Expression Of BCL2L1 Research Articles

A BCL2L1 Armoured BCMA Targeting CAR T Cell to Overcome Exhaustion and Enhance Persistence in Multiple Myeloma

Chimeric antigen receptor (CAR) T cells targeting the B-cell maturation antigen (BCMA) have resulted in deep responses in patients with relapsed MM however most remissions are not sustained. While cellular and molecular mediators of relapse post CAR T therapy in MM are not fully delineated, current data suggest three possible mechanisms including the lack of persistence of the CAR T cell product, acquired exhaustion and less commonly loss of BCMA expression.Using CITE-seq we measured the expansion of variable T cell subsets, T cell specific activation and inhibitor markers and their functional states in serial blood and marrow samples (n=10) collected from patients treated with BCMA targeting CAR T cells. CAR T cells were identified by the expression of the chimeric CAR T cell transcript. With the exception of one patient where biallelic loss of BCMA was identified at relapse, CAR T cells of resistant patients were enriched with terminally exhausted CD45RA+ cells with loss of CD28, low BCL2L1 (gene encoding BCL-XL) expression, high CD57 with co-expression of checkpoint inhibitors (LAG3, TIGIT and PD1). The lack of persistence of the CAR T cells product was notable in all relapsing patients consistent with an activation induced cells death (AICD) specially in the setting of chronic antigenic stimulation. Cognizant of the role BCL-XL plays in T cells survival in response to CD28 co-stimulatory signaling, we postulated that increasing BCL-XL expression is a feasible strategy to enhance CAR T cell resistant to AICD, improve their persistence and anti-BCMA reactivity. To this goal, we designed a 2nd generation lentiviral CAR construct where the anti-BCAM scFV-41BBz CAR and the BCL2L1 cDNA were linked with self-cleaving 2A sequence. The efficiency in eradicating MM cells of this BCL-XL armored CAR (BCMA_BCL2L1_CAR) was compared to that of non-unarmored CAR (BCMA_CAR) in vitro and i n vivo studies. While BCMA_BCL2L1_CAR and BCMA_CAR were equally cytotoxic to OPM2 MM cells, in MM cell lines expressing the FAS death receptor ligand FASLG (MM1S, OCMY5 and H929) BCMA_BCL2L1_CAR viability and cytolytic activity was significantly superior to that of unarmored BCMA_CAR. Of note, the expression of FASLG, a known interferon response gene, was upregulated in H929 cells when co-cultured with CAR T cells. Importantly, under chronic antigenic stimulation conditions (FIG 1A), where CAR T cells were stimulated every 6 days over a 28 days period with irradiated OPM2 cells, we found no phenotypic difference between BCMA_BCL2L1_CAR and BCMA_CAR with respect to the composition of effector memory T cells (Tem: CCR7− CD45RO+ CD45RA−) or central memory T cells (Tcm: CCR7+CD45RO+CD45RA−) or terminal effector / exhausted T cells. However, under these chronic antigenic stimulation conditions, the CAR T cells viability, proliferation (FIG 1B) and anti-MM cytotoxic activities (FIG 1C) of the BCMA_CAR were dramatically reduced compared to that of the BCL2L1 armored CAR. Furthermore, in initial animal studies where NOD-SCID mice were tail vein injected with 2e6 OPM2 MM cells transduced with a luciferin reporter gene, followed 10 days later by control T cells, BCMA_CAR or BCMA_BCL2L1_CAR T cells IV injection, and despite a skewing to a larger initial disease burden in the BCMA-BCL2L1-CAR group, BCL2L1 armored CAR T cells resulted in more prolonged disease control and animal survival compared to the BCMA_CAR treated mice (FIG 1D).Our studies indicate that BCL2L1 blockade of AICD not only enhanced the viability and proliferation of BCMA targeting CAR T cells but surprisingly also reduced their functional exhaustion. Our findings provide an novel approach for CAR T optimization and overcoming disease relapse resulting from lack of persistence and/or T cells exhaustion. [Display omitted] DisclosuresNeri: Amgen: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria. Bahlis: Sanofi: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Genentech: Consultancy; Janssen: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; GlaxoSmithKline: Consultancy, Honoraria; BMS/Celgene: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria.

OAB-018: A BCL2L1 armoured BCMA-targeting CAR T cells to overcome exhaustion and enhance persistence in multiple myeloma

