Supplementary Figure S1 shows a scatter plot of the GDC-0623 IC70 values across cell lines sorted by cancer entities.

Five supplementary tables 1 to 5 giving more information about:- the culture conditions and molecular profile of the 294 cell lines tested.- the IC70 range per cancer type.- the number of "sensitive" and "less sensitive to resistant" cell lines per cancer type.- the IC70 values of BI-847325 and GDC-0623 per cell line.- the genomic alterations of MAPK pathway-related genes in the cell lines sensitive to BI-847325.

In vivo antitumor growth efficacy of BI-847325 in 11 solid tumor models grown as subcutaneous xenografts in nude mice. A, Tumor growth curves over time are shown as the relative tumor volumes (%). BI-847325 (either at 80 or 40 mg/kg/dose), and the vehicle were administered orally once per week for 3 or 4 weeks. Black curves: vehicle control at 10 mL/kg/dose; light blue: BI-847325 at 40 mg/kg/day and dark blue: BI-847325 at 80 mg/kg/day; green curve: capecitabine administered orally at 200 mg/kg/day on days 1–7; violet curve: GDC-0623 administered orally at 40 mg/kg/day on days 1–14. CXF 1103 and RKO: colorectal cancer; GXA 3011 and GXA 3023: gastric cancer; MAXFTN 401 and MDA-MB-231: triple-negative mammary cancer; MIA-PaCa-2: pancreatic cancer; RT112: bladder cancer. B, antitumor activity of BI-847325 in human tumor models tested at a dose level below the MTD, in vivo tumor growth inhibition, and KRAS, BRAF, NRAS, MAP2K1 mutational status are shown. Rating of BI-847325 antitumor activity is defined in the Materials and Methods section.

<p>Comparison between the <i>in vitro</i> antiproliferative activities of BI-847325 and the MEK inhibitor GDC-0623. <b>A,</b> Contingency table with counts of CLs per class of sensitivity to BI-847325 and GDC-0623 (cutoff for Abs IC₇₀ values for both compounds: ≤ 1 µmol/L; statistics: Fisher exact test, significance: <i>P</i> < 0.05). <b>B,</b> Correlation plot of BI-847325 and GDC-0623 Abs IC₇₀ values for 276 CLs. Dark blue circles: CLs derived from solid tumors, Dark red circles: CLs derived from hematologic cancers. <b>C,</b> Counts of CLs per tumor type being sensitive to GDC-0623 and/or BI-847325 (Abs IC₇₀ ≤ 1µmol/L). Dark blue bars: CLs sensitive to both GDC-0623 and BI-847325, Green bars: CLs sensitive to BI-847325 only. The total number of CLs per tumor type is indicated in black on the right of each bar.</p>

Human cell line panel (n = 294) and cancer types investigated for BI-847325 efficacy

AbstractBI-847325 is an ATP-competitive inhibitor of MEK/Aurora kinases with the potential to treat a wide range of cancers. In a panel of 294 human tumor cell lines in vitro, BI-847325 was found to be a highly selective inhibitor that was active in the submicromolar range. The most sensitive cancer types were acute lymphocytic and myelocytic leukemia, melanomas, bladder, colorectal, and mammary cancers. BI-847325 showed a broader range of activity than the MEK inhibitor GDC-0623. The high efficacy of BI-847325 was associated with but not limited to cell lines with oncogenic mutations in NRAS, BRAF, and MAP2K1.The high antiproliferative activity of BI-847325 was validated in vivo using subcutaneous xenograft models. After oral administration of 80 and 40 mg/kg once weekly for 3 or 4 weeks, BI-847325 was highly active in four of five colorectal, two of two gastric, two of two mammary, and one of one pancreatic cancer models (test/control < 25%), and tumor regressions were observed in five of 11 cancer models. The treatment was well tolerated with no relevant lethality or body weight changes. In combination with capecitabine, BI-847325 displayed synergism over single-agent therapies, leading to complete remission in the triple-negative mammary model MAXFTN 401, partial regression in the colon model CXF 1103, and stasis in the gastric models GXA 3011 and GXA 3023. In conclusion, dual MEK/Aurora kinase inhibition shows remarkable potential for treating multiple types of hematologic and solid tumors. The combination with capecitabine was synergistic in colorectal, gastric, and mammary cancer.Significance:We report the preclinical evaluation of BI-847325, a MEK/Aurora kinase inhibitor. Our data demonstrate that BI-847325 has potent antitumor activity in a broad range of human solid and hematologic cancer models in vitro and in vivo and is well tolerated in animal models. It also shows synergistic effect when combined with capecitabine. These findings provide a strong rationale for further development of BI-847325 as a potential therapeutic for patients with cancer.

