common site of metastasis in prostate cancer (PCa) and results in significant morbidity and poor prognosis. Disseminated PCa cells conquer hematopoietic stem cell niches, and during outgrowth they induce osteoblasts to form new, disorganized bone. Also osteoclasts are activated at a varying degree. PI3K activation has important role in survival and proliferation of PCa cells, mediating critical signaling pathways involving tumor and stromal cell interaction in the bone microenvironment. BAY 1082439 is a highly selective and potent PI3K inhibitor currently in Phase I, with equipotent activity against PI3Kα and PI3Kβ isoforms. Radium-223 dichloride (ra-223/Xofigo) is alpha-emitting calcium mimetic that via efficient osteoaffinity provides targeted radiation therapy against bone metastases. We have previously reported that BAY 1082439 and ra-223 showed synergistic anti-proliferative and apoptosis-inductive effects in vitro in LNCaP PCa cells (Suominen et al, AACR Annual Meeting 2014). Here, we investigated the effects of BAY 1082439 and ra-223 as single agents and in combination in the osteoblastic LNCaP tumors in tibia of male nod.scid mice. Methods: Tumors were established 6 weeks after inoculation of LNCaP cells. Mice were randomized into four treatment groups (vehicle, ra-223; BAY 1082439 and ra-223 + BAY 1082439) based on serum PSA and bone lesion score. Treatments were continued for six weeks and the efficacy was assessed by the following endpoints biweekly and/or at the end of the study: PSA, bone formation marker PINP, bone lesions and bone volume (measured by microCT), and tumor and bone area by histological analysis. Results: Both ra-223 and BAY 1082439 monotherapies inhibited tumor growth (reduction 67.9%, p = 0.029 and 67.4%, p = 0.009, respectively). Furthermore, BAY 1082439 monotherapy treatment group displayed 68.8% of necrotic tumor, compared to 6.5% in the control group (p = 0.009). In the combination group, tumor growth was further suppressed (tumor reduction 89%, p = 0.005) and it is noteworthy that 60% of animals had no detectable tumors in histology. All treatments inhibited the tumor-induced bone formation compared to vehicle, and the combination treatment inhibited progression of bone lesions nearly completely. In conclusion, BAY 1082439 and ra-223 as monotherapies decreased tumor burden and tumor-induced bone reaction, and the observed effects were further enhanced by the combination treatment. Citation Format: Mari I. Suominen, Jukka Morko, Katja M. Fagerlund, Esa Alhoniemi, Dominik Mumberg, Karl Ziegelbauer, Jussi M. Halleen, Arne Scholz, Ningshu Liu. PI3K inhibitor BAY 1082439 and radium-223 dichloride decrease tumor burden and tumor-induced bone formation in an established bone metastatic prostate cancer model in mice. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 381.

The PI3K-AKT-mTOR signaling cascade is one of the major drivers in the development of cancer. It is constitutively activated in many types of cancers and is one of the prominent pathways that promote tumor cell survival and confers resistance to antihormonal therapies for patients with breast cancer. Breast cancer has been classified into at least four distinct subtypes, based on molecular profiling. Luminal-B breast cancer, although still expressing the hormone receptor, has been identified as relatively insensitive to endocrine therapy and is an entity with highest need for novel treatments and combination approaches. Despite the notable improvements in endocrine therapy, the invariable appearance of endocrine resistance, either primary or secondary, remains an important issue in this type of tumor. Main cancer signaling pathways, including PI3K/Akt/mTOR and CCND1/CDK4-6, are thought to play an important role in development of this resistance. Therefore AKT is considered an attractive drug target for the treatment of breast cancer. BAY 1125976, an orally active, potent, highly selective, allosteric AKT1/2 inhibitor is currently in phase I clinical development (NCT01915576). BAY 1125976 is particularly effective in preclinical models with PI3K-AKT pathway aberrations and luminal B status as shown by profiling in a panel of tumor cell lines as well as respective in vivo studies. The efficacy of BAY 1125976 in inhibition of cell proliferation is correlated with luminal status of the tumor as shown in several cell line panels. In vitro combination with anti-hormonal therapeutics showed synergistic anti-proliferative effects and rendered resistant cell lines sensitive towards tamoxifen or fulvestrant treatment. In the MCF-7 cell line tamoxifen combined with BAY 1125976 resulted in a 14 fold reduction of the IC50 for inhibition of cell proliferation compared to monotherapy. This translated into additive to synergistic activity in combination with tamoxifen in a ER+ MCF7 (PIK3CAE545K) BC model and enabled the use of alternative dosing schedules with improved efficacy versus monotherapy. BAY 1125976 also showed potent inhibition of tumor cell growth in a tamoxifen- and fulvestrant-resistant derivate of MCF-7 enabling a reduction of the therapeutic dose of BAY 1125976 and thereby improving tolerability while keeping efficacy. Combination of the allosteric AKT inhibitor BAY 1125976 therefore provides an interesting opportunity in improving efficacy of antihormonal therapy in luminal B type breast cancer. Citation Format: Oliver Politz, Lars Baerfacker, Stuart Ince, Andrea Haegebarth, Ningshu Liu, Roland Neuhaus, Ulf Boemer, Martin Michels, Karl Ziegelbauer, Dominik Mumberg. Allosteric AKT1/2-inhibitor BAY 1125976 as potent inhibitor in luminal breast cancer resistant to antihormone therapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 379.

One hallmark of cancer is the accelerated metabolism, high energy requirements, and increased glucose uptake by the tumor cells, the latter being the first and rate-limiting step for glucose metabolism. Glucose transport into the tumor cell is mediated by facilitative high-affinity glucose transporter (GLUT) proteins. Among the 14 GLUT proteins, expression of GLUT1 in normal organs is nearly exclusively restricted to the blood brain barrier, while other GLUTs are also expressed in a wide variety of vital organs such as liver and heart. Interestingly, GLUT1 expression is highly regulated by hypoxia-inducible factor (HIF)-1α, a key driver of tumor progression. In line with this finding, GLUT1 over-expression was found to be associated with tumor progression and poor overall survival in various tumor indications. Consequently, GLUT1 represents a potential target for cancer treatment. Therefore, we have developed a highly-selective GLUT1 inhibitor, namely BAY-876, with selectivity over GLUT2, 3, and 4 of 4700-, 800-, and 135-fold, respectively. We here show for the first time the pharmacological characterization of BAY-876, comprising inhibition of glucose-uptake, anti-proliferative activity in vitro, and anti-tumor efficacy in vivo in models of different tumor indications in monotherapy as well as first results on the combinability of BAY-876. Furthermore, at the therapeutic dose, BAY-876 treatment did not show any relevant finding on the behavior of treated mice in the Irwin test, assuming no or only minor effects on brain function. In conclusion, BAY-876 is the first GLUT1-selective inhibitor which reduces glucose uptake and growth of tumor cells with sufficient tolerability at the efficacious dose in preclinical models. Citation Format: Charlotte Kopitz, Luisella Toschi, Carolyn Algire, Mélanie Héroult, Anna-Lena Frisk, Kirstin Meyer, Arndt Schmitz, Eleni Lagkadinou, Heike Petrul, Iring Heisler, Roland Neuhaus, Bernd Buchmann, Herbert Himmel, Marcus Bauser, Andrea Haegebarth, Karl Ziegelbauer. Pharmacological characterization of BAY-876, a novel highly selective inhibitor of glucose transporter (GLUT)-1 in vitro and in vivo. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4746.

