Establishing a Preclinical Multidisciplinary Board for Brain Tumors Birgit V

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Published OnlineFirst January 4, 2018; DOI: 10.1158/1078-0432.CCR-17-2168

Cancer Therapy: Preclinical Clinical Cancer Research Establishing a Preclinical Multidisciplinary Board for Brain Tumors Birgit V. Nimmervoll1, Nidal Boulos2, Brandon Bianski3, Jason Dapper4, Michael DeCuypere5, Anang Shelat6, Sabrina Terranova1, Hope E. Terhune4, Amar Gajjar7, Yogesh T. Patel8, Burgess B. Freeman9, Arzu Onar-Thomas10, Clinton F. Stewart11, Martine F. Roussel12, R. Kipling Guy6,13, Thomas E. Merchant3, Christopher Calabrese14, Karen D. Wright15, and Richard J. Gilbertson1

Abstract

Purpose: Curing all children with brain tumors will require an Results: Mouse models displayed distinct patterns of response understanding of how each subtype responds to conventional to surgery, irradiation, and chemotherapy that varied with tumor treatments and how best to combine existing and novel therapies. subtype. Repurposing screens identified 3-hour infusions of It is extremely challenging to acquire this knowledge in the clinic gemcitabine as a relatively nontoxic and efficacious treatment of alone, especially among patients with rare tumors. Therefore, we SEP and CPC. Combination neurosurgery, fractionated irradia- developed a preclinical brain tumor platform to test combination, and gemcitabine proved significantly more effective than tions of conventional and novel therapies in a manner that closely surgery and irradiation alone, curing one half of all animals with recapitulates clinic trials. aggressive forms of SEP. Experimental Design: A multidisciplinary team was established Conclusions: We report a comprehensive preclinical trial to design and conduct neurosurgical, fractionated radiotherapy platform to assess the therapeutic activity of conventional and chemotherapy studies, alone or in combination, in accurate and novel treatments among rare brain tumor subtypes. It mouse models of supratentorial ependymoma (SEP) subtypes and also enables the development of complex, combination choroid plexus carcinoma (CPC). Extensive drug repurposing treatment regimens that should deliver optimal trial designs screens, pharmacokinetic, pharmacodynamic, and efficacy studies for clinical testing. Postirradiation gemcitabine infusion were used to triage active compounds for combination preclinical should be tested as new treatments of SEP and CPC. Clin trials with "standard-of-care" surgery and radiotherapy. Cancer Res; 24(7); 1654–66. 2018 AACR.

1Cancer Research UK Cambridge Institute and Department of Oncology, Uni- versity of Cambridge, Cambridge, England, United Kingdom. 2Department of Introduction Hematology, St. Jude Children's Research Hospital, Memphis, Tennessee. Despite decades of research, the treatment of brain tumors 3 Department of Radiological Sciences, St. Jude Children's Research Hospital, has remained largely unchanged. These cancers are treated with Memphis, Tennessee. 4Department of Developmental Neurobiology, St. Jude 5 an aggressive combination of neurosurgery, radiotherapy, and Children's Research Hospital, Memphis, Tennessee. Department of Surgery, St. fl fi Jude Children's Research Hospital, Memphis, Tennessee. 6Department of Chem- chemotherapy that frequently fails to cure but in icts signi - ical Biology and Therapeutics, St. Jude Children's Research Hospital, Memphis, cant side effects (1–4). This limited progress has occurred Tennessee. 7Department of Oncology, St. Jude Children's Research Hospital, despite an active clinical trials effort: more than 2,580 brain Memphis, Tennessee. 8Department of Pharmaceutical Sciences, St. Jude Chil- tumor trials are currently registered with clinicaltrials.gov, but 9 dren's Research Hospital, Memphis, Tennessee. Preclinical Pharmacokinetics only six drugs are approved for treatment of brain tumors, of Core, St. Jude Children's Research Hospital, Memphis, Tennessee. 10Department which only two—Everolimus, an inhibitor of the mTOR (5), of Biostatistics, St. Jude Children's Research Hospital, Memphis, Tennessee. — 11Department of Pharmaceutical Sciences, St. Jude Children's Research Hospital, and Bevacizumab, an inhibitor of VEGFA (1) are molecular- Memphis, Tennessee. 12Department of Tumor Cell Biology, St. Jude Children's targeted treatments. Research Hospital, Memphis, Tennessee. 13University of Kentucky College of So why have we failed to identify effective new brain tumor Pharmacy, Lexington, Kentucky. 14Preclinical Imaging Core, St. Jude Children's therapies? One possibility is that the preclinical systems used to 15 Research Hospital, Memphis, Tennessee. Boston Children's Hospital and Har- select drugs for clinical trial do not predict therapeutic activity in vard Medical School, Boston, Massachusetts. patients (6). This explanation is plausible when one considers that Note: Supplementary data for this article are available at Clinical Cancer most preclinical studies are conducted in mice harboring subcu- Research Online (http://clincancerres.aacrjournals.org/). taneous brain tumor xenografts that cannot recapitulate accurate- B.V. Nimmervoll and N. Boulos contributed equally to this article. ly the pharmacology or biology of brain tumor treatment. Fur- Corresponding Authors: Richard J. Gilbertson, Cambridge University, Cam- thermore, although brain tumor patients receive complex, multi- bridge CB2 0RE, England, United Kingdom. Phone: 01223769590; E-mail: modality therapy, mice in preclinical studies usually receive drugs [email protected]; and Karen D. Wright, Boston Children's as monotherapies. Such studies are unlikely to predict the survival Hospital and Harvard Medical School, 450 Brookline Avenue, Boston, MA benefit of a new treatment above that afforded by standard of care. 02215. E-mail: [email protected] Prioritizing treatments with the greatest potential for clinical doi: 10.1158/1078-0432.CCR-17-2168 efficacy is especially important for rare tumors that have limited 2018 American Association for Cancer Research. patient populations available for clinical trial.