Abstract The epidermal growth factor receptor (EGFR, HER1, ERBB1) is a driver of many human cancers. Standard of care treatment for colon, head and neck and non-small cell lung cancer includes drugs targeting EGFR. Numerous molecular alterations activating the oncogenic potential of the EGFR gene have been described including activating point mutations, point mutations causing resistance against EGFR-targeting drugs, activating deletions and truncations as well as overexpression of EGFR and its ligands, occasionally induced by gene amplification. In other situations, EGFR has been identified as a driver in the absence of any obvious molecular alterations of the EGFR gene. The discovery and development of EGFR-targeting agents depends on the availability of relevant tumor models. We provide an overview of our collection of EGFR-driven tumor models, including PDXs along with PDX-derived cell lines and human tumor cell lines. We compare sensitivity profiles of tumor models for EGFR-targeting agents obtained in vivo and in vitro and demonstrate that for EGFR-targeting agents, data obtained in 2D and 3D assays are predictive for the in vivo situation. We propose an optimized strategy for the preclinical profiling of EGFR-targeting anti-cancer agents. Citation Format: Markus Posch, Hagen Klett, Anne-Lise Peille, Gerhard Kelter, Armin Maier, Julia Schüler, Thomas M. Metz. Tumor models driven by EGFR: Optimizing the preclinical profiling of EGFR-targeting agents [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2813.

The BET family of bromodomain proteins (BRD2, BRD3, BRD4, and BRDT) has emerged as a promising new cancer target for small-molecule drug discovery. GSK 1324726A (I-BET726) is a highly selective inhibitor of BET family proteins that binds BRD2, BRD3 and BRD4 with IC50 of 41 nM, 31 nM, and 22 nM, respectively. In the present study, we aimed to identify sensitive tumor types for GSK 1324726A and predictive biomarkers for response or resistance. GSK 1324726A was tested in vitro in a 2D monolayer assay in 274 human tumor cell lines of 36 tumor types (50 liquid and 224 solid cancer cell lines). Antitumor activity was then tested in vivo in 5 of the most sensitive tumor cell lines transplanted sc in nude mice. Drug sensitivity results in vitro were correlated with molecular data of the tumor models, including whole exome mutations, gene copy number variations and gene expression profiles. GSK 1324726A showed a moderate anti-tumor potency (median IC70 6,6 µM, range 0.03 - >30 µM) and a pronounced selectivity in a wide range of blood and solid cancer cell lines. AML, ALL, multiple myelomas and B-cell lymphomas were 1 log more sensitive than the most sensitive solid tumors. Taking 10% of the median IC70 of all tumors as cut off (0.66 µM) 5/11 AML, 4/9 ALL, 5/11 MM and 7/13 B-NHL were sensitive. Among the solid tumor panel cervix uterus and prostate models were sensitive (2/5) followed by breast, soft tissue, CRC, ovarian cancers and melanomas (between 17 and 40%). In-vivo experiments of the most sensitive tumors are under way. The MTD of GSK 1324726A (po for 15 days) was 15 mg/kg/day as it effected a slight body weight loss of 4% and no lethality. Evaluation of tumor cell lines for molecular parameters associated with response was carried out separately for hematologic and solid cell lines. For the leukemias, lymphomas and myelomas about 100 genes were significantly associated with response to GSK 1324726A at the transcriptome level (intersection of Spearman and Limma tests, adjusted p values <0.05). JAG1 was identified as the most significant gene among them. JAG1 is known to interact with receptors in the Notch signaling pathway and regulate cell fate decisions. Solid tumor models did not show a strong predictive gene pattern. Integrative analysis with significant genomic and transcriptomic parameters is currently ongoing to develop a molecular predictor of response to GSK 1324726A. Extending evaluation of the compound in vivo and continuing investigation of predictors of response are underway. Citation Format: Heinz-Herbert Fiebig, Gerhard Kelter, Hans R Hendriks, Vincent Vuaroqueaux. Broad spectrum activity of the BET inhibitor GSK 1324726A in hematologic and solid cancer cell lines in-vitro and determination of associated predictive biomarkers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3074.

