Vemurafenib Inhibits Myeloma Cell Growth Independent Of BRAF V600E Mutations, Potentiates The Activity Of Established Anti-Myeloma Drugs, but impairs Osteogenesis Via An HGF Autocrine Loop In Bone Marrow Stromal Cells
Authors: Bolomsky Arnold , Ludwig Heinz , Zojer Niklas 
Published: October 2019

Abstract

Introduction
Vemurafenib is a small molecule inhibitor designed for the treatment of BRAF V600E mutated malignancies such as melanoma, where it shows remarkable anti-tumor activity. However, prolonged exposure usually is associated with emerging resistance, likely a consequence of upregulation of growth factor signaling. In multiple myeloma, BRAF V600E mutations have been noted in a small subset of patients providing a rationale for the use of vemurafenib in these patients.
In the current study we aimed to investigate the activity of vemurafenib in MM in vitro in relation to presence or absence of a BRAF V600E mutation, alone or in combination with other MM drugs and the role of major myeloma growth factors in modulating its activity. Furthermore the impact of vemurafenib on bone formation was investigated.

Methods
BRAF V600E mutation status was analyzed by immunohistochemistry and DNA analysis in 7 human myeloma cell lines (MMCLs) (OPM-2, KMS-12-BM, RPMI8226, SK-MM-1, U266, NCI-H929, MM.1S). Viability was assessed after a 96h treatment period in the presence or absence of vemurafenib ± lenalidomide, pomalidomide, bortezomib and carfilzomib. Apoptosis was determined by Annexin V and 7-AAD staining. For rescue experiments vemurafenib was combined with recombinant human IL-6, HGF, IGF-1 or insulin.
Human immortalized mesenchymal stromal cells (MSC TERT+) were cultured in osteogenic medium in the presence of vemurafenib and/or a specific c-met inhibitor (PHA-665752) and analyzed at day 7, 14 and 21 of differentiation by qPCR, ALP activity assessment and/or alizarin red S staining.

Results
We observed a BRAF V600E mutation verified by immunohistochemistry and DNA analysis in 1 (U266) of 7 MM cell lines tested. Treatment with vemurafenib (0-10 µM) suppressed viability in all MMCLs tested in a dose dependent manner, independent of mutation status. Induction of apoptosis was observed in 4 of 4 MMCLs (range: 14.89-37.69%; P < 0.05). Combination of vemurafenib with immunomodulatory drugs (lenalidomide, pomalidomide) and proteasome inhibitors (bortezomib, carfilzomib) led to a synergistic or additive impairment of MM cell growth. In contrast, concurrent treatment with myeloma growth factors resulted in a partial rescue from the inhibitory action of vemurafenib. We observed a significant rescue effect (range: 5.3-21.3%; P<0.05) in 1 of 7 MMCLs by IL-6, 2 of 7 by insulin and 5 of 7 by IGF-1. In BM stromal cells, we detected upregulation of HGF (2.15 fold induction; P<0.05) and RANKL (4.38 fold induction, P<0.05) under vemurafenib treatment. Moreover, vemurafenib impaired osteogenesis indicated by a significant downregulation of osteogenic transcription factors Runx2 and Dlx-5, alkaline phosphatase activity and matrix mineralization in a dose dependent manner. Of note, the inhibitory action of vemurafenib on osteogenesis was overcome by concurrently c-met inhibition (ALP activity relative to control: 0.70 ± 0.09 vs. 0.91 ± 0.1; P<0.05 and 0.48 ± 0.13 vs. 0.71 ± 0.05; P<0.05 using vemurafenib at 10 nM and 5 µM in the presence or absence of a specific c-met inhibitor, respectively) suggesting that HGF signalling is responsible, at least in part, for the inhibitory action on osteoblast development.

Conclusion
Our results indicate anti-myeloma activity of vemurafenib independent of the presence of a BRAFV600E mutation in vitro. We observed an inhibitory activity against MM cell growth and induction of apoptosis in all MM cell lines tested. Furthermore, vemurafenib showed synergistic or additive effects in combination with novel agents. The inhibitory effects of vemurafenib were partially counteracted by cytokines such as IGF-1. Targeting of MM growth factor signalling pathways might therefore further improve response rates and avoid unwanted off-target effects such as impairment of osteoblast development.

Disclosures:
No relevant conflicts of interest to declare.