PTEFb/CDK9 mediated transcription of short-lived anti-apoptotic survival proteins like Mcl-1 and Myc, plays a critical role in cancer cell growth and survival in various tumor entities including AML. In addition, these survival proteins exhibit important functions in the development of resistance to chemotherapy. In contrast to pan-CDK inhibitors, to our knowledge PTEFb selective inhibitors have not been explored for clinical utility. We report the preclinical activity of BAY 1143572, a novel selective PTEFb/CDK9 inhibitor (AACR; Cancer Res 2015;75(15 Suppl):Abstract nr DDT02-02) currently being investigated in Phase I clinical trials in advanced cancer (NCT01938638) and acute leukemia (NCT02345382) in various in vitro, ex vivo and in vivo models of AML. BAY 1143572 inhibited the proliferation of 7 MLL-rearrangements positive and negative AML cell lines with a median IC50 of 385 nM (range 230-1100 nM) and induced apoptosis. Furthermore, BAY 1143572 showed potent in vitro activity in 8 out of 10 non-MLL-rearranged patient derived AML samples incl. NPM1 mutant and Flt3-ITD positive samples derived from intermediate and high risk patients. Moreover, we elucidated the dynamic changes of the cellular proteome/phosphoproteome upon pharmacological and genetic PTEFb inhibition and identified PTEFb interaction partners in various AML in vitro models. These analyses uncover the oncogenic PTEFb-dependent signaling networks and substantiate the molecular rationale for the use of PTEFb inhibitors in this indication. When applied in vivo, BAY 1143572 exhibited single agent efficacy at tolerated doses in 4 out of 5 AML xenograft tumor models in mice and in 2 out of 2 AML xenograft tumor models in rats upon once daily oral administration. Of note, partial or even complete remissions could be achieved in several models. Furthermore, intermittent dosing schedules with up to 4 days treatment pauses were feasible in terms of efficacy and tolerability. Using MOLM-13 xenografts in mice and rats to address the in vivo MoA of BAY 1143572, a transient inhibition of RNA polymerase II phosphorylation, MYC mRNA and protein levels, MCL-1 mRNA and protein levels, and an induction of apoptosis was documented. In conclusion, our data provide the rationale for the initiation of clinical development of BAY 1143572 as a potent and highly selective inhibitor of PTEFb/CDK9 with first-in-class potential for the treatment of AML patients. A phase I clinical trial to determine the safety, tolerability and recommended Phase 2 dose in this indication is ongoing (NCT02345382). Citation Format: Arne Scholz, Thomas Oellerich, Akhtar Hussain, Sarah Lindner, Ulrich Luecking, Annette O. Walter, Peter Ellinghaus, Ray Valencia, Franz von Nussbaum, Dominik Mumberg, Michael Brands, Stuart Ince, Hubert Serve, Karl Ziegelbauer. BAY 1143572, a first-in-class, highly selective, potent and orally available inhibitor of PTEFb/CDK9 currently in Phase I, shows convincing anti-tumor activity in preclinical models of acute myeloid leukemia (AML). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3022.

Background: Mutations affecting the Wnt/β-catenin pathway have been found in 26-40% of HCC samples. Aberrant constitutive activation of this pathway leads to uncontrolled cell proliferation growth and survival of tumor cells. Thus, identification of Wnt/β-catenin pathway inhibitors might lead to a new treatment option for a large subset of HCC patients. In the present study, we aim to investigate the effects of Sorafenib plus the allosteric MEK1/2 inhibitor Refametinib (Sorafenib/Refametinib) on Wnt/β-catenin signaling in vitro and in PDX models of HCC. Methods: We treated 15 patient-derived HCC xenograft models with i) 10 mg/kg Sorafenib QD, ii) 15 mg/kg Refametinib QD, and iii) Sorafenib/Refametinib. Western blotting was employed to determine pharmacodynamic changes in biomarkers relevant Wnt/β-catenin signaling. Apoptosis, cell proliferation and localization of β-catenin were analyzed by immunohistochemistry. In-vitro explant culture from 3 patient-derived HCC lines was also tested. Results: In both wild-type and activated-mutated β-catenin HCC primary cells, Refametinib and Sorafenib/Refametinib potently inhibited cell proliferation and caused cell death, which were correlated with downregulation of Dvl3 and β-catenin target genes (Axin2, c-myc, cyclin D1 and survivin). Inhibition of p-LRP6 at Ser1490 and p-β-catenin at Tyr142 and elevation of E-cadherin were also observed. These changes were not observed in Sorafenib-treated samples. In addition, the Sorafenib/Refametinib combination also suppressed Wnt3a-induced p-LRP6 at Ser1490 and β-catenin accumulation in cells. In vivo, significant reductions in p-LRP6 at Ser1490 and p-β-catenin at Tyr142 were observed in tumor samples from the Sorafenib/Refametinib treatment group and to a lesser extent, samples from the Refametinib. Although the overall protein levels of β-catenin were not significantly changed, non-phosphorylated (active) β-catenin Ser33/37/Thr41 and WNT/β-catenin target genes in Refametinib- and Sorafenib/Refametinib-treated tumors were significantly reduced. While vehicle-treated β-catenin-mutated tumors showed strong cytoplasmatic and nuclear β-catenin localization, most of β-catenin in Sorafenib/Refametinib- and, to a lesser extent, Refametinib-treated tumors was accumulated at the membrane. Conclusion: Sorafenib/Refametinib and, to a lesser extent, Refametinib inhibit the growth of patient derived explant cultures and PDX models. They also inactivate β-catenin signaling. Since activating mutations in the Wnt/β-catenin pathway are detected in a high proportion of HCC, combination of Sorafenib and Refametinib may represent an alternative treatment for β-catenin-dependent HCC. Citation Format: Huynh T. Hung, Richard Ong, Florian Puehler, Arne Scholz, Oliver Politz, Dieter Zopf, Dominik Mumberg, Karl Ziegelbauer. Sorafenib/Refametinib potently inhibits Wnt/β-catenin in vitro and patient-derived xenograft models of human hepatocellular carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2276.

Radium-223 dichloride (radium-223), an alpha particle-emitting calcium-mimetic, improves overall survival in prostate cancer patients with symptomatic bone metastases. We have defined radium-223 mode-of-action and efficacy in two clinically relevant prostate cancer xenograft models demonstrating PSA expression and osteoblastic growth upon intratibial inoculation of cancer cells. Immunocompromised male mice were inoculated with human LNCaP or patient-derived LuCaP 58 prostate cancer cells in the intratibial compartment and subsequently stratified into treatment groups based on lesion grade and/or serum PSA levels. Radium-223 (300 kBq/kg) or vehicle was administered intravenously, two times at 4-week intervals during the experiment. X-rays and serum samples were obtained biweekly and at sacrifice. Soft tissue tumors were examined macroscopically at sacrifice and tissue samples were collected and processed for γ-counter measurements, micro-CT, autoradiography and histology. Radium-223 treatment inhibited tumor-induced osteoblastic bone growth as indicated by reduced bone volume and surface in LNCaP and LuCaP 58 prostate cancer mouse models. In addition, radium-223 treatment suppressed metabolic activity in bone as evidenced by decreased number of osteoblasts and osteoclasts relative to bone surface and reduced levels of the bone formation marker PINP. Radium-223 resulted in lower PSA values as early as two weeks after the first dose, indicating constrained tumor growth following treatment. This phenomenon was further supported by reduced total bone lesion tissue and tumor area in LNCaP and LuCaP 58 models and increased percentage of necrotic tumor area in the LuCaP 58 model in radium-223-treated mice as compared to vehicle-treated mice. Moreover, DNA double-strand breaks were increased in cancer cells 24 hours post radium-223 treatment in the LuCaP 58 model providing further evidence of anti-tumor effects. Radium-223-treated mice exhibited less visceral metastases in the LuCaP 58 model (not significant). Based on autoradiography, radium-223 was deposited in the intratumoral bone matrix and in conjunction with osteoblasts in osteoblastic metastases. Our results demonstrate that radium-223 dichloride is successfully incorporated into the intratumoral bone matrix and inhibits tumor growth in both cell line- and patient-derived osteoblastic prostate cancer metastasis models. Given the α-particle range of 50-80 μm, potent radiation effects on the tumor microenvironment are evident whereas relevant effects on the more distant bone marrow are not expected. Taken together, radium-223 therapy exhibits a dual mode-of-action that impacts tumor growth and tumor-induced bone reaction, both important players in the destructive vicious cycle of osteoblastic bone metastasis in prostate cancer. Citation Format: Mari I. Suominen, Katja M. Fagerlund, Dominik Mumberg, Karl Ziegelbauer, Sanna-Maria Käkönen, Jussi M. Halleen, Robert L. Vessella, Arne Scholz. Radium-223 dichloride inhibits tumor growth and tumor-induced bone growth in osteoblastic prostate cancer models. [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 A109.

