Heiner Fiebig

In vitro effect of organotin-substituted steroids in human tumor cell lines

Abstract Four steroidal organotin compounds have been prepared and compared in vitro with a parent steroid and two model compounds in a series of human tumor cell lines. The organotin steroids (compounds 1 and 2) showed promising in vitro activity. Another compound with important characteristics in terms of bond angles and stereochemistry (compound 3) was a highly effective antitumor agent. This compound may serve as a model for further investigation on the structure-activity relationship in antitumor organotin compounds.

Detection of monosomy in interphase nuclei and identification of marker chromosomes using biotinylated alpha-satellite DNA probes

Nonradioactive in situ hybridization with chromosome-specific highly repetitive DNA probes is a fast and easy method for the detection of the number of chromosome copies in nonmitotic cells. In this study, we report the use of four biotinylated probes of the human alpha-satellite family recognizing the (peri)centromeric regions of chromosomes 3, 10, 16, and 17. The reliability of the probes was tested by hybridizations to metaphase chromosomes and interphase nuclei of normal blood lymphocytes, which showed a two signal score in 85%-94% and 82%-86% of the cells, respectively. In situ hybridization experiments with nuclei and metaphase spreads derived from the LXFS-650 cell line indicated monosomy for chromosomes 10 and 16 and the presence of two derivative chromosomes 17. These results were in accordance with the cytogenetic data obtained with GTG-banding and confirmed the monoclonality of the cell line. Furthermore, with this method the origin of an unclassified marker chromosome could be identified as a derivative of chromosome 3. Our results show that fluorescence in situ hybridization can be a useful tool in cancer cytogenetics for the detection of numerical aberrations in interphase nuclei and for the classification of marker chromosomes in addition to conventional cytogenetic techniques.

Lung Cancer: EGFR Inhibitors with Low Nanomolar Activity against a Therapy-Resistant L858R/T790M/C797S Mutant

The treatment of non-small-cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) inhibitors is made challenging by acquired resistance caused by somatic mutations. Third-generation EGFR inhibitors have been designed to overcome resistance through covalent binding to the Cys 797 residue of the enzyme, and these inhibitors are effective against most clinically relevant EGFR mutants. However, the high dependence of these recent EGFR inhibitors on this particular interaction means that additional mutation of Cys 797 results in poor inhibitory activity, which leads to tumor relapse in initially responding patients. A new generation of irreversible and reversible mutant EGFR inhibitors was developed with strong noncovalent binding properties, and these compounds show high inhibitory activities against the cysteine-mutated L858R/T790M/C797S EGFR.

The phospholipid- and calcium-dependent protein kinase as a target in tumor chemotherapy

Evidence for a constitutive activation of protein kinase C (EC 2.7.1.37) in Ha-ras transformed 3T3 cells is presented. Several compounds which inhibit protein kinase C in vitro have been studied with regard to their antiproliferative activity in cultured tumor cells. The following agents were investigated: 3-hexadecyl-mercapto-2-methoxy-methyl-propyl-1- phosphocholine (BM 41440); 1-octadecyl-2-methyl-sn-glycero-3-phosphocholine (ET-18-OCH3); quercetin, tamoxifen and staurosporine. All compounds decrease protein kinase C activity in vitro as well as in intact cells and inhibit cell multiplication within the same dose range. The results suggest a causal relation between the antiproliferative effects and the inhibition of protein kinase C. All inhibitors of protein kinase C synergistically enhance the antiproliferative activity of cis-diamminedichloroplatinum(II). Available data suggest that the effects of protein kinase C inhibitors should be exploitable for tumor chemotherapy.

Abstract 2480: MI130004, a new antibody-drug conjugate, induces strong, long-lasting antitumor effect in HER2 expressing breast tumor models

