Glycerol tributylate (Triacylglycerol tributanoate) promoted the liver lipid metabolism by cultivating the intestinal flora of grass carp (Ctenopharyngodon idellus).

To investigate the effects of glycerol tributyrin (TB) (Triacylglycerol tributanoate) on the regulation of liver lipid metabolism by intestinal flora of grass carp (Ctenopharyngodon idellus). The compound feed with soybean oil 2.8% + fish oil 1.8%, soybean oil 6.3% + fish oil 1.8%, and soybean oil 6.2% + fish oil 1.8% + TB 0.1% was added to the basal diet as a fat source and fed to the basal (control) group, high lipid (HL) group, and tributyrin (TB) group for 12 weeks. We tested the growth performance, fat content, diversity, and abundance of gut flora and other related indexes of grass carp by Soxhlet extraction, liver tissue enzyme activity, oil red O staining, and 16S rRNA high-throughput sequencing. The results showed that the liver fat number and liver fat content of grass carp in the TB group were lower than those in the HL group, while the fattening degree was significantly higher than those in the other two groups; according to the indices such as Shannon, Ace, and Coverage, it was found that the grass carp in the TB group had the highest abundance and diversity of intestinal microflora; at the portal level, Proteobacteria and Fusobacteria were the main dominant flora in the TB group, with the number of unique OUTs accounting for about 59. 9% of the total number measured; at the genus level, the relative abundance of lipase-producing, short-chain fatty acid-associated bacteria, such as Bacillus-Lactobacillus and Bifidobacterium, was significantly lower (p < 0.05). Thus, we conclude that the addition of TB to high-fat diets can alter the structure of the intestinal microbial community and promote hepatic lipid metabolism in grass carp. TB can alleviate fatty liver in grass carp by increasing the relative abundance of short-chain fatty acids in the intestine. Meanwhile, TB inhibits the conversion of primary bile acids to secondary bile acids in the host, which can block intestinal FXR signaling and the hepatic FXR-SHP pathway, thus slowing down fat synthesis and alleviating the accumulation of liver lipids in grass carp.

Reduced Mitochondrial Apoptotic Priming Drives Resistance to BH3 Mimetics in Acute Myeloid Leukemia.

Acquired resistance to BH3 mimetic antagonists of BCL-2 and MCL-1 is an important clinical problem. Using acute myelogenous leukemia (AML) patient-derived xenograft (PDX) models of acquired resistance to BCL-2 (venetoclax) and MCL-1 (S63845) antagonists, we identify common principles of resistance and persistent vulnerabilities to overcome resistance. BH3 mimetic resistance is characterized by decreased mitochondrial apoptotic priming as measured by BH3 profiling, both in PDX models and human clinical samples, due to alterations in BCL-2 family proteins that vary among cases, but not to acquired mutations in leukemia genes. BCL-2 inhibition drives sequestered pro-apoptotic proteins to MCL-1 and vice versa, explaining why in vivo combinations of BCL-2 and MCL-1 antagonists are more effective when concurrent rather than sequential. Finally, drug-induced mitochondrial priming measured by dynamic BH3 profiling (DBP) identifies drugs that are persistently active in BH3 mimetic-resistant myeloblasts, including FLT-3 inhibitors and SMAC mimetics.

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.

Genome-wide CRISPR screening reveals genetic modifiers of mutant EGFR dependence in human NSCLC.

EGFR-mutant NSCLCs frequently respond to EGFR tyrosine kinase inhibitors (TKIs). However, the responses are not durable, and the magnitude of tumor regression is variable, suggesting the existence of genetic modifiers of EGFR dependency. Here, we applied a genome-wide CRISPR-Cas9 screening to identify genetic determinants of EGFR TKI sensitivity and uncovered putative candidates. We show that knockout of RIC8A, essential for G-alpha protein activation, enhanced EGFR TKI-induced cell death. Mechanistically, we demonstrate that RIC8A is a positive regulator of YAP signaling, activation of which rescued the EGFR TKI sensitizing phenotype resulting from RIC8A knockout. We also show that knockout of ARIH2, or other components in the Cullin-5 E3 complex, conferred resistance to EGFR inhibition, in part by promoting nascent protein synthesis through METAP2. Together, these data uncover a spectrum of previously unidentified regulators of EGFR TKI sensitivity in EGFR-mutant human NSCLC, providing insights into the heterogeneity of EGFR TKI treatment responses.