<h3>Background</h3> Chimeric antigen receptor (CAR) T cells targeting BCMA have resulted in deep responses in patients with relapsed MM however most remissions are not sustained. While cellular and molecular mediators of relapse post CAR T therapy are not fully delineated, current data suggest three possible mechanisms including the lack of persistence of the CAR T cell product, acquired exhaustion and less commonly loss of BCMA expression. <h3>Methods</h3> Using CITE-seq we measured the expansion of variable T cell subsets, T cell specific activation and inhibitor markers and their functional states in serial blood and marrow samples (n=10) collected from patients treated with anti BCMA CAR T cells. <h3>Results</h3> CAR T cells were identified by the expression of the chimeric CAR T cell transcript. With the exception of one patient where biallelic loss of BCMA was identified at relapse, CAR T cells of resistant patients were enriched with terminally exhausted CD45RA+ cells with loss of CD28, low BCL2L1 (gene encoding BclxL) expression, high CD57 with co-expression of checkpoint inhibitors (LAG3, TIGIT and PD1). The lack of persistence of the CAR T cells product was notable in all relapsing patients consistent with an activation induced cells death (AICD). <h3>Conclusions</h3> Cognizant of the role BclxL plays in T cells survival in response to CD28 co-stimulatory signaling, we postulated that increasing BclxL expression is a feasible strategy to enhance CAR T cell resistant to AICD, improve their persistence and anti-BCMA reactivity. To this goal, we designed a 2nd generation lentiviral CAR construct where the anti-BCAM scFV-41BBz CAR and the BCL2L1 cDNA were linked with self-cleaving 2A sequence. The efficiency in eradicating MM cells of this BclxL armored CAR (BCL2L1_CAR) was compared to that of non-unarmored CAR (BCMA_CAR) in vitro and in vivo studies. While BCL2L1_CAR and BCMA_CAR were equally cytotoxic to OPM2 MM cells, in MM cell lines expressing the FAS death receptor ligand FASLG (MM1S, OCMY5 and H929) BCL2L1_CAR viability and cytotoxic activity was significantly superior to that of unarmored BCMA_CAR. Of note, the expression of FASLG was upregulated in H929 cells when co-cultured with CAR T cells. Importantly, under chronic antigenic stimulation conditions, where CAR T cells were stimulated every 2 days over a 28 days period with irradiated OPM2 cells, we found no phenotypic difference between BCL2L1_CAR and BCMA_CAR with respect to the composition of Tem cells (CCR7–CD45RO+CD45RA–) or Tcm cells (CCR7+CD45RO+CD45RA–). However, under these chronic antigenic stimulation conditions, the CAR T cells viability, proliferation and anti-MM cytotoxic activities of the BCMA_CAR were dramatically reduced compared to that of the BCL2L1 armored CAR. Therefore BCL2L1 blockade of AICD not only enhanced the viability and cytotoxicity of CAR-T cells but surprisingly also reduced their functional exhaustion. Our findings provide a novel approach for CAR-T optimization and overcoming relapse resulting from lack of persistence.

Effect of retinol as antioxidant on the post-thaw viability and the expression of apoptosis and developmental competence-related genes of vitrified preantral follicles in buffalo (Bubalus bubalis).

The present study evaluated the effect of supplementation of retinol in the vitrification solution on the viability, apoptosis and development-related gene expression in vitrified buffalo preantral follicles. Preantral follicles isolated from cortical slices of ovaries were randomly assigned into three groups: Group1-Control fresh preantral follicles; Group 2-Vitrification treatment (Vitrification solution 1 (VS1) -TCM-199+25mM HEPES+Foetal bovine serum (FBS) 10%, Ethylene glycol (EG): 10%, Dimethyl sulphoxide (DMSO): 10%, Sucrose-0.3M for 4min; VS2- TCM-199+25mM HEPES+FBS10%, EG:25%, DMSO: 25%, Sucrose:0.3M for 45s); Group3-vitrification treatment +5μM of Retinol. Preantral follicles were placed in corresponding vitrification medium and plunged into liquid nitrogen (-196°C). After a week, the follicles were thawed and analysed for follicular viability and gene expression. There was no significant difference in the viability rates among the Group 1(Fresh preantral follicles) (91.46±2.39%), Group 2 (89.59±2.46%) and Group 3 (87.19±4.05%). There was a significantly (p<.05) higher mRNA expression of BCL2L1, GDF-9 and BMP-15 in the vitrification+retinol group compared with the control group. There was a significantly (p<.05) higher expression of Caspase-3 and Annexin-5 in the vitrification group and Vitrification+retinol group compared with control group of follicles. It is concluded that the supplementation of 5μM of Retinol in Vitrification solution was an efficient vitrification procedure for the vitrification of buffalo preantral follicles.

P–288 Changes in gene and protein expression in human endometrial cancer cell lines after low dose metformin treatment over time

