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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 spindle assembly checkpoint represents a highly conserved surveillance mechanism which safeguards correct chromosome segregation by delaying anaphase onset until all chromosomes are properly bi-oriented on the spindle apparatus. Non-catalytic functions of the mitotic kinase BUB1 (budding uninhibited by benzimidazoles 1) were reported to be essential for spindle assembly checkpoint activation. In contrast, the catalytic function of BUB1 plays a minor role in spindle assembly checkpoint activation but is required for chromosome arm resolution and positioning of the chromosomal passenger complex for resolution of spindle attachment errors. Here, we disclose for the first time the structure and functional characterization of a novel, first-in-class Bub1 kinase inhibitor. Medicinal chemistry efforts resulted in BAY 1816032 featuring high potency, long target residence time and good oral bioavailablity. It inhibits BUB1 enzymatic activity with an IC50 of 7 nanomol/L, shows slow dissociation kinetics resulting in a long target residence time of 87 min, and an excellent selectivity on a panel of 395 kinases. Mechanistically BAY 1816032 abrogated nocodazole-induced Thr-120 phosphorylation of the major BUB1 target protein histone H2A in HeLa cells with an IC50 of 29 nanomol/L, induced lagging chromosomes and mitotic delay. Persistent lagging chromosomes and missegregation were observed upon combination with low concentrations of paclitaxel. Single agent BAY 1816032 inhibited proliferation of various tumor cell lines with a median IC50 of 1.4 micromol/L and demonstrated synergy or additivity with paclitaxel or docetaxel in almost all cell lines evaluated (minimal combination index 0.3). In tumor xenograft studies BAY 1816032 only marginally inhibited tumor growth as single agent upon oral administration, however, upon combination with paclitaxel or docetaxel a strong and statistically significant reduction of tumor size as compared to the respective monotherapy was observed. Intratumoral levels of phospho-Thr120 H2A were found to be strongly reduced, and no hints on drug-drug interactions were found. In line with the good tolerability in xenograft studies, no relevant findings from non-GLP 2 weeks toxicological studies in rat and dog were reported. Our findings validate the innovative concept of interference with mitotic checkpoints and justify clinical proof of concept studies evaluating BUB1 inhibitor BAY 1816032 in combination with taxanes in order to enhance their efficacy and potentially overcome resistance. Citation Format: Gerhard Siemeister, Anne Mengel, Wilhelm Bone, Jens Schröder, Sabine Zitzmann-Kolbe, Hans Briem, Amaury E. Fernández-Montalván, Simon Holton, Ursula Mönning, Oliver von Ahsen, Sandra Johanssen, Arwed Cleve, Marion Hitchcock, Kirstin Meyer, Franz von Nussbaum, Michael Brands, Dominik Mumberg, Karl Ziegelbauer. BAY 1816032, a novel BUB1 kinase inhibitor with potent antitumor activity [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 287. doi:10.1158/1538-7445.AM2017-287

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, suppression of general translation, induction of DNA repair, cell survival or even 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 (e.g. in oncogene-driven tumor cells). 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. Herein we report the identification of the potent, highly selective and orally available ATR inhibitor BAY 1895344 by a collaborative effort involving medicinal chemistry, pharmacology, DMPK and computational chemistry. The chemical structures of lead compound BAY-937 and clinical candidate BAY 1895344 as well as the main SAR trends within this novel class of naphthyridine derivatives will be disclosed for the first time. The novel lead compound BAY-937 revealed promising inhibition of ATR (IC50 = 78 nM) and high kinase selectivity in vitro. In cellular mechanistic assays BAY-937 inhibited hydroxyurea-induced H2AX phosphorylation (IC50 = 380 nM) demonstrating the anticipated mode of action. Moreover, BAY-937 was shown to inhibit proliferation of a variety of tumor cell lines with low- to sub-micromolar IC50 values. In initial xenograft studies, BAY-937 revealed moderate activity in monotherapy and in combination with cis-platin. However, BAY-937 also revealed low aqueous solubility, low bioavailability (rat) and activity in the hERG patch clamp assay. Extensive lead optimization efforts led to the identification of the novel, orally available ATR inhibitor BAY 1895344. In vitro, BAY 1895344 was shown to be a very potent and highly selective ATR inhibitor (IC50 = 7 nM), which potently inhibits proliferation of a broad spectrum of human tumor cell lines (median IC50 = 78 nM). In cellular mechanistic assays BAY 1895344 potently inhibited hydroxyurea-induced H2AX phosphorylation (IC50 = 36 nM). Moreover, BAY 1895344 revealed significantly improved aqueous solubility, bioavailability across species and no activity in the hERG patch-clamp assay. BAY 1895344 also demonstrated very promising efficacy in monotherapy in DNA damage deficient tumor models as well as combination treatment with DNA damage inducing therapies. The start of clinical investigation of BAY 1895344 is planned for early 2017. Citation Format: Ulrich T. Luecking, Julien Lefranc, Antje Wengner, Lars Wortmann, Hans Schick, Hans Briem, Gerhard Siemeister, Philip Lienau, Christoph Schatz, Benjamin Bader, Gesa Deeg, Franz von Nussbaum, Michael Brands, Dominik Mumberg, Karl Ziegelbauer. Identification of potent, highly selective and orally available ATR inhibitor BAY 1895344 with favorable PK properties and promising efficacy in monotherapy and combination 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 983. doi:10.1158/1538-7445.AM2017-983

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

Due to its almost universal resistance to chemotherapy, metastasized melanoma remains a major challenge in clinical oncology. Given that phosphatidyl inositol-3 kinase (PI3K) activation in melanoma cells is associated with poor prognosis, disease progression and resistance to chemotherapy, the PI3K-Akt signalling pathway is a promising therapeutic target for melanoma treatment. We analysed six human melanoma cell lines for their constitutive activation of Akt and then tested two representative lines, A375 and LOX, for their susceptibility to PI3K-inhibition by the highly specific small molecule inhibitor, BAY 80-6946. In addition, the effect of BAY 80-6946 on A375 and LOX melanoma cells was assessed in vivo in a xenotransplantation mouse model. We provide experimental evidence that specifically inhibiting the PI3K pathway and phosphorylation of Akt by this novel compound results in antitumoral activities including inhibition of proliferation, induction of apoptosis and cell cycle arrest in vitro and in vivo. However, the susceptibility did not show a clear-cut pattern and differed between the melanoma cell lines tested, resulting in in vivo growth inhibition of A375 but not LOX melanoma cells. Thus, in some cases BAY 80-6946 or related compounds may be a valuable addition to the therapeutic armamentarium.

