Patient-derived xenograft models capture genomic heterogeneity in endometrial cancer.

BACKGROUND: Endometrial cancer (EC) is a major gynecological cancer with increasing incidence. It comprises four molecular subtypes with differing etiology, prognoses, and responses to chemotherapy. In the future, clinical trials testing new single agents or combination therapies will be targeted to the molecular subtype most likely to respond. As pre-clinical models that faithfully represent the molecular subtypes of EC are urgently needed, we sought to develop and characterize a panel of novel EC patient-derived xenograft (PDX) models. METHODS: Here, we report whole exome or whole genome sequencing of 11 PDX models and their matched primary tumor. Analysis of multiple PDX lineages and passages was performed to study tumor heterogeneity across lineages and/or passages. Based on recent reports of frequent defects in the homologous recombination (HR) pathway in EC, we assessed mutational signatures and HR deficiency scores and correlated these with in vivo responses to the PARP inhibitor (PARPi) talazoparib in six PDXs representing the copy number high/p53-mutant and mismatch-repair deficient molecular subtypes of EC. RESULTS: PDX models were successfully generated from grade 2/3 tumors, including three uterine carcinosarcomas. The models showed similar histomorphology to the primary tumors and represented all four molecular subtypes of EC, including five mismatch-repair deficient models. The different PDX lineages showed a wide range of inter-tumor and intra-tumor heterogeneity. However, for most PDX models, one arm recapitulated the molecular landscape of the primary tumor without major genomic drift. An in vivo response to talazoparib was detected in four copy number high models. Two models (carcinosarcomas) showed a response consistent with stable disease and two models (one copy number high serous EC and another carcinosarcoma) showed significant tumor growth inhibition, albeit one consistent with progressive disease; however, all lacked the HR deficiency genomic signature. CONCLUSIONS: EC PDX models represent the four molecular subtypes of disease and can capture intra-tumor heterogeneity of the original primary tumor. PDXs of the copy number high molecular subtype showed sensitivity to PARPi; however, deeper and more durable responses will likely require combination of PARPi with other agents.

Bcl-2 inhibitors enhance FGFR inhibitor-induced mitochondrial-dependent cell death in FGFR2-mutant endometrial cancer.

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.

PI3K Inhibitors Synergize with FGFR Inhibitors to Enhance Antitumor Responses in FGFR2mutant Endometrial Cancers.

Improved therapeutic approaches are needed for the treatment of recurrent and metastatic endometrial cancer. Endometrial cancers display hyperactivation of the MAPK and PI3K pathways, the result of somatic aberrations in genes such as FGFR2, KRAS, PTEN, PIK3CA, and PIK3R1 The FGFR2 and PI3K pathways, have emerged as potential therapeutic targets in endometrial cancer. Activation of the PI3K pathway is seen in more than 90% of FGFR2mutant endometrial cancers. This study aimed to examine the efficacy of the pan-FGFR inhibitor BGJ398 with pan-PI3K inhibitors (GDC-0941, BKM120) and the p110α-selective inhibitor BYL719. We assessed synergy in three FGFR2mutant endometrial cancer cell lines (AN3CA, JHUEM2, and MFE296), and the combination of BGJ398 and GDC-0941 or BYL719 showed strong synergy. A significant increase in cell death and decrease in long-term survival was seen when PI3K inhibitors were combined with BGJ398. Importantly, these effects were seen at low concentrations correlating to only partial inhibition of AKT. The combination of BGJ398 and GDC-0941 showed tumor regressions in vivo, whereas each drug alone only showed moderate tumor growth inhibition. BYL719 alone resulted in increased tumor growth of AN3CA xenografts but in combination with BGJ398 resulted in tumor regression in both AN3CA- and JHUEM2-derived xenografts. These data provide evidence that subtherapeutic doses of PI3K inhibitors enhance the efficacy of anti-FGFR therapies, and a combination therapy may represent a superior therapeutic treatment in patients with FGFR2mutant endometrial cancer. Mol Cancer Ther; 16(4); 637-48. ©2017 AACR.

Paralog-Specific Kinase Inhibition of FGFR4: Adding to the Arsenal of Anti-FGFR Agents.

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.

Nilotinib and MEK inhibitors induce synthetic lethality through paradoxical activation of RAF in drug-resistant chronic myeloid leukemia.

We show that imatinib, nilotinib, and dasatinib possess weak off-target activity against RAF and, therefore, drive paradoxical activation of BRAF and CRAF in a RAS-dependent manner. Critically, because RAS is activated by BCR-ABL, in drug-resistant chronic myeloid leukemia (CML) cells, RAS activity persists in the presence of these drugs, driving paradoxical activation of BRAF, CRAF, MEK, and ERK, and leading to an unexpected dependency on the pathway. Consequently, nilotinib synergizes with MEK inhibitors to kill drug-resistant CML cells and block tumor growth in mice. Thus, we show that imatinib, nilotinib, and dasatinib drive paradoxical RAF/MEK/ERK pathway activation and have uncovered a synthetic lethal interaction that can be used to kill drug-resistant CML cells in vitro and in vivo.

Activation of forkhead box O transcription factors by oncogenic BRAF promotes p21cip1-dependent senescence.