FGF401, A First-In-Class Highly Selective and Potent FGFR4 Inhibitor for the Treatment of FGF19-Driven Hepatocellular Cancer.

Hepatocellular carcinoma (HCC) is the most common primary malignancy of the liver and it is the third leading cause of cancer-related deaths worldwide. Recently, aberrant signaling through the FGF19/FGFR4 axis has been implicated in HCC. Here, we describe the development of FGF401, a highly potent and selective, first in class, reversible-covalent small-molecule inhibitor of the kinase activity of FGFR4. FGF401 is exquisitely selective for FGFR4 versus the other FGFR paralogues FGFR1, FGFR2, FGFR3, and all other kinases in the kinome. FGF401 has excellent drug-like properties showing a robust pharmacokinetic/pharmacodynamics/efficacy relationship, driven by a fraction of time above the phospho-FGFR4 IC90 value. FGF401 has remarkable antitumor activity in mice bearing HCC tumor xenografts and patient-derived xenograft models that are positive for FGF19, FGFR4, and KLB. FGF401 is the first FGFR4 inhibitor to enter clinical trials, and a phase I/II study is currently ongoing in HCC and other solid malignancies.

Capmatinib (INC280) Is Active Against Models of Non-Small Cell Lung Cancer and Other Cancer Types with Defined Mechanisms of MET Activation.

PURPOSE: The selective MET inhibitor capmatinib is being investigated in multiple clinical trials, both as a single agent and in combination. Here, we describe the preclinical data of capmatinib, which supported the clinical biomarker strategy for rational patient selection. EXPERIMENTAL DESIGN: The selectivity and cellular activity of capmatinib were assessed in large cellular screening panels. Antitumor efficacy was quantified in a large set of cell line- or patient-derived xenograft models, testing single-agent or combination treatment depending on the genomic profile of the respective models. RESULTS: Capmatinib was found to be highly selective for MET over other kinases. It was active against cancer models that are characterized by MET amplification, marked MET overexpression, MET exon 14 skipping mutations, or MET activation via expression of the ligand hepatocyte growth factor (HGF). In cancer models where MET is the dominant oncogenic driver, anticancer activity could be further enhanced by combination treatments, for example, by the addition of apoptosis-inducing BH3 mimetics. The combinations of capmatinib and other kinase inhibitors resulted in enhanced anticancer activity against models where MET activation co-occurred with other oncogenic drivers, for example EGFR activating mutations. CONCLUSIONS: Activity of capmatinib in preclinical models is associated with a small number of plausible genomic features. The low fraction of cancer models that respond to capmatinib as a single agent suggests that the implementation of patient selection strategies based on these biomarkers is critical for clinical development. Capmatinib is also a rational combination partner for other kinase inhibitors to combat MET-driven resistance.

Combining BH3-mimetics to target both BCL-2 and MCL1 has potent activity in pre-clinical models of acute myeloid leukemia.

Improving outcomes in acute myeloid leukemia (AML) remains a major clinical challenge. Overexpression of pro-survival BCL-2 family members rendering transformed cells resistant to cytotoxic drugs is a common theme in cancer. Targeting BCL-2 with the BH3-mimetic venetoclax is active in AML when combined with low-dose chemotherapy or hypomethylating agents. We now report the pre-clinical anti-leukemic efficacy of a novel BCL-2 inhibitor S55746, which demonstrates synergistic pro-apoptotic activity in combination with the MCL1 inhibitor S63845. Activity of the combination was caspase and BAX/BAK dependent, superior to combination with standard cytotoxic AML drugs and active against a broad spectrum of poor risk genotypes, including primary samples from patients with chemoresistant AML. Co-targeting BCL-2 and MCL1 was more effective against leukemic, compared to normal hematopoietic progenitors, suggesting a therapeutic window of activity. Finally, S55746 combined with S63845 prolonged survival in xenograft models of AML and suppressed patient-derived leukemia but not normal hematopoietic cells in bone marrow of engrafted mice. In conclusion, a dual BH3-mimetic approach is feasible, highly synergistic, and active in diverse models of human AML. This approach has strong clinical potential to rapidly suppress leukemia, with reduced toxicity to normal hematopoietic precursors compared to chemotherapy.