PTEFb (positive transcription elongation factor b) is a heterodimer of the transcriptional control kinase CDK9 (Cyclin-dependent kinase 9) and Cyclin T. PTEFb phosphorylates and activates RNA polymerase II. PTEFb inhibition causes rapid depletion of short-lived mRNA transcripts and their associated protein products involved in proliferation and survival like Myc, or Mcl-1 which results in cell death of addicted tumor cells. We previously disclosed the profile of the lead compound PTEFb BAY1, a nanomolar PTEFb inhibitor with 50-fold selectivity within the CDK family and cellular potency of about 1 μM in proliferation assays on various human tumor cell lines [1]. PTEFb BAY1 also revealed in vivo efficacy in a human acute myeloid leukemia (AML) xenograft model in nude mice. However, the lead compound also displayed certain limitations in ADME properties like low aqueous solubility and a strong recognition by efflux transporters in the Caco2 assay. Based on these findings, extensive lead optimisation efforts led to the rapid identification of BAY 1143572 which is a more potent and highly selective, orally available PTEFb inhibitor with first-in-class potential. BAY 1143572 has a high aqueous solubility, reduced drug efflux and a moderate oral bioavailability across species that allows daily as well as intermittent dosing schedules in animal models. BAY 1143572 revealed strong in vitro and in vivo anti-tumor efficacy with various cell-lines. BAY 1143572 is currently being evaluated in a Phase I study to determine the safety, tolerability, pharmacokinetics and initial pharmacodynamic biomarker response in patients with advanced cancer. This presentation will highlight the key learnings from our PTEFb lead optimization program. [1]: AACR, April 5-9, 2014, San Diego, Poster Presentation, Abstract 4538, Cancer Res October 1, 2014, 74:4538; doi:10.1158/1538-7445.AM2014-4538 Citation Format: Ulrich TJ Luecking, Arne Scholz, Philip Lienau, Gerhard Siemeister, Dirk Kosemund, Rolf Bohlmann, Knut Eis, Mark Gnoth, Ildiko Terebesi, Kirstin Meyer, Katja Prelle, Ray Valencia, Stuart Ince, Franz von Nussbaum, Dominik Mumberg, Karl Ziegelbauer, Bert Klebl, Axel Choidas, Peter Nussbaumer, Matthias Baumann, Carsten Schultz-Fademrecht, Gerd Ruehter, Jan Eickhoff, Michael Brands. Rapid identification of potent and highly selective, oral PTEFb Inhibitor BAY 1143572 with first in class potential. [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 2828. doi:10.1158/1538-7445.AM2015-2828

Although targeting lipogenesis for cancer treatment appears to have a strong rationale, drug discovery in this field has not been fully explored due to the lack of understanding the mode of action as well as the therapeutic window. We reported previously on a class of novel ACC inhibitors with potent and selective activity against human ACC1, an isoform overexpressed in many cancer types. These ACC inhibitors revealed strong anti-tumor activity, including induction of tumor cell apoptosis in vitro and tumor regression in vivo in a sub-set of tumor models. To further demonstrate the therapeutic potential of ACC inhibitors, we conducted a series of studies in xenograft mice and rat to evaluate the anti-tumor efficacy of ACC inhibitors and to characterize their safety profile. We report that breast, prostate, and pancreatic cancers are among the most sensitive tumors to ACC inhibition. Interestingly, the anti-tumor kinetics correlated with reduction in palmitate levels without substantial changes in structural lipid components. In addition, a sub-type of KRAS mutation and activation of the Wnt pathway correlates with the sensitivity of tumors to ACC inhibitors. Treatment with ACC inhibitors at high doses caused an immediate decrease in food intake and followed with body weight loss. A clear correlation between the reduction of food intake and exposure of ACC inhibitor was observed. Upon withdrawing drug, the effect on food intake is restored. Therefore, we investigated intermittent dosing schedules and food effects on the tolerability and anti-tumor efficacy of ACC inhibitors. We could demonstrate that the tolerability was improved without compromising the efficacy compared to continuous treatment. Furthermore, feeding animals a high fat diet prevented body weight loss and meanwhile maintained the antitumor activity. These results indicate that strong reduction of food intake seems the cause of intolerability, which can be prevented and reversed either by intermittent dosing, or by exogenously supplementing with a high fat diet. Furthermore, for the first time we provided in vivo evidence that exogenous lipids could complement de novo lipogenesis inhibition in normal cells, while tumor growth requires lipogenesis irrespective of existing circulating lipids. In summary, these assessments provide scientific insights and strategy on how to best target tumor lipid metabolism and lipid signaling effectively and safely for cancer therapy. Citation Format: Ningshu Liu, Wilhelm Bone, Sendhil S. Velan, Krishnarao Doddapuneni, Jadegoud Yaligar, Kai Thede Thede, Ursula Moenning, Xiaohe Shi, Xianfeng Tian, Elissaveta Petrova1, Franz von Nussbaum, Dominik Mumberg, Michael Brands, Karl Ziegelbauer. How to develop ACC1 inhibitors targeting lipid metabolism and oncogenic signaling pathways effectively and safely. [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 1129. doi:10.1158/1538-7445.AM2015-1129

MKNK1 (MAP kinase-interacting serine/threonine-protein kinase, also known as Mnk1) is activated by the mitogen-activated protein kinases ERK1/2 and p38. Thus, MKNK1 signaling is involved in the cellular response to environmental stress factors and cytokines. Of particular interest, MKNK1 kinase was shown to regulate mRNA translation by phosphorylating the translation initiation factor eIF4E (eukaryotic translation initiation factor 4E), known to be critical for malignant transformation but dispensable for translation in normal cells. In addition, MKNK is involved in resistance mechanisms to cancer therapeutics. Thus, the inhibition of MKNK1 activity may provide an innovative approach for anti-cancer therapy. BAY 1143269 was identified as a potent and selective inhibitor of MKNK1 activity with an unprecedented mode of action. It inhibits the phosphorylation of eIF4E in various cancer cell lines in vitro and leads to reduced expression of MKNK-regulated translational downstream targets, including survivin, Cdc25C and cyclin B1. In addition, BAY 1143269 potently inhibits cytokine release in LPS stimulated human blood. In vivo, BAY 1143269 shows a significant monotherapy efficacy in non-small cell lung cancer (NSCLC), colorectal cancer and melanoma xenograft models after an once daily, oral application of 200 mg/kg in mice and 70 mg/kg in rats. Furthermore, combination treatment with chemotherapy and BAY 1143269 gives additive efficacy in several NSCLC cell lines and patient-derived xenograft models leading to partial response (Lu7558, A549), stable disease (Lu7187, Lu7166) or significant delay in tumor re-growth versus chemotherapy alone after stop of treatment (Lu7558). In summary, BAY 1143269 is a selective, orally available MKNK1 inhibitor that demonstrates preclinical in vivo efficacy in mono- and combination therapy and may provide therapeutic benefit for patients with solid tumors. Citation Format: Susann Santag, Franziska Siegel, Antje Margret Wegner, Claudia Schneider, Ulf Boemer, Knut Eis, Florian Puehler, Martin Michels, Franz von Nussbaum, Karl Ziegelbauer, Dominik Mumberg, Kirstin Petersen. Preclinical anti-tumor efficacy and mode of action of a novel, orally available, selective MKNK1 inhibitor [BAY 1143269]. [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 2604. doi:10.1158/1538-7445.AM2015-2604