Abstract Study question Does metformin treatment lead to temporal expression changes of specific genes and proteins in endometrial cancer cell lines? Summary answer The expression of three different genes and proteins was investigated, of which all displayed changes over time. What is known already Endometrial cancer (EC) is one of the most common malignancies among postmenopausal women. A long-term estrogen effect on the endometrium often seen in women with obesity or the polycystic ovary syndrome (PCOS), as well as type II diabetes mellitus (T2DM) are well-known risk factors for the development and progression of EC. Metformin is a biguanide used in the treatment of T2DM patients and off label in women with PCOS. Moreover, metformin displays anti-tumor and anti-proliferative effects in various cancer types, including EC. In that regards BCL2L11, CDH1 and CDKN1A play an important role in apoptotic pathways, proliferation and invasion processes. Study design, size, duration The EC cells were cultured in normal (5.5 mmol/L) or high (17 mmol/L) glucose medium supplemented with 10 nmol/L ß-estradiol. The cells were treated with low dose metformin (1.0 mmol/L) for 2, 6, 24, 48 and 168 h (7 d). In addition, EC cells were treated with a combination of metformin and insulin (100 ng/mL) or remained untreated. Five independent experiments were fulfilled and untreated cells served as controls. Participants/materials, setting, methods The study was accomplished using two different human EC cell lines. HEC–1A represents an estrogen-independent EC, whereas Ishikawa represents the more common, estrogen-dependent EC. Proteins were extracted, quantified with a BCA assay, and the protein expression of BCL2L11, CDH1 and CDKN1A was analyzed by western blots. Furthermore, total RNA was extracted, transcribed to cDNA and Taqman real-time PCR was carried out to measure the expression of the associated genes, using fold change (FC) as parameter. Main results and the role of chance The expression of the selected genes, analyzed by RT-PCR, changed in both cell lines over time as follows: After 6 h, metformin induced a decrease in the expression of BCL2L11 (FC = 0.7) and CDH1 (FC = 0.75), whereas the expression of CDKN1A slightly increased (FC = 0.95–1.35). After 24 h, BCL2L11 expression increased in normal glucose groups (FC = 1.3, high glucose: FC = 0.93) and CDH1 expression decreased in combination with metformin and high glucose (FC = 0.7, normal glucose: FC = 1.1). CDKN1A expression was increased by metformin in both cell lines after 24 h (FC = 1.2–1.8). After 48 h of metformin treatment, expression for all three genes was only slightly changed (FC = 0.9–1.0). After 7 d it was observed that the combination of high glucose and metformin (i.e. like obese T2DM patients) led to an increased expression of BCL2L11, CDH1 and CDKN1A (FC = 1.4–2.9) in the presence and absence of insulin, whereas metformin induced a decreased expression of CDH1 and CDKN1A (FC = 0.5–0.75) in normal glucose medium. BCL2L11, CDH1 and CDKN1A expression was investigated at the protein level as well. Limitations, reasons for caution The results cannot be directly transferred to metformin treatment of patients, since the study was carried out in vitro. Additionally, further studies including more timepoints would indicate a more precisely gene and protein expression over time. Wider implications of the findings: This is the first in vitro study showing the temporal changes of BCL2L11, CDH1 and CDKN1A expression, genes related to tumorigenesis due to low dose metformin over time, suggesting differentially pathways in long term metformin treatment using physiologically achievable metformin levels. Trial registration number Not applicable

Dual inhibition of FGFR4 and BCL-xL inhibits multi-resistant ovarian cancer with BCL2L1 gain.

Aim: Overexpression of BCL2L1 (BCL-xL) was associated with platinum resistance in ovarian cancer (OvCa). However, role of copy number (CN) gain of BCL2L1 in OvCa remains elusive.Methods: In silico analyses of multiple public datasets were perform. Validation was carried out in our tissue microarray (TMA) of OvCa cases. In vitro and in vivo assays was performed to explore potential targeted compound against BCL2L1-gained OvCa.Results: BCL2L1 was gained in ~60% of OvCa. BCL2L1 was differentially expressed between healthy and cancerous ovarian cases. BCL2L1 gain was not prognostic either in overall or in progression-free survival but higher BCL2L1 expression was associated with worsened survival, indicating biological distinction between CN gain and overexpression of the gene. BCL2L1 gain was associated with multi-resistance to various drug with no significant sensitivity to any single agent. Only CRISPR-mediated BCL2L1 knockout, but not shRNA could be inhibitive. Combined genetic silencing of FGFR4/NCAM and BCL2L1 with shRNA induced potent inhibition of BCL2L1-gained OvCa with durable effect. Combined inhibition of FGFR/BCL-xL was required for inhibiting BCL2L1-gained OvCa in vitro and in vivo. Only dual inhibition of FGFR/BCL-xL without platinum was tolerable in vivo.Conclusion: Gain of BCL2L1 is associated with resistance to multiple anti-cancer agents in OvCa. Dual inhibition of FGFR4 and BCL-xL showed potent effect and tolerable toxicity, holding promise to further translation.

Expression of BCL2L12and LIPOCALIN 2 in Adult Patient with Acute Myeloid Leukemia and Correlation with Clinical Response.

Background:Acute myeloid leukemia (AML) is a malignancy arising within the bone marrow (BM), in which leukemic cells proliferate uncontrollably in association with a disruption of normal hematopoiesis.Aim of the work to evaluate the expression of LCN and BCL2L2, in newly diagnosed bone marrow samples from adult with AML and to correlate their expression levels with clinical and Laboratory data of the patients especially that known to have a prognostic feature. Methods:This study was carried out on 87 consecutive newly diagnosed adult AML patients of which 75 are evaluated for both LCN, BCL2L12 (All 87 are evaluated for LCN). In addition, 20 donors of matched age and sex healthy individuals from donors for bone marrow transplantation were included as a control group. Results:No statistical significant correlation was found between LCN over-expression and the control group , there was no statistical significance between its expression and age, sex, hepatomegally, splenomegally and lymphadenopathy, also there was no statistical significance regarding peripheral blood and bone marrow findings, immunophenotyping, cytogenetics or molecular findings and MRD at day 15.No statistical significance was found between BCL2L12 expression and the control group again there was no statistical significance between its expression and age, sex, hepatomegally, splenomegally and lymphadenopathy, also there was no statistical significance regarding peripheral blood and bone marrow findings, immunophenotyping, cytogenetics or molecular findings and MRD at day 15. Conclusion:LCN and BCL2L12 were found to be expressed with non significant difference in AML as in normal subjects; however studies on large number of cases are needed to confirm our finding. The role of LCN and BCL2L12 need to be verified by further large-scale sample and further studies.

