Small Molecule Inhibitor Targeting Research Articles

The efficacy and safety of apatinib in patients with recurrent or metastatic nasopharyngeal carcinoma: a systematic review and meta-analysis.

BackgroundApatinib is a small-molecule tyrosine kinase inhibitor targeting VEGFR-2, which was recently used in a phase II clinical trial for the treatment of recurrent or metastatic nasopharyngeal carcinoma (rmNPC). However, there is no consistent conclusion on its efficacy and safety on rmNPC. This study conducted a meta-analysis of clinical research on the efficacy and safety of apatinib in the treatment of rmNPC.MethodsIn April 2022, the PubMed, Web of Science, Scopus, Chinese National Knowledge Infrastructure (CNKI), CMB, and Wanfang databases were systematically searched, and relevant research literature were screened and analyzed. The clinical trial literatures using apatinib as the main single or combined treatment for rmNPC patients were selected and combined with objective response rate (ORR), disease control rate (DCR), progression-free survival (PFS), overall survival (OS) and other efficacy and safety indicators.ResultsThe meta-analysis included 12 studies, including 408 patients with rmNPC. The methodological index for nonrandomized studies scale was used to evaluate the bias of the included literatures and found that the bias was low. A total of 408 rmNPC patients were included in the included literature, with 11 studies being a phase II single-arm trial and one being a phase II non-randomized controlled trial. The ORR of patients with rmNPC treated with apatinib was 41.5% (95% CI: 34.8%, 48.2%), and the DCR was 80.2% (95% CI: 70.9%, 89.6%). The median PFS was 6.4 months (95% CI: 5.3, 7.4), and the median OS was 14.8 months (95% CI: 10.7, 18.9). The incidence of hypertension, hand-foot skin reaction, and proteinuria was 31% (95% CI: 19–43%), 29% (95% CI: 20–39%), and 13% (95% CI: 6–20%), respectively.DiscussionThe efficacy of apatinib in the treatment of rmNPC patients is similar to that of the previous second-line chemotherapy drugs, but since most studies are phase II single-arm studies, the advantages and disadvantages of the existing second-line chemotherapy regimens cannot be determined.

Pharmacological profile of asundexian, a novel, orally bioavailable inhibitor of factor XIa

BackgroundActivated coagulation factor XI (FXIa) contributes to the development and propagation of thrombosis but plays only a minor role in hemostasis; therefore, it is an attractive antithrombotic target. ObjectivesTo evaluate the pharmacology of asundexian (BAY 2433334), a small molecule inhibitor targeting FXIa, in vitro and in various rabbit models. MethodsThe effects of asundexian on FXIa activity, selectivity versus other proteases, plasma thrombin generation, and clotting assays were evaluated. Antithrombotic effects were determined in FeCl2‐ and arterio‐venous (AV) shunt models. Asundexian was administered intravenously or orally, before or during thrombus formation, and with or without antiplatelet drugs (aspirin and ticagrelor). Potential effects of asundexian on bleeding were evaluated in ear‐, gum‐, and liver injury models. ResultsAsundexian inhibited human FXIa with high potency and selectivity. It reduced FXIa activity, thrombin generation triggered by contact activation or low concentrations of tissue factor, and prolonged activated partial thromboplastin time in human, rabbit, and various other species, but not in rodents. In the FeCl2‐injury models, asundexian reduced thrombus weight versus control, and in the arterial model when added to aspirin and ticagrelor. In the AV shunt model, asundexian reduced thrombus weight when administered before or during thrombus formation. Asundexian alone or in combination with antiplatelet drugs did not increase bleeding times or blood loss in any of the models studied. ConclusionsAsundexian is a potent oral FXIa inhibitor with antithrombotic efficacy in arterial and venous thrombosis models in prevention and intervention settings, without increasing bleeding.

