A plasma metabolite panel as biomarkers for early primary breast cancer detection.

In recent years, metabolites have attracted substantial attention as promising novel biomarkers of various diseases. However, breast cancer plasma metabolite studies are still in their infancy. Here, we investigated the potential of metabolites to serve as minimally invasive, early detection markers of primary breast cancer. We profiled metabolites extracted from the plasma of primary breast cancer patients and healthy controls using tandem mass spectrometry (UHPLC-MS/MS and FIA-MS/MS). Two metabolites were found to be upregulated, while 16 metabolites were downregulated in primary breast cancer patients compared to healthy controls in both the training and validation cohorts. A panel of seven metabolites was selected by LASSO regression analysis. This panel could differentiate primary breast cancer patients from healthy controls, with an AUC of 0.87 (95% CI: 0.81 ~ 0.92) in the training cohort and an AUC of 0.80 (95% CI: 0.71 ~ 0.87) in the validation cohort. These significantly differentiated metabolites are mainly involved in the amino acid metabolism and breast cancer cell growth pathways. In conclusion, using a metabolomics approach, we identified metabolites that have potential value for development of a multimarker blood-based test to complement and improve early breast cancer detection. The panel identified herein might be part of a prescreening tool, especially for younger women or for closely observing women with certain risks, to facilitate decision making regarding which individuals should undergo further diagnostic tests. In the future, the combination of metabolites and other blood-based molecular marker sets, such as DNA methylation, microRNA, and cell-free DNA mutation markers, will be an attractive option.

Evaluation of Promoter Methylation of RASSF1A and ATM in Peripheral Blood of Breast Cancer Patients and Healthy Control Individuals.

Breast cancer (BC) is the most common cancer among women and has high mortality rates. Early detection is supposed to be critical for the patient's prognosis. In recent years, several studies have investigated global DNA methylation profiles and gene-specific DNA methylation in blood-based DNA to develop putative screening markers for cancer. However, most of the studies have not yet been validated. In our study, we analyzed the promoter methylation of RASSF1A and ATM in peripheral blood DNA of 229 sporadic patients and 151 healthy controls by the MassARRAY EpiTYPER assay. There were no significant differences in DNA methylation levels of RASSF1A and ATM between the sporadic BC cases and the healthy controls. Furthermore, we performed the Infinium HumanMethylation450 BeadChip (450K) array analysis using 48 sporadic BC cases and 48 healthy controls (cases and controls are the same from those of the MassARRAY EpiTYPER assay) and made a comparison with the published data. No significant differences were presented in DNA methylation levels of RASSF1A and ATM between the sporadic BC cases and the healthy controls. So far, the evidence for powerful blood-based methylation markers is still limited and the identified markers need to be further validated.

MAP4K4 deletion inhibits proliferation and activation of CD4(+) T cell and promotes T regulatory cell generation in vitro.

CD4(+) T cells are critical for adaptive immunity. MAP4K4 is a key member of germinal center kinase group. However, the physiological function of MAP4K4 in primary CD4(+) T cells is still unclear. In this study, it was demonstrated that in vitro, MAP4K4 deletion remarkably suppressed CD4(+) T cell proliferation in response to phorbol 12-myristate 13-acetate (PMA) and ionomycin, which was not due to enhancing cell apoptosis. Additionally, MAP4K4 was required for the activation of CD4(+) T cells. MAP4K4 deletion significantly down-regulated expression of interleukin 2 (IL-2) and interferon-γ (IFN-γ), while notably up-regulating the expression of regulatory T cells (Treg) transcription factor Foxp3 in peripheral CD4(+) T cells. Furthermore, western blot analysis indicated that CD4(+) T cells lacking MAP4K4 failed to phosphorylate Jnk, Erk, p38 and PKC-θ. Thus, our results provide the evidence that MAP4K4 is essential for CD4(+) T cell proliferation, activation and cytokine production.

Lisaftoclax in Combination with Alrizomadlin Overcomes Venetoclax Resistance in Acute Myeloid Leukemia and Acute Lymphoblastic Leukemia: Preclinical Studies.