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discussed in detail in the Supplementary Methods, host mice for Translational Relevance all allografts and xenografts were CD-1 nude mice (strain code: Existing drug development pipelines have failed to bring 086; Charles River). All preclinical surgery, radiotherapy, and new treatments to children with brain tumors. A lack of chemotherapy studies were performed among randomized faithful preclinical models has prevented the discovery and cohorts of mice harboring tumors with 1e107 photons/sec prioritization of potential new therapies, and the rarity of these bioluminescence (16). Tumor progression and treatment diseases presents an insurmountable hurdle for drug devel- response were assessed clinically and by weekly bioluminescence opment through clinical trial alone. Therefore, we established (16). Mice displaying signs of excessive clinical morbidity (20% a preclinical multidisciplinary tumor board comprising biol- weight loss and/or neurological impairment) were euthanized. ogists, statisticians, pharmacologists, and clinicians to conduct preclinical studies that mimic the clinic. Mouse models includ- Preclinical neurosurgery, radiotherapy, and chemotherapy ed those of specific ependymoma and choroid plexus carci- Following baseline bioluminescence imaging, mice were noma subtypes—two rare pediatric brain tumors. In contrast appropriately anaesthetized, a craniotomy fashioned over the with previous brain tumor preclinical platforms, our approach site of maximum bioluminescence, and tumors resected using enables the testing of potential new treatments of very rare a small suction tip. Postoperative hemostasis was achieved tumors, in the context of "standard-of-care" neurosurgery and with thrombin-soaked gel foam prior to skin closure. Mice fractionated irradiation. This approach enables assessment of were reimaged in the immediate postoperative period, mon- the potential therapeutic "value added" of candidate treat- itored on heating pads, and treated for 3 days with ibuprofen- ments and thereby prioritizes novel treatment combinations supplemented drinking water, dexamethasone (0.6 mg/kg/ for clinical trial. 6 hours), and mannitol (100 mg/kg/6 hours). Note that 54 Gy of radiotherapy was delivered to appropriately anesthetized mice as 2 Gy/day fractions via an orthovoltage irradiator or image-guided rodent irradiator (SARRP, Xstrahl). Drugs were Modifying long-established treatment regimens that have delivered via tail vein bolus injections or using Alzet m evolved empirically over many years is also challenging. For pumps (2001D, mean pumping rate 8.0 L/h; loaded with example, the treatment of supratentorial ependymoma (SEP) and 150 mg/mL gemcitabine solution prepared in 50:50 PEG300: choroid plexus carcinoma (CPC)—two rare pediatric brain propylene glycol; Supplementary Methods; Supplementary – tumors—has evolved over decades to include maximum surgical Table S2). For combination surgery radiotherapy or sur- – – resection and postoperative cranial irradiation (7–14). These gery radiotherapy chemotherapy studies, mice were rested for treatments are effective, but evidence suggests that this efficacy 72 hours in between therapeutic modalities. varies with tumor subtype. For example, although most SEPs containing the C11ORF95-RELA translocation (hereon, SEP- Pharmacokinetic and pharmacodynamics studies CR[þ]) resist combination surgery and irradiation, the majority Pharmacokinetic studies are described in detail in the Supple- fl of SEP-CR(–) tumors are cured with this therapy (11, 15, 16). mentary Methods. Brie y, blood samples were collected from Despite these differences in treatment sensitivity, ongoing clinical euthanized mice via cardiac stick into tubes containing tetrahy- fi m trials are testing whether classic histology SEPs, regardless of drouridine (THU, nal concentration 150 g/mL). Plasma was molecular tumor subtype, can be cured with total tumor resection separated and samples were stored at 80 C until analysis. alone (NCT01096368). Thus, there is a pressing need to deter- Intracranial microdialysis studies were performed as described mine if SEP-CR(þ) resists surgery, radiotherapy, or both. Such previously (19). A guide cannula (MD-2255, BASi) and allo- knowledge is also important if we are to combine conventional grafted tumor cells were implanted stereotactically in the cortex and novel therapies to better treat these tumors; however, this of immunocompromised mice. Once tumors formed, a precali- knowledge is unlikely to be acquired solely in the clinic, especially brated microdialysis probe (MD-2211, BASi; 38 KDa MWCO given the rarity of disease variants. Therefore, to better understand membrane) was implanted through the microdialysis guide can- fi fl m the response of SEP and CPC subtypes to surgery and radiother- nula and perfused with arti cial cerebrospinal uid (0.5 L/min). apy, and to design clinical trials that integrate conventional and Mice were dosed with gemcitabine and plasma samples collected new treatments, we established a preclinical multidisciplinary via retro-orbital bleeds. Drug levels were measured using a val- – team (pMDT) with the capacity to conduct randomized, multi- idated high-performance liquid chromatography mass spec- modality trials in mice harboring accurate models of SEP or CPC. trometry method. Tumor cell proliferation and apoptosis were assessed by immunohistochemical quantification of Ki67 and Caspase 3, respectively (Supplementary Methods). Materials and Methods Tumor cells and implants In vitro drug testing The isolation, culture, and orthotopic implantation of all High-throughput screens were performed by seeding tumor mouse and human tumor cells was described previously cells in 384-well plates as described in the Supplementary (15–18). The nomenclature, species, tumor type, driver oncogene, Methods and previously (19). Each plate included dilution series and implanted cell number of each xenograft and allograft are of test compounds (8.3 mmol/L to 0.5 nmol/L), DMSO-only provided in Supplementary Table S1. All cells were maintained as negative controls and cycloheximide or bortezomib single point in vivo grafts and confirmed by ELISA as mycoplasma negative (0.5 mmol/L) and dose-response (0.5 mmol/L to 0.01 nmol/L)– prior to and following in vitro studies. All animal studies were positive controls. Cell number was determined in each well using approved by the Animal Care and Usage Committees at St Jude the Cell Titer Glo reagent (Promega). All assays were conducted in Children's Research Hospital and the University of Cambridge. As triplicate. Wash-out studies were similarly performed to assess the