Infigratinib Mediates Vascular Normalization, Impairs Metastasis, and Improves Chemotherapy in Hepatocellular Carcinoma.

The fibroblast growth factor (FGF) signaling cascade is a key signaling pathway in hepatocarcinogenesis. We report high FGF receptor (FGFR) expression in 17.7% (11 of 62) of hepatocellular carcinoma (HCC) models. Infigratinib, a pan-FGFR inhibitor, potently suppresses the growth of high-FGFR-expressing and sorafenib-resistant HCCs. Infigratinib inhibits FGFR signaling and its downstream targets, cell proliferation, the angiogenic rescue program, hypoxia, invasion, and metastasis. Infigratinib also induces apoptosis and vessel normalization and improves the overall survival of mice bearing FGFR-driven HCCs. Infigratinib acts in synergy with the microtubule-depolymerizing drug vinorelbine to promote apoptosis, suppress tumor growth, and improve the overall survival of mice. Increased expression levels of FGFR-2 and FGFR-3 through gene amplification correlate with treatment response and may serve as potential biomarkers for patient selection. Conclusion: Treatments with Infigratinib alone or in combination with vinorelbine may be effective in a subset of patients with HCC with FGFR-driven tumors.

S55746 is a novel orally active BCL-2 selective and potent inhibitor that impairs hematological tumor growth.

Escape from apoptosis is one of the major hallmarks of cancer cells. The B-cell Lymphoma 2 (BCL-2) gene family encodes pro-apoptotic and anti-apoptotic proteins that are key regulators of the apoptotic process. Overexpression of the pro-survival member BCL-2 is a well-established mechanism contributing to oncogenesis and chemoresistance in several cancers, including lymphoma and leukemia. Thus, BCL-2 has become an attractive target for therapeutic strategy in cancer, as demonstrated by the recent approval of ABT-199 (Venclexta™) in relapsed or refractory Chronic Lymphocytic Leukemia with 17p deletion. Here, we describe a novel orally bioavailable BCL-2 selective and potent inhibitor called S55746 (also known as BCL201). S55746 occupies the hydrophobic groove of BCL-2. Its selectivity profile demonstrates no significant binding to MCL-1, BFL-1 (BCL2A1/A1) and poor affinity for BCL-XL. Accordingly, S55746 has no cytotoxic activity on BCL-XL-dependent cells, such as platelets. In a panel of hematological cell lines, S55746 induces hallmarks of apoptosis including externalization of phosphatidylserine, caspase-3 activation and PARP cleavage. Ex vivo, S55746 induces apoptosis in the low nanomolar range in primary Chronic Lymphocytic Leukemia and Mantle Cell Lymphoma patient samples. Finally, S55746 administered by oral route daily in mice demonstrated robust anti-tumor efficacy in two hematological xenograft models with no weight lost and no change in behavior. Taken together, these data demonstrate that S55746 is a novel, well-tolerated BH3-mimetic targeting selectively and potently the BCL-2 protein.

An allosteric PRC2 inhibitor targeting the H3K27me3 binding pocket of EED.

