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BACKGROUND: Aspirin reduces the incidence of conventional adenomas driven by APC mutation and thus colorectal cancer. The effect of aspirin on the ~20% of colorectal cancers arising via BRAF mutation is yet to be established. METHODS: Braf(V637E/+);Villin-Cre(ERT2/+) mice were allocated to a control (n = 86) or aspirin-supplemented (n = 83) diet. After 14 months the incidence of murine serrated lesions, carcinoma and distant metastases were measured by histological examination. RNA was extracted from carcinomas from each cohort and subjected to sequencing to identify differentially expressed genes and molecular pathways. RESULTS: Aspirin did not reduce the incidence of murine serrated lesions or carcinoma when compared to control, however, did significantly reduce lesion size (P = 0.0042). Among the mice with carcinoma there was a significant reduction in the incidence of distant metastasis with aspirin treatment (RR 0.69, 95% CI 0.48–0.90, P = 0.0134). Key pathways underlying metastasis of carcinoma cells include NOTCH, FGFR and PI3K signalling, were significantly downregulated in carcinomas sampled from mice on an aspirin-supplemented diet. CONCLUSIONS: Aspirin reduces the incidence of metastatic Braf mutant carcinoma, although this is not due to a reduction in primary disease. The reduction in metastasis could be attributed to a delay or prevention of molecular changes within the primary site driving metastatic growth.

Purpose: The two most common molecular subtypes of endometrial cancers, mismatch repair deficient (MMRd) and p53 wild-type (p53wt) comprise the majority of endometrial cancers and have intermediate prognoses where additional risk stratification biomarkers are needed. Isoform switching of FGFR2 from FGFR2b to FGFR2c (normally expressed in mesenchymal cells), has been reported in other solid carcinomas. The objective of this study was to investigate the role of FGFR2c in risk stratification of endometrial cancer. Experimental Design: We have developed and optimized a BaseScope RNA ISH assay to detect FGFR2c. FGFR2c expression was determined in a preliminary screening cohort of 78 endometrial cancers and a clinically and molecularly annotated Vancouver cohort (n = 465). Cox regression model analyses were performed to assess the prognostic value of FGFR2c. Results: Univariate and multivariate analyses revealed FGFR2c expression was significantly associated with shorter disease-specific survival (DSS) and progression-free survival (PFS) in endometrioid endometrial cancer (EEC, n = 302). Notably, FGFR2c expression was significantly associated with shorter PFS and DSS in patients with grade 3 EECs (P < 0.003 and P < 0.002) and the European Society Medical Oncology (ESMO) high-risk group (P < 0.0001 and P < 0.002), respectively. Moreover, within the MMRd subtype, FGFR2c expression was significantly associated with shorter PFS (P < 0.048) and DSS (P < 0.001). Conclusions: FGFR2c expression appears an independent prognostic biomarker in patients with EEC and further discerns the outcomes within grade 3 tumors, ESMO high-risk groups, as well as within the MMRd and p53wt subtypes. FGFR2c inclusion into future molecular subtyping can further refine risk stratification of EEC.

Introduction/Background* The molecular endometrial cancer (EC) classification has proven prognostic value and can direct adjuvant treatment decisions. Despite this, a relatively large group of EC is still molecularly unclassified (NSMP-EC). In this study we aimed to identify biomarkers among high-risk NSMP-EC patients with prognostic and/or predictive relevance. Methodology Paraffin-embedded tumour material (n=423) from the PORTEC-3 HREC trial were available for analysis. All patients with NSMP-EC were selected, hence those without pathogenic POLE mutations, mismatch repair deficiency and p53-abnormal immunohistochemistry (IHC). Protein expression of L1CAM, ER and PR (ongoing) were analysed by IHC using a 10% threshold for positivity. Tumour DNA was analysed for pathogenic somatic mutations using a next generation sequencing (NGS) cancer hotspot panel. Time to recurrence was analysed using the Kaplan-Meier method, log-rank tests and Cox’s proportional hazard models. Result(s)* In total, 126 NSMP-EC were identified in PORTEC-3, the majority were hormone receptor positive (ER n=106/121, 87.6%, PR will be presented at ESGO2021). L1CAM overexpression was observed in 11.2% (n=14/125) and mutations in CTNNB1-exon-3 were identified in 34.3% (n=36/105). Clustering showed that ER-positive NSMP-EC were predominantly endometrioid EC (n=99, 93.4%), low grade (n=88, 83.0%) and L1CAM-negative (n=102, 97.1%) (figure 1). PIK3CA and KRAS mutations were present in 27.3% (n=24) and 19.3% (n=17), respectively. ER-negative NSMP-EC were frequently non-endometrioid (n=11, 73.3%), L1CAM-positive (n=11, 73.3%) and rarely harboured PTEN and CTNNB1 mutations (n=1, 7.1% and 0%, respectively). ER-positivity was associated with lower risk of recurrence (HR 0.32 [95%CI 0.14-0.70]; figure 2A), while L1CAM-overexpression and CTNNB1-exon-3 mutations were not (HR 2.25 [95%CI 0.93-5.43] and HR 1.20 [95%CI 0.57-2.54], respectively). Multivariable analysis confirmed independent favourable prognostic impact of ER-positivity and LVSI. Figure 2B shows impact of LVSI on time to recurrence among patients with ER-positive NSMP-EC. Conclusion* The vast majority of NSMP-HREC are ER-positive (87.6%) and are likely sensitive to hormonal therapy. Other treatment targets might be found in this subgroup too as 27.3% had PIK3CA and 19.3% had KRAS mutations. NSMP-EC with loss of ER-expression were often of non-endometrioid histology and had a high risk of recurrence. Future studies should investigate whether this subgroup would benefit from other systemic therapies.

Endometrial cancer is the most commonly diagnosed gynaecological malignancy. Unfortunately, 15-20% of women demonstrate persistent or recurrent tumours that are refractory to current chemotherapies. We previously identified activating mutations in fibroblast growth factor receptor 2 (FGFR2) in 12% (stage I/II) to 17% (stage III/IV) endometrioid ECs and found that these mutations are associated with shorter progression-free and cancer-specific survival. Although FGFR inhibitors are undergoing clinical trials for treatment of several cancer types, little is known about the mechanism by which they induce cell death. We show that treatment with BGJ398, AZD4547 and PD173074 causes mitochondrial depolarization, cytochrome c release and impaired mitochondrial respiration in two FGFR2-mutant EC cell lines (AN3CA and JHUEM2). Despite this mitochondrial dysfunction, we were unable to detect caspase activation following FGFR inhibition; in addition, the pan-caspase inhibitor Z-VAD-FMK was unable to prevent cell death, suggesting that the cell death is caspase-independent. Furthermore, while FGFR inhibition led to an increase in LC3 puncta, treatment with bafilomycin did not further increase lipidated LC3, suggesting that FGFR inhibition led to a block in autophagosome degradation. We confirmed that cell death is mitochondrial-dependent as it can be blocked by overexpression of Bcl-2 and/or Bcl-XL. Importantly, we show that combining FGFR inhibitors with the BH3 mimetics ABT737/ABT263 markedly increased cell death in vitro and is more effective than BGJ398 alone in vivo, where it leads to marked tumour regression. This work may have implications for the design of clinical trials to treat a wide range of patients with FGFR-dependent malignancies.

Women with atypical hyperplasia (AH) or well-differentiated early-stage endometrioid endometrial carcinoma (EEC) who wish to retain fertility and/or with comorbidities precluding surgery, are treated with progestin. Clinically approved predictive biomarkers for progestin therapy remain an unmet need. The objectives of this study were to document the overall response rate (ORR) of levonorgestrel intrauterine device (LNG-IUD) treatment, and determine the association of FGFR2b and FGFR2c expression with treatment outcome. BaseScope RNA ISH assay was utilized to detect expression of FGFR2b and FGFR2c mRNA in the diagnostic biopsies of 89 women (40 AH and 49 EEC) treated with LNG-IUD. Detailed clinical follow-up was available for 69 women which revealed an overall response rate (ORR) of 44% (30/69) with a higher ORR seen in AH (64%) compared to EEC (23%). The recurrence rate in women who initially responded to LNG-IUD was 10/30 (33.3%). RNA ISH was successful in 72 patients and showed FGFR2c expression in 12/72 (16.7%) samples. In the 59 women with detailed clinical follow-up and RNA-ISH data, women with tumours expressing FGFR2c were 5-times more likely to have treatment failure in both univariable (HR 5.08, p &lt, 0.0001) and multivariable (HR 4.5, p &lt, 0.002) Cox regression analyses. In conclusion, FGFR2c expression appears to be strongly associated with progestin treatment failure, albeit the OOR is lower in this cohort than previously reported. Future work to validate these findings in an independent multi-institutional cohort is needed.

PURPOSE The randomized Adjuvant Chemoradiotherapy Versus Radiotherapy Alone in Women With High-Risk Endometrial Cancer (PORTEC-3) trial investigated the benefit of combined adjuvant chemotherapy and radiotherapy (CTRT) versus radiotherapy alone (RT) for women with high-risk endometrial cancer (EC). Because The Cancer Genome Atlas defined an EC molecular classification with strong prognostic value, we investigated prognosis and impact of chemotherapy for each molecular subgroup using tissue samples from PORTEC-3 trial participants. METHODS Paraffin-embedded tissues of 423 consenting patients were collected. Immunohistochemistry for p53 and mismatch repair (MMR) proteins, and DNA sequencing for POLE exonuclease domain were done to classify tumors as p53 abnormal (p53abn), POLE-ultramutated ( POLEmut), MMR-deficient (MMRd), or no specific molecular profile (NSMP). The primary end point was recurrence-free survival (RFS). Kaplan-Meier method, log-rank test, and Cox model were used for analysis. RESULTS Molecular analysis was successful in 410 high-risk EC (97%), identifying the 4 subgroups: p53abn EC (n = 93; 23%), POLEmut (n = 51; 12%), MMRd (n = 137; 33%), and NSMP (n = 129; 32%). Five-year RFS was 48% for patients with p53abn EC, 98% for POLEmut EC, 72% for MMRd EC, and 74% for NSMP EC ( P < .001). The 5-year RFS with CTRT versus RT for p53abn EC was 59% versus 36% ( P = .019); 100% versus 97% for patients with POLEmut EC ( P = .637); 68% versus 76% ( P = .428) for MMRd EC; and 80% versus 68% ( P = .243) for NSMP EC. CONCLUSION Molecular classification has strong prognostic value in high-risk EC, with significantly improved RFS with adjuvant CTRT for p53abn tumors, regardless of histologic type. Patients with POLEmut EC had an excellent RFS in both trial arms. EC molecular classification should be incorporated in the risk stratification of these patients as well as in future trials to target specific subgroups of patients.