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minimum time–concentration exposure required to inhibit cell some animals (Fig. 2F and G). Thus, the relatively poor prognosis growth by 50% by replacing drug-containing medium with fresh of patients with SEP-CR(þ) may in part reflect the failure of medium 1, 3, 6, 10, 24, or 72 hours after dosing. Tumor cell surgery to control these tumors (7). Gross total resection is apoptosis was assessed by fluorescence-activated cell sorting to generally regarded as optimal therapy of CPC, although this has detect Annexin V staining (apoptosis) and DAPI staining (DNA not been demonstrated definitively because the disease is so rare integrity). (12, 13). In support of this notion, total resection significantly reduced tumor burden for around 1 week and marginally, but fi Results signi cantly, extended the survival of mice with mCPC (Fig. 2H). Preclinical multidisciplinary brain tumor board Preclinical fractionated radiotherapy We recruited from our clinical MDT, a pMDT comprising Postoperative cranial irradiation has been a mainstay of statisticians, biologists, chemists, pharmacologists, and clinicians. ependymoma therapy for decades and is used to treat some The pMDT met weekly to design, conduct, and review preclinical patients with CPC (7–9, 11–13). To test the efficacy of radio- studies that closely recapitulate multimodality clinical trials therapy in our mouse models, we randomized mice with (Fig. 1). Trial statisticians ensured appropriate randomization of equally sized mSEP-CR(þ), xSEP-CR(þ), mSEP-CR(–)RTBDNa, tumor-bearing animals and statistical powering of study arms; mSEP-CR(–)RTBDNb, or mCPC to receive 27 daily fractions of neurosurgeons performed all mouse neurosurgery; radiation 2 Gy cranial irradiation (mimicking that given to patients) or oncologists prescribed and delivered fractionated radiotherapy mock treatment (Fig. 3A). In stark contrast with surgery, radio- to mice; clinical pharmacologists and oncologists guided trial therapy significantly impaired the growth of all SEP-CR(þ)and drug doses and schedules; and radiologists and small animal SEP-CR(–) models relative to controls for between 2 and 10 imaging specialists evaluated treatment response. The pMDT weeks, resulting in a significant survival advantage for treated adhered to strict, pre-agreed, standard operating procedures that mice (Fig. 3B–E). Notably, regrowth of mSEP-CR(þ)and dictated the progress of therapies through the preclinical pipeline mSEP-CR(–)RTBDN was observed before the end of radiother- (Fig. 1). Preclinical trial data were accessible to all pMDT members apy, suggesting the emergence of resistant clones, potentially in real time via a centralized electronic mouse medical record. explaining why this treatment ultimately failed. Conversely, Using cross-species functional genetic screens, we previously and in agreement with the limited radiosensitivity of infant generated a series of orthotopic, genetic mouse (m), and human CPC, radiotherapy only transiently impaired mCPC growth and xenograft (x) models that recapitulate the histology, transcrip- had no therapeutic efficacy against this tumor (Fig. 3F). tome, and growth of SEP-CR(þ), SEP-CR(–), and CPC tumors Having evaluated the efficacy of surgery and radiotherapy RTBDN EPHB2 (mSEP-CR[þ], xSEP-CR[þ], mSEP-CR[-] , mSEP-CR[-] , independently, we conducted a series of combination studies to mCPC; Supplementary Table S1; refs. 15–18). Because clinical determine the benefit of combining these modalities (Fig. 3G). trials frequently employ MRI to assess treatment response, we first Although surgery alone did not benefit mice harboring mSEP-CR confirmed that bioluminescence (our preferred method of imag- (þ), postoperative irradiation significantly prolonged tumor ing) and MRI provide equivalent measures of tumor volume in control in these animals relative to radiotherapy alone, resulting 2 our mouse models (R ¼ 0.96, P < 0.0001; Fig. 2A and B). Armed in cures for almost half of all treated mice (Fig. 3H). In contrast, with these data and the survival rates of 294 tumor-bearing mice, surgical resection of xSEP-CR(þ) did not prolong the survival of pMDT statisticians then employed the Wilcoxon rank-sum test mice with this tumor relative to those treated with irradiation and Noether's power formula to design studies with a >83% alone, possibly reflecting the rapid regrowth of these tumors power to detect a significant survival difference between animals following surgical debulking, resulting in a shorter period receiving test or control treatment. of tumor control overall (Fig. 3I). However, combination surgery and irradiation significantly impaired the growth of Preclinical neurosurgery mSEP-CR(–)RTBDNa relative to surgery alone and extended the To test the therapeutic value of surgery in our models, we es- survival of mice with these tumors beyond that achieved with tablished cohorts of mice harboring mSEP-CR(þ), xSEP-CR(þ), either therapy alone (Fig. 3J). Combination surgery and radio- mSEP-CR(–)RTBDNa, mSEP-CR(–)RTBDNb, or mCPC as described therapy were not attempted in mCPC because this tumor resisted previously (15, 16, 18). Mice bearing equivalent sized tumors both treatments. were then randomized to undergo microscope-guided tumor resection by neurosurgeons or anesthesia alone (Fig. 2C). Gross Repurposing of chemotherapy total resection (10% residual postoperative bioluminescence) Having established the value of surgery and radiotherapy was achieved in 100% (n ¼ 14/14), 64% (n ¼ 9/14), 51% (n ¼ 24/ among our models of SEP and CPC, and shown that the pattern 47), 71% (n ¼ 15/21), and 43% (n ¼ 13/30) of mice harboring of response to these treatments approximates that observed mSEP-CR(þ), xSEP-CR(þ), mSEP-CR(–)RTBDNa, mSEP-CR in patients, we looked to see if our models might be useful (–)RTBDNb, or mCPC, respectively, recapitulating the total resec- for developing chemotherapies. Using an integrated in vitro tion rates of these tumors in children (Fig. 2D–H; refs. 11–14). and in vivo screen that we deployed previously to identify potential Surgical resection of mSEP-CR(þ) and xSEP-CR(þ) produced brain tumor treatments for clinical trial, we screened 114 drugs only transient, significant reductions in tumor volume, and these that are FDA approved or currently in clinical trial (19, 20). mSEP- tumors regrew rapidly following total resection, resulting in no CR(–)RTBDNb and mCPC cells were chosen for these studies overall survival advantage (Fig. 2D and E). In contrast, total because they represent relatively responsive and resistant tumor resection produced sustained, significant reductions in the vol- types, respectively. In line with their relative resistance to treat- ume of mSEP-CR(–)RTBDNa and mSEP-CR(–)RTBDNb and ments, 40 drugs inhibited the proliferation of mSEP-CR(–)RTBDNb increased the survival of mice harboring these tumors, curing cells by 50% (IC50) at concentrations 1 mmol/L after 72 hours

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Preclinical brain tumor board

Statistician Mouse Radiologist Neurosurgeon Radiation Chemist Clinical Pathologist models small animal oncologist clinical oncologist biologist imaging pharmacologist

Drug selection 1 high through-put drug screen; predicted to be BBB-penetrant; cancer active

Drug selection 2 in vitro wash-out studies; BBB penetration in vivo pharmacokinetics

Figure 1. Composition of the preclinical Multi-Disciplinary Tumor Board and the multistep approach taken to develop new treatment approaches. BBB, blood–brain Drug selection 3 barrier; R, randomization. modelled ‘optimal’ dose in vivo efficacy studies

Preclinical trial design multimodality trial relative to conventional treatment

VVVVV

Electronic medical record of real-time results feasibility, efficacy, toxicity in vitro compared with only 26 drugs against mCPC cells: 22 (Fig. 4B). Exposure to < 1 mmol/L of cabazitaxel, pralatrexed, of these drugs had IC50 1 mmol/L against both cell types (P < gemcitabine, panobinostat, carﬁlzombib, or vosaroxin for just 0.0001, Fisher exact for overlap; Fig. 4A). 1 hour inhibited the proliferation of both mSEP-CR(–)RTBDNb Thirteen drugs with IC50 values 1 mmol/L at 72 hours were and mCPC cells by >50%; chaetocin was similarly active against then subjected to "washout" studies to determine the minimum mCPC, whereas the IC50 of acivicin after 1-hour exposure concentration–time exposure required to inhibit cell proliferation almost achieved 50% inhibition in the 1 mmol/L range.

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A B 10 10 1,000 Day: 71421 Bioluminescence MRI volume

9 10 MRI volume (mm 100

MRI 10 8 10 10 7 3 1 ) 10 6 R2 = 0.96 P < 0.0001 Bioluminescence (photons/sec) 5

Bioluminescence 10 0.1 7142171421 Day post allograft C Time [days]: 0171421

=Allograft R =Randomised =Bioluminescence =Surgical resection D 10 10 100 Figure 2. 10 9 75 Preclinical brain imaging and neurosurgery. 10 8 A, Concurrent MRI (top) and 50 10 7 bioluminescence (bottom) imaging of mSEP-CR(+) 25 mSEP-CR(+) the same mouse with a SEP-CR(þ) tumor. 10 6 Percent surviving Control Vehicle (n = 5) n P >90% surgical resection (d7−12) >90% resection ( = 14); = ns B, Correlation of MRI and bioluminescence 5 0 Luminescence (p/s ± SE) 10 0 5 10 15 20 25 30 35 40 45 50 0204060 imaging of the same cohort of 5 mice with E Time post implant [days] Survival [days] SEP-CR(þ) tumors. C, Preclinical mouse 108 100 neurosurgical protocol. D–H (left), Bioluminescence measurement of tumor 75 107 growth. In parentheses are the days (d) 50 when treated tumor volume was signiﬁcantly

6 P < 10 xSEP-CR(+) xSEP-CR(+) ( 0.05, nonparametric) less than control. Control 25 Vehicle (n = 6) Percent surviving (Right) survival curves of mice with indicated <90% surgical resection (ns) >90% surgical resection (n = 9); P = ns n P >90% surgical resection (d22) <90% surgical resection ( = 5); = ns 5 animal numbers and tumor types treated

Luminescence (p/s ± SE) 10 0 020406080100 0 50 100 150 with surgery or control. P value ¼ log-rank Time post implant [days] Survival [days] F relative to control. 10 10 100

10 9 75

10 8 50 10 7 mSEP-CR(-)RTBDNa mSEP-CR(-)RTBDNa Control 25 Vehicle (n = 29) 10 6 Percent surviving <90% surgical resection (ns) >90% surgical resection (n = 24); P < 0.0001 >90% surgical resection (d16−58) <90% surgical resection (n = 23); P = ns

Luminescence (p/s ± SE) 10 5 0 0 20 40 60 80 100 120 140 0 100 200 300 400 Time post implant [days] Survival [days]