Sy-1365, a Potent and Selective CDK7 Inhibitor, Exhibits Anti-Tumor Activity in Preclinical Models of Hematologic Malignancies, and Demonstrates Interactions with the BCL-XL/BCL2 Mitochondrial Apoptosis Signaling Pathway in Leukemia

Introduction: The transcriptional kinase CDK7 has been implicated in the pathogenesis of multiple malignancies, including hematologic malignancies, and may play important roles in regulation of oncogenic transcriptional dependencies and in regulation of the mitochondrial apoptosis machinery in tumors. SY-1365, a selective inhibitor of CDK7, was developed to exploit tumor dependencies driven by CDK7 and is in clinical development in patients with advanced solid tumors. We have previously reported that SY-1365 selectively induces apoptosis in leukemic cells relative to non-malignant cells in vitro, and demonstrates anti-tumor activity in AML xenografts. Here we report that SY-1365 has potent inhibitory activity against a broad panel of hematologic malignancies in vitro and ex vivo, induces regressions in AML xenografts using a clinically relevant dosing regimen, and demonstrates synergy with the BCL2 inhibitor venetoclax in AML cells in vitro.Methods: SY-1365 dose-response curves were measured using the ATP-lite assay in a panel of 85 leukemia and lymphoma cell lines in vitro, and in a panel of 226 patient-derived tumor samples from 5 leukemic indications cultured ex vivo. In vivo tumor inhibitory activity was evaluated using once weekly (QW) and twice weekly (BIW) dosing regimens in ML-2 and Kasumi-1 AML xenografts. Growth-rate (GR) adjusted dose response curves were used to classify cell lines into low and high sensitivity groups, and were evaluated relative to RNA expression data to identify markers predictive of sensitivity to SY-1365. Synergy between SY-1365 and venetoclax was evaluated in vitro using KG-1 AML cells.Results: In 42 leukemia lines, SY-1365 had a median IC50 of 11nM (range 1-79nM), with 48% (20/42) demonstrating particularly low IC50s (<10nM). In 43 lymphoma lines, the median IC50 was 6nM (range 1-39nM), with 59% (27/46) demonstrating low IC50s (<10nM). Similarly, SY-1365 demonstrated potent inhibitory activity against patient-derived leukemia samples cultured ex vivo (median IC50 of 28nM), with 82% (185/226) of the samples showing IC50 below 100nM and 8% (19/226) under 10nM. Furthermore, analysis of growth rate (GR) in hematologic tumor cell lines showed that <10nM SY-1365 induced cytotoxicity (GR<0) in 19% (8/42) of leukemia and 21% (9/43) of lymphoma lines. To evaluate SY-1365 anti-tumor activity in vivo, 2 AML cell lines that demonstrate cytotoxic responses to SY-1365 in vitro (Kasumi-1 and ML-2) were grown as subcutaneous xenografts in mice. QW and BIW dosing of 40mg/kg SY-1365 induced tumor regressions in Kasumi-1 xenografts, and BIW dosing induced tumor stasis in ML-2 xenografts. Tumor growth inhibition was detected with a 20mg/kg BIW dosing regimen in both models. Comparison of SY-1365 sensitivity with RNA expression in hematologic tumor cell lines revealed that lower expression of BCL2L1, which encodes the mitochondrial apoptosis inhibitor BCL-XL, was highly predictive of sensitivity in leukemia cell lines (Accuracy=89%, FDR<0.05); no such relationship was observed in lymphoma lines. Considering the established role of BCL2 and the mitochondrial apoptosis pathway in leukemia, we reasoned that BCL-XL low-expressing leukemias, in addition to being sensitive to SY-1365, would also be reliant on BCL2 for survival. Indeed, in vitro combination studies demonstrated synergistic inhibitory activity of SY-1365 with the BCL2 inhibitor venetoclax in KG-1 AML cells. A potential synergistic interaction with SY-1365 and venetoclax is currently being explored in vivo with AML xenografts.Conclusions: SY-1365 shows potent in vitro inhibitory activity in multiple hematological indications and can induce AML xenograft tumor regression in mice. In vitro exploratory biomarker and tumor cell inhibitory combination studies in AML suggest a role for mitochondrial apoptosis signaling in mediating sensitivity to SY-1365 and support the evaluation of a combination with venetoclax. SY-1365 is currently being assessed in a phase 1 trial in adult patients with advanced solid tumors (NCT03134638). The results described here support the potential for additional exploration in patients with hematological malignancies. DisclosuresHodgson:Syros Pharmaceuticals: Employment, Equity Ownership. Johannessen:Syros Pharmaceuticals: Employment, Equity Ownership. Rajagopal:Syros Pharmaceuticals: Employment, Equity Ownership. Hu:Syros Pharmaceuticals: Employment, Equity Ownership. Orlando:Syros Pharmaceuticals: Employment, Equity Ownership. Eaton:Syros Pharmaceuticals: Employment, Equity Ownership. McKeown:Syros Pharmaceuticals: Employment. Tyner:Takeda Pharmaceutical Company: Research Funding; Agios Pharmaceuticals: Research Funding; Incyte Corporation: Research Funding; Gilead: Research Funding; Janssen Pharmaceutica: Research Funding; Syros: Research Funding; Aptose Biosciences: Research Funding; Seattle Genetics: Research Funding; Constellation Pharmaceuticals: Research Funding; Genentech: Research Funding; Array Biopharma: Research Funding; AstraZeneca: Research Funding; Leap Oncology: Consultancy. Sprott:Syros Pharmaceuticals: Employment, Equity Ownership. Fritz:Syros Pharmaceuticals: Employment, Equity Ownership. Di Tomaso:Syros Pharmaceuticals: Employment.

