BRAF status in personalizing treatment approaches for pediatric … · 2016. 5. 21. · Genomics...

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TITLE: BRAF status in personalizing treatment approaches for pediatric gliomas

Aleksandra Olow1*, Sabine Mueller2,3,4,5*, Xiaodong Yang2, Rintaro Hashizume6, Justin

Meyerowitz2, William Weiss2,3,4, Adam C. Resnick7, Angela J. Waanders7, Lukas J. A.

Stalpers8, Mitchel S. Berger 2,5, Nalin Gupta2,5, C. David James6, Claudia Petritsch5, and

Daphne A. Haas-Kogan2,5,9.

1 Department of Laboratory Medicine, University of California, San Francisco, 2340

Sutter St, San Francisco, CA 94115-3024

2Brain Tumor Research Center, University of California, San Francisco, Helen-Diller

Cancer Diller Cancer Research Center, 1450 3rd Street, San Francisco, CA 94158

3Department of Neurology, University of California, San Francisco, 675 Nelson Rising

Lane, San Francisco, CA 94143-0449

4Department of Pediatrics, University of California, San Francisco, 505 Parnassus

Avenue, San Francisco, CA 94117-0106

5Department of Neurological Surgery, University of California, San Francisco, 505

Parnassus Avenue, San Francisco, CA 94117-0106

6Department of Neurological Surgery, Biochemistry and Molecular Genetics, Feinberg

School of Medicine, Northwestern University, 300 East Superior St, Chicago, IL 60611

7Department of Pediatrics, University of Pennsylvania Perelman School of Medicine, The

Children’s Hospital of Philadelphia, Philadelphia, PA 19104

8 Department of Radiotherapy, Academic Medical Center, University of Amsterdam, 1105

AZ Amsterdam, The Netherlands

Research. on December 29, 2020. © 2016 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on May 23, 2016; DOI: 10.1158/1078-0432.CCR-15-1101

http://clincancerres.aacrjournals.org/

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9Department of Radiation Oncology, Department of Radiation Oncology, Brigham and

Women's Hospital, Dana-Farber Cancer Institute, Boston Children’s Hospital, Harvard

Medical School, 450 Brookline Ave, Boston MA, 02215

* These authors contributed equally to this work.

Running title: BRAF alterations in pediatric low-grade glioma

Keywords: Pediatric low-grade glioma, MEK inhibition, PI3-Kinase pathway, mTOR,

BRAFV600E, resistance

Funding: This research was supported in part by the National Center for Advancing

Translational Sciences, National Institutes of Health, through UCSF-CTSI Grant Number

KL2TR000143 (SM), NIH Brain Tumor SPORE grant P50 CA097257 (SM, DHK, MP,

MSB), NINDS NS080619 (CDJ), NINDS 1R01NS091620 (DHK, WW, ACR), the

Matthew Larson Foundation (SM), Grand Philanthropic Fund (DHK), and University of

California Cancer Research Coordinating Committee (SM, DHK).

Corresponding author:

Daphne Haas-Kogan, MD

Professor and Chair

Department of Radiation Oncology, Brigham and Women's Hospital, Dana-Farber Cancer

Institute, Boston Children’s Hospital, Harvard Medical School

450 Brookline Ave. D1622

Boston, MA 02215-5418




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Tel: (617) 632-2291

FAX: (617) 632-2290

Email: [email protected]

Disclosure of potential conflicts of interest

None of the authors have any conflict of interest to report.

Total Word Count:

Number of Tables and Figures: 5 Figures, 3 supplemental Figures, 1 Supplemental

Table

Statement of translational relevance:

Pediatric low-grade gliomas (PLGG) constitute the most common group of central

nervous system tumors in children. Although the molecular underpinnings of PLGGs

represent an ideal platform for personalized approaches, precision medicine has yet to

impact PLGG therapy. Targeted therapeutics are available for many of the implicated

pathways; however, follow up biological studies defining the appropriate agents for each

alteration are lacking. Here we define the most effective combinations of discrete mTOR

and BRAF/MEK/MAPK inhibitors for PLGGs harboring the most common BRAF

genotypes. Significant limitations and challenges are posed by the dearth of PLGG

models and in fact the only patient-derived PLGG in this study is BT40; therefore, these

results and their application to PLGG treatment must be interpreted with caution and

efforts should be renewed to generate faithful models that truly recapitulate PLGG cellular

context. The work provides the pre-clinical rationale for targeted combinatorial




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approaches for the treatment of PLGGs and defines the framework for the next generation

of clinical trials for PLGGs, in which molecular features of individual tumors guide

rational therapies.




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Abstract

Purpose: Alteration of the BRAF/MEK/MAPK pathway is the hallmark of pediatric low-

grade gliomas (PLGG) and mTOR activation has been documented in the majority of

these tumors. We investigated combinations of MEK1/2, BRAFV600E and mTOR inhibitors

in gliomas carrying specific genetic alterations of the MAPK pathway.

Experimental Design:

We used human glioma lines containing BRAFV600E (adult high-grade: AM-38, DBTRG,

PLGG: BT40), or wild-type BRAF (pediatric high-grade: SF188, SF9427, SF8628) and

isogenic systems of KIAA1549:BRAF-expressing NIH/3T3 cells and BRAFV600E-

expressing murine brain tumors. Signaling inhibitors included everolimus (mTOR),

PLX4720 (BRAFV600E), and AZD6244 (MEK1/2). Proliferation was determined using

ATP-based assays. In vivo inhibitor activities were assessed in the BT40 PLGG xenograft

model.