Targeted Covalent Inhibition of Small CTD Phosphatase‐1 to Promote the Degradation of REST Transcription Factor in Human Cells

The repressor element‐1 silencing transcription factor (REST) represses neuronal gene expression, whose dysregulation is implicated in brain tumors and neurological diseases. High level of REST protein drives the tumor growth in some glioblastoma cells. While transcription factors like REST are challenging targets for small molecule inhibitors, the inhibition of a regulatory protein, Small CTD Phosphatase 1 (SCP1), promotes REST degradation and reduces transcriptional activity. This study rationally designed a series of α,β‐unsaturated sulfones to serve as potent and selective covalent inhibitors against SCP1. The compounds inactivate SCP1 via covalent modification of Cys181 located at the active site entrance. Cellular studies showed that the inhibitors inactivate SCP1 in a time‐ and dose‐dependent manner with an EC50 ~1.5 µM, reducing REST protein levels and activating specific REST‐suppressed genes. These compounds represent a promising line of small molecule inhibitors as a novel lead for glioblastoma whose growth is driven by REST transcription activity.

Small Molecule Inhibitor Targeting CDT1/Geminin Protein Complex Promotes DNA Damage and Cell Death in Cancer Cells.

DNA replication initiation requires the loading of MCM2-7 complexes at the origins of replication during G1. Replication licensing renders chromatin competent for DNA replication and its tight regulation is essential to prevent aberrant DNA replication and genomic instability. CDT1 is a critical factor of licensing and its activity is controlled by redundant mechanisms, including Geminin, a protein inhibitor of CDT1. Aberrant CDT1 and Geminin expression have been shown to promote tumorigenesis in vivo and are also evident in multiple human tumors. In this study, we developed an in vitro AlphaScreen™ high-throughput screening (HTS) assay for the identification of small-molecule inhibitors targeting the CDT1/Geminin protein complex. Biochemical characterization of the most potent compound, AF615, provided evidence of specific, dose-dependent inhibition of Geminin binding to CDT1 both in-vitro and in cells. Moreover, compound AF615 induces DNA damage, inhibits DNA synthesis and reduces viability selectively in cancer cell lines, and this effect is CDT1-dependent. Taken together, our data suggest that AF615 may serve as a useful compound to elucidate the role of CDT1/Geminin protein complex in replication licensing and origin firing as well as a scaffold for further medicinal chemistry optimisation.

Protein Tyrosine Phosphatase Receptor Type D Regulates Neuropathic Pain After Nerve Injury via the STING-IFN-I Pathway.

Neuropathic pain is usually caused by injury or dysfunction of the somatosensory system, and medicine is a common way of treatment. Currently, there are still no satisfactory drugs, like opioids and lidocaine, which carry a high risk of addiction. Protein tyrosine phosphatase receptor type D (PTPRD) is a known therapeutic target in addiction pathways and small molecule inhibitors targeting it, such as 7-butoxy illudalic acid analog (7-BIA), have recently been developed to tackle addition. PTPRD is also upregulated in the dorsal root ganglion (DRG) in a rat model of neuropathic pain, but is not yet clear whether PTPRD contributes to the development of neuropathic pain. Here, we established a chronic constriction injury (CCI) and evaluated PTPRD expression and its association with neuropathic pain. PTPRD expression was found to gradually increase after CCI in DRGs, and its expression was concomitant with the progressive development of hypersensitivity as assessed by both mechanical and thermal stimuli. Both PTPRD knockdown and administration of PTPRD inhibitor 7-BIA alleviated CCI-induced neuropathic pain while upregulating STING and IFN-α in the DRG. Treatment with H-151, a STING inhibitor, abolished the analgesic effects of PTPRD knockdown. Taken together, our study suggests that increased levels of PTPRD in the DRG following CCI are involved in the development of neuropathic pain via the STING-IFN-I pathway. 7-BIA, a small molecule inhibitor of PTPRD with anti-addiction effects, may represent a novel and safe therapeutic strategy for the clinical management of neuropathic pain without the risk of addiction.

High-Throughput Screening to Identify Inhibitors of Plasmodium falciparum Importin α.