With an estimated 700,000 new cases per year, hepatocellular carcinoma (HCC) represents the fifth commonest cancer and is the third leading cause of cancer death worldwide. Its 5-year survival rate after surgery is only 5-6%. Although HCC cases occur predominantly in the East and Southeast Asia and Sub-Saharan Africa, the incidence is rising in the West, largely due to an increasing incidence of Hepatitis C virus infection, alcoholic liver disease, and non-alcoholic steatohepatitis related to obesity and type II diabetes. Besides sorafenib, an oral multikinase inhibitor approved for the treatment of HCC, there is no other pharmacological agent in the treatment of HCC. Cancer cells undergo dramatic metabolic reprogramming to sustain uncontrolled proliferation. Accumulating evidence supports abnormal metabolic pathways as an emerging hallmark of cancer. To identify metabolic genes required for HCC tumorigenesis, we compared gene expression profiles of a rat orthotopic and a mouse DEN (Diethylnitrosamine)-induced HCC model to a liver regeneration model and to normal liver by PCR arrays, transcriptomic analysis, and metabolomic profiling. Serine and glycine levels were increased in tumors of the HCC models as determined by NMR spectroscopy. PSAT1 (phosphoserine aminotransferase 1), an enzyme in the serine biosynthesis pathway, was identified as one of the top up-regulated genes in the HCC models. An increase in PSAT1 protein levels in tumor samples versus normal liver tissue was confirmed by Western blot. Knockdown of PSAT1 in rat hepatoma cells (MH3924a) led to a dramatic reduction in cell proliferation in vitro and in vivo. Moreover, a doxycycline-inducible knockdown approach confirmed the anti-proliferation effect of PSAT1 depletion. Interestingly, depletion of PSAT1 showed a variable effect on other HCC cells (e.g. Hep3B and Huh-7), suggesting the involvement of other genetic and metabolic factors. Detailed analysis of PSAT1 as a potential drug target for HCC is currently being addressed in in vivo studies. Citation Format: Zhaobing Ding, Kenneth Wee, Eung-Sam Kim, Animesh Samanta, Michael Steckel, Marian Raschke, Sendhil Velan, Young-Tae Chang, Andrea Haegebarth, Karl Ziegelbauer, Sylvia Gruenewald, Weiping Han. Phosphoserine aminotransferase 1 (PSAT1) as a novel anti-tumor target in hepatocellular carcinoma. [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 1163. doi:10.1158/1538-7445.AM2015-1163

Antibody-drug conjugates (ADC) represent a promising therapeutic approach for treatment of cancer. We have developed a novel ADC directed against fibroblast growth factor receptor 2 (FGFR2). FGFR2 is overexpressed in several cancer indications, such as gastric, breast, and ovarian cancer and thus represents a potential therapeutic target for treatment of FGFR2-positive cancer patients with ADC-based therapy. FGFR2-ADC consists of a fully human anti-FGFR2-Ab (BAY 1179470) conjugated via a stable linker to a novel auristatin cytotoxic agent licensed from Seattle Genetics. FGFR2-ADC exhibits low nM to sub-nM potency in vitro in a panel of FGFR2-positive cancer cells lines (SNU-16, MFM-223, NCI-H716) while being inactive against FGFR2-low or -negative cell lines (MDA-MB-231, HEK-293, BaF/3) and highly selective versus a control ADC. FGFR2-ADC is highly efficacious in monotherapy and results in tumor growth inhibition in the gastric cancer xenograft model SNU-16 and tumor regression in the breast cancer xenograft model MFM-223. FGFR2-ADC induces tumor stasis in the colorectal cancer xenograft model NCI-H716 and regrown tumors are sensitive to a second treatment cycle of FGFR2-ADC. FGFR2-ADC shows high efficacy in vivo in monotherapy in patient-derived (PDX) FGFR2-positive murine xenograft models, e.g. in the ovarian cancer model OV30-0511A. FGFR2-ADC is also efficacious in the gastric cancer PDX model GC10-0608 and the breast cancer model MAXF857. The toxophore metabolite of FGFR2-ADC was more than 30-fold enriched in tumors versus other organs (liver, spleen, kidneys) in NCI-H716 tumor-bearing mice. Based on the preclinical efficacy, PK and tolerability data, evaluation of FGFR2-ADC in cancer patients appears warranted. A Phase I study is planned. Citation Format: Anette Sommer, Charlotte Kopitz, Christoph Schatz, Ruprecht Zierz, Joachim Schuhmacher, Sabine Wittemer-Rump, Klaas Prins, Manuela Braun, Frank Reetz, Bertolt Kreft, Hung T. Huynh, Karl Ziegelbauer. Preclinical anti-tumor efficacy of FGFR2-ADC BAY 1187982 in patient-derived gastric, breast and ovarian cancer models. [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 1684. doi:10.1158/1538-7445.AM2015-1684

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Gastric cancer is the second most common cause of cancer-related mortality worldwide, thus new treatment options are urgently needed. In a subset of gastric cancers, over-expression of fibroblast growth factor receptor 2 (FGFR2), a receptor tyrosine kinase, has been described and may represent a potential therapeutic target for the treatment of FGFR2-positive gastric cancer patients. To this end, we have generated a fully human anti-FGFR2 antibody (BAY 1179470) using the BioInvent Phage Display library. BAY 1179470 binds to a unique FGFR2-specific epitope that is present in all FGFR2 isoforms. Upon binding to FGFR2, BAY 1179470 induces receptor dimerization, internalization and degradation, resulting in significant tumor growth inhibition in vivo in cell line-based and patient-derived gastric cancer models overexpressing FGFR2. Additive anti-tumor efficacy in vivo was achieved by combining BAY 1179470 with either cisplatin or paclitaxel. BAY 1179470 is fully cross-reactive with FGFR2 orthologues of mouse, rat, pig, cynomolgus monkey and rhesus macaque. No significant safety findings have been seen in animal studies. Thus, BAY 1179470 represents a novel anti-FGFR2 antibody with high anti-tumor activity in gastric cancer models and an excellent preclinical safety profile. BAY 1179470 is currently being tested in a first-in-man study in all-comers ([NCT01881217][1]) in Japan. Citation Format: Charlotte Kopitz, Anette Sommer, Stefanie Hammer, Axel Harrenga, Beatrix Stelte-Ludwig, Frank Dittmer, Frank Reetz, Ekkehard May, Ruprecht Zierz, Sabine Wittemer-Rump, Christoph Schatz, H. T. Huynh, Karl Ziegelbauer, Bertolt Kreft. In vitro and in vivo characterization of a novel anti-fibroblast growth factor receptor (FGFR) 2 antibody (BAY 1179470) for the treatment of gastric cancer. [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 DDT02-01. doi:10.1158/1538-7445.AM2014-DDT02-01 [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT01881217&atom=%2Fcanres%2F74%2F19_Supplement%2FDDT02-01.atom

