Hepatocellular Carcinoma Xenograft Model Research Articles

High spatiotemporal heterogeneity, clonal selection and neoantigen evolution in acquired sorafenib-resistant patient-derived xenograft models of hepatocellular carcinoma.

e15641 Background: Patient-derived xenograft (PDX) models have been regarded as valuable preclinical models for oncology drug development and exploring underlying mechanism of drug resistance. As the standard treatment option for advanced hepatocellular carcinoma (HCC), the survival benefit of sorafenib is modest. Even among those who initially responded well to sorafenib treatment, most patients ultimately develop progressive disease owing to acquired drug resistance. Understanding how selective pressure from sorafenib directs the evolution of HCC and shapes its clonal architecture is a central biological question with important clinical implications to combat acquired drug resistance. Methods: Three sorafenib-resistant PDX models were generated by continuous sorafenib treament for more than six months. Whole exome sequencing (WES) and RNA sequencing were performed. The subclone of mutation, fusion genes, and the evolving landscape of tumor neoantigens were further explored. Finally, the antitumor efficacy of recombinant mouse PD-1 antibody was evaluated in sorafenib resistance syngeneic tumors model by immune-competent C57BL/6J mice with Hepa 1-6. Results: High spatiotemporal heterogeneity was observed in engraftment among pre- and post- sorafenib resistance in established acquired-resistance PDX models. Clonal selection on engraftment was observed in established acquired-resistance PDX models. There are many high expressed genes, which showed subclone expansion with the emergence of sorafenib resistance. There is vary difference of neoantigens between pre- and post-resistance engraftment as well as sampling points in same tissues. It was presented that a high burden of clonal neoantigens in sorafenib resistant tissues (P < 0.05), while there has no difference in tumor mutation burden. Moreover, the intra-tumor heterogeneity upon the neoantigen landscape was much bigger than that upon tumor mutation burden. We found that fusion subclone sizes were increased significantly after sorafenib resistance (P < 0.05). Finally, the antitumor efficacy of recombinant mouse PD-1 antibody was confirmed in sorafenib resistance syngeneic tumors mice model (P < 0.001). Conclusions: High spatiotemporal heterogeneity, clonal selection and neoantigen evolution was observed in acquired sorafenib-resistant PDX model of HCC. Immunotherapy such as immune checkpoint inhibitors might be a promising strategy for HCC patients with acquired sorafenib resistance. .

Antitumor and Antiangiogenic Activities of Lenvatinib in Mouse Xenograft Models of Vascular Endothelial Growth Factor-Induced Hypervascular Human Hepatocellular Carcinoma

High expression of vascular endothelial growth factor (VEGF) in patients with hepatocellular carcinoma (HCC) is associated with poor prognosis. Here, we investigated the antitumor activity of lenvatinib, a multiple receptor tyrosine kinase inhibitor, in VEGF-overexpressing HCC models. In human umbilical vein endothelial cells, lenvatinib showed potent inhibitory activities against VEGF-induced proliferation and VEGF/basic fibroblast growth factor-induced tube formation. In VEGF-overexpressing HCC xenograft models, characterized by aggressive tumor growth and hypervascularity, lenvatinib had significant antitumor and antiangiogenic activities. These results suggest that potent activity of lenvatinib against VEGF signaling underlies its antitumor and antiangiogenic activities in the hypervascular HCC models.

KIAA1199 promotes sorafenib tolerance and the metastasis of hepatocellular carcinoma by activating the EGF/EGFR-dependent epithelial-mesenchymal transition program.

Patients with advanced hepatocellular carcinoma (HCC) will almost always develop acquired tolerance after sorafenib therapy, and the molecular mechanism of sorafenib tolerance remains poorly characterized. Here, using our established sorafenib-resistant HCC cell and xenograft models, we identified a novel gene, KIAA1199, which was markedly elevated among the differentially expressed genes involved in sorafenib tolerance. Moreover, elevated expression of KIAA1199 was positively correlated with a high risk of recurrence and metastasis and advanced TNM stage in HCC patients. Functionally, loss- and gain-of-function studies showed that KIAA1199 promoted the migration, invasion, and metastasis of sorafenib-resistant HCC cells. Mechanistically, KIAA1199 is required for EGF-induced epithelial-mesenchymal transition (EMT) in sorafenib-resistant HCC cells by aiding in EGFR phosphorylation. In summary, our data uncover KIAA1199 as a novel sorafenib-tolerant promoting gene that plays an indispensable role in maintaining sorafenib-resistant HCC cell metastasis.

