FGFR2 Inhibition in Cholangiocarcinoma.

Biliary tract cancer (BTC) is the second most common primary liver cancer after hepatocellular carcinoma and accounts for 2% of cancer-related deaths. BTCs are classified according to their anatomical origin into intrahepatic (iCCA), perihilar, or distal cholangiocarcinoma, as well as gall bladder carcinoma. While the mutational profiles in these anatomical BTC subtypes overlap to a large extent, iCCA is notable for the high frequency of IDH1/2 mutations (10-22%) and the nearly exclusive occurrence of FGFR2 fusions in 10-15% of patients. In recent years, FGFR2 fusions have become one of the most promising targets for precision oncology targeting BTC, with FGFR inhibitors already approved in Europe and the United States for patients with advanced, pretreated iCCA. While the therapeutic potential of nonfusion alterations is still under debate, it is expected that the field of FGFR2-directed therapies will be subject to rapid further evolution and optimization. The scope of this review is to provide an overview of oncogenic FGFR signaling in iCCA cells and highlight the pathophysiology, diagnostic testing strategies, and therapeutic promises and challenges associated with FGFR2-altered iCCA.

Charting co-mutation patterns associated with actionable drivers in intrahepatic cholangiocarcinoma.

BACKGROUND & AIMS: In recent years, intrahepatic cholangiocarcinoma (iCCA) has evolved as a "role model" for precision oncology in gastrointestinal cancers. However, its rarity, paired with its genomic heterogeneity, challenges the development and evolution of targeted therapies. Interrogating large datasets drives better understanding of the characteristics of molecular subgroups of rare cancers and enables the identification of genomic patterns that remain unrecognized in smaller cohorts. METHODS: We performed a retrospective analysis of 6,130 patients diagnosed with iCCA from the FoundationCORE database who received diagnostic panel sequencing on the FoundationOne platform. Short variants/fusion-rearrangements and copy number alterations in >300 tumor-associated genes were evaluated, and the tumor mutational burden (TMB) as well as the microsatellite instability (MSI) status were available for the majority of the cohort. RESULTS: We provide a highly representative cartography of the genomic landscape of iCCA and outline the co-mutational spectra of seven therapeutically relevant oncogenic driver genes: IDH1/2, FGFR2, ERBB2, BRAF, MDM2, BRCA1/2, MET and KRASG12C. We observed a negative selection of RTK/RAS/ERK pathway co-alterations, and an enrichment of epigenetic modifiers such as ARID1A and BAP1 in patients with IDH1/2 and FGFR2 alterations. RNF43 as well as KMT2D occurred with high frequency in MSIhigh and TMBhigh tumors. CONCLUSION: Detailed knowledge of the most prevalent genomic constellations is key to the development of effective treatment strategies for iCCA. Our study provides a valuable resource that could be used to assess the feasibility of clinical trials and subgroup analyses, spurs the development of translationally relevant preclinical models, and serves as a knowledge base to predict potential mechanisms of resistance to targeted therapies in genomically defined subgroups. IMPACT AND IMPLICATIONS: Due to the high frequency of targetable alterations, molecular diagnostics is recommended in patients with biliary tract cancers, and especially in those with iCCA. The identification of an actionable lesion, however, does not guarantee therapeutic success, and the co-mutational spectrum may act as a critical modifier of drug response. Using a large dataset of comprehensive panel sequencing results from 6,130 patients with iCCA, we provide a detailed analysis of the co-mutational spectrum of the most frequent druggable genetic alterations, which is meant to serve as a reference to establish genetically relevant preclinical models, develop hypothesis-driven combination therapies and identify recurrent genetic profiles.

FGFR2 fusion proteins drive oncogenic transformation of mouse liver organoids towards cholangiocarcinoma.