The global burden of malaria and toxoplasmosis has been limited by the use of efficacious anti-parasitic agents, however, emerging resistance in Plasmodium species and Toxoplasma gondii threatens disease control worldwide, implying that new agents/therapeutic targets are urgently needed. Nuclear localization signal (NLS)-dependent transport into the nucleus, mediated by members of the importin (IMP) superfamily of nuclear transporters, has shown potential as a target for intervention to limit viral infection. Here, we show for the first time that IMPα from P. falciparum and T. gondii have promise as targets for small molecule inhibitors. We use high-throughput screening to identify agents able to inhibit P. falciparum IMPα binding to a P. falciparum NLS, identifying a number of compounds that inhibit binding in the µM-nM range, through direct binding to P. falciparum IMPα, as shown in thermostability assays. Of these, BAY 11-7085 is shown to be a specific inhibitor of P. falciparum IMPα-NLS recognition. Importantly, a number of the inhibitors limited growth by both P. falciparum and T. gondii. The results strengthen the hypothesis that apicomplexan IMPα proteins have potential as therapeutic targets to aid in identifying novel agents for two important, yet neglected, parasitic diseases.

Design and development of photoswitchable DFG-Out RET kinase inhibitors

REarranged during Transfection (RET) is a transmembrane receptor tyrosine kinase that is required for development of multiple human tissues, but which is also an important contributor to human cancers. RET activation through rearrangement or point mutations occurs in thyroid and lung cancers. Furthermore, activation of wild type RET is an increasingly recognized mechanism promoting tumor growth and dissemination of a much broader group of cancers. RET is therefore an attractive therapeutic target for small-molecule kinase inhibitors. Non-invasive control of RET signaling with light offers the promise of unveiling its complex spatiotemporal dynamics in vivo. In this work, photoswitchable DFG-out RET kinase inhibitors based on heterocycle-derived azobenzenes were developed, enabling photonic control of RET activity. Based on the binding mode of DFG-out kinase inhibitors and using RET kinase as the test model, we developed a photoswitchable inhibitor with a quinoline “head” constituting the azoheteroarene. This azo compound was further modified by three different strategies to increase the difference in biological activity between the E-isomer and the light enriched Z-isomer. Stilbene-based derivatives were used as model compounds to guide in the selection of substituents that could eventually be introduced to the corresponding azo compounds. The most promising quinoline-based compound showed more than a 15-fold difference in bioactivity between the two isomers in a biochemical assay. However, the same compound showed a decreased Z/E (IC50) ratio in the cellular assay, tentatively assigned to stability issues. The corresponding stilbene compound gave a Z/E (IC50) ratio well above 100, consistent with that measured in the biochemical assay. Ultimately, a 7-azaindole based photoswitchable DFG-out kinase inhibitor was shown to display more than a 10-fold difference in bioactivity between the two isomers, in both a biochemical and a cell-based assay, as well as excellent stability even under reducing conditions.

Characterization of a small-molecule inhibitor targeting NEMO/IKKβ to suppress colorectal cancer growth

NEMO/IKKβ complex is a central regulator of NF-κB signaling pathway, its dissociation has been considered to be an attractive therapeutic target. Herein, using a combined strategy of molecular pharmacological phenotyping, proteomics and bioinformatics analysis, Shikonin (SHK) is identified as a potential inhibitor of the IKKβ/NEMO complex. It destabilizes IKKβ/NEMO complex with IC50 of 174 nM, thereby significantly impairing the proliferation of colorectal cancer cells by suppressing the NF-κB pathway in vitro and in vivo. In addition, we also elucidated the potential target sites of SHK in the NEMO/IKKβ complex. Our study provides some new insights for the development of potent small-molecule PPI inhibitors.