G 10 10 100

10 9 75

108 50 mSEP-CR(-)RTBDNb mSEP-CR(-)RTBDNb Vehicle (n = 15) 10 7 Control 25 >90% surgical resection (n = 15); P = 0.0007 Percent surviving n P <90% surgical resection (d6) <90% surgical resection ( = 6); = 0.008 >90% surgical resection (d6−10) 0 Luminescence (p/s ± SE) 10 6 01020304050 0 1020304050 Time post implant [days] Survival [days] H 10 10 100

10 9 75

10 8 50 mCPC mCPC Vehicle (n = 18) 10 7 25 n P

Control Percent surviving >90% surgical resection ( = 13); = 0.03 <90% surgical resection (ns) <90% surgical resection (n = 17); P = ns >90% surgical resection (d18−23) 6 0

Luminescence (p/s ± SE) 10 15 20 25 30 35 40 0204060 Time post implant [days] Survival [days]

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A Time [days]: 017142128 35 I I I I I I I I I I I I I I I I I I I I I I I I I I I R

=Allograft R =Randomized =Bioluminescence I I I I I =2 x 5 Gy cranial irradiation BC (d10−85) (d13−28) 10 9 10 11 100 100 SE) mSEP-CR(+) SE) xSEP-CR(+) ± ± 8 10 10 10 75 75 10 9 10 7 50 50 10 8 10 6 n = 10 n = 9 n = 5 n = 5 25 7 Percent surviving 25 5 10 10 Percent surviving mSEP-CR(+) P < 0.001 xSEP-CR(+) P = 0.03

Luminescence (p/s 6 10 0 Luminescence (p/s 10 4 0 020406080 0 20406080 0 50 100 150 0 50 100 150 200 250 Time [days] Time [days] Time [days] Time [days]

DE(d7-22) (d16-60) 10 10 100 10 9 100 SE) SE) ± ± 10 9 75 10 8 75 n = 9 n = 10 n = 15 n = 9 10 8 50 10 7 50

RTBDNa 107 25 mSEP-CR(-) 6 25 RTBDNb Percent surviving 10 mSEP-CR(-) Percent surviving mSEP-CR(-)RTBDNa P = 0.0002 mSEP-CR(-)RTBDNb P < 0.0001 106 0 5 Luminescence (p/s 10 0 0 50 100 150 0255075100 Luminescence (p/s 0 102030 010203040 Figure 3. Time [days] Time [days] Time [days] Time [days] F (d17) Preclinical fractionated surgery and 10 10 100 irradiation. A, Preclinical mouse SE) n = 8 ± 10 9 75 radiation protocol. B–F (left), n 10 8 = 8 Panels A−F Bioluminescence measurement of 50 Control 10 7 Radiation tumor growth. In parentheses are the 25 mCPC 10 6 days (d) when treated tumor volume mCPC Percent surviving P = ns 105 0 was signiﬁcantly (P < 0.05, Luminescence (p/s 010203040 010203040 Time [days] nonparametric) less than control. Time [days] (Right) survival curves of mice with indicated tumor types treated with G fractionated irradiation or control. Time [Days]: 0 1 31421283542 49 G, Preclinical combination mouse I I I I I I I I I I I I I I I I I I I I I I I I I I I surgery and radiotherapy protocol. R H–J, Bioluminescence measures of tumor growth (left) and survival curves (right) of mice with the =Allograft R =Randomized =Bioluminescence =Surgical resection I I I I I =2 x 5 Gy cranial irradiation indicated tumor types treated with H 11 surgery with or without fractionated 10 100 mSEP-CR(+) n irradiation (P values are log-rank 10 10 Vehicle ( = 16) n 75 >90% resection ( = 14) **** relative to control). Comparisons 10 9 Cranial irradiation (n = 10) *** between treatments are log-rank >90% resection + cranial irradiation (n = 7) 10 8 50 not signiﬁcant (ns), P < 0.005 7 10 mSEP-CR(+) 25 ( ), P < 0.0005 ( ), and Control − Percent surviving 10 6 Cranial radiation (d7 50) P < >90% resection (d7−10) 0.00005 ( ). Luminescence [p/s+SE] >90% resection + radiation (d7−50) 0 10 5 0 50 100 150 200 0 10 20 30 40 60 80 100 Time [Days] I Time [Days] 10 9 100 xSEP-CR(+) n 10 8 Vehicle ( = 11) >90% resection (n = 16) 75 *** Cranial irradiation (n = 5) n.s. 10 7 >90% resection + cranial irradiation (n = 13) 50 10 6 xSEP-CR(+) Control 25 5 10 Cranial radiation (dy32−85) Percent surviving

Luminescence [p/s+SE] >90% resection >90% resection + radiation (dy57−85) 0 10 4 0 50 100 150 200 250 0 50 100 150 Time [days] J Time [days] 11 10 100 mSEP-CR(-)RTBDNa 10 10 Vehicle (n = 18) >90% resection (n = 15) 9 75 *** 10 Cranial irradiation (n = 10) ** n 10 8 >90% resection + cranial irradiation ( = 5) 50 10 7 mSEP-CR(-)RTBDNa 10 6 Control 25

Cranial radiation (d16−60) Percent surviving 10 5 >90% resection (d10−30) Luminescence [p/s+SE] >90% resection + radiation (d10−37) 0 10 4 0 100 200 300 400 0 10 20 30 40 50 100 150 Time [days] Time [days]

Published pharmacokinetic data indicated that cabazitaxel, gem- scheduling for vosaroxin, chaetocin, and acivicin (20–32). There- citabine, pralatrexate, and pemetrexed would penetrate the cen- fore, to select which of these drugs might be suitable for further tral nervous system (CNS) and provided appropriate doses and preclinical development, the pMDT designed and conducted a