Tumor-associated macrophages modulate resistance to oxaliplatin via inducing autophagy in hepatocellular carcinoma

BackgroundOxaliplatin-based chemotherapy is widely used to treat hepatocellular carcinoma (HCC). Recent studies suggested that therapeutic resistance of tumors was affected by tumor microenvironment (TME). As a major component of TME, the role of tumor-associated macrophages (TAMs) on drug resistance in HCC is largely unknown.Methods26 HCC samples were obtained from patients who had underwent transarterial chemoembolization (TACE) within 3 months before receiving curative resections. Immunohistochemistry was applied to detect the density of TAMs in these tissues. SMMC-7721 and Huh-7 cell lines were used to co-culture with THP-1 derived macrophages. Under oxaliplatin treatment, cell death was measured using MTT and annexin V/propidium iodide assays. Autophagy activation was evaluated by GFP-LC3 redistribution and LC3 conversion in SMMC-7721 and Huh-7. Short-interfering RNA against ATG5 gene was applied to inhibit autophagy. In vivo validation was conducted in Huh-7 with or without macrophages using an HCC xenograft model in nude mice after oxaliplatin administration.ResultsWe found that the density of TAMs in HCC samples was associated with the efficacy of TACE. Macrophages inhibited cell death induced by oxaliplatin in HCC cells. Autophagy was functionally activated in HCC cells after co-culturing with macrophages. Suppression of autophagy using RNA interference of ATG5 in HCC cells promoted the oxaliplatin cytotoxicity in the co-culture system. Critically, co-implantation with macrophages in HCC xenografts weakens cytotoxic effect of oxaliplatin through inducing autophagy to avoid apoptosis.ConclusionsOur results suggest that TAMs induce autophagy in HCC cells which might contribute to oxaliplatin resistance. Targeting TAMs is a promising therapeutic strategy to enhance the effects of chemotherapy oxaliplatin in HCC patients.

Evaluation of a locked nucleic acid form of antisense oligo targeting HIF-1α in advanced hepatocellular carcinoma.

BACKGROUNDHypoxia-inducible factor 1α (HIF-1α) is a gene that regulates tumor survival, neovascularization and invasion. Overexpression of HIF-1α correlates with poor prognosis in hepatocellular carcinoma (HCC). RO7070179 is a HIF-1α inhibitor that decreases HIF-1α mRNA and its downstream targets, it could be a potential treatment in HCC.AIMTo evaluate safety and preliminary activity of RO7070179 in patients with previously treated HCC, with focus on a patient with prolonged response to RO7070179.METHODSIn the preclinical study of RO7070179 in a HCC xenograft model, the mice were separated into 4 groups with each group received doses of 0, 3, 10 and 30 mg/kg for total 10 doses. HCC patients who failed at least one line of systemic treatment, received RO7070179 as a weekly infusion, each cycle is 6 wk. We evaluated the safety and HIF-1α mRNA levels of RO7070179.RESULTSPreclinical evaluation of RO7070179 in orthotopic HCC xenograft model showed no significant differences in HCC tumor weight between the 3 and 10 mg/kg groups. However, dose of 10 mg/kg of RO7070179, has shown 76% reduction of the amount of HIF-1α mRNA in HCC tissue. In the phase 1b study of RO7070179 in previously treated HCC patients, 8 out of 9 were evaluable: 1 achieved PR and 1 SD. The patient with PR responded after 2 cycles treatments, which has been maintained for 12 cycles. This patient also showed reduction in perfusion of dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) after 1 cycle of treatment. After 1 cycle of treatment, both patients with PR and SD showed decrease in HIF-1α mRNA at the root of biopsies (each biopsy was divided into 2 specimens, the tip and the root).CONCLUSIONRO7070179 can reduce HIF-1α mRNA level in HCC patients with SD or PR. It is well tolerated at 10 mg/kg, with transaminitis as the dose of increased toxicity. This study indicates that RO7070179 might benefit HCC patients, and an early signal for clinical benefit can potentially be predicted through changes in either mRNA level or DCE-MRI within 1 cycle of therapy.

DEAD Box Protein 5 Inhibits Liver Tumorigenesis by Stimulating Autophagy via Interaction with p62/SQSTM1.