YPD-30, a prodrug of YPD-29B, is an oral small-molecule inhibitor targeting PD-L1 for the treatment of human cancer

PD-1 and PD-L1 antibodies have brought about extraordinary clinical benefits for cancer patients, and their indications are expanding incessantly. Currently, most PD-1/PD-L1 agents are administered intravenously, which may be uncomfortable for some cancer patients. Herein, we develop a novel oral-delivered small molecular, YPD-29B, which specifically targets human PD-L1. Our data suggested that YPD-29B could potently and selectively block the interaction between PD-L1 and PD-1, but did not inhibit any other immune checkpoints. Mechanistically, YPD-29B induced human PD-L1 dimerization and internalization, which subsequently activated T lymphocytes and therefore overcomes immunity tolerance in vitro. YDP-29B was modified as the YPD-30 prodrug to improve druggability. Using humanized mice with human PD-1 xenografts of human PD-L1 knock-in mouse MC38 cancer cells, we demonstrated that YPD-30 exhibited significant antitumor activity and was well tolerated in vivo. Taken together, our results indicate that YPD-30 serves as a promising therapeutic candidate for anti-human PD-L1 cancer immunotherapy.

Development of VPC-70619, a Small-Molecule N-Myc Inhibitor as a Potential Therapy for Neuroendocrine Prostate Cancer.

The Myc family of transcription factors are involved in the development and progression of numerous cancers, including prostate cancer (PCa). Under the pressure of androgen receptor (AR)-directed therapies resistance can occur, leading to the lethal form of PCa known as neuroendocrine prostate cancer (NEPC), characterized among other features by N-Myc overexpression. There are no clinically approved treatments for NEPC, translating into poor patient prognosis and survival. Therefore, there is a pressing need to develop novel therapeutic avenues to treat NEPC patients. In this study, we investigate the N-Myc-Max DNA binding domain (DBD) as a potential target for small molecule inhibitors and utilize computer-aided drug design (CADD) approaches to discover prospective hits. Through further exploration and optimization, a compound, VPC-70619, was identified with notable anti-N-Myc potency and strong antiproliferative activity against numerous N-Myc expressing cell lines, including those representing NEPC.

Abstract P2-13-15: VRN101396, a brain-permeable HER2 inhibitor, shows the anti-tumor activity in preclinical HER2-positive cancer models

Abstract Breast cancer (BC) is the second most common cancer associated with brain metastasis (BM) and among patients with breast cancer brain metastasis (BCBM), human epidermal growth factor receptor 2 (HER2)-positive breast cancer is most likely to develop BM. The overall prognosis of HER2-positive BCBM remains dismal with median survival time after BM of less than a year. Drugs like T-DM1, T-DXd and Tucatinib are reported to deliver a progression-free survival benefit in HER2-positive BCBM patients, but there are still unmet needs for a drug that crosses the blood brain barrier more effectively. We develop VRN101396, a brain penetrant, orally available, and irreversible small molecule inhibitor targeting HER2 to offer a new therapeutic option for the treatment of HER2-positive BCBM. VRN101396 potently inhibits catalytic activity of HER2 but spares wild-type EGFR. In HER2-positive breast cancer cell lines BT474 and SK-BR3 and gastric cancer cell line NCI-N87, VRN101396 inhibited the cell proliferation and phosphorylation of HER2 with single- or double-digit nanomolar IC50 values while inhibiting wild type EGFR with an IC50 value of >100 nM. In both subcutaneous and intracranial efficacy mouse models, once-daily oral dose of VRN101396 significantly inhibited tumor growth and achieved tumor regression. In N87 subcutaneous xenograft model, VRN101396 alone showed superior efficacy to the combination of Tucatinib and Trastuzumab. Additionally, in intracranial xenograft model, VRN101396 showed superior efficacy to Tucatinib. Pharmacokinetics of VRN101396 displayed higher brain exposure than tucatinib and lapatinib, indicating better target engagement in the brain. Pharmacodynamic analysis revealed that VRN101396 reduced phosphorylation of HER2, HER3, AKT, and ERK compared to vehicle group after administration up to 24 hours. In conclusion, VRN101396 is a therapeutic candidate for the treatment of HER2-positive BC, BCBM and gastric cancer. Citation Format: Hong-ryul Jung, Sunghwan Kim, Jihye Yoo, Chan mi Park, Somi Lee, Hyoju Lee, Youngyi Lee, Jinhee Park, Kyungah Seo, Dong-Hyuk Seo, Eunhwa Ko, Jung Beom Son, Deakwon Kim, Hwan Geun Choi, Nam Doo Kim. VRN101396, a brain-permeable HER2 inhibitor, shows the anti-tumor activity in preclinical HER2-positive cancer models [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-13-15.

