Delivery of a BET protein degrader via a CEACAM6-targeted antibody-drug conjugate inhibits tumour growth in pancreatic cancer models.

Pancreatic ductal adenocarcinoma (PDAC) has the worst prognosis of all cancers. To improve PDAC therapy, we establish screening systems based on organoid and co-culture technologies and find a payload of antibody-drug conjugate (ADC), a bromodomain and extra-terminal (BET) protein degrader named EBET. We select CEACAM6/CD66c as an ADC target and developed an antibody, #84.7, with minimal reactivity to CEACAM6-expressing normal cells. EBET-conjugated #84.7 (84-EBET) has lethal effects on various PDAC organoids and bystander efficacy on CEACAM6-negative PDAC cells and cancer-associated fibroblasts. In mouse studies, a single injection of 84-EBET induces marked tumor regression in various PDAC-patient-derived xenografts, with a decrease in the inflammatory phenotype of stromal cells and without significant body weight loss. Combination with standard chemotherapy or PD-1 antibody induces more profound and sustained regression without toxicity enhancement. Our preclinical evidence demonstrates potential efficacy by delivering BET protein degrader to PDAC and its microenvironment via CEACAM6-targeted ADC.

ER-851, a Novel Selective Inhibitor of AXL, Overcomes Resistance to Antimitotic Drugs.

Innate and adaptive resistance to cancer therapies, such as chemotherapies, molecularly targeted therapies, and immune-modulating therapies, is a major issue in clinical practice. Subpopulations of tumor cells expressing the receptor tyrosine kinase AXL become enriched after treatment with antimitotic drugs, causing tumor relapse. Elevated AXL expression is closely associated with drug resistance in clinical samples, suggesting that AXL plays a pivotal role in drug resistance. Although several molecules with AXL inhibitory activity have been developed, none have sufficient activity and selectivity to be clinically effective when administered in combination with a cancer therapy. Here, we report a novel small molecule, ER-851, which is a potent and highly selective AXL inhibitor. To investigate resistance mechanisms and identify driving molecules, we conducted a comprehensive gene expression analysis of chemoresistant tumor cells in mouse xenograft models of genetically engineered human lung cancer and human triple-negative breast cancer. Consistent with the effect of AXL knockdown, cotreatment of ER-851 and antimitotic drugs produced an antitumor effect and prolonged relapse-free survival in the mouse xenograft model of human triple-negative breast cancer. Importantly, when orally administered to BALB/c mice, this compound did not induce retinal toxicity, a known side effect of chronic MER inhibition. Together, these data strongly suggest that AXL is a therapeutic target for overcoming drug resistance and that ER-851 is a promising candidate therapeutic agent for use against AXL-expressing antimitotic-resistant tumors.

Oxa-Michael-based divergent synthesis of artificial glutamate analogs.

Herein we report stereoselective generation of two skeletons, 1,3-dioxane and tetrahydropyranol, by oxa-Michael reaction as the key reaction from δ-hydroxyenone. The construction of the 1,3-dioxane skeleton, achieved through hemiacetal formation followed by oxa-Michael reaction from δ-hydroxyenone, was exploited to access structurally diverse heterotricyclic artificial glutamate analogs. On the other hand, formation of a novel tetrahydro-2H-pyranol skeleton was accomplished by the inverse reaction order: oxa-Michael reaction followed by hemiacetal formation. Thus, this study succeeded in showing that structural diversity in a compound collection can be acquired by interchanging the order of just two reactions. Among the skeletally diverse, heterotricyclic artificial glutamate analogs synthesized in this study, a neuronally active compound named TKM-50 was discovered in the mice in vivo assay.

Discovery of 5,6,7,8-tetrahydropyrido[3,4-d]pyrimidine derivatives as novel selective Axl inhibitors.

Axl and Mer are members of the TAM (Tyro3-Axl-Mer) family of receptor tyrosine kinases. Previously, we reported that enzyme-mediated inhibition of Mer by an Axl/Mer dual inhibitor led to retinal toxicity in mice, whereas selective Axl inhibition by compound 1 did not. On the other hand, compound 1 showed low membrane permeability. Here, we designed and synthesized a novel series of 5,6,7,8-tetrahydropyrido[3,4-d]pyrimidine derivatives and evaluated their Axl and Mer inhibitory activities, leading to identification of ER-001259851-000 as a potent and selective Axl inhibitor with drug-likeness and a promising pharmacokinetic profile in mice.