In hepatocellular carcinoma (HCC), dysregulated expression of DDX5 (DEAD box protein 5) and impaired autophagy have been reported separately. However, the relationship between them has not been explored. Here we present evidence to show that, by interacting with autophagic receptor p62, DDX5 promotes autophagy and suppresses tumorigenesis. DDX5 inversely correlated with p62/sequestosome 1 (SQSTM1) expression in hepatitis B virus (HBV)-associated and non-HBV-associated HCCs. Patients with low DDX5 expression showed poor prognosis after tumor resection. We found that DDX5 overexpression induced, while DDX5 knockdown attenuated, autophagic flux in HepG2 and Huh7 cells. DDX5 promoted p62 degradation and markedly reduced the half-life of p62. Moreover, DDX5 overexpression dramatically reduced, while DDX5 knockdown promoted, cancer cell growth and tumorigenesis in vitro and in vivo. We found that DDX5 bound to p62 and interfered with p62/TRAF6 (tumor necrosis factor receptor-associated factor 6) interaction. Further findings revealed that the N-terminal domain of DDX5, involved in the interaction with p62, was sufficient to induce autophagy independent of its RNA binding and helicase activity. DDX5 overexpression decreased p62/TRAF6-mediated lysine 63-linked ubiquitination of mammalian target of rapamycin (mTOR) and subsequently inhibited the mTOR signaling pathway. Knockdown of TRAF6 blocked DDX5-induced autophagy. Furthermore, we showed that miR-17-5p downregulated DDX5 and impaired autophagy. Inhibition of miR-17-5p promoted autophagic flux and suppressed tumor growth in HCC xenograft models. Conclusion: Our findings define a noncanonical pathway that links miR-17-5p, DDX5, p62/TRAF6, autophagy, and HCC. These findings open an avenue for the treatment of HCC.

Sorafenib/MEK inhibitor combination inhibits tumor growth and the Wnt/β‑catenin pathway in xenograft models of hepatocellular carcinoma.

Mutations affecting the Wnt/β‑catenin pathway have been identified in 26‑40% of hepatocellular carcinoma (HCC) cases. Aberrant activation of this pathway leads to uncontrolled cell proliferation and survival. Thus, identifying Wnt/β‑catenin pathway inhibitors may benefit a subset of patients with HCC. In the present study, the effects of sorafenib and a MEK inhibitor on tumor growth and Wnt/β‑catenin signaling in HCC models were evaluated. A β‑catenin mutant and β‑catenin wild‑type HCC models were treated once daily with i)10mg/kg sorafenib, ii)15mg/kg refametinib (or 25mg/kg selumetinib), or iii)sorafenib/refametinib. Western blotting was employed to determine changes in biomarkers relevant to Wnt/β‑catenin signaling. Apoptosis, cell proliferation and β‑catenin localization were analyzed by immunohistochemistry. Sorafenib/refametinib markedly inhibited tumor growth and cell proliferation, and caused cell death in naïve and sorafenib‑resistant HCC models. Despite similar total β‑catenin levels, significant reductions in phosphorylated (p)‑RanBP3 Ser58, p‑β‑catenin Tyr142, active β‑catenin and β‑catenin target genes were observed in sorafenib/refametinib‑treated tumors. Greater levels of β‑catenin in sorafenib/refametinib‑treated tumors were accumulated at the membrane, as compared with in the control. Invitro, sorafenib/refametinib inhibited the Wnt/β‑catenin pathway and suppressed Wnt‑3A‑induced p‑low‑density lipoprotein receptor‑related protein6 Ser1490, p‑RanBP3 Ser58 and p‑β‑catenin Tyr142 in HCC cells. Combination of sorafenib and refametinib inhibits the growth of naïve and sorafenib resistant HCC tumors in association with active suppression of β‑catenin signaling regardless of β‑catenin mutational status. Thus, the sorafenib/MEK inhibitor combination may represent an alternative treatment for patients with HCC whose tumors develop resistance to sorafenib therapy.

Developed operation method for patient-drived orthotopic xenograft model of hepatocellular carcinoma.

Background: Patient-derived xenograft(PDX) model is a biological organized platform and a patient substitute. Recent studies use the ectopic model for hepatocellular carcinoma(HCC) but this model don’t reflect likewise the tumor state of patient. For exact prognosis, it is need to establish patient-derived orthotopic xenograft(PDOX) mouse model of HCC using direct injection technique. Additionally, secretion of growth factor from bone marrow stem cells is promoted after partial hepatectomy. Based on former PDX research, we develop the new protocol using direct injection technique. Methods: From January 2015 to June 2016, consent about getting tumor pieces to the patient of HCC. The pieces is stored in RPMI1640 and moved from operation room to research room immediately. For PDOX, 8∼10weeks old NOD SCID and NSG male mouse is used. The tumor pieces cutted to the 1mm cube. For stop the bleeding, inferior vena cava is blocked using Bulldog Clamp and the cube is directly injected median lobe to 3 places randomly. After clear the Bulldog Clamp, a left lobe is removed using tie. After 3 months, the PDOX mouse is checked for cancer growth using MRI or PET-CT. Successful PDOX model is sacrificed and succeeded a generation to another Balb/C-nude male mouse. Results: The rate of success of PDOX model is about 10% respectively. Formed tumor is verified by H&E staining. Conclusion: Based on protocol established, further study plan is required for improving on the rate of success to establish PDOX model and clinical application.