TRIP13, identified as a hub gene of tumor progression, is the target of microRNA-4693-5p and a potential therapeutic target for colorectal cancer

Colorectal cancer (CRC) is one of the digestive tract malignancies whose early symptoms are not obvious. This study aimed to identify novel targets for CRC therapy, especially early-stage CRC, by reanalyzing the publicly available GEO and TCGA databases. Thyroid hormone receptor interactor 13 (TRIP13) correlated with tumor progression and prognosis of patients after several rounds of analysis, including weighted gene correlation network analysis (WGCNA), and further chosen for experimental validation in cancer cell lines and patient samples. We identified that mRNA and protein levels of TRIP13 increased in CRC cells and tumor tissues with tumor progression. miR-4693-5p was significantly downregulated in CRC tumor tissues and bound to the 3′ untranslated region (3′UTR) of TRIP13, downregulating TRIP13 expression. DCZ0415, a small molecule inhibitor targeting TRIP13, induced anti-tumor activity in vitro and in vivo. DCZ0415 markedly suppressed CRC cell proliferation, migration, and tumor growth, promoted cell apoptosis, and resulted in the arrest of the cell cycle. Our research suggests that TRIP13 might play a crucial role in CRC progression and could be a potential target for CRC therapy.

Analysis of human Tribbles 2 (TRIB2) pseudokinase.

Human Tribbles 2 (TRIB2) is a cancer-associated pseudokinase with a broad human protein interactome, including the well-studied AKT, C/EBPα and MAPK modules. Several lines of evidence indicate that human TRIB2 promotes cell survival and drug-resistance in solid tumors and blood cancers and is therefore of interest as a potential therapeutic target, although its physiological functions remain relatively poorly understood. The unique TRIB2 pseudokinase domain lacks the canonical 'DFG' motif, and subsequently possesses very low affinity for ATP in both the presence and absence of metal ions. However, TRIB2 also contains a unique cysteine-rich αC-helix, which interacts with a conserved peptide motif in its own carboxyl-terminal tail. This regulatory flanking region drives regulated interactions with distinct E3 ubiquitin ligases that serve to control the stability and turnover of TRIB2 client proteins. TRIB2 is also a low-affinity target of several known small-molecule protein kinase inhibitors, which were originally identified using purified recombinant TRIB2 proteins and a thermal shift assay. In this chapter, we discuss laboratory-based procedures for purification, stabilization and analysis of human TRIB2, including screening procedures that can be used for the identification of both reversible and covalent small molecule ligands.

Characterizing fenestration size in sodium channel subtypes and their accessibility to inhibitors

Voltage-gated sodium channels (Nav) underlie the electrical activity of nerve and muscle cells. Humans have nine different subtypes of these channels, which are the target of small-molecule inhibitors commonly used to treat a range of conditions. Structural studies have identified four lateral fenestrations within the Nav pore module that have been shown to influence Nav pore blocker access during resting-state inhibition. However, the structural differences among the nine subtypes are still unclear. In particular, the dimensions of the four individual fenestrations across the Nav subtypes and their differential accessibility to pore blockers is yet to be characterized. To address this, we applied classical molecular dynamics simulations to study the recently published structures of Nav1.1, Nav1.2, Nav1.4, Nav1.5, and Nav1.7. Although there is significant variability in the bottleneck sizes of the Nav fenestrations, the subtypes follow a common pattern, with wider DI-II and DIII-IV fenestrations, a more restricted DII-III fenestration, and the most restricted DI-IV fenestration. We further identify the key bottleneck residues in each fenestration and show that the motions of aromatic residue sidechains govern the bottleneck radii. Well-tempered metadynamics simulations of Nav1.4 and Nav1.5 in the presence of the pore blocker lidocaine also support the DI-II fenestration being the most likely access route for drugs. Our computational results provide a foundation for future in vitro experiments examining the route of drug access to sodium channels. Understanding the fenestrations and their accessibility to drugs is critical for future analyses of diseases mutations across different sodium channel subtypes, with the potential to inform pharmacological development of resting-state inhibitors and subtype-selective drug design.