PURPOSE: Despite approval of B-cell lymphoma (BCL)-2 inhibitor venetoclax for certain hematologic malignancies, its broader clinical benefit is curtailed by resistance. Our study aimed to determine if treatment with novel anticancer agents targeting BCL-2 and mouse double minute 2 (MDM2) could overcome venetoclax resistance in preclinical models. EXPERIMENTAL DESIGN: Venetoclax-sensitive and venetoclax-resistant acute myeloid leukemia (AML) and acute lymphoblastic leukemia cells and xenograft models were used to evaluate antitumor effects and underlying mechanisms associated with combined BCL-2 inhibitor lisaftoclax (APG-2575) and MDM2 inhibitor alrizomadlin (APG-115). RESULTS: The combination exhibited synergistic antiproliferative and apoptogenic activities in TP53 wild-type AML cell lines in vitro. This synergy was further exemplified by deep antitumor responses and prolonged survival in AML cell line-derived and patient-derived xenograft models. Interestingly, the combination treatment resensitized (to apoptosis) venetoclax-resistant cellular and mouse models established via chronic drug exposure or genetically engineered with clinically relevant BCL-2 gene mutations. Synergistic effects in reducing cellular viability and proliferation were also demonstrated in primary samples of patients with venetoclax-resistant AML treated with lisaftoclax and alrizomadlin ex vivo. Mechanistically, alrizomadlin likely primes cancer cells to BCL-2 inhibition-induced cellular apoptosis by downregulating expression of antiapoptotic proteins myeloid cell leukemia-1 and BCL-extra-large and upregulating pro-death BCL-2-associated X protein. CONCLUSIONS: Lisaftoclax in combination with alrizomadlin overcomes venetoclax resistance mediated by various mechanisms, including BCL-2 mutations. In addition, we posit further, putative molecular mechanisms. Our data rationalize clinical development of this treatment combination in patients with diseases that are insensitive or resistant to venetoclax.

FLT3 inhibition by olverembatinib (HQP1351) downregulates MCL-1 and synergizes with BCL-2 inhibitor lisaftoclax (APG-2575) in preclinical models of FLT3-ITD mutant acute myeloid leukemia.

INTRODUCTION: FLT3-ITD mutations occur in approximately 25% of patients with acute myeloid leukemia (AML) and are associated with poor prognosis. Despite initial efficacy, short duration of response and high relapse rates limit clinical use of selective FLT3 inhibitors. Combination approaches with other targeted therapies may achieve better clinical outcomes. MATERIALS AND METHODS: Anti-leukemic activity of multikinase inhibitor olverembatinib (HQP1351), alone or in combination with BCL-2 inhibitor lisaftoclax (APG-2575), was evaluated in FLT3-ITD mutant AML cell lines in vitro and in vivo. A patient-derived FLT3-ITD mutant AML xenograft model was also used to assess the anti-leukemic activity of this combination. RESULTS: HQP1351 potently induced apoptosis and inhibited FLT3 signaling in FLT3-ITD mutant AML cell lines MV-4-11 and MOLM-13. HQP1351 monotherapy also significantly suppressed growth of FLT3-ITD mutant AML xenograft tumors and prolonged survival of tumor-bearing mice. HQP1351 and APG-2575 synergistically induced apoptosis in FLT3-ITD mutant AML cells and suppressed growth of MV-4-11 xenograft tumors. Combination therapy improved survival of tumor bearing-mice in a systemic MOLM-13 model and showed synergistic anti-leukemic effects in a patient-derived FLT3-ITD mutant AML xenograft model. Mechanistically, HQP1351 downregulated expression of myeloid-cell leukemia 1 (MCL-1) by suppressing FLT3-STAT5 (signal transducer and activator of transcription 5) signaling and thus enhanced APG-2575-induced apoptosis in FLT3-ITD mutant AML cells. CONCLUSIONS: FLT3 inhibition by HQP1351 downregulates MCL-1 and synergizes with BCL-2 inhibitor APG-2575 to potentiate cellular apoptosis in FLT3-ITD mutant AML. Our findings provide a scientific rationale for further clinical investigation of HQP1351 combined with APG-2575 in patients with FLT3-ITD mutant AML.

MDM2 inhibitor APG-115 exerts potent antitumor activity and synergizes with standard-of-care agents in preclinical acute myeloid leukemia models.