Background: Antibody-drug conjugates (ADC`s) have emerged as powerful tools for the treatment of cancer, combining the potency of cytotoxic molecules with the selectivity of antibodies targeted towards specific antigens. Marine compounds represent an interesting opportunity worth exploring as they possess the requirements needed to be considered promising payloads. The in vivo results obtained with a new ADC, MI130004, in HER2 expressing breast tumors are presented here. Materials and Methods: To evaluate the long lasting antitumor effect induced by MI130004, immunodefficient female mice were subcutaneously implanted with HER2 expressing breast lines namely, JIMT-1, BT-474 and MAXF574.Tumor (ca. 100 mm 3 ) bearing animals were randomly allocated (Day 0) to receive MI130004 (10mg/kg), trastuzumab (30 or 10 mg/kg) or placebo (N = 8-10/group). Intravenous treatments were weekly administered for 5 consecutive weeks and then, tumor volume growth was recorded 2-3 times per week. For survival evaluation, time to endpoint was define as the time from Day 0 to death as a results of tumor growth (>2000 mm 3 ) or any other cause (e.g., tumor necrosis). Statistical differences were assessed by Kaplan-Meier curves with the log rank test. The follow-up period was extended until 90 (MAXF574) or 120 days (JIMT-1 and BT-474) after the initiation day (Day 0). Then, surviving animals were sacrificed, tumor (or skin around the former tumor site) dissected free, formalin fixed and paraffin embedded for histopathology evaluations. Results: The treatment with MI130004 induced a long lasting antitumor effect with statistically significant increases in median survival time compared to either placebo (P = 0.0336, P = 0.0001 and P 3 ). All animals originally xenografted with BT-474 experienced complete tumor remissions by Day 120. On Day 90, 90.0% of the population xenografted with MAXF574 survived and 55.6% of these experienced complete tumor remission. Conclusions: The treatment with MI130004 on mice bearing HER2 expressing breast tumors resulted in strong, long lasting antitumor activity, including complete tumor remissions. These results strongly suggest that MI130004 is a new ADC with extraordinary therapeutic anti-cancer properties. Citation Format: Pablo M. Aviles, Maria Jose J. Guillen, Alberto Gallardo, Maria Virtudes Cespedes, Ramon Mangues, Heiner Fiebig, Natalie Hartman, Juan Manuel Dominguez, Luis Francisco Garcia, Carlos Galmarini, Carmen Cuevas. MI130004, a new antibody-drug conjugate, induces strong, long-lasting antitumor effect in HER2 expressing breast tumor 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 2480. doi:10.1158/1538-7445.AM2015-2480

Abstract B178: A functional mutational profile of a compendium of 350 patient-derived tumor xenografts (PDXs).

Introduction: PDXs are an increasingly popular tool for drug discovery. One reason is that well-characterized tumor models enable streamlining of testing procedures, as molecular markers and drug efficacy can be assessed simultaneously. At Oncotest we have >350 well-characterized PDXs representing many tumor types including niche tumors. The aim of this study was to define PDXs by functionally distinct mutational profiles. Methods: Mutational analyses were conducted for well-studied oncogenes and tumor suppressor genes by Sequenom (OncoCarta panels 1, 2 and 3), the results of which were validated and extended by Sanger sequencing (targeted sequencing of exons containing hotspots of mutations). Results: We categorized mutational profiles of tumor types well-represented in the PDX collection by identifying common driver mutations and then dividing the mutation carrying tumors into sub-groups based on additional mutations. For example, we identified melanoma PDXs (MEXFs) with activating BRAF mutations and MEXFs with activating NRAS mutations. We then analyzed whether BRAF or NRAS mutant MEXFs also carried a PTEN mutation. We found that of 13 BRAF-mutant MEXFs, 8 had a PTEN mutation. Of the 7 NRAS mutant MEXFs, 2 carried a PTEN mutation. When testing a drug in melanoma, it will be of interest to include representative models from each of the sub-groups with distinct mutational profiles, as they can be expected to respond differently to treatment. We were also interested in screening all tumor types for mutations primarily present in distinct histologies and identifying mutations in new histologies. This could lead to a new histology being of interest for therapeutic intervention with a targeted agent, or the definition of tumors by a specific mutation or mutational profile, rather than by histology, as a potential concept for patient selection for future trials. For example, we analyzed IDH1 and IDH2 mutations, which are mostly studied in glioma and myeloproliferative disease in PDXs from >25 histologies. We found five PDXs with mutations in IDH1 and IDH2 known to produce the oncometabolite R(-)-2-hydroxyglutarate: Two melanoma PDXs, one liver PDX, two colon cell-line derived xenografts (CDXs) and one NSCLC PDX. The mutations are rare events in each of the histologies, but, if broad mutational screening is done, patients carrying druggable mutations regardless of the tumor histology may benefit from treatment targeting these mutations. Conclusions: Compiling mutation data of several genes leads to the identification of genetic sub-groups of a tumor histology, which is a useful basis for the efficacy testing of novel drugs, as potential biomarker information is simultaneously collected. Screening different histologies for well-characterized mutations can potentially identify additional histologies or individual tumors for the further use of drugs targeting such mutations. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B178. Citation Format: Rebekka Krumbach, Jagatheswari Virajah, Thomas Metcalfe, Heiner Fiebig, Vincent Vuaroqueaux. A functional mutational profile of a compendium of 350 patient-derived tumor xenografts (PDXs). [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B178.

Abstract B111: Molecular and chemosensitivity profiling of melanoma cell lines with acquired resistance to BRAF inhibitor Vemurafenib.