Background: Breast cancer (BC) is a heterogeneous disease for which the commonly used chemotherapeutic agents primarily include the anthracyclines (doxorubicin, epirubicin), microtubule inhibitors (paclitaxel, docetaxel, eribulin), and alkylating agents (cyclophosphamide). While these drugs can be highly effective, metastatic tumours are frequently refractory to treatment or become resistant upon tumour relapse. Methods: We undertook a cell polarity/epithelial mesenchymal plasticity (EMP)-enriched short hairpin RNA (shRNA) screen in MDA-MB-468 breast cancer cells to identify factors underpinning heterogeneous responses to three chemotherapeutic agents used clinically in breast cancer: Doxorubicin, docetaxel, and eribulin. shRNA-transduced cells were treated for 6 weeks with the EC10 of each drug, and shRNA representation assessed by deep sequencing. We first identified candidate genes with depleted shRNA, implying that their silencing could promote a response. Using the Broad Institute&rsquo, s Connectivity Map (CMap), we identified partner inhibitors targeting the identified gene families that may induce cell death in combination with doxorubicin, and tested them with all three drug treatments. Results: In total, 259 shRNAs were depleted with doxorubicin treatment (at p &lt, 0.01), 66 with docetaxel, and 25 with eribulin. Twenty-four depleted hairpins overlapped between doxorubicin and docetaxel, and shRNAs for TGFB2, RUNX1, CCDC80, and HYOU1 were depleted across all the three drug treatments. Inhibitors of MDM/TP53, TGFBR, and FGFR were identified by CMap as the top pharmaceutical perturbagens and we validated the combinatorial benefits of the TGFBR inhibitor (SB525334) and MDM inhibitor (RITA) with doxorubicin treatment, and also observed synergy between the inhibitor SB525334 and eribulin in MDA-MB-468 cells. Conclusions: Taken together, a cell polarity/EMP-enriched shRNA library screen identified relevant gene products that could be targeted alongside current chemotherapeutic agents for the treatment of invasive BC.

Purpose: Endometrial cancer (EC) is the most frequently diagnosed gynaecological cancer. The majority of women with EC are treated surgically and have a good outcome, however 25-30% of patients presenting with metastases or recurrent disease do not have effective therapies and have Citation Format: Asmerom T Sengal, Vanessa Bonazzi, Olga Kondrashova, Lewis Perrin, Naven Chetty, Deborah Smith, Antonio Gil-Moreno, Eva Colas, Pamela M Pollock. Targeting FGFR2c isoform, a novel therapeutic target with FGFR inhibitor in endometrial cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr LBA020.

Background The morphological classification of high-risk endometrial cancer is of limited prognostic value. Recent attempts to stratify tumours according to molecular signatures have shown considerable promise. Here we attempted to further refine molecular classifications using markers of the p53 pathway. Methods We analysed the expression of p53 as well as three downstream markers of the p53 pathway, p21, mdm2 and phospho-p63 ( p p63), by immunohistochemistry in a series of 114 endometrial cancers (86 endometrioid, 28 non-endometrioid subtype) with high-risk features (such as high tumour grade and deep myometrial invasion) and correlated results with clinical outcome. The Cancer Genome Atlas (TCGA) data were used to analyse TP63 mutations and copy-number alterations using cBioPortal. TP53 was silenced in two endometrial cancer cell lines to study its effect on p21 and p63. Results About half of the tumours showed a p53 mutant phenotype and there was a strong negative correlation with p21 expression. Being marker positive for p p63 or mdm2 was associated with a significantly increased likelihood of dying, [hazard ratios 5.93 (95% CI 2.37–7.27) and 7.48 (95% CI 3.04–9.39), respectively]. These findings were seen in both p53 wildtype and p53 mutant tumours. Only 11% of TCGA endometrial cancers had a functional TP63 alteration. Upon silencing of TP53, p21 expression was decreased in one cell line, but no effects on p63 were observed. Conclusion Markers of the p53 pathway improve stratification of endometrial cancers and provide novel insights into the role of this pathway in the disease.

Purpose Activating FGFR2 mutations have been identified in ~ 10% of endometrioid endometrial cancers (ECs). We have previously reported that mutations in FGFR2 are associated with shorter disease free survival (DFS) in stage I/II EC patients. Here we sought to validate the prognostic importance of FGFR2 mutations in a large, multi-institutional patient cohort. Methods Tumors were collected as part of the GOG 210 clinical trial “Molecular Staging of Endometrial Cancer” where samples underwent rigorous pathological review and had more than three years of detailed clinical follow-up. DNA was extracted and four exons encompassing the FGFR2 mutation hotspots were amplified and sequenced. Results Mutations were identified in 144 of the 973 endometrioid ECs, of which 125 were classified as known activating mutations and were included in the statistical analyses. Consistent with FGFR2 having an association with more aggressive disease, FGFR2 mutations were more common in patients initially diagnosed with stage III/IV EC (29/170;17%) versus stage I/II EC (96/803; 12%; p = 0.07, Chi-square test). Additionally, incidence of progression (progressed, recurred or died from disease) was significantly more prevalent (32/125, 26%) among patients with FGFR2 mutation versus wild type (120/848, 14%; p < 0.001, Fishers exact test). Using Cox regression analysis adjusting for known prognostic factors, patients with FGFR2 mutation had significantly (p < 0.025) shorter progression-free survival (PFS; HR 1.903; 95% CI 1.177–3.076) and endometrial cancer specific survival (ECS; HR 2.013; 95% CI 1.096–3.696). Conclusion In summary, our findings suggest that clinical trials testing the efficacy of FGFR inhibitors in the adjuvant setting to prevent recurrence and death are warranted.

Large-scale genome-wide association studies (GWAS) conducted by the Endometrial Cancer Association Consortium have identified candidate endometrial cancer risk variants at 16 loci. The vast majority of these candidate endometrial cancer risk variants are located in non-coding genomic regions. Studies of other GWAS have shown enrichment of trait-associated variants in enhancer elements suggesting that they may regulate gene expression through chromatin looping. In the current study, we analysed chromatin looping to identify the genes that may mediate the effects of these variants. We used a modified HiChIP technique to capture chromatin loops associated with H3K27Ac (characteristic of active enhancers and promoters) in a normal immortalized and three tumoral endometrial cell lines. Duplicate HiChIP libraries were analysed using paired-end sequencing and bioinformatic pipelines (HiC-Pro, hichipper and IDR2D) to identify replicating cis chromatin loops. The replicating loops were intersected with gene promoter regions to identify promoter-associated chromatin loops and we found significant enrichment of endometrial cancer risk variation in these loops. We identified 74 candidate target genes at 13 endometrial cancer risk loci by intersecting promoter-associated loops with candidate risk variants and prioritising genes with matching RNA-seq expression. The candidate target genes were more conserved than other genes on average, nearly three times more likely to be differentially regulated in endometrial tumours and their encoded proteins interacted more amongst themselves than would be expected by chance. Moreover, functional prioritisation of the wider protein-protein interaction network revealed a set of interacting proteins that was 9-fold enriched for somatic endometrial cancer drivers. In summary, our findings indicate that endometrial cancer risk variation at least partly mediates its effects through enhancer-promoter chromatin looping and corresponding data reveal a set of biologically relevant candidate target genes. Citation Format: Dylan M. Glubb, Pamela Pollock, Amanda Spurdle, Tracy A. O'Mara. Enhancer-promoter chromatin looping is enriched for endometrial cancer GWAS risk variation and reveals biologically relevant candidate susceptibility genes [abstract]. In: Proceedings of the AACR Virtual Special Conference: Endometrial Cancer: New Biology Driving Research and Treatment; 2020 Nov 9-10. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(3_Suppl):Abstract nr PO004.

Introduction: Women with atypical hyperplasia (AH) or well-differentiated endometrioid endometrial carcinoma (EEC) who wish to retain fertility and/or with severe comorbidities that preclude surgery, are treated with progestin. Previously, we reported FGFR2c was associated with aggressive tumour behaviour and poor survival outcome in EEC. The purpose of this study was to estimate the response rate of Levonorgestrel IUD treatment and determine the association of FGFR2b and FGFR2c expression with outcome in women with AH/borderline histology (BL) and EEC treated with progestin. Method: BaseScope RNA ISH and immunohistochemistry assays were deployed to detect (FGFR2b and FGFR2c mRNA) and (FGFR2 protein and progesterone receptors), respectively in 89 diagnostic biopsies of patients with AH/BL and EEC treated with progestin. Kaplan Meier Curve and Cox regression model analyses were performed to evaluate the predictive value of FGFR2b and FGFR2c in this cohort. Results: The complete resolution rate (CRR) and overall response rate (ORR) at any time point in the whole cohort were 26/82 (32%) and 35/82 (43%), respectively. When stratified by histologic diagnosis CRR was 47.5% and 17% and ORR 63% and 24% for AH/BL and EEC, respectively. The recurrence rate was 28.7%. FGFR2c expression was documented in 12/72 (15%) cases. Both univariable and multivariable Cox regression analyses showed women with FGFR2c expression were 5-fold more likely to progress compared with FGFR2b positive women (HR 5.4, P Conclusion: Progestin treatment is less effective in patients with EEC. Notably, FGFR2c expression appears to be strongly associated with progestin treatment failure. Citation Format: Asmerom Sengal, Deborah Smith, Rebecca Rogers, Cameron Snell, Elizabeth Williams, Pamela Pollock. FGFR2 isoform switching strongly associates with progestin treatment failure in atypical hyperplasia and well-differentiated endometrial cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference: Endometrial Cancer: New Biology Driving Research and Treatment; 2020 Nov 9-10. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(3_Suppl):Abstract nr PO033.

