Dual MAPK Inhibition Is an Effective Therapeutic Strategy for a Subset of Class II BRAF

Home , Binimetinib

Author Manuscript Published OnlineFirst on June 14, 2018; DOI: 10.1158/1078-0432.CCR-17-3384 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Dual MAPK inhibition is an effective therapeutic strategy for a subset of class II BRAF

mutant melanomas

Matthew Dankner1,2, Mathieu Lajoie1,3, Dan Moldoveanu1,4, Tan-Trieu Nguyen 1,3, Paul

Savage1,2, Shivshankari Rajkumar1,3, Xiu Huang7, Maria Lvova7, Alexei Protopopov7, Dana

Vuzman7,8,9, David Hogg 10, Morag Park1,2,3, Marie-Christine Guiot5,6, Kevin Petrecca5, Catalin

Mihalcioiu11, Ian R. Watson1,3, Peter M. Siegel1,2,3, April A. N. Rose 12,*

1Goodman Cancer Research Centre, Departments of 2Medicine, 3Biochemistry, 4General Surgery, 5Neurology and Neurosurgery, and 6Pathology, McGill University, Montréal, Québec, Canada, 7KEW Inc., Cambridge, Massachusetts, USA, 8Division of Genetics, Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts, USA. 9Broad Institute of Harvard and MIT, Cambridge, Massachusetts, USA. 10Princess Margaret Cancer Centre, Toronto, Ontario, Canada, 11McGill University Health Centre, McGill University, Montréal, Québec, Canada, 12Department of Medicine, Division of Medical

Oncology, University of Toronto, Toronto, Ontario, Canada.

* Corresponding Author: University of Toronto

Princess Margaret Cancer Center, OPG Building

700 University Avenue

Work Station 7W460

Toronto, Ontario, Canada

M5G 1Z5

E-mail: [email protected]

Keywords: BRAF, MEK, MAPK, Class II mutations, L597, Melanoma, Encorafenib,

Dabrafenib, PDX, Brain Metastasis, non-V600

Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on June 14, 2018; DOI: 10.1158/1078-0432.CCR-17-3384 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Running Title: Dual MAPK inhibition for class II BRAF mutations

Disclosure of Potential Conflicts of Interest: XH, ML, AP and DV are employed by a commercial diagnostic company, KEW Inc.

Word count: (excluding abstract, translational relevance, figure legends and references): 5048

Abstract word count: 250

Translational relevance: 99

Total number of figures/tables: 6

Supplemental figures/tables: 6 figures, 2 tables

Abstract

Background: Dual MAPK pathway inhibition (dMAPKi) with BRAF and MEK inhibitors improves survival in BRAF V600E/K mutant melanoma, but the efficacy of dMAPKi in non-

V600 BRAF mutant tumors is poorly understood. We sought to characterize the responsiveness

of class II (enhanced kinase activity, dimerization dependent) BRAF mutant melanoma to

dMAPKi.

Methods: Tumors from patients with BRAF WT, V600E (class I) and L597S (class II)

metastatic melanoma were used to generate patient-derived xenografts (PDX). We assembled a

panel of melanoma cell lines with class IIa (activation segment) or IIb (p-loop) mutations and

compared these to wild-type or V600E/K BRAF mutant cells. Cell lines and PDXs were treated

with BRAFi (vemurafenib, dabrafenib, encorafenib, LY3009120), MEKi (cobimetinib,

trametinib, binimetinib) or the combination. We identified two patients with BRAF L597S

metastatic melanoma who were treated with dMAPKi.

Results: BRAFi impaired MAPK signaling and cell growth in class I and II BRAF mutant cells.

dMAPKi was more effective than either single MAPKi at inhibiting cell growth in all class II

BRAF mutant cells tested. dMAPKi caused tumor regression in two melanoma PDXs with class

II BRAF mutations, and prolonged survival of mice with class II BRAF mutant melanoma brain

metastases. Two patients with BRAF L597S mutant melanoma clinically responded to dMAPKi.

Conclusions: Class II BRAF mutant melanoma are growth inhibited by dMAPKi. Responses to

dMAPKi have been observed in two patients with class II BRAF mutant melanoma. This data

provides rationale for clinical investigation of dMAPKi in patients with class II BRAF mutant metastatic melanoma.

Translational Relevance:

Class II BRAF mutations are commonly recurring mutations that confer enhanced BRAF activity and MAPK pathway hyper-activation akin to class I (V600E/K) mutations. In this study, we employ various melanoma cell lines and PDX models that endogenously express class II BRAF mutations to demonstrate that these tumors are indeed sensitive to targeted therapy with dual

BRAF+MEK inhibition. Furthermore, we present data on two melanoma patients with class II

BRAF mutations that achieved objective clinical responses to BRAF + MEK inhibition. This represents a viable therapeutic strategy for this emerging subgroup of patients and warrants further investigation in clinical trials.

Introduction

BRAF is a constituent of the mitogen-activated protein kinase (MAPK) signaling pathway and is

one of the most commonly mutated oncogenes in human tumors [1]. The most prevalent BRAF

mutations occur at codon V600, constitutively activating BRAF’s kinase domain and enhancing

MAPK signaling [2]. Given the importance of this hyper-activated pathway in cancer, several

MAPK inhibitors have been developed for targeted treatment of V600 BRAF mutant tumors,

including BRAF inhibitors (BRAFi; vemurafenib, dabrafenib and encorafenib), and MEK

inhibitors (MEKi; cobimetinib, trametinib and binimetinib) [3, 4]. BRAFi and MEKi used as

single agents, or in combination, have been shown to improve survival in BRAF V600 mutant

melanoma and non-small cell lung cancer (NSCLC) [4-6].

Data from large-scale sequencing efforts have identified many additional hotspot BRAF mutations existing outside of the V600 codon [1, 7]. Recently, a new classification system of

BRAF mutations has been proposed [8, 9]. V600 mutations are referred to as class I BRAF

mutations and signal constitutively as RAS-independent monomers. Class II mutations are also

BRAF-activating, but signal as RAS-independent dimers [9-11]. Herein we draw a distinction

between class II BRAF mutations based on their location; class IIa mutations occur within the

activation segment (i.e L597, K601), and class IIb mutations occur within the glycine rich p-loop

(i.e G464, G469) (Fig. 1A). Class III is comprised of “low activity” or kinase dead BRAF

mutations [9, 12].