Molecular mechanisms underlying the development of resistance to platinum-based treatment in patients with ovarian cancer remain poorly understood. This is mainly due to the lack of appropriate in vivo models allowing the identification of resistance-related factors. In this study, we used human whole-genome microarrays and linear model analysis to identify potential resistance-related genes by comparing the expression profiles of the parental human ovarian cancer model A2780 and its platinum-resistant variant A2780cis before and after carboplatin treatment in vivo. Growth differentiation factor 15 (GDF15) was identified as one of five potential resistance-related genes in the A2780cis tumor model. Although A2780-bearing mice showed a strong carboplatin-induced increase of GDF15 plasma levels, the basal higher GDF15 plasma levels of A2780cis-bearing mice showed no further increase after short-term or long-term carboplatin treatment. This correlated with a decreased DNA damage response, enhanced AKT survival signaling and abrogated cell cycle arrest in the carboplatin-treated A2780cis tumors. Furthermore, knockdown of GDF15 in A2780cis cells did not alter cell proliferation but enhanced cell migration and colony size in vitro. Interestingly, in vivo knockdown of GDF15 in the A2780cis model led to a basal-enhanced tumor growth, but increased sensitivity to carboplatin treatment as compared to the control-transduced A2780cis tumors. This was associated with larger necrotic areas, a lobular tumor structure and increased p53 and p16 expression of the carboplatin-treated shGDF15-A2780cis tumors. Furthermore, shRNA-mediated GDF15 knockdown abrogated p27 expression as compared to control-transduced A2780cis tumors. In conclusion, these data show that GDF15 may contribute to carboplatin resistance by suppressing tumor growth through p27. These data show that GDF15 might serve as a novel treatment target in women with platinum-resistant ovarian cancer.

Mitochondrial energy metabolism and Krebs cycle activities are developmentally regulated in the life cycle of the protozoan parasite Trypanosoma brucei. Here we report cloning of a T. brucei aconitase gene that is closely related to mammalian iron-regulatory protein 1 (IRP-1) and plant aconitases. Kinetic analysis of purified recombinant TbACO expressed inEscherichia coli resulted in a K m(isocitrate) of 3 ± 0.4 mm, similar to aconitases of other organisms. This was unexpected since an arginine conserved in the aconitase protein family and crucial for substrate positioning in the catalytic center and for activity of pig mitochondrial aconitase (Zheng, L., Kennedy, M. C., Beinert, H., and Zalkin, H. (1992)J. Biol. Chem. 267, 7895–7903) is substituted by leucine in the TbACO sequence. Expression of the 98-kDa TbACO was shown to be lowest in the slender bloodstream stage of the parasite, 8-fold elevated in the stumpy stage, and increased a further 4-fold in the procyclic stage. The differential expression of TbACO protein contrasted with only minor changes in TbACO mRNA, indicating translational or post-translational mechanisms of regulation. Whereas animal cells express two distinct compartmentalized aconitases, mitochondrial aconitase and cytoplasmic aconitase/IRP-1, TbACO accounts for total aconitase activity in trypanosomes. By cell fractionation and immunofluorescence microscopy, we show that native as well as a transfected epitope-tagged TbACO localizes in both the mitochondrion (30%) and in the cytoplasm (70%). Together with phylogenetic reconstructions of the aconitase family, this suggests that animal IRPs have evolved from a multicompartmentalized ancestral aconitase. The possible functions of a cytoplasmic aconitase in trypanosomes are discussed.

We have developed a cellular, target-specific high-throughput assay for the detection of topoisomerase I inhibitors. Topoisomerase I is a nonessential enzyme involved in controlling DNA topology. Topoisomerase I is the target of anticancer drugs such as camptothecin as well as a candidate target for new antifungal drugs. A wild-type Saccharomyces cerevisiae strain and its isogenic topoisomerase I deletion mutant (DeltatopI) were labeled with S65T and wild-type green fluorescent protein (GFP), respectively. We showed that the growth of such a pair of S. cerevisiae strains labeled with this GFP combination can be independently quantified after both strains were mixed. When growth of the mixture of wild-type and DeltatopI strain was monitored in the presence of compounds, only growth of the wild-type strain was inhibited by the topoisomerase I-specific drug camptothecin. In contrast, amphotericin B, a broad-spectrum antifungal drug, inhibited growth of both strains. The two strains were used to screen compound libraries. While 0.9% of all compounds inhibited growth of both strains, only 0.06% inhibited the wild-type but not the DeltatopI strain. Thus, by using a DeltatopI strain as internal control in the same assay mixture, the number of candidate topoisomerase I inhibitors to be retested could be reduced by more than 90%. Further applications of this type of S. cerevisiae-based cellular high-throughput assays will be discussed.

BAY 10-8888, a cyclic β-amino acid, exerts its antifungal activity by inhibition of isoleucyl-tRNA synthetase activity after accumulation to a millimolar concentration inside the cell. We have selected and characterized BAY 10-8888-resistant Candida albicans mutants. Reduced BAY 10-8888 accumulation as well as increased isoleucyl-tRNA synthetase activity was observed in these mutants. Some of the mutants were cross-resistant to cispentacin, a structurally related β-amino acid, while sensitivities to 5-fluorocytosine and fluconazole remained unchanged in all mutants. All except two in vitro-resistant mutants were pathogenic in a murine candidiasis model, and BAY 10-8888 failed to cure the infection. Furthermore, we have characterized BAY 10-8888 transport and isoleucyl-tRNA synthetase activity in several Candida tropicalis strains which showed MICs higher than those of other Candida strains. An analysis of the C. tropicalis strains revealed that intracellular concentrations of BAY 10-8888 were in the millimolar range, comparable to those for C. albicans . However, these isolates expressed isoleucyl-tRNA synthetase activities about fourfold higher than those for C. albicans . To test the possibility of resistance modeling, we determined the correlations between the intracellular concentration of BAY 10-8888, the specific activity of isoleucyl-tRNA synthetase, the number of free, i.e., noninhibited, isoleucyl-tRNA synthetase molecules/cell, and growth, assuming a linear relation. We found significant correlations between growth and the intracellular concentration of BAY 10-8888 and between growth and the number of free isoleucyl-tRNA synthetase molecules/cell, but not between growth and the specific activity of isoleucyl-tRNA synthetase.

In contrast to other eukaryotic cells the pathogenic yeast Candida albicans is resistant to many structurally unrelated metabolic inhibitors. Reduced permeability due to the cell wall and/or altered plasma membrane composition is thought to be at least partly responsible for this phenomenon. To study the uptake of low molecular weight compounds into C. albicans we developed a dual labelling method. Intact cells, metabolically inactivated cells, spheroplasts or membrane fragments of C. albicans were incubated with various [14C]-labelled compound in the presence of [3H]-labelled water. After separation of cells and supernatant isotope ratios [3H]/[14C] were determined. Quotients of the isotope ratios from cells and supernatant, called enrichment coefficients, were calculated under all four conditions. The enrichment coefficients indicated whether a compound can enter C. albicans cells, is trapped within the cell wall, is enriched in the lipophilic membrane compartment, is actively accumulated or actively exported by multidrug resistance carriers. We used six structurally unrelated compounds to test our method. We found no evidence for a general impermeability of C. albicans.