Interaction between Ras and Bcl2L12 in B cells suppresses IL-10 expression

The pathogenesis of recurrent tonsillitis is to be further investigated. B cell-derived interleukin (IL)-10 plays a critical role in immune regulation. Ras activation plays an important role in cancer and many immune disorders. This study aims to investigate the role of Ras activation in down regulating IL-10 expression in tonsillar B cells. Surgically removed tonsil tissues were collected from patients with recurrent acute tonsillar inflammation; B cells were isolated from the tonsillar tissues by flow cytometry sorting to be analyzed by the Ras-specific enzyme-linked immunosorbent assay and pertinent immunological approaches. We found that, compared to peripheral B cells (pBC), B cells isolated from the tonsillar tissues with recurrent inflammation (tBC) showed higher Ras activation, lower IL-10 expression and higher Bcl2L12 expression. Bcl2L12 formed a complex with GAP (GTPase activating protein) to prevent Ras from deactivating. The Ras activation triggered the MAPK/Sp1 pathway to promote the Bcl2L12 expression in B cells. Bcl2L12 prevented the IL-10 expression in tBCs, that was counteracted by inhibition of Ras or the Ras signal transduction pathway. In conclusion, Bcl2L12 interacts with Ras activation to compromise immune tolerance in the tonsils by inhibiting the IL-10 expression in tBCs. Inhibition of Bcl2L12 can restore the IL-10 expression in tBCs.

Targeting NF-κB–dependent alkaliptosis for the treatment of venetoclax-resistant acute myeloid leukemia cells

Venetoclax is a highly selective BCL2 inhibitor widely used in the treatment of leukemia, especially chronic lymphocytic leukemia and acute myeloid leukemia (AML). However, long-term use of venetoclax may lead to secondary drug resistance, which constitutes an important obstacle to prolonging the duration of the therapeutic response. Here, we show that the acquired resistance to venetoclax in human AML cell lines depends on NF-κB activation rather than on the upregulation of anti-apoptotic BCL2L1 expression. Moreover, alkaliptosis induced by the small molecular compound JTC801, but not necroptosis and ferroptosis, inhibits the growth of venetoclax-resistant AML cells in vitro and in xenograft mouse models. Mechanistically, NF-κB–mediated CA9 downregulation is required for intracellular pH upregulation, thereby inducing alkaliptosis in venetoclax-resistant cells. These findings provide a new strategy to selectively remove venetoclax-resistant AML cells.

NOXA-mediated degradation of MCL1 and BCL2L1 causes apoptosis of daunorubicin-treated human acute myeloid leukemia cells.

Daunorubicin (DNR) is used clinically to treat acute myeloid leukemia (AML), while the signaling pathways associated with its cytotoxicity are not fully elucidated. Thus, we investigated the DNR-induced death pathway in the human AML cell lines U937 and HL-60. DNR-induced apoptosis in U937 cells accompanied by downregulation of MCL1 and BCL2L1, upregulation of Phorbol-12-myristate-13-acetate-induced protein 1 (NOXA), and mitochondrial depolarization. DNR induced NOX4-mediated reactive reactive oxygen species (ROS) production, which in turn inactivated Akt and simultaneously activated p38 mitogen-activated protein kinase (MAPK). Activated p38 MAPK and inactivated Akt coordinately increased GSK3β-mediated cAMP response element-binding protein (CREB) phosphorylation, which promoted NOXA transcription. NOXA upregulation critically increased the proteasomal degradation of MCL1 and BCL2L1. The same pathway was also responsible for the DNR-induced death of HL-60 cells. Restoration of MCL1 or BCL2L1 expression alleviated DNR-induced mitochondrial depolarization and cell death. Furthermore, ABT-199 (a BCL2 inhibitor) synergistically enhanced the cytotoxicity of DNR in AML cell lines. Notably, DNR-induced DNA damage was not related to NOXA-mediated degradation of MCL1 and BCL2L1. Collectively, these results indicate that the upregulation of NOXA expression through the NOX4-ROS-p38 MAPK-GSK3β-CREB axis results in the degradation of MCL1 and BCL2L1 in DNR-treated U937 and HL-60 cells. This signaling pathway may provide insights into the mechanism underlying DNR-triggered apoptosis in AML cells.

LncRNA ACTA2-AS1 suppress colon adenocarcinoma progression by sponging miR-4428 upregulation BCL2L11