Discovery of a potent and selective Axl inhibitor in preclinical model.

Axl and Mer are a members of the TAM (Tyro3-Axl-Mer) family of receptor tyrosine kinases, which, when activated, can promote tumor cell survival, proliferation, migration, invasion, angiogenesis, and tumor-host interactions. Chronic inhibition of Mer leads to retinal toxicity in mice. Therefore, successful development of an Axl targeting agent requires ensuring that it is safe for prolonged treatment. Here, to clarify whether enzyme inhibition of Mer by a small molecule leads to retinal toxicity in mice, we designed and synthesized Axl/Mer inhibitors and Axl-selective inhibitors. We identified an Axl/Mer dual inhibitor 28a, which showed retinal toxicity at a dose of 100 mg/kg in mice. Subsequent derivatization of a pyridine derivative led to the discovery of a pyrimidine derivative, 33g, which selectively inhibited the activity of Axl over Mer without retinal toxicity at a dose of 100 mg/kg in mice. Additionally, the compound displayed in vivo anti-tumor effects without influencing body weight in a Ba/F3-Axl isogenic subcutaneous model.

Menthyl esterification allows chiral resolution for the synthesis of artificial glutamate analogs.

Herein, we report the enantiospecific synthesis of two artificial glutamate analogs designed based on IKM-159, an antagonist selective to the AMPA-type ionotropic glutamate receptor. The synthesis features the chiral resolution of the carboxylic acid intermediate by the esterification with ʟ-menthol, followed by a configurational analysis by NMR, conformational calculation, and X-ray crystallography. A mice in vivo assay showed that (2R)-MC-27, with a six-membered oxacycle, is neuroactive, whereas the (2S)-counterpart is inactive. It was also found that TKM-38, with an eight-membered azacycle, is neuronally inactive, showing that the activity is controlled by the ring C.

Valproic acid-exposed astrocytes impair inhibitory synapse formation and function.

Valproic acid (VPA) is widely prescribed to treat epilepsy. Maternal VPA use is, however, clinically restricted because of the severe risk that VPA may cause neurodevelopmental disorders in offspring, such as autism spectrum disorder. Understanding the negative action of VPA may help to prevent VPA-induced neurodevelopmental disorders. Astrocytes play a vital role in neurodevelopment and synapse function; however, the impact of VPA on astrocyte involvement in neurodevelopment and synapse function has not been examined. In this study, we examined whether exposure of cultured astrocytes to VPA alters neuronal morphology and synapse function of co-cultured neurons. We show that synaptic transmission by inhibitory neurons was small because VPA-exposed astrocytes reduced the number of inhibitory synapses. However, synaptic transmission by excitatory neurons and the number of excitatory synapses were normal with VPA-exposed astrocytes. VPA-exposed astrocytes did not affect the morphology of inhibitory neurons. These data indicate that VPA-exposed astrocytes impair synaptogenesis specifically of inhibitory neurons. Our results indicate that maternal use of VPA would affect not only neurons but also astrocytes and would result in perturbed astrocyte-mediated neurodevelopment.

Epigallocatechin-3-O-gallate up-regulates microRNA-let-7b expression by activating 67-kDa laminin receptor signaling in melanoma cells.

MicroRNAs (miRNAs) are non-coding RNAs involved in various biological processes by regulating their target genes. Green tea polyphenol (-)-epigallocatechin-3-O-gallate (EGCG) inhibits melanoma tumor growth by activating 67-kDa laminin receptor (67LR) signaling. To examine the effect of EGCG on miRNA expression in melanoma cells, we performed miRNA microarray analysis. We showed that EGCG up-regulated miRNA-let-7b expression through 67LR in melanoma cells. The EGCG-induced up-regulation of let-7b led to down-regulation of high mobility group A2 (HMGA2), a target gene related to tumor progression. 67LR-dependent cAMP/protein kinase A (PKA)/protein phosphatase 2A (PP2A) signaling pathway activation was involved in the up-regulation of let-7b expression induced by EGCG. These findings provide a basis for understanding the mechanism of miRNA regulation by EGCG.

