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• W1963673975 abstract "•Group3 medulloblastoma neurospheres were screened with FDA-approved drugs•Pemetrexed and gemcitabine inhibited mouse and human G3 and mouse SHH MBs in vitro•Pemetrexed and gemcitabine increased survival of mice bearing mouse and human G3 MB•Pemetrexed and gemcitabine did not affect survival of mice bearing mouse SHH MB We devised a high-throughput, cell-based assay to identify compounds to treat Group3 medulloblastoma (G3 MB). Mouse G3 MBs neurospheres were screened against a library of approximately 7,000 compounds including US Food and Drug Administration-approved drugs. We found that pemetrexed and gemcitabine preferentially inhibited G3 MB proliferation in vitro compared to control neurospheres and substantially inhibited G3 MB proliferation in vivo. When combined, these two drugs significantly increased survival of mice bearing cortical implants of mouse and human G3 MBs that overexpress MYC compared to each agent alone, while having little effect on mouse MBs of the sonic hedgehog subgroup. Our findings strongly suggest that combination therapy with pemetrexed and gemcitabine is a promising treatment for G3 MBs. We devised a high-throughput, cell-based assay to identify compounds to treat Group3 medulloblastoma (G3 MB). Mouse G3 MBs neurospheres were screened against a library of approximately 7,000 compounds including US Food and Drug Administration-approved drugs. We found that pemetrexed and gemcitabine preferentially inhibited G3 MB proliferation in vitro compared to control neurospheres and substantially inhibited G3 MB proliferation in vivo. When combined, these two drugs significantly increased survival of mice bearing cortical implants of mouse and human G3 MBs that overexpress MYC compared to each agent alone, while having little effect on mouse MBs of the sonic hedgehog subgroup. Our findings strongly suggest that combination therapy with pemetrexed and gemcitabine is a promising treatment for G3 MBs. Despite the recent identification of four molecular groups of human MB, patients are currently treated with similar chemotherapies independent of classification. G3 MB has a high incidence of metastasis and poor prognosis. Thus, more effective therapeutic approaches for these patients are desperately needed. The development of a mouse model of G3 MB enabled production of cultured neurospheres that provided an ideal platform to identify additional chemotherapies. We found two FDA-approved drugs that significantly inhibited mouse and human G3 MB neurosphere cultures, mouse allografts, and xenografts from G3 MB primary patient samples, but not mouse SHH MBs. These findings provide a strong rationale for combination therapy with pemetrexed and gemcitabine to treat patients with G3 MB. Medulloblastoma (MB), a tumor of the posterior fossa, is primarily a pediatric disease, although it occasionally occurs in adults (Ellison et al., 2011Ellison D.W. Kocak M. Dalton J. Megahed H. Lusher M.E. Ryan S.L. Zhao W. Nicholson S.L. Taylor R.E. Bailey S. Clifford S.C. Definition of disease-risk stratification groups in childhood medulloblastoma using combined clinical, pathologic, and molecular variables.J. Clin. Oncol. 2011; 29: 1400-1407Crossref PubMed Scopus (220) Google Scholar). MB is classified into four major subgroups based on clinical and molecular profiles (Taylor et al., 2012Taylor M.D. Northcott P.A. Korshunov A. Remke M. Cho Y.J. Clifford S.C. Eberhart C.G. Parsons D.W. Rutkowski S. Gajjar A. et al.Molecular subgroups of medulloblastoma: the current consensus.Acta Neuropathol. 