The receptor tyrosine kinase Tie2 is predominantly expressed in the endothelium but has also been identified on primitive hematopoietic stem cells, monocyte and macrophage subclasses, as well as on glioma or hematological tumor cells. Based on its expression in many patient-derived leukemic blasts inhibition of the Tie2 pathway may provide an attractive opportunity for therapeutic intervention in leukemias. In this study we report the pharmacological profile of a novel, highly potent and orally available Tie2 inhibitor (BAY-Tie2). The discovery and design process leading to BAY-Tie2 was performed with the goal of sparing other angiogenic RTKs, such as VEGFRs, FGFRs or PDGFRs. BAY-Tie2 is based on a novel imidazopyrazole core, combined with a SF5-substituted phenyl ring that fills the deep DFG-out pocket. BAY-Tie2 binds to Tie2 with a Kd value of 1.6 nM and is selective against VEGFR2 (Kd of 1600 nM), FGFR1 ( In subcutaneous xenograft models of highly angiogenic tumors, BAY-Tie2 reduced tumor growth and showed evidence for potential combination benefit with anti-VEGF therapy. In order to explore the potential of a Tie2 inhibitor beyond affecting angiogenesis, we established disseminated leukemia models, using Tie2-expressing cell lines, such as the CML cell lines MEG-01 and EM-2. Both cell lines engrafted predominantly in bone marrow and spleen. Treatment started 3 days after i.v. cell implantation with either BAY-Tie2 or cytarabine and was well tolerated. Efficacy was monitored by a) inhibition of disease progression, b) weekly fluorescence-based in vivo imaging (IVI) using an Alexa750-labeled anti-human CD33 antibody, and c) q-RT-PCR specific for BCR-ABL and hCD45 in murine peripheral blood. BAY-Tie2 inhibited disease progression comparable to cytarabine. Tumor load measured by IVI was reduced in BAY-Tie2 treated groups by 45% in the MEG-01 and by 65% in the EM-2 model compared with the untreated control, very similar to the cytotoxic treatment with cytarabine. Quantitative RT-PCR on peripheral blood revealed that BAY-Tie2 and cytarabine delayed the appearance of circulating tumor cells in both CML models. These data demonstrate that BAY-Tie2 is an orally active Tie2 inhibitor that may have therapeutic benefit not only in angiogenic tumors but also in hematological, Tie2-expressing malignancies. Citation Format: Sylvia Gruenewald, Julia Schueler, Michael Haerter, Frank Suessmeier, Kerstin Klingner, Ulf Boemer, Stefan Kaulfuss, Alexander Walter, Mario Lobell, Ingo V. Hartung, Bernd Buchmann, Dieter Heldmann, Holger Hess-Stumpp, Karl Ziegelbauer. Novel Tie2 inhibitor with in vivo efficacy in disseminated hematological tumor models in 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 1026. doi:10.1158/1538-7445.AM2014-1026

Fibroblast growth factors (FGFs) orchestrate a variety of cellular functions by binding to their transmembrane tyrosine-kinase receptors (FGFR1-4) and activating downstream signaling pathways. FGF signaling has been demonstrated to be altered in a high proportion of cancers, with activating mutations and/or overexpression of FGFRs frequently observed in lung, gastric, breast and urothelial tumors. Therefore, targeting FGFRs using selective FGFR inhibitors is an attractive therapeutic approach to treat cancer patients. BAY 1163877 is as an orally available, selective and potent inhibitor of FGFR-1, -2 and -3 kinase activity. BAY 1163877 has been advanced through preclinical development and we disclose here the first details of its preclinical profile. BAY 1163877 inhibited FGFR-1, -2, -3 kinase activity in the nanomolar range and demonstrated a kinase selectivity profile for FGFR-1, -2 and -3 over 222 kinases tested. BAY 1163877 inhibited proliferation of various cancer cell lines in vitro and phosphorylation of downstream signaling molecules. BAY 1163877 was also tested in vivo in monotherapy and combination therapy on various human xenografts and syngeneic tumors and inhibited growth of tumors presenting at least one FGFR alteration.Overall, the in vitro and in vivo studies confirm that the FGFR inhibitor BAY 1163877 is a potent and selective inhibitor of altered FGFRs pathways in cancer models. A Phase 1 clinical trial (NCT01976741) has been initiated. Citation Format: Melanie Heroult, Peter Ellinghaus, Christian Sieg, Dirk Brohm, Sylvia Gruenewald, Marie-Pierre Collin, Ulf Boemer, Mario Lobell, Walter Huebsch, Matthias Ocker, Stuart Ince, Andrea Haegebarth, Rolf Jautelat, Holger Hess-Stumpp, Michael Brands, Karl Ziegelbauer. Preclinical profile of BAY 1163877 - a selective pan-FGFR inhibitor in phase 1 clinical trial. [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 1739. doi:10.1158/1538-7445.AM2014-1739

C4.4a (LYPD3) has been identified previously as a cancer- and metastasis-associated internalizing cell surface protein. Targeting C4.4a with a specific antibody-drug conjugate (ADC) represents an unique opportunity to treat tumors with high unmet medical need such as squamous cell carcinomas SCC, in particular lung SCC. We have generated an anti-C4.4a ADC consisting of a fully human monoclonal antibody linked to a non cell-permeable tubulin-binding auristatin cytotoxic agent (technology licensed from Seattle Genetics). In vitro, anti-C4.4a ADC showed an anti-proliferative efficacy (IC50) in the nanomolar range in cell lines endogenously expressing C4.4a (e.g. human lung cancer cell lines NCI-H292 and NCI-H322). High ADC stability and selectivity was observed in transfected A549 lung cancer cells over-expressing C4.4a compared to mock-transfected cells. In vivo, anti-C4.4a ADC exhibited a potent and selective antitumor activity in various human xenograft models (NCI-H292, NCI-H322, SCC-4) as well as in two SCC (Lu7433, Lu7343) and one pleomorphic (Lu7064) patient-derived lung cancer xenograft models. The in vivo efficacy is strictly target-dependent and selective as no efficacy was observed in C4.4a negative models (Fadu, Lu 7700) or using a non-specific isotype antibody ADC (NCI-H292, NCI-H322). A minimal effective dose (MED) as low as 1.9 mg/kg, response rates of up to 100%, and additive anti-tumor efficacy in combination with cisplatin were observed in the NCI-H292 xenograft model. Furthermore, it has been demonstrated that NCI-H292 were still sensitive to ADC treatment when tumors were allowed to regrow after the initial treatment cycle(). The anti-C4.4a ADC, which is fully cross-reactive with the mouse orthologue of C4.4a, was well tolerated at efficacious doses. Reversible skin reddening was observed only at doses markedly higher than the MED. In summary, anti-C4.4a ADC is a promising therapeutic candidate for the treatment of C4.4a-expressing squamous cell carcinomas, andpreclinical development has been initiated. Citation Format: Joerg Willuda, Lars Linden, Hans-Georg Lerchen, Charlotte Kopitz, Sven Golfier, Ute Bach, Joachim Schumacher, Beatrix Stelte-Ludwig, Oliver Von Ahsen, Claudia Schneider, Frank Dittmer, Rudolf Beier, Sherif El-Sheik, Jan Tebbe, Gabriele Leder, Heiner Apeler, Rolf Jautelat, Bertolt Kreft, Karl Ziegelbauer. Preclinical anti-tumor efficacy of an anti-C4.4a (LYPD3) antibody drug conjugate for the treatment of lung squamous cell carcinoma. [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 5445. doi:10.1158/1538-7445.AM2014-5445

About 80% of advanced prostate, breast and lung cancer patients develop bone metastases, which cause significant morbidity including chronic pain and pathologic fractures. Patients with bone metastases have a poor prognosis and currently there is no curative treatment. This is largely due to the unique biological microenvironments of bone where a wide range of growth factors promote tumor cell survival and tumor cells stimulate bone remodeling. Activation of PI3K plays important role in prostate and breast cancer cell proliferation, survival and metastasis. PI3K also mediates critical signaling pathways involving tumor and stromal cell interaction, including cancer-induced bone turnover and osteolysis. BAY 1082439 is a highly selective and potent PI3K inhibitor with balanced activity against PI3kα and PI3Kβ isoforms and currently being evaluated in a phase I clinical trial (N. Liu, et al. AACR 2012 Abstract #2799). Radium-223 dichloride (radium-223), a novel therapy recently approved for treatment of castration-resistant prostate cancer (CRPC) with bone metastases, selectively targets bone and kills metastatic cancer cells by the emission of alpha particles (Parker et al. N Engl J Med. 2013; 369:213). To identify more effective therapy to treat bone metastases, we evaluated the combination of PI3K inhibitor BAY 1082439 and radium-223 in vitro and in the syngeneic 4T1 metastatic breast cancer model in mice. BAY 1082439 and radium-223 showed synergistic anti-proliferative effects in vitro in both hormone receptor positive (HR+, MCF7 and LNCaP) and negative (4T1 and PC3) breast and prostate tumor cell lines. Pronounced synergistic effects on apoptosis induction were observed in MCF7 and LNCaP cell lines. In vivo, combination of BAY 1082439 at the maximum tolerable dose and radium-223 at the efficacious dose was well tolerated. BAY 1082439 significantly decreased whole body tumor burden as single agent (67% reduction, p=0.00156) and more effectively in combination with radium-223 (81% tumor reduction, p=0.0012), while the effect of radium-223 alone was not statistically significant. Furthermore, BAY 1082439 strongly inhibited tumor-induced osteolysis measured by radiography and led to 53.2% (p=0.16) and 84% (p=0.00698) reduction in total osteolytic area as single agent and in combination with radium-223, respectively. The frequency of soft tissue metastases was also decreased by treatment with BAY 1082439 alone (e.g. 73% reduction in kidney, p=0.012) and more pronounced with radium-223 combination treatment (e.g. complete inhibition of kidney metastasis, p=0.0003). In summary, our data indicate synergistic effects of the PI3K inhibitor BAY 1082439 and radium-223 in inhibiting tumor cell proliferation, survival, total and bone marrow tumor burden, and tumor-induced osteolysis, and warrant further clinical evaluation of this promising combination therapy for the treatment of cancer with bone metastases. Citation Format: Mari I. Suominen, Katja Fagerlund, Enrico Stasik, Andrea Haegebarth, Arne Scholz, Jukka P. Rissanen, Martin Kornacker, Dominik Mumberg, Jussi M. Halleen, Karl Ziegelbauer, Ningshu Liu. Combination of PI3K inhibitor BAY 1082439 with radium-223 is a promising treatment of cancer with bone metastases. [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 4022. doi:10.1158/1538-7445.AM2014-4022