BACKGROUND & AIMS: About 15% of intrahepatic cholangiocarcinomas (iCCAs) express fibroblast growth factor receptor 2 (FGFR2) fusion proteins (FFs), usually alongside mutational inactivation of TP53, CDKN2A or BAP1. In FFs, FGFR2 residues 1-768 fuse to sequences encoded by a diverse array of partner genes (>60) causing oncogenic FF activation. While FGFR-specific tyrosine kinase inhibitors (F-TKI) provide clinical benefit in FF+ iCCA, responses are partial and/or limited by resistance mechanisms, such as the V565F substitution in the FGFR2 gatekeeper residue. Improving on FF targeting in iCCA therefore remains a critical unmet need. Herein, we aimed to generate a murine model of FF-driven iCCA and use this to uncover actionable FF-associated dependencies. METHODS: Four iCCA FFs carrying different fusion sequences were expressed in Tp53-/- mouse liver organoids. Tumorigenic properties of genetically modified liver organoids were assessed by transplantation into immuno-deficient mice. Cellular models derived from neoplastic lesions were exploited for pre-clinical studies. RESULTS: Transplantation of FF-expressing liver organoids yielded tumors diagnosed as CCA based on histological, phenotypic and transcriptomic analyses. The penetrance of this tumorigenic phenotype was influenced by FF identity. Tumor organoids and 2D cell lines derived from CCA lesions were addicted to FF signaling via Ras-Erk, regardless of FF identity or V565F mutation. Dual blockade of FF and the Ras-Erk pathway by concomitant pharmacological inhibition of FFs and Mek1/2 provided greater therapeutic efficacy than single agent F-TKI in vitro and in vivo. CONCLUSIONS: FF-driven iCCA pathogenesis was successfully modeled on a Tp53-/- murine background, revealing biological heterogeneity among structurally different FFs. Double blockade of FF-ERK signaling deserves consideration for precision-based approaches against human FF+ iCCA. LAY SUMMARY: Intrahepatic cholangiocarcinoma (iCCA) is a rare cancer that is difficult to treat. A subtype of iCCA is caused by genomic alterations that generate oncogenic drivers known as FGFR2 fusions. Patients with FGFR2 fusions respond to FGFR inhibitors, but clinical responses are often of modest duration. We used animal and cellular models to show that FGFR2 fusions require the activity of a downstream effector named Mek1/2. We found that dual blockade of FGFR2 fusions and Mek1/2 was more effective than isolated inhibition of FGFR2 fusions, pointing to the potential clinical utility of dual FGFR2-MEK1/2 blockade in patients with iCCA.

Making sense of Cbp/p300 loss of function mutations in skin tumorigenesis.

CBP and p300 are highly homologous lysine acetyltransferases involved in cell cycle regulation, DNA synthesis and DNA repair. Loss of function mutations of CBP and p300 are found in about one-third of cutaneous squamous cell carcinoma (cSCC) and often co-occur, yet their role in cSCC pathogenesis is unclear. Writing in The Journal of Pathology, Ichise and colleagues modeled combined heterozygous loss of Cbp/p300 in mouse keratinocytes expressing a transgenic HrasS35 allele that allows selective coupling of Hras to the Erk pathway. Epidermal thickening caused by expression of HrasS35 was exacerbated by reduced dosage of Cbp/p300 and eventually resulted in development of skin papillomas. This phenotype was associated with reduced expression of Mig6, an Egfr feedback inhibitor, and attendant enhancement of Egfr signaling to the Ras-Erk pathway. This model provides a mechanistic framework for understanding how Cbp/p300 loss of function mutations impact on skin tumorigenesis and suggests potential therapeutic options in CBP/p300 mutated human cSCC. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Current and novel therapeutic opportunities for systemic therapy in biliary cancer.

Biliary tract cancers (BTCs) are a group of rare and aggressive malignancies that arise in the biliary tree within and outside the liver. Beyond surgical resection, which is beneficial for only a small proportion of patients, current strategies for treating patients with BTCs include chemotherapy, as a single agent or combination regimens, in the adjuvant and palliative setting. Increased characterisation of the molecular landscape of these tumours has facilitated the identification of molecular vulnerabilities, such as IDH mutations and FGFR fusions, that can be exploited for the treatment of BTC patients. Beyond targeted therapies, active research avenues explore the development of novel therapeutics that target the crosstalk between cancer and stroma, the cellular pathways involved in the regulation of cell death, the chemoresistance phenotype and the dysregulation of RNA. In this review, we discuss the therapeutic opportunities currently available in the management of BTC patients, and explore the strategies that can support the implementation of precision oncology in BTCs, including novel molecular targets, liquid biopsies and patient-derived predictive tools.