Acute myeloid leukemia (AML) is a clinically and genetically heterogeneous clonal disease associated with unmet medical needs. Paralleling the pathology of other cancers, AML tumorigenesis and propagation can be ascribed to dysregulated cellular processes, including apoptosis. This function and others are regulated by tumor suppressor P53, which plays a pivotal role in leukemogenesis. Opposing P53-mediated activities is the mouse double minute 2 homolog (MDM2), which promotes P53 degradation. Because the TP53 mutation rate is low, and MDM2 frequently overexpressed, in patients with leukemia, targeting the MDM2-P53 axis to restore P53 function has emerged as an attractive AML treatment strategy. APG-115 is a potent MDM2 inhibitor under clinical development for patients with solid tumors. In cellular cultures and animal models of AML, we demonstrate that APG-115 exerted substantial antileukemic activity, as either a single agent or when combined with standard-of-care (SOC) hypomethylating agents azacitidine (AZA) and decitabine (DAC), or the DNA-damaging agent cytarabine (Ara-C). By activating the P53/P21 pathway, APG-115 exhibited potent antiproliferative and apoptogenic activities, and induced cell cycle arrest, in TP53 wild-type AML lines. In vivo, APG-115 significantly reduced tumor burden and prolonged survival. Combinations of APG-115 with SOC treatments elicited synergistic antileukemic activity. To explain these effects, we propose that APG-115 and SOC agents augment AML cell killing by complementarily activating the P53/P21 pathway and upregulating DNA damage. These findings and the emerging mechanism of action afford a sound scientific rationale to evaluate APG-115 (with or without SOC therapies) in patients with AML.

MDM2 inhibitor APG-115 synergizes with PD-1 blockade through enhancing antitumor immunity in the tumor microenvironment.

BACKGROUND: Programmed death-1 (PD-1) immune checkpoint blockade has achieved clinical successes in cancer therapy. However, the response rate of anti-PD-1 agents remains low. Additionally, a subpopulation of patients developed hyperprogressive disease upon PD-1 blockade therapy. Combination therapy with targeted agents may improve immunotherapy. Recent studies show that p53 activation in the myeloid linage suppresses alternative (M2) macrophage polarization, and attenuates tumor development and invasion, leading to the hypothesis that p53 activation may augment antitumor immunity elicited by anti-PD-1 therapy. METHOD: Using APG-115 that is a MDM2 antagonist in clinical development as a pharmacological p53 activator, we investigated the role of p53 in immune modulation and combination therapy with PD-1 blockade. RESULTS: In vitro treatment of bone marrow-derived macrophages with APG-115 resulted in activation of p53 and p21, and a decrease in immunosuppressive M2 macrophage population through downregulation of c-Myc and c-Maf. Increased proinflammatory M1 macrophage polarization was observed in the spleen from mice treated with APG-115. Additionally, APG-115 has co-stimulatory activity in T cells and increases PD-L1 expression in tumor cells. In vivo, APG-115 plus anti-PD-1 combination therapy resulted in enhanced antitumor activity in Trp53wt, Trp53mut, and Trp53-deficient (Trp53-/-) syngeneic tumor models. Importantly, such enhanced activity was abolished in a syngeneic tumor model established in Trp53 knockout mice. Despite differential changes in tumor-infiltrating leukocytes (TILs), including the increases in infiltrated cytotoxic CD8+ T cells in Trp53wt tumors and M1 macrophages in Trp53mut tumors, a decrease in the proportion of M2 macrophages consistently occurred in both Trp53wt and Trp53mut tumors upon combination treatment. CONCLUSION: Our results demonstrate that p53 activation mediated by APG-115 promotes antitumor immunity in the tumor microenvironment (TME) regardless of the Trp53 status of tumors per se. Instead, such an effect depends on p53 activation in Trp53 wild-type immune cells in the TME. Based on the data, a phase 1b clinical trial has been launched for the evaluation of APG-115 in combination with pembrolizumab in solid tumor patients including those with TP53mut tumors.

Preclinical development of HQP1351, a multikinase inhibitor targeting a broad spectrum of mutant KIT kinases, for the treatment of imatinib-resistant gastrointestinal stromal tumors.