2012; 123: 465-472Crossref PubMed Scopus (1245) Google Scholar). Two subgroups exhibit constitutive activation of the sonic hedgehog (SHH) or wingless (WNT) developmental pathways. The other two are referred to as group 3 (G3) and G4. Importantly, the molecular subgrouping of these tumors also relates to distinct patient demographics, histologic classification, somatic genetic variations, and clinical outcome. For example, patients with WNT MB tend to be older, female, and to uniformly survive with current therapy (Northcott et al., 2012Northcott P.A. Korshunov A. Pfister S.M. Taylor M.D. The clinical implications of medulloblastoma subgroups.Nat Rev Neurol. 2012; 8: 340-351Crossref PubMed Scopus (222) Google Scholar). In contrast, patients with G3 MB tend to be younger, male, have anaplastic histology, exhibit a higher incidence of metastatic disease, and have a poor prognosis (Dubuc et al., 2013Dubuc A.M. Remke M. Korshunov A. Northcott P.A. Zhan S.H. Mendez-Lago M. Kool M. Jones D.T. Unterberger A. Morrissy A.S. et al.Aberrant patterns of H3K4 and H3K27 histone lysine methylation occur across subgroups in medulloblastoma.Acta Neuropathol. 2013; 125: 373-384Crossref PubMed Scopus (126) Google Scholar, Kool et al., 2012Kool M. Korshunov A. Remke M. Jones D.T. Schlanstein M. Northcott P.A. Cho Y.J. Koster J. Schouten-van Meeteren A. van Vuurden D. et al.Molecular subgroups of medulloblastoma: an international meta-analysis of transcriptome, genetic aberrations, and clinical data of WNT, SHH, Group 3, and Group 4 medulloblastomas.Acta Neuropathol. 2012; 123: 473-484Crossref PubMed Scopus (719) Google Scholar, Parsons et al., 2011Parsons D.W. Li M. Zhang X. Jones S. Leary R.J. Lin J.C. Boca S.M. Carter H. Samayoa J. Bettegowda C. et al.The genetic landscape of the childhood cancer medulloblastoma.Science. 2011; 331: 435-439Crossref PubMed Scopus (582) Google Scholar, Pugh et al., 2012Pugh T.J. Weeraratne S.D. Archer T.C. Pomeranz Krummel D.A. Auclair D. Bochicchio J. Carneiro M.O. Carter S.L. Cibulskis K. Erlich R.L. et al.Medulloblastoma exome sequencing uncovers subtype-specific somatic mutations.Nature. 2012; 488: 106-110Crossref PubMed Scopus (585) Google Scholar, Rausch et al., 2012Rausch T. Jones D.T. Zapatka M. Stütz A.M. Zichner T. Weischenfeldt J. Jäger N. Remke M. Shih D. Northcott P.A. et al.Genome sequencing of pediatric medulloblastoma links catastrophic DNA rearrangements with TP53 mutations.Cell. 2012; 148: 59-71Abstract Full Text Full Text PDF PubMed Scopus (620) Google Scholar, Robinson et al., 2012Robinson G. Parker M. Kranenburg T.A. Lu C. Chen X. Ding L. Phoenix T.N. Hedlund E. Wei L. Zhu X. et al.Novel mutations target distinct subgroups of medulloblastoma.Nature. 2012; 488: 43-48Crossref PubMed Scopus (621) Google Scholar). One characteristic feature of G3 MB is their high MYC expression in >75% of cases. Indeed, one study shows that MYC expression is elevated in 20 of 26 (77%) G3 MBs (named group C), compared to 1 of 35 (3%) G4 MBs (named group D; Northcott et al., 2011Northcott P.A. Korshunov A. Witt H. Hielscher T. Eberhart C.G. Mack S. Bouffet E. Clifford S.C. Hawkins C.E. French P. et al.Medulloblastoma comprises four distinct molecular variants.J. Clin. Oncol. 2011; 29: 1408-1414Crossref PubMed Scopus (975) Google Scholar). Whole genome sequencing studies on 17 G3 MBs reveals only one tumor (5.9%) with true MYC amplification, whereas two other tumors display an aberrant copy number gain of the MYC gene. However, 15 of these 17 (88%) G3 MBs demonstrate high MYC expression (Robinson et al., 2012Robinson G. Parker M. Kranenburg T.A. Lu C. Chen X. Ding L. Phoenix T.N. Hedlund E. Wei L. Zhu X. et al.Novel mutations target distinct subgroups of medulloblastoma.Nature. 