Genetic alterations in the fibroblast growth factor receptor (FGFR) pathway are promising therapeutic targets in many cancers, including intrahepatic cholangiocarcinoma (ICC). The FGFR inhibitor BGJ398 displayed encouraging efficacy in patients with FGFR2 fusion-positive ICC in a phase II trial, but the durability of response was limited in some patients. Here, we report the molecular basis for acquired resistance to BGJ398 in three patients via integrative genomic characterization of cell-free circulating tumor DNA (cfDNA), primary tumors, and metastases. Serial analysis of cfDNA demonstrated multiple recurrent point mutations in the FGFR2 kinase domain at progression. Accordingly, biopsy of post-progression lesions and rapid autopsy revealed marked inter- and intra-lesional heterogeneity, with different FGFR2 mutations in individual resistant clones. Molecular modeling and in vitro studies indicated that each mutation lead to BGJ398 resistance and was surmountable by structurally distinct FGFR inhibitors. Thus, polyclonal secondary FGFR2 mutations represent an important clinical resistance mechanism that may guide development of future therapeutic strategies.

ATP-competitive fi broblast growth factor receptor (FGFR) kinase inhibitors, including BGJ398 and Debio 1347, show antitumor activity in patients with intrahepatic cholangiocarcinoma (ICC) harboring activating FGFR2 gene fusions. Unfortunately, acquired resistance develops and is often associated with the emergence of secondary FGFR2 kinase domain mutations. Here, we report that the irreversible pan-FGFR inhibitor TAS-120 demonstrated effi cacy in 4 patients with FGFR 2 fusion-positive ICC who developed resistance to BGJ398 or Debio 1347. Examination of serial biopsies, circulating tumor DNA (ctDNA), and patient-derived ICC cells revealed that TAS-120 was active against multiple FGFR2 mutations conferring resistance to BGJ398 or Debio 1347. Functional assessment and modeling the clonal outgrowth of individual resistance mutations from polyclonal cell pools mirrored the resistance profi les observed clinically for each inhibitor. Our fi ndings suggest that strategic sequencing of FGFR inhibitors, guided by serial biopsy and ctDNA analysis, may prolong the duration of benefi t from FGFR inhibition in patients with FGFR2 fusion-positive ICC. SIGNIFICANCE: ATP-competitive FGFR inhibitors (BGJ398, Debio 1347) show effi cacy in FGFR2-altered ICC; however, acquired FGFR2 kinase domain mutations cause drug resistance and tumor progression. We demonstrate that the irreversible FGFR inhibitor TAS-120 provides clinical benefi t in patients with resistance to BGJ398 or Debio 1347 and overcomes several FGFR2 mutations in ICC models.

Genetic alterations in the fibroblast growth factor receptor (FGFR) pathway are promising therapeutic targets in a broad range of cancers and occur in ~20% of ICCs. As seen with other targeted therapies, however, acquired resistance has limited the efficacy of selective FGFR kinase inhibitors such as BGJ398. In a phase II trial of patients with advanced refractory cholangiocarcinoma harboring an FGFR gene alteration, BGJ398 displayed an overall response rate of 22%, but the durability of response was short in some patients. We report the molecular basis of acquired resistance in 4 patients with advanced FGFR2-fusion positive ICC via integrative genomic characterization of cell-free circulating tumor DNA (cfDNA), the primary tumor, and metastases. Each patient enjoyed an initial response, but all subsequently progressed within 10 months. Serial analysis of cfDNA revealed multiple point mutations in the FGFR2 kinase domain at progression (Table 1). The gatekeeper mutation, p. V564F, sterically hinders drug binding and was identified in 3 of 4 patients. In patient #1, five different FGFR2 mutations were detected in the post-progression cfDNA but only one, p. K641R, was identified in the post-progression biopsy. A rapid autopsy was performed, and genomic characterization of 12 metastatic lesions revealed marked inter- and intra-lesional heterogeneity, with different FGFR2 mutations in individual resistant clones. Molecular modeling and in vitro studies indicated that each mutation lead to BGJ398 resistance that was surmountable by structurally distinct FGFR inhibitors. Thus, our report provides the first genetic evidence of clinical acquired resistance to FGFR inhibitor therapy in patients and informs future strategies for detecting mechanisms of resistance and promoting more durable remissions. Clinical and Molecular Data on Patients with Advanced Refractory CCA treated with BGJ398Patient IDSexAge at Diagnosis (years)FGFR2 Fusion partnerMaximum ResponseProgression Free Survival on BGJ398 (months)Overall Survival since Diagnosis (months)FGFR2 Mutations on cfDNA at progression1F47OPTN28%3.632V564F, N549H, K641R, E565A, L617V2F59ZMYMY450%5.631V564F, N549H/K, E565A, K659M3F69SORBS168%12.650K659M, K714R4M43BICC137%7.418V564F Note: This abstract was not presented at the meeting. Citation Format: Lipika Goyal, Supriya K. Saha, Leah Y. Liu, Giulia Siravegna, Ignaty Leshchiner, Leanne G. Ahronian, Jochen K. Lennerz, Phuong Vu, Benedetta Mussolin, Stephanie Reyes, Pascal Furet, A. John Iafrate, Gad Getz, Diana G. Porta, Ralph Tiedt, Alberto Bardelli, Dejan Juric, Ryan B. Corcoran, Nabeel Bardeesy, Andrew X. Zhu. Polyclonal secondary FGFR2 mutations drive acquired resistance to FGFR inhibition in FGFR2 fusion-positive cholangiocarcinoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4114. doi:10.1158/1538-7445.AM2017-4114

