Sporadic ERK pulses drive non-genetic resistance in drug-adapted BRAF^V600Emelanoma cells

Abstract

Anti-cancer drugs commonly target signal transduction proteins activated by mutation. In patients with BRAF V600E melanoma, small molecule RAF and MEK kinase inhibitors cause dramatic but often transient tumor regression. Emerging evidence suggests that cancer cells adapting by non-genetic mechanisms constitute a reservoir for the development of drug-resistant tumors. Here, we show that few hours after exposure to RAF/MEK inhibitors, BRAF V600E melanomas undergo adaptive changes involving disruption of negative feedback and sporadic pulsatile reactivation of the MAPK pathway, so that MAPK activity is transiently high enough in some cells to drive proliferation. Quantitative proteomics and computational modeling show that pulsatile MAPK reactivation is possible due to the co-existence in cells of two MAPK cascades: one driven by BRAF V600E that is drug-sensitive and a second driven by receptors that is drug-resistant. Paradoxically, this may account both for the frequent emergence of drug resistance and for the tolerability of RAF/MEK therapy in patients.

Variability within rare cell states enables multiple paths towards drug resistance

“…We also observed that within individual clusters of closely related primed cells, not all cells contained higher levels of pERK, which may reflect pulsatile changes in pERK as documented elsewhere (Supp. Fig 8D) (Gerosa et al, 2019) . These results suggest that primed cells are able to maintain residual MAPK signaling following vemurafenib treatment that may allow them to continue proliferating in the face of drug.…”

“…Shown is one such example. We speculate that this variability may be a result of pulsatile MAPK signaling, which has been documented in other melanoma cell lines (Gerosa et al 2019), and our snapshot measurement of ERK phosphorylation via immunofluorescence E. After identifying primed cells in situ, we performed both single-molecule RNA FISH and immunofluorescence to measure gene expression and phosphorylated ERK in the same single cells. Shown is the relationship between phosphorylated ERK levels and AXL (left) or SOX10 (right) expression in individual primed (green points) and non-primed (gray points) cells.…”

“…The processes involved in the acquisition of stable drug resistance act both on short timescales (such as signaling) and on longer timescales (transcription). For instance, vemurafenib acts by inhibiting MAPK signaling, but the vemurafenib treatment itself relieves negative feedback on growth factor receptor signalling and allows ERK reactivation via BRAF V600E -independent routes (Gerosa et al, 2019;Lito et al, 2012) . Single cell analysis of ERK signaling has shown that individual cells vary dramatically in ERK activity following vemurafenib treatment with rare cells reactivating ERK to levels comparable to untreated cells.…”

Cracking the context-specific PI3K signaling code

“…These include (i) the use of CRISPR-mediated tagging of endogenous effector proteins, such as AKT or FOXO, to follow their dynamic translocation live and without stoichiometric changes and (ii) the integration of quantitative, multiplexed immunofluorescence in time course studies that seek to assess a wider repertoire of signaling responses at the single-cell level (65)(66)(67). Proof-of-concept studies from Sorger and his team illustrate the detailed cell signaling insight that can be obtained with some of these approaches and the translational potential of the acquired knowledge (62,68). Adoption of these methodologies will likely be instrumental in closing the PI3K signaling knowledge gaps that will be discussed next.…”

PI3K in stemness regulation: from development to cancer

“…Recent technological advances in high content imaging and -omics technologies are offering novel ways in which future studies may address the complexity of PI3K-mediated stemness, through a combination of conventional mechanistic studies and emerging systems biology strategies applied successfully in other areas [124][125][126][127]. To succeed in providing a unifying picture of PI3K-mediated stemness in development and cancer, such systems biology approaches will necessitate better interdisciplinary 'cross-talk' to combine the multifaceted mechanistic data on this pathway already available into comprehensive computational models.…”