Targeted Covalent Inhibition of Small CTD Phosphatase 1 to Promote the Degradation of the REST Transcription Factor in Human Cells.

The repressor element-1 silencing transcription factor (REST) represses neuronal gene expression, whose dysregulation is implicated in brain tumors and neurological diseases. A high level of REST protein drives the tumor growth in some glioblastoma cells. While transcription factors like REST are challenging targets for small-molecule inhibitors, the inactivation of a regulatory protein, small CTD phosphatase 1 (SCP1), promotes REST degradation and reduces transcriptional activity. This study rationally designed a series of α,β-unsaturated sulfones to serve as potent and selective covalent inhibitors against SCP1. The compounds inactivate SCP1 via covalent modification of Cys181 located at the active site entrance. Cellular studies showed that the inhibitors inactivate SCP1 in a time- and dose-dependent manner with an EC50 ∼1.5 μM, reducing REST protein levels and activating specific REST-suppressed genes. These compounds represent a promising line of small-molecule inhibitors as a novel lead for glioblastoma whose growth is driven by REST transcription activity.

Efficacy and Synergy of Small Molecule Inhibitors Targeting FLT3-ITD+ Acute Myeloid Leukemia.

Simple SummaryFLT3-ITD mutations belong to the most frequent yet most detrimental genetic alterations in AML. Next-generation FLT3 inhibitors are potent therapeutics and often effective in AML patients carrying the FLT3-ITD driver kinase. However, AML cells are particularly quick in acquiring resistance to FLT3 kinase blockers. We aimed to identify novel therapeutic options for FLT3-ITD+ AML, to investigate possible emerging resistance mechanisms to FLT3 inhibitors and to explore alternative targeting strategies. We applied a kinase-focused drug screen to find alternative therapeutics. We identified ispinesib, a kinesin spindle blocker, and kinase blockers WS6, ponatinib and cabozantinib, as very efficacious agents against FLT3-ITD+ AML cells. Importantly, we identify the combination of cabozantinib and ispinesib as particularly potent against FLT3-ITD+ AML. We suggest that a combinatorial treatment with these drugs could overcome resistance mechanisms and kill FLT3-ITD+ AML blasts.Constitutive activation of FLT3 by ITD mutations is one of the most common genetic aberrations in AML, present in ~1/3 of cases. Patients harboring FLT3-ITD display worse clinical outcomes. The integration and advancement of FLT3 TKI in AML treatment provided significant therapeutic improvement. However, due to the emergence of resistance mechanisms, FLT3-ITD+ AML remains a clinical challenge. We performed an unbiased drug screen to identify 18 compounds as particularly efficacious against FLT3-ITD+ AML. Among these, we characterized two investigational compounds, WS6 and ispinesib, and two approved drugs, ponatinib and cabozantinib, in depth. We found that WS6, although not yet investigated in oncology, shows a similar mechanism and potency as ponatinib and cabozantinib. Interestingly, ispinesib and cabozantinib prevent activation of AXL, a key driver and mechanism of drug resistance in FLT3-ITD+ AML patients. We further investigated synergies between the selected compounds and found that combination treatment with ispinesib and cabozantinib or ponatinib shows high synergy in FLT3-ITD+ AML cell lines and patient samples. Together, we suggest WS6, ispinesib, ponatinib and cabozantinib as novel options for targeting FLT3-ITD+ AML. Whether combinatorial tyrosine kinase and kinesin spindle blockade is effective in eradicating neoplastic (stem) cells in FLT3-ITD+ AML remains to be determined in clinical trials.

Small Molecule Inhibitors Targeting Nuclear Factor κB Activation Markedly Reduce Expression of Interleukin-2, but Not Interferon-γ, Induced by Phorbol Esters and Calcium Ionophores.

