Syk Inhibition Reprograms Tumor-Associated Macrophages and Overcomes Gemcitabine-Induced Immunosuppression in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is an insidious disease with a low 5-year survival rate. PDAC is characterized by infiltration of abundant tumor-associated macrophages (TAM), which promote immune tolerance and immunotherapeutic resistance. Here we report that macrophage spleen tyrosine kinase (Syk) promotes PDAC growth and metastasis. In orthotopic PDAC mouse models, genetic deletion of myeloid Syk reprogrammed macrophages into immunostimulatory phenotype, increased the infiltration, proliferation, and cytotoxicity of CD8+ T cells, and repressed PDAC growth and metastasis. Furthermore, gemcitabine (Gem) treatment induced an immunosuppressive microenvironment in PDAC by promoting protumorigenic polarization of macrophages. In contrast, treatment with the FDA-approved Syk inhibitor R788 (fostamatinib) remodeled the tumor immune microenvironment, "re-educated" protumorigenic macrophages towards an immunostimulatory phenotype and boosted CD8+ T-cell responses in Gem-treated PDAC in orthotopic mouse models and an ex vivo human pancreatic slice culture model. These findings illustrate the potential of Syk inhibition for enhancing the antitumor immune responses in PDAC and support the clinical evaluation of R788 either alone or together with Gem as a potential treatment strategy for PDAC. SIGNIFICANCE: Syk blockade induces macrophage polarization to an immunostimulatory phenotype, which enhances CD8+ T-cell responses and improves gemcitabine efficacy in pancreatic ductal adenocarcinoma, a clinically challenging malignancy.

Targeting macrophage Syk enhances responses to immune checkpoint blockade and radiotherapy in high-risk neuroblastoma.

Background: Neuroblastoma (NB) is considered an immunologically cold tumor and is usually less responsive to immune checkpoint blockade (ICB). Tumor-associated macrophages (TAMs) are highly infiltrated in NB tumors and promote immune escape and resistance to ICB. Hence therapeutic strategies targeting immunosuppressive TAMs can improve responses to ICB in NB. We recently discovered that spleen tyrosine kinase (Syk) reprograms TAMs toward an immunostimulatory phenotype and enhances T-cell responses in the lung adenocarcinoma model. Here we investigated if Syk is an immune-oncology target in NB and tested whether a novel immunotherapeutic approach utilizing Syk inhibitor together with radiation and ICB could provide a durable anti-tumor immune response in an MYCN amplified murine model of NB. Methods: Myeloid Syk KO mice and syngeneic MYCN-amplified cell lines were used to elucidate the effect of myeloid Syk on the NB tumor microenvironment (TME). In addition, the effect of Syk inhibitor, R788, on anti-tumor immunity alone or in combination with anti-PDL1 mAb and radiation was also determined in murine NB models. The underlying mechanism of action of this novel therapeutic combination was also investigated. Results: Herein, we report that Syk is a marker of NB-associated macrophages and plays a crucial role in promoting immunosuppression in the NB TME. We found that the blockade of Syk in NB-bearing mice markedly impairs tumor growth. This effect is facilitated by macrophages that become immunogenic in the absence of Syk, skewing the suppressive TME towards immunostimulation and activating anti-tumor immune responses. Moreover, combining FDA-approved Syk inhibitor, R788 (fostamatinib) along with anti-PDL1 mAb provides a synergistic effect leading to complete tumor regression and durable anti-tumor immunity in mice bearing small tumors (50 mm3) but not larger tumors (250 mm3). However, combining radiation to R788 and anti-PDL1 mAb prolongs the survival of mice bearing large NB9464 tumors. Conclusion: Collectively, our findings demonstrate the central role of macrophage Syk in NB progression and demonstrate that Syk blockade can "reeducate" TAMs towards immunostimulatory phenotype, leading to enhanced T cell responses. These findings further support the clinical evaluation of fostamatinib alone or with radiation and ICB, as a novel therapeutic intervention in neuroblastoma.

Rapid detection of FMO3 single nucleotide polymorphisms using a pyrosequencing method.