Developed operation method for patient-drived orthotopic xenograft model of hepatocellular carcinoma

Developed operation method for patient-drived orthotopic xenograft model of hepatocellular carcinoma

KPNA3 Confers Sorafenib Resistance to Advanced Hepatocellular Carcinoma via TWIST Regulated Epithelial-Mesenchymal Transition.

Sorafenib, a multikinase inhibitor, is a new standard treatment for patients with advanced hepatocellular carcinoma (HCC). However, resistance to this regimen is frequently observed in clinical practice, and the molecular basis of this resistance remains largely unknown. Herein, the antitumor activity of sorafenib was assessed in 16 patient-derived xenograft (PDX) models of HCC. Gene expression analysis was conducted to identify factors that promote sorafenib resistance. Quantitative RT-PCR and immunoblotting were used to determine gene expression and activation of signaling pathways. Cell proliferation, clone formation, and transwell assays were conducted to evaluate drug-sensitivity, proliferation, and invasiveness, respectively. Kaplan-Meier analysis was used to evaluate the predictive power of biomarkers for sorafenib response. Differential gene expression analysis suggested that sorafenib resistance correlated with high karyopherin subunit alpha 3 (KPNA3) expression. Overexpression of KPNA3 in HCC cells enhanced tumor cell growth and invasiveness. Interestingly, KPNA3 was found to trigger epithelial-mesenchymal transition (EMT), a key process mediating drug resistance. On a mechanistic level, KPNA3 increased phosphorylation of AKT, which then phosphorylated ERK, and ultimately promoted TWIST expression to induce EMT and sorafenib resistance. Moreover, retrospective analysis revealed that HCC patients with low KPNA3 expression had remarkably longer survival after sorafenib treatment. Finally, we have identified a novel KPNA3-AKT-ERK-TWIST signaling cascade that promotes EMT and mediates sorafenib resistance in HCC. These findings suggest that KPNA3 is a promising biomarker for predicting patient responsiveness to sorafenib. Targeting KPNA3 may also contribute to resolving sorafenib resistance in HCC.

DM1 Loaded Ultrasmall Gold Nanoparticles Display Significant Efficacy and Improved Tolerability in Murine Models of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the sixth most common cancer worldwide with poor prognosis and limited options for treatment. Life expectancy after diagnosis is short; the currently available treatments are not well tolerated and have limited clinical benefit. There is a clear unmet clinical need for the development of new treatments. In this study, ultrasmall, 2 nm gold core nanoparticles (MidaCore) conjugated with the potent maytansine analogue DM1 (MTC-100038) were assessed as a systemic nanomedicine for the treatment of hepatocellular carcinoma. The platform improved overall tolerability of DM1, permitting ∼3-fold higher levels of drug to be administered compared to free drug. Dose for dose, MTC-100038 also facilitated delivery of ∼2.0-fold higher ( p = 0.039) levels of DM1 to the tumor compared to free DM1. MTC-100038 produced significant efficacy (tumor growth index ∼102%; p = <0.0001), in several murine xenograft models of HCC, and was superior to both free DM1 and the current standard of care, sorafenib. Furthermore, MTC-100038 displayed potent (nM) in vitro activity in various HCC primary patient derived cell lines and across various other different cancer cell types. These data demonstrate the potential of MidaCore nanoparticles to enhance tumor delivery of cytotoxic drugs and indicate MTC-100038 is worthy of further investigation as a potential treatment for HCC and other cancer types.

New insight into BIRC3: A novel prognostic indicator and a potential therapeutic target for liver cancer.