Targeted therapy of BRCA-deficient cancers has been achieved using poly(ADP-ribose) polymerase (PARP) inhibitors, which block BRCA-independent DNA repair. With first approval in 2014 of Olaparib the concept of tumor-specific synthetic lethality was added to the treatment portfolio of cancer patients. Although the effects of PARPi have shown promising results in multiple cancer types, how and whether patients might benefit from combination with compounds modulating the immune landscape of a tumor is largely unknown. In the current study, we investigate the cross-talk between PARPi and immune checkpoint inhibition, in particular, anti PD-1 and anti CTLA-4, as the most advanced targets in the field. PARP inhibitors Niraparib, Rucaparib, Talazoparib and Olaparib were investigated in vivo using the murine EMT6/BRCA1-/- model in monotherapy as well as in combination with anti-CTLA-4 or anti-PD1 treatment. The four PARPi showed distinct activity profiles in the two breast cancer models. Talazoparib was the most active compound in the BRCA1-/- model (optimal T/C (test/control) of 60%), followed by Niraparib and Rucaparib (65% and 67%, respectively). Olaparib was considered inactive with a T/C value of 80% in monotherapy. The EMT6/BRCA1-/- turned out to be sensitive towards anti CTLA-4 treatment (optimal T/C of 35% - 38% in three independent experiments). Anti PD-1 treatment in monotherapy induced no significant reduction in tumor growth (optimal T/C of 80% - 82%, in two independent experiments). The combination of PARPi and anti CTLA-4 induced a transient but significant reduction of tumor load early in the treatment phase (p< 0.002, one-way ANOVA, day 8). However, anti PD-1 treatment significantly prolonged overall survival in combination with Talazoparib (p< 0.011 log-rank test). The analysis of tumor infiltrating lymphocytes (TILs) by flow cytometry revealed that Talazoparib as monotherapy and in combination with checkpoint (CP) inhibitors enhanced the number of gMDSC as well as the number of CD3/CD11b double positive T cells. Weekly cytokine analysis in the serum of tumor bearing mice will elucidate possible interactions between PARP and CP inhibitors and give guidance to optimal combination and schedules. First results indicate that PARPi as well as CPi induce unique cytokine profiles correlating well with the modified TIL composition of the respective treatment groups. Further mechanistic studies as well as comparative studies with the EMT6/BRCAwt model will elucidate the tumor biology behind these observations and might lead to beneficial combination strategies in patients suffering from triple negative and BRCA1-/- breast cancer.Citation Format: Anya Avrutskaya, Cordula Tschuch, Astrid Jensen, William Durham, Maycee Robinson, Charles Krause, Emily O'Koren, Gerhard Kelter, Anne-Lise Peille, Armin Maier, Anne-Marie Zuurmond, Julia Schüler. Modulation of the tumor-infiltrating lymphocyte population by PARP inhibitor talazoparib in combination with anti-PD1 treatment significantly enhances overall survival in a murine BRCA1-/- breast cancer model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2709.

