Results
Case 1, a 16 year-old female with a 2.5 cm rapidly growing subcutaneous
thigh mass was diagnosed with a malignant histiocytic neoplasm (“M
group”)8, with high-grade morphologic features and a
phenotype spanning histiocytic sarcoma (CD163/CD14/CD68+) and Langerhans
cell sarcoma (CD1a/Langerin/S100) with a modestly elevated Ki-67
proliferation index (up to 20%) (Figure 1A-F). Targeted RNA-sequencing
identified a MTAP-BRAF fusion transcript. Resection margins were
negative. The patient is disease-free three years post-resection. Case
2, a 12-year-old female with a 5.3 cm rapidly enlarging heel mass
invading the calcaneus was originally diagnosed with a juvenile
xanthogranuloma (JXG) family lesion (CD163/CD68/CD14/fascin/Factor
XIIIa+) (Figure 1G-M). Despite low-grade morphologic features and lack
of cytologic atypia or increased mitotic rate by H&E stain, a high
proliferation rate (up to 40%) was noted by Ki-67 proliferation index
stain. Targeted RNA-seq identified a MS4A6A-BRAF fusion
transcript. During staging, the patient was found to have PET-avid
dissemination to lymph nodes and lung (Figure 1N-P). While the initial
morphologic features were consistent with a low-grade histiocytic lesion
of JXG phenotype, the integration of the high Ki-67 proliferation index
and aggressive clinical behavior with lymphatic/metastatic-like spread,
along with a novel molecular BRAF-fusion, at the time of diagnosis,
suggested an atypical JXG family neoplasm with uncertain biological
behavior. The patient was treated with 12 cycles of clofarabine with
clinical remission of metastatic sites and near clinical remission at
primary site now 18 months off therapy.
MTAP-BRAF and MS4A6A-BRAF fusions are predicted to contain all
functional domains of MTAP and MS4A6A, respectively, along with the BRAF
kinase domain but no N-terminal regulatory, RAS-binding domain (Figure
1Q). For molecular and therapeutic characterization, MTAP-BRAFand MS4A6A-BRAF were cloned and stably expressed in a
heterologous cell model since patient-derived cell lines were lacking.
The NIH/3T3 cells model system was utilized for its ability to reliably
discern oncogenic fusion profiles9-11. In soft agar
assays, both MTAP-BRAF and MS4A6A-BRAF expressing NIH3T3
showed a significant increase in colony count over control
(p<0.0001, Figure 1R). Next, we tested activation of
downstream MAPK and PI3K/mTOR pathways. Upon serum starvation, we
observed elevated levels of phosphorylated-ERK and -S6 in both
BRAF- fusion expressing cells compared to controls, indicating
aberrant activation of the MAPK and PI3K/mTOR pathways, respectively
(Figure 1S). Slightly higher PI3K/mTOR pathway activation levels in
MTAP-BRAF versus MS4A6A-BRAF cells are partly explained by higher
MTAP-BRAF protein expression (Figure 1S, Myc-tag blot).
A single report on BRAF- fusions in LCH4 has
shown unresponsiveness to first generation specific BRAF-V600E
inhibitors (RAFi) such as vemurafenib, but observed suppression by
second-generation RAFi, PLX8394, and downstream MEK inhibition, similar
to other pediatric glioma studies on BRAF-fusions9,11.
Herein, we evaluated the responsiveness of MTAP-BRAF and MS4A6A-BRAF to
such targeted inhibitors. Upon targeting the NIH3T3 models with
first-generation RAFi PLX4720, as expected, no suppression of
BRAF-fusion driven signaling or growth was observed (Supplemental Figure
S1A). Interestingly, second-generation RAFi PLX8394 also showed no
suppression in MTAP- or MS4A6A-BRAF driven soft agar growth despite
targeting MAPK/PI3K signaling (Figure 2A-B). This is in contrast to
PLX8394-mediated suppression of BRAF-fusion driven growth in the
previously described LCH4 and other cancers, such as
the KIAA1549-BRAF fusion in pediatric glioma9-11.
PLX8394 suppressed FAM131B-BRAF (a pediatric glioma derived
fusion12,13) and BRAF-V600E driven growth and
signaling as well as actively disrupted FAM131B-BRAF dimers
(Supplemental Figures S1B-D), highlighting therapeutic differences
between MTAP-/MS4A6A-BRAF, BRAF-V600E and other BRAF-fusions.
BRAF- fusions function as active homo- and heterodimers (with
wild-type BRAF) to mediate cell signaling9,11. We
found that MTAP- and MS4A6A-BRAF also mediate such protein-protein
interactions in co-immunoprecipitation assays (Figure 2C-D, DMSO lanes).
PLX8394 blocks BRAF kinase activity via disrupting BRAF
dimerization14 but we observed no disruption of MTAP-
and MS4A6A-BRAF fusion dimerization with PLX8394 (Figure 2C-D, PLX8394
lanes), thereby providing a plausible explanation for PLX8394
unresponsiveness in soft agar assays though MAPK/PI3K signaling remains
discordantly suppressed by some unknown mechanism. This distinct
unresponsiveness to pan-RAFi represents a significant departure from the
current view that BRAF -fusions and other BRAF mutations
should respond to second-generation RAFi such as
PLX83949,15. We found that this difference arises due
to the contribution of N-terminal partners, MTAP (exons 1-7) and MS4A6A
(exons 1-6), to respective fusion dimerization that is unaffected by
PLX8394 (Figure 2C-D, lanes 3,7). Similar role of N-terminal partner
accounts for differential response of CRAF- fusions to
PLX839410. Furthermore, we observed that Trunc-MTAP
(exons 1-7) competitively substituted MTAP-BRAF homo-dimerization in a
dose-dependent manner, suggesting preferential and potent protein
interactions mediated by the N-terminal partner in these
histiocytic-specific BRAF-fusions (Figure 2E).
To target dimerization-dependent oncogenicity of MTAP- and MS4A6A-BRAF
via a different mechanism, we tested LY3009120, a pan-RAF dimer
inhibitor that binds and stabilizes the BRAF dimer in an inactive
conformation16. LY3009120 showed robust suppression of
both fusion-mediated signaling and colony transformation (Figure 2B)
while stabilizing the MTAP- or MS4A6A-BRAF in inactive conformation
(Figure 2C-D, respectively, lanes 9-11). We also tested the effect of
FDA-approved MEK inhibitors (MEKi)17, selumetinib and
trametinib. We observed dose-dependent decrease in phospho-ERK and
growth with trametinib (Figure 2F) and selumetinib (Supplemental Figure
S2) in both BRAF-fusion models suggesting downstream MEKi as a
therapeutic alternative to RAFi.