Endometrial cancer is a major gynaecological cancer with a high incidence resulting in over 500 Australian women dying every year. Good pre-clinical models are urgently needed to study the biology of this disease, and to develop and test novel treatment strategies. Most current pre-clinical research is conducted in cell lines that often do not resemble the disease on the molecular level and do not recapitulate tumour heterogeneity, which results in untranslatable research findings. Patient-derived xenograft (PDX) models overcome the issues of representing tumour molecular landscape and heterogeneity, making them a translatable pre-clinical cancer model. Here, we generated and genomically characterised 11 PDX models by performing whole genome or whole exome sequencing of primary patient tumours and matched PDX samples. We performed a detailed genomic characterisation of these models to determine their suitability as pre-clinical models, study tumour heterogeneity and identify biomarkers of response and resistance. PDX models were successfully generated from all four molecular subtypes of EC and uterine carcinosarcomas, and they recapitulated morphology and the molecular landscape of primary tumors without major genomic drift. We also observed a wide range of inter-tumor and intra-tumor heterogeneity, well captured by different PDX lineages, which could lead to different treatment responses. An in vivo response to talazoparib was detected in two p53mut models consistent with stable disease, however both lacked the HR deficiency genomic signature. EC PDX models represent the four molecular subtypes of disease and can capture intra-tumoral heterogeneity of the original primary tumor. PDXs of the p53mut molecular subtype showed sensitivity to PARPi, however, deeper and more durable responses will likely require combination of PARPi with other agents. Citation Format: Olga Kondrashova, Vanessa F. Bonazzi, Deborah Smith, Katia Nones, Asmerom Sengal, Robert Ju, Leisl M. Packer, Lambros T Koufariotis, Stephen H. Kazakoff, Aimee L. Davidson, Priya Ramarao-Milne, Vanessa Lakis, Felicity Newell, Rebecca Rogers, Claire Davies, Naven Chetty, Lewis Perrin, John Pearson, Ann-Marie Patch, Andrea Garrett, Nicola Waddell, Pamela Pollock, James Nicklin. Genomic characterization of endometrial cancer patient-derived xenografts reveals intratumor heterogeneity [abstract]. In: Proceedings of the AACR Virtual Special Conference: Endometrial Cancer: New Biology Driving Research and Treatment; 2020 Nov 9-10. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(3_Suppl):Abstract nr PR007.

Background The PORTEC-3 trial investigated the benefit of combined adjuvant chemotherapy and radiotherapy (CTRT) versus radiotherapy alone (RT) for women with stage I-III endometrial cancer with high-risk features (HREC). Since the TCGA defined 4 molecular subgroups of EC with strong prognostic value, we investigated outcomes and impact of chemotherapy for each molecular subgroup using tissue samples from PORTEC-3 trial participants. Methods Paraffin-embedded tissues of 423 consenting patients (64% of 660) were collected. Immunohistochemistry for p53 and mismatch repair (MMR) proteins, and DNA sequencing for POLE pathogenic exonuclease domain mutations were done to classify tumours as p53 mutant staining (p53abn), POLE ultramutated (POLEmut), MMR deficient (MMRd), or no specific molecular profile (NSMP). The Kaplan-Meier method, log-rank test and Cox model were used for analysis. Results Molecular analysis was successful in 410 HREC (97%), identifying the four subgroups; p53abn (n = 92, 22%), POLEmut (n = 52, 13%), MMRd (n = 137, 33%) and NSMP (n = 129, 32%). Five-year recurrence free survival (RFS) was 50% for patients with p53abn HREC, 98% for POLEmut, 74% for MMRd and 76% for NSMP (p Table: LBA63 . Recurrence-free survival by molecular subgroup Events 5-year estimate % HR (95% CI) P value of HR p53abn EC RT 28 37,2 1 CTRT 20 61,1 0,50 (0,28-0,88) 0,017 POLEmut EC RT 1 96,6 1 CTRT 0 100 0,02 ( 104) 0,632 MMRd EC RT 17 75,8 1 CTRT 18 72,4 1,15 (0,59-2,22) 0,687 NSMP EC RT 19 68,9 1 CTRT 17 81,2 0,71 (0,37-1,37) 0,311 Conclusions Molecular EC classification has a strong prognostic value in HREC and better identifies those who benefit from adjuvant CTRT than clinicopathological factors. Patients with p53abn HREC had significantly improved RFS with adjuvant CTRT, while those with MMRd did not seem to benefit from chemotherapy. Patients with POLEmut HREC had an excellent RFS in both arms. Future trials should incorporate the molecular classification and target specific subgroups. Clinical trial identification NCT00411138. Legal entity responsible for the study Leiden University Medical Center. Funding Koningin Wilhelmina Fonds voor de Nederlandse Kankerbestrijding (KWF). Disclosure A. Leary: Travel / Accommodation / Expenses, Officer / Board of Directors: AZ; Officer / Board of Directors: Tesaro; Officer / Board of Directors: Clovis; Officer / Board of Directors: MSD; Officer / Board of Directors: Grisdstone; Officer / Board of Directors: Seattle Genetics; Officer / Board of Directors: Gamamabs; Officer / Board of Directors: Biocad; Travel / Accommodation / Expenses: Roche . H.W. Nijman: Leadership role, Founder: SME Vicinivax; Advisory / Consultancy: Aduro; Advisory / Consultancy: TRON ; Advisory / Consultancy: Merck. All other authors have declared no conflicts of interest.

The overall 5-year survival for melanoma is 91%. However, if distant metastasis occurs (stage IV), cure rates are, Highlights • A seven-miRNA panel (MELmiR-7) detected the presence of melanoma with high sensitivity (93%) and specificity (≥ 82%). • In serially collected stage IV specimens, members of the ‘MELmiR-7’ panel confirmed tumour progression in 100% of cases. • The ‘MELmiR-7’ panel is superior to currently used serological markers for melanoma progression, recurrence, and survival.

// Mitchell S. Stark 1,2 , Vanessa F. Bonazzi 3 , Glen M. Boyle 1 , Jane M. Palmer 1 , Judith Symmons 1 , Catherine M. Lanagan 1 , Christopher W. Schmidt 1 , Adrian C. Herington 2 , Robert Ballotti 3 , Pamela M. Pollock 2 and Nicholas K. Hayward 1 1 QIMR Berghofer Medical Research Institute, Herston, Brisbane, QLD, Australia 2 School of Biomedical Sciences, Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, Australia 3 Inserm U1065, Centre Mediterraneen de Medecine Moleculaire, Equipe 1, Biologie et pathologies des melanocytes, Nice, France Correspondence to: Mitchell S. Stark, email: // Keywords : miRNA, microRNA, miR-514a, BRAFi, NF1 Received : March 16, 2015 Accepted : April 07, 2015 Published : April 23, 2015 Abstract To identify ‘melanoma-specific’ microRNAs (miRNAs) we used an unbiased microRNA profiling approach to comprehensively study cutaneous melanoma in relation to other solid malignancies, which revealed 233 differentially expressed (≥ 2 fold, p < 0.05) miRNAs. Among the top 20 most significantly different miRNAs was hsa-miR-514a-3p. miR-514a is a member of a cluster of miRNAs (miR-506-514) involved in initiating melanocyte transformation and promotion of melanoma growth. We found miR-514a was expressed in 38/55 (69%) melanoma cell lines but in only 1/34 (3%) other solid cancers. To identify miR-514a regulated targets we conducted a miR-514a-mRNA ‘pull-down’ experiment, which revealed hundreds of genes, including: CTNNB1 , CDK2 , MC1R , and NF1 , previously associated with melanoma. NF1 was selected for functional validation because of its recent implication inacquired resistance to BRAFV600E -targeted therapy. Luciferase-reporter assays confirmed NF1 as a direct target of miR-514a and over-expression of miR-514a in melanoma cell lines inhibited NF1 expression, which correlated with increased survival of BRAFV600E cells treated with PLX4032. These data provide another mechanism for the dysregulation of the MAPK pathway which may contribute to the profound resistance associated with current RAF-targeted therapies. Mitchell S. Stark

High-risk endometrial cancer (EC) is an aggressive disease for which new therapeutic options are needed. Aims of this study were to validate the enhanced immune response in highly mutated ECs and to explore immune profiles in other EC subgroups. We evaluated immune infiltration in 116 high-risk ECs from the TransPORTEC consortium, previously classified into four molecular subtypes: (i) ultramutated POLE exonuclease domain-mutant ECs (POLE-mutant); (ii) hypermutated microsatellite unstable (MSI); (iii) p53-mutant; and (iv) no specific molecular profile (NSMP). Within The Cancer Genome Atlas (TCGA) EC cohort, significantly higher numbers of predicted neoantigens were demonstrated in POLE-mutant and MSI tumors compared with NSMP and p53-mutants. This was reflected by enhanced immune expression and infiltration in POLE-mutant and MSI tumors in both the TCGA cohort (mRNA expression) and the TransPORTEC cohort (immunohistochemistry) with high infiltration of CD8+ (90% and 69%), PD-1+ (73% and 69%) and PD-L1+ immune cells (100% and 71%). Notably, a subset of p53-mutant and NSMP cancers was characterized by signs of an antitumor immune response (43% and 31% of tumors with high infiltration of CD8+ cells, respectively), despite a low number of predicted neoantigens. In conclusion, the presence of enhanced immune infiltration, particularly high numbers of PD-1 and PD-L1 positive cells, in highly mutated, neoantigen-rich POLE-mutant and MSI endometrial tumors suggests sensitivity to immune checkpoint inhibitors.

