Search

The incidence and biochemical consequences of rare tumor subtypes are often hard to study. Fibrolamellar liver cancer (FLC) is a rare malignancy affecting adolescents and young adults. To better characterize the incidence and biochemical consequences of this disease, we combined a comprehensive analysis of the electronic medical record and national payer data and found that FLC incidence is likely five to eight times higher than previous estimates. By employing unsupervised learning on clinical laboratory data from patients with hyperammonemia, we find that FLC-associated hyperammonemia mirrors metabolic dysregulation in urea cycle disorders. Our findings demonstrate that advanced computational analysis of rich clinical datasets can provide key clinical and biochemical insights into rare cancers.

FGFR inhibitors are approved for the treatment of advanced cholangiocarcinoma harboring FGFR2 fusions. However, the response rate is moderate, and resistance emerges rapidly due to acquired secondary FGFR2 mutations or due to other less-defined mechanisms. Here, we conducted high-throughput combination drug screens, biochemical analysis, and therapeutic studies using patient-derived models of FGFR2 fusion-positive cholangiocarcinoma to gain insight into these clinical profiles and uncover improved treatment strategies. We found that feedback activation of EGFR signaling limits FGFR inhibitor efficacy, restricting cell death induction in sensitive models and causing resistance in insensitive models lacking secondary FGFR2 mutations. Inhibition of wild-type EGFR potentiated responses to FGFR inhibitors in both contexts, durably suppressing MEK/ERK and mTOR signaling, increasing apoptosis, and causing marked tumor regressions in vivo. Our findings reveal EGFR-dependent adaptive signaling as an important mechanism limiting FGFR inhibitor efficacy and driving resistance and support clinical testing of FGFR/EGFR inhibitor therapy for FGFR2 fusion-positive cholangiocarcinoma.SignificanceWe demonstrate that feedback activation of EGFR signaling limits the effectiveness of FGFR inhibitor therapy and drives adaptive resistance in patient-derived models of FGFR2 fusion-positive cholangiocarcinoma. These studies support the potential of combination treatment with FGFR and EGFR inhibitors as an improved treatment for patients with FGFR2-driven cholangiocarcinoma. This article is highlighted in the In This Issue feature, p. 1171.

The androgen receptor (AR) is a central driver of aggressive prostate cancer. After initial treatment with androgen receptor signaling inhibitors (ARSi), reactivation of AR signaling leads to resistance. Alternative splicing of AR mRNA yields the AR-V7 splice variant, which is currently an undruggable mechanism of ARSi resistance: AR-V7 lacks a ligand binding domain, where hormones and anti-androgen antagonists act, but still activates AR signaling. We reveal PKCβ as a druggable regulator of transcription and splicing at the AR genomic locus. We identify a clinical PKCβ inhibitor in combination with an FDA-approved anti-androgen as an approach for repressing AR genomic locus expression, including expression of AR-V7, while antagonizing full-length AR. PKCβ inhibition reduces total AR gene expression, thus reducing AR-V7 protein levels and sensitizing prostate cancer cells to current anti-androgen therapies. We demonstrate that this combination may be a viable therapeutic strategy for AR-V7-positive prostate cancer.

Cancers have been associated with a diverse array of genomic alterations. To help mechanistically understand such alterations in breast-invasive carcinoma, we applied affinity purification–mass spectrometry to delineate comprehensive biophysical interaction networks for 40 frequently altered breast cancer (BC) proteins, with and without relevant mutations, across three human breast cell lines. These networks identify cancer-specific protein-protein interactions (PPIs), interconnected and enriched for common and rare cancer mutations, that are substantially rewired by the introduction of key BC mutations. Our analysis identified BPIFA1 and SCGB2A1 as PIK3CA-interacting proteins, which repress PI3K-AKT signaling, and uncovered USP28 and UBE2N as functionally relevant interactors of BRCA1. We also show that the protein phosphatase 1 regulatory subunit spinophilin interacts with and regulates dephosphorylation of BRCA1 to promote DNA double-strand break repair. Thus, PPI landscapes provide a powerful framework for mechanistically interpreting disease genomic data and can identify valuable therapeutic targets.

