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Anti-tumor efficacy of C4.4A-ADC in NSCLC PDX models in mice.

Supplementary Materials and Methods. Supplemental Table S1. Combination effects of BAY 80-6946 and lapatinib on caspase 9 activation in BT474 cells. Supplemental Table S2. Single dose i.v. pharmacokinetic parameter of BAY 80-6946 in female nude rats and ICR mice (n=3 per timepoint). Supplemental Table S3. Combination effects of BAY 80-6946 and paclitaxel in patient-derived NSCLC xenograft tumor model. Supplemental Figure S1: Chemical structure of reference compounds. Supplemental Figure S2. Nuclear translocation of FOXO1A in U2OS cells. Supplemental Figure S3. Caspase 9 activity in BT474 and BT20 cells measured 24 hours after exposure to BAY 80-6946 and reference drugs. Supplemental Figure S4. Single and multiple dose i.v. plasma concentration versus time profile of BAY 80-6946 in female nude rats (n=2 per timepoint).

PDF - 118K, Supplementary Table S1. Mesothelin staining intensity in FFPE tissues of human primary ovarian cancer, pancreatic cancer and mesothelioma.

Detailed method descriptions

Effect of BAY 1217389 in monotherapy and combination therapy with paclitaxel on total KNL1 in A2780Cis tumors in nude mice

Mesothelin is a tumor differentiation antigen frequently overexpressed in tumors such as mesothelioma, ovarian, pancreatic, and lung adenocarcinomas while showing limited expression in nonmalignant tissues. Mesothelin is therefore an attractive target for cancer therapy using antibody–drug conjugates (ADC). This study describes the detailed characterization of anetumab ravtansine, here referred to as BAY 94-9343, a novel ADC consisting of a human anti-mesothelin antibody conjugated to the maytansinoid tubulin inhibitor DM4 via a disulfide-containing linker. Binding properties of the anti-mesothelin antibody were analyzed using surface plasmon resonance, immunohistochemistry, flow cytometry, and fluorescence microscopy. Effects of BAY 94-9343 on cell proliferation were first studied in vitro and subsequently in vivo using subcutaneous, orthotopic, and patient-derived xenograft tumor models. The antibody binds to human mesothelin with high affinity and selectivity, thereby inducing efficient antigen internalization. In vitro, BAY 94-9343 demonstrated potent and selective cytotoxicity of mesothelin-expressing cells with an IC50 of 0.72 nmol/L, without affecting mesothelin-negative or nonproliferating cells. In vivo, BAY 94-9343 localized specifically to mesothelin-positive tumors and inhibited tumor growth in both subcutaneous and orthotopic xenograft models. In addition, BAY 94-9343 was able to induce a bystander effect on neighboring mesothelin-negative tumor cells. Antitumor efficacy of BAY 94-9343 correlated with the amount of mesothelin expressed and was generally superior to that of standard-of-care regimen resulting in complete tumor eradication in most of the models. BAY 94-9343 is a selective and highly potent ADC, and our data support its development for the treatment of patients with mesothelin-expressing tumors. Mol Cancer Ther; 13(6); 1537–48. ©2014 AACR.

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

PDF file, 54K, Cell line panel, cell viability results upon BAY 1000394 treatment.

Tables S1-S4

PDF file, 137K, Cellular mode of action investigations.

PDF file, 57K, Pharmacokinetic data.

Multinuclearity in U2OS cells induced by Mps1 inhibition

H&E stained A2780cis tumors after treatment with vehicle paclitaxel alone BAY 1161909 alone or in combination with BAY 1161909 and paclitaxel

PDF - 117K, Supplementary materials and methods, figure legends for supplementary figures and table.

PDF file, 46K, Exposure kinetics of BAY 1000394.

PDF file, 63K, Cell line panels.