Breast cancer metastasis to bone results in significant morbidity and poor prognosis. Radium-223 dichloride is an alpha-emitting calcium mimetic that localizes to bone and provides targeted radiation therapy. A phase III clinical study on prostate cancer patients with bone metastases showed that radium-223 dichloride improved overall survival (ALSYMPCA, Parker et al. ECCO/ESMO 2011). We have previously reported that radium-223 decreases osteolysis and tumor burden in bone in a mouse model of breast cancer bone metastasis in preventive and micro-metastatic settings (Suominen et al. AACR Annual Meeting 2012), as well as, in mice with established bone metastases (Suominen et al. AACR Annual Meeting 2011). Here, we investigated the effects of radium-223 dichloride monotherapy compared to and in combination with either doxorubicin or zoledronic acid on survival in a mouse model of established breast cancer bone metastasis. Human MDA-MB-231(SA)/GFP cells were inoculated intracardially into nude mice, and 15 days later, a single dose of vehicle, radium-223 dichloride (300 kBq/kg, iv injection) and/or zoledronic acid (0.1 mg/kg, sc injection) was administered. Doxorubicin (5 mg/kg, ip injection) was administered once weekly. Radium-223 dichloride monotherapy extended time to sacrifice (P = 0.039), unlike doxorubicin or zoledronic acid monotherapy which did not improve survival as compared to the vehicle group. Radium-223 dichloride in combination with zoledronic acid (P = 0.004) or doxorubicin (P < 0.001) also extended time to sacrifice as compared to the vehicle but did not provide additional survival benefit as compared to the radium-223 dichloride monotherapy. Histological examination revealed that radium-223 dichloride treatment induced tumor cell necrosis in bone metastases. Therefore, the effect of radium-223 dichloride in inducing double-strand breaks in cancer cells was evaluated by immunohistochemical staining of γ-H2AX molecules. A 3-fold increase in the number of tumor cells with double-strand breaks in the radium-223 dichloride-treated as compared to the vehicle control mice was observed (P < 0.001). This finding supports our previous observations that radium-223 dichloride has an effect on both tumor cells and osteoclasts. In conclusion, radium-223 dichloride therapy alone or in combination with doxorubicin or zoledronic acid increases survival in breast cancer bone metastasis mouse model via dual action by targeting tumor growth and osteolysis, both important players in the destructive vicious cycle of bone metastasis. Our findings strongly support the development of radium-223 dichloride for the treatment of patients with bone metastatic breast cancer. Citation Format: Mari I. Suominen, Jukka P. Rissanen, Rami Kakonen, Katja M. Fagerlund, Esa Alhoniemi, Dominik Mumberg, Karl Ziegelbauer, Jussi M. Halleen, Sanna-Maria Kakonen, Arne Scholz. Radium-223 dichloride monotherapy and combination therapy with zoledronic acid or doxorubicin improve survival in a mouse model of breast cancer bone metastasis. [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 LB-105. doi:10.1158/1538-7445.AM2013-LB-105

Targeted therapies in combination with cytotoxic agents are promising future treatment regimens for women with resistant ovarian cancer. Current standard treatment of serous ovarian cancer is a platinum- (carboplatin) based chemotherapy, usually combined with taxanes. A 90% cure rate is observed in early stage ovarian cancers, however, in advanced stages, about 20-30% of patients never have a remission or will suffer from recurrent disease within 5 years. One major reason is the development of a resistance against the platinum-based chemotherapy. In order to identify possible novel therapeutic targets for platinum-resistant ovarian cancer, we compared gene expression and oncogenic kinase phosphorylation patterns in the human ovarian cancer cell line A2780 and its platinum-resistant variant A2780cis in vitro and in vivo.In vivo, the untreated A2780cis-derived tumor model showed reduced proliferation activity and enhanced apoptosis, while carboplatin treatment failed to induce tumor reduction, as compared to the sensitive A2780-derived tumor model. Analysis of 47 kinase phosphorylation sites in carboplatin-treated A2780 and A2780cis derived tumors revealed that Akt was hyperphosphorylated at Ser273 but not altered during treatment in resistant tumors, whereas Akt phosphorylation was weak and further reduced during treatment in sensitive tumors. This was associated with an unchanged p53 phosphorylation status in resistant tumors during treatment. In line with this, the novel allosteric Akt inhibitor BAY 10001931 resensitized A2780 and A2780cis cells to carboplatin in vitro. To investigate if the Akt phosphorylation status correlated with an activation of downstream targets, RNA of A2780 and A2780cis vehicle- or carboplatin treated tumors was hybridized onto an Illumina Bead Chip array. Here, linear model analysis identified GDF15 as one of four p53-induced target genes possibly involved in the development of chemoresistance. GDF15 protein expression was highly induced during carboplatin treatment in both cell lines, whereas basal GDF15 expression was higher in resistant cells than in sensitive A2780 cells in vitro and in vivo. This was correlated with a higher secretion of GDF15 into the serum of A2780cis-tumor-bearing mice. Similar tendencies were observed in vitro. Interestingly, simultaneous Akt inhibition with BAY 10001931 markedly reduced the carboplatin-induced upregulation of GDF15 in A2780 and A2780cis cells. Additionally, shRNA-mediated knockdown of GDF15 in resistant A2780cis cells also abrogated the phosphorylation of Akt in vitro. These results indicate that a causal relationship exists between Akt activation and GDF15 expression in resistant ovarian cancer cells. Together, a combination treatment with an Akt inhibitor and carboplatin of GDF15-expressing ovarian cancer cells might represent a novel approach to treat resistant ovarian cancer. Citation Format: Julia C. Meier, Bernard Haendler, Anette Sommer, Andrea Hägebarth, Georg Beckmann, Bertolt Kreft, Karl Ziegelbauer, Charlotte C. Kopitz. Inhibition of Akt signaling: a strategy to overcome platinum resistance in ovarian 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 3383. doi:10.1158/1538-7445.AM2013-3383