Summary: In this issue of Cancer Discovery, Hagel and colleagues report the design and the in vitro and in vivo activity of a novel, irreversible, paralog-specific kinase inhibitor of FGFR4, BLU9931. This compound binds covalently to a cysteine residue in the hinge region of FGFR4 but not in FGFR1–3. BLU9931 induces tumor shrinkage in hepatocellular carcinoma models that express a functioning ligand/receptor complex consisting of FGF19/FGFR4/KLB and adds to a growing list of anti-FGFR4 agents. Cancer Discov; 5(4); 355–7. ©2015 AACR. See related article by Hagel et al., p. 424

Mebendazole (MBZ) was identified as a promising therapeutic on the basis of its ability to induce apoptosis in melanoma cell lines through a B-cell lymphoma 2 (BCL2)-dependent mechanism. We now show that in a human xenograft melanoma model, oral MBZ is as effective as the current standard of care temozolomide in reducing tumor growth. Inhibition of melanoma growth in vivo is accompanied by phosphorylation of BCL2 and decreased levels of X-linked inhibitor of apoptosis (XIAP). Reduced expression of XIAP on treatment with MBZ is partially mediated by its proteasomal degradation. Furthermore, exposure of melanoma cells to MBZ promotes the interaction of SMAC/DIABLO with XIAP, thereby alleviating XIAP's inhibition on apoptosis. XIAP expression on exposure to MBZ is indicative of sensitivity to MBZ as MBZ-resistant cells do not show reduced levels of XIAP after treatment. Resistance to MBZ can be reversed partially by siRNA knockdown of cellular levels of XIAP. Our data indicate that MBZ is a promising antimelanoma agent on the basis of its effects on key antiapoptotic proteins.

PTEN loss and constitutive activation of the class I phosphoinositide 3-kinase (PI3K) pathway are key drivers of endometrial tumorigenesis. In some cancer types, PTEN-deficient tumors are reliant on class I PI3K p110β (encoded by PIK3CB) activity but little is known about this contribution in endometrial tumorigenesis. In this study, we find that p110β is overexpressed in a panel of 7 endometrial cancer cell lines compared to non-transformed cells. Furthermore, in 234 clinically annotated patient samples, PIK3CB mRNA levels increase significantly in the early phase of tumorigenesis from precursors to low grade primary malignant lesions whereas PIK3CA levels are higher in non-endometrioid compared to endometrioid primary tumors. While high levels of either PIK3CA or PIK3CB associate with poor prognosis, only elevated PIK3CB mRNA levels correlate with a high cell cycle signature score in clinical samples. In cancer cell lines, p110α inhibition reduces cell viability by inducing cell death in PIK3CA mutant cells while p110β inhibition delayed proliferation in PTEN-deficient cells, but not in WT cells. Taken together, our findings suggest that PIK3CB/p110β contributes to some of the pleiotropic functions of PI3K in endometrial cancer, particularly in the early steps by contributing to cell proliferation. publishedVersion

p53 is the central member of a critical tumor suppressor pathway in virtually all tumor types, where it is silenced mainly by missense mutations. In melanoma, p53 predominantly remains wild type, thus its role has been neglected. To study the effect of p53 on melanocyte function and melanomagenesis, we crossed the ‘high-p53’Mdm4+/− mouse to the well-established TP-ras0/+ murine melanoma progression model. After treatment with the carcinogen dimethylbenzanthracene (DMBA), TP-ras0/+ mice on the Mdm4+/− background developed fewer tumors with a delay in the age of onset of melanomas compared to TP-ras0/+ mice. Furthermore, we observed a dramatic decrease in tumor growth, lack of metastasis with increased survival of TP-ras0/+: Mdm4+/− mice. Thus, p53 effectively prevented the conversion of small benign tumors to malignant and metastatic melanoma. p53 activation in cultured primary melanocyte and melanoma cell lines using Nutlin-3, a specific Mdm2 antagonist, supported these findings. Moreover, global gene expression and network analysis of Nutlin-3-treated primary human melanocytes indicated that cell cycle regulation through the p21WAF1/CIP1 signaling network may be the key anti-melanomagenic activity of p53.

Although molecularly targeted therapies have been effective in some cancer types, no targeted therapy is approved for use in endometrial cancer. The recent identification of activating mutations in fibroblast growth factor receptor 2 (FGFR2) in endometrial tumors has generated a new avenue for the development of targeted therapeutic agents. The majority of the mutations identified are identical to germline mutations in FGFR2 and FGFR3 that cause craniosynostosis and hypochondroplasia syndromes and result in both ligand-independent and ligand-dependent receptor activation. Mutations that predominantly occur in the endometrioid subtype of endometrial cancer, are mutually exclusive with KRAS mutation, but occur in the presence of PTEN abrogation. In vitro studies have shown that endometrial cancer cell lines with activating FGFR2 mutations are selectively sensitive to a pan-FGFR inhibitor, PD173074. Several agents with activity against FGFRs are currently in clinical trials. Investigation of these agents in endometrial cancer patients with activating FGFR2 mutations is warranted.

We report that 10% of melanoma tumors and cell lines harbor mutations in the fibroblast growth factor receptor 2 (FGFR2) gene. These novel mutations include three truncating mutations and 20 missense mutations occurring at evolutionary conserved residues in FGFR2 as well as among all four FGFRs. The mutation spectrum is characteristic of those induced by UV radiation. Mapping of these mutations onto the known crystal structures of FGFR2 followed by in vitro and in vivo studies show that these mutations result in receptor loss of function through several distinct mechanisms, including loss of ligand binding affinity, impaired receptor dimerization, destabilization of the extracellular domains, and reduced kinase activity. To our knowledge, this is the first demonstration of loss-of-function mutations in a class IV receptor tyrosine kinase in cancer. Taken into account with our recent discovery of activating FGFR2 mutations in endometrial cancer, we suggest that FGFR2 may join the list of genes that play context-dependent opposing roles in cancer. (Mol Cancer Res 2009;7(1):41–54)

Purpose: Hepatocellular carcinoma (HCC) is the fifth most common primary neoplasm; surgery is the only curative option but 5-year survival rates are only 25% to 50%. Vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) are known to be involved in growth and neovascularization of HCC. Therefore, agents that target these pathways may be effective in the treatment of HCC. The aim of this study was to determine the antineoplastic activity of brivanib alaninate, a dual inhibitor of VEGF receptor (VEGFR) and FGF receptor (FGFR) signaling pathways. Experimental Design: Six different s.c. patient-derived HCC xenografts were implanted into mice. Tumor growth was evaluated in mice treated with brivanib compared with control. The effects of brivanib on apoptosis and cell proliferation were evaluated by immunohistochemistry. The SK-HEP1 and HepG2 cells were used to investigate the effects of brivanib on the VEGFR-2 and FGFR-1 signaling pathways in vitro. Western blotting was used to determine changes in proteins in these xenografts and cell lines. Results: Brivanib significantly suppressed tumor growth in five of six xenograft lines. Furthermore, brivanib–induced growth inhibition was associated with a decrease in phosphorylated VEGFR-2 at Tyr1054/1059, increased apoptosis, reduced microvessel density, inhibition of cell proliferation, and down-regulation of cell cycle regulators. The levels of FGFR-1 and FGFR-2 expression in these xenograft lines were positively correlated with its sensitivity to brivanib-induced growth inhibition. In VEGF-stimulated and basic FGF stimulated SK-HEP1 cells, brivanib significantly inhibited VEGFR-2, FGFR-1, extracellular signal-regulated kinase 1/2, and Akt phosphorylation. Conclusion: This study provides a strong rationale for clinical investigation of brivanib in patients with HCC.

KRAS activation and PTEN inactivation are frequent events in endometrial tumorigenesis, occurring in 10% to 30% and 26% to 80% of endometrial cancers, respectively. Because we have recently shown activating mutations in fibroblast growth factor receptor 2 (FGFR2) in 16% of endometrioid endometrial cancers, we sought to determine the genetic context in which FGFR2 mutations occur. Analysis of 116 primary endometrioid endometrial cancers revealed that FGFR2 and KRAS mutations were mutually exclusive, whereas FGFR2 mutations were seen concomitantly with PTEN mutations. Here, we show that shRNA knockdown of FGFR2 or treatment with a pan-FGFR inhibitor, PD173074, resulted in cell cycle arrest and induction of cell death in endometrial cancer cells with activating mutations in FGFR2. This cell death in response to FGFR2 inhibition occurred within the context of loss-of-function mutations in PTEN and constitutive AKT phosphorylation, and was associated with a marked reduction in extracellular signal-regulated kinase 1/2 activation. Together, these data suggest that inhibition of FGFR2 may be a viable therapeutic option in endometrial tumors possessing activating mutations in FGFR2, despite the frequent abrogation of PTEN in this cancer type. [Cancer Res 2008;68(17):6902–7]