Results: In BRAFV600E cells, the three possible doublet combinations of AZD6244,

everolimus and PLX4720 exhibited significantly greater effects on cell viability. In

BRAFWT cells, everolimus+AZD6244 was superior compared to respective

monotherapies. Similar results were found using isogenic murine cells. In

KIAA1549:BRAF cells, MEK1/2 inhibition reduced cell viability and S-phase content,

effects that were modestly augmented by mTOR inhibition. In vivo experiments in the

BRAFV600E pediatric xenograft model BT40 showed the greatest survival advantage in

mice treated with AZD6244+PLX4720 (p<0.01).

Conclusions: In BRAFV600E tumors, combination of AZD6244+PLX4720 is superior to

monotherapy and to other combinatorial approaches. In BRAFWT pediatric gliomas




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everolimus+AZD6244 is superior to either agent alone. KIAA1549:BRAF-expressing

tumors display marked sensitivity to MEK1/2 inhibition. Application of these results to

PLGG treatment must be exercised with caution since the dearth of PLGG models

necessitated only a single patient-derived PLGG (BT40) in this study.




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Introduction

Pediatric low-grade gliomas (PLGG) constitute the most common group of central

nervous system tumors in children (1). Although they exhibit rare malignant

transformation and relatively slow growth, PLGGs pose great morbidity including vision

loss, endocrine dysfunction and poor cognition. Viewed as a chronic disease, ideal therapy

that would carry limited side effects can be best achieved through selective, targeted

therapy. Despite the known heterogeneity of PLGGs and characterized driver mutations

that together offer the ideal, timely platform for personalized approaches to therapy, most

children are still treated with standard chemotherapy protocols in a “one treatment fits all”

approach.

Genomic discoveries in recent years have significantly enhanced our

understanding of the molecular pathogenesis of PLGGs. The most frequent aberration

identified in PLGG implicates BRAF, a key regulatory kinase of the mitogen-activated

protein kinase (MAPK) pathway. The majority of pilocytic astrocytomas (PA) carry the

BRAF fusion protein KIAA1549:BRAF leading to MAPK signaling activation (2, 3). The

impact of this fusion protein on clinical outcome remains controversial (4, 5). Clinical

studies are currently investigating effects of specific inhibitors of the MAPK pathway,

such as the MEK1/2 inhibitor AZD62444, for the treatment of PLGGs (ClinicalTrials.gov;

Identifier NCT01089101). Activating BRAFV600E point mutations are present in 6-10% of

PAs with higher incidences reported in other LGG subtypes such as pleomorphic

xanthoastrocytoma (51-66%) and ganglioglioma (11-21%) (6-12). As this specific

BRAFV600E mutation is also common in adult patients with melanoma, several inhibitors

are now clinically available, including vermurafenib and dabrafenib. Ongoing clinical




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trials are testing these inhibitors in children with recurrent/progressive BRAFV600E-

mutated tumors (ClinicalTrials.gov Identifier NCT01748149).

Another important signaling cascade implicated in the tumorigenesis of PLGGs is

the phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR)

pathway (13, 14). Recent studies have indicated that KIAA1549:BRAF regulates PLGG

cell growth in an mTOR-dependent manner (15). Ongoing clinical studies are examining

the specific mTOR inhibitor everolimus for the treatment of recurrent/progressive PLGGs

(ClinicalTrials.gov Identifier NCT01734512, NCT01158651).

Although the molecular underpinnings of PLGGs represent an ideal platform for

pathway-driven treatment approaches, and targeted therapeutics are entering the clinic,

follow-up biological studies defining the appropriate, most effective agents for each

alteration are lacking. We address this impediment by assessing effects of MEK,

BRAFV600E and mTOR inhibitors as single agents and as combination therapies in models

of pediatric gliomas carrying the most commonly found genetic alterations.




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Methods

Cell lines, xenografts, and inhibitors

DBTRG, AM-38 and BT40 (BRAFV600E), as well as SF188, SF9427 and SF8628

(BRAFWT) were obtained from the Brain Tumor Research Center (BTRC) Tissue Bank at

the University of California, San Francisco (UCSF) and authenticated by the UCSF

Genomics Core using Promega Powerplex 1.2 System (Promega, Madison, WI). SF9427

and SF8628 were established from pediatric high-grade gliomas as described previously

(16) (Supplemental Table1). BRAFV600E mutations were confirmed by the UCSF genomics

core by DNA sequencing. The BRAFWT and KIAA1549-BRAF NIH/3T3 fibroblast

isogenic cell model was generated as previously described (17). The isogenic system of

murine brain tumors differing only in BRAFV600E status was generated as described (18).

The genotypes used in this study are BRafCA/WT Ink4a-arf lox/lox (6390, 6392) and

BRafWT/WT Ink4a-arf lox/lox (6387, 6393). Mouse neurosphere cultures from the

subventricular zone of P60 littermates were established and cultured as previously

described (19). In brief, cells were maintained in neurobasal medium (Invitrogen)

supplemented with human basic fibroblast growth factor, epidermal growth factor

(Peprotech) and B27 (Invitrogen). Cultures were maintained at 37°C and 5% CO2. To

induce BRAFV600E expression and deletion of Ink4a-arf, SVZ neurospheres were

transduced in vitro with adenovirus-Cre-GFP (Vector Biolabs) for 12-18 hours. AZD6244

and everolimus were obtained from Selleckchem (Houston, TX) and PLX4720 from

Plexxikon Inc. (Berkeley, CA).