BackgroundLong non-coding RNA is considered to be essential to modulate the development and progression of human malignant cancers. And long non-coding RNA can act as crucial modulators by sponging the corresponding microRNA in tumorigenesis. We aimed to elucidate the function of ACTA2-AS1 and its molecular mechanism in colon adenocarcinoma.Materials and methodsThe expression of ACTA2-AS1, miR-4428 and BCL2L11 in colon adenocarcinoma tissues were detected via qRT-PCR. SW480 and HT29 cells were transfected with shRNA ACTA2-AS1, OE ACTA2-AS1, miRNA mimics of miR-4428, miR-4428 inhibitor, si-BCL2L11 and over-expression of si-BCL2L11. Cell proliferation, colony formation and apoptosis were respectively assessed using CCK-8 assay, colony assay and flow cytometry. Luciferase reporter assay was performed to verify the targets of ACTA2-AS1 and miR-4428. Tumor subcutaneous xenograft mode was constructed to explore tumor growth in vivo.ResultsACTA2-AS1 was obviously downregulated in human colon adenocarcinoma tissues and colon adenocarcinoma cell lines. Silence or over-expression of ACTA2-AS1 promoted or inhibited cell proliferation and colony formation abilities, and regulated apoptosis. The silence of ACTA2-AS1 resulted in the decrease of Bax and increase of Bal2, while restored in OE ACTA2-AS1 group when compared with the control transfected cells. In addition, luciferase reporter assay revealed that ACTA2-AS1 interacted with miR-4428 and suppressed its expression. miR-4428 could bind to 3ʹ untranslated region of BCL2L11 and modulated the expression of BCL2L11 negatively. Knockdown of ACTA2-AS1 and over-expression of BCL2L11 reversed the biological function that ACTA2-AS1 mediated by knockdown ACTA2-AS1 alone.ConclusionOur data demonstrated that ACTA2-AS1 could suppress colon adenocarcinoma progression via sponging miR-4428 to regulate BCL2L11 expression.

Effects of novel brain-penetrating oxime acetylcholinesterase reactivators on sarin surrogate-induced changes in rat brain gene expression.

Past assassinations and terrorist attacks demonstrate the need for a more effective antidote against nerve agents and other organophosphates (OP) that cause brain damage through inhibition of acetylcholinesterase (AChE). Our lab has invented a platform of phenoxyalkyl pyridinium oximes (US patent 9,277,937) that demonstrate the ability to cross the blood-brain barrier in in vivo rat tests with a sarin surrogate nitrophenyl isopropyl methylphosphonate (NIMP) and provide evidence of brain penetration by reducing cessation time of seizure-like behaviors, accumulation of glial fibrillary acidic protein (GFAP), and hippocampal neuropathology, as opposed tothe currently approved oxime, 2-pyridine aldoxime methyl chloride (2-PAM). Using two of the novel oximes (Oximes 1 and 20), this project examined whether gene expression changes might help explain this protection. Expression changes in the piriform cortex were examined using polymerase chain reaction arrays for inflammatory cytokines and receptors. The hippocampus was examined via quantitative polymerase chain reaction for the expression of immediate-early genes involved in brain repair (Bdnf), increasing neurotoxicity (Fos), and apoptosis control (Jdp2, Bcl2l1, Bcl2l11). In the piriform cortex, NIMP significantly stimulated expression for the macrophage inflammatory proteins CCL4, IL-1A, and IL-1B. Oxime 20 by itself elicited the most changes. When it was given therapeutically post-NIMP, the largest change occurred: a 310-fold repression of the inflammatory cytokine, CCL12. In the hippocampus, NIMP increased the expression of the neurotoxicity marker Fos and decreased the expression of neuroprotective Bdnf and antiapoptotic Bcl2l1. Compared with 2-PAM, Oxime 20 stimulated Bcl2l1 expression more and returned expression closer to the vehicle control values.

Melatonin protects against defects induced by Enniatin B1 during porcine early embryo development.

Exogenous factors influence embryo development. Enniatin B1 (EB1), one emerging mycotoxin of Fusarium fungi, can cause damage to cells and mouse blastocysts. However, the toxicity of EB1 on porcine embryo development and whether melatonin can eliminate the detrimental effects of EB1 on embryos remain unclear. Here, this work demonstrated that EB1 significantly decreased the cleavage and blastocyst rates and blastocyst cell number of embryos in a dose and time dependent manner. Further study displayed that EB1 obviously destroyed nuclear remodeling dynamics. Importantly, EB1 triggered embryo apoptosis through downregulating the expression of Sod1, Gpx4, Cat and Bcl2l1 while upregulating the transcription of Bax and Caspase3. Moreover, EB1 significantly disrupted the transcription of Dnmt1, Dnmt3a, Tet1 and Tet3, further leading to incomplete DNA demethylation of CenRep, Oct4, Nanog and Sox2, thus, the expression of Eif1a, Oct4, Nanog and Sox2 remarkably decreased. Whereas EB1-exposed embryos were treated with melatonin, these disorders were obviously ameliorated, and the development ability of embryos was also rescued. In conclusion, EB1 exerted detrimental effects on porcine early embryos, while melatonin effectively rescued EB1-mediated defects in embryos. This work provides a novel insight into the improvement of embryo quality and the promotion of human and animal reproduction.

