https://openworks.mdanderson.org/sumexp22/1092/thumbnail.jp

Cheng, Donghang

Diallo, Habibatou

Gopalakrishnan, Vidya, PhD

Maegawa, Shinji, PhD

Singh, Sandeep

English

Habibatou Diallo

Sandeep Singh

Shinji Maegawa

Donghang Cheng

Vidya Gopalakrishnan

Crossref

Cytotoxic Potential of MIthramycin against DIPG cell lines

OpenWorks @ MD Anderson

Summary of ResultsConclusionsMithramycin is able to induce cell death in DIPG cell lines. REST-SET DB1 interaction axis is need to be evaluated for its potential impact on DIPG tumor survival.Detailed mechanism of Mithramycin action is needed to be elucidated.ReferencesHauser B, Gröger M, Ehrmann U, Albicini M, Brückner UB, Schelzig H, Venkatesh B, Li H, Szabó C, Speit G, Radermacher P, Kick J. The parp-1 inhibitor ino-1001 facilitates hemodynamic stabilization without affecting DNA repair in porcine thoracic aortic cross-clamping-induced ischemia/reperfusion. Shock. 2006 Jun;25(6):633-40. doi: 10.1097/01.shk.0000209561.61951.2e. PMID: 16721272.Kumar P, Nagarajan A, Uchil PD. Analysis of Cell Viability by the MTT Assay. Cold Spring Harb Protoc. 2018 Jun 1;2018(6). doi: 10.1101/pdb.prot095505. PMID: 29858338.Vanan MI, Eisenstat DD. DIPG in Children - What Can We Learn from the Past? Front Oncol. 2015 Oct 21;5:237. doi: 10.3389/fonc.2015.00237. PMID: 26557503; PMCID: PMC4617108.Aziz-Bose R, Monje M. Diffuse intrinsic pontine glioma: molecular landscape and emerging therapeutic targets. Curr Opin Oncol. 2019 Nov;31(6):522-530. doi: 10.1097/CCO.0000000000000577. PMID: 31464759; PMCID: PMC7242222.Srikanthan D, Taccone MS, Van Ommeren R, Ishida J, Krumholtz SL, Rutka JT. Diffuse intrinsic pontine glioma: current insights and future directions. Chin Neurosurg J. 2021 Jan 11;7(1):6. doi: 10.1186/s41016-020-00218-w. PMID: 33423692; PMCID: PMC7798267.MethodsCell CultureTSM base media was prepared by mixing equal volumes of Neurobasal-A and DMEM/F12 mix along with growth factors. Fresh culture media was prepared by adding EGF, FGC, PDGF, Heparin and other growth factors in TSM base medium and used to culture all three cell lines.TreatmentsMithramycin was dissolved in DMSO at 10mM concentration followed by serial dilutions in complete media. 10 & 100uMStocks were used to administer treatments in 96 well plates for 120 hours. MTT AssayCells were plated in 96-well plates for an incubation period of 120 hours. For adherent cells, media is suctioned from the plate. For suspension cells, cells are centrifuged at 4 degrees Celsius for 5 minutes before media is sanctioned. 50uL of media per well followed by 50uL of MTT solution per well is added to 96-well plates. Cells are incubated at 37 degrees Celsius for 4 hours. Following incubation, 150 uL of MTT solvent is added to each well. Absorbance is read at OD=590nm within 1 hour.Fiigure 2. (A) REST and SET DB1 interaction shown via IP (B) SET DB1 expression is also elevated in DIPG and (C) SET DB1 knockout decreases DIPG proliferation. ChIP experiment showing REST (D), SET DB1 (E) and H3K9me3 (F) on caveolin-1 promoter. Cytotoxic potential of  Mithramycin against DIPG cell linesHabibatou Diallo, Sandeep Singh, PhD, Shinji Maegawa, PhD, Donghang Cheng, PhD, Vidya Gopalakrishnan, PhD. Department of Pediatrics, The University of Texas- MD Anderson Cancer Center, Houston, TXResearch BackgroundDiffuse Intrinsic Pontine Glioma (DIPG) is a glial tumor occurringprimarily in children with extremely poor prognosis. Occurringoutside of the pons, surgery following diagnosis is not viable forDIPG patients as the location of the tumor is vital for motorfunctions. There has yet to be a significant improvement in thesurvival median rate upon diagnosis outside of the effects of radiationtherapy. Though radiation therapy has proven to improve neurologicalsymptoms of DIPG patients, the results of this treatment have provento be transitory at best. Currently, there is an interest in looking atspecific oncogenic targets to find the most efficacious biologicalagent(s) to decrease tumor cell growth.Mithramycin is a relatively new anticancer drug reported to beeffective against variety of cancers. It blocks Specificity protein-1(Sp1) from binding to DNA through its guanine-cytosine(G-C)-specific DNA-binding ability and selectively downregulatesan X-linked inhibitor of apoptosis protein (XIAP) levels, thereforeincreasing levels of apoptosis in cancer cells. SET Domain BifurcatedHistone Lysine Methyltransferase 1 (coded by SETDB1 gene) is anenzyme that reversibly catalyzes methylation in Histone 3 K9(H3K9). SETDB1 is heavily involved in the downregulation of vitaltumor-suppressive genes, adding to its pro-oncogenic nature and isrecently found to be overexpressed in DIPGs by our lab. Mithramycinwas reported to be a SETDB1 inhibitor by blocking the interactionbetween SP-1 and SETDB1 regulatory sites. REST and SET DB1 are overexpressed in DIPG tumors and contribute to proliferative potential.Interaction of REST and SET DB1 indicates towards cooperative oncogenic behavior in DIPGs.REST and SET DB 1 occupies Caveolin -1 promoter and induce trimethylation of H3K9.Targeting SET DB1 expression using Mithramycin results in significant decrease in cell proliferation in all DIPG cell lines.Slightly lower toxicity in REST high cell lines indicate REST might be rescuing against loss of SET DB1 expression.Cell Cycle analysis showed decreased population in G2/M phase for DIPG7 cells while in DIPG 7REST, there is significant decrease in cell populations in S and G2/M phase.REST 971 ChIP-Seq targetsNES=-7.70., FDR=0.0DIPG13scr SETDB1siRNADIPG7scr SETDB1A BC DEA BDIPG7DIPG13DIPG13DIPG13DIPG7DIPG7HypothesisSince oncogenic proteins REST and SET DB1 interacts with each other and are overexpressed in DIPGs, targeting their expression/blocking interaction may prove a novel therapeutic approach. Using mithramycin may result in interfering with SET DB1 and REST oncogenic interaction  and inhibit DIPG proliferation.Figure 2: Cytotoxicity analysis of DIPG 7 (A), DIPG 4 (B), DIPG  13 (C), DIPG 7REST (D), DIPG 4REST (E) and DIPG 13REST (F) cell lines upon treatmentwith different doses of Mithramycin for 120 hours. Figure 3: Cell cycle analysis of DIPG 7 and DIPG 7REST cells upon 72 hours treatmentWith Mithramycin. Figure 1: (A) REST complex & its binding to RE1 sites. (B) REST expressionin DIPGs. (C) ChIPseq shows decreased expression of genes wherepromoters are bound by REST in DIPGs. (D) ATACseq and RNAseq combined analysis revealed REST and SETDB1 are hyperactive TFs in DIPGs. (E) REST knockdown block DIPG tumors and increase survival in mice models. DIPG7DIPG13C D E FA B CD E F

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Cytotoxic Potential of MIthramycin against DIPG cell lines

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