Abstract 4HF Biotec recently developed an in-silico platform dedicated to cancer data investigations. It connects cancer-related molecular and drug sensitivity information enabling target and biomarker discovery. We used the platform to perform an integrative pharmacogenomic study for the CDK4/6 inhibitor Palbociclib. CDK4/6 inhibitors are molecules blocking E2F1 mediated G1/S cell cycle progression. Palbociclib is already approved in the clinic for treatment of HR-positive, ERBB2 negative advanced and metastatic breast cancer. Clinical trials in other tumor types such as leukemia, head and neck, ovarian and non-small cell lung cancer are also ongoing. In this context, large preclinical studies may help for identification of companion diagnostics improving patient selection. In a data-driven approach, we searched for molecular events associated with cancer response and resistance to Palbociclib. A publicly available dataset was used that contains 843 tumor cell lines which were tested for response to Palbociclib in a 2D monolayer assay in vitro (GDSC). Palbociclib IC50s values were integrated to our platform and were evaluated for associations with the molecular features of the cell lines including mutated genes (whole exome sequencing), gene deletions or amplifications (SNP6.0 microarrays) and gene expression (Affymetrix HGU133 plus 2.0) or combinations. The resulting biomarker candidates were analyzed for predictivity in an independent dataset of 142 tumor cell lines obtained from 4HF Biotec (internal data). In vitro, the best efficacies were observed in cell lines from hematological cancers (leukemia and lymphoma) and solid tumors such as renal, neuroblastoma, head and neck cancers. Very sensitive cell lines were also seen in most of the other tumor types tested. Surprisingly, the panel of breast cancer cell lines was among those with the lowest proportion of sensitive cell lines (6 out of 43) with those derived from small cell lung and uterus cancers. Statistical analyses identified alterations in RB1 gene by far the most prominent biomarker associated with resistance to Palbociclib (adjusted p value 2.53.10-9) and that can occur by gene deletions, mutations or loss of expression. Other genomic alterations such as the loss of chromosome 9 containing CDKN2A and CDKN2B were associated with increased response rates. In contrast, cell lines with chromosome amplification in 17q containing ERBB2 or chromosome 1 containing MYCL were mainly resistant to the compound (p<0.05). At the gene expression level, CDK6 and RB1 were among the most significant markers of sensitivity. Focusing on cell lines not altered for RB1, pathway analysis revealed additionally that the genes of the mesenchymal or poorly differentiated phenotypes were the strongest predictors of response. The present pharmacogenomic study allowed identification of a relevant panel of biomarkers predicting Palbociclib response or resistance. Their validation should continue for clinical use in different tumor types and a predictive algorithm could be constructed to improve identification of candidate cancer patients for such treatment. We believe such approach should be more systematically done in programs of drug development. Citation Format: Vincent Vuaroqueaux, Hoor Al Hasani, Gerhard Kelter, Hans R Hendriks, Heinz-Herbert Fiebig. The use of 4HF Cancer Data Miner platform for an in Silico pharmacogenomic study predicting tumor response to the CDK4/6 inhibitor Palbociclib [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C075. doi:10.1158/1535-7163.TARG-19-C075

Boshramycinones A-C (1-3), three new anthracyclinones, were isolated from the culture broth of the marine-derived Streptomyces sp. Mei 16-1,2 together with 2-acetyl-1,8-dihydroxy-3-methyl-anthraquinone (4) and bafilomycins B1, B2, and C1-amide. The isolated compounds were identified by NMR spectroscopy and mass spectrometry, the absolute configuration of 3 was determined by comparison of experimental and ab initio-calculated chiroptical data. The antimicrobial activity of the bacterial extract and the isolated compounds were assayed using a set of microorganisms, and cytotoxic activities were determined against 36 human cancer cell lines.