A neutral trehalase gene, NTC1, from the human pathogenic yeast Candida albicans was isolated and characterized. An ORF of 2724 bp was identified encoding a predicted protein of 907 amino acids and a molecular mass of 104 kDa. A single transcript of approxymately 3·2 kb was detected by Northern blot analysis. Comparison of the deduced amino acid sequence of the C. albicans NTC1 gene product with that of the Saccharomyces cerevisiae NTH1 gene product revealed 57% identity. The NTC1 gene was localized on chromosome 1 or R. A null mutant (Δntc1/Δntc1) was constructed by sequential gene disruption. Extracts from mutants homozygous for neutral trehalase deletion had only marginal neutral trehalase activity. Extracts from heterozygous mutants showed intermediate activities between extracts from the wild-type strain and from the homozygous mutants. The null mutant showed no significant differences in pathogenicity as compared to the wild-type strain in a mouse model of systemic candidiasis. This result indicates that the neutral trehalase of C. albicans is not a potential target for antifungal drugs.

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.

Isocitrate dehydrogenase 1 (IDH1) is a metabolic enzyme that is frequently mutated in certain cancers, with incidence rates ranging from 7-90% for glioma, chondrosarcoma, intrahepatic cholangiocarcinoma and AML. Wildtype IDH1 (wtIDH1) catalyzes the conversion of isocitrate to α-ketoglutarate (αKG), while tumor-associated mutant IDH1 (mIDH1) catalyzes a rogue reaction: the production of 2-hydroxyglutarate (2-HG) from αKG. 2-HG therefore represents an “oncometabolite” that is believed to play a role in cancer by interfering with αKG-dependent enzymes, which in turn causes hypermethylation of histones/DNA and a block of normal cellular differentiation. Mutant IDH1 is a “driver” oncogene and the inhibition of this altered enzyme will decrease the growth of mIDH1 dependent tumors. We report for the first time the preclinical profile and structure of BAY 1436032, a novel selective mIDH1 inhibitor. An optimization program based on a high throughput screening resulted in the identification of the clinical candidate BAY 1436032 for the treatment of mIDH1 dependent cancer. BAY 1436032 is a double-digit nanomolar and selective pan-inhibitor of the enzymatic activity of various IDH1-R132X mutants in vitro and displayed potent inhibition of 2-HG release (nanomolar range) in patient derived and engineered cell lines expressing different IDH1 mutants. In line with the proposed mode of action, a concentration-dependent lowering of 2-HG was observed in vitro accompanied by differentiation and maturation of mIDH1 tumor cells. Furthermore, BAY 1436032 showed a favourable selectivity profile against wtIDH1/2 and a large panel of off-targets in vitro. To the best of our knowledge we were able to show for the first time single agent in vivo efficacy in mIDH1 patient derived glioma and intrahepatic cholangiocarcinoma solid tumor models with this clinical candidate along with monitoring of intratumoral 2-HG levels as a predictive biomarker. The BBB penetration profile of BAY 1436032 is further supported by preclinical data on in vivo brain-plasma ratios. In conclusion, our data provide in vitro and in vivo proof of concept for BAY 1436032 as a potent and highly selective inhibitor of mutant forms of IDH1. The start of a Phase I study with BAY 1436032 is currently in preparation to determine the safety, tolerability, pharmacokinetics and preliminary anti-tumor and pharmacodynamic biomarker responses in patients with solid tumors. Citation Format: Olaf Panknin, Stefan Pusch, Lena Herbst, Stefan Kaulfuss, Katja Zimmermann, Hartmut Rehwinkel, Roland Neuhaus, Sven Ring, Michael Brüning, Claudia Stark, Katja Prelle, Martin Michels, Michael Jeffers, Holger Hess-Stumpp, Karl Ziegelbauer, Michael Brands, Alwin Krämer, Andreas von Deimling. BAY 1436032: A highly selective, potent and orally available inhibitor of mutant forms of IDH1. [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 2645.

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.

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. Alterations in FGFR encoding genes are frequently observed in a variety of solid tumors including lung, gastric, breast and urothelial cancer. Therefore, targeting FGFRs using selective FGFR inhibitors is an attractive therapeutic approach to treat cancer patients. BAY 1163877 is an orally active, highly potent and selective small molecule FGFR-1, -2 and -3 kinase inhibitor. We disclose for the very first time its discovery and chemical structure. BAY 1163877 was derived from a de novo structure-based design approach and medicinal chemistry optimization. Data on the structure activity relationship and the pharmacokinetic profile of the benzothiophenyl-pyrrolotriazine structure class will be presented. Based on its favorable preclinical profile, BAY 1163877 is currently being investigated in a Phase 1 clinical trial (NCT01976741). Citation Format: Marie-Pierre L. Collin, Mario Lobell, Walter Huebsch, Dirk Brohm, Mélanie Héroult, Klemens Lustig, Sylvia Gruenewald, Ulf Boemer, Rolf Jautelat, Holger Hess-Stump, Stefan Jaroch, Michael Brands, Karl Ziegelbauer. Discovery of BAY 1163877 - A pan-FGFR inhibitor: De novo structure-based design and lead optimization of benzothiophenyl-pyrrolotriazines. [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 4332.