Cancer therapy is evolving to a detailed molecular analysis of the patient's tumor followed by treatment with selective drug(s) targeting the individual properties of the tumor based upon identification of prognostic and predictive biomarkers. This requires comprehensive, highly sensitive test systems. We therefore sought to develop a sensitive diagnostic test for functionally profiling a spectrum of signaling pathway proteins in tumor samples. Here, we evaluated and compared two assay platforms: CEER (Collaborative Enzyme Enhanced Reactive-Immunoassay; Prometheus Labaratories) and MSD (Meso Scale Discovery). Breast, lung, and prostate cancers as well as glioblastoma and melanoma model cell lines (in all 10 cell lines) with various oncogenic pathway signatures were treated with different concentrations of PI3K inhibitor (BAY 806946) or an inhibitor targeting HER1 and HER2 (Lapatinib). Cells were lysed and the activation status as well as abundance of ten pathway proteins (HER1, HER2, cMET, PI3K, AKT, ERK, MEK, PRAS40, RPS6, and P70S6K) was measured by MSD at Bayer in Berlin and by CEER in a blinded fashion at Prometheus Laboratories. Target-specific inhibition (IC50) and downstream signal modulations were determined and compared. Overall we found a high concordance between the two assays. While target specific inhibitions were observed in relevant cell lines, varying mechanisms of treatment resistance due to redundant pathway activation, feedback loop or pathway cross talks were observed between the PI3K/AKT and RAS/ERK pathways. This study shows that sensitive immunoassays are a suitable tool for the detection and monitoring of biomarkers and provides new insights into the mode of action of targeted agents. Comprehensive profiling of signaling pathways holds promise as an approach to personalize selection of anticancer therapy. Citation Format: Antje Stresemann, Oliver von Ahsen, Oliver Politz, Phillip Kim, Sharat Singh, Khusru Asadullah, Karl Ziegelbauer, Thomas Krahn. Pathway profiling for personalized medicine: Comparison of two immunoassays. [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 3497. doi:10.1158/1538-7445.AM2013-3497

Biliary tract cancer (BTC) includes a spectrum of genetically diverse invasive adenocarcinomas consisting intra- and extrahepatic cholangiocarcinoma (CC), and carcinoma arising from the gall bladder (GBC). Only 10% of patients diagnosed with early-stage disease are considered candidates for surgical resection, which offers the only chance for cure. The prognosis is poor for the majority of patients with locally advanced or metastatic BTC with a median survival of under a year. Recently, high incidence of oncogenic mutations such as KRAS, BRAF, PIK3CA and EGFR were found in BTC patients, potentially rendering these patients sensitive to inhibitors of these pathways. We report on preclinical evaluation of highly potent and selective PI3K inhibitor BAY 80-6946 and allosteric MEK inhibitor BAY 86-9766 in BTC as single agent and in combination. In vitro profiling a panel of BTC tumor cell lines indicated that KRASmut and PIK3CAmut cell lines were sensitive to MEKi (BAY 86-9766) and PI3Ki (BAY 80-6946), respectively, compared to the cell lines with wild type KRAS and PIK3CA. However, although MEKi BAY 86-9766 inhibited proliferation with IC50s of 147 nM in HuCCT-1 (KRASG12D) and 137 nM in EGI-1 (KRASG12D) cell lines, it can neither reach IC90 nor induce tumor apoptosis at concentration up to10 µM, indicating activation of additional survival pathways in these KRASmut BTC cell lines. When combining MEKi BAY 86-9766 with PI3Ki BAY 80-6946 in KRASmut EGI-1, IC90 could be reached at much lower concentrations, e.g. 108 nM of MEKi with 81 nM of PI3Ki in EGI-1 cells. Furthermore, strong activation (10-fold) of caspase3/7 was observed when EGI-1 cells were treated with 700 nM of MEKi with 230 nM of PI3Ki. To confirm the observed in vitro synergies, anti-tumor efficacy of BAY 80-6946 and BAY 86-9766 in mono- as well as combination therapy was evaluated in a set of human BTC xenograft models. Similar to the in vitro results, robust in vivo antitumor activities were observed in the PI3Ki and MEKi combination groups compared to monotherapy arms. Further detailed molecular mechanism on the synergistic inhibition of BTC tumor growth by combination of PI3K and MEK inhibitors, as well as in vitro and in vivo comparison to the chemotherapeutics will be presented. In conclusion, PI3Ki and MEKi combination therapy might open a new treatment paradigm for BTC to be developed in clinic. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 869. doi:1538-7445.AM2012-869

Radium-223 chloride is an alpha-emitting radiopharmaceutical that has been shown to improve overall survival in the phase III clinical study (ALSYMPCA) in the treatment of castration resistant prostate cancer with bone metastases (Parker et al. ECCO/ESMO 2011, abstract LBA1). As a calcium mimetic, radium-223 localizes to bone, where the emission of alpha-particles provide an efficient and localized radiation treatment to metastatic skeletal tumor lesions. As previously reported, radium-223 decreases osteolysis and also tumor burden in established model of breast cancer bone metastasis in nude mice (Suominen et al. AACR 2011, abstract 2664). In this study, we investigated the effects of radium-223 on the development of breast cancer bone metastases when administered in the preventive and micro-metastastic settings. The effects of radium-223 were studied in a breast cancer bone metastatic model using intra-cardiac injection of human MDA-MB-231SA/GFP cells in nude mice at day 0. In the first study, the animals were randomized into four groups (n=7) and dosed with either vehicle or a single dose of radium-223 (300 kBq/kg) at days -1, 2 or 15. Three mice were also sacrificed at day 2 for immunohistochemical (pan-cytokeratin and GFP) detection of tumor cells in the bone marrow. Radiography and fluorescence imaging were performed at sacrifice (day 25). Tumor burden was also analysed from mid-sagittal sections of both hind limbs. The second study was a survival study, in which the animals were randomized to three groups (n=12) and dosed with either vehicle or a single dose of radium-223 (300 kBq/kg) at days -1 or 2. Radiography and fluorescence imaging were performed only on animals surviving to day 50. In the first study there were disseminated tumor cells in the bone marrow of all three animals (altogether 18/18 positive sections) sacrificed at day 2, as detected by IHC staining. Radium-223 decreased whole body tumor burden by 81, 84 and 55% and osteolysis by 98, 99.6 and 82%, when administered at days -1, 2 or 15, respectively. No tumor foci were detected in the bone sections of mice treated at days -1 and 2, and tumor area was decreased by 65% in animals treated at day 15. In the survival study, median survival was 24.5 days in the control group, 39.5 days (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5712. doi:1538-7445.AM2012-5712

BAY 94-9343 is a novel antibody drug conjugate consisting of a fully human monoclonal antibody directed against mesothelin conjugated with the maytansinoid derivative DM4 by a linker containing a disulfide bridge (SPDB) and currently in Phase I clinical testing. Mesothelin is expressed in a range of tumor types, such as mesothelioma, ovarian cancer, pancreatic cancer and lung cancer. Membrane-anchored forms may play a role in cellular adhesion and metastasis. Here we report on the correlation of tumor mesothelin antigen expression levels, as determined by a newly developed mesothelin IHC staining protocol, and the efficacy of BAY 94-9343 in 13 preclinical tumor models. Further, mesothelin levels in xenograft tumors were compared to those in clinical samples of human tumors. Efficacy of BAY 94-9343 in tumor models with high mesothelin expression levels were generally higher compared to models with low mesothelin expression. OVCAR6677, OVCAR6668 and MIAPaCa-meso models characterized by high expression of mesothelin showed 90-100 % tumor growth inhibition, when treated with 2.5 mg/kg BAY 94-9343. PAXF736 and PAXF1881, tumor models with no mesothelin expression, did not show tumor growth inhibition at 2.5 mg/kg mesothelin-ADC, but responded to higher doses. Analysis of mesothelin expression by IHC using human tumor samples (mesothelioma, ovarian and pancreatic cancer) demonstrated that tumor cell mesothelin expression varied both within and between tumor types. IHC scoring revealed that 92% of mesothelioma tumors, 80% of ovarian tumors, and 63% of pancreatic cancer tumors assayed were mesothelin positive. Mesothelin expression was stable when comparing primary ovarian cancers with relapsing tumor tissue from the same patient. Maximum mesothelin expression in ovarian cancer is at least 400,000 antigenic sites per cell determined by Quantibrite technology. In preclinical models analysis of serum mesothelin extracellular domain (ECD) by ELISA revealed an initial rise in serum mesothelin ECD as early as 1 days after 10 mg/kg BAY 94-9343 followed by a decrease to below-baseline levels. Increased levels of the apoptosis marker CK18 were observed following BAY 94-9343 administration. Changes in serum mesothelin ECD levels as well as CK18 will be explored as an early pharmacodynamic (PD) response markers in clinical trials for BAY 94-9343. In summary, these results demonstrate that targeted delivery of DM4 resulted in higher efficacy of BAY 94-9343 in tumor models with high mesothelin expression. Responsive xenograft models were representative of clinical samples with respect to mesothelin expression scored by IHC. Quantification of mesothelin by IHC is a candidate stratification biomarker for the antibody drug conjugate BAY 94-9343 currently in Phase I testing. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2726. doi:1538-7445.AM2012-2726