Altered mRNA expression levels of autophagy- and apoptosis-related genes in the FOXO pathway in schizophrenia patients treated with olanzapine

This study attempted to analyze the alterations in the mRNA expression levels of autophagy- and apoptosis-related genes in the forkhead box transcription factor O (FOXO) pathway in schizophrenia patients before and after olanzapine treatment. For a total of 32 acute schizophrenic inpatients, clinical data with PANSS were obtained before and after four weeks of olanzapine treatment (mean dose 14.24 ± 4.35 mg/d) along with data from 32 healthy volunteers. The mRNA expression levels of the FOXO pathway genes were measured by real-time qPCR after fasting venous blood was collected and analyzed. The mRNA expression levels of FOXO1, FOXO3A, FASLG, and BCL2L11 were observed to be significantly decreased in acute schizophrenia patients. After four weeks of olanzapine treatment, the expression levels of the first three genes were further reduced, but BCL2L11 expression levels were not significantly changed. The pairwise correlations between the mRNA expression level of FASLG and those of the other three genes were not observed in acute schizophrenia patients, while these relationships were observed in healthy controls. After olanzapine treatment, the FASLG mRNA expression level was restored and exhibited a pairwise correlation with the FOXO3A and BCL2L11 mRNA expression levels but not with the FOXO1 mRNA expression level, and FASLG mRNA expression was also correlated with the duration of the disease. The statuses and correlations of the mRNA expression levels of FOXO pathway-related genes were altered in schizophrenia patients and were affected by olanzapine treatment and the duration of the disease.

Study on mechanism of matrine in treatment of COVID-19 combined with liver injury by network pharmacology and molecular docking technology

The aim of the present study was to investigate the pharmacological mechanism of matrine in treatment of COVID-19 combined with liver injury. Potential targets related to matrine, COVID-19 and liver injury were identified from several databases. We constructed PPI network and screened the core targets according to the degree value. Then, GO and KEGG enrichment were carried out. Molecular docking technology was used to verify the affinity between matrine and the crystal structure of core target protein. Finally, real-time RT-PCR was used to detect the effects of matrine on hub gene expression in liver tissue of liver injury mice and lung tissue of lung injury mice to further confirm the results of network pharmacological analysis. The results show that six core targets including AKT1, TP53, TNF, IL6, BCL2L1 and ATM were identified. The potential therapeutic mechanism of matrine on COVID-19 combined with liver injury is closely related to regulate antiviral process, improve immune system and regulate the level of inflammatory factors. Molecular docking showed that matrine could spontaneously bind to the receptor protein and had strong binding force. Real-time RT-PCR demonstrated that matrine could significantly reduce the expression of AKT1, TP53, TNF, IL6 and ATM in mice with liver injury or lung injury (P < 0.05), and increase the expression of BCL2L1 to a certain extent (P > 0.05). Our results indicate that matrine can achieve simultaneous intervention of COVID-19 combined with liver injury by multi-dimensional pharmacological mechanism.

Determining molecular and genetic markers for severe EBV-mononucleosis

Epstein—Barr virus (EBV) is one of the etiological agents causing infectious mononucleosis. A severe form of the disease can result in developing serious complications, which risk might also depend on the state of patient’s immune system. To date, no specific tests for assessing a risk of developing severe disease form are available. Our study was aimed at identifying molecular genetic markers of severe EBV-infectious mononucleosis (EBV-IM) in immunocompetent peripheral blood cells. Expression of 483 genes and gene transcripts regulating apoptosis, proliferation and differentiation of immunocompetent cells was measured in the peripheral blood leukocytes from patients with severe and moderate EBV-IM as well as apparently healthy donors. A DNA-microarray designed by us and subsequent data processing were carried out by using custom-made “MiDA” software. To identify markers of a severe form of the pathology, expression of each gene and transcript was compared in EBV-IM patients and apparently healthy donors. For each gene and transcript, the level of expression fold change and significance for binary classification were determined. Genes and transcripts, characterized by the maximum values of two determined parameters while comparing patients with severe infection and healthy donors, as well as patients with severe and moderate EBV-IM forms, were selected as markers of severe EBV-IM. Genes and transcripts with differed expression in patients with moderate EBV-IM and healthy donors, were excluded from the list of markers. In addition, sex- and age-linked markers with differed expression were excluded as well. The markers for severe EBV-IM consisted of apoptosis regulators (BCL2L11, BIRC3 genes and XIAP.NM_001167 transcript) and splicing factors (CELF6 gene and SF1.NM_201995 transcript). Compared with donors and patients with a moderate form of the disease, a decreased expression of BCL2L11, BIRC3 genes, transcripts SF1.NM_201995 and XIAP.NM_001167, as well as increased expression of the CELF6 gene were detected in the blood of patients with severe EBV-IM. The functional role of identified molecular markers suggests that severe EBV-IM is characterized by suppressed mitochondrial and activated TRAF-dependent apoptosis pathways in immunocompetent cells. The expression pattern for select markers is specific for severe EBV-MI, not associated with patient sex and age. Thus, study data may be used to develop specific tools for assessing a risk of developing complications of EBV mononucleosis.

Bcl2 like protine-12 (Bcl2L12) facilitates experimental airway allergic inflammation by inducing autocrine eotaxin in eosinophils

BackgroundThe pathogenesis of airway allergic disorders (AAD) needs to be further investigated. Eosinophils (Eos) are the canonical effector cells in AAD attacks. Bcl2 like protein-12 (Bcl2L12) is an apoptosis inhibitor and an immune regulator. Eos have the defects of apoptosis. This study aims to investigate the role of Bcl2L12 in the AAD pathogenesis by regulating Eo activities. MethodsHuman nasal lavage fluids (NLF) and mouse bronchoalveolar lavage fluids (BALF) was collected. Eos in NLF and BALF were analyzed by flow cytometry. A murine AAD model was developed with ovalbumin as a specific antigen. ResultsWe found that Eos isolated from NLF or BALF of AAD subjects expressed high levels of Bcl2L12 and showed defects of apoptosis. The Bcl2L12 expression in Eos was positively correlated with the AAD response. High lipopolysaccharide levels were detected in the AAD airways, that promoted the Bcl2L12 expression in Eos. Bcl2L12 mediated the LPS-induced autocrine eotaxin 1 expression in Eos through activating the MAPK p38/STAT6/NF-κB signal pathway. Depletion of Bcl2L12 in Eos suppressed experimental AAD in mice. ConclusionsAAD Eos express high levels of Bcl2L12, the latter is associated with AAD response by regulating the autocrine eotaxin 1 in Eos. Depletion of Bcl2L12 in Eos attenuates experimental AAD, suggesting that to suppress the Bcl2L12 Eos has the translational potential in the treatment of AAD.

