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The chemokine system plays an important role in mediating a proinflammatory microenvironment for tumor growth in hepatocellular carcinoma (HCC). The CXCR6 receptor and its natural ligand CXCL16 are expressed at high levels in HCC cell lines and tumor tissues and receptor expression correlates with increased neutrophils in these tissues contributing to poor prognosis in patients. Availability of pharmacologcal tools targeting the CXCR6/CXCL16 axis are needed to elucidate the mechanism whereby neutrophils are affected in the tumor environment. We report the discovery of a series of small molecules with an exo-[3.3.1]azabicyclononane core. Our lead compound 81 is a potent (EC50 = 40 nM) and selective orally bioavailable small molecule antagonist of human CXCR6 receptor signaling that significantly decreases tumor growth in a 30-day mouse xenograft model of HCC.

Introduction Ikaros family zinc finger protein 1 and 3 (IKZF1/3) are essential transcription factors (TF) for terminal differentiation of B and T cells. Depletion of IKZF1/3 in MM cells inhibits growth, confirming their dependency on IKZF1/3. IMiDs (lenalidomide[len], pomalidomide[pom]) are effective therapies for treatment of MM and promote degradation of IKZF1/3 via their interaction with CRL4-CRBN E3 ligase. Most patients treated with len or pom eventually develop progressive disease due to acquired resistance, underscoring an unmet medical need. CFT7455 is a novel IKZF1/3 degrader optimized for high affinity cereblon (CRBN) binding, rapid IKZF1/3 degradation, and potent in vivo efficacy. Results CFT7455 demonstrated an 800 to 1600-fold improvement in CRBN binding compared to pom in biochemical and cellular NanoBRET assays, respectively. In H929 MM cells expressing HiBiT-tagged IKZF1, CFT7455 induced >75% degradation of IKZF1 within 1.5 hr. The high binding affinity and degradation catalysis shown with CFT7455 enabled potent antiproliferative activity across a panel of MM cell lines as well as H929 cells made resistant to IMiDs. In RPMI-8226 MM mice xenografts, CFT7455 (0.1 mg/kg/day) resulted in deep, durable degradation of IKZF3 (21% and 9.5% of vehicle levels at 4 and 24 hr, respectively). Protein levels for IRF4, an essential TF in MM, declined over 7 days with daily CFT7455 treatment to 8% of vehicle levels. Dose dependent efficacy ranged from 0.003-0.1 mg/kg/day, with tumor regression evident at doses ≥0.01 mg/kg/day. Pom was inactive in this model at a human equivalent dose (3 mg/kg/day), with no observed tumor shrinkage in these mice following 17 days of dosing. Switching pom treatment to CFT7455 (0.1 mg/kg/day) on Day 18 led to tumor regression in 67% of animals on Day 28 and 100% tumor regression on Day 35, demonstrating that CFT7455 penetrates large tissues and is efficacious in rapidly growing, IMiD resistant tumors. In the H929 tumor xenograft model, administration of CFT7455 (0.1 mg/kg/day) promoted tumor regression (95% tumor growth inhibition by 7 days); dosing was stopped after 21 days. On Day 63, half the tumors remained below their starting tumor volume. Additionally, CFT7455 demonstrated durable tumor regression in the aggressive MM1.S systemic MM tumor model. In mice bearing RPMI-8226 xenograft tumors, the combination of CFT7455 (QD) and dexamethasone (QW) was more active, and demonstrated a significant survival improvement, compared to either agent alone. Conclusions CFT7455 is a highly potent, catalytic degrader of IKZF1/3, with marked antitumor activity as a single agent and in combination with dexamethasone. Importantly, CFT7455 retains activity in models resistant or insensitive to IMiDs. These results warrant investigation of CFT7455 as a therapeutic approach for MM and a clinical study is planned. Citation Format: James A. Henderson, R. Jason Kirby, Samantha Perino, Roman V. Agafonov, Prasoon Chaturvedi, Bradley Class, David Cocozziello, Scott J. Eron, Andrew Good, Ashley A. Hart, Christina Henderson, Marta Isasa, Brendon Ladd, Matt Schnaderbeck, Michelle Mahler, Roy M. Pollock, Adam S. Crystal, Christopher G. Nasveschuk, Andrew J. Phillips, Stewart L. Fisher, David A. Proia. CFT7455: A novel, IKZF1/3 degrader that demonstrates potent tumor regression in IMiD-resistant multiple myeloma (MM) xenograft models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB007.