Figures S1-S4

Additional data showing pharmacological characterization of Mps1 Inhibitors BAY 1161909 and BAY 1217389; Suppl. Table 1: Kinase selectivity profile of BAY 1161909; Suppl. Table 2: Kinase selectivity profile of BAY 1217389; Suppl. Table 3: Kinase selectivity profiles of BAY 1161909 compared to SP600125 and Reversine; Suppl. Table 4: Inhibition of tumor cell proliferation by BAY 1161909 or BAY 1217389; Suppl. Table 5: Summary of in vivo activity of BAY 1161909 in human xenograft tumor models in mice; Suppl. Table 6: Summary of in vivo activity of BAY 1217389 in human xenograft tumor models in mice.

PDF file, 80K.

Effect of BAY 1161909 in monotherapy on the frequencies of peripheral blood reticulocytes and micronuclei-harboring reticulocytes in nude mice

PDF - 3895K, Supplementary Figure S1. Immunohistochemical detection of mesothelin expression in FFPE tissues of human primary ovarian cancer, pancreatic cancer, and mesothelioma. Supplementary Figure S2. Immunohistochemical detection of mouse mesothelin on Cryo embedded peritoneum sections. Supplementary Figure S3. Immunohistochemical detection of rat mesothelin on Cryo embedded tissue sections. Supplementary Figure S4. Immunohistochemical detection of cynomolgus monkey mesothelin on Cryo embedded tissue sections Supplementary Figure S5. Confirmation of mesothelin expression by IHC in vector or mesothelin-transfected tumor cell lines. Supplementary Figure S6. Anti-tumor activity of BAY 94-9343 in vivo.

PDF file, 93K, Overview on xenograft data.

PDF file, 7K, Kinase inhibition data.

PDF file, 64K, Antiproliferation data.

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.

PDF file, 63K, Cell line sources and authentication.

Exposure data from xenograft studies

'Supplementary Figure S1 shows the efficacy of radium-223 in an abiraterone-resistant LuCaP 58 prostate cancer PDX model; Supplementary Table S1 shows the characteristics of LNCaP and LuCaP 58 prostate cancer growth in bone models in mice and responses to radium-223; Supplementary Table S2 shows responses of radium-223 and abiraterone in LuCaP 58 prostate cancer PDX model in mice.'

The file contains description of Supporting Methods.

The file contains Supporting Figures.

The file contains Supporting Tables.

Purpose:The catalytic function of BUB1 is required for chromosome arm resolution and positioning of the chromosomal passenger complex for resolution of spindle attachment errors and plays only a minor role in spindle assembly checkpoint activation. Here, we present the identification and preclinical pharmacologic profile of the first BUB1 kinase inhibitor with good bioavailability.Experimental Design:The Bayer compound library was screened for BUB1 kinase inhibitors and medicinal chemistry efforts to improve target affinity and physicochemical and pharmacokinetic parameters resulting in the identification of BAY 1816032 were performed. BAY 1816032 was characterized for kinase selectivity, inhibition of BUB1 signaling, and inhibition of tumor cell proliferation alone and in combination with taxanes, ATR, and PARP inhibitors. Effects on tumor growth in vivo were evaluated using human triple-negative breast xenograft models.Results:The highly selective compound BAY 1816032 showed long target residence time and induced chromosome mis-segregation upon combination with low concentrations of paclitaxel. It was synergistic or additive in combination with paclitaxel or docetaxel, as well as with ATR or PARP inhibitors in cellular assays. Tumor xenograft studies demonstrated a strong and statistically significant reduction of tumor size and excellent tolerability upon combination of BAY 1816032 with paclitaxel or olaparib as compared with the respective monotherapies.Conclusions:Our findings suggest clinical proof-of-concept studies evaluating BAY 1816032 in combination with taxanes or PARP inhibitors to enhance their efficacy and potentially overcome resistance.