Prognosis of hepatocellular carcinoma (HCC) remains poor due to high recurrence rate and ineffective treatment options, highlighting the need to better understand the mechanism of recurrence and metastasis in HCC. We first collected messenger RNA (mRNA) expression data from 442 cases of HCC patients from The Cancer Genome Atlas (TCGA) database as well as 251 HCC patients from Zhongshan Hospital during 2009 and 2010 to analyze the expression pattern from tissue microarray (TMA) of baculoviral IAP repeat containing 3 (BIRC3). Then, we used BIRC3 gain-of-function (overexpression) and loss-of-function (knockdown) studies to examine the effect of BIRC3 on HCC cell proliferation and invasion. In addition, we also investigated the undying mechanism by which BIRC3 contributes to HCC tumor progression. Functionally, we also used a BIRC3-specific inhibitor AT-406 in HCC xenograft model to explore the potential therapeutic benefit of targeting BIRC3 in liver cancer. BIRC3 serves as a novel prognostic indicator for HCC patients undergoing curative resection. BIRC3 promotes HCC epithelial-mesenchymal transition (EMT), cell migration, and metastasis via upregulating MAP3K7, therefore, inducing ERK1/2 phosphorylation. The specific BIRC3 inhibitor AT-406 can inhibit HCC cell proliferation and reduce pulmonary metastases. BIRC3 induces tumor proliferation and metastasis in vitro and in vivo. BIRC3 may serve as a novel therapeutic target for liver cancer.

Combination therapy of sorafenib with mesenchymal stem cells as a novel cancer treatment regimen in xenograft models of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the most common liver malignancy and the second leading cause of cancer-related deaths in the world. Sorafenib is the first-line treatment of HCC. Although sorafenib has positive effects on the survival of patients, novel therapeutic strategies are needed to extend survival and improve the efficacy of sorafenib. This study combines sorafenib with mesenchymal stem cells (MSCs) as a new approach to enhance the efficacy of sorafenib. A subcutaneous xenograft model of HCC, established by human HepG2 cell lines, was implanted into the flank of nude miceand was used to evaluate tumor growth after treatment with sorafenib alone or in combination with MSCs. The aspartate aminotransferase, alanine aminotransferase, blood urea nitrogen, and creatinine levels were measured for safety assessment. Histopathological studies were performed using hematoxylin and eosin staining, and immunohistochemistry tests were performed to evaluate proliferation (Ki67) and angiogenesis (CD34). The TUNEL assay was used to detect apoptosis and measure the expression of major inflammatory cytokines (IL-1a, IL-10, and TNF-α) with real-time polymerase chain reaction. Sorafenib, in combination with MSCs, strongly inhibited tumor growth in the xenograft model. Furthermore, the combination therapy significantly inhibited HCC cell proliferation, decreased tumor angiogenesis, and induced apoptosis and maintained antitumor-associated anti-inflammatory effects of MSCs. This combination therapy strategy could be used as a new therapeutic approach to the treatment of HCC that significantly improves upon the results achieved using sorafenib as monotherapy.

CRISPR/Cas9‑mediated hypoxia inducible factor‑1α knockout enhances the antitumor effect of transarterial embolization in hepatocellular carcinoma.

Transarterial embolization (TAE) is a palliative option commonly used for the treatment of advanced, unresectable hepatocellular carcinoma (HCC). However, patient prognosis in regards to overall survival has not improved with this method, mainly due to hypoxia-inducible factor-1α (HIF-1α)-induced angiogenesis and invasiveness. Thus, it is hypothesized that HIF-1α may be an ideal knockout target for the treatment of HCC in combination with TAE. Thus, in the present study, HIF-1α knockout was conducted in human liver cancer SMMC-7721 cells and a xenograft HCC model was established using a lentivirus-mediated CRISPR/Cas system (LV-Cas) with small guide RNA-721 (LV-H721). Furthermore, hepatic artery ligation (HAL) was used to mimic human transarterial chemoembolization in mice. The results revealed that HIF-1α was highly expressed in both HCC patient tissues and SMMC-7721-induced tumor tissues. The HIF-1α knockout in SMMC-7721 cells significantly suppressed cell invasiveness and migration, and induced cell apoptosis under CoCl2-mimicking hypoxic conditions. Compared with the control groups, HAL + LV-H721 inhibited SMMC-7721 tumor growth in orthotopic HCC and markedly prolonged the survival of HCC-bearing mice, which was accompanied by a lower CD31 expression (tumor angiogenesis) and increased apoptosis in the tumor cells. These findings demonstrated a valuable antitumor synergism in combining CRISPR/Cas9-mediated HIF-1α knockout with TAE in mice and highlighted the possibility that HIF-1α may be an effective therapeutic knockout target in combination with TAE for HCC treatment.