Hepatic steatosis, a reversible state of metabolic dysregulation, can promote the onset of nonalcoholic steatohepatitis (NASH), and its transition is thought to be critical in disease evolution. The association between endoplasmic reticulum (ER) stress response and hepatocyte metabolism disorders prompted us to characterize ER stress-induced hepatic metabolic dysfunction in human induced pluripotent stem cell-derived hepatocytes (hiPSC-Hep), to explore regulatory pathways and validate a phenotypic in vitro model for progression of liver steatosis. We treated hiPSC-Hep with a ratio of unsaturated and saturated fatty acids in the presence of an inducer of ER stress to synergistically promote triglyceride accumulation and dysregulate lipid metabolism. We monitored lipid accumulation by high-content imaging and measured gene regulation by RNA sequencing and reverse transcription quantitative PCR analyses. Our results show that ER stress potentiated intracellular lipid accumulation by 5-fold in hiPSC-Hep in the absence of apoptosis. Transcriptome pathway analysis identified ER stress pathways as the most significantly dysregulated of all pathways affected. Obeticholic acid dose dependently inhibited lipid accumulation and modulated gene expression downstream of the farnesoid X receptor. We were able to identify modulation of hepatic markers and gene pathways known to be involved in steatosis and nonalcoholic fatty liver disease (NAFLD), in support of a hiPSC-Hep disease model that is relevant to clinical data for human NASH. Our results show that the model can serve as a translational discovery platform for the understanding of molecular pathways involved in NAFLD, and can facilitate the identification of novel therapeutic molecules based on high-throughput screening strategies., Summary: Our study demonstrates expanded use of human induced pluripotent stem cell-derived hepatocytes for molecular studies and drug screening, to evaluate new therapeutics with an antisteatotic mechanism of action for nonalcoholic fatty liver disease.

Modulation of autotaxin (ATX), the lysophospholipase D enzyme that produces lysophosphatidic acid, with small-molecule inhibitors is a promising strategy for blocking the ATX-lysophosphatidic acid signaling axis. Although discovery campaigns have been successful in identifying ATX inhibitors, many of the reported inhibitors target the catalytic cleft of ATX. A recent study provided evidence for an additional inhibitory surface in the hydrophobic binding pocket of ATX, confirming prior studies that relied on enzyme kinetics and differential inhibition of substrates varying in size. Multiple hits from previous high-throughput screening for ATX inhibitors were obtained with aromatic sulfonamide derivatives interacting with the hydrophobic pocket. Here, we describe the development of a ligand-based strategy and its application in virtual screening, which yielded novel high-potency inhibitors that target the hydrophobic pocket of ATX. Characterization of the structure-activity relationship of these new inhibitors forms the foundation of a new pharmacophore model of the hydrophobic pocket of ATX.

Branching morphogenesis of the metanephric kidney is critically dependent on the delicate orchestration of diverse cellular processes including proliferation, apoptosis, migration, and differentiation. Sphingosine-1-phosphate (S1P) is a potent lipid mediator influencing many of these cellular events. We report increased expression and activity of both sphingosine kinases and S1P phosphatases during development of the mouse metanephric kidney from induction at embryonic day 11.5 to maturity. Sphingosine kinase activity exceeded S1P phosphatase activity in embryonic kidneys, resulting in a net accumulation of S1P, while kinase and phosphatase activities were similar in adult tissue, resulting in reduced S1P content. Sphingosine kinase expression was greater in the metanephric mesenchyme than in the ureteric bud, while the S1P phosphatase SPP2 was expressed at greater levels in the ureteric bud. Treatment of cultured embryonic kidneys with sphingosine kinase inhibitors resulted in a dose-dependent reduction of ureteric bud tip numbers and increased apoptosis. Exogenous S1P rescued kidneys from apoptosis induced by kinase inhibitors. Ureteric bud tip number was unaffected by exogenous S1P in kidneys treated with N, N-dimethylsphingosine, although tip number increased in those treated with d,l- threo-dihydrosphingosine. S1P1 and S1P2 were the predominant S1P receptors expressed in the embryonic kidney. S1P1 expression increased during renal development while expression of S1P2 decreased, and both receptors were expressed predominantly in the metanephric mesenchyme. These results demonstrate dynamic regulation of S1P homeostasis during renal morphogenesis and suggest that differential expression of S1P metabolic enzymes and receptors provides a novel mechanism contributing to the regulation of kidney development.

