Targeted inhibition of FGF19/FGFR cascade improves antitumor immunity and response rate in hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is the most common liver cancer globally, claiming nearly 1 million lives each year. The overexpression of fibroblast growth factor (FGF) receptors (FGFRs) signaling cascade has been shown to contribute to tumorigenesis, metastasis, and poor prognosis in HCC. Therefore, targeted inhibition of the FGF/FGFR cascade may represent a new treatment strategy for HCC patients. METHODS: HCC patient-derived xenograft (PDX) models were implanted into either severe combined immunodeficient (SCID) or CD34+hu-NSG (humanized) mice and subsequently treated with vehicle, infigratinib (FGFR1-3 inhibitor), FGF401 (FGFR4 inhibitor), or the combination of infigratinib and FGF401. Tumor progressions, overall survival of mice, lung metastasis, and drug resistance were monitored, and samples collected at the end of the treatment cycle were subjected to Western blot analyses and immunohistochemistry. RESULTS: HCC PDX models expressing high levels of FGF19/FGFR4 or FGFR2/3 showed favorable initial treatment response to FGF401 and infigratinib, respectively. However, progressive disease due to acquired resistance was observed. Combination infigratinib/FGF401 augmented the antitumor activity, response rate, and overall survival of mice. This combination significantly increased the infiltration of B cells, macrophages, CD8+ T cells, and CD4+ T cells associated with granzyme-B-mediated apoptosis, delayed onset of resistance, and inhibited metastasis by potently inhibiting several critical signaling pathways involved in proliferation and metastasis. CONCLUSIONS: Our findings suggest that HCC patients with high FGFR2/3 or FGF19/FGFR4 expressing tumors might benefit from a combination infigratinib/FGF401; thus, supporting its evaluation in clinical trials.

Upregulation of the ErbB family by EZH2 in hepatocellular carcinoma confers resistance to FGFR inhibitor.

PURPOSE: Hepatocellular carcinoma (HCC), the most common manifestation of liver cancer, is one of the leading causes of cancer-related mortality worldwide with limited treatment options. Infigratinib, a pan-FGFR inhibitor, has shown a potent antitumour effect in HCC. However, drug resistance is often observed in long-term treatment. In this study, we examined the potential feedback mechanism(s) leading to infigratinib and explored a combination therapy to overcome resistance in HCC. METHODS: Patient-derived xenograft (PDX) tumours were subcutaneously implanted into SCID mice and were subsequently treated with infigratinib. Tumour growth was monitored over time, and tumour samples were subjected to immunohistochemistry and Western blotting. For drug combination studies, mice were treated with infigratinib and/or varlitinib. Gene overexpression and knockdown studies were conducted to investigate the relationship between EZH2 and ErbB activity in infigratinib resistance. RESULTS: Infigratinib-resistant tumours exhibited higher levels of p-ErbB2 and p-ErbB3, concomitant with an increase in EZH2 expression. Gene overexpression and knockdown studies revealed that EZH2 directly regulates the levels of p-ErbB2 and p-ErbB3 in acquired resistance to infigratinib. The addition of varlitinib effectively overcame infigratinib resistance and prolonged the antitumour response, with minimal toxicity. CONCLUSION: The upregulation of the ErbB family by EZH2 appears to contribute to infigratinib resistance. The combination of infigratinib and varlitinib showed a potent antitumour effect and did not result in additional toxicity, warranting further clinical investigation.

Dynamic Contrast-Enhanced Magnetic Resonance Imaging as Imaging Biomarker for Vascular Normalization Effect of Infigratinib in High-FGFR-Expressing Hepatocellular Carcinoma Xenografts.