2012; 488: 43-48Crossref PubMed Scopus (621) Google Scholar). Despite these pronounced differences, patients with MB are typically treated with uniform surgery, radiotherapy, and adjuvant chemotherapy including vincristine, cisplatin, and cyclophosphamide (Packer et al., 2013Packer R.J. Zhou T. Holmes E. Vezina G. Gajjar A. Survival and secondary tumors in children with medulloblastoma receiving radiotherapy and adjuvant chemotherapy: results of Children's Oncology Group trial A9961.Neuro. Oncol. 2013; 15: 97-103Crossref PubMed Scopus (186) Google Scholar) or lomustine and carboplatin (Massimino et al., 2012Massimino M. Cefalo G. Riva D. Biassoni V. Spreafico F. Pecori E. Poggi G. Collini P. Pollo B. Valentini L. et al.Long-term results of combined preradiation chemotherapy and age-tailored radiotherapy doses for childhood medulloblastoma.J. Neurooncol. 2012; 108: 163-171Crossref PubMed Scopus (17) Google Scholar). These therapies fail to cure one-third of all patients and carry widespread morbidities that impair survivor’s quality of life. Molecular subgrouping has the potential to improve risk stratification and tailor therapy to reduce toxicities to potential survivors. Paramount to this strategy is the development of accurate models that recapitulate the subgroups for preclinical therapeutic testing. We developed a mouse model of G3 MB through the orthotopic transplantation of transgenic cerebellar granule neuronal progenitors (GNPs) in the cortices of naive recipient animals. GNPs were purified by percoll density gradient from the cerebella of 5- to 7-day-old Trp53−/−;Cdkn2c−/− mice and infected with retroviruses encoding MYC (Kawauchi et al., 2012Kawauchi D. Robinson G. Uziel T. Gibson P. Rehg J. Gao C. Finkelstein D. Qu C. Pounds S. Ellison D.W. et al.A mouse model of the most aggressive subgroup of human medulloblastoma.Cancer Cell. 2012; 21: 168-180Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar). MB tumors develop within 30 days of transplantation with as few as 100 tumor cells that recapitulate the high level of trimethylation of histone H3 at lysine 27 (H3K27me3) seen in human G3 and G4 (Robinson et al., 2012Robinson G. Parker M. Kranenburg T.A. Lu C. Chen X. Ding L. Phoenix T.N. Hedlund E. Wei L. Zhu X. et al.Novel mutations target distinct subgroups of medulloblastoma.Nature. 2012; 488: 43-48Crossref PubMed Scopus (621) Google Scholar). Tumors grow as neurospheres that, when transplanted into cortices of recipient mice, induce secondary MBs that mimic the primary tumors (Kawauchi et al., 2012Kawauchi D. Robinson G. Uziel T. Gibson P. Rehg J. Gao C. Finkelstein D. Qu C. Pounds S. Ellison D.W. et al.A mouse model of the most aggressive subgroup of human medulloblastoma.Cancer Cell. 2012; 21: 168-180Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar). Because neurospheres can be passaged repeatedly while maintaining their functional and molecular properties, they provide a unique platform to conduct screens of compounds to identify those with therapeutic potential against human G3 MB. We here report the outcome of screening a library of compounds that included US Food and Drug Administration (FDA)-approved drugs and candidate compounds in development. Tumor cells purified from several independently derived primary mouse G3 MBs were grown as neurospheres for four to five passages providing lines with comparable cell proliferation characteristics. Two lines, derived from independent tumors and infections, were hereafter referred to as “Myc1” and “Myc2.” Neurospheres from the cerebellum of 7-day-old (P7) Trp53−/−;Cdkn2c−/− mice (hereafter referred to as Trp53-null) were used as control. To determine the number of cells necessary for exponential growth 4 days after plating, mouse Trp53-null and Myc1 were plated at different densities (Figure S1A available online). Other control cells included TERT− human fibroblasts (BJ) to identify compounds with