// Mitchell S. Stark 1, 2 , Lisa N. Tom 1 , Glen M. Boyle 2 , Vanessa F. Bonazzi 3 , H. Peter Soyer 1 , Adrian C. Herington 3 , Pamela M. Pollock 3 , Nicholas K. Hayward 2 1 Dermatology Research Centre, The University of Queensland, School of Medicine, Translational Research Institute, Brisbane, QLD, Australia 2 QIMR Berghofer Medical Research Institute, Herston, Brisbane, QLD, Australia 3 School of Biomedical Sciences, Institute of Health and Biomedical Innovation, Queensland University of Technology, at The Translational Research Institute, Brisbane, QLD, Australia Correspondence to: Mitchell S. Stark, email: m.stark@uq.edu.au Keywords: SSX, microRNA, melanoma, PMP22, miR-4731 Received: May 02, 2015 Accepted: June 01, 2016 Published: June 16, 2016 ABSTRACT We previously identified miR-4731-5p (miR-4731) as a melanoma-enriched microRNA following comparison of melanoma with other cell lines from solid malignancies. Additionally, miR-4731 has been found in serum from melanoma patients and expressed less abundantly in metastatic melanoma tissues from stage IV patients relative to stage III patients. As miR-4731 has no known function, we used biotin-labelled miRNA duplex pull-down to identify binding targets of miR-4731 in three melanoma cell lines (HT144, MM96L and MM253). Using the miRanda miRNA binding algorithm, all pulled-down transcripts common to the three cell lines (n=1092) had potential to be targets of miR-4731 and gene-set enrichment analysis of these (via STRING v9.1) highlighted significantly associated genes related to the ‘cell cycle’ pathway and the ‘melanosome’. Following miR-4731 overexpression, a selection (n=81) of pull-down transcripts underwent validation using a custom qRT-PCR array. These data revealed that miR-4731 regulates multiple genes associated with the cell cycle (e.g. CCNA2 , ORC5L , and PCNA ) and the melanosome (e.g. RAB7A , CTSD , and GNA13 ). Furthermore, members of the synovial sarcoma X breakpoint family (SSX) (melanoma growth promoters) were also down-regulated (e.g. SSX2 , SSX4 , and SSX4B ) as a result of miR-4731 overexpression. Moreover, this down-regulation of mRNA expression resulted in ablation or reduction of SSX4 protein, which, in keeping with previous studies, resulted in loss of 2D colony formation. We therefore speculate that loss of miR-4731 expression in stage IV patient tumours supports melanoma growth by, in part; reducing its regulatory control of SSX expression levels.

Once melanoma metastasizes, no effective treatment modalities prolong survival in most patients. This notorious refractoriness to therapy challenges investigators to identify agents that overcome melanoma resistance to apoptosis. Whereas many survival pathways contribute to the death-defying phenotype in melanoma, a defect in apoptotic machinery previously highlighted inactivation of Apaf-1, an apoptosome component engaged after mitochondrial damage. During studies involving Notch signaling in melanoma, we observed a γ-secretase tripeptide inhibitor (GSI; z-Leu-Leu-Nle-CHO), selected from a group of compounds originally used in Alzheimer's disease, induced apoptosis in nine of nine melanoma lines. GSI only induced G2-M growth arrest (but not killing) in five of five normal melanocyte cultures tested. Effective killing of melanoma cells by GSI involved new protein synthesis and a mitochondrial-based pathway mediated by up-regulation of BH3-only members (Bim and NOXA). p53 activation was not necessary for up-regulation of NOXA in melanoma cells. Blocking GSI-induced NOXA using an antisense (but not control) oligonucleotide significantly reduced the apoptotic response. GSI also killed melanoma cell lines with low Apaf-1 levels. We conclude that GSI is highly effective in killing melanoma cells while sparing normal melanocytes. Direct enhancement of BH3-only proteins executes an apoptotic program overcoming resistance of this lethal tumor. Identification of a p53-independent apoptotic pathway in melanoma cells, including cells with low Apaf-1, bypasses an impediment to current cytotoxic therapy and provides new targets for future therapeutic trials involving chemoresistant tumors.

Endometrial cancer (EC) is the most commonly diagnosed malignancy of the female reproductive tract. Unfortunately, 15-20% of women demonstrate persistent or recurrent tumors that are refractory to current chemotherapies with an associated poor prognosis. Our laboratory identified activating mutations in Fibroblast Growth Factor Receptor 2 (FGFR2) in 12% (stage I/II) to 17% (stage III/IV) endometrioid endometrial tumors and have since shown in a large (n=970) multi-institutional cohort they are associated with shorter progression free and cancer specific survival. Although FGFR inhibitors are in clinical trials in several cancer types, no detailed study of the mechanism of cell death has been published. We now show that treatment with BGJ398, AZD4547 and PD173074 leads to the induction of mitochondrial depolarization and changes in metabolic flux in two endometrial cancer cell lines (JHUEM2 and AN3CA) carrying activating mutations (C383R and N550K respectively). Despite this mitochondrial dysfunction, we have convincingly shown that the cell death following FGFR inhibition was caspase-independent, as evidenced by the lack of caspase-3, -7, and -9 activation, absence of PARP cleavage, and the inability of the broad-spectrum caspase inhibitor, Z-VAD-FMK, to prevent cell death. Knockdown of EndoG and AIF, common mediators of caspase-independent death, had no effect. Detailed quantification of LC3 positive puncta shows an increase in autophagy in JHUEM2 and AN3CA cells treated with all FGFR inhibitors. Knockdown of ATG3, ATG7 and ATG12 resulted in a slight increase in Annexin positive cell death indicating that the autophagy was cytoprotective in this context. We have now confirmed this novel caspase-independent cell death is mitochondrial dependent as it can be blocked by overexpression of Bcl-2 and/or Bcl-XL. Importantly we have shown that the combination of FGFR inhibitors with the BH3 mimetic ABT737 can markedly augment this caspase-independent cell death which may have implications for the design of more effective clinical trials. Citation Format: Leisl Packer, Sara Byron, Samantha Stehbens, Vanessa Bonazzi, David Loch, Andreas Wortmann, Mike Gartside, Nigel Waterhouse, Jennifer Gunter, Pamela M. Pollock. FGFR inhibition in endometrial cancer induces caspase-independent cell death that can be augmented with ABT-737 [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 LB-B31.

Deletions detected in cytogenetic and loss of heterozygosity (LOH) studies indicate that at least one tumour suppressor gene maps to the long arm of chromosome 10. Previous deletion mapping studies have observed LOH on 10q in about 30% of melanomas analysed. The PTEN gene, mapping to chromosome band 10q23.3, encodes a protein with both lipid and protein phosphatase activity. Somatic mutations and deletions in have been detected in a variety of cell lines and tumours, including melanoma samples. We performed mutation analyses and extensive allelic loss studies to investigate the role this gene plays in melanoma pathogenesis. We found that a total of 34 out of 57 (60%) melanoma cell lines carried hemizygous deletions of chromosome 10q encompassing the PTEN locus. A further three cell lines carried smaller deletions excluding PTEN. Inactivation of both PTEN alleles by exon-specific homozygous deletion or mutation was observed in 13 out of 57 (23%) melanoma cell lines. The mutation spectrum observed does not indicate an important role for ultraviolet radiation in the genesis of these mutations, and evidence from three cell lines supports the acquisition of PTEN aberrations in culture. Ten out of 49 (20%) matched melanoma tumour/normal samples harboured hemizygous deletions of either the whole chromosome or most of the long arm. Mutations within were detected in only one of the 10 tumours demonstrating LOH at 10q23 that were analysed. These results suggest that PTEN inactivation may be important for the propagation of melanoma cells in culture, and that another chromosome 10 tumour suppressor gene may be important for melanoma pathogenesis.

Brivanib, an oral, multi-targeted tyrosine kinase inhibitor with activity against vascular endothelial growth factor (VEGF) and fibroblast growth factor receptor (FGFR) was investigated as a single agent in a phase II trial to assess the activity and tolerability in recurrent or persistent endometrial cancer (EMC).Eligible patients had persistent or recurrent EMC after receiving one to two prior cytotoxic regimens, measurable disease, and performance status of ≤2. Treatment consisted of brivanib 800 mg orally every day until disease progression or prohibitive toxicity. Primary endpoints were progression-free survival (PFS) at six months and objective tumor response. Expression of multiple angiogenic proteins and FGFR2 mutation status was assessed.Forty-five patients were enrolled. Forty-three patients were eligible and evaluable. Median age was 64 years. Twenty-four patients (55.8%) received prior radiation. Median number of cycles was two (range 1-24). No GI perforations but one rectal fistula were seen. Nine patients had grade 3 hypertension, with one experiencing grade 4 confusion. Eight patients (18.6%; 90% CI 9.6%-31.7%) had responses (one CR and seven PRs), and 13 patients (30.2%; 90% CI 18.9%-43.9%) were PFS at six months. Median PFS and overall survival (OS) were 3.3 and 10.7 months, respectively. When modeled jointly, VEGF and angiopoietin-2 expression may diametrically predict PFS. Estrogen receptor-α (ER) expression was positively correlated with OS.Brivanib is reasonably well tolerated and worthy of further investigation based on PFS at six months in recurrent or persistent EMC.

The CDKN2A gene encodes p16 (CDKN2A), a cell-cycle inhibitor protein which prevents inappropriate cell cycling and, hence, proliferation. Germ-line mutations in CDKN2A predispose to the familial atypical multiple-mole melanoma (FAMMM) syndrome but also have been seen in rare families in which only 1 or 2 individuals are affected by cutaneous malignant melanoma (CMM). We therefore sequenced exons 1alpha and 2 of CDKN2A using lymphocyte DNA isolated from index cases from 67 families with cancers at multiple sites, where the patterns of cancer did not resemble those attributable to known genes such as hMLH1, hMLH2, BRCA1, BRCA2, TP53 or other cancer susceptibility genes. We found one mutation, a mis-sense mutation resulting in a methionine to isoleucine change at codon 53 (M531) of exon 2. The individual tested had developed 2 CMMs but had no dysplastic nevi and lacked a family history of dysplastic nevi or CMM. Other family members had been diagnosed with oral cancer (2 persons), bladder cancer (1 person) and possibly gall-bladder cancer. While this mutation has been reported in Australian and North American melanoma kindreds, we did not observe it in 618 chromosomes from Scottish and Canadian controls. Functional studies revealed that the CDKN2A variant carrying the M531 change was unable to bind effectively to CDK4, showing that this mutation is of pathological significance. Our results have confirmed that CDKN2A mutations are not limited to FAMMM kindreds but also demonstrate that multi-site cancer families without melanoma are very unlikely to contain CDKN2A mutations.