Gastric cancer is the 3rd most common cause of cancer-related mortality worldwide, thus new treatment options are urgently needed. In gastric cancer, over-expression of the tumor-promoting tyrosine kinase receptor fibroblast growth factor receptor 2 (FGFR2) has been described, representing a potential therapeutic target. FGFR2 expression in gastric cancer has been reported to be heterogeneous. Most methods for detection of RNA in FFPE (formalin fixed paraffin embedded) do not provide spatial resolution for assessment of tumor heterogeneity. Here we used a novel RNA in situ technique called RNAscope. The technology allows visual investigation of FGFR2 transcription on FFPE slides. Samples from 1036 gastric adenocarcinoma patients who underwent surgery in National Cancer Center Hospital East (Chiba, Japan) were assembled in tissue micro-arrays and analyzed by RNAscope. A total of 578 samples were also analyzed by DISH to determine gene amplification. Using these tissue based methods we were able to assess the localization and heterogeneity of both FGFR2 gene amplification and RNA expression. Strong FGFR2 gene amplification (FGFR2:CEN10 >10) was detected in 2% of 578 samples. Moderate FGFR2 gene amplification (FGFR2:CEN10 between 2 and 10) was seen in 8% of the samples. High FGFR2 RNA expression (score 4+) was seen in 4% of 718 evaluable samples. Gene amplification and RNA expression were closely correlated. All samples with dense clusters of score 4+ RNA showed FGFR2:CEN10 ratios >10. The identical tumor areas showed high gene amplification and RNA expression. FGFR2 RNA and gene amplification was highly heterogeneous in the tissue. Only 0.4% of the samples showed homogeneous FGFR2 expression in >80% of the tumor cells. High RNA expression intensity was associated with a more homogeneous expression pattern compared to moderate FGFR2 expression. In early stage I/II gastric cancer samples with score 4+ RNA expression are mostly of the differentiated type. RNA score 4+ patients of grade III/IV gastric cancer were mostly of undifferentiated histology. In a subset of 195 samples with available data cMet IHC 3+ and Her2 positivity status were not mutually exclusive with regards to FGFR2 RNA Score 4+ expression. In a multivariant analysis FGFR2 RNA expression was associated with patient outcome. Gastric cancer patients with score 4+ RNA expression had a shorter progression free survival compared to patients with score 0-3. Interestingly, there was a trend for patients with homogeneous score 3+ RNA expression, but not heterogeneous score 3+ expression, to show shorter PFS and OS suggesting that patients with high intensity or homogeneous FGFR2 RNA expression may define a subgroup of patients with gastric cancer. Patients with homogenous or strong focal FGFR2 expression might therefore be candidates for FGFR2 directed therapies in gastric cancer. Citation Format: Yasutoshi Kuboki, Christoph A. Schatz, Sabine Jabusch, Karl Koechert, Janine Feng, Sabine Wittemer-Rump, Karl Ziegelbauer, Thomas Krahn, Akiko Nagatsuma, Atsushi Ochiai. In Situ analysis of FGFR2 RNA and comparison with FGFR2 gene copy number by dual-color in situ hybridization in a large cohort of gastric cancer patients. [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 4933.

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.

BUB1 (budding uninhibited by benzimidazoles 1) is a serine/threonine protein kinase. The protein is bound to kinetochores and plays a key role in the establishment of the mitotic spindle checkpoint and chromosome congression prior to anaphase. Inhibition of BUB1 kinase represents a novel approach for cancer treatment: whereas cell cycle arrest is the predominant mode of action of a number of antimitotic cancer drugs (e.g. taxanes and vinca alkaloids), BUB1 inhibition results in aneuploidy and cell death by driving cells through mitosis irrespective of DNA damage and misattached chromosomes. Here, we report the characterization of a novel benzylpyrazole lead-structure series inhibiting BUB1 exemplified by BAY-320, a novel, first-in-class small molecule inhibitor of BUB1 kinase. This structure class was initially discovered as a single hit in a high-throughput screen, and resulted in a lead compound by chemical optimization. Benzylpyrazole BAY-320 is highly selective for BUB1 with single digit nanomolar biochemical potency and single-digit micromolar cellular potency (HeLa proliferation assay). Synergistic effects can be observed when BUB1 inhibitor BAY-320 is combined with low doses of paclitaxel affecting chromosome segregation and cell proliferation. X-ray data of benzylpyrazoles allowed for a better understanding the binding mode for rational property design. Further data on structure-activity relationship including pharmacokinetic, drug metabolism and the synthesis of BAY-320 and analogues will be presented. These results validate the benzylpyrazoles as novel selective BUB1 inhibitors and BUB1 as a promising approach for cancer treatment. Citation Format: Marion Hitchcock, Gerhard Siemeister, Hans Briem, Amaury Ernest Fernandez-Montalvan, Simon Holton, Anne Mengel, Ursula Mönning, Michael Brands, Karl Ziegelbauer, Dominik Mumberg, Franz von Nussbaum. Synthesis and characterization of novel benzylpyrazole-based BUB1 kinase inhibitors with anti-tumor activity. [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 2718.

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.

Deregulated activity of cyclin-dependent kinases (CDK) results in loss of cell-cycle checkpoint function and increased expression of antiapoptotic proteins, which has been directly linked to the molecular pathology of cancer. BAY 1000394 inhibits the activity of cell-cycle CDKs CDK1, CDK2, CDK3, CDK4, and of transcriptional CDKs CDK7 and CDK9 with IC50 values in the range between 5 and 25 nmol/L. Cell proliferation was inhibited at low nanomolar concentration in a broad spectrum of human cancer cell lines. In cell-based assays, the inhibition of phosphorylation of the CDK substrates retinoblastoma protein, nucleophosmin, and RNA polymerase II was shown. Cell-cycle profiles were consistent with inhibition of CDK 1, 2, and 4 as shown in cell-cycle block and release experiments. The physicochemical and pharmacokinetic properties of BAY 1000394 facilitate rapid absorption and moderate oral bioavailability. The compound potently inhibits growth of various human tumor xenografts on athymic mice including models of chemotherapy resistance upon oral dosing. Furthermore, BAY 1000394 shows more than additive efficacy when combined with cisplatin and etoposide. These results suggest that BAY 1000394 is a potent pan-CDK inhibitor and a novel oral cytotoxic agent currently in phase I clinical trials. Mol Cancer Ther; 11(10); 2265–73. ©2012 AACR.

Surface proteins of the mammalian stage of the parasitic protozoan, Trypanosoma brucei, were biotinylated with sulfosuccinimidyl 6-(biotinamido) hexanoate. Since the predominant protein labeled by this reagent is the membrane form of the variant surface glycoprotein (mfVSG), a procedure was developed to convert mfVSG to its soluble form by the endogenous glycosylphosphatidylinositol-specific phospholipase C while retaining other biotinylated surface proteins in a membrane-bound state. From these membranes, three novel glycoproteins of 60, 65, and 75 kDa could be isolated by a combination of Triton X-114 phase separation and precipitations by streptavidin and concanavalin A coupled to solid supports. These polypeptides were detected in trypanosomes expressing different mfVSGs and are thus considered to be invariant. In a variant clone in which the mfVSG is trypsin-sensitive, the invariant surface glycoproteins of 65 and 75 kDa, designated ISG65 and ISG75, respectively, were proteolytically degraded with similar kinetics as the mfVSG. Neither ISG65 nor ISG75 could be detected in procyclic trypanosomes, the stage of the parasite characteristic for the insect midgut. Gene cloning reported in the accompanying paper (Ziegelbauer, K., Multhaup, G., and Overath, P. (1992) J. Biol. Chem. 267, 10797-10803) suggests that ISG65 and ISG75 are transmembrane proteins.