It has been previously reported that only tumors with class I BRAF mutations are sensitive to

approved BRAFi [10]. However, several other studies report that cell lines endogenously

expressing non-V600 BRAF mutants are sensitive to BRAFi [13-15]. This evidence, combined

with case reports of patients with BRAF non-V600 expressing tumors responding to BRAFi

suggests that the established paradigm for non-V600 BRAF mutants may be incomplete [8, 13,

16, 17].

In this study, we employ cell lines, patient derived xenograft (PDX) models and report on clinical responses in two patients to demonstrate that dMAPKi with approved BRAFi + MEKi is an effective therapeutic strategy for some patients with class II BRAF mutant melanoma. These results provide the rationale for clinical trials to assess the efficacy of dMAPKi in these patients.

Materials and Methods

Sequencing of patient samples and PDX models

A next-generation sequencing-based test was performed by the CANCERPLEX assay [18]. The

CANCERPLEX data analysis pipeline was applied to report single nucleotide variants,

insertions, deletions, structural variants, and copy number variations. For each patient tumor, the

reported mutations in the primary metastatic tumor sample and the PDX sample were intersected

to identify common variants. Variant Allele Frequencies (VAF) were compared and plotted

using R (www.R-project.org). Variants of interest were manually reviewed in BAM files using

IGV [19].

Cell growth assays

For long term growth assays, cells were seeded into 12-well plates and treated with inhibitors at

the following concentrations for 10 and 15 day assays, respectively: vemurafenib (1500nM,

2000nM), dabrafenib (150nM, 300nM), encorafenib (150nM, 300nM), LY3009120 (100 nM),

cobimetinib (25nM) trametinib (5nM), binimetinib (50nM, 100nM). Media with drug was

replaced every 4-5 days. At experimental endpoint (10 days for Fig. 3A and Fig. S4C, 15 days

for Fig. 3B and 3C) cells were fixed in 10% formalin, incubated in crystal violet (Sigma-Aldrich,

Cat # HT90132-1L), and washed in water. Five representative images were taken of each well

and quantified using Scion Image Software. Positive pixel count was acquired from these

images, representing the area covered by tumor cells. Experiments were repeated in 3 wells per

experiment and performed in triplicate for a total of 9 wells.

In vivo experiments

For subcutaneous tumor growth experiments, 5 x 105 tumor cells were injected bilaterally. For

cranial tumor growth experiments, 1 x 105 tumor cells were injected into the right frontal lobe using a guide screw technique [20]. All in vivo subcutaneous and cranial PDX experiments were performed with passage 2 or earlier, or passage 5 or earlier, respectively. For subcutaneous xenografts, tumors were measured with calipers (ASICSA cat # 19600). For brain metastasis measurements, lesions were measured with IVIS Spectrum (Perkin Elmer). For each mouse prior to imaging, 50 µl of luciferin was injected intra-peritoneally. Quantification of signal intensity

was performed with Living Image software. For cranial injection experiments, treatment was

initiated when all mice exhibited clear detectable lesions by IVIS Spectrum imaging. Mice were

treated by daily oral gavage with vehicle of hydroxypropyl methylcellulose, trametinib (LC

Laboratories T-8123) at 0.5mg/kg mouse body weight, dabrafenib at 25 or 50 mg/kg, as

indicated in the figure legends (LC Laboratories D-5699), encorafenib (Array Biopharma) at 75

mg/kg, and binimetinib (Array Biopharma) at 15 mg/kg. All animal studies and protocols were

pre-approved by the McGill Comparative Medicine and Animal Resources Centre.

Patient information

Patient clinical information and tissue were received after obtaining written informed consent

from patients in accordance with the Declaration of Helsinki and after studies were approved by

an institutional review board.

Results

Classifying BRAF mutations in melanoma

To assess the prevalence of class II mutations across tumor types, we accessed the AACR

GENIE project [21]. Within this dataset, among tumor types with at least 5 BRAF mutant tumors present, the prevalence of BRAF mutations varied substantially across tumor types from 0.4% in breast cancer to 40.4% melanoma (Fig. 1B). Among melanoma samples, class I mutations

comprised 65.9% of all BRAF mutations, whereas class II and III comprised 11.4% and 9.5%,

respectively (Fig. 1C). A further 13.2% were mutants of unknown function that did not belong to any of the three classes. A similar distribution of class II and III BRAF mutations were observed in the TCGA melanoma dataset [22]. Class II mutations occurred within the activation segment

(i.e L597, K601; class IIa) and in the glycine-rich P-loop (i.e G464, G469; class IIb) (Fig. 1A).

An additional subset of class II mutations is comprised of BRAF fusions (class IIc) that have also

been reported to signal as RAS-independent BRAF dimers [10, 23, 24]. All non-V600 mutations

identified in the AACR GENIE dataset with known function are indicated in Table S1.

It has been reported that class III BRAF mutations are commonly associated with RAS mutations

in melanoma [9]. Indeed, we found that 47% of class III mutant melanoma within the GENIE

dataset co-expressed activating RAS mutations (Table S1, Fig. 1D). In contrast, we found that

class II mutant tumors were similar to class I mutant tumors, in that they rarely co-expressed

activating RAS mutations, (2.8% and 1.4%, respectively). This data supports the notion that

BRAF class II mutations, like class I mutations, are kinase activating in a RAS-independent manner.

In datasets published before the widespread approval of BRAFi and MEKi, melanoma patients with BRAF V600 mutations who did not receive MAPKi had worse prognosis than those with

BRAF wild-type (WT) tumors [25]. We asked whether melanoma patients with other, potentially targetable mutations also experienced poor prognosis. Indeed, metastatic melanoma patients with class II/III and/or NRAS mutations in the TCGA data set experienced inferior overall survival compared to patients with class I mutations (Fig. 1E). The improved survival of melanoma

patients with class I BRAF mutations due to the development of targeted therapies highlights the

need for the identification of similarly effective targeted therapy strategies for patients with class

II/III BRAF mutant and NRAS mutant melanoma [4].

