Expression Of Inhibitory Factors Research Articles

Abstract 5583: SUV39H1 disruption enhances the persistence and anti-tumor efficacy of CD28-costimulated human CAR T cells

Abstract Retrovirally encoded CD19-specific CARs that incorporate CD28 and CD3z signaling motifs (Rv-1928z) have induced remarkable responses in patients with refractory leukemia and lymphoma. These CARs induce a strong effector differentiation program in T cells that can limit their persistence and result in T cell dysfunction. This induction of terminal effector differentiation is accompanied by transcriptional and epigenetic changes resulting in induction of effector transcriptional factors, inhibitory receptors, and suppression of memory associated genes. In this study, we examine the effect of modulating the epigenome of human Rv-1928z CAR T cells by disruption of the histone methyl transferase, SUV39H1, which has been implicated in regulating memory to effector transition in murine T cells (Pace et al. 2018). To assess the impact of SUV39H1 on human Rv-1928z CAR T cells, we treated immune deficient mice bearing the human ALL cell line, NALM6, with limiting doses of SUV39H1-edited T cells. SUV39H1 editing (SUV39H1etd) significantly enhanced the anti-tumor efficacy of Rv-1928z CAR T cells relative to non-edited counterparts, with 9/10 NALM6 bearing mice treated with SUV39H1etd Rv-1928z surviving over the duration of observation (90 days) as compared to 1/12 mice treated with WT Rv-1928z. This enhanced tumor control in SUV39H1etd Rv-1928z CAR T cells was associated with greater initial CAR T cell proliferation upon tumor encounter and enhanced long-term CAR T cell persistence (> day 50). To assess whether the persisting SUV39H1etd Rv-1928z CAR T cells can mount an effective effector response upon tumor rechallenge, we modified the stress test model such that post primary tumor clearance (day 17), mice were rechallenged by tumor 5 times over 70 days. SUV39H1etd Rv-1928z CAR T cells outperformed WT Rv-1928z CAR T cells in eliminating NALM6 upon rechallenge. Paired genome accessibility (ATACseq) and transcriptional analysis revealed epigenetic changes associated with SUV39H1 loss in Rv-1928z CAR T cells that promote expression of memory associated transcription factors and receptors while reducing expression of inhibitory receptors and transcriptional factors associated with T cell dysfunction. In summary, we find that loss of SUV39H1 in human Rv-1928z CAR T cells enhances their anti-tumor efficacy by bringing about changes in their epigenome that enhance their functional persistence. Citation Format: Nayan Jain, Zeguo Zhao, Richard Koche, Yosi Gozlan, David Brocks, Tali Raveh-Sadka, Danny Wells, Anton Dobrin, Yuzhe Shi, Michael Lopez, Gertrude Gunset, Michel Sadelain. SUV39H1 disruption enhances the persistence and anti-tumor efficacy of CD28-costimulated human CAR T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5583.

Inhibition of ABCC9 by zinc oxide nanoparticles induces ferroptosis and inhibits progression, attenuates doxorubicin resistance in breast cancer