Tubulin binding drugs are approved for the treatment of many cancer types, without the use of molecular markers to select patients likely to respond. Plinabulin binds β-tubulin in a differentiated pocket and is being tested in a Phase 3 clinical study for the treatment of NSCLC. Additional indications are being considered for plinabulin and an algorithm for selecting especially responsive cancers and patient subgroups would be of significant value. With this in mind, Affymetrix HG-U133 Plus 2.0 array mRNA expression data for 43 human breast, lung, prostate, ovarian or CNS cancer cell lines were utilized to develop mathematical models to predict in vitro plinabulin potency against the same cell lines. Cells were treated for only 24 hours with plinabulin and then cultured for another 48 hours without plinabulin. Viable cell number was then measured with a Cell Titer-Blue Assay, and the plinabulin concentration causing a 70% reduction in viable cells (IC70) versus vehicle treated controls was derived. Cell lines were clearly separable into plinabulin Active (21 cell lines with IC70<1.0 μM) and Inactive (IC70>9.5 μM) groups. Log2 transformed Affymetrix gene probeset signal values, preprocessed with the GeneChip robust multi-array average analysis algorithm, were selected and ranked as predictors of plinabulin activity with a bootstrap forest partitioning technique, utilizing JMP 14.1 statistical software. 56 HIT probesets were identified that also had significantly different expression in responding versus non-responding cell lines (p<0.01, uncorrected t-test). For probesets with gene annotation, only the probeset for each gene with the highest Jetset score was utilized (Li et al., 2011). Top HIT predictor genes include CTNNB1 (β-catenin; oncogene), CALD1 (caldesmon; inhibits myosin ATPase activity), ERI1 (RNA processing), LGR5 (adult stem cell biomarker), SECISBP2L (SLAN; prolongs mitosis), and TRAK1 (mitochondrial/endosome trafficking). Models were constructed from HIT gene probesets in JMP to identify plinabulin responding cell lines, utilizing either one-layer TanH multimode fit neural networks or binary logistic regression. Surprisingly, models incorporating approximately 4-10 probeset values were derived that perfectly predicted plinabulin activity. Importantly, the cell lines tested in the above analyses were not those known to express high levels of multi-drug resistant (MDR) transporters. The importance of MDR status was therefore evaluated separately. Plinabulin activity, unlike that of taxanes, was not significantly affected by the MDR transporter inhibitor verapamil (10 μM), in ovarian cancer cell lines with a known MDR phenotype. To conclude, our work provides novel algorithms that may be of value in selecting cancer patients with tumor cells that are particularly susceptible to the direct cytotoxic effects of plinabulin. Citation Format: James R. Tonra, Hagen Klett, Chenghao Shen, Gerhard Kelter, Ramon W. Mohanlal, G. Kenneth Lloyd, Lan Huang. Predictive models for tumor cell targeting with plinabulin, derived from in vitro screening and Affymetrix mRNA expression data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1254.