Polycomb repressive complex 2 (PRC2) consists of three core subunits, EZH2, EED and SUZ12, and plays pivotal roles in transcriptional regulation. The catalytic subunit EZH2 methylates histone H3 lysine 27 (H3K27), and its activity is further enhanced by the binding of EED to trimethylated H3K27 (H3K27me3). Small-molecule inhibitors that compete with the cofactor S-adenosylmethionine (SAM) have been reported. Here we report the discovery of EED226, a potent and selective PRC2 inhibitor that directly binds to the H3K27me3 binding pocket of EED. EED226 induces a conformational change upon binding EED, leading to loss of PRC2 activity. EED226 shows similar activity to SAM-competitive inhibitors in blocking H3K27 methylation of PRC2 target genes and inducing regression of human lymphoma xenograft tumors. Interestingly, EED226 also effectively inhibits PRC2 containing a mutant EZH2 protein resistant to SAM-competitive inhibitors. Together, we show that EED226 inhibits PRC2 activity via an allosteric mechanism and offers an opportunity for treatment of PRC2-dependent cancers.

Discovery of First-in-Class, Potent, and Orally Bioavailable Embryonic Ectoderm Development (EED) Inhibitor with Robust Anticancer Efficacy.

Overexpression and somatic heterozygous mutations of EZH2, the catalytic subunit of polycomb repressive complex 2 (PRC2), are associated with several tumor types. EZH2 inhibitor, EPZ-6438 (tazemetostat), demonstrated clinical efficacy in patients with acceptable safety profile as monotherapy. EED, another subunit of PRC2 complex, is essential for its histone methyltransferase activity through direct binding to trimethylated lysine 27 on histone 3 (H3K27Me3). Herein we disclose the discovery of a first-in-class potent, selective, and orally bioavailable EED inhibitor compound 43 (EED226). Guided by X-ray crystallography, compound 43 was discovered by fragmentation and regrowth of compound 7, a PRC2 HTS hit that directly binds EED. The ensuing scaffold hopping followed by multiparameter optimization led to the discovery of 43. Compound 43 induces robust and sustained tumor regression in EZH2MUT preclinical DLBCL model. For the first time we demonstrate that specific and direct inhibition of EED can be effective as an anticancer strategy.

Enhanced targeting of CML stem and progenitor cells by inhibition of porcupine acyltransferase in combination with TKI.

Tyrosine kinase inhibitor (TKI) treatment of chronic myeloid leukemia (CML) has limited efficacy against leukemia stem cells (LSC) responsible for disease propagation, and most CML patients require continued TKI treatment to maintain remission. LSC maintenance is related, at least in part, to signals from the bone marrow microenvironment (BMM). Our previous studies have shown that Wnt signaling from the BMM contributes to preservation of CML LSC following TKI treatment. Secretion of Wnt ligands requires their modification by the O-acyl transferase Porcupine (PORCN). Here we investigated the activity of a potent and selective PORCN inhibitor, WNT974, against CML stem and progenitor cells. WNT974 efficiently antagonized Wnt signaling in human CML CD34+ cells, and in combination with the TKI nilotinib (NIL) significantly enhanced inhibition of proliferation and colony-forming potential of CML stem and progenitor cells and reduced their growth in immunodeficient mice in vivo, in comparison with NIL alone. Treatment of transgenic CML mice in vivo with NIL in combination with WNT974 significantly reduced leukemic stem and progenitor cell numbers, reduced regeneration of leukemic long-term hematopoietic stem cells in secondary transplant recipients, and enhanced survival of mice after discontinuation of treatment, in comparison with NIL alone. CML progenitors demonstrated enhanced sensitivity to Wnt stimulation, associated with increased expression of the FZD4 receptor. FZD4 knockdown inhibited CML progenitor growth. These results support further investigation of PORCN targeting to inhibit Wnt secretion and signaling and enhance targeting of CML stem cells while sparing their normal counterparts.

High-throughput screening using patient-derived tumor xenografts to predict clinical trial drug response.