Development and characterization of WT, class I and class II BRAF mutant PDX models

We established patient derived xenografts (PDXs) from four patients with metastatic melanoma,

including two with class II BRAF mutations (both BRAF L597S) (Fig. 2A). All PDXs retained similar genomic landscapes compared to the tumor from which they were derived. Genomic

analyses included copy number alterations (CNA) (Fig. 2B, Fig. S1A), somatic missense variants

(Fig. 2C,D) and variant allele frequencies (VAF) (Fig. S1b). An exception to this trend was the

expected discrepancy in the CNA and VAF between the clinical specimen and GCRC2073 PDX

(Fig. S1A,B). This was due to a low purity of the patient sample that can be seen in the

representative H&E from this specimen (Fig. 2E). Importantly, the known driver mutations that

result in gain (BRAF, NRAS, RET) or loss of function (PTEN, ARID2, CDKN2A), were

conserved in the corresponding PDX models (Fig. 2D). Immunohistochemical staining of three

PDX models and corresponding patient tissues revealed that PDXs maintain similar expression of melanoma markers (Melan-A, BRAF V600E, HMB-45) compared to their tumor of origin

(Fig. 2E). Taken together, these profiles demonstrate the high fidelity of these PDX model systems to the metastatic tumor from which they were derived.

We also obtained a variety of cancer cell lines bearing WT, class I mutant and class II mutant

BRAF. Among these cell lines and PDX models, co-occurring RAS mutations were present in

2/7 melanoma cell lines that expressed class II BRAF mutations (Table S2). This is consistent

with the notion that activating RAS mutations are commonly found in class III but less

frequently in class I or class II BRAF mutant melanomas (Fig. 1C) [9]. Both class II BRAF

mutant melanoma cell lines that co-expressed activating RAS mutations were of the class IIb

type. Class IIb activating mutations have been reported to enhance mutant BRAF:CRAF

dimerization [26], and therefore the presence of an activating RAS mutation may facilitate their

signaling capacity in this manner.

Class II BRAF mutant cancer cells respond to single agent BRAFi or MEKi

Clinically indicated BRAFi, such as vemurafenib and dabrafenib cause paradoxical activation of

the MAPK pathway in cells with WT BRAF [27, 28], but it is unclear whether the same is true

for class II BRAF mutant tumors [7, 10, 11]. Therefore, we employed cells derived from the

aforementioned PDX and cell line models (Table S2) to determine whether cells with class II

mutations are responsive to BRAF or MEK inhibitors in vitro.

Short-term treatment with MEKi (cobimetinib and trametinib) universally inhibited the MAPK

pathway, irrespective of BRAF class (Fig. 3A, S2A). Short-term treatment with BRAFi

(vemurafenib, dabrafenib) induces paradoxical activation of the MAPK pathway in BRAF WT

cells, while class I (BRAF V600) and IIa (K601E and L597S) mutant cells exhibit marked

inhibition of the MAPK pathway (Fig. 3B, S2A). In contrast, the class IIb mutant cancer cells tested were neither paradoxically activated nor inhibited by single agent BRAFi (Fig. 3B). This

result highlights the marked difference between class IIa and class IIb cells with respect to their

biochemical response to BRAFi. These differences may be based on the location of the mutation

within the BRAF protein or by the RAS mutation status of the cell lines tested (Table S2).

One of the key determinants of BRAFi efficacy is the speed of pERK recovery following drug

treatment [10]. We sought to compare the dynamics of pERK recovery between melanoma cells

of different BRAF mutant classes treated with physiologically relevant doses of encorafenib.

Encorafenib is an emerging BRAFi that is a promising candidate to become a front-line targeted therapy for class I BRAF mutant melanoma [3]. Cells were treated with encorafenib for 1 hour, washed with drug-free media and then lysed at defined time points post-washout. In WM3918

BRAF WT cells, we observe paradoxical activation of the MAPK pathway at 1 hour on treatment, which returns to baseline levels within minutes post-treatment (Fig. 3C). In A375

class I BRAF mutant melanoma cells, we observe strong pERK inhibition that recovers to baseline levels by 8 hours post removal of drug. In class IIa mutant cells, pERK levels returned to baseline at earlier time points (1-2 hours) following drug removal. By contrast, pERK levels in class IIb HMV-II and M619 melanoma cells are not significantly decreased by encorafenib after

1 hour of treatment. This data indicates that class I, and to a lesser extent, class IIa mutant BRAF dimers are effectively inhibited by single agent encorafenib, while WT and class IIb BRAF dimers are not.

Another important indicator of MAPKi efficacy is the extent to which the inhibitory signal is propagated to down-stream effector molecules. Such signals include cell cycle regulators such as

Cyclin D1 (CCND1) [29], which in turn phosphorylates the retinoblastoma (Rb) tumor suppressor protein to promote cell survival and proliferation [30]. In addition to these transcriptionally regulated targets of the MAPK pathway, ERK is itself a kinase that phosphorylates and stabilizes a number of effector proteins with critical functions, including

FRA-1 [31].

We examined the effects of either MEKi (trametinib) (Fig. 3D) or BRAFi (encorafenib) (Fig. 3E) on these downstream effectors of the MAPK pathway. Trametinib inhibited phosphorylation of

ERK, FRA-1 and Rb. Total levels of CCND1, FRA-1, and Rb were also diminished in all cell lines treated with trametinib. Encorafenib similarly inhibited these downstream signaling components to a comparable extent in class I and class IIa mutant melanoma cells. This demonstrates that cell proliferation and survival pathways are inhibited in class IIa mutant cells treated with either BRAFi or MEKi.