HSP90 Inhibition Drives Degradation of FGFR2 Fusion Proteins: Implications for Treatment of Cholangiocarcinoma.

About 15% of intrahepatic cholangiocarcinomas (ICCs) express constitutively active fibroblast growth factor receptor 2 (FGFR2) fusion proteins (FFs) generated by chromosomal translocations. FFs have been nominated as oncogenic drivers because administration of FGFR tyrosine kinase inhibitors (F-TKIs) can elicit meaningful objective clinical responses in patients carrying FF-positive ICC. Thus, optimization of FF targeting is a pressing clinical need. Herein, we report that three different FFs, previously isolated from ICC samples, are heat shock protein 90 (HSP90) clients and undergo rapid degradation upon HSP90 pharmacological blockade by the clinically advanced HSP90 inhibitor ganetespib. Combining catalytic suppression by the F-TKI BGJ398 with HSP90 blockade by ganetespib suppressed FGFR2-TACC3 (transforming acidic coiled-coil containing protein 3) signaling in cultured cells more effectively than either BGJ398 or ganetespib in isolation. The BGJ398 + ganetespib combo was also superior to single agents when tested in mice carrying subcutaneous tumors generated by transplantation of FGFR2-TACC3 NIH3T3 transformants. Of note, FF mutants known to enforce clinical resistance to BGJ398 in ICC patients retained full sensitivity to ganetespib in cultured cells. Conclusion: Our data provide a proof of principle that upfront treatment with the BGJ398 + ganetespib combo improves therapeutic targeting of FGFR2 fusions in an experimental setting, which may be relevant to precision medicine approaches to FF-driven ICC.

Signalling networks in cholangiocarcinoma: Molecular pathogenesis, targeted therapies and drug resistance.

Cholangiocarcinoma (CCA) is a deadly disease. While surgery may attain cure in a minor fraction of cases, therapeutic options in either the adjuvant or advanced setting are limited. The possibility of advancing the efficacy of therapeutic approaches to CCA relies on understanding its molecular pathogenesis and developing rational therapies aimed at interfering with oncogenic signalling networks that drive and sustain cholangiocarcinogenesis. These efforts are complicated by the intricate biology of CCA, which integrates not only the driving force of tumour cell-intrinsic alterations at the genetic and epigenetic level but also pro-tumorigenic cues conveyed to CCA cells by different cell types present in the rich tumour stroma. Herein, we review our current understanding of the mechanistic bases underpinning the activation of major oncogenic pathways causative of CCA pathogenesis. We subsequently discuss how this knowledge is being exploited to implement rationale-based and genotype-matched therapeutic approaches that predictably will radically transform CCA clinical management in the next decade. We conclude by highlighting the mechanisms of therapeutic resistance in CCA and reviewing innovative approaches to combat resistance at the preclinical and clinical level.

Regulation of the ErbB network by the MIG6 feedback loop in physiology, tumor suppression and responses to oncogene-targeted therapeutics.

The ErbB signaling network instructs the execution of key cellular programs, such as cell survival, proliferation and motility, through the generation of robust signals of defined strength and duration. In contrast, unabated ErbB signaling disrupts tissue homeostasis and leads to cell transformation. Cells oppose the threat inherent in excessive ErbB activity through several mechanisms of negative feedback regulation. Inducible feedback inhibitors (IFIs) are expressed in the context of transcriptional responses triggered by ErbB signaling, thus being uniquely suited to regulate ErbB activity during the execution of complex cellular programs. This review focuses on MIG6, an IFI that restrains ErbB signaling by mediating ErbB kinase suppression and receptor down-regulation. We will review key issues in MIG6 function, regulation and tumor suppressor activity. Subsequently, the role for MIG6 loss in the pathogenesis of tumors driven by ErbB oncogenes as well as in the generation of cellular addiction to ErbB signaling will be discussed. We will conclude by analyzing feedback inhibition by MIG6 in the context of therapies directed against ErbB and non-ErbB oncogenes.

Regulation of epidermal growth factor receptor signalling by inducible feedback inhibitors.