Sphingosine Kinase-1 Protects Multiple Myeloma from Apoptosis Driven by Cancer-Specific Inhibition of RTKs.

Activation of acid sphingomyelinase (ASM) leads to ceramide accumulation and induces apoptotic cell death in cancer cells. In the present study, we demonstrate that the activation of ASM by targeting cancer-overexpressed 67-kDa laminin receptors (67LR) induces lipid raft disruption and inhibits receptor tyrosine kinase (RTK) activation in multiple myeloma cells. Sphingosine kinase 1 (SphK1), a negative regulator of ceramide accumulation with antiapoptotic effects, was markedly elevated in multiple myeloma cells. The silencing of SphK1 potentiated the apoptotic effects of the green tea polyphenol epigallocatechin-3-O-gallate (EGCG), an activator of ASM through 67LR. Furthermore, the SphK1 inhibitor safingol synergistically sensitized EGCG-induced proapoptotic cell death and tumor suppression in multiple myeloma cells by promoting the prevention of RTK phosphorylation and activation of death-associated protein kinase 1 (DAPK1). We propose that targeting 67LR/ASM and SphK1 may represent a novel therapeutic strategy against multiple myeloma.

Green tea polyphenol epigallocatechin-O-gallate induces cell death by acid sphingomyelinase activation in chronic myeloid leukemia cells.

An epidemiological study showed that green tea consumption is associated with a reduced risk of hematopoietic malignancy. The major green tea polyphenol epigallocatechin­3-O-gallate (EGCG) is reported to have anticancer effects. Chronic myeloid leukemia (CML) is a major hematopoietic malignancy characterized by expansion of myeloid cells. In the present study, we showed EGCG-induced acid sphingomyelinase (ASM) activation and lipid raft clustering in CML cells. The ASM inhibitor desipramine significantly reduced EGCG-induced cell death. Protein kinase Cδ is a well­known kinase that plays an important role in ASM activation. We observed EGCG-induced phosphorylation of protein kinase Cδ at Ser664. Importantly, EGCG-induced ASM activation was significantly reduced by pretreatment of CML cells with the soluble guanylate cyclase inhibitor NS2028, suggesting that EGCG induced ASM activation through the cyclic guanosine monophosphate (cGMP)-dependent pathway. Indeed, pharmacological inhibition of a cGMP-negative regulator enhanced the anti-CML effect of EGCG. These results indicate that EGCG-induced cell death via the cGMP/ASM pathway in CML cells.

67-kDa laminin receptor-dependent protein phosphatase 2A (PP2A) activation elicits melanoma-specific antitumor activity overcoming drug resistance.

The Ras/Raf/MEK/ERK pathway has been identified as a major, druggable regulator of melanoma. Mutational activation of BRAF is the most prevalent genetic alteration in human melanoma, resulting in constitutive melanoma hyperproliferation. A selective BRAF inhibitor showed remarkable clinical activity in patients with mutated BRAF. Unfortunately, most patients acquire resistance to the BRAF inhibitor, highlighting the urgent need for new melanoma treatment strategies. Green tea polyphenol (-)-epigallocatechin-3-O-gallate (EGCG) inhibits cell proliferation independently of BRAF inhibitor sensitivity, suggesting that increased understanding of the anti-melanoma activity of EGCG may provide a novel therapeutic target. Here, by performing functional genetic screening, we identified protein phosphatase 2A (PP2A) as a critical factor in the suppression of melanoma cell proliferation. We demonstrated that tumor-overexpressed 67-kDa laminin receptor (67LR) activates PP2A through adenylate cyclase/cAMP pathway eliciting inhibitions of oncoproteins and activation of tumor suppressor Merlin. Activating 67LR/PP2A pathway leading to melanoma-specific mTOR inhibition shows strong synergy with the BRAF inhibitor PLX4720 in the drug-resistant melanoma. Moreover, SET, a potent inhibitor of PP2A, is overexpressed on malignant melanoma. Silencing of SET enhances 67LR/PP2A signaling. Collectively, activation of 67LR/PP2A signaling may thus be a novel rational strategy for melanoma-specific treatment.