nonspecific toxicities and HepG2 (a human hepatocellular carcinoma cell line) to eliminate highly cytotoxic compounds. We performed a primary screen of a “bioactive” library using a luminescence-based assay that measures cell proliferation via ATP. The library contained 7,389 compounds (6,568 unique) obtained from different sources, including 830 FDA-approved drugs (Figure S1B; see Supplemental Experimental Procedures for details). Compounds were tested at a single concentration (10 μM) in triplicate. Z-prime and other assay diagnostics were acceptable (Figure S1C) and the scatterplot of controls and compound activities showed adequate separation between signal and noise for both Myc1 and Trp53-null (Figure S1D, left). Receiver operator characteristic analysis indicated that the assay demonstrated acceptable discriminatory power between true-positive and true-negative results, with the area under the curve (AUC) >0.8 for both lines, and that an assay cutoff of >50% returned ∼70% of all true-positive results for Myc1 (Figure S1D, right). A total of 690 of the 7,389 compounds, including all with inhibition >50% in the primary screen with Myc1, analogs of these hits, and other compounds of interest, were tested in dose-response experiments in triplicate using concentrations ranging from 4 nM to 10 μM. Of the 690 hits, we identified 65 compounds with potency <1 μM against Myc 1 (Figure 1; Table S1). To accelerate the transition of potential therapeutics into the clinic, we prioritized the FDA-approved drugs with oncology indications and potencies below or near 1 μM for further study. We conducted dose-response experiments on 35 FDA-approved drugs in triplicate on Myc1, Trp53-null, HepG2, and BJ cell lines (Figure S2A; Table 1). Drugs were grouped into nine activity classes: folate pathway inhibitors, other inhibitors of DNA/RNA synthesis, purine antimetabolites, microtubule inhibitors, sterol biosynthesis inhibitors, topoisomerase inhibitors, epigenetic regulators, proteasome inhibitors, and RNA polymerase inhibitors. Myc1 was extremely sensitive to folate pathway inhibitors with pemetrexed, methotrexate, and raltitrexed being the most selective. The DNA/RNA synthesis inhibitor gemcitabine was highly potent in both Myc1 and Trp53-null, but achieved 100% efficacy in only Myc1. The purine antimetabolite cladribine showed promising activity against Myc1, but a narrower therapeutic window against the control cell lines HepG2 and BJ compared to other drugs. The proteasome inhibitor bortezomib and the RNA polymerase inhibitor dactinomycin were equipotent and equally efficacious against both Myc1 and Trp53 null. Microtubule inhibitors, including vincristine and vinblastine, and topoisomerase inhibitors, such as doxorubicin, etoposide, and topotecan, were active in G3 MB. Because they are already used in the clinic for the treatment of MB, they were not considered further. Cerivastatin and fluvastatin, two sterol biosynthesis inhibitors targeting 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase, had high efficacy but low potency relative to the other compound classes. Decitabine, a drug that causes both DNA damage and alterations in DNA methylation, was moderately potent, and had much higher efficacy for Myc1 over Trp53-null.Table 1EC50 Values for FDA-Approved Drugs on Myc1, Trp53-null, and Control Cell LinesCompoundMyc1 (μM)Trp53-null (μM)HepG2 (μM)BJ (μM)Amsacrine0.16 (0.097–0.27)0.55 (0.18–1.7)11 (5.6–23)13 (7.6–21)Ancitabine0.031 (0.024–0.04)0.55 (0.29–1)NDNDBortezomib0.0063 (0.0051–0.0078)0.0027 (0.0018–0.004)0.026 (0.0076–0.087)0.023 (0.0086–0.061)Cerivastatin0.77 (0.65–0.9)2.5 (1.3–4.7)0.92 (0.55–1.6)NDCladrabine0.0043 (0.0024–0.0078)0.25 (0.026–2.4)3.3 (2.6–4.3)1.4 (0.13–17)Clofarabine0.15 (0.13–0.18)0.34 (0.28–0.4)NDNDCycloguanil0.11 (0.059–0.21)0.18 (0.14–0.23)NDNDCytarabine0.083 (0.066–0.1)0.2 (0.092–0.42)0.078 (0.041–0.15)aIndicates regression curve failed to reach 50% efficacy.NDDactinomycin9e-04 (0.00016–0.005)0.0024 (0.0012–0.005)0.0084 (0.005–0.014)NDDaunorubicin0.0075 (0.0059–0.0097)0.036 (0.016–0.079)1.7 (0.11–25)0.1 (0.02–0.52)aIndicates regression curve failed to reach 50% efficacy.Decitabine1.3 (0.72–2.2)0.032 (0.014–0.073)aIndicates regression curve failed to reach 50% efficacy.NDNDDoxorubicin0.028 (0.015–0.052)0.11 (0.058–0.2)0.58 (0.14–2.3)0.14 (0.027–0.77)aIndicates regression curve failed to reach 50% efficacy.Etoposide0.24 (0.17–0.36)0.046 (0.013-0.15)NDNDFloxuridine0.0016 (0.0013–0.002)0.0001bIndicates questionable EC50 due to regression artifacts.NDNDFluorouracil0.36 (0.3–0.43)0.22 (0.17–0.29)NDNDFluvastatin4.5 (1.6–13)NDNDNDGemcitabine0.0021 (0.0018–0.0025)0.00032 (0.00012–0.00087)NDNDLovastatin4 (3.3–4.8)2.1 (0.11–38)aIndicates regression curve failed to reach 50% efficacy.13 (4.7–38)NDMethotrexate0.0052 (0.0024–0.012)0.15 (0.05–0.46)NDNDMitoxanthrone0.18 (0.11–0.29)0.19 (0.12–0.31)0.12 (0.072–0.2)aIndicates regression curve failed to reach 50% efficacy.NDNocodazole0.11 (0.036–0.32)0.094 (0.037–0.23)aIndicates regression curve failed to reach 50% efficacy.0.015 (0.0073–0.03)aIndicates regression curve failed to reach 50% efficacy.NDPemetrexed0.035 (0.027–0.046)15 (0.57–410)NDNDPitavastatin3.5 (1.5–8.6)ND5 (2.8–8.7)NDPodofilox0.0064 (0.0015–0.028)0.0074 (0.0048–0.012)aIndicates regression curve failed to reach 50% efficacy.0.0039 (0.0025–0.0059)aIndicates regression curve failed to reach 50% efficacy.NDPyrimethamine4.9 (3–8.1)0.87 (0.2–3.9)NDNDRaltiterxed0.003 (0.0024–0.0038)0.34 (0.18–0.63)NDNDRosuvastatin2.6 (2.3–3)NDNDNDSimvastatin3 (2.6–3.5)ND9.4 (6–15)5.8 (4.7–7.2)Tenoposide0.052 (0.003–0.89)0.11 (0.0097–1.2)7.7 (1.5–39)29 (3.8–230)Thioguanine0.44 (0.082–2.4)0.8 (0.52–1.2)11 (2.3–52)NDTopotecan0.075 (0.05–0.11)0.18 (0.06–0.53)0.09 (0.026–0.31)NDTrifluridine0.038 (0.034–0.043)0.0029 (0.0022–0.0039)NDNDTrimetrexate0.035 (0.0028–0.45)0.0047 (0.0033–0.0066)NDNDVinblastine0.012 (0.0028–0.053)0.015 (0.011–0.021)aIndicates regression curve failed to reach 50% efficacy.NDNDVincristine0.0094 (0.0044–0.02)0.01 (0.0071–0.014)aIndicates regression curve failed to reach 50% efficacy.NDNDConcentration range in parentheses. ND, EC50 could not be determined in the concentration range tested.a Indicates regression curve failed to reach 50% efficacy.b Indicates questionable EC50 due to regression artifacts. Open table in a new tab Concentration range in parentheses. ND, EC50 could not be determined in the concentration range tested. We selected decitabine, pemetrexed, and gemcitabine for further study because of their selectivity and diversity in mechanism of action. As noted earlier, mouse and human G3 MBs are marked by a high level of H3K27me3. Decitabine is an S-adenosyl methionine and cytidine analog. At low dose and prolonged exposure, decitabine targets DNA and histone methylation, whereas it induces DNA damage at high doses (Figure 2A; Palii et al., 2008Palii S.S. Van Emburgh B.O. Sankpal U.T. Brown K.D. Robertson K.D. DNA methylation inhibitor 5-Aza-2′-deoxycytidine induces reversible genome-wide DNA damage that is distinctly influenced by DNA methyltransferases 1 and 3B.Mol. Cell. Biol. 2008; 28: 752-771Crossref PubMed Scopus (284) Google Scholar). After 72 hr of treatment, the half-maximal effective concentration (EC50) for decitabine was 1.3 μM against Myc1 with 100% efficacy (Figure S2A, blue curve; Table 1), whereas efficacy against Trp53-null never exceeded 35% (Figure S2A, red curve; Table 1). Treatment of Myc1 with 0.5 μM decitabine for 72 hr significantly decreased H3K27me3 levels while 