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mSEP-CR(-)RTBDNb mCPC A RTBDNb B time (hours) time (hours) 136102472 1 3 6 102472 mSEP-CR(-)mCPC 0.03 0.09 PRALATREXATE Cabazitaxel LUMINESPIB CABAZITAXEL GANETESPIB Pralatrexate 0.16 0.08 DELANZOMIB OLTIPRAZ DOXORUBICIN Gemcitabine 0.25 0.16 IXABEPILONE GEMCITABINE VINORELBINE Panobinostat 0.19 0.39 HOMOHARRINGTONINE OPROZOMIB PEMETREXED Carfilzomib 0.11 0.83 ACIVICIN UCN-01 CARFILZOMIB Vosaroxin 0.56 0.62 CHAETOCIN DACTOLISIB CERIVASTATIN DINACICLIB Cladribine 5.70 1.22 PHENOXODIOL SEPANTRONIUM NVP-BGT226 Pemetrexed 2.00 10.0 SELINEXOR RIGOSERTIB TALAZOPARIB GMX-1778 10.2 11.1 IXAZOMIB QUISINOSTAT Rigosertib Chaetocin ETOPOSIDE CUDC-907 9.07 0.11 VOSAROXIN <0.1 0.5 1 >1,000 TANESPIMYCIN III I CLADRIBINE Selinexor Acivicin ROMIDEPSIN µ 1.32 1.89 BMS-754807 Washout IC50 ( mol/L) PANOBINOSTAT AMUVATINIB LESTAURTINIB 10 GMX-1778 C 10 mSEP-CR(-)RTBDNb AZD7762 LY2801653 BMS-911543 10 9 MK-8776 Figure 4. AXITINIB PALIFOSFAMIDE Preclinical repurposing of chemotherapies. GSK-923295 10 8 RABUSERTIB MK-2206 A, Repurposing screen of 114 FDA-approved VOLASERTIB 7 MK-1775 10 and/or clinical trial drugs. Heatmap (left) PF562271 Vehicle AZD1480 Pemetrexed (400 mg/kg; ns) reports 72-hour IC50 against the indicated cell Cabazitaxel (ns) DOCETAXEL 10 6 TOFACITINIB Luminescence [p/s ± SE] Pralatrexate (15 mg/kg; ns) type; gray bars (middle) indicate FDA-approved EVODIAMINE Vosaroxin (20 mg/kg; ns) DANUSERTIB Gemcitabine (120 mg/kg; d11−18) drugs; graph (right) reports number of OMIPALISIB 10 5 completed trials of each drug. Arrows denote ENMD-2076 0 10203040 BARDOXOLONE GANDOTINIB Time post implant [Days] drugs selected for further study in B. B, CUDC-101 TERAMEPROCOL 100 "Washout studies": heatmaps report IC50 values VORINOSTAT BARICITINIB after timed exposures of the indicated cell types GEDATOLISIB TEMSIROLIMUS to drug. C–D (top), Bioluminescence TIVANTINIB 75 PACRITINIB measurement of tumor growth. In parentheses ERIBULIN TAFENOQUINE are the days (d) when treated tumor volume CRIZOTINIB 50 RTBDNb NAVITOCLAX mSEP-CR(-) was signiﬁcantly (P < 0.05, nonparametric) less ARTESUNATE FEDRATINIB Vehicle (n = 36) than control. (Bottom) survival curves of mice LCL161 Pemetrexed (n = 9) P = ns Percent surviving MEFLOQUINE 25 Cabazitaxel (n = 9) P = ns with indicated tumor types treated with the PF-477736 Pralatrexate (n = 10) P = ns NIRAPARIB Vosaroxin (n = 10) P = 0.01 indicated drug monotherapy. Comparisons COBIMETINIB Gemcitabine (n = 8) P = 0.002 GSK461364 0 between treatments are log-rank not signiﬁcant ALISERTIB REGORAFENIB 0 10203040 (ns) and P < 0.0005 ( ). GSK-2636771 Time post implant [days] TH-302 D ERASTIN 10 10 TRAMETINIB mCPC MLN 4924 LINSITINIB 9 RUXOLITINIB 10 GDC-0032 RG7112 DIMETHYL FUMARATE 10 8 SULFORAPHANE AZD5363 BORTEZOMIB OLAPARIB 10 7 ARRY-520 Vehicle RIBAVIRIN Pemetrexed (400 mg/kg; ns) SELUMETINIB Cladribine (75 mg/kg; ns) 10 6 Pralatrexate (15 mg/kg; ns) OLMESARTAN MEDOXOMIL Luminescence (p/s ± SE) SIROLIMUS Chaetocin (0.2 mg/kg; ns) VELIPARIB Acivicin (0.4 mg/kg; ns) Gemcitabine (120 mg/kg; ns) VEMURAFENIB 10 5 LOMEGUATRIB PX-478 0 10203040 ABT-199 Time post implant [days] BARASERTIB TAK-960 Vehicle (n = 60) AT-406 100 Pemetrexed (n = 9) P = ns BIRINAPANT Cladribine (n = 13) P = ns FG-4592 Pralatrexate (n = 8) P = ns PS-1145 Chaetocin (n = 6) P = ns LY2157299 Acivivin (n = 8) P = ns VISTUSERTIB 75 Gemcitabine (n = 15) P < 0.0001

IC50 0- 500- <0.1 15 >10 1,000- 1,500- 50 III I Active/completed FDA approved FDA clinical trials 72 hr IC50 (µmol/L) Percent surviving 25

mCPC 0 0 20406080100 Time post implant [days]

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series of monotherapy preclinical trials in mice harboring mSEP- 200 mg/kg bolus and 6-hour infusions (n 10 mice per CR(–)RTBDNb or mCPC. The goal of these studies was to look for cohort; Fig. 5D–F). Of all regimens tested, 3- and 6-hour infusions any evidence of antitumor activity (growth and/or survival). of gemcitabine were most efficacious, producing similar degrees Doses and schedules of each drug were designed to mimic those of tumor growth suppression and enhanced overall survival; achievable in patients. We also conducted a monotherapy however, 3-hour infusions proved the least toxic. Additional 3- study of cladribine as a "negative control" compound because hour gemcitabine infusion monotherapy trials identified signif- this drug was relatively inactive in vitro and was predicted not to icant active against mSEP-CR(þ), xSEP-CR(þ), and a mSEP-CR penetrate the CNS. Of all agents tested, only gemcitabine (120 (–) model driven by EPHB2 (Fig. 5G–I; ref. 17). Three-hour mg/kg intravenous bolus) displayed significant activity against gemcitabine infusions were more efficacious than combination both mSEP-CR(–)RTBDNb and mCPC: this treatment was the only cisplatin/cyclophosphamide or cisplatin/etoposide/vincristine monotherapy to significantly impair the growth of mSEP-CR that approximates "standard of care" chemotherapy regimens (–)RTBDNb and to prolong the survival of mice with these tumors; that have been tested against ependymoma and CPC, respectively, therefore, this drug was selected for further repurposing studies in the clinic (Fig. 5J and K; refs. 7, 35). Therefore, we selected 3- (Fig. 4C and D). Vosaroxin—a topoisomerase II inhibitor causing hour infusions of gemcitabine for our final phase of preclinical site-selective DNA damage—also produced a modest but signif- repurposing. icant survival advantage for mice harboring mSEP-CR(-)RTBDNb tumors (Fig. 4C). These data underscore that drugs with relatively Combining gemcitabine and conventional therapy potent activity in vitro may lack efficacy in vivo when administered The efficacy of gemcitabine monotherapy in our model at clinically relevant doses. In light of the considerable systems suggests it may have value as an adjuvant therapy in activity of gemcitabine, we selected this drug for further preclinical the clinic. Therefore, the pMDT designed a combination development. study aimed at testing the value of adding 3-hour gemcitabine infusions to "standard-of-care" surgery and fractionated radio- Optimization of gemcitabine therapy therapy (Fig. 6A). With regard to ependymoma, we focused on Gemcitabine can be administered as an intravenous bolus or SEP-CR(þ) disease because this tumor type is the most aggres- infusion, resulting in very different pharmacokinetic profiles sive form of SEP. The mSEP-CR(þ) rather than xSEP-CR(þ) (33). Therefore, we treated mice bearing mSEP-CR(–)RTBDNb or model was chosen for these studies because these models mCPC with various gemcitabine regimens and simultaneously displayed similar responses to surgery, radiotherapy, and gem- measured concentrations of the drug in plasma and brain citabine as monotherapies, but the more rapid growth profile of tumor extracellular fluid (tECF) using intratumoral microdia- mSEP-CR(þ) enabled completion of these large combination lysis. Mice were treated initially with two clinically relevant studies in a timely manner. Mice were treated with GTR fol- gemcitabine regimens: 60 mg/kg i.v. bolus that is active against lowed by 54 Gy fractionated irradiation and then 3 weeks of a mouse model of group 3 medulloblastoma; or continuous 3- consecutive 3-hour gemcitabine infusions. This treatment was hour infusion via subcutaneous Alzet pumps (19, 20). Note tolerated remarkably well. Although the average tumor burden that 60 mg/kg i.v. bolus gemcitabine produced a plasma of mice receiving gemcitabine was lower than that of animals AUC0–6hr of 25.9 mmol/L hr that is equivalent to that observed treated with surgery and irradiation alone, this difference in children treated with 1,200 mg/m2 (Fig. 5A; ref. 34). The was not significant(Fig.6A);however,theadditionofgemci- tumor-to-plasma partition coefficient for unbound gemcita- tabine doubled the median survival (183 days) of mice relative bine (Kp,uu) at this dose was 0.51 and 0.18 for mice bearing to those treated with surgery and irradiation alone (96 days), mSEP-CR(–)RTBDNb and mCPC tumors, respectively. The gem- and cured 50% of animals (P < 0.00001; Fig. 6B). These data citabine concentration in tECF produced by this regimen only underscore the need to assess both tumor volume and animal remained above the in vitro washout IC50 of each tumor type survival as response metrics to preclinical therapy because for less than 3 hours (compare Figs. 4B and 5A). In contrast, tumor imaging in small animals may operate at the limits 3-hour infusions of gemcitabine produced plasma exposures of resolution. We next assessed the value of adding serial of 95.1 24.1 mmol/Lhr—equivalent to treating children postoperative, 3-hour gemcitabine infusions to the treatment with 2,000 mg/m2—and in both models maintained a tECF of mCPC (Fig. 6C and D). As observed previously, gross total concentration above the IC50 in washout studies for 7hours tumor resection alone produced a modest but significant sur- (compare Figs. 4B and 5B). To determine if these in vivo vival advantage for mice harboring mCPC (median survival exposures produce the antitumor cell effects predicted in vitro, surgery ¼ 34 days vs. control ¼ 22 days; P < 0.003; Fig. 6D); we harvested tumors from mice at 3, 8, 24, and 48 hours and gemcitabine therapy alone markedly extended the survival following initiation of gemcitabine therapy and estimated of mice with these tumors (median survival gemcitabine ¼ 42 levels of tumor cell proliferation and apoptosis. Three-hour days vs. control ¼ 22 days; P < 0.0001; Fig. 6D). Notably, no infusions of gemcitabine induced significantly greater and significant difference in survival was observed between mice more sustained levels of tumor cell apoptosis in mSEP-CR undergoing surgery or gemcitabine therapy alone; however, (–)RTBDNb and mCPC than did 60 mg/kg i.v. bolus treatment, surgical resection followed by gemcitabine significantly extend- and 3-hour infusions produced a more significant and ed survival above that of animals receiving surgery alone sustained reduction in tumor cell proliferation, although this (median survival surgery alone ¼ 44 vs. surgeryþgemcitabine was only observed in mSEP-CR(–)RTBDNb (Fig. 5C; Supple- ¼ 46.5 days; P < 0.0001; Fig. 6D). Together, these data suggest mentary Fig. S1). that 3-hour infusions of gemcitabine may add therapeutic value As a final step to select the optimal dose and schedule of to "standard-of-care" surgery and radiation in the treatment of gemcitabine for preclinical assessment, we further expanded the SEP and may improve the results of surgical resection of CPC. repertoire of gemcitabine regimens to assess the relative activity of We propose that these regimens should be tested in the clinic.

