Amide-Linked C4″-Saccharide Modification of KRN7000 Provides Potent Stimulation of Human Invariant NKT Cells and Anti-Tumor Immunity in a Humanized Mouse Model.

Activation of invariant natural killer T (iNKT) cells by α-galactosylceramides (α-GalCers) stimulates strong immune responses and potent anti-tumor immunity. Numerous modifications of the glycolipid structure have been assessed to derive activating ligands for these T cells with altered and potentially advantageous properties in the induction of immune responses. Here, we synthesized variants of the prototypical α-GalCer, KRN7000, with amide-linked phenyl alkane substitutions on the C4″-position of the galactose ring. We show that these variants have weak iNKT cell stimulating activity in mouse models but substantially greater activity for human iNKT cells. The most active of the C4″-amides in our study showed strong anti-tumor effects in a partially humanized mouse model for iNKT cell responses. In silico analysis suggested that the tether length and degree of flexibility of the amide substituent affected the recognition by iNKT cell antigen receptors of the C4″-amide substituted glycolipids in complex with their antigen presenting molecule CD1d. Our findings establish the use of stable C4″-amide linked additions to the sugar moiety for further exploration of the immunological effects of structural modifications of iNKT cell activating glycolipids and highlight the critical need for more accurate animal models to assess these compounds for immunotherapeutic potential in humans.

Spindle Assembly Disruption and Cancer Cell Apoptosis with a CLTC-Binding Compound.

AK3 compounds are mitotic arrest agents that induce high levels of γH2AX during mitosis and apoptosis following release from arrest. We synthesized a potent AK3 derivative, AK306, that induced arrest and apoptosis of the HCT116 colon cancer cell line with an EC50 of approximately 50 nmol/L. AK306 was active on a broad spectrum of cancer cell lines with total growth inhibition values ranging from approximately 25 nmol/L to 25 µmol/L. Using biotin and BODIPY-linked derivatives of AK306, binding to clathrin heavy chain (CLTC/CHC) was observed, a protein with roles in endocytosis and mitosis. AK306 inhibited mitosis and endocytosis, while disrupting CHC cellular localization. Cells arrested in mitosis by AK306 showed the formation of multiple microtubule-organizing centers consisting of pericentrin, γ-tubulin, and Aurora A foci, without apparent centrosome amplification. Cells released from AK306 arrest were unable to form bipolar spindles, unlike nocodazole-released cells that reformed spindles and completed division. Like AK306, CHC siRNA knockdown disrupted spindle formation and activated p53. A short-term (3-day) treatment of tumor-bearing APC-mutant mice with AK306 increased apoptosis in tumors, but not normal mucosa. These findings indicate that targeting the mitotic CHC complex can selectively induce apoptosis and may have therapeutic value.Implication: Disruption of clathrin with a small-molecule inhibitor, AK306, selectively induces apoptosis in cancer cells by disrupting bipolar spindle formation. Mol Cancer Res; 16(9); 1361-72. ©2018 AACR.

Dual Modifications of α-Galactosylceramide Synergize to Promote Activation of Human Invariant Natural Killer T Cells and Stimulate Anti-tumor Immunity.

Glycosylceramides that activate CD1d-restricted invariant natural killer T (iNKT) cells have potential therapeutic applications for augmenting immune responses against cancer and infections. Previous studies using mouse models identified sphinganine variants of α-galactosylceramide as promising iNKT cell activators that stimulate cytokine responses with a strongly proinflammatory bias. However, the activities of sphinganine variants in mice have generally not translated well to studies of human iNKT cell responses. Here, we show that strongly proinflammatory and anti-tumor iNKT cell responses were achieved in mice by a variant of α-galactosylceramide that combines a sphinganine base with a hydrocinnamoyl ester on C6″ of the sugar. Importantly, the activities observed with this variant were largely preserved for human iNKT cell responses. Structural and in silico modeling studies provided a mechanistic basis for these findings and suggested basic principles for capturing useful properties of sphinganine analogs of synthetic iNKT cell activators in the design of immunotherapeutic agents.