Green tea extract containing a highly absorbent catechin prevents diet-induced lipid metabolism disorder.

We investigated the effects of extracts of Benifuuki (a tea cultivar that contains methylated catechins such as epigallocatechin-3-O-(3-O-methyl) gallate (EGCG3"Me)) in mice fed a high-fat/high-sucrose (HF/HS) diet. This tea cultivar was then compared with an extract of Yabukita (a popular tea cultivar that lacks methylated catechins). For 6 weeks, C57BL/6J mice were fed either HF/HS diet with or without tea extracts from tea cultivars, which contained almost identical ingredients except for methylated catechins (i.e., Yabukita (0.2% and 1%) or Benifuuki (0.2% and 1%) extract powders). Supplementation with Benifuuki 0.2% markedly lowered plasma levels of TG and NEFAs compared with mice supplemented with Yabukita 0.2%. The diet containing Benifuuki 1% decreased adipose tissue weights, liver TG, and expression of lipogenic genes in the liver. These results suggested that Benifuuki had much greater lipid-lowering effects than Yabukita. Taken together, these data suggest that methylated catechins direct the strong lipid-lowering activity of Benifuuki.

Phosphodiesterase 5 inhibitor acts as a potent agent sensitizing acute myeloid leukemia cells to 67-kDa laminin receptor-dependent apoptosis.

(-)-Epigallocatechin-3-O-gallate (EGCG), a polyphenol in green tea, induces apoptosis in acute myeloid leukemia (AML) cells without affecting normal cells. In this study, we observed that cGMP acts as a cell death mediator of the EGCG-induced anti-AML effect through acid sphingomyelinase activation. EGCG activated the Akt/eNOS axis, a well-known mechanism in vascular cGMP upregulation. We also observed that a major cGMP negative regulator, phosphodiesterase 5, was overexpressed in AML cells, and PDE5 inhibitor, an anti-erectile dysfunction drug, synergistically enhanced the anti-AML effect of EGCG. This combination regimen killed AML cells via overexpressed 67-kDa laminin receptors.

67-kDa laminin receptor increases cGMP to induce cancer-selective apoptosis.

The 67-kDa laminin receptor (67LR) is a laminin-binding protein overexpressed in various types of cancer, including bile duct carcinoma, colorectal carcinoma, cervical cancer, and breast carcinoma. 67LR plays a vital role in growth and metastasis of tumor cells and resistance to chemotherapy. Here, we show that 67LR functions as a cancer-specific death receptor. In this cell death receptor pathway, cGMP initiated cancer-specific cell death by activating the PKCδ/acid sphingomyelinase (PKCδ/ASM) pathway. Furthermore, upregulation of cGMP was a rate-determining process of 67LR-dependent cell death induced by the green tea polyphenol (-)-epigallocatechin-3-O-gallate (EGCG), a natural ligand of 67LR. We found that phosphodiesterase 5 (PDE5), a negative regulator of cGMP, was abnormally expressed in multiple cancers and attenuated 67LR-mediated cell death. Vardenafil, a PDE5 inhibitor that is used to treat erectile dysfunction, significantly potentiated the EGCG-activated 67LR-dependent apoptosis without affecting normal cells and prolonged the survival time in a mouse xenograft model. These results suggest that PDE5 inhibitors could be used to elevate cGMP levels to induce 67LR-mediated, cancer-specific cell death.

Isoflavones suppress the expression of the FcεRI high-affinity immunoglobulin E receptor independent of the estrogen receptor.

Isoflavones found in soybeans and soy products possess clinically relevant properties. However, the anti-allergic effect of isoflavones has been poorly studied. We examined the effects of isoflavones, genistein, daidzein, and equol, on the expression of the high-affinity immunoglobulin E (IgE) receptor, FcεRI, which plays a central role in IgE-mediated allergic response. Flow cytometric analysis showed that all of these isoflavones reduced the cell surface expression of FcεRI on mouse bone-marrow-derived mast cells and human basophilic KU812 cells. All isoflavones decreased the levels of the FcεRIα mRNA in the cells. Genistein reduced the mRNA expression of the ß chain, and daidzein and equol downregulated that of the γ chain. The suppressive effects of isoflavones on FcεRI expression were unaffected by ICI 182,780, an estrogen receptor antagonist, suggesting that these effects were independent of estrogen receptors.