We previously identified miR-4731-5p (miR-4731) as a melanoma-enriched microRNA following comparison of melanoma with other cell lines from solid malignancies. Additionally, miR-4731 has been found in serum from melanoma patients and expressed less abundantly in metastatic melanomas from stage IV patients relative to stage III patients. As miR-4731 has no known function, we used biotin-labelled miRNA duplex pull-down to identify binding targets of miR-4731 in three melanoma cell lines. Using the miRanda miRNA binding algorithm, all pulled-down transcripts common to the three cell lines (n = 1092) were predicted to be targets of miR-4731 and gene-set enrichment analysis of these (via STRING v9.1) highlighted significantly associated genes related to the ‘cell cycle’ and ‘melanosome’ pathways. Following miR-4731 overexpression, a selection (n = 81) of pull-down transcripts underwent validation using a custom qRT-PCR array. These data revealed that miR-4731 regulates multiple genes associated with the cell cycle (e.g. CCNA2, ORC5L, and PCNA) and melanosome (e.g. RAB7A, CTSD, and GNA13). Furthermore, members of the synovial sarcoma X breakpoint family (SSX) (melanoma growth promoters) were also down-regulated (e.g. SSX2, SSX4, and SSX4B) as result of miR-4731 overexpression. We therefore speculate that loss of miR-4731 expression supports melanoma growth by, in part; reducing its regulatory control of SSX expression levels together with members of the cell cycle pathway, which warrants further investigation. Citation Format: Mitchell S. Stark, Lisa Tom, Glen M. Boyle, Vanessa F. Bonazzi, Adrian C. Herington, Pamela M. Pollock, Nicholas K. Hayward. The melanoma-enriched microRNA miR-4731 regulates genes involved in cell cycle and the melanosome. [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 1093.

Summary: In this issue of Cancer Discovery, Guagnano and colleagues use a large and diverse annotated collection of cancer cell lines, the Cancer Cell Line Encyclopedia, to correlate whole-genome expression and genomic alteration datasets with cell line sensitivity data to the novel pan-fibroblast growth factor receptor (FGFR) inhibitor NVP-BGJ398. Their findings underscore not only the preclinical use of such cell line panels in identifying predictive biomarkers, but also the emergence of the FGFRs as valid therapeutic targets, across an increasingly broad range of malignancies. Cancer Discov; 2(12); 1081–3. ©2012 AACR. Commentary on Guagnano et al., p. 1118

OBJECTIVE: The fibroblast growth factor (FGF) family of signaling molecules has been associated with chemoresistance and poor prognosis in a number of cancer types, including lung, breast, ovarian, prostate, and head and neck carcinomas. Given the identification of activating mutations in the FGF receptor 2 (FGFR2) receptor tyrosine kinase in a subset of endometrial tumors, agents with activity against FGFRs are currently being tested in clinical trials for recurrent and progressive endometrial cancer. Here, we evaluated the effect of FGFR inhibition on the in vitro efficacy of chemotherapy in endometrial cancer cell lines. METHODS: Human endometrial cancer cell lines with wild-type or activating FGFR2 mutations were used to determine any synergism with concurrent use of the pan-FGFR inhibitor, PD173074, and the chemotherapeutics, doxorubicin and paclitaxel, on cell proliferation and apoptosis. RESULTS: FGFR2 mutation status did not alter sensitivity to either chemotherapeutic agent alone. The combination of PD173074 with paclitaxel or doxorubicin showed synergistic activity in the 3 FGFR2 mutant cell lines evaluated. In addition, although nonmutant cell lines were resistant to FGFR inhibition alone, the addition of PD173074 potentiated the cytostatic effect of paclitaxel and doxorubicin in a subset of FGFR2 wild-type endometrial cancer cell lines. CONCLUSIONS: Together these data suggest a potential therapeutic benefit to combining an FGFR inhibitor with standard chemotherapeutic agents in endometrial cancer therapy particularly in patients with FGFR2 mutation positive tumors.

Background Melanoma is the most lethal form of skin cancer, but recent advances in molecularly targeted agents against the Ras/Raf/MAPK pathway demonstrate promise as effective therapies. Despite these advances, resistance remains an issue, as illustrated recently by the clinical experience with vemurafenib. Such acquired resistance appears to be the result of parallel pathway activation, such as PI3K, to overcome single-agent inhibition. In this report, we describe the cytotoxicity and anti-tumour activity of the novel MEK inhibitor, E6201, in a broad panel of melanoma cell lines (n = 31) of known mutational profile in vitro and in vivo. We further test the effectiveness of combining E6201 with an inhibitor of PI3K (LY294002) in overcoming resistance in these cell lines. Results The majority of melanoma cell lines were either sensitive (IC50 PTEN and mutant BRAF status, whereas mutant RAS and PI3K pathway activation were associated with resistance. Although MEK inhibitors predominantly exert a cytostatic effect, E6201 elicited a potent cytocidal effect on most of the sensitive lines studied, as evidenced by Annexin positivity and cell death ELISA. Conversely, E6201 did not induce cell death in the two resistant melanoma cell lines tested. E6201 inhibited xenograft tumour growth in all four melanoma cell lines studied to varying degrees, but a more pronounced anti-tumour effect was observed for cell lines that previously demonstrated a cytocidal response in vitro. In vitro combination studies of E6201 and LY294002 showed synergism in all six melanoma cell lines tested, as defined by a mean combination index Conclusions Our data demonstrate that E6201 elicits a predominantly cytocidal effect in vitro and in vivo in melanoma cells of diverse mutational background. Resistance to E6201 was associated with disruption of PTEN and activation of downstream PI3K signalling. In keeping with these data we demonstrate that co-inhibition of MAPK and PI3K is effective in overcoming resistance inherent in melanoma.

Mutations in multiple oncogenes including KRAS, CTNNB1, PIK3CA and FGFR2 have been identified in endometrial cancer. The aim of this study was to provide insight into the clinicopathological features associated with patterns of mutation in these genes, a necessary step in planning targeted therapies for endometrial cancer. 466 endometrioid endometrial tumors were tested for mutations in FGFR2, KRAS, CTNNB1, and PIK3CA. The relationships between mutation status, tumor microsatellite instability (MSI) and clinicopathological features including overall survival (OS) and disease-free survival (DFS) were evaluated using Kaplan-Meier survival analysis and Cox proportional hazard models. Mutations were identified in FGFR2 (48/466); KRAS (87/464); CTNNB1 (88/454) and PIK3CA (104/464). KRAS and FGFR2 mutations were significantly more common, and CTNNB1 mutations less common, in MSI positive tumors. KRAS and FGFR2 occurred in a near mutually exclusive pattern (p = 0.05) and, surprisingly, mutations in KRAS and CTNNB1 also occurred in a near mutually exclusive pattern (p = 0.0002). Multivariate analysis revealed that mutation in KRAS and FGFR2 showed a trend (p = 0.06) towards longer and shorter DFS, respectively. In the 386 patients with early stage disease (stage I and II), FGFR2 mutation was significantly associated with shorter DFS (HR = 3.24; 95% confidence interval, CI, 1.35-7.77; p = 0.008) and OS (HR = 2.00; 95% CI 1.09-3.65; p = 0.025) and KRAS was associated with longer DFS (HR = 0.23; 95% CI 0.05-0.97; p = 0.045). In conclusion, although KRAS and FGFR2 mutations share similar activation of the MAPK pathway, our data suggest very different roles in tumor biology. This has implications for the implementation of anti-FGFR or anti-MEK biologic therapies.

Using a genome-scanning approach to search for oncogenes, a recent report identifies somatic mutations in the signaling gene BRAF that are particularly prevalent in melanoma.

Objective Ovarian cancer is the leading cause of death from gynecologic malignancies in the Western world. Fibroblast growth factor receptor (FGFR) signaling has been implicated to play a role in ovarian tumorigenesis. Mutational activation of one member of this receptor family, FGFR2, is a frequent event in endometrioid endometrial cancer. Given the similarities in the histologic and molecular genetics of ovarian and endometrial cancers, we hypothesized that activating FGFR2 mutations may occur in a subset of endometrioid ovarian tumors, and possibly other histotypes. Methods Six FGFR2 exons were sequenced in 120 primary ovarian tumors representing the major histologic subtypes. Results FGFR2 mutation was detected at low frequency in endometrioid (1/46, 2.2%) and serous (1/41, 2.4%) ovarian cancer. No mutations were detected in clear cell, mucinous, or mixed histology tumors or in the ovarian cancer cell lines tested. Functional characterization of the FGFR2 mutations confirmed that the mutations detected in ovarian cancer result in receptor activation. Conclusions Despite the low incidence of FGFR2 mutations in ovarian cancer, the two FGFR2 mutations identified in ovarian tumors (S252W, Y376C) overlap with the oncogenic mutations previously identified in endometrial tumors, suggesting activated FGFR2 may contribute to ovarian cancer pathogenesis in a small subset of ovarian tumors.