Next, we sought to determine whether class II BRAF mutant cells were growth inhibited by these

targeted therapies using standard BRAFi or MEKi doses that achieved >50% growth inhibition

of BRAF V600 mutant cells in short-term proliferation assays (Fig. S2B,C). In clonogenic

growth assays, MEKi effectively inhibited the growth of class I and IIa mutant cancer cells and,

to a lesser extent, BRAF WT and class IIb cells (Fig. 4A). BRAFi inhibited growth of class I and

IIa BRAF mutant melanoma cells but did not significantly impair the growth of wild-type and

class IIb mutant cancer cells (Fig. 4A). Representative images of clonogenic assays from each class are shown in Fig. S3A. While class I BRAF mutant cells responded similarly to all 3

BRAFi, we observed a marked contrast in class IIa mutant cells between the marginal efficacy

seen with vemurafenib and stronger inhibition of cell proliferation in the presence of dabrafenib

and encorafenib. Class IIb mutant cells were not significantly growth inhibited by single agent

BRAFi (Fig. 4A).

LY3009120 (LY), a pan-RAF and BRAF dimer inhibitor that is in early stage clinical

development, was also tested to assess its efficacy in class II BRAF mutant cells. LY inhibited

pERK in class I and II cell lines at low doses, but induced modest paradoxical activation in the

BRAF WT cell line, WM3918 (Fig. S4A). Using short term cell growth assays, we determined

the dose of LY3009120 that achieved >50% growth inhibition of BRAF V600 mutant cells at

two days (Fig. S4B). In clonogenic growth assays, LY3009120 at this dose (100 nM) moderately

inhibited growth of WM3918 but substantially inhibited growth of class I and II BRAF mutant

cells, including the class IIb cell line, HMV-II (Fig. S4C,D). These data demonstrate that while

LY3009120 is only marginally effective in BRAF WT cells, it may also be effective for patients

with class II BRAF mutant tumors.

Enhanced efficacy of dMAPK inhibition in class II BRAF mutant cancer cells

To assess efficacy of a combined therapeutic strategy employing BRAFi and MEKi, cells were

treated with the standard clinical BRAFi/MEKi combinations (vemurafenib/cobimetinib, dabrafenib/trametinib, encorafenib/binimetinib). We observe augmented growth inhibition when

either dabrafenib, encorafenib or LY3009120 were added to a MEKi in class I or IIa BRAF

mutant cells (Fig. 4A, S4C,D). While dMAPKi did significantly inhibit the growth of BRAF WT

cells compared to DMSO, combined BRAFi + MEKi was less effective than single agent MEKi

in most WT cells. In particular, we observed significantly enhanced growth of BRAF wild-type

cells treated with vemurafenib (SkMel2 P=0.009; WM3918 P=0.005; CHL1 P=0.024) or

dabrafenib (SkMel2 P=0.047, WM3918 P=0.001) in addition to a MEKi, compared to MEKi

alone (Fig. 4A). Encorafenib did not significantly enhance the growth of any BRAF WT cells

treated with a MEKi. Conversely, encorafenib significantly inhibited the growth of binimetinib

treated triple wild-type CHL1 cells (P=0.047). In class IIb mutant cell lines, we consistently

observed further growth inhibition only with encorafenib, but not with vemurafenib, when added

to a MEKi (Fig. 4A).

Next, we sought to directly compare the effects of specific BRAFi when added to the same

MEKi. To do so, we tested each BRAFi in combination with either trametinib or binimetinib. In long term growth assays where all cells were grown in the presence of trametinib +/- BRAFi

(Figs. 4B, S3B), vemurafenib potentiated the growth of BRAF WT, NRAS mutant SkMel2 and

GCRC1987 cells, and all class IIb mutant cells tested. Meanwhile, vemurafenib modestly augmented growth inhibition of class I A375 and class IIa mutant cells. Dabrafenib potentiated the growth of trametinib treated SkMel2 and GCRC1987 cells but inhibited the growth of all trametinib treated class I, IIa, and IIb cells, with the sole exception of class IIb mutant MDA-

MB-231 breast cancer cells, which were unaffected by the addition of dabrafenib. When added to trametinib, encorafenib potently inhibited the growth of BRAF WT, NRAS mutant SkMel2 cells and all class I, IIa, and IIb BRAF mutant cells tested (Fig. 4A). Similar BRAFi effects were observed when binimetinib was used as the MEKi (Fig. 4C, Fig. S3C).

In all classes of cells, 48-hour treatment with trametinib led to sustained inhibition of ERK phosphorylation (Fig. 4D). In class I BRAF mutant melanoma, dMAPK inhibition further impairs the MAPK pathway [32, 33]. Therefore, we asked if the addition of BRAFi will have a similar effect in MEKi treated class II cells. In class I and class IIa mutant cells, the addition of any BRAFi further exacerbated ERK inhibition. Meanwhile, in class IIb mutant cells, only encorafenib led to more profound ERK inhibition than trametinib alone (Fig. 4D).

dMAPKi induces regression of two melanoma PDXs expressing class II BRAF mutations

To determine whether BRAF + MEK inhibitor combinations cause tumor regression in vivo, we employed our melanoma PDX models bearing class IIa, BRAF L597S mutations (GCRC2015 and GCRCMel1). Tumors were implanted subcutaneously and treated with vehicle or MAPKi.

In the GCRCMel1 model, treatment with either single agent dabrafenib or single agent trametinib was insufficient to induce shrinkage in any of the tumors, but 17/19 (89%) tumors

treated with dabrafenib + trametinib had shrunk by day 4 (Fig. 5A). This result was corroborated with immunoblots that demonstrated decreased pERK in dabrafenib + trametinib 4 day treated tumors, compared to vehicle, dabrafenib or trametinib treated tumors (Fig. 5E). Both dabrafenib and trametinib, when used as single agents, were capable of delaying the growth of GCRCMel1 tumors over time. Meanwhile, tumors treated with dMAPKi were significantly more growth inhibited than tumors treated with either single agent (Fig. 5C). By day 15, all tumors in the study had begun to progress, implying that they had acquired resistance to MAPKi (Fig. 5C).

Phosphorylated ERK levels were uniform between all arms at experimental endpoint (Fig. 5E).

Resistant dabrafenib + trametinib treated tumors demonstrated increased expression of the HER3 receptor tyrosine kinase (RTK), as well as increased pAKT and pCRAF (Fig. 5E), implying potential mechanisms of resistance to dMAPKi in class II mutant tumors.