Using unbiased kinase profiling, we identified protein kinase A (PKA) as an active kinase in small cell lung cancer (SCLC). Inhibition of PKA activity genetically, or pharmacologically by activation of the PP2A phosphatase, suppresses SCLC expansion in culture and in vivo. Conversely, GNAS (G-protein α subunit), a PKA activator that is genetically activated in a small subset of human SCLC, promotes SCLC development. Phosphoproteomic analyses identified many PKA substrates and mechanisms of action. In particular, PKA activity is required for the propagation of SCLC stem cells in transplantation studies. Broad proteomic analysis of recalcitrant cancers has the potential to uncover targetable signaling networks, such as the GNAS/PKA/PP2A axis in SCLC.

http://aasldpubs.onlinelibrary.wiley.com/hub/journal/10.1002/(ISSN)2046-2484/video/15-2-reading-huppert a video presentation of this article Answer questions and earn https://www.wileyhealthlearning.com/Activity/7036144/disclaimerspopup.aspx.

Small nucleolar RNAs (snoRNAs) are a diverse group of non-coding RNAs that direct chemical modifications at specific residues on other RNA molecules, primarily on ribosomal RNA (rRNA). SnoRNAs are altered in several cancers; however, their role in cell homeostasis as well as in cellular transformation remains poorly explored. Here, we show that specific subsets of snoRNAs are differentially regulated during the earliest cellular response to oncogenic RASG12V expression. We describe a novel function for one H/ACA snoRNA, SNORA24, which guides two pseudouridine modifications within the small ribosomal subunit, in RAS-induced senescence in vivo. We find that in mouse models, loss of Snora24 cooperates with RASG12V to promote the development of liver cancer that closely resembles human steatohepatitic hepatocellular carcinoma (HCC). From a clinical perspective, we further show that human HCCs with low SNORA24 expression display increased lipid content and are associated with poor patient survival. We next asked whether ribosomes lacking SNORA24-guided pseudouridine modifications on 18S rRNA have alterations in their biophysical properties. Single-molecule Fluorescence Resonance Energy Transfer (FRET) analyses revealed that these ribosomes exhibit perturbations in aminoacyl-transfer RNA (aa-tRNA) selection and altered pre-translocation ribosome complex dynamics. Furthermore, we find that HCC cells lacking SNORA24-guided pseudouridine modifications have increased translational miscoding and stop codon readthrough frequencies. These findings highlight a role for specific snoRNAs in safeguarding against oncogenic insult and demonstrate a functional link between H/ACA snoRNAs regulated by RAS and the biophysical properties of ribosomes in cancer.

Inhibitors targeting KRASG12C, a mutant form of the guanosine triphosphatase (GTPase) KRAS, are a promising new class of oncogene-specific therapeutics for the treatment of tumors driven by the mutant protein. These inhibitors react with the mutant cysteine residue by binding covalently to the switch-II pocket (S-IIP) that is present only in the inactive guanosine diphosphate (GDP)-bound form of KRASG12C, sparing the wild-type protein. We used a genome-scale CRISPR interference (CRISPRi) functional genomics platform to systematically identify genetic interactions with a KRASG12C inhibitor in cellular models of KRASG12C mutant lung and pancreatic cancer. Our data revealed genes that were selectively essential in this oncogenic driver-limited cell state, meaning that their loss enhanced cellular susceptibility to direct KRASG12C inhibition. We termed such genes "collateral dependencies" (CDs) and identified two classes of combination therapies targeting these CDs that increased KRASG12C target engagement or blocked residual survival pathways in cells and in vivo. From our findings, we propose a framework for assessing genetic dependencies induced by oncogene inhibition.