Apatinib targets both tumor and endothelial cells in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most commonly diagnosed malignancies worldwide with poor prognosis and tends to be hypervascular. Aberrant expression of the vascular endothelial growth factor 2 (VEGFR‐2) has been implicated in the progression of HCC and represents a valid target for anticancer therapy. Apatinib, a small molecule inhibitor of VEGFR‐2 tyrosine kinase, shows strong antitumor activity in various tumors. This study is designed to evaluate the activity of apatinib on both human umbilical vein vascular endothelial cells (HUVECs) and HCC cell lines (in vitro and in vivo), and also to investigate the characteristics and possible mechanisms underlying these effects by molecular biology methods. Following the results in our study, apatinib inhibited phosphorylation of VEGFR‐2 in HUVECs and blocked in vitro endothelial cell migration and tube formation. Concentration‐dependent antiproliferative effects of apatinib were also observed in all 6 HCC cell lines including SK‐Hep‐1, HepG2, Hep3B, Huh‐7, PLC/PRF/5, SMMC‐7721. Moreover, response to apatinib of HCC cell lines was significantly correlated with VEGFR‐2 expression level. Additionally, apatinib significantly inhibit VEGF‐triggered VEGFR‐2 phosphorylation and activation of downstream signaling molecules such as Akt and ERK1/2 in HCCs. Apatinib can also induce a cell cycle arrest at G2/M phase and promote HCC apoptosis tested in vitro. In vivo data showed that apatinib can effectively inhibit tumor growth, decreased angiogenesis, as well as induced HCC apoptosis (in some tumors), and thus prolonged animal survival in a mouse xenograft model of human HCC. Our findings suggested that apatinib is a highly potent, oral active anti‐angiogenic, and anti‐HCC agent. The results from current study provide a clear biological rationale to evaluate apatinib as a new agent in HCC in clinical setting, especially for the VEGFR‐2 overexpression ones.

In vivo acquired sorafenib-resistant patient-derived tumor model displays alternative angiogenic pathways, multi-drug resistance and chromosome instability.

Acquired resistance to targeted therapies is an important clinical challenge. Research focusing on acquired resistance is hindered by the lack of relevant model systems. In the present study, the generation and characterization of an in vivo acquired sorafenib-resistant hepatocellular carcinoma (HCC) xenograft model derived from a patient tumor is reported. A cancer cell line (LIXC-004SR) was generated from a tumor that had developed following ~100 days of sorafenib treatment of a HCC patient-derived xenograft (PDX) model (LIX004). The xenograft tumors derived from this cell line demonstrated sorafenib-resistance in vivo. By contrast, a cell line (LIXC-004NA) generated from a vehicle-treated LIX004 PDX model remained sensitive to sorafenib in vivo. Following treatment with sorafenib in vivo, angiogenesis was significantly elevated in the LIXC-004SR tumors when compared with that in the LIXC-004NA tumors. The LIXC-004SR cell culture supernatant stimulated human umbilical vein endothelial cell proliferation and extracellular-signal-regulated kinase and protein kinase B phosphorylation, which can only be inhibited by the combination of sorafenib and a fibroblast growth factor receptor 1 (FGFR1) inhibitor, AZD4547. The tumor growth of the sorafenib-resistant LIXC-004SR xenograft was inhibited by the FGFR1 inhibitor in vivo, suggesting that one of the underlying mechanisms of the acquired resistance is likely due to activation of alternative angiogenic pathways. The LIXC-004SR cell line also exhibited signs of multi-drug resistance and genetic instability. Taken together, these data suggest that this in vivo model of acquired resistance from a PDX model may reflect sorafenib-resistance in certain patients and may facilitate drug resistance research, as well as contributing to the clinical prevention and management of drug resistance.

Abstract LB-272: Discovery and characterization of a novel FGFR4 Inhibitor for the treatment of hepatocellular carcinoma