In the GCRC2015 model, after 4 days of treatment, 83.3% (10/12) of vehicle treated tumors were progressively growing. Trametinib monotherapy induced tumor shrinkage in 75% (8/12) of subcutaneous tumors. Meanwhile, dMAPKi with dabrafenib and trametinib induced tumor shrinkage in 100% (13/13) of tumors (Fig. 5B). Immunoblot analysis revealed that early into treatment, dabrafenib augmented the inhibitory effect of trametinib on the MAPK pathway (Fig.

5F). All treatment groups eventually began to acquire MAPK inhibitor resistance, as evidenced by the reactivation of pERK (Fig. 5F) and increasing tumor growth (Fig. 5D) at the experimental endpoint of 14 days. However, 88.9% (8/9) of dabrafenib + trametinib compared to 0% (0/8) of trametinib treated tumors maintained an overall reduction in tumor size at endpoint.

Immunoblots from GCRC2015 resistant tumors demonstrate the same resistance mechanisms as the GCRCMel1 model, in that RTKs (HER2 and HER3) were up-regulated, coinciding with

increased pAKT in all MAPKi treated tumors. In both PDX models, we only observed enhanced pCRAF in tumors that had acquired resistance to dMAPKi with dabrafenib + trametinib (Figs.

5E,5F). Together this suggests that activation of CRAF is a mechanism of resistance that is unique to dMAPKi in class II BRAF mutant melanoma.

Treatment with encorafenib, binimetinib, or encorafenib + binimetinib produced similar results to dabrafenib + trametinib, causing shrinkage of 8% (1/12), 25% (3/12), and 67% (8/12) of

GCRC2015 tumors respectively, whereas all of the vehicle tumors were progressively growing by day 4 (Fig. S5A). Immunoblot analysis of tumors treated for 4 days demonstrated that both encorafenib and binimetinib robustly inhibit ERK phosphorylation as single agents, while the encorafenib + binimetinib combination further inhibited pERK compared to either agent alone

(Fig. S5B). Both encorafenib and binimetinib, when used as single agents, delayed GCRC2015 tumor growth. Combined encorafenib + binimetinib elicited tumor shrinkage and more significant tumor growth delay compared to either single agent (Fig. S5C).

Importantly, the patient from whom the GCRCMel1 PDX was derived presented with stage IV

(M1a) metastatic melanoma, with disease involving the inguinal lymph nodes, muscle and adjacent soft tissues. This patient was treated with dabrafenib + trametinib and achieved an objective radiographic response, with a 34% reduction in tumor size at two months on treatment

(Fig. 5G). After several months of treatment, the patient began to experience drug toxicity

(pyrexia, hepatotoxicity) despite dose reductions, and was switched to immunotherapy. These observations of an objective partial response provide proof-of-principle demonstrating that dabrafenib + trametinib has clinical activity in class IIa BRAF mutant melanoma.

BRAF + MEK inhibition is effective in class II BRAF mutant brain metastases

GCRC2015 was derived from a melanoma brain metastasis. In light of recent data indicating that dMAPKi can effectively shrink brain metastases in patients with BRAF V600E mutant melanoma [34], we asked whether dabrafenib + trametinib would be similarly effective in class

II BRAF mutant brain metastases. The GCRC2015 PDX model was propagated as an intracranial xenograft and infected with pHIV-Luc-ZsGreen virus to allow longitudinal bioluminescence imaging in vivo (Fig. 6A). We observed that both trametinib monotherapy and dabrafenib + trametinib slowed growth of GCRC2015 intracranial tumors. At the experimental endpoint, the change in in vivo bioluminescence from the time treatment was initiated was significantly smaller in the dabrafenib + trametinib group compared to trametinib or vehicle (Fig. 6B, Fig.

S6A). Indeed, after 9 days of treatment the average size of the intracranial tumors from dMAPKi was smaller than tumors treated with trametinib alone or vehicle (Fig. S6B,C). Furthermore, immunohistochemistry for pERK revealed decreased staining in dabrafenib + trametinib, but not in trametinib treated brain metastases (Fig. 6C-D). In longer-term survival analyses, trametinib alone did not significantly prolong survival compared to vehicle treatment of mice bearing class

II BRAF mutant melanoma brain metastases (Fig. 6E). However, dMAPKi treatment did significantly improve survival of mice compared to either trametinib monotherapy or vehicle.

Finally, we retrospectively identified a patient with class II BRAF (L597S) mutant melanoma with brain metastases. This patient received treatment with dabrafenib + trametinib, and experienced a dramatic response in metastases in the brain, lung, liver, and adrenal gland (Fig.

6F). After 4 months of treatment, this patient developed progressive brain metastases. She went on to receive additional brain radiation and immunotherapy but eventually died of her disease.

These observations provide further validation that dMAPKi can induce objective responses in visceral and brain metastases of patients with class II BRAF mutant melanoma.

Discussion

We initiated this study after encountering two melanoma patients with BRAF L597S mutations in clinical practice. At the time of their presentation, little was known about class II BRAF mutations and their responsiveness to targeted therapy. We sought to better characterize this emerging class of BRAF mutant tumors and to inquire whether patients with class II BRAF mutant melanoma are responsive to therapeutic intervention with approved targeted therapies.

Data from CRC [35] or NSCLC [36] indicates that patients with non-V600 BRAF mutations tend to experience improved overall survival than those with V600 BRAF mutations. In contrast, we report here that advanced melanoma patients with potentially targetable NRAS and/or class II/III

BRAF mutations experience worse survival than those with class I BRAF mutations. This finding highlights the need for improved therapeutic strategies for melanoma patients with non-

V600 BRAF mutations.

We draw the distinction between class IIa mutations within the activation segment and class IIb mutations within the glycine rich p-loop. Class IIa and IIb mutations have been shown to engender enhanced kinase activity that is RAS-independent and dimerization dependent [10].