PURPOSE: Overexpression of fibroblast growth factor receptor (FGFR) contributes to tumorigenesis, metastasis, and poor prognosis of hepatocellular carcinoma (HCC). Infigratinib-a pan-FGFR inhibitor-potently suppresses the growth of high-FGFR-expressing HCCs in part via alteration of the tumor microenvironment and vessel normalization. In this study, we aim to assess the utility of dynamic contrast-enhanced (DCE) magnetic resonance imaging (MRI) as a non-invasive imaging technique to detect microenvironment changes associated with infigratinib and sorafenib treatment in high-FGFR-expressing HCC xenografts. PROCEDURES: Serial DCE-MRIs were performed on 12 nude mice bearing high-FGFR-expressing patient-derived HCC xenografts to quantify tumor microenvironment pre- (day 0) and post-treatment (days 3, 6, 9, and 15) of vehicle, sorafenib, and infigratinib. DCE-MRI data were analyzed using extended generalized kinetic model and two-compartment distributed parameter model. After treatment, immunohistochemistry stains were performed on the harvested tumors to confirm DCE-MRI findings. RESULTS: By treatment day 15, infigratinib induced tumor regression (70 % volume reduction from baseline) while sorafenib induced relative growth arrest (185 % volume increase from baseline versus 694 % volume increase from baseline of control). DCE-MRI analysis revealed different changes in microcirculatory parameters upon exposure to sorafenib versus infigratinib. While sorafenib induced microenvironment changes similar to those of rapidly growing tumors, such as a decrease in blood flow (F), fractional intravascular volume (vp), and permeability surface area product (PS), infigratinib induced the exact opposite changes as early as day 3 after treatment: increase in F, vp, and PS. CONCLUSIONS: Our study demonstrated that DCE-MRI is a reliable non-invasive imaging technique to monitor tumor microcirculatory response to FGFR inhibition and VEGF inhibition in high-FGFR-expressing HCC xenografts. Furthermore, the microcirculatory changes from FGFR inhibition manifested early upon treatment initiation and were reliably detected by DCE-MRI, creating possibilities of combinatorial therapy for synergistic effect.

Ribociclib enhances infigratinib-induced cancer cell differentiation and delays resistance in FGFR-driven hepatocellular carcinoma.

BACKGROUND & AIMS: Infigratinib is a pan-FGFR (fibroblast growth factor receptor) inhibitor that has shown encouraging activity in FGFR-dependent hepatocellular carcinoma (HCC) models. However, long-term treatment results in the emergence of resistant colonies. We sought to understand the mechanisms behind infigratinib-induced tumour cell differentiation and resistance and to explore the potential of adding the CDK4/6 inhibitor ribociclib to prolong cell differentiation. METHODS: Nine high and three low FGFR1-3-expressing HCC patient-derived xenograft (PDX) tumours were subcutaneously implanted into SCID mice and subsequently treated with either infigratinib alone or in combination with ribociclib. Tumour tissues were then subjected to immunohistochemistry to assess cell differentiation, as indicated by the cytoplasmic-to-nuclear ratio and markers such as CYP3A4, HNF4α and albumin. Western blot analyses were performed to investigate the signalling pathways involved. RESULTS: Infigratinib induced cell differentiation in FGFR1-3-dependent HCC PDX models, as indicated by an increase in the cytoplasmic/nuclear ratio and an increase in CYP3A4, HNF4α and albumin. Resistant colonies emerged in long-term treatment, characterised by a reversal of differentiated cell morphology, a reduction in the cytoplasmic-to-nuclear ratio and a loss of differentiation markers. Western blot analyses identified an increase in the CDK4/Cdc2/Rb pathway. The addition of ribociclib effectively blocked this pathway and reversed resistance to infigratinib, resulting in prolonged cell differentiation and growth inhibition. CONCLUSIONS: Our findings demonstrate that the combined inhibition of FGFR/CDK4/6 pathways is highly effective in providing long-lasting tumour growth inhibition and cell differentiation and reducing drug resistance. Therefore, further clinical investigations in patients with FGFR1-3-dependant HCC are warranted.

Bevacizumab Augments the Antitumor Efficacy of Infigratinib in Hepatocellular Carcinoma.