Signalling by the epidermal growth factor receptor (EGFR) controls morphogenesis and/or homeostasis of several tissues from worms to mammals. The correct execution of these programmes requires the generation of EGFR signals of appropriate strength and duration. This is obtained through a complex circuitry of positive and negative feedback regulation. Feedback inhibitory mechanisms restrain EGFR activity in time and space, which is key to ensuring that receptor outputs are commensurate to the cell and tissue needs. Here, we focus on the emerging field of inducible negative feedback regulation of the EGFR in mammals. In mammalian cells, four EGFR inducible feedback inhibitors (IFIs), namely LRIG1, RALT (also known as MIG6 and ERRFI1), SOCS4 and SOCS5, have been discovered recently. EGFR IFIs are expressed de novo in the context of early or delayed transcriptional responses triggered by EGFR activation. They all bind to the EGFR and suppress receptor signalling through several mechanisms, including catalytic inhibition and receptor downregulation. Here, we review the mechanistic basis of IFI signalling and rationalise the function of IFIs in light of gene-knockout studies that assign LRIG1 and RALT an essential role in restricting cell proliferation. Finally, we discuss how IFIs might participate in system control of EGFR signalling and highlight the emerging roles for IFIs in the suppression of EGFR-driven tumorigenesis.

Mig-6 controls EGFR trafficking and suppresses gliomagenesis.

Glioblastoma multiforme (GBM) is the most common and lethal primary brain cancer that is driven by aberrant signaling of growth factor receptors, particularly the epidermal growth factor receptor (EGFR). EGFR signaling is tightly regulated by receptor endocytosis and lysosome-mediated degradation, although the molecular mechanisms governing such regulation, particularly in the context of cancer, remain poorly delineated. Here, high-resolution genomic profiles of GBM identified a highly recurrent focal 1p36 deletion encompassing the putative tumor suppressor gene, Mig-6. We show that Mig-6 quells the malignant potential of GBM cells and dampens EGFR signaling by driving EGFR into late endosomes and lysosome-mediated degradation upon ligand stimulation. Mechanistically, this effect is mediated by the binding of Mig-6 to a SNARE protein STX8, a protein known to be required for late endosome trafficking. Thus, Mig-6 functions to ensure recruitment of internalized receptor to late endosomes and subsequently the lysosomal degradation compartment through its ability to specifically link EGFR and STX8 during ligand-stimulated EGFR trafficking. In GBM, the highly frequent loss of Mig-6 would therefore serve to sustain aberrant EGFR-mediated oncogenic signaling. Together, these data uncover a unique tumor suppression mechanism involving the regulation of receptor trafficking.

Criteria for preclinical models of cholangiocarcinoma: scientific and medical relevance.

Cholangiocarcinoma (CCA) is a rare malignancy that develops at any point along the biliary tree. CCA has a poor prognosis, its clinical management remains challenging, and effective treatments are lacking. Therefore, preclinical research is of pivotal importance and necessary to acquire a deeper understanding of CCA and improve therapeutic outcomes. Preclinical research involves developing and managing complementary experimental models, from in vitro assays using primary cells or cell lines cultured in 2D or 3D to in vivo models with engrafted material, chemically induced CCA or genetically engineered models. All are valuable tools with well-defined advantages and limitations. The choice of a preclinical model is guided by the question(s) to be addressed; ideally, results should be recapitulated in independent approaches. In this Consensus Statement, a task force of 45 experts in CCA molecular and cellular biology and clinicians, including pathologists, from ten countries provides recommendations on the minimal criteria for preclinical models to provide a uniform approach. These recommendations are based on two rounds of questionnaires completed by 35 (first round) and 45 (second round) experts to reach a consensus with 13 statements. An agreement was defined when at least 90% of the participants voting anonymously agreed with a statement. The ultimate goal was to transfer basic laboratory research to the clinics through increased disease understanding and to develop clinical biomarkers and innovative therapies for patients with CCA.

Combination therapies for targeting FGFR2 fusions in cholangiocarcinoma.

Fibroblast growth factor receptor 2 (FGFR2) fusion proteins (FFs) are oncogenic drivers in 10-15% of intrahepatic cholangiocarcinoma (iCCA). FGFR-specific inhibitors provide temporary benefit in FF+ unresectable patients. Recent work with mouse iCCA models has documented the necessary role of RAS-ERK downstream to FFs and provided examples of preclinical experimentation aimed at improving FF targeting in iCCA.

miR-205 mediates adaptive resistance to MET inhibition via ERRFI1 targeting and raised EGFR signaling.