This study compared the physiological process of cholesterol absorption in different strains of inbred mice with the goal of identifying novel mechanism(s) by which cholesterol absorption can be controlled. The rate and amount of cholesterol absorption were evaluated based on [14C]cholesterol appearance in plasma after feeding a meal containing [14C]cholesterol and by the percentage of [14C]-cholesterol absorbed over a 24 h period. Results showed that the rate of [14C]cholesterol appearance in plasma was slower in 129P3/J mice than in SJL/J mice. However, more dietary cholesterol was absorbed over a 24 h period by 129P3/J mice than by SJL/J mice. In both strains of mice, cholesterol delivered with medium-chain triglyceride was absorbed less efficiently than cholesterol delivered with olive oil. The strain- and vehicle-dependent differences in cholesterol absorption efficiency correlated negatively with stomach-emptying rates. Furthermore, inhibition of gastric emptying with nitric oxide synthase inhibitor increased cholesterol absorption efficiency in SJL/J mice. These results document that stomach-emptying rate contributes directly to the rate of dietary cholesterol absorption, which is inversely correlated with the total amount of cholesterol absorbed from a single meal. Additionally, genetic factor(s) that influence gastric emptying may be an important determinant of cholesterol absorption efficiency.

Bile salt-stimulated carboxyl ester lipase (CEL), also called cholesterol esterase, is one of the major proteins secreted by the pancreas. The physiological role of CEL was originally thought to be its mediation of dietary cholesterol absorption. However, recent studies showed no difference between wild type and CEL knockout mice in the total amount of cholesterol absorbed in a single meal. The current study tests the hypothesis that CEL in the intestinal lumen may influence the type of lipoproteins produced. A lipid emulsion containing 4 mm phospholipid, 13.33 mm[3H]triolein, and 2.6 mm[14C]cholesterol in 19 mm taurocholate was infused into the duodenum of lymph fistula CEL(+/+) and CEL(−/−) mice at a rate of 0.3 ml/h. Results showed no difference between CEL(+/+) and CEL(−/−) mice in the rate of cholesterol and triglyceride transport from the intestinal lumen to the lymph. However, CEL(−/−) mice produced predominantly smaller lipoproteins, whereas the CEL(+/+) mice produced primarily large chylomicrons and very low density lipoprotein. The proximal intestine of CEL(−/−) mice was also found to possess significantly less ceramide hydrolytic activity than that present in CEL(+/+) mice. By using Caco2 cells grown on Transwell membranes as a model, sphingomyelinase treatment inhibited the secretion of larger chylomicron-like lipoproteins without affecting total cholesterol secretion. In contrast, the addition of CEL to the apical medium increased the amount of large lipoproteins produced and alleviated the inhibition induced by sphingomyelinase. Taken together, this study identified a novel and physiologically significant role for CEL, namely the promotion of large chylomicron production in the intestine. The mechanism appears to be mediated through CEL hydrolysis of ceramide generated during the lipid absorption process.

Autotaxin (ATX), a lysophospholipase D, plays an important role in cancer invasion, metastasis, tumor progression, tumorigenesis, neuropathic pain, fibrotic diseases, cholestatic pruritus, lymphocyte homing, and thrombotic diseases by producing the lipid mediator lysophosphatidic acid (LPA). A high-throughput screen of ATX inhibition using the lysophosphatidylcholine-like substrate fluorogenic substrate 3 (FS-3) and ∼10,000 compounds from the University of Cincinnati Drug Discovery Center identified several small-molecule inhibitors with IC50 vales ranging from nanomolar to low micromolar. The pharmacology of the three most potent compounds: 918013 (1; 2,4-dichloro-N-(3-fluorophenyl)-5-(4-morpholinylsulfonyl) benzamide), 931126 (2; 4-oxo-4-{2-[(5-phenoxy-1H-indol-2-yl)carbonyl]hydrazino}-N-(4-phenylbutan-2-yl)butanamide), and 966791 (3; N-(2,6-dimethylphenyl)-2-[N-(2-furylmethyl)(4-(1,2,3,4-tetraazolyl)phenyl)carbonylamino]-2-(4-hydroxy-3-methoxyphenyl) acetamide), were further characterized in enzyme, cellular, and whole animal models. Compounds 1 and 2 were competitive inhibitors of ATX-mediated hydrolysis of the lysophospholipase substrate FS-3. In contrast, compound 3 was a competitive inhibitor of both FS-3 and the phosphodiesterase substrate p-nitrophenyl thymidine 5′-monophosphate. Computational docking and mutagenesis suggested that compounds 1 and 2 target the hydrophobic pocket, thereby blocking access to the active site of ATX. The potencies of compounds 1–3 were comparable to each other in each of the assays. All of these compounds significantly reduced invasion of A2058 human melanoma cells in vitro and the colonization of lung metastases by B16-F10 murine melanoma cells in C57BL/6 mice. The compounds had no agonist or antagonist effects on select LPA or sphingosine 1-phosphate receptors, nor did they inhibit nucleotide pyrophosphatase/phosphodiesterase (NPP) enzymes NPP6 and NPP7. These results identify the molecular surface of the hydrophobic pocket of ATX as a target-binding site for inhibitors of enzymatic activity.