Cell Viability Assay




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Cell viability was measured by CellTiter-Glo Luminescent Cell Viability Assay

(Promega, Madison, WI) per the manufacturer’s guidelines. Assays were performed after

72 hours of treatment with each inhibitor or combination. Each experiment was set up in

six replicates and repeated at least three times. Measurements were normalized to DMSO

control. Pairwise comparisons were determined by Student’s t-test using GraphPad Prism

6.0 Software (GraphPad Software, La Jolla, CA). To evaluate drug synergy, combinatorial

index (CI) values were calculated using the software CalcuSyn (Biosoft Inc, UK) (20). CI

< 1 signifies synergistic effect, CI = 1 indicates additive effect, and CI > 1 defines drug

antagonism.

Cell cycle analysis

Cell cycle distributions were determined using flow cytometry following staining

with BrdU and 7-amino-actinomycin D (7-AAD). Cells were grown exponentially in

appropriate media containing 10% FBS or as specifically indicated. Subsequently cells

were treated with different inhibitors, fixed and stained 24 hours after treatment according

to the manufacturer’s instructions (BD Pharmingen FITC BrdU Flow Kit). For

combination treatments, both compounds were administered concurrently. Fluorescence

was measured on a FAC-sort flow cytometer (FACSCalibur, Becton Dickson), and data

analyzed using FlowJo (TreeStar, Inc). All measurements are represented as an average of

at least four replicates. Error bars represent standard error. Statistical significance (p <

0.05) was determined with a one-way analysis of variance (ANOVA) and Bonferroni’s

multiple comparisons correction using GraphPad Prism 6.0 Software (GraphPad Software,

La Jolla, CA).

Annexin-V cytometric analysis




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Proportions of apoptotic cells were quantified by PI/FITC Annexin V flow

cytometry (BD Bioscience, San Jose, CA), 72 hours after treatment. Samples were

processed and stained as described in the manufacturer’s protocol. All measurements

represent an average of at least four replicates. FITC fluorescence was measured on a

FAC-sort flow cytometer (Becton Dickson) and data analyzed using FlowJo (TreeStar,

Inc).

Western blot analysis

To assess modulation of pathway specific signaling molecules, we performed

western blot analyses. Total protein extracts from cells were prepared using cell lysis

buffer (50 mM HEPES pH 7.0, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 0.5%

sodium deoxycholate, 1% NP-40, 1.5 mM MgCl2, and 10 mM EDTA) containing a

complete protease and phosphatase inhibitor cocktail (Roche Diagnostics Corporation,

Indianapolis, IN). Protein lysates were separated by SDS-PAGE and transferred to

polyvinyllidene difluoride membranes, then incubated with antibodies against p-Akt, total

Akt, p-MEK1/2, p-Erk, p-S6, total S6, β-actin (Cell Signaling,Beverly, MA). Band

intensities were visualized by ECL western detection reagent (GE Healthcare, Little

Chalfont, Buckinghamshire, UK).

Immunohistochemistry of tumor samples

For immunohistochemical analyses of in vivo drug responses, nude mice harboring

subcutaneous BT40 (BRAFV600E) xenografts were treated for 10 consecutive days. Tumors

were harvested one hour after the last treatment, fixed in 4% paraformaldehyde overnight

and processed through ethanol dehydration series prior to paraffin embedding. Five μm

sections were cut, rehydrated with decreasing ethanol concentrations, and hybridized




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using anti-Ki67 (Dako, Carpinteria, CA), p-S6, p-Erk (R&D Systems, Minneapolis, MN),

detected with DAB substrate (Vector Labs, CA) and counterstained with hematoxylin.

Image-based quantification was performed for each stain. Five 20x pictures were taken per

tumor (Leica, DMLS microscope), with positively stained cells scored both manually and

by the ImageJ based ImmunoRatio plugin (Tampere, Finland).

Animals and surgical procedures

The UCSF Institutional Animal Care and Use Committee approved all animal

protocols. Four-six-week-old female NOD SCID mice were purchased from Jackson

Laboratories (Maine, USA). Animals were housed under aseptic conditions. BT40 tumor

cells were implanted into the flank of mice as described previously (21). For each

condition 10 mice were injected but not all animals developed tumors. For the final

analysis the following numbers of animals were included: control n=6, AZD6244 n=7,

everolimus n=7, PLX4720 n=7, everolimus + PLX4720=9, AZD6244 + PLX4720=7, and

AZD6244 + everolmius n=7.

Treatments were initiated when tumors reached 100-200 mm3 in size. Tumor

bearing mice were randomized to vehicle control (OraPlus: Paddock Laboratories),

AZD6244, everolimus, PLX4720 or combinations thereof. Mice were treated for a total of

10 days (Mo-Fri) with the following dosing schedule: 10 mg/kg of AZD6244 and/or 10

mg/kg everolimus once daily by oral administration and/or 10 mg/kg PLX4720 once daily

by intraperitoneal (ip) administration. For combination treatment inhibitors were given

concurrently. All mice were examined daily for the development of symptoms related to

tumor growth. Mice were euthanized when they exhibited symptoms indicative of

significant impairment. In addition to the mice used for survival analyses, mice from each




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cohort were sacrificed one hour after completion of the treatment, with tumors resected

and placed in 4% paraformaldehyde (PFA), then processed for IHC analysis.