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

Supplementary Materials and Methods and Supplementary Figure Legends

Supplementary Figure Legends, Methods and References

The fibroblast growth factor receptor FGFR2 is overexpressed in a variety of solid tumors, including breast, gastric, and ovarian tumors, where it offers a potential therapeutic target. In this study, we present evidence of the preclinical efficacy of BAY 1187982, a novel antibody–drug conjugate (ADC). It consists of a fully human FGFR2 monoclonal antibody (mAb BAY 1179470), which binds to the FGFR2 isoforms FGFR2-IIIb and FGFR2-IIIc, conjugated through a noncleavable linker to a novel derivative of the microtubule-disrupting cytotoxic drug auristatin (FGFR2-ADC). In FGFR2-expressing cancer cell lines, this FGFR2-ADC exhibited potency in the low nanomolar to subnanomolar range and was more than 100-fold selective against FGFR2-negative cell lines. High expression levels of FGFR2 in cells correlated with efficient internalization, efficacy, and cytotoxic effects in vitro. Pharmacokinetic analyses in mice bearing FGFR2-positive NCI-H716 tumors indicated that the toxophore metabolite of FGFR2-ADC was enriched more than 30-fold in tumors compared with healthy tissues. Efficacy studies demonstrated that FGFR2-ADC treatment leads to a significant tumor growth inhibition or tumor regression of cell line–based or patient-derived xenograft models of human gastric or breast cancer. Furthermore, FGFR2 amplification or mRNA overexpression predicted high efficacy in both of these types of in vivo model systems. Taken together, our results strongly support the clinical evaluation of BAY 1187982 in cancer patients and a phase I study (NCT02368951) has been initiated. Cancer Res; 76(21); 6331–9. ©2016 AACR.

The anti-tumor efficacy of different FGFR2-ADC BAY 1187982 treatment schedules on MFM-223 human TNBC mouse model.

Cell cycle analyses

The expression of FGFR2 on tumor cell surface and half maximal inhibitory concentration of cell viability for FGFR2-ADC BAY 1187982 and control ADC.

MCL1 is an anti-apoptotic protein that is frequently amplified in cancer and confers resistance to current standard of care. Therefore, MCL1 is an attractive anti-cancer target. Here we describe ANJ810 as a novel, potent, and selective MCL1 inhibitor and its key design principle of a rapid systemic clearance to potentially minimize AUC-driven toxicities associated with MCL1 inhibition. ANJ810 induced rapid cell killing within 4 hours in vitro but in the same 4-hour window had no impact on cell viability or troponin I release in hiPSC-derived cardiomyocytes, even at supra-pharmacologic concentrations. In vivo ANJ810 induced efficacy in xenograft hematological and solid tumor models despite the short residence time of ANJ810 in plasma. In totality our data support the hypothesis that short-term inhibition of MCL1 with ANJ810 can induce cell apoptosis in tumor cells while maintaining an acceptable safety profile. We therefore intend to advance ANJ810 to clinical trials.

Inhibition of monopolar spindle 1 (MPS1) kinase represents a novel approach to cancer treatment: instead of arresting the cell cycle in tumor cells, cells are driven into mitosis irrespective of DNA damage and unattached/misattached chromosomes, resulting in aneuploidy and cell death. Starting points for our optimization efforts with the goal to identify MPS1 inhibitors were two HTS hits from the distinct chemical series "triazolopyridines" and "imidazopyrazines". The major initial issue of the triazolopyridine series was the moderate potency of the HTS hits. The imidazopyrazine series displayed more than 10-fold higher potencies; however, in the early project phase, this series suffered from poor metabolic stability. Here, we outline the evolution of the two hit series to clinical candidates BAY 1161909 and BAY 1217389 and reveal how both clinical candidates bind to the ATP site of MPS1 kinase, while addressing different pockets utilizing different binding interactions, along with their synthesis and preclinical characterization in selected in vivo efficacy models.