BACKGROUND Increasing the efficacy of chemotherapeutics by reducing chemoresistance may be a useful strategy in cancer therapy. Constitutive activation of nuclear factor-kappa B (NF-kappaB) is a hallmark of various cancers, including melanoma, which is almost universally resistant to chemotherapy. NF-kappaB is regulated by inhibitory kappaB (IkappaB) proteins, which are in turn phosphorylated by the IkappaB kinase (IKK) complex. METHODS The effect on NF-kappaB activity of a novel small-molecule inhibitor of the beta subunit of IKK (KINK-1; kinase inhibitor of nuclear factor-kappaB-1) was assessed by measuring phosphorylation of the alpha subunit of IkappaB by immunoblotting, DNA binding by electrophoretic mobility shift assays, and nuclear translocation of NF-kappaB using immunofluorescence. Regulation of NF-kappaB-dependent gene expression was determined by microarray analysis, real-time and semiquantitative reverse transcription polymerase chain reaction (RT-PCR), and Western blot analyses. The effects of KINK-1 (alone and in combination with cytostatic agents) on melanoma cells were characterized by assessing proliferation, soft agar colony formation, and markers of apoptosis. The antitumoral efficacy of KINK-1 in combination with the cytostatic agents doxorubicin or camptothecin (all injected intraperitoneally) was tested in vivo by measuring lung weight and counting metastases in C57BL6 mice (groups of six) bearing metastases of melanoma cells. All statistical tests were two-sided. Results KINK-1 strongly suppressed both constitutive and induced NF-kappaB activity in melanoma cells. It reduced the expression of NF-kappaB-dependent gene products that regulate proliferation, cytokine production, and antiapoptotic responses but exhibited little antiproliferative or proapoptotic activity at the cellular level. However, KINK-1 markedly increased the activities of some cytostatic agents in vitro and abrogated doxorubicin-induced NF-kappaB activation. Combined treatment of C57BL6 mice that had been injected with melanoma cells with KINK-1 and doxorubicin or camptothecin reduced metastases and pulmonary tumor mass compared with either treatment alone (mean lung weight 19 days after injection of melanoma cells of mice treated with 3 mg/kg KINK-1 alone, 1 mg/kg doxorubicin alone, and 1 mg/kg doxorubicin plus 3 mg/kg KINK-1 = 260 mg, 95% confidence interval (CI) = 216 to 305 mg; 268 mg, 95% CI = 224 to 313 mg; and 181 mg, 95% CI = 171 to 192 mg, respectively, P < .001 from t tests comparing mean lung weight of double-treated mice to that in mice treated with either compound alone). CONCLUSION Inhibition of constitutive and induced IKKbeta-activity through treatment with KINK-1 might increase tumor susceptibility to chemotherapy.

Chronic myelogenous leukemia is a malignant disease of the hematopoietic stem cell compartment, which is characterized by expression of the BCR-ABL fusion protein. Expression of BCR-ABL allows myeloid cells to grow in the absence of the growth factors interleukin-3 and granulocyte-macrophage colony-stimulating factor. The tyrosine kinase activity of BCR-ABL constitutively activates signaling pathways associated with Ras and its downstream effectors and with the Jak/STAT pathway. Additionally, we reported previously that BCR-ABL activates the transcription factor nuclear factor-κB (NF-κB) in a manner dependent on Ras and that inhibition of NF-κB by expression of a modified form of IκBα blocked BCR-ABL-driven tumor growth in a xenograft model. Here, we show that a highly specific inhibitor of IκB kinase β, a key upstream regulator of the NF-κB pathway, induces growth suppression and death in cells expressing wild-type, Imatinib-resistant, or the T315I Imatinib/Dasatinib-resistant forms of BCR-ABL. Cell cycle variables were not affected by this compound. These data indicate that blockage of BCR-ABL-induced NF-κB activation via IκB kinase β inhibition represents a potential new approach for treatment of Imatinib- or Dasatinib-resistant forms of chronic myelogenous leukemia. [Mol Cancer Ther 2008;7(2):391–7]

A close look at the biology and pharmacology of monoclonal antibodies reveals both their continuing promise as therapeutic agents to address unmet medical needs, as well as a number of challenges to the future discovery and development of this unique class of biologics. A remarkably consistent experience of reliable clinical efficacy and safety ensures that Biotech and Pharma have strong incentives to accelerate the antibody drug discovery process. Their attractive commercial potential invites consideration of potential challenges to the future expansion of the monoclonal antibody drug market. Four challenges arise from scientific and technical aspects of the antibody drug format: drug target limitations, biodistribution limitations, species specificity issues, and limitations to the route of administration and four challenges are based in the commercial and clinical use of antibody drugs: cost of goods, product differentiation within the antibody market, competition from small molecule drugs, and price sensitivity of clinical acceptance. Despite these challenges and recent setbacks, such as the withdrawal and subsequent relaunch of Tysabri and the TGN1412 Phase I disaster, the prevailing opinion is that monoclonal antibodies will continue to be safe and effective medicines that are worthy of commercialisation.

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

SMYD2 (SET and MYND domain-containing protein 2) is a protein lysine methyltransferase (PKMT) which was initially described as a histone H3K36 and H3K4 methyltransferase involved in transcriptional regulation. SMYD2 has recently been reported to methylate and regulate several non-histone cancer relevant proteins such as p53, retinoblastoma protein (Rb) and the estrogen receptor alpha. Given the reports that overexpression of SMYD2 is linked to poor prognosis in certain cancers, SMYD2 is proposed to be an oncogene and an attractive cancer drug target. Here we report the discovery of a novel potent and selective SMYD2 inhibitor, SMYD2-BAY-01, by high throughput screening and extensive biophysical validation. The co-crystal structure revealed that SMYD2-BAY-01 binds to the substrate binding site and occupies the hydrophobic pocket for lysine binding using an unprecedented hydrogen bond pattern. The competitive behavior of the inhibitor in biochemical assays is consistent with the binding mode observed in the crystal structure. Further optimization generated SMYD2-BAY-02, which shows improved low nanomolar potency and is selective against kinases and other PKMTs. Furthermore, SMYD2-BAY-02 specifically inhibits SMYD2 methylation activity in a cellular assay with similar potency and reduces methylation of the tumor suppressor protein p53. Based on promising in vitro and in vivo DMPK data, SMYD2-BAY-02 was further characterized in vivo for SMYD2-specific methylation inhibition. In vivo activity could be shown upon in vivo administration at doses as low as 30 mg/kg p.o. in a SMYD2 overexpressing esophageal squamous cell carcinoma model. In summary, SMYD2-BAY-02 is a promising selective and potent SMYD2 inhibitor in vitro and in vivo and may represent a new treatment option for cancers overexpressing SMYD2. Citation Format: Carlo Stresemann, Ingo Hartung, Timo Stellfeld, Naomi Barak, Jeffrey Mowat, Clara Christ, Antonius ter Laak, Silke Koehr, Jörg Weiske, Roman Hillig, Volker Badock, Detlef Stoeckigt, Karl Ziegelbauer, Hilmar Weinmann, Volker Gekeler. Discovery and in vitro and in vivo characterization of aminopyrazoline-based SMYD2 inhibitors. [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 2829. doi:10.1158/1538-7445.AM2015-2829