Introduction: One of the main limitations of patient benefit from targeted therapies is the rapid development of resistance. The BRAF inhibitor Vemurafenib is approved for BRAF mutant melanoma. Here we generated four Vemurafenib resistant melanoma cell lines and found that each had a unique profile of receptor tyrosine kinase over or underexpression, compared to the parent cell lines. Methods: Cell lines used were the commercially available HT-144 and three Oncotest patient-derived xenograft-derived cell lines MEXF 276L, MEXF 394L and MEXF 520L, all sensitive to Vemurafenib and carrying the BRAF V600E or V600K mutation. Each was treated continuously with increasing Vemurafenib concentration up to 6 µM. Results: Upon continuous Vemurafenib treatment all four melanoma cell lines developed Vemurafenib resistance. The resistant cell lines had between 19 and 168 fold higher IC50 values compared to the mock-treated lines. These IC50 values are comparable with IC50 values for cell lines without BRAF mutations, meaning that these cell lines are truly resistant to Vemurafenib. Chemosensitivity profiling revealed cross-resistance to other BRAF inhibitors (n=3) as well as to MEK inhibitors (n=6), while for AKT, c-MET, Eg5, EGFR or PI3K inhibitors sensitivities were mostly similar to those of the parental cell lines. The cross-resistance with BRAF and MEK inhibitors suggests a mechanism of resistance either downstream of MEK or independent of the RAS/RAF/MEK pathway. Excess receptor tyrosine kinase (RTK) expression has been associated with resistance of BRAF mutant melanomas to Vemurafenib. Molecular analyses showed that each of the four resistant cell lines displayed an altered expression of RTKs with each showing a different combination of over- and under-expressed receptors. Downstream phosphorylation of Erk1/2, Akt and S6RP were also found to be altered in the resistant cell lines compared to their sensitive counterparts. Conclusions: Four BRAF-mutant melanoma cell lines acquired resistance to BRAF inhibitor Vemurafenib with cross- resistance to other BRAF and MEK inhibitors. However, the cross resistance seems to be the only similarity between the resistant cell lines, as changes in RTK expression and downstream Erk1/2, Akt and S6RP phosphorylation were unique to each cell line. This leads to the conclusion that each of the four resistant cell lines may represent a resistance mechanism present in a sub-group of the patient population. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B111. Citation Format: Rebekka Krumbach, Anne-Lise Peille, Torsten Giesemann, Vincent Vuaroqueaux, Heiner Fiebig, Thomas Metcalfe, Gerhard Kelter. Molecular and chemosensitivity profiling of melanoma cell lines with acquired resistance to BRAF inhibitor Vemurafenib. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B111.

Abstract C8: Functional and molecular characteristics of patient-derived xenografts of colon cancer.

Introduction: Colorectal carcinoma (CRC) is the second leading cause of cancer-related deaths worldwide. Patient-derived xenografts (PDX) subcutaneously implanted in nude mice are highly suitable tools to aid in the development of novel anti-cancer therapeutics as their response is a good predictor of clinical outcome. In the present study, a panel of colon cancer PDXs (CXFs) was characterized for their molecular profile and sensitivity to standard of care (SoC) drugs. Material and Methods: CRC samples from patients undergoing surgery were implanted subcutaneously in nude mice and sequentially passaged. Tumor material was collected for molecular profiling, including mutational analysis by Sanger sequencing and Sequenom OncoCarta panels I, II, III. The efficacy of the SoC drugs 5-fluoruracil, oxaliplatin, irinotecan, bevacizumab and cetuximab was tested in vivo. All compounds were administered at clinically relevant dose levels and schedules. The tumor load was determined by caliper measurements twice a week. Anti-tumor activity was determined by comparing the relative tumor load of test groups vs. the vehicle-treated control group. Tumors were considered sensitive if the minimal test / control (T/C [%]) value recorded during an experiment was smaller than 30%. For cytotoxic SoC drugs, sensitivity of CXFs were also analyzed in an ex vivo tumor colony assay (TCA). Results: 73 CRC patient samples were implanted within the framework of this study. Up to now, 65 of them were established as CXF corresponding to a take rate of 89%. Mutational analysis was performed for 58 CXFs. Regarding the RAS/RAF pathway, mutations were found in K-RAS (26 out of 58 CXFs, 45%), NRAS (3%) and BRAF (7%). Mutations in the AKT/PI3K pathway were also found (PTEN, 9% and PIK3CA, 17%). In addition, one CXF had an IDH1 mutation (2%). For all SoC drugs both sensitive and resistant CXFs were identified and given CXFs differed in their sensitivities to different SoC drugs. In vivo sensitivity data will be correlated with molecular characteristics of the tested CXFs and with their sensitivities determined in the TCA. Conclusion and Outlook: Due to the high take rate, our CXF panel represents a large proportion of clinical variants of CRC. We will describe mutation profiles and sensitivity profiles to SoC drugs used for colon cancer. Correlation of sensitivity data and molecular data may reveal interesting correlations and identify biomarkers predictive of response to SoC drugs and experimental compounds. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C8. Citation Format: Jianing M. Guo, Kerstin Klingner, Rebekka Krumbach, Armin Maier, Silvia Naus, Vincent Vuaroqueaux, Evelyn Lamy, Heiner Fiebig, Julia B. Schueler. Functional and molecular characteristics of patient-derived xenografts of colon cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C8.