The Kaplan-Meier estimator was used to generate survival curves and differences

between survival curves were calculated using a log-rank test. Differences between

bioluminescence growth curves were compared using a 2-tailed unpaired t-test using

Prism software (GraphPad Software, San Diego, CA, USA). Results

Combination therapy in BRAFV600E-mutant human glioma cells enhances cell cycle

arrest and apoptosis and reduces proliferation compared to monotherapy.

We assessed effects of MEK1/2- (AZD6244), mTOR- (everolimus) or BRAFV600E-

inhibitions (PLX4720) as monotherapy as well as in combination in BRAFV600E mutated

glioma cells DBTRG and AM-38. As shown in Figure 1A-C (DBTRG) and D-F (AM-38),

the three possible doublet combinations of AZD6244, everolimus and PLX4720, led to

significant decreases in cell proliferation compared to the respective single agents. The

combination index (CI) theorem of Chou-Talalay offers quantitative definitions for

additive effect (CI=1), synergism (CI<1), and antagonism (CI>1) in drug combinations

and demonstrated CI<1 for all tested BRAFV600E glioma lines when PLX4720 was

combined with AZD6244 (Supplemental Figure 1A, B). Similarly, the most pronounced

induction of apoptosis was seen after combined MEK1/2 and BRAFV600E inhibition

(Figure 1C, Supplemental Figure 2A-C). All three combinations performed equally well in

reducing S and G2 phase accumulations; of note, PLX4720 was extremely effective as a

single agent in arresting cell cycle progression and therefore cell cycle effects were not

significantly enhanced when everolimus or AZD6244 was added to PLX4720 (Figure 1A-




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C, Supplemental Figure 2A-C). Western blot analyses revealed that treatment with

everolimus significantly reduced p-S6. Treatment with PLX4720 or AZD6244 reduced

expression of p-ERK, although AZD6244 was more effective in down-regulating p-ERK.

PLX4720 reduced p-ERK at 1 hour with a rebound in expression at 6 hours, a rebound

that was prevented by the addition of AZD6244 to PLX4720. Treatment of BRAFV600E-

mutant DBTRG cells with all examined single agents and combinations led to up-

regulation of p-AKT (Figure 1G). Similar western blot results were found in BRAFV600E-

mutant AM-38 cells (data not shown).

Combination therapy in BRAFWT human glioma cells enhances cell cycle arrest and

apoptosis and reduces proliferation compared to monotherapy.

We next turned to examining responses in BRAFWT-PLGGs by assessing

proliferation, apoptosis and cell cycle distribution in SF188. We did not assess PLX4720

in BRAFWT cells since this would not be clinically relevant due to the known paradoxical

activation of the MAPK pathway in BRAFWT tumor cells (22-24). As shown in Figure 2A

combined inhibition of MEK1/2 and mTOR decreased cell proliferation significantly more

than single pathway inhibition. Similar results were also seen in SF8628 (Figure 2B) and

SF9427 BRAFWT glioma cells (Figure 2C). The combinatorial index calculation indicates

strong synergy (CI<1) in the examined BRAFWT cells SF188 and SF9427 (Supplemental

Figure 3 A, B). In addition, SF188 cells treated with AZD6244 + everolimus exhibited

significantly greater apoptosis compared to single agent treatments (Figure 2D), a finding

documented in SF8628 cells as well (Supplemental Figure 4A). In addition to

combinatorial effects on apoptosis and proliferation, we observed decreased S and G2




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phase accumulation in SF188 and SF8628 cells treated with combination of AZD6244 +

everolimus compared to the respective single agent treated cells (Figure 2E, Supplemental

Figure 4B). Western blot analyses (Figure 2F) revealed expected pathway modulations,

including down-regulation of p-ERK by AZD6244, reduction in p-S6 by everolimus, and

up-regulation of p-Akt by AZD6244, everolimus and their combination.

Combination therapy in BRAFV600E and BRAFWT isogenic mouse cells is superior to

single agent treatments.

To confirm our results that combination treatment is superior to single agents in

BRAFV600E and BRAFWT cells, we used an isogenic system of murine brain tumors

differing only in BRAFV600E-status. In this BRAFV600E-isogenic system, BRAFV600E-

gliomas were as sensitive to everolimus as BRAFWT cells, but exquisitely more sensitive

to AZD6244 than BRAFWT cells (Figure 3). Comparing inhibitor combinations, each of

the three combinations was superior to single agents in reducing cell proliferation in

BRAFV600E-mutated cells (Figure 3A). Similar to our findings in BRAFV600E-mutated

DBTRG cells (Figure 1), the combination of PLX4720 + AZD6244 was superior to the

other two doublet combinations, although AZD6244 + everolimus also showed marked

combinatorial activity. The combination index of PLX4720 + AZD6244 was indicative of

strong synergism at all dose levels. As expected, western blot analyses in BRAFV600E-

mutated cells revealed down-regulation of p-ERK by AZD6244 or PLX4720 and down-

regulation of p-S6 at 6 hours by everolimus (Figure 3B). In BRAFWT murine glioma cells,

combination of AZD6244 + everolimus enhanced anti-proliferative effects compared to

single agents (Figure 3C), with CI values below one, indicating synergistic effects of this




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combinatorial approach (data not shown). Western blot analyses revealed expected down-

regulation of p-ERK by AZD6244 and of p-S6 by everolimus (Figure 3D) and only the

combination treatment effectively inhibited both the MAPK and mTOR pathways.