Oxygen partial pressure modulates 67-kDa laminin receptor expression, leading to altered activity of the green tea polyphenol, EGCG.

(-)-Epigallocatechin-3-O-gallate (EGCG) exhibits anti-tumor activity mediated via the 67-kDa laminin receptor (67LR). In this study, we found that 67LR protein levels are reduced by exposure to low O(2) levels (5%), without affecting the expression of HIF-1α. We also found that EGCG-induced anti-cancer activity is abrogated under low O(2) levels (5%) in various cancer cells. Notably, treatment with the proteasome inhibitor, prevented down-regulation of 67LR and restored sensitivity to EGCG under 5% O(2). In summary, 67LR expression is highly sensitive to O(2) partial pressure, and the activity of EGCG can be regulated in cancer cells by O(2) partial pressure.

Green tea polyphenol EGCG sensing motif on the 67-kDa laminin receptor.

BACKGROUND: We previously identified the 67-kDa laminin receptor (67LR) as the cell-surface receptor conferring the major green tea polyphenol (-)-epigallocatechin-3-O-gallate (EGCG) responsiveness to cancer cells. However, the underlying mechanism for interaction between EGCG and 67LR remains unclear. In this study, we investigated the possible role of EGCG-67LR interaction responsible for its bioactivities. METHODOLOGY/PRINCIPAL FINDINGS: We synthesized various peptides deduced from the extracellular domain corresponding to the 102-295 region of human 67LR encoding a 295-amino acid. The neutralizing activity of these peptides toward EGCG cell-surface binding and inhibition of cancer cell growth were assayed. Both activities were inhibited by a peptide containing the 10-amino acid residues, IPCNNKGAHS, corresponding to residues 161-170. Furthermore, mass spectrometric analysis revealed the formation of a EGCG-LR161-170 peptide complex. A study of the amino acid deletion/replacement of the peptide LR161-170 indicated that the 10-amino acid length and two basic amino acids, K(166) and H(169), have a critical role in neutralizing EGCG's activities. Moreover, neutralizing activity against the anti-proliferation action of EGCG was observed in a recombinant protein of the extracellular domain of 67LR, and this effect was abrogated by a deletion of residues 161-170. These findings support that the 10 amino-acid sequence, IPCNNKGAHS, might be the functional domain responsible for the anti-cancer activity of EGCG. CONCLUSIONS/SIGNIFICANCE: Overall, our results highlight the nature of the EGCG-67LR interaction and provide novel structural insights into the understanding of 67LR-mediated functions of EGCG, and could aid in the development of potential anti-cancer compounds for chemopreventive or therapeutic uses that can mimic EGCG-67LR interactions.

Green tea polyphenol EGCG induces lipid-raft clustering and apoptotic cell death by activating protein kinase Cδ and acid sphingomyelinase through a 67 kDa laminin receptor in multiple myeloma cells.

EGCG [(-)-epigallocatechin-3-O-gallate], the major polyphenol of green tea, has cancer chemopreventive and chemotherapeutic activities. EGCG selectively inhibits cell growth and induces apoptosis in cancer cells without adversely affecting normal cells; however, the underlying molecular mechanism in vivo is unclear. In the present study, we show that EGCG-induced apoptotic activity is attributed to a lipid-raft clustering mediated through 67LR (67 kDa laminin receptor) that is significantly elevated in MM (multiple myeloma) cells relative to normal peripheral blood mononuclear cells, and that aSMase (acid sphingomyelinase) is critical for the lipid-raft clustering and the apoptotic cell death induced by EGCG. We also found that EGCG induces aSMase translocation to the plasma membrane and PKCδ (protein kinase Cδ) phosphorylation at Ser664, which was necessary for aSMase/ceramide signalling via 67LR. Additionally, orally administered EGCG activated PKCδ and aSMase in a murine MM xenograft model. These results elucidate a novel cell-death pathway triggered by EGCG for the specific killing of MM cells.