Abstract 3835: Ex vivo 3D assay: rapid and reliable replication of the in vivo anti-tumor efficacy of c-Met inhibitors.

For compounds directed against molecular targets expressed in tumor cells, the ex vivo 3D tumor clonogenic assay (TCA) is a rapid and reliable ex vivo assay with a high predictive value for in vivo tumor sensitivity. Single cell suspensions prepared from patient-derived tumor xenografts (PDX) growing subcutaneously in nude mice or from cultured human tumor cell lines are seeded in semisolid medium and tumor colony formation is monitored in the presence or absence of test compounds over a period of one to three weeks. Based on experiments with up to 70 PDX, we have demonstrated that the TCA accurately replicates the in vivo sensitivity of PDX towards cMet inhibitors across all major tumor histologies. More specifically, all three NSCLC PDX that regressed in response to cMet inhibition in in vivo efficacy tests were sensitive to several cMet inhibitors in the ex vivo TCA. By contrast, data obtained with a 2D cell proliferation and survival assay did not correlate with the 3D and in vivo situations, suggesting that cMet function does not affect cell survival and proliferation on plastic but confers the capacity for anchorage-independent growth. The correlation of 3D but not 2D data with in vivo sensitivity was confirmed using anti-cMet siRNAs in selected PDX-derived non-small cell lung cancer cell lines. Preliminary immunohistochemical analysis revealed that PDX sensitive to cMet inhibitors expressed high cMet levels while not all PDX expressing high cMet levels were sensitive to cMet inhibitors. In conclusion, for cMet inhibitors the TCA replicates in vivo sensitivities of PDX to a high degree. Due to its short duration the TCA is an excellent tool for the screening of large numbers of cMet inhibitors and PDX. As all of the PDX qualified for use in the TCA (>200) have been extensively molecularly characterised (gene expression, gene copy number variation and mutation analysis) this assay is also an excellent tool for generating high quality biomarker hypotheses during the preclinical profiling of molecules intended for oncology indications. Citation Format: Sabine Gorynia, Jianing Guo, Andreas Ackermann, Armin Maier, Rebekka Krumbach, Gerhard Kelter, Vincent Vuaroqueaux, Thomas Metz, Thomas Metcalfe, Heiner H. Fiebig. Ex vivo 3D assay: rapid and reliable replication of the in vivo anti-tumor efficacy of c-Met inhibitors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3835. doi:10.1158/1538-7445.AM2013-3835

Nuclear Components and Plasma Membrane as Targets of Chemotherapeutic Agents

Autonomous self-stimulation of cellular replication is an intrinsic property of malignant growth. Compounds which inhibit enzymes involved in autonomous growth stimulation should, therefore, be useful agents in tumor chemotherapy. This autonomy is achieved (1) by an autocrine production of growth factors, (2) by modified growth factor receptors which are active even in the absence of the corresponding ligand, or (3) by a constitutive activation of elements of the growth factor signal transduction pathways (Sporn and Todaro 1980; Goustin et al. 1986). Various growth factors cause an activation of phospholipid and Ca2+-dependent protein kinase (protein kinase C); furthermore, this enzyme has been shown to be constitutively activated in some transformed cell lines (Nishizuka 1987; Maly et al. 1988). Therefore, inhibitors of this enzyme seem to be suitable candidates for new antitumor agents. The fact that various isoenzymes have been described for protein kinase C (Coussens et al. 1986; Knopf et al. 1986) may even open up possibilities for tumor-specific effects. We have, therefore, investigated whether inhibitors of protein kinase C inhibit tumor growth and whether these inhibitions are related to a depression of the corresponding enzyme. Furthermore, it seemed of interest to investigate whether combinations of protein kinase C-inhibitors with established antitumor agents can be found in which the antiproliferative activities of the combined drugs exhibit a synergistic or additional behaviour. As protein kinase C (in its active form) is localized in the plasma membrane (Anderson et al. 1985), we have focussed on antitumor agents which are known to interfere with the plasma membrane.