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A Nonquiescent "Idling" Population State in Drug-Treated, BRAF-Mutated Melanoma.

ABSTRACT: Targeted therapy is an effective standard of care in BRAF-mutated malignant melanoma. However, the duration of tumor remission varies unpredictably among patients, and relapse is almost inevitable. Here, we examine the responses of several BRAF-mutated melanoma cell lines (including isogenic subclones) to BRAF inhibitors. We observe complex response dynamics across cell lines, with short-term responses (<100 h) varying from cell line to cell line. In the long term, however, we observe equilibration of all drug-treated populations into a nonquiescent state characterized by a balanced rate of death and division, which we term the "idling" state, and to our knowledge, this state has not been previously reported. Using mathematical modeling, we propose that the observed population-level dynamics are the result of cells transitioning between basins of attraction within a drug-modified phenotypic landscape. Each basin is associated with a drug-induced proliferation rate, a recently introduced metric of an antiproliferative drug effect. The idling population state represents a new dynamic equilibrium in which cells are distributed across the landscape such that the population achieves zero net growth. By fitting our model to experimental drug-response data, we infer the phenotypic landscapes of all considered melanoma cell lines and provide a unifying view of how BRAF-mutated melanomas respond to BRAF inhibition. We hypothesize that the residual disease observed in patients after targeted therapy is composed of a significant number of idling cells. Thus, defining molecular determinants of the phenotypic landscape that idling populations occupy may lead to "targeted landscaping" therapies based on rational modification of the landscape to favor basins with greater drug susceptibility.

SUBMITTER: Paudel BB

PROVIDER: S-EPMC5883941 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundMost mutations in melanoma affect one critical amino acid on BRAF gene, resulting in the V600E substitution. Patient management is often based on the use of specific inhibitors targeting this mutation.MethodsDNA and RNA mutation status was assessed in 15 melanoma cell lines by Sanger sequencing and RNA-seq. We tested the cell lines responsiveness to BRAF inhibitors (vemurafenib and PLX4720, BRAF-specific and sorafenib, BRAF non-specific). Cell proliferation was assessed by MTT colorimetric assay. BRAF V600E RNA expression was assessed by qPCR. Expression level of phosphorylated-ERK protein was assessed by Western Blotting as marker of BRAF activation.ResultsThree cell lines were discordant in the mutation detection (BRAF V600E at DNA level/Sanger sequencing and BRAF WT on RNA-seq). We initially postulated that those cell lines may express only the WT allele at the RNA level although mutated at the DNA level. A more careful analysis showed that they express low level of BRAF RNA and the expression may be in favor of the WT allele. We tested whether the discordant cell lines responded differently to BRAF-specific inhibitors. Their proliferation rate decreased after treatment with vemurafenib and PLX4720 but was not affected by sorafenib, suggesting a BRAF V600E biological behavior. Yet, responsiveness to the BRAF specific inhibitors was lower as compared to the control. Western Blot analysis revealed a decreased expression of p-ERK protein in the BRAF V600E control cell line and in the discordant cell lines upon treatment with BRAF-specific inhibitors. The discordant cell lines showed a lower responsiveness to BRAF inhibitors when compared to the BRAF V600E control cell line. The results obtained from the inhibition experiment and molecular analyses were also confirmed in three additional cell lines.ConclusionCell lines carrying V600E mutation at the DNA level may respond differently to BRAF targeted treatment potentially due to a lower V600E RNA expression.

Project description:BACKGROUND:Approximately one third of women who develop melanoma at childbearing age are diagnosed during gestation or the postpartum period, facing pregnancy-associated melanoma (PAM). However, only some retrospective studies with heterogeneous data have analyzed the impact of pregnancy on melanoma development, and no evidence exists about the behavior and the management of BRAF-mutated disease. SUBJECTS, MATERIALS, AND METHODS:In order to better describe the evolution of BRAF V600E-mutated PAM, we present here all consecutive cases diagnosed in our site during the last 7?years, recording oncological, obstetrical, and perinatal parameters, as well as the therapeutic decisions for both melanoma and gestation. Based on our institutional experience, we weigh the current published evidence and discuss upcoming clinical considerations about the prognosis of PAM, the role of BRAF status, and the possible treatment options during pregnancy in localized or advanced/metastatic disease. Five women were diagnosed with newly metastatic or relapsed BRAF V600E-mutated PAM (four during gestation and one in the 1st year postpartum) between 2012 and 2019. All of them developed extensive metastatic disease with multiple organ involvement, and four developed brain metastases. All cases experienced melanoma progression in less than 6 months under targeted therapy and died soon independently of the followed sequence of treatments. All the neonates were delivered alive and healthy, but one developed melanoma earlier than the second year of life. RESULTS:Reviewing the literature to confirm our unfavorable outcomes, no specific data on BRAF-mutated PAM were retrieved and current evidence still supports that the prognosis of PAM should be guided by the established risk factors, whereas the management of advanced/metastatic PAM should be evaluated on a case-by-case basis. CONCLUSION:More data are required to ascertain whether BRAF-mutated profile adversely affects PAM outcome, although the clinicians should be aware to detect any potential melanoma lesion during pregnancy as soon as possible, treating it locally, regardless of its BRAF status. IMPLICATIONS FOR PRACTICE:The prognosis and management of pregnancy-associated melanoma whether BRAF-mutated or wild type, is currently guided by the same parameters as in the nonpregnant condition. In this special nontrial subpopulation, BRAF-mutated status seems to have a detrimental effect on disease outcome, independently of the following treatments. In early stage melanoma, wide local excision with or without sentinel lymph node dissection may be curative at any trimester of gestation, while in advanced/metastatic setting, therapeutic strategy including immune-checkpoint or BRAF/MEK inhibitors, is more challenging, regardless of BRAF status, and should be based on an individualized decision in each case at a multidisciplinary level.

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A Nonquiescent "Idling" Population State in Drug-Treated, BRAF-Mutated Melanoma.

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