NIH/3T3 cells carrying the KIAA1549:BRAF fusion protein are highly sensitive to

MEK1/2 inhibition compared to isogenic BRAFWT-expressing cells.

We next turned to examining responses in BRAF-fusion cells. In contrast to

BRAFWT and BRAFV600E, no patient-derived LGG lines expressing BRAF-fusions have

been successfully developed to permit preclinical testing. This is despite significant efforts

to grow primary cells in culture by numerous laboratories including our own. To address

this, we recently generated a set of BRAF-fusion heterologous expression systems

consisting of NIH/3T3 expressing BRAFWT or the most common BRAF alteration in

PLGGs, the KIAA1549:BRAF fusion protein (Figure 4). Under 10% serum conditions,

MEK1/2 and/or mTOR inhibition produced similar decreases in cell proliferation in cells

carrying the KIAA1549:BRAF fusion protein compared to BRAFWT (Figure 4A). In

contrast, BRAFWT cells showed minimal change in cell cycle distribution in response to

MEK1/2-inhibition, mTOR-inhibition or the combination, whereas the KIAA1549:BRAF

cells showed significant reduction in S and G2 phase after MEK1/2 inhibition but not after

mTOR inhibition (Figure 4B). Western blot analyses showed no difference in basal p-

ERK and p-S6 levels between KIAA1549:BRAF and BRAFWT cells (Figure 4C, D), as

growth factors present in full serum media conditions cause persistent activation of wild-

type BRAF.




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Low serum conditions (0.5% FBS) augmented disparate activity of AZD6244 on

KIAA1549:BRAF- and BRAFWT-cells, demonstrating exquisite anti-proliferative effects of

single agent MEK1/2 inhibition on BRAF-fusion cells (Figure 4E). Growth in low serum

led to expected accumulation of cells in G1 phase that was further augmented by MEK1/2

inhibition in KIAA1549:BRAF fusion cells but not BRAFWT-cells (Figure 4F).

Western Blot analyses revealed that when grown in low serum, BRAFWT cells had

lower basal levels of p-ERK and p-S6 (Figure 4G) than KIAA1549:BRAF fusion cells

(Figure 4H). This difference was not seen when cells were grown in full serum conditions

as growth factors cause persistent activation of wild-type BRAF (Figure 4C, D), masking

differences between the isogenic lines. In KIAA1549:BRAF cells, regardless of serum

levels, AZD6244 reduced p-ERK and everolimus decreased p-S6, but complete inhibition

of both the MAPK and mTOR pathways was evident only in the combined treatment arm

(Figure 4D, H).

In vivo Efficacy of Combination Therapy in BRAFV600E Model of PLGG

To address the in vivo efficacy of combinations of PLX4720, everolimus and

AZD6244 we used the BRAFV600E-PLGG model BT40. Mice treated with a combination of

AZD6244 plus everolimus or PLX4720 showed significantly prolonged survival and

tumor volume reduction compared to single agent treatment, with AZD6244 + PLX4720

appearing superior to AZD6244 + everolimus (Figure 5A-C).

As observed in our in vitro studies, we found reduced expression of Ki67, a marker

of proliferation, in xenografts from mice treated with everolimus, AZD6244, PLX4720 or

combinations thereof, with the greatest reduction of Ki67 documented in mice treated with




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the combination of PLX4720 + AZD6244 (Figure 5D, E). Xenografts treated with

everolimus showed reduced expression of p-S6, as expected (Figure 5D, F). Treatment

with AZD6244 and PLX4720 reduced p-ERK expression in xenografts, as anticipated, an

effect that was significantly more pronounced after AZD6244 than after PLX4720

treatment (Figure 5D, G).

Discussion

In this manuscript we provide the preclinical rationale for targeted therapy options

based on the most commonly found genetic alterations in PLGG. Our studies indicate that

combination therapies with PLX4720, everolimus or AZD6244 for PLGGs carrying the

BRAFV600E mutation or BRAFWT are superior to single agent therapy. We further

demonstrate that the KIAA1549:BRAF fusion protein renders cells highly sensitive to

MEK1/2 inhibition and combination therapy is marginally superior compared to single

agent therapy.

Although BRAFV600E-specific inhibitors such as vemurafenib and dabrafenib have

only recently entered clinical practice for children whose tumors carry this mutation,

initial reports are encouraging (25-27). Case reports indicate that vemurafenib is highly

effective and have led to complete regression in BRAFV600E-expressing pediatric high-

grade gliomas (26). However, based on our experience and that of others, we also know

that some patients progress on single agent therapy with BRAFV600E inhibitors. Feedback

activation of EGFR and downstream effectors has been proposed as a potential escape

mechanism, further supporting combination strategies that address the MAPK as well as

the PI3K pathways (28, 29). Furthermore, combination of MEK- and BRAFV600E-




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inhibition improves tolerability with decreased frequency of squamous cell carcinomas, a

rare but serious side effect of vemurafenib (30).