Endocannabinoid (EC) signaling mediates psychotropic effects and regulates appetite. By contrast, potential roles in organ development and embryonic energy consumption remain unknown. Here, we demonstrate that genetic or chemical inhibition of cannabinoid receptor (Cnr) activity disrupts liver development and metabolic function in zebrafish (Danio rerio), impacting hepatic differentiation, but not endodermal specification: loss of cannabinoid receptor 1 (cnr1) and cnr2 activity leads to smaller livers with fewer hepatocytes, reduced liver-specific gene expression and proliferation. Functional assays reveal abnormal biliary anatomy and lipid handling. Adult cnr2 mutants are susceptible to hepatic steatosis. Metabolomic analysis reveals reduced methionine content in Cnr mutants. Methionine supplementation rescues developmental and metabolic defects in Cnr mutant livers, suggesting a causal relationship between EC signaling, methionine deficiency and impaired liver development. The effect of Cnr on methionine metabolism is regulated by sterol regulatory element-binding transcription factors (Srebfs), as their overexpression rescues Cnr mutant liver phenotypes in a methioninedependent manner. Our work describes a novel developmental role for EC signaling, whereby Cnr-mediated regulation of Srebfs and methionine metabolism impacts liver development and function.

SUMMARYGenome-wide association studies (GWAS) have revealed numerous associations between many phenotypes and gene candidates. Frequently, however, further elucidation of gene function has not been achieved. A recent GWAS identified 69 candidate genes associated with elevated liver enzyme concentrations, which are clinical markers of liver disease. To investigate the role of these genes in liver homeostasis, we narrowed down this list to 12 genes based on zebrafish orthology, zebrafish liver expression and disease correlation. To assess the function of gene candidates during liver development, we assayed hepatic progenitors at 48 hours post fertilization (hpf) and hepatocytes at 72 hpf using in situ hybridization following morpholino knockdown in zebrafish embryos. Knockdown of three genes (pnpla3, pklr and mapk10) decreased expression of hepatic progenitor cells, whereas knockdown of eight genes (pnpla3, cpn1, trib1, fads2, slc2a2, pklr, mapk10 and samm50) decreased cell-specific hepatocyte expression. We then induced liver injury in zebrafish embryos using acetaminophen exposure and observed changes in liver toxicity incidence in morphants. Prioritization of GWAS candidates and morpholino knockdown expedites the study of newly identified genes impacting liver development and represents a feasible method for initial assessment of candidate genes to instruct further mechanistic analyses. Our analysis can be extended to GWAS for additional disease-associated phenotypes.

Heterochronic genes function to ensure the timing of stage-specific developmental events in C. elegans. Mutations in these genes cause certain developmental programs to be executed in a precocious or retarded manner. Canonical precocious (loss-of-function) and retarded (gain-of-function) mutations in the lin-14 gene lead to elimination or reiteration of larval stage-specific cellular events. Here, we describe a hypomorphic, missense allele of lin-14, sa485. lin-14(sa485) hermaphrodites pass through normal larval stages, but exhibit asynchrony between vulval and gonadal maturation in the L4 larval stage. We show that a subtly precocious morphogenetic event in the vulva disrupts tissue synchrony and is followed by retarded vulval eversion. Additionally, uterine uv1 cell differentiation is retarded in lin-14(sa485) animals that exhibit delayed vulval eversion. Together, these experiments outline a function for LIN-14 in coordinating the temporal progression of development, which is separable from its role in regulating stage-specific events during C. elegans postembryonic development. Developmental Dynamics 238:394 -404, 2009.