Abstract Introduction: Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths worldwide and has one of the fastest growing incidence among cancers. Aberrant FGF19-FGFR4 signaling occurs in 10-30% of HCC patients and drives tumor development and progression. Pan-FGFR inhibitors are associated with hyperphosphatemia in clinic due to inhibition of FGFR1. Therefore, selectively targeting FGFR4 may render a safer treatment approach with improved clinical benefits. Using advanced computation-aided structural analysis and medicinal chemistry design, we have discovered a novel and highly selective small molecular FGFR4 inhibitor, ABSK-011, demonstrating robust anti-tumor activity in hepatocellular carcinoma models. Method: ABSK-011 was evaluated in biochemical Caliper assay and Celltiter-Glo assay for its inhibition on FGFR4 enzymatic and cellular activities. Its selectivity against other FGFR family members was analyzed through related biochemical, cellular, and KinomeScan profiling assays. The pharmacokinetics (PK) profile of ABSK-011 was characterized across various species of animals including mouse, rat and dog. Efficacy studies were conducted in two HCC xenograft models, and preliminary toxicity profile was also evaluated. Results: ABSK-011 inhibits FGFR4 with IC50 &amp;lt; 10 nM in biochemical assay and exhibits at least 50 fold selectivity against other FGFR kinases. The strong selectivity arises from the ability of ABSK-011 to irreversibly modify Cys552, a residue within the active site of FGFR4 that is not conserved among other FGFR kinases. ABSK-011 also shows greater than 800-fold selectivity against other RTK members. In HCC cells with autocrine production of FGF19, ABSK-011 inhibits the auto-phosphorylation of FGFR4 and blocks signal transduction from FGFR4 to downstream pathway activation. HCC cell lines harboring amplification and overexpression of FGF19 are uniquely sensitive to growth inhibition by ABSK-011 (EC50 &amp;lt; 50 nM) compared with cancer cell lines with FGFR1 dependence (EC50 &amp;gt; 2000 nM), confirming selectivity for FGFR4 over FGFR1. In vivo PK study demonstrated high exposure and superior DMPK profile of ABSK-011 in mouse, rat and dog. In preclinical in vivo studies, oral administration of ABSK-011 strongly inhibits the growth and induces regressions of subcutaneous xenograft tumors dependent upon FGFR4 activity. Suppression of tumor growth is dose dependent and well correlated with pharmacodynamic inhibition of FGFR4 signaling. Safety studies showed that ABSK-011 has no inhibition on CYP family members, hERG, etc. Conclusion: ABSK-011, presented here by Abbisko Therapeutics, is a highly potent, selective, and oral-available small molecular FGFR4 inhibitor. Its superior profile supports fast-track preclinical development. Citation Format: Zhui Chen. Discovery and characterization of a novel FGFR4 Inhibitor for the treatment of hepatocellular carcinoma [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-272.

Abstract 1986: IRAK1 augments cancer stemness and drug resistance via the AP-1/AKR1B10 signaling cascade in hepatocellular carcinoma

Abstract Frequent relapse and drug resistance can be attributed to the existence of tumor-initiating cells (T-ICs) within the tumor bulk. From transcriptome sequencing of 16 pairs of clinical HCC samples, we found that Interleukin-1 receptor-associated kinase 1 (IRAK1) in the TLR/IRAK pathway was significantly upregulated in hepatocellular carcinoma (HCC). IRAK1 overexpression in HCC was further confirmed at the mRNA and protein levels and correlated with advanced tumor stages and poor patents survival. Interestingly, IRAK4, an upstream regulator of IRAK1, was also found to be consistently upregulated. We demonstrated that IRAK1 regulates liver T-IC properties, including self-renewal, tumorigenicity and liver T-IC marker expression. IRAK1 inhibition sensitized the HCC cells to doxorubicin and sorafenib treatment in vitro through the suppression of the apoptotic cascade. Pharmacological inhibition of IRAK1 with a specific IRAK1/4 kinase inhibitor consistently suppressed liver T-IC populations. Through RNA sequencing analysis by comparing gene expression profiles between IRAK1-knockdown and control cells, we identified Aldo-Keto Reductase Family 1 Member 10 (AKR1B10) as a novel downstream target of IRAK1. Clinically, AKR1B10 was found to be overexpressed in HCC, which was significantly correlated with IRAK1 expression. Functional analysis demonstrated that knockdown of AKR1B10 negated the IRAK1-induced T-IC functions via modulation of the AP-1 complex. Using an HCC xenograft model, we found that an IRAK1/4 inhibitor in combination with sorafenib synergistically suppressed the tumor growth. In conclusion, targeting the IRAK4/IRAK1/AP-1/AKR1B10 signaling pathway may be a potential therapeutic strategy against HCC. Citation Format: Bowie Yik Ling Cheng, Doris Hoi Wing Leung, Eunice Yuen Ting Lau, Irene Oi Lin Ng, Kin Wah Lee. IRAK1 augments cancer stemness and drug resistance via the AP-1/AKR1B10 signaling cascade in hepatocellular carcinoma [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 1986.