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

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Cell cycle deregulation represents one of the hallmarks of cancer and consequently cell cycle arrest is the predominant mode of action for a number of antimitotic cancer drugs (e.g. taxanes and vinca alkaloids). Targeted disruption of the cell cycle checkpoint offers a novel approach to cancer treatment since tumor cells will not arrest in mitosis despite DNA damage or unattached/misattached chromosomes resulting in aneuploidy and cell death. Mps1, a mitotic kinase that is overexpressed in several human cancers, has been shown to function as the key kinase which activates the spindle assembly checkpoint (SAC) to secure proper distribution of chromosomes to daughter cells. Here, we disclose for the first time the structure and functional characterization of two novel Mps1 inhibitors, BAY 1161909 and BAY 1217389, derived from structurally distinct chemical classes. BAY 1161909 and BAY 1217389 inhibited Mps1 kinase activity with IC50 values below 10 nM while showing an excellent selectivity profile against a broad panel of kinases. In cellular mechanistic assays, BAY 1161909 and BAY 1217389 abrogated nocodazole-induced SAC activity, inducing premature exit from mitosis (“mitotic breakthrough”), which results in multinuclearity and tumor cell death. Both compounds efficiently inhibited tumor cell proliferation in vitro (IC50 values in low nanomolar range), showing a similar inhibitory pattern in a broad panel of tumor cell lines. In vivo, the Mps1 inhibitors BAY 1161909 and BAY 1217389 achieved moderate efficacy in monotherapy in tumor xenograft studies (tumor growth inhibition ∼ 50%). However, according to its unique mode of action, when combined with paclitaxel, at the maximum tolerated dose, low doses of Mps1 inhibitor reduced paclitaxel-induced mitotic arrest in line with weakening of SAC activity. Consequently, combination therapy strongly improved efficacy over paclitaxel or Mps1 inhibitor mono-treatment in a broad range of xenograft models including those being intrinsically paclitaxel-insensitive as well as those with acquired paclitaxel resistance. Both Mps1 inhibitors showed good tolerability without adding toxicity to paclitaxel monotherapy. Our findings validate the innovative concept of SAC abrogation and justify clinical proof of concept studies evaluating Mps1 inhibitors BAY 1161909 and BAY 1217389 in combination with antimitotic cancer drugs in order to enhance their efficacy and potentially overcome resistance. BAY 1161909 is currently in a phase I clinical trial ([NCT02138812][1]), start of clinical investigation of BAY 1217389 is planned. To our knowledge BAY 1161909 and BAY 1217389 are the first Mps1 inhibitors in clinical trials. Citation Format: Antje Margret Wengner, Gerhard Siemeister, Marcus Koppitz, Volker Schulze, Dirk Kosemund, Ulrich Klar, Detlef Stoeckigt, Roland Neuhaus, Philip Lienau, Benjamin Bader, Stefan Prechtl, Olaf Doehr, Marian Raschke, Oliver von Ahsen, Cem Elbi, Ingmar Bruns, Martin Michels, Bertolt Kreft, Franz von Nussbaum, Michael Brands, Dominik Mumberg, Karl Ziegelbauer. Novel Mps1 kinase inhibitors with potent anti-tumor activity. [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 3090. doi:10.1158/1538-7445.AM2015-3090 [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT02138812&atom=%2Fcanres%2F75%2F15_Supplement%2F3090.atom

PTEFb/CDK9 mediated transcription of short-lived anti-apoptotic survival proteins like MYC, a key oncogene in multiple tumors, plays a critical role in cancer cell growth and survival. In addition, these survival proteins exhibit important functions in the development of resistance to chemotherapy. In contrast to pan-CDK inhibitors which are currently evaluated in Phase I and II clinical trials, to our knowledge PTEFb selective inhibitors have not been explored for clinical utility. We report for the first time the preclinical profile and structure of BAY 1143572, a novel selective PTEFb/CDK9 inhibitor currently being investigated in a Phase I clinical trial. BAY 1143572 had potent and highly selective PTEFb-kinase inhibitory activity in the low nanomolar range against PTEFb/CDK9 and an at least 50-fold selectivity against other CDKs in enzymatic assays. Furthermore, BAY 1143572 showed a favorable selectivity against a panel of non-CDK kinases in vitro. The potent enzymatic activity on PTEFb translated into broad antiproliferative activity against a panel of tumor cell lines with sub-micromolar IC-50 values. In line with the proposed mode of action, a concentration-dependent inhibition of the phosphorylation of the RNA polymerase II and downstream reduction of MYC mRNA and protein levels was observed in vitro. This inhibition was accompanied by an induction of apoptosis in cellular assays. BAY 1143572 also showed single agent in vivo efficacy at tolerated doses in various xenograft tumor models in mice and rats upon once daily oral administration. Potent anti-tumor activity characterized with partial or even complete remissions could be documented in models showing different MYC gene alterations like amplifications and translocations. Treatment with BAY 1143572 resulted in a transient inhibition of intratumoral MYC mRNA and protein levels and an induction of apoptosis in these models. The inhibition of MYC mRNA was also observed in blood cells of BAY 1143572-treated rats indicating the potential clinical utility of MYC in blood cells as a pharmacodynamic marker in clinical development. The in vivo efficacy of BAY 1143572 was significantly enhanced in combination with several chemotherapeutics in different solid tumor models. These pharmacology data provided the rationale for the initiation of clinical development of BAY 1143572 in advanced cancer patients (NCT01938638). In conclusion, our data provide preclinical proof of concept for BAY 1143572 as a potent and highly selective inhibitor of PTEFb/CDK9 with first-in-class potential. Further clinical evaluation of BAY 1143572 for the treatment of cancers dependent on the transcription of the key oncogene MYC and other short-lived survival proteins is warranted. Citation Format: Arne Scholz, Ulrich Luecking, Gerhard Siemeister, Philip Lienau, Ulf Boemer, Peter Ellinghaus, Annette O. Walter, Ray Valencia, Stuart Ince, Franz von Nussbaum, Dominik Mumberg, Michael Brands, Karl Ziegelbauer. BAY 1143572: A first-in-class, highly selective, potent and orally available inhibitor of PTEFb/CDK9 currently in Phase I, inhibits MYC and shows convincing anti-tumor activity in multiple xenograft models by the induction of apoptosis. [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 DDT02-02. doi:10.1158/1538-7445.AM2015-DDT02-02