BackgroundZinc oxide nanoparticles (ZONs) are a type of nanomaterial that has presented anti-cancer properties in breast cancer (BC). However, the function of ABCC9 in BC and its correlation with ZONs are still elusive.MethodsHere, we identified the crucial role of ABCC9 in modulating ferroptosis and doxorubicin (Dox) resistance in BC and the targeted function of ZONs to ABCC9.ResultsThe silencing of ABCC9 significantly repressed the viability of BC cells. The knockdown of ABCC9 decreased the numbers of Edu-positive BC cells. Conversely, BC cell apoptosis was increased by the inhibition of ABCC9. Besides, the silencing of ABCC9 reduced the capability of migration and invasion of BC cells. Significantly, tumorigenicity analysis demonstrated that the tumor growth of BC cells was suppressed by the depletion of ABCC9 in the xenograft model of nude mice. Moreover, the treatment of ferroptosis activator erastin repressed cell viability of BC cells and ABCC9 overexpression rescued the repression. Similarly, the numbers of Edu-positive BC cells were inhibited by erastin and the overexpression of ABCC9 reversed the inhibitory effect of erastin. The levels of GSH were decreased and MDA, lipid ROS, and iron levels were increased by the treatment of erastin, while the ABCC9 overexpression could reverse these results in BC cells. Consistently, erastin suppressed the expression of ferroptosis inhibitory factors, including GPX4 and SLC7A11, in BC cells and the overexpression of ABCC9 rescued the expression. The IC50 value of Dox was reduced by the knockdown of ABCC9 in Dox-resistant BC cells (BC/Dox). The numbers of Edu-positive BC/Dox cells were attenuated by the depletion of ABCC9. Meanwhile, the apoptosis of BC/Dox cells was stimulated by the silencing of ABCC9. Furthermore, the treatment of ZONs attenuated Dox resistance of BC cells. ZONs remarkably repressed the expression of ABCC9 in BC/Dox cells. ZONs inhibited the cell viability of BC/Dox cells and the overexpression of ABCC9 reversed the repression. Moreover, the treatment of ZONs reduced GSH levels and enhanced MDA, lipid ROS, and iron levels in erastin-stimulated BC/Dox cells.ConclusionsIn conclusion, we discovered that the inhibition of ABCC9 by zinc oxide nanoparticles induces ferroptosis and attenuates Dox resistance in BC.

Zscan10 suppresses osteoclast differentiation by regulating expression of Haptoglobin.

Zinc finger and SCAN domain containing 10 (Zscan10) was identified as a novel transcription factor that is involved in osteoclast differentiation in our previous report. However, the biological functions of Zscan10 are not fully understood except its roles in the maintenance of genome stability and pluripotency of embryonic stem cells. Therefore, the purpose of this study was to clarify the function of Zscan10 in somatic cells, especially during osteoclast differentiation. First, Zscan10 KO RAW264 (KO) cells were established by genome editing using CRISPR/Cas9 and single cell sorting. Then, control (Ctrl) and KO cells were differentiated into osteoclasts by RANKL stimulation. We observed that TRAP activity and the expression levels of differentiation marker genes, such as Nfatc1, were significantly increased and the expression of inhibitory factors, such as Irf8, was decreased in KO cells compared to Ctrl cells. These results suggest that Zscan10 might regulate transcription of the genes that negatively control osteoclastogenesis. To understand gene expression profiles controlled by Zscan10, RNA-seq was performed and stringent analyses identified the haptoglobin gene (Hp) as a possible target of Zscan10. In addition, ChIP against Zscan10 revealed that Zscan10 could interact with its binding motif located near the Hp gene locus as well as the transcription start site of Hp, suggesting that Zscan10 can directly regulate transcription of Hp. Finally, to examine the effects of Hp on osteoclastogenesis, KO cells were treated with recombinant Hp (rHp). rHp treatment suppressed TRAP activity of KO cells without affecting cell viability. Furthermore, it has been reported that Hp KO mice exhibit decreased bone mass and increased osteoclast number. Importantly, hemolytic disease patients exhibited decreased serum level of Hp as well as low bone mineral density. Taken together, this study suggests that Zscan10 negatively regulates osteoclast differentiation through transcription of Hp.

Emerging A New Strategy for the Antitumor Immunotherapy: Pharmacological Modulation of the Ca2+/Camp Signaling Interaction

Cancer is a major public health problem and the second leading cause of mortality around the world. Antitumor immunotherapy using monoclonal antibodies is considered selective and efficient in the treatment of different types of tumors, but its cost and toxic effects limit its application. Many tumor microenvironments, including lymphoma and carcinoma, are enriched in immune suppressive cells that contribute to immune exhaustion by means expression of inhibitory ligands, suppressive cytokines, and tumor-promoting factors. Antitumor therapies targeted to reduce the induction, recruitment, or suppressive activities of the immune cells have been investigated. New antitumor strategies using drugs targeted to intracellular signaling involved in cell proliferation and survival, angiogenesis, and metastasis have become promising in recent years. Thus, our discovery of the role of functional interaction between intracellular signaling pathways mediated by calcium ions (Ca2+) and cyclic adenosine monophosphate (cAMP) (Ca2+/cAMP signaling interaction) in these cellular responses, opened a great avenue for the development of new antitumor therapeutic strategies. Here, we discuss how the combined use of monoclonal antibodies with drugs that modulate the Ca2+/cAMP signaling interaction to reduce tumor growth could be a potential strategy in the antitumor immunotherapy due to the increment of antitumor efficacy and reduction of adverse effects.