Abstract The Mouse Double Minute 2 (MDM2) proto-oncogene is a primary cellular inhibitor of p53 tumor suppressor protein. MDM2 encodes for a nuclear-localized E3 ubiquitin-protein ligase via direct interaction responsible for proteasomal degradation of p53. Its overexpression in TP53 wild type tumors leads to the loss of apoptotic function and cell survival. MDM2 inhibitors were envisioned to restore p53 activity in p53 wild type tumors, several are currently being evaluated in the clinic. MI-773 is a small molecule, that inhibits the p53-MDM2 protein-protein interaction. It has been evaluated in Phase I. We aimed to pursue characterization of MI-773’s anti-tumor activity by performing a large pharmacogenomic study to identify tumor candidates for such treatment. MI-773 was tested in vitro in a 2D monolayer assay in 274 human tumor cell lines (50 hematologic and 224 solid cancer cell lines) belonging to 36 tumor types. Drug sensitivity results in vitro were correlated with molecular data of the cell lines, including whole exome mutations, gene copy number variations and gene expression profiles, to study molecular determinants for a response. MI-773 showed a moderate anti-tumor potency (median IC50 13,5 µM, range 0.1 to up to30 µM) and pronounced selectivity was observed in several hematologic and solid cancer cell lines. Fifty tumor cell lines (18,5%) were 10 times more sensitive compared to the median IC50 of all cell lines. Among the solid cell lines, melanomas, kidney, stomach, breast and pleural mesotheliomas were the most sensitive tumor types, whereas ALL, AML, and multiple myelomas were the most sensitive hematological tumors. Confirmatory in vivo studies of MI-773 in the most sensitive tumors are planned. The Compare analysis of MI-773 with 44 targeted agents and 250 cytotoxic agents in 40 to 117 tumor cell lines showed the highest similarity with p53/MDM2 inhibitors Nutlin-3a and YH239-EE with Spearman correlation coefficients of 0,71 and 0,56, a clear indication that biological testing’s in cell line panels allows identification of the mechanism of action of novel compounds. As expected, TP53 mutation was the major determinant of tumor sensitivity to MI-773 (Wilcoxon test, adjusted p value= 2;28E-12). Sixty one out of 88 (69%) tumor cell lines wild type TP53 showed a high sensitivity to MI-773 whereas 141 out of 162 (87%) cell lines mutated for the gene were resistant. Transcriptome analysis revealed that the expression levels of 355 genes were significantly associated with sensitivity to MI-773 (LIMMA and Spearman test adjusted p values both p<0.05). MDM2, CDKN1A, SPATA18 and ZMAT3 were the strongest predictors positively associated with both TP53 integrity and sensitivity toward MI-773. Pathway analysis confirmed that overall the genes upregulated in tumor cell lines sensitive to MI-773 to be dominantly involved in negative regulation of mitotic cell cycle, regulation of cell cycle phase transition or signal transduction by p53 class mediator. The present study demonstrated that the MDM2 inhibitor MI-773 could be used in a broad range of hematological and solid tumor types, however, with the determination of TP53 as a prerequisite. MDM2 inhibitors should be evaluated in p53 wild type melanomas, mesotheliomas, kidney, stomach, and breast cancers as well as in ALL, AML, and multiple myelomas. Citation Format: Heinz-Herbert Fiebig, Gerhard Kelter, Hans R Hendriks, Hoor Al Hasani, Vincent Vuaroqueaux. Broad spectrum activity of the MDM2 inhibitor MI-773 in hematologic and solid cancer cell lines in-vitro and determination of predictive biomarkers [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C060. doi:10.1158/1535-7163.TARG-19-C060

Karamomycins A-C (2-4), the first natural 2-naphthalen-2-yl-thiazole derivatives, were isolated along with a plausible precursor molecule, 1-hydroxy-4-methoxy-2-naphthoic acid (1), uracil, 1-acetyl-β-carboline, and actinomycin C2 from the culture broth of the terrestrial actinomycete strain GW58/450, identified as Nonomuraea endophytica. These compounds were characterized by analysis of their NMR and mass spectrometry (MS) data; the absolute configurations of 2 and 4 were determined by comparison of 13C NMR, NOESY, and circular dichroism (CD) spectra with density functional theory (DFT)-calculated data. In karamomycin C (4), the thiazole of 2 is connected to an unusual iminothiazolo[4,3- c][1,4]thiazepinone, for which we proposed a biosynthetic origin from two cysteine residues. It is closely related to ulbactin F; however, the heterocycle is enantiomeric to the latter and connected to phenol instead of 4-methoxy-1-naphthol. Karamomycins A (2) and C (4) were cytotoxic.