Estrogens Determine Adherens Junction Organization and E-Cadherin Clustering in Breast Cancer Cells via Amphiregulin.

SummaryEstrogens play an important role in the development and progression of human cancers, particularly in breast cancer. Breast cancer progression depends on the malignant destabilization of adherens junctions (AJs) and disruption of tissue integrity. We found that estrogen receptor alpha (ERα) inhibition led to a striking spatial reorganization of AJs and microclustering of E-Cadherin (E-Cad) in the cell membrane of breast cancer cells. This resulted in increased stability of AJs and cell stiffness and a reduction of cell motility. These effects were actomyosin-dependent and reversible by estrogens. Detailed investigations showed that the ERα target gene and epidermal growth factor receptor (EGFR) ligand Amphiregulin (AREG) essentially regulates AJ reorganization and E-Cad microclustering. Our results not only describe a biological mechanism for the organization of AJs and the modulation of mechanical properties of cells but also provide a new perspective on how estrogens and anti-estrogens might influence the formation of breast tumors.

In Vitro Maturation with Leukemia Inhibitory Factor Prior to the Vitrification of Bovine Oocytes Improves Their Embryo Developmental Potential and Gene Expression in Oocytes and Embryos.

Oocyte cryopreservation has a significant impact on subsequent embryonic development. Herein, we investigated whether supplementing in vitro maturation medium with Leukemia Inhibitory Factor (LIF) prior to vitrification affects embryo development and gene expression at different embryo developmental stages. A panel of genes including maternal effect, epigenetics, apoptosis and heat stress was relatively quantified. The results show reduced cleavage rates after vitrification, regardless of the LIF treatment. Although not statistically different from control-vitrified oocytes, oocyte apoptosis and the blastocyst yield of LIF-vitrified oocytes were similar to their non-vitrified counterparts. Vitrification increased oocyte ZAR1, NPM2 and DPPA3 gene expression while its expression decreased in LIF-vitrified oocytes to similar or close levels to those of non-vitrified oocytes. With a few gene-specific exceptions, vitrification significantly increased the expression of DNMT3A, HDAC1, KAT2A, BAX and BCL2L1 in oocytes and most stages of embryo development, while comparable expression patterns for these genes were observed between LIF-vitrified and non-vitrified groups. Vitrification increased HSPA1A expression in oocytes and HSP90AA1 in 2-cell embryos. Our data suggest that vitrification triggers stage-specific changes in gene expression throughout embryonic development. However, the inclusion of LIF in the IVM medium prior to vitrification stimulates blastocyst development and several other developmental parameters and induces oocytes and embryos to demonstrate gene expression patterns similar to those derived from non-vitrified oocytes.

IKAROS and CK2 regulate expression of BCL-XL and chemosensitivity in high-risk B-cell acute lymphoblastic leukemia

AbstractHigh-risk B-cell acute lymphoblastic leukemia (B-ALL) is an aggressive disease, often characterized by resistance to chemotherapy. A frequent feature of high-risk B-ALL is loss of function of the IKAROS (encoded by the IKZF1 gene) tumor suppressor. Here, we report that IKAROS regulates expression of the BCL2L1 gene (encodes the BCL-XL protein) in human B-ALL. Gain-of-function and loss-of-function experiments demonstrate that IKAROS binds to the BCL2L1 promoter, recruits histone deacetylase HDAC1, and represses BCL2L1 expression via chromatin remodeling. In leukemia, IKAROS' function is impaired by oncogenic casein kinase II (CK2), which is overexpressed in B-ALL. Phosphorylation by CK2 reduces IKAROS binding and recruitment of HDAC1 to the BCL2L1 promoter. This results in a loss of IKAROS-mediated repression of BCL2L1 and increased expression of BCL-XL. Increased expression of BCL-XL and/or CK2, as well as reduced IKAROS expression, are associated with resistance to doxorubicin treatment. Molecular and pharmacological inhibition of CK2 with a specific inhibitor CX-4945, increases binding of IKAROS to the BCL2L1 promoter and enhances IKAROS-mediated repression of BCL2L1 in B-ALL. Treatment with CX-4945 increases sensitivity to doxorubicin in B-ALL, and reverses resistance to doxorubicin in multidrug-resistant B-ALL. Combination treatment with CX-4945 and doxorubicin show synergistic therapeutic effects in vitro and in preclinical models of high-risk B-ALL. Results reveal a novel signaling network that regulates chemoresistance in leukemia. These data lay the groundwork for clinical testing of a rationally designed, targeted therapy that combines the CK2 inhibitor, CX-4945, with doxorubicin for the treatment of hematopoietic malignancies.