However, the class IIa and IIb mutant cells tested are unique in terms of their sensitivity to single agent BRAFi: class IIa BRAF mutant cells were sensitive to single agent BRAFi while class IIb

BRAF mutant cells were not. The differential sensitivities of class IIa and IIb BRAF mutants to

approved BRAFi may be due, in part, to the ability of BRAFi to more effectively inhibit the

second protomer of a class IIa BRAF mutant dimer than that of IIb BRAF dimers. Alternatively,

while it is clear that BRAF L597S and K601E signal predominantly as dimers [10], class IIa

mutant BRAF may harbor some degree of monomeric signaling capacity. It is also possible that class IIa mutants more readily form BRAF homodimers, while class IIb mutants form

BRAF:CRAF heterodimers, rendering them less sensitive to BRAFi [26]. Interestingly, we found

that activated CRAF is a common resistance mechanism to dMAPKi in our two class II BRAF

L597S mutant PDX models; this result suggests that acquired resistance to dMAPKi with BRAFi

and MEKi in class II BRAF mutant melanoma results from a shift from primarily BRAF

homodimer-driven MAPK signaling towards BRAF:CRAF heterodimer or CRAF homodimer-

driven MAPKsignaling.

While all three BRAFi augmented MEKi mediated growth inhibition in class IIa mutant cells,

encorafenib was the only BRAFi that consistently augmented MEKi mediated growth inhibition

in class IIb mutant cells. The contrast between vemurafenib, dabrafenib and encorafenib in this

context may be due to differences in eliciting paradoxical activation of the MAPK pathway. This

results from differential ability of each inhibitor to bind to and inhibit the second protomer of a

BRAF dimer. It has been shown that significantly higher concentrations of vemurafenib are

required to inhibit BRAF dimers, compared to encorafenib and dabrafenib [10, 37]. Moreover,

recent findings have demonstrated the efficacy of encorafenib when used in combination with

MEKi in NRAS mutant melanoma through an ER stress pathway [38]. This may explain the

sensitivity we observe in NRAS mutant SKMel2 cells treated with MEKi + encorafenib,

highlighting unique properties of encorafenib that may support its broader utility among

melanoma patients. The encorafenib + binimetinib combination has been shown to provide a significant survival advantage over vemurafenib in BRAF V600E/K mutant melanoma, with a favorable safety profile [3]. Therefore, this combination is promising for patients with class II

BRAF mutations, and perhaps even some NRAS mutant melanoma patients.

It has been proposed that non-V600 BRAF mutant melanoma are sensitive to single agent MEKi, prompting an on-going trial recruiting non-V600 mutant melanoma patients for treatment with trametinib [39, 40]. Since these initial observations, several clinical trials investigating single agent MEKi have failed to yield sustained clinical benefit in a variety of indications [41-43]. In

BRAF V600 mutant melanoma, trametinib has a much lower overall response rate (22%) than single agent BRAFi (48-51%) [4]. These data suggest that the more effective therapeutic approach of approved agents for class II BRAF mutant melanoma would be combination therapy including a clinically viable BRAFi (i.e encorafenib) plus a MEKi. Moreover, in addition to the potential for enhanced efficacy with dMAPKi, these combination regimens are frequently better tolerated than either BRAFi or MEKi alone [3, 44].

In this study, we established two PDX models of BRAF L597S mutant melanoma in order to assess their sensitivity to MAPKi. Importantly, the PDX models established herein adequately retain the genetic features of their tumors of origin. We demonstrate in both class II BRAF mutant PDX models that dMAPKi augments inhibition of the MAPK pathway and impairs tumor growth of class II BRAF mutant melanoma compared to single-agent therapy. Single agent

MEKi produced only short-lived stable disease in the GCRC2015 BRAF L597S subcutaneous

PDX model and only modestly slowed progression of the GCRCMel1 model, while the addition

of BRAFi to MEKi resulted in sustained partial responses in the majority of tumors in both models. As an important proof of principle, we show that single agent encorafenib is able to robustly inhibit the MAPK pathway and slow tumor growth in the GCRC2015 PDX model.

Furthermore, we report a partial response to dabrafenib + trametinib in patient GCRCMel1, corroborating the results from the aforementioned PDX studies.

While our results indicate that dMAPKi is superior to single agent MAPKi for class II BRAF

L597S mutant melanomas, the duration of growth inhibition with dMAPKi was less than we

have observed in class I BRAF mutant melanoma [45]. This implies that melanoma patients with

class II BRAF mutations may be less responsive to dMAPKi than those with class I BRAF

mutations. Moreover, both of our PDX models bore a BRAF L597S mutation, and as such it is

unknown at this point whether other common class IIa mutations (ie. L597Q/R/V, K601N/T)

would derive equivalent benefit from dMAPKi in vivo. As such, investigation into combinations

of dMAPKi with antibody drug conjugates [46], ERK inhibitors [47], and immunotherapies are

also warranted for class II BRAF mutant melanoma. Further investigation of therapies targeting

the resistance pathways we identified in both PDX models, such as RTKs, PI3K/AKT signaling,

and CRAF, may also be beneficial in preventing or delaying resistance to dMAPKi.

The patient from whom the GCRC2015 PDX was derived presented with brain metastases.

Cytotoxic chemotherapies have minimal effect in intracranial metastatic disease, in part due to

limitations of the blood brain barrier [48]. However, emerging data espouses the efficacy of

systemic immunotherapies and MAPKi in the management of brain metastatic melanoma [48,

49]. In this study, dMAPKi provided a significant survival advantage to mice with class II BRAF

L597S mutant brain metastases, while single agent MEKi did not. Therefore, we speculate that a

similar approach can be applied for melanoma patients with class II BRAF mutant tumors,

including those with brain metastases. This is further supported by a patient with BRAF L597S

brain-metastatic melanoma who experienced a major intracranial response to dabrafenib + trametinib.

In summary, we have provided in vitro, in vivo and clinical evidence indicating that dMAPKi effectively impairs the growth of subsets of non-V600, class II BRAF mutant melanoma. These data provide intriguing pre-clinical rationale to support the development of clinical trials to investigate BRAFi + MEKi combinations in patients with class II BRAF mutations.

AUTHOR CONTRIBUTIONS

Conception and design of experiments: M. Dankner, P.M. Siegel, A.A.N. Rose

Acquisition of Data: M. Dankner, M. Lajoie, D. Moldoveanu, TT. Nguyen, S. Rajkumar, P.