MEK inhibitors emerged as a promising class of anti-cancer agents to inhibit KRAS/RAF driven tumors like melanoma and colorectal cancer. GDC-0623 is a potent ATP-noncompetitive allosteric MEK1 inhibitor. The compound forms a strong hydrogen bond with Ser212 of MEK1, blocking MEK feedback phosphorylation of wild-type RAF. Preclinical studies showed efficacy in vitro and in vivo and in a phase I clinical study, suggesting that the compound has clinical potential. In the present work, we aimed to define candidate tumors to GDC-0623 treatment and gain insight into biomarkers and predictive models of response.GDC-0623 was tested in vitro in a 2D monolayer assay (260 human tumor cell lines of 36 histological subtypes). Next, activity was tested in vivo in a human tumor cell line engrafted subcutaneously in nude mice. Drug sensitivity results were then bound to molecular information of the tumor models, including whole exome mutations, gene copy number variations and gene expression profiles, to identify determinants and build a predictor of response.In vitro, GDC-0623 showed a strong anti-tumor activity (abs. IC70 <1 µM) in a wide range of blood and solid tumors. IC50 values varied from 0.001 to >30 µM (Geometric mean: 2.95 µM). The majority of leukemia CML and AML cell lines were sensitive (4/7 and 6/11, respectively) whereas lymphoma cell lines were mainly resistant. In solid tumors, strong activity was seen in almost all melanoma cell lines (10/12) and similar activity was observed in 5/8 bladder, 10/16 colon, 7/12 head and neck, 5/12 pancreas, 4/12 gastric and 4/12 breast cell lines. Anti-tumor activity of GDC-0623 was confirmed in vivo in the human colon HTX model COLO-205 (BRAF V600E mutant). An oral dose of 40 mg/kg/day caused a strong inhibition of the tumor growth (T/C = 6%) without impacting animal body weight.Screening of cell lines for molecular features associated with response showed BRAF V600E, NRAS Q61K, Q61R and DUSP27 gene mutations among the strongest predictors of response (Fisher test, p <0.05). Furthermore, cell lines with EGFR and MET amplifications were almost all sensitive (4/5 and 3/4, respectively, Fisher p<0.03). At transcriptome levels, a total 2108 genes were significantly associated with response to GDC-0623 (intersection of Spearman and Limma tests, adjusted p values <0.05). Integrative analysis with significant genomic and transcriptomic parameters is currently ongoing to develop a molecular predictor of response to GDC-0623. Extending evaluation of the compound in vivo and validating predictors of response are needed to complete these preclinical investigations.Citation Format: Vincent Vuaroqueaux, Hoor Al Hasani, Gerhard Kelter, Hans R Hendriks, Heinz-Herbert Fiebig. Strong anti-tumor activity of MEK inhibitor GDC-0623 and determination of predictive biomarkers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2196.

Patient-derived tumor xenograft (PDX) models have become indispensable for the preclinical profiling of novel anti-cancer agents as they retain histological, molecular and pharmacological characteristics of the parental patient tumors and for many tumor types collectively replicate the diversity of patient tumors. To make PDX models available for in vitro assays, we have established a panel of 79 low passage cell lines derived from PDXs representing 15 different tumor histologies. Using, among others, the non-small cell lung cancer model LXFE_2478 which is driven by the exon 20 insertion EGFR mutation M766_A767insASV as an example, we demonstrate the following similarities of PDX models and the corresponding PDX-derived cell lines. (i) Subcutaneous xenografts established from PDX-derived cell lines mirror the histology of the corresponding PDX models and usually also patient tumors. (ii) For LXFE_2478 both the PDX model and the corresponding PDX-derived cell line express high levels of EGFR, suggesting that the oncogenic driver is still present. (iii) For LXFE_2478 the PDX and the PDX-derived cell line subcutaneously implanted in mice display comparable sensitivity to a variety of EGFR inhibitors and cytotoxics. (iv) For LXFE_2478 the sensitivity of the PDX-derived cell line to a variety of EGFR inhibitors in an in vitro 2D cell survival and proliferation assay in general corresponds to the EGFRi sensitivity of the corresponding PDX model in vivo. (v) For several additional PDX models representing various histotypes the sensitivity to first generation EGFR inhibitors of PDX-derived cell lines in the 2D assay is in line with the in vivo EGFR inhibitor sensitivity of the corresponding PDXs. In conclusion, at least for some mechanisms of action in vitro data obtained with PDX-derived cell lines can predict the in vivo behavior of the corresponding PDX model and can thereby accelerate, and reduce the costs of, the discovery of novel anticancer agents. Citation Format: Gerhard Kelter, Anne-Lise Peille, Jutta Fehr, Hagen Klett, Armin Maier, Markus Posch, Thomas M Metz. Characterization of a panel of 79 PDX-derived cell lines with a focus on the EGFR exon 20 insertion mutation-driven NSCLC model LXFE 2478 [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr LB-B05. doi:10.1158/1535-7163.TARG-19-LB-B05