The importance of mitogen-activated protein kinase signaling in melanoma is underscored by the prevalence of activating mutations in N-Ras and B-Raf, yet clinical development of inhibitors of this pathway has been largely ineffective, suggesting that alternative oncogenes may also promote melanoma. Notch is an interesting candidate that has only been correlated with melanoma development and progression; a thorough assessment of tumor-initiating effects of activated Notch on human melanocytes would clarify the mounting correlative evidence and perhaps identify a novel target for an otherwise untreatable disease. Analysis of a substantial panel of cell lines and patient lesions showed that Notch activity is significantly higher in melanomas than their nontransformed counterparts. The use of a constitutively active, truncated Notch transgene construct (NIC) was exploited to determine if Notch activation is a “driving” event in melanocytic transformation or instead a “passenger” event associated with melanoma progression. NIC-infected melanocytes displayed increased proliferative capacity and biological features more reminiscent of melanoma, such as dysregulated cell adhesion and migration. Gene expression analyses supported these observations and aided in the identification of MCAM, an adhesion molecule associated with acquisition of the malignant phenotype, as a direct target of Notch transactivation. NIC-positive melanocytes grew at clonal density, proliferated in limiting media conditions, and also exhibited anchorage-independent growth, suggesting that Notch alone is a transforming oncogene in human melanocytes, a phenomenon not previously described for any melanoma oncogene. This new information yields valuable insight into the basic epidemiology of melanoma and launches a realm of possibilities for drug intervention in this deadly disease. [Cancer Res 2009;69(13):5312–20]

Background: Our lab has identified FGFR2 mutations in 12% of over 1500 endometrioid endometrial cancers (ECs) analyzed. The majority of these tumors harbor genetic aberrations in the PI3K/AKT pathway resulting in constitutive activation. In other tissue types, PTEN loss has been shown to provide resistance to specific PIK3CA inhibition. We have previously reported that FGFR inhibition with PD173074 induced cell death despite loss of PTEN. We hypothesize that dual inhibition of FGFR2 and the PI3K pathway will lead to increased cell death and more effective tumor growth inhibition. Materials and Methods: Two FGFR2 mutant EC cell lines were treated with BGJ398 (pan FGFR inhibitor) alone or in combination with either a pan-PI3K inhibitor (GDC0941) or a specific PI3KA inhibitor (BYL719). Synergy was assessed using the fixed ratio method proposed by Chou and Talalay. Cell death was determined by Annexin V staining and colony formation assays using low clinically relevant drug concentrations. Downstream signaling pathways were analyzed by Western blot analysis and BGJ398 in combination with either GDC0941 or BYL719 was assessed in vivo. Results: Synergy was observed between BGJ398 and either the pan-PI3K inhibitor (GDC0941), or the specific PI3KA inhibitor (BYL719). Regression of tumor xenografts was observed in mice treated with BGJ398 and GDC0941 as well as BGJ398 and BYL719, despite PTEN inactivation. Conclusions: Dual targeting of the FGFR and PI3K pathways is more effective than targeting either of them alone. PTEN loss does not provide resistance to PIK3CA inhibition in our EC models suggesting context dependent differences in PI3K signaling. Citation Format: Pamela M. Pollock, Xinyan Geng, Vanessa F. Bonazzi, Clare Mahon, Sally Stephenson, Leisl Packer. Combination of BGJ398 with either a pan-PI3K inhibitor or a specific PIK3CA inhibitor shows synergy in FGFR2 mutant endometrial cancer cell lines. [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 B114.

Patients with metastatic melanoma or multiple myeloma have a dismal prognosis because these aggressive malignancies resist conventional treatment. A promising new oncologic approach uses molecularly targeted therapeutics that overcomes apoptotic resistance and, at the same time, achieves tumor selectivity. The unexpected selectivity of proteasome inhibition for inducing apoptosis in cancer cells, but not in normal cells, prompted us to define the mechanism of action for this class of drugs, including Food and Drug Administration–approved bortezomib. In this report, five melanoma cell lines and a myeloma cell line are treated with three different proteasome inhibitors (MG-132, lactacystin, and bortezomib), and the mechanism underlying the apoptotic pathway is defined. Following exposure to proteasome inhibitors, effective killing of human melanoma and myeloma cells, but not of normal proliferating melanocytes, was shown to involve p53-independent induction of the BH3-only protein NOXA. Induction of NOXA at the protein level was preceded by enhanced transcription of NOXA mRNA. Engagement of mitochondrial-based apoptotic pathway involved release of cytochrome c, second mitochondria-derived activator of caspases, and apoptosis-inducing factor, accompanied by a proteolytic cascade with processing of caspases 9, 3, and 8 and poly(ADP)-ribose polymerase. Blocking NOXA induction using an antisense (but not control) oligonucleotide reduced the apoptotic response by 30% to 50%, indicating a NOXA-dependent component in the overall killing of melanoma cells. These results provide a novel mechanism for overcoming the apoptotic resistance of tumor cells, and validate agents triggering NOXA induction as potential selective cancer therapeutics for life-threatening malignancies such as melanoma and multiple myeloma.

Mutations in exon 3 of the CTNNB1 gene encoding beta-catenin have been reported in colorectal cancer cell lines and tumours. Although one study reported mutations or deletions affecting beta-catenin in 20% of melanoma cell lines, subsequent reports detected a much lower frequency of aberrations in uncultured melanomas. To determine whether this difference in mutation frequency reflected an in vitro culturing artefact, exon 3 of CTNNB1 was screened in a panel of 62 melanoma cell lines. In addition, reverse transcription-polymerase chain reaction (RT-PCR) was performed to detect intragenic deletions affecting exon 3. One out of 62 (1.6%) cell lines was found to carry a mutation, indicating that aberration of the Wnt-1/wingless pathway through activation of beta-catenin is a rare event, even in melanoma cell lines.

Preclinical studies by our lab and others have demonstrated Fibroblast Growth Factor Receptor (FGFR) inhibition is a viable therapeutic strategy in FGFR2-mutant endometrial cancer (EC). A significant clinical issue that we aim to address is that of intrinsic and acquired resistance to anti-FGFR therapeutics in the context of EC. Using mutagenesis screens, we have identified mutations in the FGFR2 kinase domain that cause resistance to the FGFR inhibitors dovitinib and NVP-BGJ398 and explore the mechanism of action of the drug-resistant mutations. We previously reported the N550K and V565I mutations caused resistance to dovitinib. The sensitivity/resistance of these FGFR2 mutants to emerging FGFR inhibitors including AZD4547, NVP-BGJ398 and LY2874455 has now been tested. To gain insights into potential mechanisms of acquired resistance to FGFR inhibitors in EC cells we generated subclones of FGFR2mutant AN3CA, MFE280 and JHUEM-2 EC cells resistant to FGFR inhibitors PD173074, Dovitinib and NVP-BGJ398, respectively. Phospho- protein arrays, gene expression profiling, exome sequencing and SNP analysis were performed to identify genes and signalling pathways that mediate resistance to FGFR inhibitors. Sequencing of AN3CAresistant and MFE280resistant subclones excluded secondary FGFR2 mutations as the resistance mechanisms. Upregulation of downstream survival pathways such as pERK and pAKT was observed in some of the JHUEM-2resistant and AN3CAresistant subclones. To further characterise these subclones we are currently analysing data from exome sequencing and copy number analysis, to determine the molecular mechanisms of resistance in these cell lines. In the MFE280resistant cells resistance to FGFR inhibition is associated with an EMT phenotype. Signaling through pERK and pAKT is downregulated in these cells, suggesting that resistance is not mediated by activation of kinase-driven survival pathways. Analysis of expression data is underway to understand how these cells have become resistant to FGFR inhibitors. This research will provide insight into possible combination therapies that may combat drug resistance in FGFR-mutant endometrial cancer. Citation Format: Leisl M. Packer, Sara Byron, David Loch, Farhad Dehkhoda, Andreas Wortmann, Xinyan Geng, Katia Nones, Sean Grimmond, John Pearson, Nic Waddell, Pamela Pollock. Elucidating mechanisms of resistance to FGFR inhibitors in endometrial 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 1715. doi:10.1158/1538-7445.AM2014-1715

The majority of small-cell lung cancers (SCLCs) express p16 but not pRb. Given our previous study showing loss of pRb in Merkel cell carcinoma (MCC)/neuroendocrine carcinoma of the skin and the clinicopathological similarities between SCLC and MCC, we wished to determine if this was also the case in MCC. Twenty-nine MCC specimens from 23 patients were examined for deletions at 10 loci on 9p and 1 on 9q. No loss of heterozygosity (LOH) was seen in 9 patients including 2 for which tumour and cell line DNAs were examined. Four patients had LOH for all informative loci on 9p. Ten tumours showed more limited regions of loss on 9p, and from these 2 common regions of deletion were determined. Half of all informative cases had LOH at D9S168, the most telomeric marker examined, and 3 specimens showed loss of only D9S168. A second region (IFNA-D9S126) showed LOH in 10 (44%) cases, and case MCC26 showed LOH for only D9S126, implicating genes centromeric of the CDKN2A locus. No mutations in the coding regions of p16 were seen in 7 cell lines tested, and reactivity to anti-p16 antibody was seen in all 11 tumour specimens examined and in 6 of 7 cell lines from 6 patients. Furthermore, all cell lines examined reacted with anti-p14(ARF) antibody. These results suggest that neither transcript of the CDKN2A locus is the target of deletions on 9p in MCC and imply the existence of tumour-suppressor genes mapping both centromeric and telomeric of this locus.