Inhibition of beta cell growth and function by bone morphogenetic proteins.

Impairment of beta cell mass and function is evident in both type 1 and type 2 diabetes. In healthy physiological conditions pancreatic beta cells adapt to the body's increasing insulin requirements by proliferation and improved function. We hypothesised that during the development of diabetes, there is an increase in the expression of inhibitory factors that prevent the beta cells from adapting to the increased need for insulin. We evaluated the effects of bone morphogenetic protein (BMP) 2 and -4 on beta cells. The effects of BMP2 and -4 on beta cell proliferation, apoptosis, gene expression and insulin release were studied in isolated islets of Langerhans from rats, mice and humans. The expression of BMPs was analysed by immunocytochemistry and real-time PCR. The role of endogenous BMP was investigated using a soluble and neutralising form of the BMP receptor 1A. BMP2 and -4 were found to inhibit basal as well as growth factor-stimulated proliferation of primary beta cells from rats and mice. Bmp2 and Bmp4 mRNA and protein were expressed in islets and regulated by inflammatory cytokines. Neutralisation of endogenous BMP activity resulted in enhanced proliferation of rodent beta cells. The expression of Id mRNAs was induced by BMP4 in rat and human islets. Finally, glucose-induced insulin secretion was significantly impaired in rodent and human islets pre-treated with BMP4, and inhibition of BMP activity resulted in enhanced insulin release. These data show that BMP2 and -4 exert inhibitory actions on beta cells in vitro and suggest that BMPs exert regulatory roles of beta cell growth and function.

SCIRR39 Promotes Differentiation of Oligodendrocyte Precursor Cells and Regulates Expression of Myelin-Associated Inhibitory Factors

SCIRR39 is an identified upregulated gene in rat primary neuron injury and/or regeneration process. However, roles of SCIRR39 in the regeneration of central nervous system (CNS) injury are still largely unexplored. Using real-time quantitative PCR and Western blotting, SCIRR39 expression was detected in oligodendrocyte precursor cells (OPCs) and oligodendrocytes. Moreover, the results from cell proliferation and cell cycle indicated that SCIRR39 inhibited OPCs proliferation and induced cell cycle arrest in G0/G1 and G2/M phases. Importantly, SCIRR39 positively regulated OPC differentiation and the expression of myelin basic protein. We also examined the effect of SCIRR39 on expression of myelin-associated inhibitory factors, including myelin-associated glycoprotein (MAG), oligodendrocyte myelin glycoprotein (OMgp), and Nogo A. Nogo A level was markedly regulated by SCIRR39 overexpression or knockdown in oligodendrocytes and cortical neurons co-cultures, while the expression of MAG and OMgp was not obviously changed by SCIRR39 overexpression or knockdown. Taken together, our results indicate the important role of SCIRR39 either in OPC differentiation or in axon myelination, and may provide a new therapeutic target for the treatment of CNS injury.

Inhibitory effect of icariin on expression of myelin inhibitory factors in the central nervous system of rats with focal cerebral ischemia

Icariin, the major active component of Chinese medicinal herb epimedium brevicornum maxim, is used widely in traditional Chinese medicine for the treatment of neurological diseases. However, the effects of icariin on myelin inhibitory factors are as yet unclear. In the present study, administration of icariin at 20 mg/kg showed a marked reduction in neurological deficit of middle cerebral artery occlusion rats. Icariin exhibited better inhibitory effects on myelin inhibitory factors: Nogo-A, myelin-associated glycoprotein and oligodendrocyte myelin glycoprotein in ischemia regions of middle cerebral artery occlusion rats compared with monosialotetrahexosylganglioside. These results indicate that icariin exhibits potent inhibitory effects on expression of myelin inhibitors after middle cerebral artery occlusion-induced focal cerebral ischemia in vivo. This effect may be mediated, at least in part, by the inhibition of both Nogo-A, myelin-associated glycoprotein and oligodendrocyte myelin glycoprotein activation, followed by the enhancement of axonal sprouting and regeneration, resulting in neurological functional recovery.

