A brain-penetrant RAF dimer antagonist for the noncanonical BRAF oncoprotein of pediatric low-grade astrocytomas

Abstract

Background: Activating mutations or structural rearrangements in BRAF are identified in roughly 75% of all pediatric low-grade astrocytomas (PLGAs). However, first-generation RAF inhibitors approved for adult melanoma have poor blood-brain penetrance and are only effective on tumors that express the canonical BRAFV600E oncoprotein, which functions as a monomer. These drugs (type I antagonists that target the "DFG-in" conformation of the kinase) fail to block signaling via KIAA1549:BRAF, a truncation/fusion BRAF oncoprotein which functions as a dimer and is found in the most common form of PLGA.

Methods: A panel of small molecule RAF inhibitors (including type II inhibitors, targeting the "DFG-out" conformation of the kinase) was screened for drugs showing efficacy on murine models of PLGA and on authentic human PLGA cells expressing KIAA1549:BRAF.

Results: We identify a type II RAF inhibitor that serves as an equipotent antagonist of BRAFV600E, KIAA1549:BRAF, and other noncanonical BRAF oncoproteins that function as dimers. This drug (MLN2480, also known as TAK-580) has good brain penetrance and is active on authentic human PLGA cells in brain organotypic cultures.

Conclusion: MLN2480 may be an effective therapeutic for BRAF mutant pediatric astrocytomas.

Keywords: MLN2480; RAF dimers; pediatric low-grade astrocytoma.

Fig. 1

Type I and type II RAF inhibitors have differential effects on BRAF activating mutants. (A) p53^-/- neuroprogenitor cells were transduced with the indicated constructs. Control cells (with EGF and basic FGF) and oncogene-transformed cells (in factor-free medium) were treated with vemurafenib or dabrafenib for 4 days and viability at day 4 was monitored by CellTiter Glo. (B) As in (A) above, except that treatment with vemurafenib or dabrafenib was for 1 hour and phospho-ERK signaling was monitored by immunoblot. (C, D) Proliferation and signal transduction were monitored as in panels (A) and (B) above. (E) Wild-type BRAF cells were treated as in (D) and phospho-ERK assayed by immunoblot.

Fig. 2

The response of KIAA1549:BRAF expressing cells to type II RAF inhibitors requires drug interaction with the KIAA1549:BRAF oncoprotein. (A, B) Mouse neuroprogenitor cells transduced with the indicated constructs were treated with MLN2480 (MLN) at 1 µM or DMSO and examined by immunoblot. (C) p53^-/- mouse neuroprogenitor cells stably transduced with the indicated constructs, then treated with MLN2480 and assayed for cell proliferation. V600EGK, BRAFV600E with gatekeeper mutation; KIAAGK, BRAF fusion with gatekeeper mutation (D) KiNativ assay for kinase specificity was performed after 1 hour of drug treatment in KIAA1549:BRAF expressing p53 null neural progenitor cells. The circle represents the kinase families tested in the assay and the bars extending outward from the circle represent relative inhibition of these kinases by MLN2480. MLN2480 binds native BRAF and RAF1 (aka CRAF) as indicated in the dendrogram.

Fig. 3

MLN2480 can cross the blood–brain barrier and retard growth of BRAFV600E and KIAA1549:BRAF intracranial tumors in NCr-NU mice. (A) Hematoxylin and eosin (H&E) staining of a normal brain section following a single oral dose of 30 mg/kg MLN2480. Tissue was collected after 4 hours. Green and red colors indicate the distributions of MLN2480 (third isotope, m/z 508.0148) and heme (m/z 616.1763). (B) H&E staining of a normal brain section following a single oral dose of 100 mg/kg dabrafenib as in (A). White dotted area delineates normal brain regions, which were subjected to MALDI-MS imaging at 75 µm spatial resolution. Green and red colors indicate the distributions of dabrafenib and heme. (C) H&E staining of a KIAA1549:BRAF tumor bearing brain section following 41 days of twice daily oral MLN2480 treatment at 30 mg/kg. Brain tissue was collected 4 hours after the final dose. The representative images of MLN2480 and heme distribution in tumor region (“T”) highlighted in rectangle are indicated. (D) Decreased phospho-ERK signaling in KIAA1549:BRAF brain tumors treated with MLN2480 as shown by immunohistochemistry. Ki67-positive cells in MLN2480-treated tumors are also decreased. (E) Immunoblot analysis of phospho-ERK signaling in MLN2480 treated brain tumors (T) and from contralateral normal brain (NB). (F, G) p53^-/- neuroprogenitor cells were stably transduced with BRAFV600E or KIAA1549:BRAF, then intracranially injected into NCr-NU mice. Mice harboring tumors were dosed orally with MLN2480 each day (days 1–9 for the BRAFV600E and 1–28 for KIAA1549:BRAF), as indicated, and subjected to bioluminescent imaging (mean ± SEM; P < .05. One way ANOVA, n = 10). (H, I) Kaplan–Meier analysis of mice with intracranial tumors as in F, G treated (or not) with MLN2480.