Purpose: Preclinical studies by our lab and others have demonstrated fibroblast growth factor receptor (FGFR) inhibition is a viable therapeutic strategy in tumors with FGFR amplification, such as gastric and breast cancer, as well as FGFR2 mutation positive endometrial cancer (EC). A significant clinical issue that we aim to address is that of intrinsic and acquired resistance to anti-FGFR therapeutics in the context of EC. Experimental Design: Our lab performed a BaF3 screen to identify FGFR2 kinase domain mutations that would provide resistance to dovitinib, a multikinase inhibitor with anti-FGFR activity. BaF3 cells transduced with FGFR2 were treated with TKI258 at 10x and 15x the IC50 for dovitinib in these cells. Following clonal selection of dovitinib-resistant cells, the exons encoding the intracellular domain of FGFR2 were sequenced and mutations identified. Independent BaF3 stable lines were generated with wild-type FGFR2 and each of the dovitinib resistance mutations. Using these cells, sensitivity of the different FGFR2 mutants to dovitinib, ponatinib, BGJ398, AZD4547, and DCC-2036 was measured. To explore nonkinase domain mechanisms of acquired resistance to FGFR inhibition, we have also passaged AN3CA and MFE280 cells (both FGFR2 mutant) in increasing concentrations of dovitinib or PD173074. In preliminary studies, we compared the gene expression profile at baseline and 14 hours after dovitinib/PD173074 treatment of one resistant clone for both AN3CA and MFE280 versus parental clones for each. Results: Mutations in the kinase domain of FGFR2 were found in 26/63 resistant clones treated with 10X and 15X IC50. Mutations observed in individual resistant clones included M536I, M538I, I548V, N550K, N550S, V565I, E566G, L618M, Y770lfsX14 and multiple clones were found to carry the N550H mutation in FGFR2. Modeling of these mutations on FGFR2 crystal structures indicate that the majority either abrogate the “molecular brake” in the kinase hinge region resulting in receptor activation, or strengthen the hydrophobic spine, a series of interactions involved in stabilizing the activated conformation of the kinase. All but one mutation provided resistance to both dovitinib and PD173074 when analyzed in an independent BaF3 stable line. Together this data indicates that dovitinib and PD173074 predominantly bind the inactive conformation of the FGFR kinases. This is clinically significant as it suggests that cancer patients harboring activating FGFR2 mutations, such as N550K, may not respond to the anti-FGFR activity of dovitinib as a first line therapy. Significantly, ponatinib inhibited the growth of BaF3 cells transduced with all resistance mutations, except the gatekeeper mutation V565I. From our gene expression profiling of isogenic resistant EC cell lines, only a small number of significant gene expression changes were noted between resistant clones and their respective parental lines at baseline. With drug treatment, however, we observed a significant activation of pro-survival pathways, most notably BCL-XL and the WNT signaling pathway. Conclusions: In this study we identified FGFR2 mutations that confer resistance to dovitinib and show that these mutations increase FGFR2 ligand dependent and independent activity. Importantly, we found that ponatinib has strong inhibitory activity against FGFR2 activating mutations suggesting that this inhibitor may be more effective as a first-line therapy, as well as in the second line setting to target tumors with resistance to dovitinib. Moreover, it suggests that the active state of the FGFR2 kinase should be targeted for anticancer drug discovery.

Background: Melanoma's intrinsic resistance to therapy has been attributed to modulation of cellular survival pathways including apoptosis. We originally reported that mebendazole (MBZ), which acts as a microtubule-disrupting agent, is a potent inducer of apoptosis in melanoma cells in vitro via a Bcl-2 dependent mechanism (Molecular Cancer Research, 2008). Here, we show that MBZ is also able to effectively modulate another key anti-apoptotic factor, XIAP, to promote melanoma apoptosis both in vitro and in vivo. Methodology: Safety and efficacy of mebendazole was assessed using a human melanoma xenograft model established from M-14 cells. The effect of MBZ on XIAP expression and induction of apoptotic pathways was evaluated by immunoblotting and co-immunoprecipitation techniques along with densitometric quantification. The growth inhibitory effects of MBZ were determined in a panel of melanoma cell lines with the sulphorhodamine (SRB) assay. siRNA techniques were used to reduce expression of XIAP. Results: Oral MBZ is as effective as the current standard of care temozolomide (TMZ) in reducing melanoma growth in vivo, with no notable toxicities observed. Inhibition of melanoma growth in vivo is accompanied by MBZ specific reduced expression of XIAP and cleavage of caspase 9. In response to MBZ treatment, sensitive cell lines display reduced levels of XIAP expression whereas resistant cell lines do not. Moreover, down-regulation of XIAP expression by siRNA in the MBZ-resistant melanoma cell line UACC1097 was accompanied by a reduction in the IC50 for MBZ from >10 μM to 3.7 μM. Exposure of melanoma cells to MBZ promotes the interaction of SMAC/DIABLO with XIAP, thereby alleviating inhibition of apoptosis by XIAP. Conclusions: In addition to demonstrating efficacy and safety in vivo, our preclinical studies of the promising anti-melanoma agent mebendazole provide insight into characterization of an apoptotic promoting mechanism of action. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4893. doi:1538-7445.AM2012-4893

Members of the fibroblast growth factor receptor (FGFR) family are amplified or mutationally activated in a variety of cancers, including breast, endometrial, ovarian, lung, gastric, and bladder cancers, glioblastoma and rhabdomyosarcoma. Consequently FGFRs are attractive therapeutic targets in cancer, with a number of FGFR inhibitors currently progressing through clinical trials. Dovitinib, a lead FGFR kinase inhibitor exhibits activity against FLT3, FGFRs, VEGFRs, and PDGFR, and has demonstrated considerable preclinical activity in cancer models with FGFR activation. Though targeted tyrosine kinase inhibitors (TKIs) have shown dramatic clinical responses, the long-term efficacy of these agents is frequently limited by development of resistance to the targeted agent, often due to mutation of the target kinase. Here we sought to identify the mutational mechanisms of resistance to Dovitinib using a BaF3 cell line screening strategy. The BaF3 cell line is an IL-3 dependent murine pro-B cell line that is commonly employed to model TKI resistant mutations. These cells do not express any FGF ligands or receptors and introduction and activation of FGFRs has been shown to substitute for IL-3 to promote cell proliferation. BaF3 cells transduced with FGFR2 were treated with TKI258 at 5x, 10x, and 15x the cellular IC50 for Dovitinib in these cells. Following clonal selection of Dovitinib resistant cells, the exons encoding the intracellular domain of FGFR2 were sequenced. Mutations in FGFR2 kinase domain were identified in 26 out of 63 (41.2%) resistant clones screened, with an increase in frequency of mutation with increasing selective pressure. Ten distinct Dovitinib-resistant mutations in FGFR2 were identified and subsequently confirmed to result in Dovitinib-resistance and kinase activation. The binding mode of Dovitinib and the mechanisms of action of the resistance mutations were studied using the crystal structures of unphosphorylated and phosphorylated FGFR2Ks. Mutations at N550 and E566 at the kinase hinge/interlobe region are expected to drive the kinase into the active state by disengaging the molecular brake that keeps the kinase in an autoinhibited state. Five additional mutations are also predicted to stabilize the active conformation of the kinase by strengthening a network of hydrophobic interactions between N- and C-terminal lobes of the kinase, termed the hydrophobic spine, that is a hallmark of the active state of the kinase. Hence our biochemical and structural data show that the drug predominantly binds the inactive state of the FGFR2 kinase. Our data have clinical ramifications as they suggest that cancer patients harboring these FGFR2 mutations may not respond to the anti-FGFR activity of Dovitinib. Taken together our study provides the first report of TKI-resistant mutations in FGFR2 and suggests that the active state of the FGFR2 kinase should be targeted for anti-cancer drug discovery. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4733. doi:10.1158/1538-7445.AM2011-4733

e19021 Background: Mebendazole (MBZ), a microtubule binding agent, is a potent inducer of apoptosis in a panel of melanoma cells, yet largely non-toxic to melanocytes (Mol Cancer Res, Aug 2008). We...

Inhibition of the RAS/RAF/MEK/ERK signaling pathway has emerged as a promising therapeutic strategy for melanoma given the high frequency of BRAF and NRAS mutations in this tumor type. Sensitivity to E6201, an inhibitor of MEK1 and other cancer relevant kinases, was assessed in a panel of 32 cell lines for which the mutation status of common melanoma genes was known. The majority (24/32) of the melanoma cell lines were sensitive to E6201, resulting in G1-cell cycle arrest and cell death in 11 sensitive cell lines and cell cycle arrest but not cell death in two sensitive lines. Xenograft studies revealed that E6201 exhibited a cytotoxic effect in vivo, even in cell lines where only a cytostatic effect was observed in vitro. Among cell lines carrying mutations in BRAF, or NRAS, sensitivity to E6201 was associated with wildtype PTEN status (p Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B93.

Endometrial cancer is the most common gynecological malignancy and the fourth most common cancer in women in the United States. Most women present with early stage disease confined to the uterus and thus have a favorable prognosis. However, for the subset of women who present with advanced disease or that recur, prognosis is poor, with a median survival after recurrence of 10 months, reflecting a lack of effective treatment options. We identified activating mutations in the fibroblast growth factor receptor 2 (FGFR2) receptor tyrosine kinase in 10–15% of endometrial tumors, and hypothesized that FGFR inhibition may be a viable therapeutic option for patients with FGFR2 mutation positive tumors. Indeed, we have shown that treatment of FGFR2 mutation positive endometrial cancer cell lines with PD173074, a pan-FGFR small molecular inhibitor, induces cell death both in vitro and in vivo. We have shown this cell death is caspase-independent, raising the possibility that combination of an FGFR inhibitor with chemotherapeutic agents may exhibit synergistic activity due to their non-redundant mechanisms of action. Moreover, as FGF ligand expression has been associated with chemoresistance and poor prognosis in a number of other cancer types, including lung, breast, ovarian, prostate, and head and neck carcinomas, we hypothesized that FGFR inhibition may also sensitize wildtype FGFR2 endometrial cancer cell lines to chemotherapeutic agents. We therefore sought to evaluate whether FGFR inhibition synergizes with conventional chemotherapeutic agents in endometrial cancer. Using combination index and Bliss additivity analysis, we evaluated whether FGFR inhibition potentiated the effect of paclitaxel and doxorubicin in a panel of FGFR2 mutant and non-mutant endometrial cancer cell lines. FGFR2 mutation status did not alter sensitivity to either chemotherapeutic agent alone. The combination of PD173074 with paclitaxel or doxorubicin showed synergistic activity in all three FGFR2 mutant cell lines. In addition, though non-mutant cell lines were resistant to FGFR inhibition alone, the addition of PD173074 potentiated the effect of paclitaxel and doxorubicin in a subset of FGFR2-wildtype cell lines tested. Together these data suggest a therapeutic benefit to combining an FGFR inhibitor with standard chemotherapeutic agents, and, importantly, this may extend to patients whose tumors possess wildtype FGFR2. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C86.