The present study aimed to develop a reliable pyrosequencing method to detect four single nucleotide polymorphisms (SNPs) of the flavin­containing monooxygenase 3 (FMO3) gene and to compare the ethnic differences in their allelic frequencies. The pyrosequencing method was used to detect four FMO3 SNPs, namely, c.855C>T (N285N, rs909530), c.441C>T (S147S, rs1800822), c.923A>G (E308G, rs2266780) and c.472G>A (E158K, rs2266782). The allelic frequencies of these SNPs in 122 unrelated Korean subjects were as follows: i) 44.7% for c.855C>T; ii) 23.4% for c.441C>T; iii) 23.0% for c.923A>G; and iv) 27.1% for c.472G>A. Linkage disequilibrium (LD) analysis revealed that the SNPs c.923A>G and c.472G>A exhibited a strong LD (D'=0.8289, r2=0.5332). In conclusion, the pyrosequencing method developed in this study was successfully applied to detect the c.855C>T, c.441C>T, c.923A>G and c.472G>A SNPs of FMO3.

Influence of FMO3 and CYP3A4 Polymorphisms on the Pharmacokinetics of Teneligliptin in Humans.

Teneligliptin, a dipeptidyl peptidase-4 inhibitor, is used to treat type 2 diabetes mellitus. FMO3 and CYP3A4 metabolize teneligliptin into teneligliptin sulfoxide. This study examined the effects of FMO3 (rs909530, rs1800822, rs2266780, and rs2266782) and CYP3A4 (rs2242480) polymorphisms on teneligliptin pharmacokinetics at a steady state among 23 healthy participants administered 20 mg teneligliptin daily for 6 days. Subjects with FMO3 rs909530, rs2266780, and rs2266782 polymorphisms exhibited a significant gene dosage-dependent increase in maximum steady-state plasma drug concentration (Cmax,ss) and area under the drug concentration vs time curve (AUC) (p<0.05). However, the Cmax values significantly decreased but the AUC values did not significantly vary in subjects with CYP3A4 polymorphism (rs2242480). These results suggest that FMO3 and CYP3A4 polymorphisms affect teneligliptin pharmacokinetics in humans. The findings of this study provide a scientific basis for the inter-individual variation in teneligliptin disposition.

Development and validation of LC-MS/MS method for simultaneous determination of dabigatran etexilate and its active metabolites in human plasma, and its application in a pharmacokinetic study.

Dabigatran is a direct thrombin inhibitor widely used for preventing various thrombotic events. Although there are several established LC-MS/MS based methods for quantification of plasma dabigatran etexilate and its active metabolites (dabigatran and dabigatran acylglucuronide), so far, there are no studies for simultaneous quantification of dabigatran etexilate, dabigatran, and dabigatran acylglucuronide in human plasma samples. In the present study, a novel and sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed and validated for assessment of dabigatran pharmacokinetics in human plasma samples according to FDA guidelines. We used the new method to simultaneously quantify dabigatran etexilate, dabigatran, and dabigatran acylglucuronide in human plasma samples. After deproteinization using acetonitrile, the supernatants were evaporated, dissolved in a mobile phase, and finally injected onto an HPLC system with a silica-based C18 reverse-phase column. Mass spectrometer system was operated in multiple reaction monitoring mode (MRM) (dabigatran etexilate: m/z 629.464→290.100; dabigatran: m/z 472.300→289.100, and dabigatran acylglucuronide: m/z 648.382→289.100) and all the components were confirmed using positive electrospray ionization (ESI). Correlation coefficients > 0.999 were achieved for all the calibration curves with linear regression. The intra-day and inter-day accuracies of dabigatran etexilate, dabigatran, and dabigatran acylglucuronide were 95.84-109.44 %, 99.4-103.42 %, and 98.37-104.42 %, respectively, while their corresponding precisions were 3.84-9.79, 1.07-8.76 %, and 2.56-4.51 %, respectively. We successfully applied the new method to determine the pharmacokinetic profiles of dabigatran etexilate, dabigatran, and dabigatran acylglucuronide in humans. Our findings demonstrated the method as robust, reliable, and sensitive.

ABCG2 Single Nucleotide Polymorphism Affects Imatinib Pharmacokinetics in Lower Alpha-1-Acid Glycoprotein Levels in Humans.