40 nM of pemetrexed had no effect (Figure 2B). A comparative study of gene expression profiles in mouse G3 and SHH MBs and GNPs suggested that mouse G3 MBs were sensitive to inhibitors of purine (Figure 2C, top), pyrimidine (Figure 2C, middle), and folate (Figure 2C, bottom) metabolism compared to SHH MBs and GNPs. In agreement with this gene expression pattern, pemetrexed and gemcitabine targeted these pathways. Pemetrexed targets three enzymes in the folate pathway; phosphoribosylglycinamide formyltransferase (GART), dihydrofolate reductase, and thymidylate synthase (Figure 2D; Chattopadhyay et al., 2007Chattopadhyay S. Moran R.G. Goldman I.D. Pemetrexed: biochemical and cellular pharmacology, mechanisms, and clinical applications.Mol. Cancer Ther. 2007; 6: 404-417Crossref PubMed Scopus (237) Google Scholar). After 72 hr of pemetrexed treatment, the EC50 for Myc1 was 35 nM (Figure S2A, blue curve; Table 1), compared to 15 μM in Trp53-null (Figure S2A, red curve; Table 1). Gemcitabine disrupts DNA synthesis via incorporation in DNA or inhibition of the ribonucleotide reductase (Figure 2E; van Moorsel et al., 2000van Moorsel C.J. Bergman A.M. Veerman G. Voorn D.A. Ruiz van Haperen V.W. Kroep J.R. Pinedo H.M. Peters G.J. Differential effects of gemcitabine on ribonucleotide pools of twenty-one solid tumour and leukaemia cell lines.Biochim. Biophys. Acta. 2000; 1474: 5-12Crossref PubMed Scopus (60) Google Scholar). When incorporated into DNA, gemcitabine causes single-strand breaks that lead to apoptosis (Ewald et al., 2007Ewald B. Sampath D. Plunkett W. H2AX phosphorylation marks gemcitabine-induced stalled replication forks and their collapse upon S-phase checkpoint abrogation.Mol. Cancer Ther. 2007; 6: 1239-1248Crossref PubMed Scopus (158) Google Scholar). After 72 hr treatment, the Myc1 EC50 for gemcitabine was 2.1 nM with 100% efficacy, whereas efficacy against Trp53-null never exceeded 63% (Figure S2A; Table 1). Decitabine, pemetrexed, and gemcitabine were further tested on four additional mouse G3 MB lines derived from independently derived tumors and infections; all displayed comparable potency and efficacy compared to Myc1 (Figure S2B). In contrast, we found a SHH MB-derived line was as sensitive to pemetrexed as Myc1 but was greater than 5-fold less sensitive to gemcitabine compared to Myc1 (Figure S2C). To determine the concentration-time threshold required to inhibit proliferation of neurospheres in vitro, we performed ”wash-out” experiments with each of the three compounds. The EC50 values for decitabine were approximately 2.4 μM, 920 nM, and 500 nM after 1, 10, and 24 hr drug exposure, respectively (Figure 3A, top). While the EC50 for pemetrexed was 1.2 μM after 1 hr exposure, it decreased to 500 nM after 10 hr, and to 180 nM after 24 hr (Figure 3A, middle). With gemcitabine, the EC50 values were 44 nM, 13 nM, and 3.8 nM after 1, 10, and 24 hr exposure, respectively (Figure 3A, bottom). Although decitabine affected the viability of Myc1 and decreased H3K27me3 in vitro (Figure 2B), a review of the published preclinical and clinical pharmacokinetic data (Chabot et al., 1983Chabot G.G. Rivard G.E. Momparler R.L. Plasma and cerebrospinal fluid pharmacokinetics of 5-Aza-2′-deoxycytidine in rabbits and dogs.Cancer Res. 1983; 43: 592-597PubMed Google Scholar, George et al., 2010George R.E. Lahti J.M. Adamson P.C. Zhu K. Finkelstein D. Ingle A.M. Reid J.M. Krailo M. Neuberg D. Blaney S.M. Diller L. Phase I study of decitabine with doxorubicin and cyclophosphamide in children with neuroblastoma and other solid tumors: a Children’s Oncology Group study.Pediatr. Blood Cancer. 