BACKGROUND: Imatinib shows limited efficacy in patients with gastrointestinal stromal tumors (GISTs) carrying secondary KIT mutations. HQP1351, an orally bioavailable multikinase BCR-ABL inhibitor, is currently in clinical trials for the treatment of T315I mutant chronic myelogenous leukemia (CML), but the potential application in imatinib-resistant GISTs carrying secondary KIT mutations has not been explored. METHODS: The binding activities of HQP1351 with native or mutant KIT were first analyzed. Imatinib-sensitive GIST T1 and imatinib-resistant GIST 430 cells were employed to test the in vitro antiproliferative activity. Colony formation assay, cell migration assay and cell invasion assay were performed to evaluate the clonogenic, migration and invasion ability respectively. Flow cytometry and western blot analysis were used to detect cell apoptosis, cell cycle and signaling pathway. In vivo antitumor activity was evaluated in mouse xenograft models derived from GIST cell lines. RESULTS: HQP1351 potently inhibited both wild-type and mutant KIT kinases. In both imatinib-resistant and sensitive GIST cell lines, HQP1351 exhibited more potent or equivalent antiproliferative activity compared with ponatinib, a third generation BCR-ABL and KIT inhibitor. HQP1351 led to more profound inhibition of cell colony formation, cell migration and invasion, cell cycle arrest and cell apoptosis than ponatinib. Furthermore, HQP1351 also inhibited p-KIT, p-AKT, p-ERK1/2, and p-STAT3 to a higher extent than ponatinib. Finally, in xenograft tumor models derived from imatinib-resistant GIST cancer cell lines, HQP1351 exhibited antitumor activity superior to ponatinib. CONCLUSIONS: Collectively, our in vitro and in vivo results suggest that the therapeutic application of HQP1351 in imatinib-resistant GIST patients deserves further investigation in clinical trials.

Cell-Free Circulating DNA Integrity Based on Peripheral Blood as a Biomarker for Diagnosis of Cancer: A Systematic Review.

Background: Cell-free DNA integrity (cfDI) has been identified as a potential diagnostic biomarker for different types of cancer, suggesting the importance of liquid biopsy.Methods: This systematic review aims to assess the diagnostic performance of cfDI in cancer. After an extensive search of literature published through February 28, 2017, 25 articles that included 40 studies were identified. The descriptions of all the studies were analyzed. The sensitivity, specificity, positive and negative likelihood ratios, diagnostic ORs, weighted symmetric summary receiver operating characteristic curve, and the area under the curve (AUC) of cfDI in these studies were calculated.Results: Aberrant results of cfDI were observed. Some studies observed an increased cfDI in cancer patients, while some studies confirmed a decreased cfDI compared with healthy or benign controls.Conclusions: This review suggests that cfDI is controversial as a blood-based biomarker of cancers, although the sensitivity and AUC were relatively high.Impact: cfDI shows heterogeneity between different studies; more perspective studies are needed to further assess its diagnostic performance, especially with other markers in combination. Cancer Epidemiol Biomarkers Prev; 26(11); 1595-602. ©2017 AACR.

The effect of ethephon on immune system in male offspring of mice.

Ethephon can liberate ethylene which could interfere the plant growth process. The aim of the present study was to determine the effect of ethephon on developing immune system of male offspring. Ethephon could enhance NK cell activity in male mice. For 4-week-old male mice, lymphocytes of peripheral blood increased while the hemolytic plaque number decreased. Delayed type hypersensitivity(DTH) was inhibited in all groups. The expression of protein Bcl11b and p-p38 in thymus of treatment groups were lower than control group. Our results indicated that cellular immunity of male offspring is more sensitive to ethephon when exposed in pregnancy and lactation period. It should be emphasized that exposure to ethephon during the in utero stage and lactation stage still could damage the immune function of animal in the period before fully mature even in the dosage that could not influence the immune function of adult animal.

Blood-based DNA methylation as biomarker for breast cancer: a systematic review.