MCL1 is an anti-apoptotic protein inhibiting cancer cell death. It is frequently amplified or overexpressed in cancer and confers resistance to relevant standard of care. Therefore, MCL1 is an attractive target to potentially re-sensitize tumors to conventional chemotherapy and targeted agents. Here we describe ANJ810 as a novel, potent, and selective MCL1 inhibitor which specifically induces apoptosis in cancer cells. ANJ810 potently and reversibly binds to the BH3-binding groove of human MCL1 (KD = 0.3 nM), thereby inhibiting the interaction with the BH3-only protein NOXA (IC50 = 0.4 nM). ANJ810 shows a 4-log-fold greater selectivity to MCL1 over other anti-apoptotic proteins like Bcl-xL or Bcl-2. In cells, ANJ810 treatment rapidly (< 15 min) disrupts the MCL1-BAK interaction with an IC50 < 10 nM, resulting in caspase-3 activation and cancer cell death. Knockout of BAK and BAX completely rescues cells from ANJ810 induced killing, indicating that initiation of cell death occurs through the intrinsic apoptotic pathway. Screening over 750 cell lines (PRISM), we found that ANJ810 induced anti-cancer activity in 222 cell lines with an IC50 < 1 μM across multiple solid and hematological cancers, including breast, lung, melanoma, sarcoma, lymphoma, and leukemia. The top genomic feature that correlates with sensitivity to ANJ810 treatment is the ratio of Bcl-xL/BAK expression. A key design principle of ANJ810 is its rapid systemic clearance to potentially minimize exposure-driven toxicities associated with MCL1 inhibition. ANJ810 induces efficient cancer cell killing within 4 hours in vitro but has no impact on cell viability or troponin I release in hiPSC-derived cardiomyocytes at supra-pharmacologic concentrations. In vivo, i.v. bolus injections of ANJ810 lead to short plasma residence time, yet are efficacious in xenograft models of multiple myeloma, DLBCL, NSCLC and HCC. ANJ810 will test the hypothesis in human clinical trials that short-term inhibition of MCL1 can overcome tumor resistance with an acceptable safety profile to improve current standard of care. Citation Format: Ulrike Rauh, Guo Wei, Michael Serrano-Wu, Georgios Kosmidis, Stefan Kaulfuss, Franziska Siegel, Kai Thede, James McFarland, Christopher Lemke, Nicolas Werbeck, Katrin Nowak-Reppel, Sabine Pillari, Stephan Menz, Matthias Ocker, Brian Hubbard, Virendar Kaushik, Karl Ziegelbauer, Todd Golub. ANJ810 is a highly selective novel MCL1 inhibitor with optimized in vivo clearance showing robust efficacy in preclinical solid and hematological tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 506.

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

Targeted 227Th conjugates (TTCs) represent a new class of therapeutic radiopharmaceuticals for targeted α-therapy. They comprise the α-emitter 227Th complexed to a 3,2-hydroxypyridinone chelator conjugated to a tumor-targeting monoclonal antibody. The high energy and short range of the α-particles induce antitumor activity, driven by the induction of complex DNA double-strand breaks. We hypothesized that blocking the DNA damage response (DDR) pathway should further sensitize cancer cells by inhibiting DNA repair, thereby increasing the response to TTCs. Methods: This article reports the evaluation of the mesothelin (MSLN)-TTC conjugate (BAY 2287411) in combination with several DDR inhibitors, each of them blocking different DDR pathway enzymes. MSLN is a validated cancer target known to be overexpressed in mesothelioma, ovarian, lung, breast, and pancreatic cancer, with low expression in normal tissue. In vitro cytotoxicity experiments were performed on cancer cell lines by combining the MSLN-TTC with inhibitors of ataxia telangiectasia mutated, ataxia telangiectasia and Rad3-related (ATR), DNA-dependent protein kinase, and poly[adenosine diphosphate ribose] polymerase (PARP) 1/2. Further, we evaluated the antitumor efficacy of the MSLN-TTC in combination with DDR inhibitors in human ovarian cancer xenograft models. Results: Synergistic activity was observed in vitro for all tested inhibitors (inhibitors are denoted herein by the suffix “i”) when combined with MSLN-TTC. ATRi and PARPi appeared to induce the strongest increase in potency. Further, in vivo antitumor efficacy of the MSLN-TTC in combination with ATRi or PARPi was investigated in the OVCAR-3 and OVCAR-8 xenograft models in nude mice, demonstrating synergistic antitumor activity for the ATRi combination at doses demonstrated to be nonefficacious when administered as monotherapy. Conclusion: The presented data support the mechanism-based rationale for combining the MSLN-TTC with DDR inhibitors as new treatment strategies in MSLN-positive ovarian cancer.