The onset of secondary resistance represents a major limitation to long-term efficacy of target therapies in cancer patients. Thus, the identification of mechanisms mediating secondary resistance is the key to the rational design of therapeutic strategies for resistant patients. MiRNA profiling combined with RNA-Seq in MET-addicted cancer cell lines led us to identify the miR-205/ERRFI1 (ERBB receptor feedback inhibitor-1) axis as a novel mediator of resistance to MET tyrosine kinase inhibitors (TKIs). In cells resistant to MET-TKIs, epigenetically induced miR-205 expression determined the downregulation of ERRFI1 which, in turn, caused EGFR activation, sustaining resistance to MET-TKIs. Anti-miR-205 transduction reverted crizotinib resistance in vivo, while miR-205 over-expression rendered wt cells refractory to TKI treatment. Importantly, in the absence of EGFR genetic alterations, miR-205/ERRFI1-driven EGFR activation rendered MET-TKI-resistant cells sensitive to combined MET/EGFR inhibition. As a proof of concept of the clinical relevance of this new mechanism of adaptive resistance, we report that a patient with a MET-amplified lung adenocarcinoma displayed deregulation of the miR-205/ERRFI1 axis in concomitance with onset of clinical resistance to anti-MET therapy.

Loss of RALT/MIG-6 expression in ERBB2-amplified breast carcinomas enhances ErbB-2 oncogenic potency and favors resistance to Herceptin.

An emerging paradigm holds that loss of negative signalling to receptor tyrosine kinases (RTKs) is permissive for their oncogenic activity. Herein, we have addressed tumor suppression by RALT/MIG-6, a transcriptionally controlled feedback inhibitor of ErbB RTKs, in breast cancer cells. Knockdown of RALT expression by RNAi enhanced the EGF-dependent proliferation of normal breast epithelial cells, indicating that loss of RALT signalling in breast epithelium may represent an advantageous condition during ErbB-driven tumorigenesis. Although mutational inactivation of the RALT gene was not detected in human breast carcinomas, RALT mRNA and protein expression was strongly and selectively reduced in ERBB2-amplified breast cancer cell lines. Reconstitution of RALT expression in ERBB2-amplified SKBr-3 and BT474 cells inhibited ErbB-2-dependent mitogenic signalling and counteracted the ability of ErbB ligands to promote resistance to the ErbB-2-targeting drug Herceptin. Thus, loss of RALT expression cooperates with ERBB2 gene amplification to drive full oncogenic signalling by the ErbB-2 receptor. Moreover, loss of RALT signalling may adversely affect tumor responses to ErbB-2-targeting agents.

Expression of RALT, a feedback inhibitor of ErbB receptors, is subjected to an integrated transcriptional and post-translational control.

Over-expression studies have demonstrated that RALT (receptor associated late transducer) is a feedback inhibitor of ErbB-2 mitogenic and transforming signals. In growth-arrested cells, expression of endogenous RALT is induced by mitogenic stimuli, is high throughout mid to late G1 and returns to baseline as cells move into S phase. Here, we show that physiological levels of RALT effectively suppress ErbB-2 mitogenic signals. We also investigate the regulatory mechanisms that preside to the control of RALT expression. We demonstrate that pharmacological ablation of extracellular signal-regulated kinase (ERK) activation leads to blockade of RALT expression, unlike genetic and/or pharmacological interference with the activities of PKC, Src family kinases, p38 SAPK and PI-3K. Tamoxifen-dependent activation of an inducible Raf : ER chimera was sufficient to induce RALT expression. Thus, activation of the Ras-Raf-ERK pathway is necessary and sufficient to drive RALT expression. The RALT protein is labile and was found to accumulate robustly upon pharmacological inhibition of the proteasome. We were able to detect ubiquitin-conjugated RALT species in living cells, suggesting that ubiquitinylation targets RALT for proteasome-dependent degradation. Such an integrated transcriptional and post-translational control is likely to provide RALT with the ability to fluctuate timely in order to tune ErbB signals.

Feedback inhibition by RALT controls signal output by the ErbB network.