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A 1,000 Gemcitabine (60 mg/kg) 1,000 Gemcitabine (60 mg/kg) 1,000 Gemcitabine (60 mg/kg) 1,000 Gemcitabine (60mg/kg) mSEP-CR(-)RTBDNb mSEP-CR(-)RTBDNb mCPC mCPC 100 Plasma 100 Plasma 100 Plasma 100 Plasma Tumor ECF

µ mol/L) µ mol/L) Tumor ECF µ mol/L) µ mol/L) 10 10 10 10

1 1 1 1

0.1 0.1 0.1 0.1 Gemcitabine ( Gemcitabine ( Gemcitabine ( Gemcitabine ( 0.01 0.01 0.01 0.01 0246 0246 0246 0246 B Time after dosing (hr) Time after dosing (hr) Time after dosing (hr) Time after dosing (hr) 100 Gemcitabine (3 hr inf.) 100 Gemcitabine (3 hr inf.) 100 Gemcitabine (3 hr inf.) 100 Gemcitabine (3 hr inf.) mSEP-CR(-)RTBDNb mSEP-CR(-)RTBDNb mCPC mCPC 10 10 10 10 µ mol/L) µ mol/L) µ mol/L) µ mol/L) 1 1 1 1

0.1 0.1 0.1 0.1 Gemcitabine ( Gemcitabine ( Gemcitabine (

Gemcitabine ( Plasma (±95CI) Tumor ECF (±95CI) Plasma (±95CI) Tumor ECF (±95CI) 0.01 0.01 0.01 0.01 02468 02468 02468 02468 CDTime after dosing (hr) Time after dosing (hr) Time after dosing (hr) Time after dosing (hr) 20 50 30 *** mSEP-CR(-)RTBDNb * mCPC 40 ** 10 ** 20 30 * cells (±SE) cells (±SE) + + * 0 20 ** **** 10 -10 10 % Caspase 3 % Caspase 3

0 0 % Weight change (min-max) -20 3 hours 8 hours 24 hours 48 hours 3 hours 8 hours 24 hours 48 hours 1 day 4 days 7 days 8 days Time post onset of treatment Time post onset of treatment Time post onset of treatment Vehicle control Vehicle iv Vehicle pumps Gemcitabine 60 mg/kg iv 120 mg/kg iv 3 hour pumps E Gemcitabine 3 hour pump F 200 mg/kg iv 6 hour pumps

10 10 100 100 mCPC 10 9 Vehicle (n = 65) 10 9 60 mg/kg; n = 16, P = ns 75 75 200 mg/kg; n = 8, P = 0.02 10 8 6 hr inf; T = 10, P = 0.0005 10 8 3 hr inf; n = 17, P < 0.0001 RTBDNb 50 RTBDNb 50 7 mSEP-CR(-) mSEP-CR(-) mCPC 10 Vehicle Vehicle (n = 43) 10 7 Vehicle n P 60 mg/kg (ns) 60 mg/kg (ns) 25 60 mg/kg; = 9, = ns 6 n P 200 mg/kg (ns) 25 10 200 mg/kg (ns) 200 mg/kg; = 9, = 0.02 −

− n P 6 6 hr infus. (d8 48) Percent surviving 6 hr inf (d6 20) Percent surviving 6 hr inf; = 10, = 0.006 10 − Luminescence (p/s ± SE) 3 hr infus. (d10 50) 3 hr inf (d12−20) 3 hr infus; n = 9, P = 0.02 Luminescence (p/s ± SE) 5 0 0 10 0 10203040506070 0 1020304050 010203040 0 20406080100 Time (days) Time (days) Time (days) Time (days) GH

10 100 10 mSEP-CR(+) 1010 100 xSEP-CR(+) xSEP-CR(+) Vehicle (n = 18) Vehicle Vehicle (n = 6) 75 3 hr infus; n = 20 3 hr infus. (d78-88) 3 hr infus; n = 6 9 75 10 9 P < 0.0001 10 P = 0.0008 50 50 108 10 8 25 mSEP-CR(+) 25

Vehicle Percent surviving 7 3 hr infus. (d7−24) 10 Percent surviving Luminescence (p/s ± SE) 0 Luminescence (p/s ± SE) 10 7 0 20406080 0 0204060 0 50 100 150 0 100 200 300 Time (days) Time (days) Time (days) Time (days) I J

10 11 10 10 100 100 mSEP-CR(+) Vehicle (n = 18) 10 10 109 CDDP/CP (n = 9) 75 75 10 9 P < 0.001

108 108 50 50 mSEP-CR(-)EPHB2 107 n 25 Vehicle ( = 10) 107 mSEP-CR(+) mSEP-CR(-)EPHB2 n 25 6 3 hr infus; = 9 Vehicle