Multiple studies have investigated global DNA methylation profiles and gene-specific DNA methylation in blood-based DNA to develop powerful screening markers for cancer. This systematic review summarizes the current evidence on methylation studies that investigated methylation level of blood-derived DNA of breast cancer (BC) patients in comparison to healthy controls by conducting a systematic literature review in PubMed and Web of Science. Essential results, such as methylation levels of BC cases and healthy controls, p values, and odds ratios, were extracted from these studies by two investigators independently. Overall, 45 publications met the inclusion criteria for this review. DNA from whole blood, as well as cell-free DNA (cfDNA) from serum or plasma, was used in these studies. The most common method used for measuring global DNA methylation was the investigation of repetitive elements as surrogates and the application of array-based genome-wide methylation analysis. For measuring gene-specific methylation level, methylation-specific PCR and pyrosequencing were the most frequently used methods. Epigenome-wide blood DNA hypomethylation in BC patients were reported in several studies; however, the evidence is still not conclusive. The most frequently investigated gene in whole blood was BRCA1, which was found more frequently methylated in patients compared to controls. RASSF1A was the most widely investigated gene in cfDNA of serum or plasma, which was also found more frequently methylated in patients compared to controls. Several of the eligible studies reported the associations of global hypomethylation and increased BC risk. Studies investigated associations between gene-specific methylation and BC risk, while got heterogeneous results. But two studies reported that hypermethylation of ATM gene was associated with increased BC risk, which suggest the potential use of this gene for BC risk stratification. Overall, our review suggests the possibility of using blood-based DNA methylation marker as promising marker for BC risk stratification, as several studies found associations between certain methylation level in blood and BC risk. However, so far, the evidence is still quite limited. Optimal markers are yet to be developed and promising results needed to be validated in prospective study cohorts and tested in large screening populations.

Grape-seed proanthocyanidins inhibit the lipopolysaccharide-induced inflammatory mediator expression in RAW264.7 macrophages by suppressing MAPK and NF-κb signal pathways.

Grape-seed proanthocyanidins (GSPs) have been shown to function as an anti-oxidant and anti-inflammatory agent with little toxicity in vivo and in vitro. However, little is known about their anti-inflammatory properties and mechanisms of action. The specific focus being its effects on the MAP kinases and nuclear factor-kappaB (NF-κB) signal transduction pathways in lipopolysaccharide (LPS) -stimulated RAW264.7 cells. GSPs extract has been found to suppress the mRNA expression of pro-inflammatory cytokines like tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß) and inflammatory molecule of cyclooxygenase-2 (COX-2) while mRNA level of IL-10 was greatly promoted. Furthermore, GSPs extract inhibited the expression of phosphorylated ERK, JNK and P38, as well as phosphorylated IKKα/ß and NF-κB p65 subunit. In conclusion, our results show that GSPs extract showed its anti-inflammatory and immunomodulatory properties by suppressing the activation of MAP kinases and NF-κB signal transduction pathways.

DNA methylation array analysis identifies breast cancer associated RPTOR, MGRN1 and RAPSN hypomethylation in peripheral blood DNA.

DNA methylation changes in peripheral blood DNA have been shown to be associated with solid tumors. We sought to identify methylation alterations in whole blood DNA that are associated with breast cancer (BC). Epigenome-wide DNA methylation profiling on blood DNA from BC cases and healthy controls was performed by applying Infinium HumanMethylation450K BeadChips. Promising CpG sites were selected and validated in three independent larger sample cohorts via MassARRAY EpiTyper assays. CpG sites located in three genes (cg06418238 in RPTOR, cg00736299 in MGRN1 and cg27466532 in RAPSN), which showed significant hypomethylation in BC patients compared to healthy controls in the discovery cohort (p < 1.00 x 10-6) were selected and successfully validated in three independent cohorts (validation I, n =211; validation II, n=378; validation III, n=520). The observed methylation differences are likely not cell-type specific, as the differences were only seen in whole blood, but not in specific sub cell-types of leucocytes. Moreover, we observed in quartile analysis that women in the lower methylation quartiles of these three loci had higher ORs than women in the higher quartiles. The combined AUC of three loci was 0.79 (95%CI 0.73-0.85) in validation cohort I, and was 0.60 (95%CI 0.54-0.66) and 0.62 (95%CI 0.57-0.67) in validation cohort II and III, respectively. Our study suggests that hypomethylation of CpG sites in RPTOR, MGRN1 and RAPSN in blood is associated with BC and might serve as blood-based marker supplements for BC if these could be verified in prospective studies.

