Resistance to BRAF inhibition in vitro is mediated by the two distinct mechanisms of clonal outgrowth of genetically distinct resistant clones, and of transcriptional rewiring, according to a study presented at the 17th International Myeloma Workshop.
In this study, researchers modeled resistance to dabrafenib in vitro in two BRAF-mutated multiple myeloma (MM) cell lines -(MMCL) U266 (K601N-mut) and DP6 (BRAFV600E-mut). Subsequently, they performed low-pass whole genome sequencing (LPWGS), RNA sequencing, ChIP sequencing and immunoblotting for genomic, transcriptomic, epigenomic and molecular characterization. They also used CRISPR/Cas9 technology for genome editing to assess functional validation.
According to the results of the study, modeling of dabrafenib resistance in vitro exhibited an initial decline of cell numbers, and subsequently a plateau phase followed by a gradual outgrowth of resistant cells, after 80 days of treatment. The researchers observed that exposure of BRAF-mut MMCL to dabrafenib led to initial downregulation of pERK and pMEK. Moreover, at later timepoints, the results revealed the upregulation of pERK and pMEK, indicating that re-activation of the ERK/MEK pathway overcomes BRAF inhibition. The authors wrote that this outgrowth “was associated with highly distinct copy number profiles in each resistant clone. This implies clonal selection with outgrowth of genetically resistant clones as one mechanism of drug resistance in MM.”
A second step of the study found that BRAF inhibition of BRAF-mut MMCL prompts changes of the transcriptional circuitry independent from clonal outgrowth of genetically resistant clones. The researchers noted the transcriptional changes were highly homogenous, occurred as early as a week after treatment, and was correlated with dedifferentiation of MMCL into a more immature B lymphocytic phenotype, which was linked with a greater mRNA expression and an upregulation of pathway genes.
The authors wrote in their conclusion that transcriptional rewiring that leads to activation of alternative pathways is characterized by a “de-differentiated B-cell phenotype and upregulation of PI3K and CD28/CD86 signaling.”
They added that these concepts “may provide a framework for revealing therapeutic vulnerabilities and to overcome drug resistance mediated by genetic heterogeneity in MM.”
Waldschmidt J. Determining Resistance Mechanisms in BRAF-Mutated Multiple Myeloma. Presented at the 17th International Myeloma Workshop; September 12-15, 2019; Boston, MA.