10 Percent surviving Vehicle P = 0.02 − Percent surviving 3 hr infus. (d6−20) Luminescence (p/s ± SE) CDDP/CP (d11 41) Luminescence (p/s ± SE) 105 0 0 0 102030 0 10203040 0204060 0 20406080 Time (days) Time (days) Time (days) Time (days) K 10 11 100 mCPC Vehicle (n = 14) 10 10 CDDP/VP16/VCR (n = 16), P = ns 75 10 9 50 10 8 mCPC 25 10 7 Vehicle CDDP/VP16/VCR (ns) Percent surviving Luminescence (p/s ± SE) 10 6 0 0 5 10 15 20 25 30 020406080 Time (days) Time (days)

Figure 5. Pharmacokinetic, toxicity, and efﬁcacy studies of gemcitabine. Plasma and tECF concentration–time plots in the indicated tumor types treated with 60 mg/kg bolus (A) or 3-hour infusion (B) gemcitabine. C, Graphs showing induction of tumor cell apoptosis measured by cleaved Caspase 3 immunohistochemistry in mice with the indicated brain tumors treated with 60 mg/kg or 3-hour infusion of gemcitabine. D, Toxicity determined by weight loss in mice treated with the indicated doses and regimens of gemcitabine (in C and D: , P < 0.05; , P < 0.005; , P < 0.0005, Mann–Whitney). E–I, Bioluminescence measurement of tumor growth in mice treated with indicated dose and schedule of gemcitabine. In parentheses are the days (d) when treated tumor volume was signiﬁcantly (P < 0.05, nonparametric) less than control. (Right) survival curves of the same mice shown left. P value ¼ log-rank relative to control. Similar growth and survival curves are shown in J and K for mice treated with cisplatin/cyclophosphamide or cisplatin/etoposide/vincristine, respectively.

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A B 1011 100 1010 109 75 108 50 107 106 mSEP-CR(+) 25 mSEP-CR(+) Control Percent survival Vehicle (n = 16), median survival 31 days 105 Surgery/radiation (d17−50) Surgery/radiation (n = 7), median survival 96 days P < 0.0001

Luminescence (p/s ± SE) n P Surgery/radiation/gemcitabine (d6−50) Surgery/radiation/gemcitabine ( = 9), median survival 190 days < 0.0001 104 0 0 1020304060 80 100 0 50 100 150 200 250 Time (days) Time (days)

C D 1010 100 mCPC Vehicle (n = 25), med survival 22 days ***

Surgery (n = 44), med survival 34 days P < 0.003 n.s. n P n.s. 9 Surgery/gemcitabine ( = 14), med survival 46.5 days < 0.0001 10 75 Gemcitabine (n = 17), med survival 42 days P < 0.0001

108 50 mCPC Control 7 Surgery (d17−23)

10 Percent survival 25 Surgery/gemcitabine (d17−24)

Luminescence (p/s ± SE) Gemcitabine (d17−21) 10 6 0 15 20 25 30 35 40 45 50 0 255075100 Time (days) Time (days)

Figure 6. Combination surgery, fractionated irradiation, and postirradiation gemcitabine therapy. Bioluminescence measures of tumor growth (A) and survival curves (B) of mice with mSEP-CR(þ) treated with surgery and radiotherapy alone or surgery, radiation, and 3-hour infusions of gemcitabine. Bioluminescence measures of tumor growth (C) and survival curves (D) of mice with mCPC treated with surgery and 3-hour infusions of gemcitabine alone or surgery and gemcitabine. Figures in parentheses in bioluminescence plots are the days (d) when treated tumor volume was signiﬁcantly (P < 0.05, nonparametric) less than control.

Discussion through clinical trials alone, especially among patients with rare The past decade has witnessed a revolution in our understand- disease variants: the small populations of patients with these ing of human cancer. The integration of genomic and develop- tumors limit the number of drugs and regimens that can be tested mental biology has shown that morphologically similar cancers in a timely manner. The preclinical platform described here comprise subtypes, driven by different genetic alterations, which provides an evidence-based approach to guide clinical trials for likely arise within distinct cell lineages (36). These data help rare brain tumor subtypes. It is important to note that this explain why cancers once regarded histologically as homoge- platform is not designed to replace or reduce the clinical trial neous diseases display discrepant behaviors. For example, medul- platform; but rather to better triage drugs so that clinicians can loblastoma and ependymoma are now known to include sub- focus on novel regimens with the greatest potential to cure. Key types with extraordinarily good (e.g., WNT-medulloblastoma features of our approach include the use of accurate mouse and SEP-CR[-]) or bad (Group 3-medulloblastoma with MYC models of human brain tumors and the coordinated engagement amplification and SEP-CR[þ]) prognosis (11, 37). This knowl- of clinical and research professionals in regular pMDT discus- edge could pinpoint patients who might be cured with less sions, greatly facilitating the codevelopment of clinically relevant toxic therapy, as well as poor prognosis patients who need new preclinical trials. treatments. Indeed, clinical trials of decreased radiotherapy Maximal surgical resection of ependymoma followed by irra- are ongoing among patients with WNT-medulloblastoma diation is consistently associated with a better patient outcome (NCT01878617). But integrating understanding of tumor biology regardless of primary tumor site (8, 10, 11, 14). This observation into established clinical practice is enormously challenging and has led to the widespread notion that SEPs have a high probability requires a number of assumptions that are often made without of cure with surgery alone, and underpins Arm 1 of an ongoing knowledge of subtype-specific treatment efficacy. For example, Children's Oncology Group study in which children achieving reducing radiotherapy for children with WNT-medulloblastoma a gross total resection of classic histology SEP receive no further assumes that this therapy, rather than surgery or chemotherapy, treatment (NCT01096368; ACNS0831). But in our studies, is relatively redundant. And trials of new treatments for 'poor surgery alone had no therapeutic value in the treatment of prognosis' tumors often assume that relatively ineffective con- mSEP-CR(þ) or xSEP-CR(þ), only benefiting mice with mSEP- ventional therapies should be retained; this approach runs the risk CR(–)RTBDNa. However, total resection of mSEP-CR(þ)did of increasing toxicity unnecessarily. markedly improve the efficacy of irradiation, curing a signifi- So how can we integrate new understanding of cancer biology cant number of animals and also improved the survival of mice and therapy into empirical treatment regimens that have with mSEP-CR(–)RTBDNa. These data underscore the important developed over decades? It is unlikely that we will achieve this point that treatments can display surprising interactions,