The fibroblast growth factor (FGF) signaling cascade is one of the key signaling pathways in hepatocellular carcinoma (HCC). FGF has been shown to augment vascular endothelial growth factor (VEGF)-mediated HCC development and angiogenesis, as well as to potentially lead to resistance to VEGF/VEGF receptor (VEGFR)-targeted agents. Thus, novel agents targeting FGF/FGF receptor (FGFR) signaling may enhance and/or overcome de novo or acquired resistance to VEGF-targeted agents in HCC. Mice bearing high- and low-FGFR tumors were treated with Infigratinib (i.e., a pan-FGFR kinase inhibitor) and/or Bevacizumab (i.e., an angiogenesis inhibitor). The antitumor activity of both agents was assessed individually or in combination. Tumor vasculature, intratumoral hypoxia, and downstream targets of FGFR signaling pathways were also investigated. Infigratinib, when combined with Bevacizumab, exerted a synergistic inhibitory effect on tumor growth, invasion, and lung metastasis, and it significantly improved the overall survival of mice bearing FGFR-dependent HCC. Infigratinib/Bevacizumab promoted apoptosis, inhibited cell proliferation concomitant with upregulation of p27, and reduction in the expression of FGFR2-4, p-FRS-2, p-ERK1/2, p-p70S6K/4EBP1, Cdc25C, survivin, p-Cdc2, and p-Rb. Combining Infigratinib/Bevacizumab may provide therapeutic benefits for a subpopulation of HCC patients with FGFR-dependent tumors. A high level of FGFR-2/3 may serve as a potential biomarker for patient selection to Infigratinib/Bevacizumab.

FGF401 and vinorelbine synergistically mediate antitumor activity and vascular normalization in FGF19-dependent hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a lethal cancer with limited therapeutic options, and standard therapy with sorafenib provides only modest survival benefits. Fibroblast growth factor 19 (FGF19) has been proposed as a driver oncogene, and targeting its receptor, FGFR-4, may provide a better alternative to standard therapy for patients with FGF19-driven tumors. Sixty-three HCC patient-derived xenograft (PDX) models were screened for FGF19 expression. Mice bearing high and low FGF19-expressing tumors were treated with FGF401 and/or vinorelbine, and the antitumor activity of both agents was assessed individually and in combination. Tumor vasculature and intratumoral hypoxia were also examined. High FGF19 expression was detected in 14.3% (9 of 63) of the HCC models tested and may represent a good target for HCC treatment. FGF401 potently inhibited the growth of high FGF19-expressing HCC models regardless of FGF19 gene amplification. Furthermore, FGF401 inhibited the FGF19/FGFR-4 signaling pathway, cell proliferation, and hypoxia, induced apoptosis and blood vessel normalization and prolonged the overall survival (OS) of mice bearing high FGF19 tumors. FGF401 synergistically acted with the microtubule-depolymerizing drug vinorelbine to further suppress tumor growth, promote apoptosis, and prolong the OS of mice bearing high FGF19 tumors, with no evidence of increased toxicity. Our study suggests that a subset of patients with high FGF19-expressing HCC tumors could benefit from FGF401 or FGF401/vinorelbine treatment. A high level of FGF19 in a tumor may serve as a potential biomarker for patient selection.

Vinorelbine Augments Radiotherapy in Hepatocellular Carcinoma.

There is a need to improve the effectiveness of radiotherapy (RT) in hepatocellular carcinoma (HCC). Therefore, the purpose of this study was to explore the efficacy and toxicity of the anti-microtubule agent Vinorelbine as a radiosensitizer in HCC. The radio sensitivity of 16 HCC patient-derived xenograft (PDX) models was determined by quantifying the survival fraction following irradiation in vitro, and Vinorelbine radio sensitization was determined by clonogenic assay. Ectopic HCC xenografts were treated with a single dose of 8 Gy irradiation and twice-weekly 3 mg/kg Vinorelbine. Tumor growth and changes in the proteins involved in DNA repair, angiogenesis, tumor cell proliferation, and survival were assessed, and the 3/16 (18.75%), 7/16 (43.75%), and 6/16 (37.5%) HCC lines were classified as sensitive, moderately sensitive, and resistant, respectively. The combination of RT and Vinorelbine significantly inhibited tumor growth, DNA repair proteins, angiogenesis, and cell proliferation, and promoted more apoptosis compared with RT or Vinorelbine treatment alone. Vinorelbine improved HCC tumor response to standard irradiation with no increase in toxicity. HCC is prevalent in less developed parts of the world and is mostly unresectable on presentation. Vinorelbine and conventional radiotherapy are cost-effective, well-established modalities of cancer treatment that are readily available. Therefore, this strategy can potentially address an unmet clinical need, warranting further investigation in early-phase clinical trials.