Savage, M-C. Guiot, A. Protopopov, M. Lvova, M. Park, K. Petrecca, I.R. Watson, P.M. Siegel,

A.A.N. Rose

Analysis and interpretation of data: M. Dankner, M. Lajoie, X. Huang, D. Vuzman,

A.A.N.Rose

Writing and review of manuscript: M. Dankner, M. Lajoie, P. Savage, TT. Nguyen, M-C.

Guiot, X. Huang, M. Lvova, A. Protopopov, D. Vuzman, D. Moldoveanu, S. Rajkumar, D.

Hogg, M. Park, Petrecca, C. Mihalcioiu, I. R. Watson, P. M. Siegel, A.A.N.Rose

ACKNOWLEDGEMENTS

We are grateful to the patients who donated the tissues studied in this work. We acknowledge

technical assistance from the McGill/GCRC Histology core facility and the McGill Comparative

Medicine and Animal Resources Centre (CMARC). We acknowledge the support and assistance

of Valentina Muñoz Ramos and Margarita Souleimanova in biobanking and sample collection.

We are thankful to Array Biopharma for providing encorafenib and binimetinib used in in vivo

studies. We are thankful to Juan Canale, Karen Stone, Vasilios Papavasiliou, Matthew Annis and

William Muller for animal support. We are grateful to Nicholas Hayward, Antoni Ribas, Wilson

Miller, and David Dankort for providing cell lines used in this study. We thank members of the

Siegel laboratory for thoughtful discussions and critical reading of the manuscript.

GRANT SUPPORT

This research has been supported by a grant from the DOD (CA-140389 to P.M.S). M.D.

acknowledges support from the McGill University MD/PhD program and the Brain Tumour

Foundation of Canada. P.M.S. is a McGill University William Dawson Scholar. I.R.W is funded

by grants from the Melanoma Research Alliance (MRA – Grant #412429), the V Foundation

(Grant #V2016-023), and the Canadian Institute of Health Research (CIHR – Grant # PJT-

152975). A.A.N.R. acknowledges a David Cornfield Melanoma Fund Award.

FIGURE LEGENDS

FIGURE 1: Classification of BRAF mutations in cancer. A) Lollipop plot from the AACR

GENIE tumor sequencing dataset representing the incidence of BRAF mutations found in

melanoma samples (n=785). Class IIa and IIb mutations are indicated in blue and purple,

respectively and class III mutations are indicated in red. B) Incidence of BRAF mutations in

different tumor types in AACR GENIE dataset. Only those cancer types with greater than 5

samples harboring BRAF mutations were included in this analysis. Melanoma (n=785), thyroid

(n=410), histiocytosis (n=24), small bowel (n=69), colorectal (n=2081), gastrointestinal

neuroendocrine (n=92), carcinoma of unknown primary (n=367), non-small cell lung cancer

(n=2985), non-melanoma skin cancer (n=198), endometrial (n=552), cervical (n=148), leukemia

(n=344), bladder (n=638), non-hodgkin lymphoma (n=189), glioma (n=977), pancreatic (n=455),

ovarian (n=934), prostate (n=752), hepatobiliary (n=386), esophagogastric (n=528), soft tissue

sarcoma (n=635) and breast (n=2193). C) Prevalence of BRAF mutation classes among BRAF

mutant tumors in common cancer types in AACR GENIE tumor sequencing dataset: melanoma

(n=317), colorectal (n=230), non-small cell lung cancer (n=162). D) Co-occurrence of RAS

activating mutations with different BRAF mutant classes in the AACR GENIE tumor sequencing

dataset melanoma cohort. E) Survival analysis of metastatic melanoma patients whose tumors expressed BRAF WT/ NRAS WT (n=88), BRAF class I V600E/K (n=149) and class II / III and/

or NRAS mutant (n=101). In comparison between BRAF class I V600E/K and BRAF class II /

III and/ or NRAS mutant, P = 0.021, HR: 1.49, 95% CI 1.06-2.09. Data was obtained from

updated survival analysis of the melanoma TCGA dataset.

FIGURE 2: Characterization of metastatic melanoma PDX models. A) Characteristics of

patients whose tumors were used to establish patient derived xenografts B) Copy number

analysis of the GCRC2015 (BRAF L597S brain metastasis) and GCRCMel1 (BRAF L597S

lymph node metastasis) patient tumor and first-passage mouse xenograft. C) Total number of

identified mutations in GCRC 1987, 2073, 2015 and Mel1 patient tumor tissue (blue) and first-

passage xenografts (red) in 435 gene panel sequencing. D) Spectrum of mutations in clinically

actionable genes in patient and first-passage xenografts of GCRC 1987, 2073, 2015 and Mel1.

Green = gain of function, Red = loss of function, Blue = mutation of unknown significance. E)

Representative images of patient brain metastasis and matching PDX material embedded into a tissue microarray (TMA) and stained for H&E, Melan-A, BRAF V600E, and HMB-45.

FIGURE 3: BRAFi and MEKi effectively inhibit MAPK signaling in class IIa BRAF

mutant cells. Immunoblots of BRAF WT (green border), class I BRAF mutant (black), class IIa

(blue), and class IIb (purple) BRAF mutant cells treated with A) single agent MEK inhibitors

cobimetinib (5 nM, 50 nM) and trametinib (1 nM, 10 nM) for 1 hour or B) single agent BRAF

inhibitors vemurafenib (100 nM, 1000 nM) and dabrafenib (10 nM, 100 nM) for 1 hour. C)

Immunoblots of BRAF WT (green border), class I BRAF mutant (black), class IIa (blue), and

class IIb (purple) BRAF mutant cells treated for 1 hour with encorafenib (300 nM), which were

then washed three times in pre-warmed media and replaced with drug-free media. Cells were

lysed at the following time points post washout: 0 minutes, 5 minutes, 30 minutes, 60 minutes,

120 minutes, 480 minutes, 1440 minutes. D, E) Immunoblots against the indicated proteins in

BRAF WT (green border), class I BRAF mutant (black), class IIa (blue), and class IIb (purple)

BRAF mutant cells treated for 24 hours with D) DMSO or trametinib (5 nM), or E) DMSO or

encorafenib (300 nM).