In all our model systems of BRAFV600E mutated gliomas [isogenic murine,

BRAFV600E PLGG (BT40) and BRAFV600E adult high-grade glioma (DBTRG, AM-38)],

whether in vitro or in vivo, treatments with the three possible doublet combinations of

AZD6244, everolimus and PLX4720 were superior to their respective mono-therapies,

with PLX4720 + AZD6244 appearing the superior combination overall, showing

consistently reduced cell viability, increased apoptosis and cell cycle arrest in vitro and

most prominently prolonged survival and reduced tumor growth in vivo. In the in vitro

models, rebound in p-ERK expression after 6 hours of PLX4720 treatment was completely

obliterated by the addition of AZD6244 to PLX4720. These results, in addition to already

published data, provide a strong clinical rationale for combination therapies in BRAFV600E

positive gliomas. One limitation of our in vivo studies is the use of a flank model rather

than an orthotopic model. However, BT40, the only available pediatric model that carries

the BRAFV600E mutation, only grows as a flank tumor and there are no intracranial models

currently available to study PLGG. Planned clinical trials will test the combination of

BRAFV600E-specific inhibition in combination with MEK inhibition in pediatric patients

(NCT02124772).

In examining responses in BRAFWT pediatric high-grade gliomas, we show that the

combination of AZD6244 + everolimus was superior to either single agent therapy, as

assessed by cell proliferation, cell cycle arrest and induction of apoptosis. In comparison

with BRAFV600E cells, BRAFWT lines had reduced sensitivity to MEK inhibitor

monotherapy consistent with previous reports (31, 32). We have shown that treatment




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with everolimus leads to activation of the MAPK pathway; most likely through a PI3K-

dependent feedback loop. This has been shown in previously published studies and

provides a strong rationale for combination therapy using MEK- and mTOR-inhibitors for

BRAFWT gliomas (33, 34). Ongoing studies are assessing the tolerability of the

combination of such dual inhibition in adult patients with advanced cancers

(NCT01347866) and studies in pediatric patients are underway.

In contrast to existing and available BRAFWT and BRAFV600E pre-clinical models,

no patient derived glioma cells lines expressing BRAF-fusions have been successfully

established for preclinical studies. To circumvent this limitation we used a BRAF-fusion

heterologous expression system in NIH/3T3 cells that we described previously (17). We

have shown in vivo and in vitro that BRAF-fusion expressing lines display robust

resistance to and enhanced paradoxical activation by PLX4720 (17). Importantly, these

models predict the clinical responses to BRAF targeting, as a recent phase 2 study found

acceleration of PLGGs when treated with sorafenib, leading to early closure of the trial

(35). Herein we found that BRAF-fusion cells are highly sensitive to single agent MEK1/2

inhibition with a small benefit derived from combining MEK1/2 and mTOR inhibition.

Given the observed large sensitivity to MEK1/2 inhibition alone, single agent activity may

be sufficient to successfully inhibit growth of such tumors. Targeted therapy

recommendations such as the one proposed herein, will increase the need for tissue

acquisition even for tumors such as optic pathway gliomas, a tumor type that classically

has not been biopsied. The additional risk of biopsy might be justified if therapy

recommendations would change based on the individual tumor characteristics. Several




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studies have shown that tissue acquisition is feasible and relatively safe, even for optic

pathway gliomas (35, 36).

Conclusion

Using model systems of the most common PLGG BRAF alterations, we provide the

preclinical rationale for therapeutic approaches for each genotype. Our results support

treatment of BRAFV600E positive PLGGs with a combination of PLX4720 + AZD6244,

although AZD6244 + everolimus also shows marked combinatorial activity. BRAFWT

gliomas appear to benefit from the combination of mTOR and MEK inhibition. Our

studies are consistent with clinical reports of sensitivity of KIAA1549:BRAF fusion

gliomas to MEK1/2 inhibition as a single agent.




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14. Mueller S, Phillips J, Onar-Thomas A, Romero E, Zheng S, Wiencke JK, et al. PTEN promoter methylation and activation of the PI3K/Akt/mTOR pathway in pediatric gliomas and influence on clinical outcome. Neuro-oncology. 2012;14:1146-52. 15. Kaul A, Chen YH, Emnett RJ, Dahiya S, Gutmann DH. Pediatric glioma-associated KIAA1549:BRAF expression regulates neuroglial cell growth in a cell type-specific and mTOR-dependent manner. Genes & development. 2012. 16. Mueller S, Hashizume R, Yang X, Kolkowitz I, Olow AK, Phillips J, et al. Targeting Wee1 for the treatment of pediatric high-grade gliomas. Neuro-oncology. 2014;16:352-60. 17. Sievert AJ, Lang SS, Boucher KL, Madsen PJ, Slaunwhite E, Choudhari N, et al. Paradoxical activation and RAF inhibitor resistance of BRAF protein kinase fusions characterizing pediatric astrocytomas. Proceedings of the National Academy of Sciences of the United States of America. 2013;110:5957-62. 18. Huillard E, Hashizume R, Phillips JJ, Griveau A, Ihrie RA, Aoki Y, et al. Cooperative interactions of BRAFV600E kinase and CDKN2A locus deficiency in pediatric malignant astrocytoma as a basis for rational therapy. Proceedings of the National Academy of Sciences of the United States of America. 2012;109:8710-5. 19. Sugiarto S, Persson AI, Munoz EG, Waldhuber M, Lamagna C, Andor N, et al. Asymmetry-defective oligodendrocyte progenitors are glioma precursors. Cancer cell. 2011;20:328-40.