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producing high cure rates when used in combinations that not treatments through clinical trial (13). For example, the only are not apparent when the treatments are used individually. multicenter CPC clinical trial conducted to date was initiated in Our data also support the notion that irradiation is a highly 2000 (CTP-SIOP-2000), but 17 years later, the results of this trial effective treatment of SEP and suggest that avoiding radiother- are yet to be published. Our preclinical system provides an apy for all patients with totally resected SEP, regardless of alternative, evidence-based approach to prioritize combination subtype, may be inappropriate. Rather, as a minimum, we regimens for the clinic, potentially avoiding years of trials of recommend the prospective evaluation of SEP molecular sub- ineffective therapies. Of particular note, by recapitulating surgery, type in ACNS0831 to ensure that SEP-CR(þ) patients are not irradiation, and chemotherapy, our approach allows for preclin- undertreated. Reverse translation of these clinical data will be ical trials of multiple doses, delivery routes, and schedules of critical to validate the predictions made by our preclinical novel chemotherapies in the context of standard-of-care treat- system. This later point is particularly important because our ment. In this regard, sequential total tumor resection, fractionated model system is likely to be predictive but not infallible. radiotherapy, and 3-hour gemcitabine infusions doubled the Indeed,incontrastwithmSEP-CR(þ), total resection of median survival of mice with mSEP-CR(þ) relative to surgery xSEP-CR(þ) did not improve the efficacy of radiotherapy, and irradiation alone, curing half of all animals. Our studies also indicating that further iteration between preclinical and clinical provide evidence that combination surgery and gemcitabine work will be required to understand the ependymoma-sub- infusion therapy may benefit the treatment of CPC. These data type–specific relevance of combination treatment. are in keeping with the activity of gemcitabine in other chemore- The effectiveness of preirradiation surgery in our models also sistant cancers including pancreatic cancer (44, 45). We therefore supports the widely held notion that cytoreductive surgery recommend that gemcitabine infusions might prove effective as increases radiosensitivity and chemotherapy by removing thera- postsurgery and irradiation chemotherapy. Furthermore, because py-resistant, hypoxic, and highly-proliferative tumor cores (38). gemcitabine may also serve as a radiosensitizer, we are currently These data might also explain why resection and irradiation are exploring the timing of gemcitabine treatment relative to irradi- more effective than partial resection and irradiation among ation and whether gemcitabine may be added to conventional patients with posterior fossa subtype-A ependymoma—another treatment regimens in younger patients. aggressive disease subtype (14). Thus, our models provide an Although our model system provides a promising tool to opportunity to explore the biological basis of cytoreductive sur- prioritize complex combination treatment regimens for clinical gical efficacy. Our models may also facilitate the identification, trial, the accuracy of these predictions remains to be assessed. It is a isolation, and study of radiation resistant tumor clones, because hard reality that most cancer treatments that are effective in our imaging studies revealed regrowth of mSEP-CR(þ), xSEP-CR animal models fail in patients (46, 47). Indeed, although our (þ), and mSEP-CR(-) prior to completion of radiotherapy. models closely replicate the morphology and transcriptome of the In contrast with our SEP models, radiotherapy proved ineffec- corresponding human tumors, they are maintained in immuno- tive against mCPC. The radioresistance of mCPC may reflect the compromised hosts and therefore cannot account for contribu- Tp53-null status of these tumors, because this gene mediates cell tions of the host immune system to tumor biology and treatment. death mechanisms in irradiated cells (39). Notably, 60% of Thus, preclinical platforms such as the one presented here require human CPCs contain mutant Tp53: these tumors also tend to careful iterative study with clinical translation to be validated and be radioresistant, clinically aggressive, and to develop in infants refined. This important ongoing process further underscores the (40, 41). It is interesting that our mCPC model is initiated in value of convening pMDT teams comprising laboratory and embryonic choroid plexus; therefore, these tumors likely model clinical oncology professionals. radioresistant, aggressive, and TP53-mutant infant CPC (18). Although chemotherapy has been evaluated in ependymoma Disclosure of Potential Conflicts of Interest and CPC, its role remains controversial with only limited benefit B.B. Freeman is an employee of and has ownership interests (including reported (7, 13). These data are in keeping our observations that patents) at KinDynaMet LLC. M.F. Roussel is a consultant/advisory board most drugs displaying potent activity against our models in vitro member for Cold Spring Harbor Laboratories and the National Cancer Institute, fi in vivo and reports receiving commercial research support from Eli Lilly. No potential failed to produce therapeutic bene t . This notion is conflicts of interest were disclosed by the other authors. also supported by our observation that mouse models of SHH- — — medulloblastoma a more chemosensitive disease responded Authors' Contributions to treatments that were ineffective against mSEP and mCPC, e.g., Conception and design: B.V. Nimmervoll, N. Boulos, A. Shelat, A. Gajjar, pemetrexed and 60 mg/kg bolus gemcitabine (20, 42). Our Y.T. Patel, R.K. Guy, T.E. Merchant, K.D. Wright, R.J. Gilbertson preclinical in vitro and in vivo pipeline did identify 3-hour infu- Development of methodology: B.V. Nimmervoll, N. Boulos, B. Bianski, sions of gemcitabine as a potential new treatment of SEP and CPC. J. Dapper, A. Shelat, S. Terranova, A. Gajjar, Y.T. Patel, B.B. Freeman, A. Onar-Thomas, C.F. Stewart, R.K. Guy, T.E. Merchant, C. Calabrese, This regimen generated tECF concentrations above the in vitro IC50 K.D. Wright, R.J. Gilbertson for 7 hours and proved more effective against mSEP-CR(þ) and Acquisition of data (provided animals, acquired and managed patients, mCPC than combination conventional chemotherapy regimens provided facilities, etc.): B.V. Nimmervoll, N. Boulos, B. Bianski, J. Dapper, with reported activity in patients (7, 35). Thus, we suggest that M. DeCuypere, Y.T. Patel, B.B. Freeman, C.F. Stewart, T.E. Merchant, 3-hour infusions of gemcitabine should be tested in patients C. Calabrese, K.D. Wright, R.J. Gilbertson with SEP and CPC. Analysis and interpretation of data (e.g., statistical analysis, biostatistics, Fewer than 150 adults and children with all variants of SEP and computational analysis): B.V. Nimmervoll, N. Boulos, J. Dapper, A. Shelat, Y.T. Patel, B.B. Freeman, C.F. Stewart, R.K. Guy, T.E. Merchant, C. Calabrese, CPC are available for enrollment on clinical trials in the United K.D. Wright, R.J. Gilbertson States each year, severely limiting studies of new treatments (43). Writing, review, and/or revision of the manuscript: B.V. Nimmervoll, Indeed, it is widely agreed that the rarity of CPC poses an almost N. Boulos, A. Shelat, A. Gajjar, A. Onar-Thomas, C.F. Stewart, M.F. Roussel, insurmountable hurdle to the efficient development of new T.E. Merchant, K.D. Wright, R.J. Gilbertson

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A Preclinical Multidisciplinary Brain Tumor Board

Administrative, technical, or material support (i.e., reporting or organizing Y.T.Patel,B.BFreeman,A.Onar-Thomas,R.K.Guy,T.E.Merchant, data, constructing databases): B.V. Nimmervoll, N. Boulos, A. Gajjar, C. Calabrese, and K.D. Wright); Cancer Research UK (R.J. Gilbertson, B.B. Freeman, K.D. Wright, R.J. Gilbertson B.V. Nimmervoll, and S. Terranova); the Mathile Family Foundation Study supervision: B.V. Nimmervoll, N. Boulos, A. Gajjar, K.D. Wright, (R.J. Gilbertson, B.V. Nimmervoll, and S. Terranova); and Cure Search R.J. Gilbertson (R.J. Gilbertson, B.V. Nimmervoll, and S. Terranova). Other (wrote the manuscript): R.J. Gilbertson

Acknowledgments The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in This work was supported by grants from the NIH, P01CA96832 accordance with 18 U.S.C. Section 1734 solely to indicate this fact. (R.J. Gilbertson, B.V. Nimmervoll, N. Boulos, A. Gajjar, C.F. Stewart, and M.F. Roussel) and R0CA1129541 (R.J. Gilbertson and N. Boulos); the American Lebanese Syrian Associated Charities (R.J. Gilbertson, B.V. Nimmervoll, Received July 26, 2017; revised November 21, 2017; accepted December 21, N. Boulos, A. Gajjar, C.F. Stewart, M.F. Roussel, B. Bianski, J. Dapper, A. Shelat, 2017; published OnlineFirst January 4, 2018.

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1666 Clin Cancer Res; 24(7) April 1, 2018 Clinical Cancer Research

Establishing a Preclinical Multidisciplinary Board for Brain Tumors

Birgit V. Nimmervoll, Nidal Boulos, Brandon Bianski, et al.

Clin Cancer Res 2018;24:1654-1666. Published OnlineFirst January 4, 2018.

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