The T-box transcription factor Eomesodermin (Eomes) promotes the expression of interferon-γ (IFN-γ). We recently reported that the small molecule inhibitors, TPCA-1 and IKK-16, which target nuclear factor κB (NF-κB) activation, moderately reduced Eomes-dependent IFN-γ expression in mouse lymphoma BW5147 cells stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin (IM). In the present study, we investigated the direct effects of NF-κB on IFN-γ expression in mouse lymphoma EL4 cells and primary effector T cells. Eomes strongly promoted IFN-γ expression and the binding of RelA and NFATc2 to the IFN-γ promoter when EL4 cells were stimulated with PMA and IM. Neither TPCA-1 nor IKK-16 reduced IFN-γ expression; however, they markedly decreased interleukin (IL)-2 expression in Eomes-transfected EL4 cells. Moreover, TPCA-1 markedly inhibited the binding of RelA, but not that of Eomes or NFATc2 to the IFN-γ promoter. In effector CD4+ and CD8+ T cells activated with anti-CD3 and anti-CD28 antibodies, IFN-γ expression induced by PMA and A23187 was not markedly decreased by TPCA-1 or IKK-16 under conditions where IL-2 expression was markedly reduced. Therefore, the present results revealed that NF-κB is dispensable for IFN-γ expression induced by PMA and calcium ionophores in EL4 cells expressing Eomes and primary effector T cells.

H3K36 Trimethylation Mediated By SETD2 Regulates Cell Proliferation and Cell Cycle By Modulating BCMA-JNK and c-Myc Pathways in Multiple Myeloma

In addition to genetic aberrations, accumulating evidence indicates that deregulation of histone methyltransferases, such as MMSET, EZH2 and KDM6A, plays crucial roles in the oncogenic transformation and development of multiple myeloma (MM). For example, overexpression of MMSET leading to a global increase in H3K36me2, is believed to be the driving force in the pathogenesis of t (4;14) MM. However, as the histone methyltransferase is responsible for H3K36me3, the role of SETD2 is not been known in myeloma.To explore the possible clinical value of SETD2 in MM, we first examined the gene expression profile from GEO database, which indicated that the SETD2 expression was significantly decreased in MM patients when compared with monoclonal gammopathy of undetermined significance and smoldering multiple myeloma patients (GSE6477). Moreover, the expression of SETD2 decreased with the advanced international staging system stage of MM patients (GSE19784). The Kaplan-Meier analysis showed that low expression of SETD2 was significantly associated with a poor overall survival in MM patients (GSE2658, P<0.05; GSE9782, P<0.001). Thus, our analysis suggests that SETD2 might participate in cancer progression and could become a biomarker for the prognosis of patients with MM.We then investigated the biological role and the underlying mechanism of SETD2 in MM. Firstly, we used lentiviral-mediated RNA interference to knockdown SETD2 (SETD2 KD) in MM cell lines (RPMI8226 and MM.1S). CCK8 and colony-forming assays showed that reduced expression of SETD2 significantly promoted MM cell proliferation and colony growth. BrdU incorporation assay revealed increased DNA synthesis in SETD2 KD MM cells. Then, treatment with JIB-04, a small molecule inhibitor targeting H3K36me3 loss in SETD2 KD MM cells showed that H3K36me3 recovery was capable of reversing the tumor-promoting effect due to SETD2 down-regulation of MM cells in vitro. Moreover, the xenograft growth assay revealed that SETD2 down-regulation facilitated tumor growth and JIB-04 treatment exhibited anti-myeloma activity in vivo. Therefore, we conclude that SETD2 plays an important role in MM maintenance, and inhibition of H3K36me3 shows therapeutic efficacy for MM.To further explore the underlying mechanisms, we performed the RNA-Seq analysis and discovered that low H3K36me3 level was associated with reduced expression of CDKN1A and increased expression of TNFRSF17 (BCMA) and c-Myc in MM cells. The Gene Set Enrichment Analysis (GSEA) revealed that MAPK signaling pathway was enriched in SETD2 knockdown and JIB-04 treated MM cells. Subsequent Western blotting analysis further confirmed that SETD2 KD cells had increased JNK activation, while JIB-04 treated group showed decreased level of p-JNK. To investigate whether BCMA was the directly transcriptional target for H3K36me3, we carried out a dual-luciferase reporter assay in both SETD2 KD and JIB-04 treated MM cells, and confirmed that H3K36me3 inhibited the expression of BCMA through physically interacting with motifs in its promoter. Furthermore, down-regulation of BCMA in SETD2 KD MM cells could lead to a reduction of p-JNK and an up-regulation of CDKN1A, and resulting inhibited cell proliferation and cell cycle progression. Furthermore, blockage of the JNK pathway by its inhibitor SP600125 resulted in significant inhibition of MM cell proliferation induced by SETD2 knockdown. Additionally, suppression of c‐Myc using 10058‐F4 inhibited proliferation, and induced cell cycle arrest as well as CDKN1A expression in SETD2 KD MM cells. These results indicate that BCMA/JNK and c-Myc pathways were involved in STED2 and H3K36me3 mediated cell proliferation in MM.Together, our data delineate that SETD2-dependent H3K36me3 modification plays a critical role in regulation cell proliferation and cell cycle by BCMA-JNK and c-Myc pathways in MM cells. Targeting the SETD2-H3K36me3 pathway represents a promising therapy for MM. DisclosuresNo relevant conflicts of interest to declare.