Ligand-dependent oligomerization of receptor tyrosine kinases (RTKs) results in their activation through highly specific conformational changes in the extracellular and intracellular receptor domains. These conformational changes are unique for each RTK subfamily, limiting cross-activation between unrelated RTKs. The proto-oncogene MET receptor tyrosine kinase overcomes these structural constraints and phosphorylates unrelated RTKs in numerous cancer cell lines. The molecular basis for these interactions is unknown. We investigated the mechanism by which MET phosphorylates the human epidermal growth factor receptor-3 (HER3 or ERBB3), a catalytically impaired RTK whose phosphorylation by MET has been described as an essential component of drug resistance to inhibitors targeting EGFR and HER2. We find that in untransformed cells, HER3 is not phosphorylated by MET in response to ligand stimulation, but rather to increasing levels of MET expression, which results in ligand-independent MET activation. Phosphorylation of HER3 by its canonical co-receptors, EGFR and HER2, is achieved by engaging an allosteric site on the HER3 kinase domain, but this site is not required when HER3 is phosphorylated by MET. We also observe that HER3 preferentially interacts with MET during its maturation along the secretory pathway, before MET is post translationally processed by cleavage within its extracellular domain. This results in accumulation of phosphorylated HER3 in the Golgi apparatus. We further show that in addition to HER3, MET phosphorylates other RTKs in the Golgi, suggesting that this mechanism is not limited to HER3 phosphorylation. These data demonstrate a link between MET overexpression and its aberrant activation in the Golgi endomembranes and suggest that non-canonical interactions between MET and other RTKs occur during maturation of receptors. Our study highlights a novel aspect of MET signaling in cancer that would not be accessible to inhibition by therapeutic antibodies.

Cancer cells develop mechanisms to escape immunosurveillance, among which modulating the expression of immune suppressive messenger RNAs is most well-documented. However, how this is molecularly achieved remains largely unresolved. Here, we develop an in vivo mouse model of liver cancer to study oncogene cooperation in immunosurveillance. We show that MYC overexpression (MYCTg) synergizes with KRASG12D to induce an aggressive liver tumor leading to metastasis formation and reduced mouse survival compared with KRASG12D alone. Genome-wide ribosomal footprinting of MYCTg;KRASG12 tumors compared with KRASG12D revealed potential alterations in translation of mRNAs, including programmed-death-ligand 1 (PD-L1). Further analysis revealed that PD-L1 translation is repressed in KRASG12D tumors by functional, non-canonical upstream open reading frames in its 5' untranslated region, which is bypassed in MYCTg;KRASG12D tumors to evade immune attack. We show that this mechanism of PD-L1 translational upregulation was effectively targeted by a potent, clinical compound that inhibits eIF4E phosphorylation, eFT508, which reverses the aggressive and metastatic characteristics of MYCTg;KRASG12D tumors. Together, these studies reveal how immune-checkpoint proteins are manipulated by distinct oncogenes at the level of mRNA translation, which can be exploited for new immunotherapies.

PurposeIntrahepatic cholangiocarcinoma (ICC) is an aggressive cancer type, lacking effective therapies and associated with a dismal prognosis. Palbociclib is a selective CDK4/6 inhibitor, which has been shown to suppress cell proliferation in many experimental cancer models. Recently, we demonstrated that pan-mTOR inhibitors, such as MLN0128, effectively induce apoptosis, although have limited efficacy in restraining proliferation of ICC cells. Here, we tested the hypothesis that palbociclib, due to its antproliferative properties in many cancer types, might synergize with MLN0128 to impair ICC growth.Experimental designHuman ICC cell lines and the AKT/YapS127A ICC mouse model were used to test the therapeutic efficacy of palbociclib and MLN0128, either alone or in combination.ResultsAdministration of palbociclib suppressed in vitro ICC cell growth by inhibiting cell-cycle progression. Concomitant administration of palbociclib and MLN0128 led to a pronounced, synergistic growth constraint of ICC cell lines. Furthermore, while treatment with palbociclib or MLN0128 alone resulted in tumor growth reduction in AKT/YapS127A mice, a remarkable tumor regression was achieved when the two drugs were administered simultaneously. Mechanistically, palbociclib was found to potentiate MLN0128 mTOR inhibition activity, whereas MLN0128 prevented the upregulation of cyclin D1 induced by palbociclib treatment.ConclusionsOur study indicates the synergistic activity of palbociclib and MLN0128 in inhibiting ICC cell proliferation. Thus, combination of CDK4/6 and mTOR inhibitors might represent a novel, promising, and effective therapeutic approach against human ICC.See related commentary by Malumbres, p. 6.