Profiling candidate therapeutics with limited cancer models during preclinical development hinders predictions of clinical efficacy and identifying factors that underlie heterogeneous patient responses for patient-selection strategies. We established ∼1,000 patient-derived tumor xenograft models (PDXs) with a diverse set of driver mutations. With these PDXs, we performed in vivo compound screens using a 1 × 1 × 1 experimental design (PDX clinical trial or PCT) to assess the population responses to 62 treatments across six indications. We demonstrate both the reproducibility and the clinical translatability of this approach by identifying associations between a genotype and drug response, and established mechanisms of resistance. In addition, our results suggest that PCTs may represent a more accurate approach than cell line models for assessing the clinical potential of some therapeutic modalities. We therefore propose that this experimental paradigm could potentially improve preclinical evaluation of treatment modalities and enhance our ability to predict clinical trial responses.

FGFR-Mediated Reactivation of MAPK Signaling Attenuates Antitumor Effects of Imatinib in Gastrointestinal Stromal Tumors.

UNLABELLED: Activating mutations in either KIT or PDGFRA are present in approximately 90% of gastrointestinal stromal tumors (GIST). Although treatment with the KIT and PDGFR inhibitor imatinib can control advanced disease in about 80% of GIST patients, the beneficial effect is not durable. Here, we report that ligands from the FGF family reduced the effectiveness of imatinib in GIST cells, and FGF2 and FGFR1 are highly expressed in all primary GIST samples examined. The combination of KIT and FGFR inhibition showed increased growth inhibition in imatinib-sensitive GIST cell lines and improved efficacy in patient-derived GIST xenografts. In addition, inhibition of MAPK signaling by imatinib was not sustained in GIST cells. An ERK rebound occurred through activation of FGF signaling, and was repressed by FGFR1 inhibition. Downregulation of Sprouty proteins played a role in the imatinib-induced feedback activation of FGF signaling in GIST cells. SIGNIFICANCE: We here show that FGFR-mediated reactivation of the MAPK pathway attenuates the antiproliferation effects of imatinib in GISTs. The imatinib-induced ERK rebound can be repressed by the FGFR inhibitor BGJ398, and combined KIT and FGFR inhibition leads to increased efficacy in vitro and in patient-derived xenografts.

Measurement of PIP3 levels reveals an unexpected role for p110ß in early adaptive responses to p110α-specific inhibitors in luminal breast cancer.

BYL719, which selectively inhibits the alpha isoform of the phosphatidylinositol 3-kinase (PI3K) catalytic subunit (p110a), is currently in clinical trials for the treatment of solid tumors, especially luminal breast cancers with PIK3CA mutations and/or HER2 amplification. This study reveals that, even among these sensitive cancers, the initial efficacy of p110α inhibition is mitigated by rapid re-accumulation of the PI3K product PIP3 produced by the p110ß isoform. Importantly, the reactivation of PI3K mediated by p110ß does not invariably restore AKT phosphorylation, demonstrating the limitations of using phospho-AKT as a surrogate to measure PI3K activation. Consistently, we show that the addition of the p110ß inhibitor to BYL719 prevents the PIP3 rebound and induces greater antitumor efficacy in HER2-amplified and PIK3CA mutant cancers.

Characterization of the novel and specific PI3Kα inhibitor NVP-BYL719 and development of the patient stratification strategy for clinical trials.

Somatic PIK3CA mutations are frequently found in solid tumors, raising the hypothesis that selective inhibition of PI3Kα may have robust efficacy in PIK3CA-mutant cancers while sparing patients the side-effects associated with broader inhibition of the class I phosphoinositide 3-kinase (PI3K) family. Here, we report the biologic properties of the 2-aminothiazole derivative NVP-BYL719, a selective inhibitor of PI3Kα and its most common oncogenic mutant forms. The compound selectivity combined with excellent drug-like properties translates to dose- and time-dependent inhibition of PI3Kα signaling in vivo, resulting in robust therapeutic efficacy and tolerability in PIK3CA-dependent tumors. Novel targeted therapeutics such as NVP-BYL719, designed to modulate aberrant functions elicited by cancer-specific genetic alterations upon which the disease depends, require well-defined patient stratification strategies in order to maximize their therapeutic impact and benefit for the patients. Here, we also describe the application of the Cancer Cell Line Encyclopedia as a preclinical platform to refine the patient stratification strategy for NVP-BYL719 and found that PIK3CA mutation was the foremost positive predictor of sensitivity while revealing additional positive and negative associations such as PIK3CA amplification and PTEN mutation, respectively. These patient selection determinants are being assayed in the ongoing NVP-BYL719 clinical trials.