Imatinib is transported extracellularly by ABCB1 and ABCG2 efflux transporters and bound to alpha-1-acid glycoprotein (AGP) in the bloodstream. However, the clinical and pharmacokinetic effects of ABCB1 and ABCG2 on imatinib were inconsistent in the previous literature and have not been confirmed. Therefore, in the present study, we explored the effects of the ABCG2 and ABCB1 genetic polymorphisms on imatinib pharmacokinetics in association with plasma AGP levels in healthy subjects. Twenty-seven healthy individuals were recruited, genotyped for ABCG2 and ABCB1, and given a single oral dose of 400 mg imatinib. Plasma imatinib concentrations were measured and its pharmacokinetics was assessed with respect to ABCG2 (c.421C>A and c.34G>A) and ABCB1 (c.1236C>T, c.2677C>T/A, and c.3435C>T) genotypes, and plasma AGP levels. AGP levels showed a strong positive correlation with imatinib pharmacokinetics. ABCG2 c.421C>A single nucleotide polymorphism showed a statistically significant effect on imatinib pharmacokinetics in low plasma AGP levels groups (<80 mg/dl); subjects with high plasma AGP levels (n = 5, ≥80 mg/dl) were excluded. The results indicate that plasma AGP levels and ABCG2 polymorphisms modulated imatinib pharmacokinetics; however, the effects of the ABCG2 transporter was masked at high plasma AGP levels.

Radiobiological mechanisms of stereotactic body radiation therapy and stereotactic radiation surgery.

Despite the increasing use of stereotactic body radiation therapy (SBRT) and stereotactic radiation surgery (SRS) in recent years, the biological base of these high-dose hypo-fractionated radiotherapy modalities has been elusive. Given that most human tumors contain radioresistant hypoxic tumor cells, the radiobiological principles for the conventional multiple-fractionated radiotherapy cannot account for the high efficacy of SBRT and SRS. Recent emerging evidence strongly indicates that SBRT and SRS not only directly kill tumor cells, but also destroy the tumor vascular beds, thereby deteriorating intratumor microenvironment leading to indirect tumor cell death. Furthermore, indications are that the massive release of tumor antigens from the tumor cells directly and indirectly killed by SBRT and SRS stimulate anti-tumor immunity, thereby suppressing recurrence and metastatic tumor growth. The reoxygenation, repair, repopulation, and redistribution, which are important components in the response of tumors to conventional fractionated radiotherapy, play relatively little role in SBRT and SRS. The linear-quadratic model, which accounts for only direct cell death has been suggested to overestimate the cell death by high dose per fraction irradiation. However, the model may in some clinical cases incidentally do not overestimate total cell death because high-dose irradiation causes additional cell death through indirect mechanisms. For the improvement of the efficacy of SBRT and SRS, further investigation is warranted to gain detailed insights into the mechanisms underlying the SBRT and SRS.

Spermidine inhibits MMP-2 via modulation of histone acetyltransferase and histone deacetylase in HDFs.

In recent years, aging has been reported to be regulated by HAT. In this study, the inhibitory effects of spermidine on the matrix metalloproteinase-2 (MMP-2) activity and expression were investigated in human dermal fibroblasts (HDFs). It was observed that spermidine inhibits MMP-2 activity and expression. In addition, the expression levels of histone acetyltransferase (HAT), phospho-extracellular-signal related kinase (p-ERK), phospho-c-jun N-terminal kinase (p-JNK), and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) are decreased in the presence of spermidine. In contrast, the expression levels of histone deacetylase 1 (HDAC1), sirtuin 1 (SIRT1), phospho-p38 (p-p38) are increased by spermidine. In conclusion, our results suggest that spermidine could have a therapeutic potential in inhibition of metastasis through the inhibitory effects on activity and expression of MMP-2 via regulation of HAT and HDAC.

Advanced adipose-derived stem cell protein extracts with antioxidant activity modulates matrix metalloproteinases in human dermal fibroblasts.

Advanced adipose-derived stem cell protein extracts (AAPE) were used instead of live stem cells to investigate their effect on oxidative stress and matrix metalloproteinases (MMPs) related to tissue repair in human dermal fibroblasts (HDFs). In this study, it was observed that AAPE at 2µg/ml specifically exhibited scavenging activity of hydrogen peroxide and reducing power. The inhibitory effect of AAPE at 2µg/ml on MMP-2 activity was increased in the presence of phorbol myristate acetate (PMA). In the absence of PMA, AAPE significantly enhanced activities of MMP-1 and MMP-2 in HDFs, respectively. However, the level of MMP-1 expression was decreased in a dose dependent manner by AAPE. In addition, while the level of extracellular signal-regulated kinases 1 (ERK1) activation was reduced in the presence of AAPE compared to blank, the level that of ERK2 activation was not changed. The expression level of c-Fos, a part of activator protein-1 (AP-1), was increased in nucleus of HDFs. These results reveal that activation of MMPs in the presence of AAPE was increased via AP-1 in HDFs, suggesting that AAPE can be a potential candidate for tissue repair.