The ErbB-2 interacting protein receptor-associated late transducer (RALT) was previously identified as a feedback inhibitor of ErbB-2 mitogenic signals. We now report that RALT binds to ligand-activated epidermal growth factor receptor (EGFR), ErbB-4 and ErbB-2.ErbB-3 dimers. When ectopically expressed in 32D cells reconstituted with the above ErbB receptor tyrosine kinases (RTKs) RALT behaved as a pan-ErbB inhibitor. Importantly, when tested in either cell proliferation assays or biochemical experiments measuring activation of ERK and AKT, RALT affected the signalling activity of distinct ErbB dimers with different relative potencies. RALT deltaEBR, a mutant unable to bind to ErbB RTKs, did not inhibit ErbB-dependent activation of ERK and AKT, consistent with RALT exerting its suppressive activity towards these pathways at a receptor-proximal level. Remarkably, RALT deltaEBR retained the ability to suppress largely the proliferative activity of ErbB-2.ErbB-3 dimers over a wide range of ligand concentrations, indicating that RALT can intercept ErbB-2.ErbB-3 mitogenic signals also at a receptor-distal level. A suppressive function of RALT deltaEBR towards the mitogenic activity of EGFR and ErbB-4 was detected at low levels of receptor occupancy, but was completely overcome by saturating concentrations of ligand. We propose that quantitative and qualitative aspects of RALT signalling concur in defining identity, strength and duration of signals generated by the ErbB network.

Mitogen-Inducible Gene-6 Mediates Feedback Inhibition from Mutated BRAF towards the Epidermal Growth Factor Receptor and Thereby Limits Malignant Transformation.

BRAF functions in the RAS-extracellular signal-regulated kinase (ERK) signaling cascade. Activation of this pathway is necessary to mediate the transforming potential of oncogenic BRAF, however, it may also cause a negative feedback that inhibits the epidermal growth factor receptor (EGFR). Mitogen-inducible gene-6 (MIG-6) is a potent inhibitor of the EGFR and has been demonstrated to function as a tumor suppressor. As MIG-6 can be induced via RAS-ERK signaling, we investigated its potential involvement in this negative regulatory loop. Focus formation assays were performed and demonstrated that MIG-6 significantly reduces malignant transformation induced by oncogenic BRAF. Although this genetic interaction was mirrored by a physical interaction between MIG-6 and BRAF, we did not observe a direct regulation of BRAF kinase activity by MIG-6. Interestingly, a selective chemical EGFR inhibitor suppressed transformation to a similar degree as MIG-6, whereas combining these approaches had no synergistic effect. By analyzing a range of BRAF mutated and wildtype cell line models, we could show that BRAF V600E causes a strong upregulation of MIG-6, which was mediated at the transcriptional level via the RAS-ERK pathway and resulted in downregulation of EGFR activation. This feedback loop is operational in tumors, as shown by the analysis of almost 400 patients with papillary thyroid cancer (PTC). Presence of BRAF V600E correlated with increased MIG-6 expression on the one hand, and with inactivation of the EGFR and of PI3K/AKT signaling on the other hand. Importantly, we also observed a more aggressive disease phenotype when BRAF V600E coexisted with low MIG-6 expression. Finally, analysis of methylation data was performed and revealed that higher methylation of MIG-6 correlated to its decreased expression. Taken together, we demonstrate that MIG-6 efficiently reduces cellular transformation driven by oncogenic BRAF by orchestrating a negative feedback circuit directed towards the EGFR.

Epigenetic silencing of miR-145-5p contributes to brain metastasis.

Brain metastasis is a major cause of morbidity and mortality of lung cancer patients. We assessed whether aberrant expression of specific microRNAs could contribute to brain metastasis. Comparison of primary lung tumors and their matched metastatic brain disseminations identified shared patterns of several microRNAs, including common down-regulation of miR-145-5p. Down-regulation was attributed to methylation of miR-145's promoter and affiliated elevation of several protein targets, such as EGFR, OCT-4, MUC-1, c-MYC and, interestingly, tumor protein D52 (TPD52). In line with these observations, restored expression of miR-145-5p and selective depletion of individual targets markedly reduced in vitro and in vivo cancer cell migration. In aggregate, our results attribute to miR-145-5p and its direct targets pivotal roles in malignancy progression and in metastasis.