Comparative evaluation of the teratogenicity of genistein and genistin using rat whole embryo culture and limbud micromass culture methods.

Genistein (GEN) is one kind of phytoestrogen. Several studies have demonstrated the teratogenic potential of GEN in vitro by postimplantation rat whole embryo culture (WEC) assay, but GEN showed no teratogenic effects in vivo even at a dose up to 1000 mg/kg bw/day. The mechanism of such discrepancy is still unclear. Because more than 80% of total genistein (free plus glycoside form) in circulation is its glycoside metabolite, genistin (GIN), we thus hypothesize that genistin is non-teratogenic. To prove this hypothesis, rat whole embryo culture (WEC) and limbud micromass culture methods were applied to compare the teratogenic effects of GEN and GIN on developing embryos in vitro. In WEC assay, we found that the development of embryos was affected by GEN treatment dose-dependently, while GIN-treated embryos displayed slight developmental defects only at the highest dose (222 µM). In micromass culture assay, the IC50 of cell proliferation and differentiation for GEN were 15.6 and 37.2 µM, respectively, while neither was influenced by GIN treatment up to 111 µM. Collectively, our study indicated that GEN showed no teratogenic effects in vivo probably due to its transformation to the non-teratogenic metabolite, GIN.

Grape-seed proanthocyanidins ameliorate contact hypersensitivity induced by 2,4-dinitrofluorobenzene (DNFB) and inhibit T cell proliferation in vitro.

Contact hypersensitivity (CHS) is a delayed-type hypersensitivity reaction which is mediated by hapten-specific T cells. Strong haptens, such as 2, 4-dinitrofluorobenzene (DNFB) can induce it. Grape seed proanthocyanidins extract (GSPs), which is an antioxidant derived from grape seeds, has been reported to possess a variety of potent properties. However, few reports demonstrated the effects of GSPs on contact hypersensitivity. Therefore, the present study was devised to describe the role of GSPs on a mouse model of experimental CHS induced by DNFB and try to explore the possible underlying mechanisms. We observed that, GSPs when orally administrated into the CHS mice, inhibited the aggravation of inflammation. After administration of GSPs, there was obvious fewer inflammatory cell infiltration in the inflamed ears. Ear swelling after challenge was significantly reduced. In addition, we investigated the effects of GSPs on T cells in vitro, which play critical role during the progress of CHS. It was found that GSPs inhibited proliferative activity of T cells by blocking the activation of mitogen-activated protein kinases (MAPK) and NF-кB signaling pathways. Collectively, these results showed that GSPs has protective effect on CHS induced by DNFB and it also could inhibit the proliferation ability of T cells in vitro, suggesting the potential of GSPs as new and effective compound for the treatment of T-cell mediated inflammatory diseases.

Let-7 g is involved in doxorubicin induced myocardial injury.

OBJECTIVES: To investigate whether let-7 g (miRNA) was involved in doxorubicin-induced cardiotoxicity. METHODS: Rats were treated with doxorubicin at increasing doses (0mg/kg, 6 mg/kg, 12 mg/kg, 18 mg/kg). Heart rate, pulse pressure and plasma cardiac troponin T concentrations were measured. Primary cultured myocardial cells were incubated with DOX at increasing concentrations (0 µmol/l, 0.004 µmol/l, 0.02 µmol/l, 0.1 µmol/l, 0.5 µmol/l) for 24h. Cellular viability and the beat frequency were measured. For both rats and cultured cells, miRNA content was measured by real-time reverse-transcription PCR. RESULTS: All DOX-treated rats had a decrease in heart rate, an increase in pulse pressure compared with control group after injections (p<0.05). Concentration of cTnT was increased significantly in 18 mg/kg group. Content of let-7 g decreased significantly (p<0.05) in 18 mg/kg group in vivo and all the doxorubicin treated group in vitro. CONCLUSIONS: The down regulation of let-7 g in the myocardial-injury model suggests that let-7 g may play an important role in the development of cardiac disease.