2010; 55: 629-638Crossref PubMed Scopus (50) Google Scholar) strongly suggested that the maximally achievable decitabine brain concentrations in humans would be well below those required to induce significant inhibition of proliferation. Therefore, decitabine was not considered for further in vivo studies. For gemcitabine and pemetrexed, total plasma and tumor extracellular fluid (tECF) drug concentrations were assessed in separate groups of mice bearing Myc1-induced G3 MB after single intravenous (i.v.) injections of pemetrexed (200 mg/kg) and gemcitabine (60 mg/kg). A three-compartment pharmacokinetic model adequately described the plasma and tECF concentration-time data for each drug. The murine plasma pharmacokinetics for each agent differed only modestly from previously published reports, with the clearance of gemcitabine and pemetrexed being approximately 2-fold higher in our studies (Rocchetti et al., 2007Rocchetti M. Simeoni M. Pesenti E. De Nicolao G. Poggesi I. Predicting the active doses in humans from animal studies: a novel approach in oncology.Eur. J. Cancer. 2007; 43: 1862-1868Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, Wang et al., 2004Wang H. Li M. Rinehart J.J. Zhang R. Pretreatment with dexamethasone increases antitumor activity of carboplatin and gemcitabine in mice bearing human cancer xenografts: in vivo activity, pharmacokinetics, and clinical implications for cancer chemotherapy.Clin. Cancer Res. 2004; 10: 1633-1644Crossref PubMed Scopus (101) Google Scholar, Woodland et al., 1997Woodland J.M. Barnett C.J. Dorman D.E. Gruber J.M. Shih C. Spangle L.A. Wilson T.M. Ehlhardt W.J. Metabolism and disposition of the antifolate LY231514 in mice and dogs.Drug Metab. Dispos. 1997; 25: 693-700PubMed Google Scholar). The plasma exposure of each drug, quantified by the area under the plasma concentration-time curve (pAUC), was similar to that achieved in humans at clinically relevant dosages (Figure 3B, upper and middle; Malempati et al., 2007Malempati S. Nicholson H.S. Reid J.M. Blaney S.M. Ingle A.M. Krailo M. Stork L.C. Melemed A.S. McGovern R. Safgren S. et al.Children’s Oncology GroupPhase I trial and pharmacokinetic study of pemetrexed in children with refractory solid tumors: the Children’s Oncology Group.J. Clin. Oncol. 2007; 25: 1505-1511Crossref PubMed Scopus (23) Google Scholar, Reid et al., 2004Reid J.M. Qu W. Safgren S.L. Ames M.M. Krailo M.D. Seibel N.L. Kuttesch J. Holcenberg J. Phase I trial and pharmacokinetics of gemcitabine in children with advanced solid tumors.J. Clin. Oncol. 2004; 22: 2445-2451Crossref PubMed Scopus (80) Google Scholar). We found that the tECF concentrations of pemetrexed and gemcitabine exceeded the in vitro EC50 versus time threshold, suggesting that these compounds should have in vivo efficacy in G3 MB (Figure 3B, bottom). To address how much drug crosses the normal blood-brain barrier, studies of pemetrexed and gemcitabine were conducted in six non-tumor-bearing mice. Pemetrexed (200 mg/kg i.v.) or gemcitabine (60 mg/kg i.v.) was administered, the brain was harvested, and the drug concentration was measured in the brain parenchyma and in the plasma. The brain-to-plasma ratio of pemetrexed and gemcitabine in these samples was 7.3% and 45%, respectively, indicating that both drugs cross a normal blood-brain barrier in a mouse model. To assess whether pemetrexed and gemcitabine inhibited proliferation of human G3 MB in vitro, we generated patient-derived xenografts (PDXs) from primary G3 MBs overexpressing the MYC protein with or without MYC amplification and derived neurospheres for two of them. Gene expression profiling of Icb-1572 (Zhao et al., 2012Zhao X. Liu Z. Yu L. Zhang Y. Baxter P. Voicu H. Gurusiddappa S. Luan J. Su J.M. Leung H.C. Li X.N. Global gene expression profiling confirms the molecular fidelity of primary tumor-based orthotopic xenograft mouse models of medulloblastoma.Neuro-oncol. 