High-mobility-group protein 2 regulated by microRNA-127 and small heterodimer partner modulates pluripotency of mouse embryonic stem cells and liver tumor initiating cells.

High-mobility-group protein 2 (HMGB2) expression is upregulated in human liver cancer. However, little is known about its regulatory function. Here we establish HMGB2 as a new modulator of the pluripotency of mouse embryonic stem cells (ESCs). Similar to OCT4 and SOX2, HMGB2 protein is highly expressed in undifferentiated CGR8 cells, whereas it undergoes rapid decline during embryonic body (EB) formation. HMGB2 interacts with OCT4, increases protein expression of OCT4 and SOX2, and enhances their transcriptional activities. We also show that miRNA-127 is a translational repressor of HMGB2 protein expression by targeting its 3'UTR. We further elucidate a trancrptional mechanism controlling HMGB2 mRNA expression by nuclear receptor SHP and transcription factor E2F1. Diminishing HMGB2 expression by ectopic expression of miR-127 or SHP, or treatment with a small molecule inhibitor inflachromene (ICM), decreases OCT4 and SOX2 expression and facilitates CGR8 differentiation. In addition, HMGB2 is markedly induced in liver tumor initiating cells (TICs). Diminishing HMGB2 expression by shHMGB2, miR-127 or SHP impaires spheroid formation. Importantly, HMGB2 expression is elevated in various human cancers. Conclusion: HMGB2 acts upstream of the OCT4/SOX2 signaling to control ESCs pluripotency. Diminishing HMGB2 expression by miR-127 or SHP may provide a potential means to decrease the pluripotency of tumor initiating cells.

Toward a formal synthesis of laureatin: unexpected rearrangements involving cyclic ether nucleophiles.

Laureatin, a metabolite of the red algae Laurencia nipponica, has shown potent activity as a mosquito larvicide. The two previously published syntheses of laureatin involved an initial preparation of the 8-membered cyclic ether, followed by formation of the oxetane ring. Our strategy was the reverse, i.e., to utilize an oxetane as the framework to construct the larger ring. During this work, attempted N-bromosuccinimide (NBS)-mediated cyclization of oxetane alcohol 17, prepared from readily accessible 2-methyleneoxetane 12, yielded epoxytetrahydrofuran 19 rather than the expected laureatin core. Further derivatization of 19 yielded trans fused bis-tetrahydrofuran 32. The synthesis of 19 and 32, as well as structural and stereochemical elucidation studies, are described.

Adaptability of the semi-invariant natural killer T-cell receptor towards structurally diverse CD1d-restricted ligands.

The semi-invariant natural killer (NK) T-cell receptor (NKTcr) recognises structurally diverse glycolipid antigens presented by the monomorphic CD1d molecule. While the alpha-chain of the NKTcr is invariant, the beta-chain is more diverse, but how this diversity enables the NKTcr to recognise diverse antigens, such as an alpha-linked monosaccharide (alpha-galactosylceramide and alpha-galactosyldiacylglycerol) and the beta-linked trisaccharide (isoglobotriaosylceramide), is unclear. We demonstrate here that NKTcrs, which varied in their beta-chain usage, recognised diverse glycolipid antigens with a similar binding mode on CD1d. Nevertheless, the NKTcrs recognised distinct epitopic sites within these antigens, including alpha-galactosylceramide, the structurally similar alpha-galactosyldiacylglycerol and the very distinct isoglobotriaosylceramide. We also show that the relative roles of the CDR loops within the NKTcr beta-chain varied as a function of the antigen. Thus, while NKTcrs characteristically use a conserved docking mode, the NKTcr beta-chain allows these cells to recognise unique aspects of structurally diverse CD1d-restricted ligands.