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

1 Pulmonary inflammatory diseases such as asthma are characterized by chronic, cell-mediated inflammation of the bronchial mucosa. 2 Recruitment and activation of inflammatory cells is orchestrated by a variety of mediators such as cytokines, chemokines, or adhesion molecules, the expression of which is regulated via the transcription factor nuclear factor kappa B (NF-kappaB). 3 NF-kappaB signaling is controlled by the inhibitor of kappa B kinase complex (IKK), a critical catalytic subunit of which is IKK-beta. 4 We identified COMPOUND A as a small-molecule, ATP-competitive inhibitor selectively targeting IKK-beta kinase activity with a K(i) value of 2 nM. 5 COMPOUND A inhibited stress-induced NF-kappaB transactivation, chemokine-, cytokine-, and adhesion molecule expression, and T- and B-cell proliferation. 6 COMPOUND A is orally bioavailable and inhibited the release of LPS-induced TNF-alpha in rodents. 7 In mice COMPOUND A inhibited cockroach allergen-induced airway inflammation and hyperreactivity and efficiently abrogated leukocyte trafficking induced by carrageenan in mice or by ovalbumin in a rat model of airway inflammation. 8 COMPOUND A was well tolerated by rodents over 3 weeks without affecting weight gain. 9 Furthermore, in mice COMPOUND A suppressed edema formation in response to arachidonic acid, phorbol ester, or edema induced by delayed-type hypersensitivity. 10 These data suggest that IKK-beta inhibitors offer an effective therapeutic approach for inhibiting chronic pulmonary inflammation.

Phenylalanyl (Phe)-tRNA synthetase (Phe-RS) is an essential enzyme which catalyzes the transfer of phenylalanine to the Phe-specific transfer RNA (tRNA Phe ), a key step in protein biosynthesis. Phenyl-thiazolylurea-sulfonamides were identified as a novel class of potent inhibitors of bacterial Phe-RS by high-throughput screening and chemical variation of the screening hit. The compounds inhibit Phe-RS of Escherichia coli , Haemophilus influenzae , Streptococcus pneumoniae , and Staphylococcus aureus , with 50% inhibitory concentrations in the nanomolar range. Enzyme kinetic measurements demonstrated that the compounds bind competitively with respect to the natural substrate Phe. All derivatives are highly selective for the bacterial Phe-RS versus the corresponding mammalian cytoplasmic and human mitochondrial enzymes. Phenyl-thiazolylurea-sulfonamides displayed good in vitro activity against Staphylococcus , Streptococcus , Haemophilus , and Moraxella strains, reaching MICs below 1 μg/ml. The antibacterial activity was partly antagonized by increasing concentrations of Phe in the culture broth in accordance with the competitive binding mode. Further evidence that inhibition of tRNA Phe charging is the antibacterial principle of this compound class was obtained by proteome analysis of Bacillus subtilis . Here, the phenyl-thiazolylurea-sulfonamides induced a protein pattern indicative of the stringent response. In addition, an E. coli strain carrying a relA mutation and defective in stringent response was more susceptible than its isogenic relA + parent strain. In vivo efficacy was investigated in a murine S. aureus sepsis model and a S. pneumoniae sepsis model in rats. Treatment with the phenyl-thiazolylurea-sulfonamides reduced the bacterial titer in various organs by up to 3 log units, supporting the potential value of Phe-RS as a target in antibacterial therapy.

Syk is a tyrosine kinase which is indispensable in immunoglobulin Fc receptor- and B cell receptor-mediated signal transduction in various immune cells. This pathway is important in the pathophysiology of allergy. In this study we established a quantitative nonradioactive kinase assay to identify inhibitors of Syk. We used recombinant GST-tagged Syk purified from baculovirus-infected insect cells. As a substrate, biotinylated peptide corresponding to the activation loop domain of Syk, whose tyrosine residues are autophosphorylated upon activation, was employed to screen both ATP- and substrate-competitive inhibitors. After the kinase reaction in solution phase, substrate was trapped on a streptavidin-coated plate, followed by detection of the phosphorylated tyrosine with europium-labeled anti-phosphotyrosine antibody. The kinase reaction in solution phase greatly enhanced phosphorylation of substrate compared to that of plate-coated substrate. High signal-to-background ratio and low data scattering were obtained in the optimized high-throughput screening (HTS) format. Further, several kinase inhibitors showed concentration-dependent inhibition of recombinant Syk kinase activity with almost the same efficacy for immunoprecipitated Syk from a human cell line. These data suggest that this assay is useful to screen Syk kinase inhibitors in HTS.

Malignant cells are known for their accelerated metabolism, high energy requirements, and increased glucose uptake. Transport of glucose across the plasma membrane is the first and rate-limiting step for glucose metabolism and is mediated by facilitative glucose transporter (GLUT) proteins. Increased glucose uptake in malignant cells has been associated with upregulated expression of glucose transporters, mainly overexpression of GLUT1 and/or GLUT3. There is limited knowledge about how selective (e.g. GLUT1) versus broad (multi-) GLUT inhibition affects glucose homeostasis in tumor bearing mice. Using potent small molecule inhibitors, we compared [14C]-2-Deoxy-D-Glucose (2-DG) distribution after selective GLUT1 versus GLUT1, 3 and 4 [multi-GLUT] inhibition and versus control in human NSCLC NCI-H460 tumor bearing mice. A single dose of a GLUT1 selective and a multi-GLUT inhibitor were administered to NCI-H460 tumor bearing NMRI nu/nu mice. At the respective Cmax concentrations, a bolus of 2-DG was rapidly injected intra-peritoneally and the distribution of metabolically stable 2-DG was obtained using whole-body autoradiography after 15 min and 120 min. With the multi-GLUT inhibitor only a very short inhibition of 2-DG uptake was observed in the NCI-H460 tumors while a long-lasting inhibition was detected in heart, brain and brown fat tissue. In contrast, a long-lasting inhibition of 2-DG uptake was observed in NCI-H460 tumors for the selective GLUT1 inhibitor. 2-DG concentrations were reduced in the brain following administration of the selective GLUT1 inhibitor at 15 min and returned to normal levels at 120 min while the tumor 2-DG concentration stayed low. The 2-DG findings go in parallel with the histopathological findings present in the brain and heart after treatment with the multi-GLUT inhibitor. However, similar histopathological findings have not been observed in the brain and heart after treatment with the selective GLUT-1 inhibitor. Therefore, selective GLUT1 inhibition is associated with a sustained low 2-DG concentration in the NCI-H460 tumors while only minor changes in glucose homeostasis were observed in other organ systems. Citation Format: Melanie Heroult, Wolfram Steinke, Anna-Lena Frisk, Sandra Borkowski, Kirstin Meyer, Heike Petrul, Iring Heisler, Maria Quanz, Roland Neuhaus, Bernd Buchmann, Thomas Mueller, Marcus Bauser, Andrea Haegebarth, Michael Brands, Karl Ziegelbauer. Effects of selective and broad glucose transporter (GLUT) inhibition on glucose distribution in tumor bearing 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 1442. doi:10.1158/1538-7445.AM2014-1442