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28. Nazarian R, Shi H, Wang Q, Kong X, Koya RC, Lee H, et al. Melanomas acquire resistance to B-RAF(V600E) inhibition by RTK or N-RAS upregulation. Nature. 2010;468:973-7. 29. Poulikakos PI, Persaud Y, Janakiraman M, Kong X, Ng C, Moriceau G, et al. RAF inhibitor resistance is mediated by dimerization of aberrantly spliced BRAF(V600E). Nature. 2011;480:387-90. 30. Flaherty KT, Infante JR, Daud A, Gonzalez R, Kefford RF, Sosman J, et al. Combined BRAF and MEK inhibition in melanoma with BRAF V600 mutations. The New England journal of medicine. 2012;367:1694-703. 31. Davies H, Bignell GR, Cox C, Stephens P, Edkins S, Clegg S, et al. Mutations of the BRAF gene in human cancer. Nature. 2002;417:949-54. 32. Solit DB, Garraway LA, Pratilas CA, Sawai A, Getz G, Basso A, et al. BRAF mutation predicts sensitivity to MEK inhibition. Nature. 2006;439:358-62. 33. Carracedo A, Ma L, Teruya-Feldstein J, Rojo F, Salmena L, Alimonti A, et al. Inhibition of mTORC1 leads to MAPK pathway activation through a PI3K-dependent feedback loop in human cancer. J Clin Invest. 2008;118:3065-74. 34. Chen XG, Liu F, Song XF, Wang ZH, Dong ZQ, Hu ZQ, et al. Rapamycin regulates Akt and ERK phosphorylation through mTORC1 and mTORC2 signaling pathways. Molecular carcinogenesis. 2010;49:603-10. 35. Karajannis MA, Legault G, Fisher MJ, Milla SS, Cohen KJ, Wisoff JH, et al. Phase II study of sorafenib in children with recurrent or progressive low-grade astrocytomas. Neuro-oncology. 2014.




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Figure Legends

Figure 1: In Vitro Response to Co-inhibition of MAPK and mTOR pathways in

BRAFV600E mutated DBTRG and AM-38 Glioma Cells

Combination of (A) AZD6244 + everolimus, (B) PLX4720 + everolimus and (C)

AZD6244 + PLX4720 in DBTRG cells. For each combination we display results of cell

viability, apoptosis and cell cycle distribution (left to right). For cell viability, means of six

replicates with standard errors are displayed. Measurements are normalized to DMSO

control. Cell viability was assessed 72 hours after treatment. Pairwise comparisons were

determined by Student’s t-test; * marks p-value ≤ 0.05; ** p-value ≤ 0.01, *** p-value ≤

0.001, and **** p-value 0.0001. Apoptosis was measured by flow cytometry. The

proportions of apoptotic cells were quantified 72 hours after treatment. All data points

represent averages of at least four replicates. Statistical analysis include ANOVA and

Bonferroni multiple comparisons. Cell cycle analyses were performed 24 hours after

treatment and data points represent averages of four replicates. Bar graphs display

distribution of cells within each cell cycle phase. Error bar indicate standard error. (D-F)

Corresponding cell viability measurements in AM-38 for (D) AZD6244 + everolimus, (E)

PLX4720 + everolimus and (F) AZD6244 + PLX4720. (G) Western blot analyses of p-

Akt, p-ERK and p-S6 in BRAFV600E human glioma cells (DBTRG). β-Actin was used as

loading control. Levels of p-Akt, p-ERK and p-S6 were determined 1 and 6 hours after

treatment. For all of the above results, in samples receiving combination therapy,

inhibitors were administered simultaneously.




29


BRAFWT Glioma Cells

(A-C) Cell viability responses to AZD6244 + everolimus assessed in (A) SF188

BRAFWT, (B) SF8628 BRAFWT and (C) SF9427 BRAFWT. For cell viability means of six

replicates with standard errors are displayed. Measurements are normalized to DMSO

control. Cell viability was assessed 72 hours after treatment. Pairwise comparisons were

determined by Student’s t-test; * marks p-value ≤ 0.05, ** p-value ≤ 0.01, *** p-value ≤

0.001, and **** p-value ≤ 0.0001. (D) Apoptotic response of SF188 BRAFWT cells to

combined inhibition of AZD6244 + everolimus. Apoptosis was measured by flow

cytometry. The proportions of apoptotic cells were quantified 72 hours after treatment. All

data points represent averages of at least four replicates. Statistical analysis includes

ANOVA and Bonferroni multiple comparisons. (E) Cell cycle distribution of SF188

BRAFWT cells in response to combined inhibition of AZD6244 + everolimus. Analyses

were performed 24 hours after treatment and depicted are averages of four replicates. Bar

graphs display distribution of cells within each cell cycle phase. Error bars indicate

standard error. (F) Western blot analyses of p-Akt, p-ERK and p-S6 in SF188 BRAFWT

cells. β-actin was used as loading control. Levels of p-Akt, p-ERK and p-S6 were

determined 1 and 6 hours after treatment. For all of the above results, in samples receiving

combination therapy, inhibitors were administered simultaneously.


Isogenic Mouse BRAFV600E and BRAFWT Cells




30

Assessments of isogenic murine (A-B) BRAFV600E and (C-D) BRAFWT cells in response

to treatment with AZD6244 + everolimus, everolimus + PLX4720 or AZD6244 +

PLX4720. (A, C) Represent cell viability data assessed 72 hours after treatment. Means of

six replicates with standard errors are displayed. Measurements are normalized to DMSO

controls. (B, D) Western blot analyses of p-Akt, p-ERK and p-S6 in isogenic murine (B)

BRAFV600E and (D) BRAFWT cells. β-actin was used as loading control. Levels of p-Akt,

p-ERK and p-S6 were determined 1 and 6 hours after treatment. For all of the above

results, in samples receiving combination therapy, inhibitors were administered

simultaneously.


