Antiproliferative Effects of Continued Mitogen-Activated Protein Kinase Pathway Inhibition following Acquired Resistance to BRAF and/or MEK Inhibition in Melanoma

Abstract

Inhibitors of the mitogen-activated protein kinases (MAPK), BRAF, and MAP-ERK kinase (MEK) induce tumor regression in the majority of patients with BRAF-mutant metastatic melanoma. The clinical benefit of MAPK inhibitors is restricted by the development of acquired resistance with half of those who benefit having progressed by 6 to 7 months and long-term responders uncommon. There remains no agreed treatment strategy on disease progression in these patients. Without published evidence, fears of accelerated disease progression on inhibitor withdrawal have led to the continuation of drugs beyond formal disease progression. We now show that treatment with MAPK inhibitors beyond disease progression can provide significant clinical benefit, and the withdrawal of these inhibitors led to a marked increase in the rate of disease progression in two patients. We also show that MAPK inhibitors retain partial activity in acquired resistant melanoma by examining drug-resistant clones generated to dabrafenib, trametinib, or the combination of these drugs. All resistant sublines displayed a markedly slower rate of proliferation when exposed to MAPK inhibitors, and this coincided with a reduction in MAPK signaling, decrease in bromodeoxyuridine incorporation, and S-phase inhibition. This cytostatic effect was also associated with diminished levels of cyclin D1 and p-pRb. Two short-term melanoma cultures generated from resistant tumor biopsies also responded to MAPK inhibition, with comparable inhibitory changes in proliferation and MAPK signaling. These data provide a rationale for the continuation of BRAF and MEK inhibitors after disease progression and support the development of clinical trials to examine this strategy.

Mitogen-activated protein kinase signaling pathway in oral cancer (Review)

“…For instance, it is helpful to add a Ras inhibitor while keeping the patient on the Raf inhibitor with which they were already being treated. This was partially confirmed when the clinical benefits of continued ERK1/2 sub-pathway inhibition following cancer progression were demonstrated (77). However, several preclinical models revealed that discontinuation of a drug based on the inhibitors or intermittent scheduling may also slow tumor growth (78).…”

Increased MAPK reactivation in early resistance to dabrafenib/trametinib combination therapy of BRAF-mutant metastatic melanoma

“…The clarified cell suspension was maintained in DMEM/10% FCS containing penicillin and streptomycin (Gibco). Contaminating fibroblasts were removed by treating cultures with 100 mg ml À 1 G418 (Gibco) as required 58 . Cells were grown in DMEM with 10% FCS and glutamine (Gibco BRL) and cultured in a 37°C incubator with 5% CO 2 .…”

“…Pharmacological growth inhibition assays were performed using 1-2 Â 10 3 cells per well in 96-well plates and 24 h after seeding, serial dilutions of each inhibitor were added to cells. Cells were incubated for an additional 72 h and cell viability was measured using the Cell proliferation Aqueous MTS assay (Promega) on a VICTOR 2 Multilabel counter (PerkinElmer) 58 .…”

BRAF Inhibitor Resistance Mechanisms in Metastatic Melanoma: Spectrum and Clinical Impact

“…Cell culture, constructs, and lentivirus transductions SKMel28 and A375 melanoma cells were obtained from Prof. P. Hersey (Kolling Institute, University of Sydney, New South Wales) and short-term cultures were established from a subset of patients as previously described (30). Cells were grown in Dulbecco's Modified Eagle Medium (DMEM) with 10% FBS and glutamine (Gibco-BRL) and cultured in a 37 C incubator with 5% CO 2 .…”

“…Total cellular proteins were extracted and analyzed as previously described (30). Western blots were probed with the following antibodies: total ERK (137F5; Cell Signaling Technology), phosphorylated ERK (E-4; Santa Cruz Biotechnology), MYC (A-14; Santa Cruz Biotechnology), FLAG (Sigma-Aldrich), MEK1/2 (L38C12; Cell Signaling Technology), ATM (D2E2; Cell Signaling Technology) and b-actin (AC-74; Sigma-Aldrich).…”