Oncogene-induced senescence (OIS) is a potent tumor-suppressive mechanism that is thought to come at the cost of aging. The Forkhead box O (FOXO) transcription factors are regulators of life span and tumor suppression. However, whether and how FOXOs function in OIS have been unclear. Here, we show a role for FOXO4 in mediating senescence by the human BRAF(V600E) oncogene, which arises commonly in melanoma. BRAF(V600E) signaling through mitogen-activated protein kinase/extracellular signal-regulated kinase kinase resulted in increased reactive oxygen species levels and c-Jun NH(2) terminal kinase-mediated activation of FOXO4 via its phosphorylation on Thr(223), Ser(226), Thr(447), and Thr(451). BRAF(V600E)-induced FOXO4 phosphorylation resulted in p21(cip1)-mediated cell senescence independent of p16(ink4a) or p27(kip1). Importantly, melanocyte-specific activation of BRAF(V600E) in vivo resulted in the formation of skin nevi expressing Thr(223)/Ser(226)-phosphorylated FOXO4 and elevated p21(cip1). Together, these findings support a model in which FOXOs mediate a trade-off between cancer and aging.

Identification of direct transcriptional targets of (V600E)BRAF/MEK signalling in melanoma.

Oncogenic mutations in BRAF are common in melanoma and drive constitutive activation of the MEK/ERK pathway. To elucidate the transcriptional events downstream of (V600E)BRAF/MEK signalling we performed gene expression profiling of A375 melanoma cells treated with potent and selective inhibitors of (V600E)BRAF and MEK (PLX4720 and PD184352 respectively). Using a stringent Bayesian approach, we identified 69 transcripts that appear to be direct transcriptional targets of this pathway and whose expression changed after 6 h of pathway inhibition. We also identified several additional genes whose expression changed after 24 h of pathway inhibition and which are likely to be indirect transcriptional targets of the pathway. Several of these were confirmed by demonstrating their expression to be similarly regulated when BRAF was depleted using RNA interference, and by using qRT-PCR in other BRAF mutated melanoma lines. Many of these genes are transcription factors and feedback inhibitors of the ERK pathway and are also regulated by MEK signalling in NRAS mutant cells. This study provides a basis for understanding the molecular processes that are regulated by (V600E)BRAF/MEK signalling in melanoma cells.

Gene expression profiling in melanoma identifies novel downstream effectors of p14ARF.

p14ARF is inactivated by deletions/mutations in many cancer types and can suppress cell growth by both p53-dependent and p53-independent mechanisms. To identify novel downstream effectors of p14ARF, we used gene expression profiling as a primary screening tool to select candidates for follow up validation studies using in vitro cell-based assays. Gene expression profiles of a panel of 35 melanoma cell lines with either wild-type (n = 12) or mutant (n = 23) p14ARF were compared to identify genes associated with inactivation of p14ARF. Analysis of the microarray data identified 1,316 probe sets that were significantly (p < 0.01) differentially expressed between the p14ARF wild-type and mutant cell lines. Pathway analysis of these genes showed an overrepresentation of many receptor-mediated signal transduction pathways, e.g. TGFbeta, EGF, HGF, PDGF, MAPK, Wnt and integrin pathways. A number of components of these pathways, including FLRT3, RUNX2, MIG-6 and SMURF2 were confirmed as downstream targets of p14ARF using p14ARF-inducible cell lines and RNAi. We propose that regulation of these genes may contribute to melanoma development when p14ARF function is lost.

Expression of the cell surface mucin gene family in adenocarcinomas.

Cell surface mucins are complex glycoproteins expressed on the apical membrane surface of mucosal epithelial cells. In malignant epithelial cells they are thought to influence cell adhesion, and are clinical targets for tumor immunotherapy and serum tumor marker assays. We have compared expression of MUC1, MUC3, MUC4, MUC11, MUC12 and MUC13 mRNA in epithelial cancers and/or cell lines with non-malignant tissues. In non-malignant tissues, MUC3, 4, 11, 12 and 13 were expressed at highest levels in gastrointestinal tissues, whereas MUC1 was more widely distributed. Significant down-regulation of the MUC4, MUC12 and MUC13 genes was observed in colonic cancers compared with normal tissue, whereas MUC1 was upregulated. In rectal cancers, levels of all six mucin genes were not significantly different to those in normal rectal tissues. Both MUC1 and MUC4 were down-regulated in gastric cancers, whereas cancer and normal tissue levels were similar for MUC3, 11, 12 and 13. In esophageal cancers there was a general trend toward higher levels than in normal tissue for MUC1, 3, 12 and 13. In ovarian cancers MUC1 levels were very high, whereas only low levels of all other mucins were observed. We also report expression in renal cell carcinomas, bladder carcinomas and breast cancer cell lines. The reported expression profiles of the cell surface mucin gene family will help direct biological and clinical studies of these molecules in mucosal biology, and in malignant and inflammatory diseases of epithelial tissues.

Expression of MUC1 splice variants in benign and malignant ovarian tumours.

MUC1 is expressed on the surface of ovarian cancer cells. Nine different splice variants of MUC1 have been described, but no study has reported on the expression of MUC1 isoforms in human ovarian cancer. Our study compares patterns of expression of MUC1 splice variants of malignant and benign ovarian tumours. Ovarian tissue samples were taken from patients with benign ovarian tumours (n = 34) and from patients who had surgery for primary (n = 47) or recurrent (n = 8) ovarian cancer. RT-PCR for MUC1 splice variants A, B, C, D, X, Y, Z, REP and SEC was performed and their expression compared to clinical and histopathologic parameters. Variants A, D, X, Y and Z were more frequently expressed in malignant than in benign tumours. All primary ovarian cancer cases were positive for variant REP but negative for variant SEC. No significant association of the expression of MUC1 splice variants with the response to chemotherapy or patient survival could be demonstrated. Expression of MUC1 splice variants A, D, X, Y, Z and REP is associated with the presence of malignancy, whereas expression of MUC1/SEC is associated with the absence of malignancy.