2012; 14: 574-583Crossref PubMed Scopus (116) Google Scholar) and TB-12-5950 (St. Jude Children’s Research Hospital [SJCRH]) confirmed clustering with previously published human G3 MB (Robinson et al., 2012Robinson G. Parker M. Kranenburg T.A. Lu C. Chen X. Ding L. Phoenix T.N. Hedlund E. Wei L. Zhu X. et al.Novel mutations target distinct subgroups of medulloblastoma.Nature. 2012; 488: 43-48Crossref PubMed Scopus (621) Google Scholar), and demonstrated that a similar profile was maintained through several passages in mice (Figure S3A). Fluorescence in situ hybridization analysis revealed that MYC was not amplified in Icb-1572 (Figure S3B, left; Shu et al., 2008Shu Q. Wong K.K. Su J.M. Adesina A.M. Yu L.T. Tsang Y.T. Antalffy B.C. Baxter P. Perlaky L. Yang J. et al.Direct orthotopic transplantation of fresh surgical specimen preserves CD133+ tumor cells in clinically relevant mouse models of medulloblastoma and glioma.Stem Cells. 2008; 26: 1414-1424Crossref PubMed Scopus (118) Google Scholar) but was amplified in TB-12-5950 that also displayed leptomeningeal dissemination (Figure S3B, right). PDX OA-2012-1 overexpressed the MYC protein without amplification, as measured by aCGH (O.A., unpublished data), whereas PDX Med-511-FH was confirmed to have MYC amplification by nanostring analysis (J.M.O., unpublished data). TB-12-5950, OA-2012-1, and human neural stem cells H9 formed neurospheres in vitro, allowing us to test the effects of pemetrexed and gemcitabine in vitro. Neurospheres were treated for 72 hr at doses ranging from 1 nM to 10 μM. Cell viability was measured. For TB-12-5950 and OA-2012, EC50 for pemetrexed were 160 nM and 100 nM (Figure 3C, top), whereas those for gemcitabine were 5.1 nM and 11 nM, respectively (Figure 3C, bottom). These EC50 values were similar to those found for Myc1 (within 2- to 5-fold; Table 1). For H9 cells, the EC50 for pemetrexed was 0.29 μM and for gemcitabine 0.0015 μM (Figure S3C), which corresponded to the response seen in mouse control Trp53-null (Figure S2A, Table 1). To determine how efficacious pemetrexed and gemcitabine were in suppressing proliferation of mouse G3 MB in vivo, we stereotactically transplanted 1 × 105 purified tumor cells, retrovirally transduced with luciferase, in the cortices of CD1 mice. We previously determined that this cell number induces MBs that kill the animals within 15 days after transplant and recapitulate the primary tumors (Kawauchi et al., 2012Kawauchi D. Robinson G. Uziel T. Gibson P. Rehg J. Gao C. Finkelstein D. Qu C. Pounds S. Ellison D.W. et al.A mouse model of the most aggressive subgroup of human medulloblastoma.Cancer Cell. 2012; 21: 168-180Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar). Bioluminescence detection of tumor progression correlated with tumor volume measured with magnetic resonance imaging (Figures S4A–S4C). Hematoxylin and eosin (H&E) staining of tumor sections performed 3 days after transplant confirmed the presence of an organized tumor mass that was vascularized and surrounded by blood vessels (Figure S4D). The schedule and dosage of drug delivery was calculated based upon modeling and simulation of data from our pharmacokinetic studies, and related to pAUC values tolerable in pediatric clinical trials (Malempati et al., 2007Malempati S. Nicholson H.S. Reid J.M. Blaney S.M. Ingle A.M. Krailo M. Stork L.C. Melemed A.S. McGovern R. Safgren S. et al.Children’s Oncology GroupPhase I trial and pharmacokinetic study of pemetrexed in children" @default.
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• W1963673975 title "Pemetrexed and Gemcitabine as Combination Therapy for the Treatment of Group3 Medulloblastoma" @default.
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