FIGURE 4: Dual MAPKi effectively inhibits the growth of class II BRAF mutant cancer

cells in vitro. A) Quantification of 10-day cell growth clonogenic assay using cell lines

endogenously expressing BRAF class I mutant (black), WT (green), class IIa (blue) and IIb

(purple) BRAF mutants that were treated with BRAF inhibitors vemurafenib (1500 nM),

dabrafenib (150 nM), encorafenib (150 nM), cobimetinib (25 nM), trametinib (5 nM) and

binimetinib (50 nM). In comparisons between DMSO and BRAFi, MEKi or BRAFi + MEKi, *

represents p<0.05, ** represents <0.0005 and # represents not significant. B,C) Quantification of

15 day cell growth assay with cell lines expressing BRAF class I mutant (black), WT (green),

class IIa (blue) and class IIb BRAF mutants (purple) treated with (B) trametinib (5 nM) , or (C)

binimetinib (100nM), plus vemurafenib (2000 nM), dabrafenib (300 nM) or encorafenib (300

nM). In comparisons between MEKi and BRAFi+ MEKi, * represents p<0.05, ** represents

<.0005 and # represents not significant. For A, B, and C, adjacent scale bars represent positive

pixel count (i.e area covered by cancer cells) at quantification, compared to either (A) DMSO, or

(B,C) MEKi controls. Representative images from A, B, and C for each condition, taken at

experimental end point, are shown in Fig. S3. D) Immunoblots of class I BRAF mutant (black),

BRAF WT (green), class IIa (blue), and class IIb BRAF mutant (purple) cells treated with

DMSO, the MEK inhibitor trametinib, or trametinib in combination with BRAF inhibitors

vemurafenib, dabrafenib and encorafenib, at the same doses as (B).

FIGURE 5: Dual MAPK inhibition induces tumor regression in class II BRAF L597S

mutant PDX melanoma models. A) Waterfall plot demonstrating responses of individual

GCRCMel1 tumors grown subcutaneously; treatment was initiated when tumors reached an

average volume of 180 mm3. Mice were treated with vehicle (V; n=18), dabrafenib (D; 50 mg/kg, n=16), trametinib (T; 0.5 mg/kg, n=16) or dabrafenib (50 mg/kg) + trametinib (0.5mg/kg)

(DT; n=19). N=4 tumors were removed from each cohort at day 4. B) Waterfall plot demonstrating responses of individual GCRC2015 tumors grown subcutaneously; treatment was

initiated when tumors reached an average volume of 460 mm3. Mice were treated with vehicle

(V; n=12), trametinib (T; 0.5 mg/kg, n=12) or dabrafenib (50 mg/kg) + trametinib (0.5mg/kg)

(DT; n=13). N=4 tumors were removed from each cohort at day 4. C) Growth curves plotted

from GCRCMel1 subcutaneous tumor measurements. V vs. D: P = 0.2, V vs. T: P = 0.0164, V vs. DT: P < 0.0001 , D vs. T: P = 0.4, D vs. DT: P < 0.0001, T vs. DT: P < 0.0001. D) Growth

curves plotted from GCRC2015 subcutaneous tumor measurements. V vs. T: P = 0.0018, V vs.

DT: P = 0.0002, T vs. DT: P = 0.0001. E) Immunoblots against the indicated proteins from

GCRCMel1 tumor lysates at 4 days and 14 days on treatment. F) Immunoblots against the

indicated proteins from GCRC2015 tumor lysates at 4 days and 14 days on treatment. G) Patient

GCRCMel1 scan results before and after treatment with dabrafenib + trametinib. Prior to

treatment, target lesions #1 and #2 measured 1.8 x 1.8cm and 2.4 x 1.7cm, respectively. After 6

weeks of treatment target lesions #1 and #2 measured 1.0 x 0.8cm and 1.8 x 1.2cm, respectively.

Target lymph node lesions are delineated by green cross hairs, and outlined in red. Together this

represents a 33% reduction in size, according to RECIST1.1 criteria.

FIGURE 6: dMAPK inhibition improves survival in class II BRAF L597S mutant

melanoma brain metastases. A) Experimental pipeline outlining development of patient-

derived xenograft models of brain metastasis. Brain metastatic tissue is retrieved from the

operating room and grown in the flank of immune-compromised mice. The resulting tumors are

enzymatically dissociated and injected into the brains of new mice, and are labeled with pHIV-

Luc-ZsGreen for longitudinal imaging in vivo. These tumor cells were then used for in vivo

treatment experiments. B) GCRC2015 PDX expressing pHIV-Luc-ZsGreen were injected intra-

cranially. Tumor growth was monitored with in vivo bioluminescent imaging. Representative

images are shown from three treatment groups: vehicle (n=6), trametinib (0.5mg/kg, n=7),

dabrafenib (50mg/kg) + trametinib (0.5mg/kg) (n=6) at day 9. C, D) IHC was performed for

pERK on n=5 brain metastases per treatment arm. Quantification of staining was performed for

nuclear pERK positivity (C), and representative images (D) are shown. * = p<0.05. E) Mice were

injected intracranial with GCRC2015 cells, and treated with vehicle (n=11), T (0.5mg/kg)

(n=12), or D (25mg/kg) + T (0.5mg/kg) (n=11). Mice were monitored until they showed signs of

neurologic decompensation or poor body condition, at which point they were recommended for

euthanasia by blinded animal health technicians. Overall survival for each group is presented in a

Kaplan-Meier plot. T vs. V, HR 0.723, 95% CI 0.253 – 2.064; DT vs. V, HR 0.272, 95% CI

0.107 – 0.697; DT vs. T, HR 0.376, 95% CI 0.167 – 0.848. F) A patient presenting with BRAF

L597S metastatic melanoma was treated with dabrafenib + trametinib. Before and on-treatment scan results are shown, demonstrating a profound response in the brain, lung and adrenal.

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Dual MAPK inhibition is an effective therapeutic strategy for a subset of class II BRAF mutant melanoma

Matthew Dankner, Mathieu Lajoie, Dan Moldoveanu, et al.

Clin Cancer Res Published OnlineFirst June 14, 2018.

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