Abstract
BackgroundKinase inhibition in the mitogen activated protein kinase (MAPK) pathway is a standard therapy for cancer patients with activating BRAF mutations. However, the anti-tumorigenic effect and clinical benefit are only transient, and tumors are prone to treatment resistance and relapse. To elucidate mechanistic insights into drug resistance, we have established an in vitro cellular model of MAPK inhibitor resistance in malignant melanoma.MethodsThe cellular model evolved in response to clinical dosage of the BRAF inhibitor, vemurafenib, PLX4032. We conducted transcriptomic expression profiling using RNA-Seq and RT-qPCR arrays. Pathways of melanogenesis, MAPK signaling, cell cycle, and metabolism were significantly enriched among the set of differentially expressed genes of vemurafenib-resistant cells vs control. The underlying mechanism of treatment resistance and pathway rewiring was uncovered to be based on non-genomic adaptation and validated in two distinct melanoma models, SK-MEL-28 and A375. Both cell lines have activating BRAF mutations and display metastatic potential.ResultsDownregulation of dual specific phosphatases, tumor suppressors, and negative MAPK regulators reengages mitogenic signaling. Upregulation of growth factors, cytokines, and cognate receptors triggers signaling pathways circumventing BRAF blockage. Further, changes in amino acid and one-carbon metabolism support cellular proliferation despite MAPK inhibitor treatment. In addition, treatment-resistant cells upregulate pigmentation and melanogenesis, pathways which partially overlap with MAPK signaling. Upstream regulator analysis discovered significant perturbation in oncogenic forkhead box and hypoxia inducible factor family transcription factors.ConclusionsThe established cellular models offer mechanistic insight into cellular changes and therapeutic targets under inhibitor resistance in malignant melanoma. At a systems biology level, the MAPK pathway undergoes major rewiring while acquiring inhibitor resistance. The outcome of this transcriptional plasticity is selection for a set of transcriptional master regulators, which circumvent upstream targeted kinases and provide alternative routes of mitogenic activation. A fine-woven network of redundant signals maintains similar effector genes allowing for tumor cell survival and malignant progression in therapy-resistant cancer.
Highlights
Kinase inhibition in the mitogen activated protein kinase (MAPK) pathway is a standard therapy for cancer patients with activating B-Raf proto-oncogene (BRAF) mutations
In MAPK signaling, combinations of specific inhibitors have proven to be superior to singleagent regimens: BRAF inhibitors (BRAFi) in combination with MEK inhibitors (MEKi) improved survival compared to single MAPK inhibitors (MAPKi) [7–10]
Generation of BRAFi-resistant melanoma cell lines The parental melanoma cell lines SK-MEL-28 and A375 were exposed to incrementally increasing concentrations of the mutant-BRAF inhibitor vemurafenib (Fig. 1a)
Summary
Kinase inhibition in the mitogen activated protein kinase (MAPK) pathway is a standard therapy for cancer patients with activating BRAF mutations. Therapy resistance in cancer Cancer drug resistance is a major obstacle in achieving durable clinical responses with targeted therapies. This highlights a need to elucidate the underlying mechanisms responsible for resistance and identify strategies to overcome this challenge. In malignant melanoma, activating point-mutations in the mitogen activated protein kinase (MAPK) pathway in BRAF kinase (B-Raf protooncogene, serine/threonine kinase, Gene ID: 673) [1–3] made it possible to develop potent kinase inhibitors matched to genotyped kinase mutations in precision medicine approaches [4–6]. Trametinib and cobimetinib target MAP2K7 (MEK, mitogen-activated protein kinase kinase 7, Gene ID: 5609), the BRAF target and downstream effector molecule. Disease progression will take place and patients relapse with lethal drug-resistant disease
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