Abstract 4779: Rogaratinib, a small molecule pan-FGFR inhibitor potently inhibits FGFR4-phosphorylation and exerts anti-tumor efficacy in vivo and in vitro

Abstract Fibroblast growth factor signaling plays an essential role in many physiological processes, including cell proliferation, survival, differentiation, migration, and apoptosis Over-activation of the pathway can lead to the development of various cancers. There are four different FGFR subtypes with over 20 ligands providing a very complex network of interactions and signaling. FGFR4 plays an important role in normal physiology such as myogenesis muscle regeneration and bile acid synthesis Activating mutations in the kinase domain of FGFR4 have been shown to mediate cancer development, for example, breast cancer, hepatocellular carcinoma (HCC) or rhabdomyosarcoma. Rogaratinib is a potent inhibitor of all FGFR subtypes with low nanomolar binding kinetics towards the four kinase domains. We explored the effect of rogaratinib on cell lines with high expression levels of FGFR4 mRNA, such as breast cancer cell line MDA-MB453 and rhabdomyosarcoma cell line SH30. We could show that the proliferation of these cell lines is potently inhibited by rogaratinib and accompanied with induction of apoptosis. The Mode-of-action analysis of rogaratinib activity in these cell lines showed inhibition of FGFR4-phosphorylation and downstream signaling at clinical relevant doses. The in vitro efficacy of rogaratinib in FGFR4 dependent cell lines was further observed as in vivo efficacy in xenograft models of rhabdomyosarcoma and HCC. Elevation of FGFR4 expression has been associated with resistance development in several cancer types such as breast cancer. In line with the observed anti-tumor efficacy of rogartinib in vitro and in vivo in breast cancer, sarcoma and HCC models, RNA in situ hybridization by RNAscope, revealed high FGFR4 mRNA expression in TMA's from these tumor types..) The combination of rogaratinib with standard-of-care provides further options for addressing resistance mechanisms mediated by elevated FGFR4 expression. In addition we have profiled a number of patient derived HCC xenograft models revealing the high efficacy of rogaratinib in models with elevated FGFR expression including FGFR4. Citation Format: Oliver Politz, Sylvia Gruenewald, Alexander Walter, Franziska Siegel, Arne Scholz, Sebastian Bender, Christoph Kneip, Peter Ellinghaus. Rogaratinib, a small molecule pan-FGFR inhibitor potently inhibits FGFR4-phosphorylation and exerts anti-tumor efficacy in vivo and in vitro [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 4779.

Abstract 4780: A novel, potent and selective FGFR4 inhibitor, HM81422 in hepatocellular carcinoma with FGFR4-driven pathway activation

Abstract Introduction: Hepatocellular carcinoma (HCC) is the sixth most prevalent cancer and the second most frequent cause of cancer-related death, however, treatment options are very limited. In recent studies, aberrant signaling through FGFR4 and its ligand, FGF19 have been identified as the oncogenic driver in a subset of HCCs and reported to be associated with poor prognosis. About 30% of HCC patients have altered FGF19/FGFR4 pathway signaling, therefore, the treatment with FGFR4 inhibitor may produce benefit. Materials and Method: Using the structure-based design, we have generated a novel, potent and selective FGFR4 inhibitor, HM81422 with irreversible-covalent binding mode, and evaluated its anti-tumor activity in a variety of HCC cell lines, HCC cell line xenografts and orthotopic grafts. Results: Biochemical selectivity assays demonstrated that HM81422 is highly selective towards FGFR4 compared to other FGFR isotypes as well as a panel of several kinases. The treatment of HM81422 to FGF19 amplified and overexpressed HCC cell lines led to suppression of FGF19/FGFR4 signaling pathway and concomitant reduction in cell viability in dose-dependent manner. Oral administration of HM81422 to mice bearing FGF19 altered HCC cells showed dose-dependent pharmacokinetics, pharmacodynamic modulation of FGFR4 signaling and antitumor efficacy in xenograft models. And HM81422 demonstrated inhibition of tumor growth in an orthotopic liver xenograft model of FGF19 altered HCC in nude mice. Conclusion: In conclusion, the treatment of HCC patients with a potent and selective FGFR4 inhibitor, HM81422, can be an attractive approach targeting approximately 30% of HCC patients by inhibiting altered FGF19-mediated signaling cascade. Further preclinical studies with HM81422 will be performed and reported soon. Citation Format: JaeHo Lee, Hyunjeong Kang, Kyounghwa Koo, Youngeun Ha, Sun Young Lim, Joo-Yun Byun, Hyunkyung Yu, Taehun Song, Moonsub Lee, Seung Hyun Jung, Taewoo Kim, Hyojeong Bang, Eunyoung Kim, Jahoon Kang, Ho Jeong Lee, YoungHoon Kim, YoungGil Ahn, KweeHyun Suh, Sun-Jin Kim. A novel, potent and selective FGFR4 inhibitor, HM81422 in hepatocellular carcinoma with FGFR4-driven pathway activation [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 4780.