Purpose: The catalytic function of BUB1 is required for chromosome arm resolution and positioning of the chromosomal passenger complex for resolution of spindle attachment errors and plays only a minor role in spindle assembly checkpoint activation. Here, we present the identification and preclinical pharmacologic profile of the first BUB1 kinase inhibitor with good bioavailability. Experimental Design: The Bayer compound library was screened for BUB1 kinase inhibitors and medicinal chemistry efforts to improve target affinity and physicochemical and pharmacokinetic parameters resulting in the identification of BAY 1816032 were performed. BAY 1816032 was characterized for kinase selectivity, inhibition of BUB1 signaling, and inhibition of tumor cell proliferation alone and in combination with taxanes, ATR, and PARP inhibitors. Effects on tumor growth in vivo were evaluated using human triple-negative breast xenograft models. Results: The highly selective compound BAY 1816032 showed long target residence time and induced chromosome mis-segregation upon combination with low concentrations of paclitaxel. It was synergistic or additive in combination with paclitaxel or docetaxel, as well as with ATR or PARP inhibitors in cellular assays. Tumor xenograft studies demonstrated a strong and statistically significant reduction of tumor size and excellent tolerability upon combination of BAY 1816032 with paclitaxel or olaparib as compared with the respective monotherapies. Conclusions: Our findings suggest clinical proof-of-concept studies evaluating BAY 1816032 in combination with taxanes or PARP inhibitors to enhance their efficacy and potentially overcome resistance.

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

Background Fibroblast growth factor receptor (FGFR2) has been proposed as a target in gastric cancer. However, appropriate methods to select patients for anti-FGFR2 therapies have not yet been established. Methods We used in situ techniques to investigate FGFR2 mRNA expression and gene amplification in a large cohort of 1036 Japanese gastric cancer patients. FGFR2 mRNA expression was determined by RNAscope. FGFR2 gene amplification was determined by dual-color in situ hybridization (DISH). Results We successfully analyzed 578 and 718 samples by DISH and RNAscope, respectively; 2% (12/578) showed strong FGFR2 gene amplification (FGFR2:CEN10 >10); moderate FGFR2 gene amplification (FGFR2:CEN10 10 dots/cell and >10% of positive cells with dot clusters under a 20× objective) was seen in 4% (29/718). For 468 samples, both mRNA and DISH data were available. FGFR2 mRNA expression levels were associated with gene amplification; FGFR2 mRNA levels were highest in the highly amplified samples (n = 12). All highly amplified samples showed very strong FGFR2 mRNA expression (dense clusters of the signal visible under a 1× objective). Patients with very strong FGFR2 mRNA expression showed more homogeneous FGFR2 mRNA expression compared to patients with lower FGFGR2 mRNA expression. Gastric cancer patients with tumors that had an FGFR2 mRNA expression score of 4 had shorter RFS compared with score 0–3 patients. Conclusion RNAscope and DISH are suitable methods to evaluate FGFR2 status in gastric cancer. Formalin-fixed paraffin-embedded (FFPE) tissue slides allowed evaluation of the intratumor heterogeneity of these FGFR2 biomarkers. Electronic supplementary material The online version of this article (doi:10.1007/s10120-017-0758-x) contains supplementary material, which is available to authorized users.

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