Abstract The p38 MAPK pathway, also called stress activated protein kinase pathway, is involved in basic cellular processes of inflammation and acts on cell growth, proliferation, differentiation, migration, and apoptosis. p38 MAP kinase activation occurs by environmental stress and proinflammatory cytokines signaling and is frequently deregulated in cancers. p38 MAP kinase inhibition has been suggested for treatment of the cancer; a number of inhibitors have been developed and some are in an early phase of clinical evaluation. The present study aimed to investigate the antitumor activity and discover potential predictive biomarkers for the novel p38 inhibitor LN1222/AD736 in a large and diverse series of tumor models. Cancer cell inhibition was tested in vitro by means of a propidium-based monolayer assay in a total of 222 human tumor cell lines, covering 29 different types of solid tumors including 70 derived from PDX. To illuminate molecular determinants of drug sensitivity, we conducted a biomarker discovery study using the molecular profiles of cell lines, which included whole exome mutations, gene copy number variations, and gene expression profiles as determined by next-generation sequencing, Affymetrix SNP6.0 and Affymetrix U133 2plus. Throughout the 222 cell lines, the IC50 and IC70 geometric means were 0.33 μM and 1.3 μM, respectively. High antitumor potency (IC70 <500 nΜ) was found in 121 out of 222 cell lines (54%), and the cell line sensitivity toward LN1222/AD736 was well dichotomized into very sensitive (<500 nM) and very resistant (>30 μM) models. Very sensitive cell lines were observed in leukemia and lymphoma as well as a wide variety of solid tumors including sarcoma (soft tissues and osteosarcoma), prostate, bladder, breast, melanoma, colon, NSCLC, and kidney tumor models, irrespective of the cancer type. Molecular analyses showed that tumor models had comparable expression patterns of the four different isoforms of p38 mRNA (MAPK11, 12, 13, and 14) with those of the patient’s tumors (TCGA data set). None of the four isoforms was found significantly predictive for tumor sensitivity to LN1222/AD736 (Lima test and Spearman correlation analyses). Interestingly, the biomarker discovery screening associated KRAS gene mutations (24 of 127 model tested (20%)) as a strong factor for resistance toward LN1222/AD736, only wild type KRAS models were sensitive (Wilcoxon test p<0.0001). In parallel, we identified a subset of 80 differentially expressed genes that discriminates groups of tumors with a high response rate by using unsupervised hierarchical clustering (p<0.0001). The ingenuity pathway analysis indicates that part of these genes are related to inflammation and cell survival. These biomarker data will be evaluated in an independent data set of tumors treated with p38 inhibitors. Our results demonstrate that the p38 inhibitor LN1222/AD736 had antitumor activity in a wide range of tumors and may represent a relevant treatment option for tumors with wild type KRAS. Extending the analysis in vitro and in vivo in tumor types in which activity was seen and testing combinations of LN1222/AD736 and other compounds targeting the MAPK pathway are needed to refine the evaluation. The biomarker findings should be tested and validated in larger cohort. Citation Format: Vincent Vuaroqueaux, Gerhard Kelter, Hans R Hendriks, Stefan Laufer, Heinz-Herbert Fiebig. KRAS gene mutations are associated with tumor resistance toward the LN1222/AD736 p38 inhibitor [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B167.