FGF10/FGF17 as prognostic and drug response markers in acute myeloid leukemia

BackgroundFibroblast growth factors (FGFs) play important roles in solid tumor progression. Little is known about the function and the prognostic value of distinct FGFs in acute myeloid leukemia (AML). MethodsWe used dataset from Beat AML to screen the FGFs family in AML by log-rank test. Subsequently, we identified the biological functions and the crucial signaling pathways associated with these screened FGFs using gene set enrichment analysis (GSEA). In addition, IC50 from 122 small-molecule inhibitors was used to explore the relationship between these signaling pathways and targets of sensitive inhibitors. ResultsAmong the FGFs family, over expressions of FGF10/FGF17 were found to be significantly associated with poor prognosis. FGF10 over expression was related to FLT3 and NPM1 mutations, and FGF17 over expression was linked to MUC12 and ZRSR2 mutations. Some cancer-related pathways such as PI3K-Akt, MAPK were significantly enriched by GSEA, and these pathways were concordant with sensitive inhibitors targeted pathways. ConclusionOur results indicated that FGF10 and FGF17 could be prognostic biomarkers for survivals of AML patients, and potential therapeutic targets for small-molecule inhibitors.

Acetyltransferase p300 Is a Putative Epidrug Target for Amelioration of Cellular Aging-Related Cardiovascular Disease.

Cardiovascular disease is the leading cause of accelerated as well as chronological aging-related human morbidity and mortality worldwide. Genetic, immunologic, unhealthy lifestyles including daily consumption of high-carb/high-fat fast food, lack of exercise, drug addiction, cigarette smoke, alcoholism, and exposure to environmental pollutants like particulate matter (PM)-induced stresses contribute profoundly to accelerated and chronological cardiovascular aging and associated life threatening diseases. All these stressors alter gene expression epigenetically either through activation or repression of gene transcription via alteration of chromatin remodeling enzymes and chromatin landscape by DNA methylation or histone methylation or histone acetylation. Acetyltransferase p300, a major epigenetic writer of acetylation on histones and transcription factors, contributes significantly to modifications of chromatin landscape of genes involved in cellular aging and cardiovascular diseases. In this review, the key findings those implicate acetyltransferase p300 as a major contributor to cellular senescence or aging related cardiovascular pathologies including vascular dysfunction, cardiac hypertrophy, myocardial infarction, cardiac fibrosis, systolic/diastolic dysfunction, and aortic valve calcification are discussed. The efficacy of natural or synthetic small molecule inhibitor targeting acetyltransferase p300 in amelioration of stress-induced dysregulated gene expression, cellular aging, and cardiovascular disease in preclinical study is also discussed.