Biliary tract cancers such as cholangiocarcinoma represent a heterogeneous group of cancers that can be difficult to diagnose. Recent comprehensive genomic analyses in large cholangiocarcinoma cohorts have defined important molecular subgroups within cholangiocarcinoma that may relate to anatomic location and etiology [1], [2], [3], [4] and may predict responsiveness to targeted therapies in development [5], [6], [7]. These emerging data highlight the potential for tumor genomics to inform diagnosis and treatment options in this challenging tumor type. We report the case of a patient with a germline BRCA1 mutation who presented with a cholangiocarcinoma driven by the novel YWHAZ-BRAF fusion. Hybrid capture-based DNA sequencing and copy number analysis performed as part of clinical care demonstrated that two later-occurring tumors were clonally derived from the primary cholangiocarcinoma rather than distinct new primaries, revealing an unusual pattern of late metachronous metastasis. We discuss the clinical significance of these genetic alterations and their relevance to therapeutic strategies.Key pointsHybrid capture-based next-generation DNA sequencing assays can provide diagnostic clarity in patients with unusual patterns of metastasis and recurrence in which the pathologic diagnosis is ambiguous.To our knowledge, this is the first reported case of a YWHAZ-BRAF fusion in pancreaticobiliary cancer, and a very rare case of cholangiocarcinoma in the setting of a germline BRCA1 mutation.The patient's BRCA1 mutation and YWHAZ-BRAF fusion constitute potential targets for future therapy.

Dysregulation of the PI3K-AKT-mTOR signaling network is a prominent feature of breast cancers. However, clinical responses to drugs targeting this pathway have been modest, possibly because of dynamic changes in cellular signaling that drive resistance and limit drug efficacy. Using a quantitative chemoproteomics approach, we mapped kinome dynamics in response to inhibitors of this pathway and identified signaling changes that correlate with drug sensitivity. Maintenance of AURKA after drug treatment was associated with resistance in breast cancer models. Incomplete inhibition of AURKA was a common source of therapy failure, and combinations of PI3K, AKT or mTOR inhibitors with the AURKA inhibitor MLN8237 were highly synergistic and durably suppressed mTOR signaling, resulting in apoptosis and tumor regression in vivo. This signaling map identifies survival factors whose presence limits the efficacy of targeted therapies and reveals new drug combinations that may unlock the full potential of PI3K-AKT-mTOR pathway inhibitors in breast cancer.

Advances in single-cell technologies have highlighted the prevalence and biological significance of cellular heterogeneity. A critical question researchers face is how to design experiments that faithfully capture the true range of heterogeneity from samples of cellular populations. Here we develop a data-driven approach, illustrated in the context of image data, that estimates the sampling depth required for prospective investigations of single-cell heterogeneity from an existing collection of samples.

Protein kinase A (PKA) is a basophilic kinase implicated in the modulation of many cellsignaling and physiological processes. PKA also contributes to cancer-relevant events such as growth factor action, cell cycle control, cell migration and tumor metabolism. Germline and somatic mutations in PKA, gene amplifications, and chromosome rearrangements that encode kinase fusions, are linked to a growing number of malignant neoplasms. Mislocalization of PKA by exclusion from A-Kinase Anchoring Protein (AKAP) signaling islands further underlies cancer progression. This article highlights the influence of AKAP signaling and local kinase action in selected hallmarks of cancer. We also feature the utility of kinase inhibitor drugs as frontline and future anti-cancer therapies. [ABSTRACT FROM AUTHOR]