Introduction: BAY 869766 (RDEA119) is an orally available, allosteric MEK1/2 inhibitor, targeting the central switch in the MAP kinase pathway. BAY 869766 demonstrates potent in vitro and in vivo activity in different human cancer cell lines and preclinical models. BAY 869766 is currently in clinical phase I/II studies in the indications HCC (hepatocellular carcinoma) and pancreatic cancer (PaCa). In the described preclinical studies BAY 869766 was studied in two orthotopic, syngeneic HCC and PaCa models and five subcutaneous PaCa xenografts as a single agent and combination therapy. In contrast to subcutaneous xenografts, orthotopic syngeneic models provide additional important information about microenvironment-dependent tumor growth, metastatic spread, immune response and survival. Results: To establish a syngeneic, orthotopic HCC model, the rat HCC cell line MH3924a was first evaluated in vitro. BAY 869766 induced antiproliferative effects in MH3924a HCC cells in monotherapy and showed synergistic effects in combination with sorafenib. In the corresponding rat allograft model efficacy of BAY 869766 in mono- and combination therapy with sorafenib was confirmed. Primary tumor growth and metastatic spread were strongly reduced. These effects were accompanied by a reduction of pERK levels, proliferation index and microvessel density. Finally, the median overall survival in combination treatment was increased by 100% in comparison with vehicle treatment. To characterize BAY 869766 in an orthotopic syngenic PaCa model, the Panc02 mouse allograft model was established. BAY 869766 was tested in mono- and combination therapy with Gemcitabine. Especially, combination treatment led to significant lower tumor weight, reduction of metastatic spread and increase of median survival by 135%. To test BAY 869766 in further clinical relevant pancreatic cancer models, five patient-derived pancreatic cancer tumor samples were subcutaneously implanted in mice and subsequently treated with BAY 869766. Significant tumor growth inhibition in these patient-derived pancreatic cancer xenograft models was observed (Treatment/Control ratios from 23,5% − 44%). Conclusion: These preclinical results support the clinical development of the MEK inhibitor BAY 869766 in HCC and pancreatic cancer. Inhibition of Ras-Raf-MEK-Erk-signaling, direct antiproliferative effects on tumor cells, reduction of metastatic spread and anti-angiogenic properties were demonstrated in these studies. In combination with the respective SoC drugs a significant benefit in survival could be achieved in the orthotopic models. 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 B247.

Monoclonal antibodies have proven to be very effective in the treatment of various cancers, including solid tumors. For example, HERCEPTIN® and Erbitux® are successfully used to treat HER2-positive breast cancer and EGFR-positive colorectal cancer, respectively. Conjugation of cytotoxic drugs to antibodies represents a promising approach to improve cancer therapy. Antibody-drug conjugates (ADCs) are able to deliver highly potent toxophores to tumors while at the same time reducing systemic toxicity. Promising efficacy and tolerability profiles of ADCs have been observed in clinical trials including Hodgkin lymphoma (brentuximab vedotin) and breast cancer (trastuzumab-DM1), thus, development of new ADCs targeting tumor- associated antigens has potential to identifiy novel cancer therapeutics. Mesothelin, a glycoprotein expressed in mesothelial cells found in the membrane lining of the peritoneal and pleural cavities, is overexpressed in all mesotheliomas as well as many ovarian and pancreatic cancers. Due to its limited expression on normal tissues and higher expression in a number of tumor types, mesothelin represents an attractive ADC target. BAY 94-9343 consists of a fully human anti-mesothelin IgG1 antibody conjugated to the potent tubulin-binding drug DM4 with an average of 3.2 drug molecules per antibody. The resulting ADC bound to human recombinant mesothelin with high affinity (Kd = 15nM) leading to antigen-dependent internalization and potent cytotoxicity (nanomolar range in vitro IC50) in tumor cells that express mesothelin either endogenously or exogenously, but not in mesothelin-negative cells. In vivo, BAY 94-9343 demonstrated dose-dependent, mesothelin-specific anti-tumor efficacy in subcutaneous and orthotopic xenograft models at doses between 2.5 and 10 mg/kg using a Q3Dx3 schedule. Endogenously expressing mesothelin tumor models included sc and orthotopic OVCAR3 (ovarian), sc BxPC-3 (pancreatic) and sc NCI-H226 (mesothelioma). Furthermore, in mesothelin-positive patient-derived preclinical tumor models of both platinum-resistant ovarian cancer and gemcitabine-resistant pancreatic cancer, BAY 94-9343 exhibited high anti-tumor efficacy leading to partial and complete tumor regressions with a 10mg/kg Q3Dx3 dosing schedule. This ADC was well tolerated in mice at 10mg/kg (Q3Dx3) without any evidence of body weight loss, compared to either cisplatin or gemcitabine treatments. In summary, BAY 94-9343 is a mesothelin-targeted ADC with promising preclinical anti-tumor activity for mesothelin-positive tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1754. doi:10.1158/1538-7445.AM2011-1754

Alpharadin is an alpha-emitting radium isotope currently in phase III clinical study for the treatment of castration resistant prostate cancer bone metastases. As a calcium mimetic Alphapharmaceutical, radium localizes to bone, where the emitted alpha-particles provide an efficient and localized radiation treatment to the metastatic skeletal tumor lesion. While promising clinical data in predominately osteoblastic bone metastases such as prostate cancer have already been obtained (Nilsson, Lancet Oncol 2007), robust clinical evidence for the activity of Alpharadin in osteolytic bone metastases associated with other cancer entities is lacking. As previously reported (Henriksen, Cancer Res 2002), Alpharadin increases symptom-free survival in an osteolytic breast cancer bone metastasis model in nude rats. Besides this study on the therapeutic efficacy in a preclinical skeletal metastasis tumor model it is still unclear how Alpharadin affects the vicious cycle of tumor growth and osteolysis. We report here the effects of Alpharadin on differentiation and activity of human osteoclasts in vitro and on treatment of established breast cancer bone metastases in vivo. The in vitro effects of Alpharadin (50-1600 Bq/ml, n=8/group) on osteoclasts were studied using human osteoclast precursor cells cultured on bovine bone slices. Alpharadin was added in the osteoclast differentiation assay at day 0, and in the activity assay at day 7. Secreted tartrate-resistant acid phosphatase isoform 5b (TRACP 5b) was measured at day 7 as a marker of osteoclast differentiation and secreted C-terminal cross-linked telopeptides of type I collagen (CTX) at day 10 as a marker of resorption during days 7-10. The in vivo effects were studied using a model where human MDA-MB-231SA/GFP cells were inoculated intracardially in nude mice. The animals were randomized to two groups (n=15) based on body weight and presence of osteolytic lesions at day 14. Either vehicle or Alpharadin (300 kBq/kg) were administered iv at day 15. Radiography and fluorescence imaging were performed at day 14 and at sacrifice (day 25). Alpharadin inhibited dose dependently differentiation of osteoclasts in vitro at all tested concentrations, but had no effect on resorption activity of mature osteoclasts. In vivo, Alpharadin decreased whole body tumor burden by 43% as quantified by fluorescence imaging and osteolysis by 56% compared to the vehicle control group. Furthermore, Alpharadin treatment completely prevented tumor associated cachexia observed in the control group. In summary, Alpharadin inhibits differentiation of osteoclasts and progression of established, osteolytic breast cancer bone metastases. These preclinical findings strongly support the clinical development of Alpharadin in additional cancer indications which form predominantely osteolytic bone metastasis, e.g. breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2664. doi:10.1158/1538-7445.AM2011-2664