Assessment of PARP4 as a candidate breast cancer susceptibility gene.

PURPOSE: PARP4 has been proposed as a candidate breast cancer susceptibility gene. However, its function and involvement in breast carcinogenesis is unclear. We sought to determine the variant frequency of PARP4 in BRCA-negative women referred for genetic testing from Singapore and to perform functional analyses of PARP4. METHODS: Next-generation sequencing of PARP4 was conducted for 198 BRCA-negative cases from Singapore. Three independent case-control association analyses of PARP4 were performed for (1) our Singaporean cohort, (2) three dbGaP datasets, and (3) cases from TCGA, with controls from the Exome Aggregation Consortium (ExAC). PARP4 knockout cells were generated utilizing the CRISPR-Cas9 approach in MDA-MB-231 (breast cancer) and MCF10A (normal breast) cell lines, and colony formation, cell proliferation, and migration assays carried out. RESULTS: Candidate variants in PARP4 were identified in 5.5% (11/198) of our Singapore cohort. Case-control association studies for our cases and the dbGaP datasets showed no significant association. However, a significant association was observed for PARP4 variants when comparing 988 breast cancer cases from the TCGA provisional data and 53,105 controls from ExAC (ALL) (OR 0.249, 95% CI 0.139-0.414, P = 2.86 × 10-11). PARP4 knockout did not affect the clonogenicity, proliferation rate, and migration of normal breast cells, but appeared to decrease the proliferation rate and clonogenicity of breast cancer cells. CONCLUSIONS: Taken together, our results do not support that PARP4 functions as a cancer susceptibility gene. This study highlights the importance of performing functional analyses for candidate cancer predisposition genes.

Breast cancer in women with neurofibromatosis type 1 (NF1): a comprehensive case series with molecular insights into its aggressive phenotype.

PURPOSE: The purpose of the study was to improve the understanding of NF1-associated breast cancer, given the increased risk of breast cancer in this tumour predisposition syndrome and the limited data. METHODS: We identified 18 women with NF1 and breast cancer at our institution. Clinical and pathologic characteristics of NF1-associated breast cancers were compared with 7132 breast cancers in patients without NF1 from our institutional database. Next generation sequencing was performed on DNA from blood and breast cancer specimens available. Blood specimens negative for NF1 mutation were subjected to multiplex ligation-dependent probe amplification (MLPA) to identify complete/partial deletions or duplications. Expression of neurofibromin in the NF1-associated breast cancers was evaluated using immunohistochemistry. RESULTS: There was a higher frequency of grade 3 (83.3% vs 45.4%, p = 0.005), oestrogen receptor (ER) negative (66.7% vs 26.3%, p < 0.001) and human epidermal growth factor receptor 2 (HER2)-positive (66.7% vs 23.4%, p < 0.001) tumours among NF1 patients compared to non-NF1 breast cancers. Overall survival was inferior in NF1 patients in multivariable analysis (hazard ratio 2.25, 95% CI 1.11-4.60; p = 0.025). Apart from germline NF1 mutations (11/16; 69%), somatic mutations in TP53 (8/10; 80%), second-hit NF1 (2/10; 20%), KMT2C (4/10; 40%), KMT2D (2/10; 20%), and PIK3CA (2/10; 20%) were observed. Immunohistochemical expression of neurofibromin was seen in the nuclei and/or cytoplasm of all specimens, but without any consistent pattern in the intensity or extent. CONCLUSIONS: This comprehensive series of NF1-associated breast cancers suggests that their aggressive features are related to germline NF1 mutations in cooperation with somatic mutations in TP53, KMT2C and other genes.