Signals of Apoptotic Pathways in Several Types of Meningioma

Meningiomas are intracranial tumour derived from meningothelial cells, which aggressive behaviour has been frequently associated to cell apoptosis. In this paper activation of several factors involved in apoptosis has been investigated on biopsies of primary, non recurrent meningiomas. Benign (meningotheliomatous, transitional, fibrous, angiomatous), atypical and anaplastic meningiomas were analysed by immunohistochemistry and western blot, to visualize the occurring of different apoptotic pathways and their association with clinical grading. Apoptotic cell have been detected by a double colorimetric staining for TUNEL and caspase-3 active form. Apoptotic signal positive cells have been detected in all type of meningiomas analysed, with exception of meningotheliomatous meningiomas. Differences have been found in the activation of apoptotic pathways between several types of grade I meningiomas and among benign, anaplastic and atypical meningiomas. An intense expression of several apoptotic inhibitor occurred in grade I meningiomas. The correlation among expression of apoptotic and inhibitory factors and cell proliferation index may suggest that in grade I meningiomas apoptosis may be related to mechanisms involved into tumor cells surviving. Instead in grade II and III meningiomas the same correlation seems indicate an high turnover of tumor cells that might be useful as index of cell proliferation and tumor mass growth.

The Effects of Shenyankangfupian on the expression of macrophage migration inhibitory factors in children with henoch-schoeulein purpura nephritis

Objective To investigate the effects of Shenyankangfupian on the expression of macrophage migration inhibitory factors(MIF) in children with henoch-schoenlein purpura nephritis(HSPN).Methods Sixty children with HSPN were divided into two groups randomly.Patients in the control group(n=30) receivded routine therapy for 3 months,and those in the treatment group(n=30) receivded routine therapy plus Shenyankangfupian.Clinical symptoms and the MIF variation were assessed.Results Blcod and protein in urine following Shenyankangfupian therapy were markedly reduced in the treatment group (P＜0.05 ).Compared with the control group (0.68 ± 0.23 ) g/24 h,protein in urine in the treatment group (0.31 ± 0.16 ) g/24 h indicated a statistical significance (P＜0.05 );There is also a statistical difference(P＜0.05) in MIF between the treatment group(2.54 ± 1.06) ng/L and the control group(2.97 ± 1.14) ng/L.Conclusion Shenyankangfupian could markedly relieve symptoms in children with HSPN by mitigating the expression of MIF thus reducing protein in urine. Key words: Nephritis; Maerophage migration inhibitory factors; Shenyankangfupian

Enhanced expression of growth factors and imbalance between hepatocyte proliferation and apoptosis in the livers of rats fed a choline-deficient, ?-amino acid-defined diet

Abstract In a rat model of hepatocarcinogenesis induced by a choline-deficient, l -amino acid-defined (CDAA) diet, hepatocellular carcinoma (HCC) occurs in conjunction with fatty liver, hepatocyte injury and regeneration, fibrosis and cirrhosis. This is similar to human HCC development with cirrhosis. The aim of this study is to clarify sequential changes in the expression of growth and growth inhibitory factors, and hepatocyte proliferation and apoptosis during development of preneoplastic nodules in rats fed a CDAA diet. The expression of hepatocyte growth factor was stimulated at about the same time as CDAA diet-induced liver injury within 1 week. Hepatocyte growth factor reached a maximum level of expression from 4 to 8 weeks. Transforming growth factor (TGF)-α expression increased from 4 to 40 weeks. Although TGF-β, a growth inhibitory factor for hepatocytes, was also expressed with a peak from 4 to 8 weeks followed by a gradual decrease until 48 weeks, expression of cyclin D1 and hepatocyte proliferation continued to be stimulated throughout the experimental period. Additionally, the number of apoptotic hepatocytes was markedly reduced after peaking at 8 weeks. These results suggest that some hepatocytes in the livers of rats fed a CDAA diet may escape from TGF-β-induced growth inhibition and apoptosis, leading to development of preneoplastic nodules.