NIH/3T3 BRAFWT and KIAA1549:BRAF fusion cells.

(A, E) Cell viability in BRAFWT and KIAA1549:BRAF-expressing NIH/3T3 cells in

response to AZD6244, everolimus or combination thereof under (A) high (10% DBS) and

(E) low (0.5% DBS) serum conditions. Means of six replicates with standard error are

displayed. Measurements are normalized to DMSO control. Cell viability was assessed 72

hours after treatment. (B, F) Cell cycle distribution of BRAFWT and KIAA1549:BRAF-

expressing NIH/3T3 cells after treatment with AZD6244 and/or everolimus under (B) high

(10%) and (F) low (0.5%) serum condition. Cell cycle analyses were performed 24 hours

after treatment and depicted are averages of four experiments. Bar graphs display

distributions of cells within each cell cycle phase. Error bars indicate standard error.

Western blot analyses of p-Akt, p-ERK and p-S6 in (C, G) NIH/3T3 BRAFWT cells and

(D, H) KIAA1549:BRAF-expressing NIH/3T3 cells after treatment with AZD6244,




31

everolimus or combination thereof. β-actin was used as loading control. Levels of p-Akt,

p-ERK and p-S6 were determined 1 and 6 hours after treatment. For all of the above

results, in samples receiving combination therapy, inhibitors were administered

simultaneously.

Figure 5: In Vivo Response to Co-inhibition of MAPK and mTOR pathways in

BRAFV600E Pediatric Pilocytic Astrocytoma (BT40) Model

BT40 xenografts were treated with AZD6244, everolimus, PLX4720, or combinations

thereof. For each treatment group 7 animals were included in the analysis except for

control (n=6), and everolimus+PLX4720 (n=9). Animals were treated for a total of 10

days (Mo-Fri) with 10 mg/kg AZD6244 or everolimus by oral gavage once daily and/or

10 mg/kg PLX4720 administered by intraperitoneal injection. For combination therapies

inhibitors were given concurrently. (A) Tumor volumes on day 27; (B) Median survival

(days) and (C) Kaplan-Meier survival curves stratified by treatment group. For the

immunohistochemical analyses, 2 mice were sacrificed 1 hour after completion of therapy.

(D) IHC results for Ki67, p-ERK and p-S6 in above xenografts from mice treated with

either monotherapy or combination therapy, (E-G) Boxplots of IHC results for Ki67 (E),

pS6 (F) and pERK (G).




Figure 1

A

B

C

G

Hour c 1h 6h 1h 6h 1h 6h 1h 6h 1h 6h 1h 6h

pERK

β-Actin

p-Akt (S473)

p-S6 (S240/244)

c Everolimus (4nM)

PLX4720 (5µM)

AZD6244 Everolimus

AZD6244 (1uM)

Everolimus PLX4720

AZD6244 PLX4720

DBTRG (BRAFV600E)

D E F

AM-38 (BRAFV600E)




Figure 2

A

Hour c 1h 6h 1h 6h 1h 6h

pERK

β-Actin

p-Akt (S473)

p-S6 (S240/244)

c Everolimus (4nM)

AZD6244 Everolimus

AZD6244 (1uM)

SF188 (BRAFWT)

B

C

F

D E

SF8628 (BRAFWT)

SF9427 (BRAFWT)

SF188 (BRAFWT)




A C

BRAF WT

Figure 3

BRAFV600E

p-Akt (S473)

p-S6 (S240/244)

p-ERK

β-Actin

Hour c 1h 6h 1h 6h 1h 6h 1h 6h 1h 6h 1h 6h

B c Everolimus

(4nM)

PLX4720 (5µM)

AZD6244 Everolimus

AZD6244 (1uM)

Everolimus PLX4720

AZD6244 PLX4720

BRAF V600E

D BRAFWT


p-Akt (S473)

p-S6 (S240/244)

p-ERK

β-Actin

c Everolimus (4nM)

AZD6244 Everolimus

AZD6244 (1uM)




A

Figure 4

KIAA1549: BRAF BRAF WT B

10

% D

BS

KIAA1549: BRAF BRAF WT

C D KIAA1549: BRAF BRAF WT

0.5

% D

BS

E F

G H KIAA1549: BRAF BRAF WT

pERK

β-Actin

p-Akt (S473)

p-S6 (S240/244)

KIAA1549: BRAF BRAF WT KIAA1549: BRAF BRAF WT


c Everolimus (4nM)

AZD6244 Everolimus

AZD6244 (1uM)

pERK

β-Actin

p-Akt (S473)

p-S6 (S240/244)


c Everolimus (4nM)

AZD6244 Everolimus

AZD6244 (1uM)


c Everolimus (4nM)

AZD6244 Everolimus

AZD6244 (1uM)


c Everolimus (4nM)

AZD6244 Everolimus

AZD6244 (1uM)

pERK

β-Actin

p-Akt (S473)

p-S6 (S240/244)

pERK

β-Actin

p-Akt (S473)

p-S6 (S240/244)




Figure 5

A

E

B C

Ki67 pS6 pERK F G

Control PLX4720 Everolimus PLX4720 + Everolimus

AZD6244 PLX4720 + AZD6244

Everolimus + AZD6244

Ki6

7

pER

K

p-S

6

(S2

40

/24

4)

D




Published OnlineFirst May 23, 2016.Clin Cancer Res Aleksandra Olow, Sabine Mueller, Xiaodong Yang, et al. pediatric gliomasBRAF status in personalizing treatment approaches for

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