Fibroblast growth factor receptor (FGFR) inhibitors are currently in clinical trials in a variety of cancers including lung, breast, urothelial, gastric, and uterine cancers and intrahepatic cholangiocarcinoma (ICC). The selective and potent ATP-competitive FGFRi BGJ398 has shown a 19% response rate in advanced refractory FGFR2-fusion positive ICC, but acquired resistance rapidly limits its efficacy. We previously reported the emergence of polyclonal FGFR2 kinase domain mutations in patients with ICC progressing on BGJ398. TAS120, a covalently-binding FGFRi, demonstrates in vitro activity against wildtype FGFR2 and multiple FGFR2 mutations shown to confer resistance to BGJ398. Here we report the ability of TAS120 to overcome resistance to BGJ398 in the clinic. Three patients with FGFR2 fusion positive ICC treated on a phase II trial of BGJ398 experienced a partial response, progressed, and 2 of 3 subsequently had a PR on a phase I trial of TAS120 (NCT02052778) and one had stable disease for 7.3 months. Baseline and post-progression plasma samples and tissue biopsies were obtained on BGJ398, and sequencing results from post-progression cell-free DNA (Guardant360) and tissue (whole exome sequencing or FoundationOne) were compared to results from baseline samples (see Table 1). In Patient #1, three of 5 mutations detected upon progression on BGJ398 (i.e. FGFR2 K659M, E565A, and N549H) became undetectable on Guardant360 during treatment with TAS120, as did the only 2 FGFR2 mutations – K659M and K714R - that emerged on BGJ398 in Patient #2. In vitro studies validated TAS-120's ability to suppress the activity of FGFR2-fusion alleles carrying these secondary FGFR2 mutations. In summary, TAS120 demonstrates preliminary activity in overcoming resistance to prior FGFRi, and it is currently under investigation in a clinical trial in this setting. Table 1: Clinical benefit and emergence of FGFR resistance on BGJ398 with subsequent clinical benefit on TAS120IDFGFR2 Fusion detected in tissueBOR on BGJ398PFS on BGJ398New FGFR2 Mutations Identified in post-BGJ398 ctDNANew FGFR2 Mutations Identified on post-BGJ398 biopsyIntervening therapies and Duration between last day of BGJ398 and first day of TAS120BOR on TAS120Time on TAS1201FGFR2-ZMYM4-49.9%5.6 monthsV564F N549K/H E565A K659MV564F K659MNone, 48 days+8.3%7.3 months2FGFR2-SORBS1-68.2%12.4 monthsK659M K714RNoneNone, 36 days-76.7%15.2+ months, ongoing3FGFR2-NRAP-40.0%7.1 monthsNot performedNot performedPembrolizumab, FOLFOX, 225 days-45.9%8.3+ months, ongoingBOR=Best Overall Response; PFS = Progression Free Survival Citation Format: Lipika Goyal, Leah Y. Liu, Jochen K. Lennerz, James J. Harding, Jerry Huang, Robert Winkler, Hirai Hiroshi, David T. Ting, Dejan Juric, Ryan B. Corcoran, Nabeel El-Bardeesy, Andrew X. Zhu. TAS120, a covalently-binding FGFR inhibitor (FGFRi), overcomes resistance to BGJ398 in patients with FGFR2 fusion positive cholangiocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-092.

<div>Abstract<p>ATP-competitive fibroblast growth factor receptor (FGFR) kinase inhibitors, including BGJ398 and Debio 1347, show antitumor activity in patients with intrahepatic cholangiocarcinoma (ICC) harboring activating <i>FGFR2</i> gene fusions. Unfortunately, acquired resistance develops and is often associated with the emergence of secondary <i>FGFR2</i> kinase domain mutations. Here, we report that the irreversible pan-FGFR inhibitor TAS-120 demonstrated efficacy in 4 patients with <i>FGFR</i>2 fusion–positive ICC who developed resistance to BGJ398 or Debio 1347. Examination of serial biopsies, circulating tumor DNA (ctDNA), and patient-derived ICC cells revealed that TAS-120 was active against multiple FGFR2 mutations conferring resistance to BGJ398 or Debio 1347. Functional assessment and modeling the clonal outgrowth of individual resistance mutations from polyclonal cell pools mirrored the resistance profiles observed clinically for each inhibitor. Our findings suggest that strategic sequencing of FGFR inhibitors, guided by serial biopsy and ctDNA analysis, may prolong the duration of benefit from FGFR inhibition in patients with <i>FGFR2</i> fusion–positive ICC.</p>Significance:<p>ATP-competitive FGFR inhibitors (BGJ398, Debio 1347) show efficacy in <i>FGFR2</i>-altered ICC; however, acquired <i>FGFR2</i> kinase domain mutations cause drug resistance and tumor progression. We demonstrate that the irreversible FGFR inhibitor TAS-120 provides clinical benefit in patients with resistance to BGJ398 or Debio 1347 and overcomes several FGFR2 mutations in ICC models.</p><p><i>This article is highlighted in the In This Issue feature, p. 983</i></p></div>