Novel neutralizing hedgehog antibody MEDI-5304 exhibits antitumor activity by inhibiting paracrine hedgehog signaling.

The hedgehog pathway has been implicated in the tumorigenesis, tumor progression, and metastasis of numerous human cancers. We generated the first fully human hedgehog antibody MEDI-5304 and characterized its antitumor activity and preclinical toxicology. MEDI-5304 bound sonic hedgehog (SHH) and Indian hedgehog (IHH) with low picomolar affinity and neutralized SHH and IHH activity in cellular mGLI1 reporter assays. The antibody inhibited transcription of hedgehog target genes and osteoblast differentiation of C3H10T1/2 cells. We evaluated the activity of MEDI-5304 in vivo in model systems that allowed us to evaluate two primary hypotheses of hedgehog function in human cancer, paracrine signaling between tumor and stromal cells and cancer stem cell (CSC) self-renewal. MEDI-5304 displayed robust pharmacodynamic effects in stromal cells that translated to antitumor efficacy as a single agent in an HT-29/MEF coimplantation model of paracrine hedgehog signaling. MEDI-5304 also improved responses to carboplatin in the HT-29/MEF model. The antibody, however, had no effect as a single agent or in combination with gemcitabine on the CSC frequency or growth of several primary pancreatic cancer explant models. These findings support the conclusion that hedgehog contributes to tumor biology via paracrine tumor-stromal signaling but not via CSC maintenance or propagation. Finally, the only safety study finding associated with MEDI-5304 was ondontodysplasia in rats. Thus, MEDI-5304 represents a potent dual hedgehog inhibitor suitable for continued development to evaluate efficacy and safety in human patients with tumors harboring elevated levels of SHH or IHH.

NSD2 is recruited through its PHD domain to oncogenic gene loci to drive multiple myeloma.

Histone lysine methyltransferase NSD2 (WHSC1/MMSET) is overexpressed frequently in multiple myeloma due to the t(4;14) translocation associated with 15% to 20% of cases of this disease. NSD2 has been found to be involved in myelomagenesis, suggesting it may offer a novel therapeutic target. Here we show that NSD2 methyltransferase activity is crucial for clonogenicity, adherence, and proliferation of multiple myeloma cells on bone marrow stroma in vitro and that NSD2 is required for tumorigenesis of t(4;14)+ but not t(4;14)- multiple myeloma cells in vivo. The PHD domains in NSD2 were important for its cellular activity and biological function through recruiting NSD2 to its oncogenic target genes and driving their transcriptional activation. By strengthening its disease linkage and deepening insights into its mechanism of action, this study provides a strategy to therapeutically target NSD2 in multiple myeloma patients with a t(4;14) translocation.

Monoclonal antibody lead characterization: in vitro and in vivo methods.

This chapter describes in vitro and in vivo methods to characterize a lead monoclonal antibody candidate in the drug discovery setting. Approaches to characterize monoclonal antibody specificity, heavy and light chain composition, and antibody mode of action including the ability to mediate secretion of effector molecules, inhibit cell proliferation, induce apoptosis, or elicit antibody effector function are described. ELISA and flow cytometry based methods, as well as in vitro assays to assess for cell proliferation, ADCC, and CDC are detailed.In addition, both subcutaneous and orthotopic in vivo tumor xenograft models to assess antibody efficacy are described. The xenograft tumor model is a valuable tool for assessing the therapeutic activity of a monoclonal antibody drug candidate. Xenograft models are generated by the implantation of tumor cells or tumor fragments of human origin into immune-compromised mice or rats. This allows for fast and efficient in vivo evaluation of an antibody drug candidate in human cancer models. Here, we describe the procedures for generating preclinical animal tumor models frequently employed in the preclinical drug discovery setting.