Background: Early changes in alpha-fetoprotein (AFP) are a promising surrogate endpoint for systemic treatment outcomes in hepatocellular carcinoma (HCC). Objectives: We sought to investigate the utility of AFP response across first-line sorafenib (1L SOR) and later-line checkpoint inhibitor (CPI) therapies. Design: We conducted a multicenter, retrospective cohort study of patients with advanced HCC who received 1L SOR and any subsequent CPI. Methods: The primary outcomes were overall survival (OS) and time on treatment (TOT). Pre-treatment AFP and the lowest AFP within 3 months of treatment initiation were used to calculate the percent change in AFP for each treatment. AFP response was defined as an AFP reduction by ⩾20% within 3 months, and AFP progression was defined as an increase in AFP by ⩾20% within 3 months. Patients with baseline AFP < 20 ng/mL were considered not evaluable for AFP change. Results: Of 176 study patients, 46 (28%) received CPI after SOR, and 125 (71%) had a baseline AFP ⩾ 20. Patients who experienced AFP response on SOR had significantly longer OS and TOT than those who did not and those who were not evaluable (OS: median 689 vs 320 vs 452 days, log-rank p < 0.001; TOT: median log of days 5.2 vs 4.5 vs 4.9, p < 0.001). Patients with AFP progression following SOR had significantly shorter OS than those who did not and those who were not evaluable (median 304 vs 557 vs 452, log-rank p = 0.008). Similarly, patients with AFP response following CPI therapy had a significantly reduced risk of death compared with those who did not have an AFP response (hazard ratio 0.13, 95% confidence interval 0.03–0.60, p = 0.009). Conclusion: Early AFP response with 1L SOR and any subsequent CPI was associated with longer OS and TOT, and early AFP progression was associated with shorter OS and TOT. These data support utilizing longitudinal AFP changes as a surrogate endpoint in HCC systemic therapy. [ABSTRACT FROM AUTHOR]

Reliable quantitation of protein abundances in defined sets of cellular proteins is critical to numerous biological applications. Traditional immunodetection-based methods are limited by the quality and availability of specific antibodies, especially for site-specific post-translational modifications. Targeted proteomic methods, including the recently developed parallel reaction monitoring (PRM) mass spectrometry, have enabled accurate quantitative measurements of up to a few hundred specific target peptides. However, the degree of practical multiplexing in label-free PRM workflows remains a significant limitation for the technique. Here we present a strategy for significantly increasing multiplexing in label-free PRM that takes advantage of the superior separation characteristics and retention time stability of meter-scale monolithic silica-C18 column-based chromatography. We show the utility of the approach in quantifying kinase abundances downstream of previously developed active kinase enrichment methodology based on multidrug inhibitor beads. We examine kinase activation dynamics in response to three different MAP kinase inhibitors in colorectal carcinoma cells and demonstrate reliable quantitation of over 800 target peptides from over 150 kinases in a single label-free PRM run. The kinase activity profiles obtained from these analyses reveal compensatory activation of TGF-β family receptors as a response to MAPK blockade. The gains achieved using this label-free PRM multiplexing strategy will benefit a wide array of biological applications.

UnlabelledIntrahepatic cholangiocarcinoma (ICC) is an aggressive liver bile duct malignancy exhibiting frequent isocitrate dehydrogenase (IDH1/IDH2) mutations. Through a high-throughput drug screen of a large panel of cancer cell lines, including 17 biliary tract cancers, we found that IDH mutant (IDHm) ICC cells demonstrate a striking response to the multikinase inhibitor dasatinib, with the highest sensitivity among 682 solid tumor cell lines. Using unbiased proteomics to capture the activated kinome and CRISPR/Cas9-based genome editing to introduce dasatinib-resistant "gatekeeper" mutant kinases, we identified SRC as a critical dasatinib target in IDHm ICC. Importantly, dasatinib-treated IDHm xenografts exhibited pronounced apoptosis and tumor regression. Our results show that IDHm ICC cells have a unique dependency on SRC and suggest that dasatinib may have therapeutic benefit against IDHm ICC. Moreover, these proteomic and genome-editing strategies provide a systematic and broadly applicable approach to define targets of kinase inhibitors underlying drug responsiveness.SignificanceIDH mutations define a distinct subtype of ICC, a malignancy that is largely refractory to current therapies. Our work demonstrates that IDHm ICC cells are hypersensitive to dasatinib and critically dependent on SRC activity for survival and proliferation, pointing to new therapeutic strategies against these cancers. Cancer Discov; 6(7); 727-39. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 681.