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

The family of cyclin-dependent kinase (CDK) proteins consists of multiple cell cycle regulating CDK members as well as members involved in the regulation of gene transcription like CDK9/PTEFb (positive transcription elongation factor b). Inhibition of PTEFb and its direct downstream target RNA polymerase II is thought to cause rapid depletion of short-lived mRNA transcripts of important survival proteins like c-myc and Mcl-1 and thereby to induce growth delay and apoptosis in addicted tumor cells. In contrast to pan-CDK inhibitiors which are currently evaluated in Phase I and II clinical trials, PTEFb selective inhibitors have not been explored for clinical utility. BAY 1112054 is a potent and highly selective PTEFb-kinase inhibitor with low nanomolar activity against PTEFb/CDK9 and an at least 50-fold selectivity against other CDKs in enzymatic assays. Furthermore, BAY 1112054 shows a favourable selectivity against non-CDK kinases in vitro. The compound exhibits broad anti-proliferative activity against a panel of tumor cell lines with sub-micromolar IC-50 values. In line with the proposed mode of action, a concentration-dependent inhibition of the phosphorylation of the RNA polymerase II was observed in A549 tumor cells. This inhibition was accompanied by a reduction of intracellular Mcl-1 protein levels. Furthermore, BAY 1112054 increased DNA fragmentation in synchronized HeLa cells upon compound treatment for 24 hours. BAY 1112054 showed convincing in vivo efficacy at tolerated doses in two xenograft models in mice. Once daily oral treatment led to complete tumor stasis in established MOLM-13 AML xenografts. Pharmacokinetic analysis revealed that unbound plasma levels were 8 to 12 hours above the cellular IC50 in this model. In vivo efficacy and tolerability of the once daily po schedule of BAY 1112054 was confirmed in NCI-H82 SCLC xenografts. Xenografted tumors of this model showed lower levels of RNA polymerase II phosphorylation and Mcl-1 upon treatment with BAY 1112054. In conclusion, our data provides in vitro and in vivo proof of concept for BAY 1112054, a potent and highly selective inhibitor of PTEFb/CDK9 with first-in-class potential, and warrant further clinical evaluation of PTEFb selective inhibitors for the treatment of cancers addicted to the transcription of short-lived anti-apoptotic survival proteins. Citation Format: Arne Scholz, Ulrich Lücking, Gerhard Siemeister, Philip Lienau, Knut Eis, Antje Wengner, Kirstin Petersen, Ulf Bömer, Peter Nussbaumer, Axel Choidas, Gerd Rühter, Jan Eickhoff, Carsten Schultz-Fademrecht, Bert Klebl, Stuart Ince, Franz von Nussbaum, Dominik Mumberg, Michael Brands, Karl Ziegelbauer. BAY 1112054, a highly selective, potent and orally available inhibitor of PTEFb/CDK9, shows convincing anti-tumor activity. [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 4538. doi:10.1158/1538-7445.AM2014-4538

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

Cell cycle deregulation represents one of the classical hallmarks of cancer and consequently cell cycle arrest is the predominant mode of action of a number of antimitotic cancer drugs (e.g. taxanes and vinca alkaloids). Targeted disruption of the cell cycle checkpoint offers a novel approach to cancer treatment: driving tumor cells into cell division despite DNA damage or unattached/misattached chromosomes resulting in a lethal degree of DNA damage or aneuploidy. MPS1, a mitotic kinase that is overexpressed in several human cancers, has been shown to function as the key kinase which activates the spindle assembly checkpoint (SAC) to secure proper distribution of chromosomes to daughter cells. Here, we report the identification and functional characterization of novel inhibitors of MPS1 from two structurally unrelated chemical classes. In biochemical assays, these molecules inhibited the kinase activity of MPS1 with IC50 values in the single digit nanomolar range and have shown an exquisite selectivity against a panel of kinases. In cellular assays the compounds were able to abrogate nocodazole-induced SAC, to reduce time in mitosis, to induce multinuclearity and apoptotic cell death, and to inhibit tumor cell proliferation with IC50 values in the low nanomolar range. In combination experiments MPS1 inhibitors showed cooperativity with low concentrations of paclitaxel. MPS1 inhibitors showed limited efficacy in monotherapy (T/C ca. 0.6) in tumor xenograft studies. However, when combined with paclitaxel dosed at the maximal tolerated dose, low doses of MPS1 inhibitor strongly improved efficacy over paclitaxel or MPS inhibitor monotreatment in intrinsically paclitaxel-insensitive xenograft models. The combination treatment was well tolerated. These results validate the concept of SAC abrogation preclinically and pave the way to a clinical proof of concept. Citation Format: Dominik Mumberg, Gerhard Siemeister, Antje M. Wengner, Marcus Koppitz, Volker Schulze, Benjamin Bader, Stefan Prechtl, Bertolt Kreft, Karl Ziegelbauer. SAC abrogation by MPS1 kinase inhibition: preclinical proof of concept of a novel approach to tumor treatment. [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 4540. doi:10.1158/1538-7445.AM2014-4540

This paper describes the development of a new, malachite green based, enzymatic assay for the identification of specific inhibitors of inorganic pyrophosphatase (iPPase) from Saccharomyces cerevisiae for antifungal drug discovery. The human iPPase was used as counterscreen. The coding regions of both enzymes were amplified, cloned into a vector providing a His-tag at the C-terminus, expressed in Escherichia coli, and purified by metal chelate affinity chromatography. Since the complete human sequence had not been published previously, the human iPPase was cloned on the basis of expressed sequence tag data. The human sequence was confirmed and showed about 55% amino acid identity with the yeast enzyme and 95% identity with an already published bovine enzyme. Both recombinant iPPases were characterized with regard to their biochemical properties, showing that the His-tag did not influence the specific activity, pH optimum, inhibitor profile, or dimerization. The enzyme activity was determined by quantifying released phosphate by complex formation with malachite green. The resulting complex was quantified spectrophotometrically. The assay was adapted to a microtiter plate format. Thus, it is possible to screen a large compound pool for iPPase inhibitors in a short period of time.

We describe a 96-well microtiter plate format assay to detect changes in proton permeability in membranes of the pathogenic yeast, Candida albicans. Candida albicans cells were incubated with the lipophilic ester of 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF), a pH-sensitive fluorescein derivative. Inside the cells, BCECF was released and trapped in the vacuole. Compounds that destroyed membrane integrity increased the pH value of the vacuole due to proton leakage into the cytoplasm. This was paralleled by an increase in BCECF fluorescence intensity, which could be quantified. The test assay was validated with amphotericin B, as well as with other membrane-active compounds known to increase membrane permeability. Possible applications and limitations of this assay in the field of antifungal drug discovery are discussed.