Fig. 4

A type II RAF inhibitor is active on BRAF mutant human PLGAs. (A) Freshly isolated human PLGA cells expressing the BRAF truncation/fusion mutation (BT728, see Supplementary Table S2) were seeded onto laminin-coated microscope coverslips and incubated overnight in growth factor-free medium. The cells were then treated with 1 µM MLN2480 or vemurafenib (VMRF) for 1 hr, fixed and processed for immunofluorescence of phospho-ERK (P-ERK) or OLIG2. (B) Quantification of the P-ERK and OLIG2 images (mean ± SEM; ***P < .001; one way ANOVA with Bonferroni test; n = 441 cells). (C) Example of organotypic slice overlay assay using p53^-/- murine neural progenitor cells transduced with KIAA1549:BRAF. Cells were labeled with Cm-Dil, seeded onto brain slices (see “Materials and Methods”) and then incubated with vehicle control (DMSO) or with 1 µM MLN2480 or vemurafenib (VMRF) as indicated for 7 days. Slices were imaged for Cm-Dil at days 1 and 7. (D) Quantification of drug responses on slice overlay cultures. KIAA1549:BRAF-transformed mouse cells or, alternatively, cells from 8 different juvenile pilocytic astrocytomas bearing the truncation/fusion BRAF mutation were processed as in panel (C) above. The overlay cultures were incubated with DMSO or with drugs at 1 mM (filled symbols) or at 100 nM (open symbols). For the human samples, a total of 5 different tumors were tested for MLN2480 and 4 different tumors were tested for vemurafenib (Table S2). The data shown depict cumulative cell counts (day 7/day 1) for 7 biological replicate experiments (mouse). Each symbol represents the average cell counts (day 7/day 1) of a single tumor. MLN2480 suppressed proliferation of the KIAA1549:BRAF murine model cells (mean ± SEM;*P < .05. One way ANOVA with Tukey’s multiple comparison test, 7 slices for each group) and their human tumor counterparts (mean ± SEM; DMSO vs. MLN, ** P = .0006, paired t-test, n = 7 tumors;). Vemurafenib is without effect on the mouse cells (mean ± SEM; P > .05, one way ANOVA with Tukey’s multiple comparison test) and has no discernible effect on the human tumor cells (DMSO vs VMRF, P = .185, paired t-test, n = 4 tumors; *MLN vs VMRF, P = .0305, paired t-test, n = 3 tumors). Scale bar, 50 uM.

Fig. 5

Type II RAF inhibitors block BRAF catalytic activity. BRAF cDNAs were genetically engineered to encode activators (rectangles) or receivers (ovals). The gatekeeper mutation (T529N) was introduced into either activator or receiver mutants to prevent drug binding (red rectangles or ovals). Both vemurafenib (VMRF) and MLN2480 (MLN) were used at 1 µM. (A) Optimum signaling requires receiver and activator present, as indicated by increased P-ERK. (B) Same as (A) but with gatekeeper mutation in the receiver subunit. (C) MLN2480 (MLN) but not vemurafenib (VMRF) can block signaling via BRAF dimers. The action of MLN2480 is unaffected by insertion of the gatekeeper mutation into the activator subunit. (D) Introduction of the gatekeeper mutation into the receiver subunit prevents the action of MLN2480.

Fig. 6

Differential interaction of type I and type II RAF inhibitors with BRAF monomers and dimers. (A) Indicated constructs were cotransfected into HEK293 cells. The gatekeeper mutation variants were used as internal controls, * denotes BRAFV600E. (B) Interaction of drugs with BRAF monomers (V600E). Representative immunoblots are shown to left and quantification of triplicate experiments is to right. (C, D) Data for the N-terminal truncation mutant and for the KIAA1549:BRAF, respectively, as in (B).