UnlabelledThere is an urgent need in oncology to link molecular aberrations in tumors with therapeutics that can be administered in a personalized fashion. One approach identifies synthetic-lethal genetic interactions or dependencies that cancer cells acquire in the presence of specific mutations. Using engineered isogenic cells, we generated a systematic and quantitative chemical-genetic interaction map that charts the influence of 51 aberrant cancer genes on 90 drug responses. The dataset strongly predicts drug responses found in cancer cell line collections, indicating that isogenic cells can model complex cellular contexts. Applying this dataset to triple-negative breast cancer, we report clinically actionable interactions with the MYC oncogene, including resistance to AKT-PI3K pathway inhibitors and an unexpected sensitivity to dasatinib through LYN inhibition in a synthetic lethal manner, providing new drug and biomarker pairs for clinical investigation. This scalable approach enables the prediction of drug responses from patient data and can accelerate the development of new genotype-directed therapies.SignificanceDetermining how the plethora of genomic abnormalities that exist within a given tumor cell affects drug responses remains a major challenge in oncology. Here, we develop a new mapping approach to connect cancer genotypes to drug responses using engineered isogenic cell lines and demonstrate how the resulting dataset can guide clinical interrogation.

Despite the development of potent RAF/mitogen-activated protein kinase (MAPK) pathway inhibitors, only a fraction of BRAF-mutant patients benefit from treatment with these drugs. Using a combined chemogenomics and chemoproteomics approach, we identify drug-induced RAS-RAF-MEK complex formation in a subset of BRAF-mutant cancer cells characterized by primary resistance to vemurafenib. In these cells, autocrine interleukin-6 (IL-6) secretion may contribute to the primary resistance phenotype via induction of JAK/STAT3 and MAPK signaling. In a subset of cell lines, combined IL-6/MAPK inhibition is able to overcome primary resistance to BRAF-targeted therapy. Overall, we show that the signaling plasticity exerted by primary resistant BRAF-mutant cells is achieved by their ability to mimic signaling features of oncogenic RAS, a strategy that we term "oncogene mimicry." This model may guide future strategies for overcoming primary resistance observed in these tumors.

Fibrolamellar carcinoma (FLC) is a rare liver cancer that disproportionately affects adolescents and young adults. Currently, no standard of care is available and there remains a dire need for new therapeutics. Most patients harbor the fusion oncogene DNAJB1-PRKACA (DP fusion), but clinical inhibitors are not yet developed and it is critical to identify downstream mediators of FLC pathogenesis. Here, we identify long noncoding RNA LINC00473 among the most highly upregulated genes in FLC tumors and determine that it is strongly suppressed by RNAi-mediated inhibition of the DP fusion in FLC tumor epithelial cells. We show by loss- and gain-of-function studies that LINC00473 suppresses apoptosis, increases the expression of FLC marker genes, and promotes FLC growth in cell-based and in vivo disease models. Mechanistically, LINC00473 plays an important role in promoting glycolysis and altering mitochondrial activity. Specifically, LINC00473 knockdown leads to increased spare respiratory capacity, which indicates mitochondrial fitness. Overall, we propose that LINC00473 could be a viable target for this devastating disease. Schematic was created with BioRender.com. Author summary: Fibrolamellar carcinoma (FLC) is a lethal liver cancer that is characterized by the DNAJB1-PRKACA (DP fusion) oncogene. This disease is particularly challenging to treat as it lacks non-invasive diagnostic biomarkers, known risk factors, and effective therapeutic options. Patients with FLC have a median age of 22 years and the prognosis is poor. In this study, we examined FLC tumors from independent patient cohorts and multiple disease models and found consistently elevated levels of a primate-specific long noncoding RNA named LINC00473. By leveraging single-cell analysis on patient tumors, we found that LINC00473 is enriched in tumor epithelial cells. Using multiple FLC models, we show that the DP fusion drives LINC00473 expression and that LINC00473 promotes FLC growth by mitigating cell death. We also show that LINC00473 modulates FLC energetics by increasing glycolysis and altering mitochondrial fitness. Together, this study unveils an important mechanism downstream of the signature DP fusion oncogene in FLC pathogenesis. [ABSTRACT FROM AUTHOR]