Targeting CDK4/6 Represents a Therapeutic Vulnerability in Acquired BRAF/MEK Inhibitor-Resistant Melanoma

Abstract

There is a clear need to identify targetable drivers of resistance and potential biomarkers for salvage therapy for patients with melanoma refractory to the combination of BRAF and MEK inhibition. In this study, we performed whole-exome sequencing on BRAF-V600E-mutant melanoma patient tumors refractory to the combination of BRAF/MEK inhibition and identified acquired oncogenic mutations in NRAS and loss of the tumor suppressor gene CDKN2A We hypothesized the acquired resistance mechanisms to BRAF/MEK inhibition were reactivation of the MAPK pathway and activation of the cell-cycle pathway, which can both be targeted pharmacologically with the combination of a MEK inhibitor (trametinib) and a CDK4/6 inhibitor (palbociclib). In vivo, we found that combination of CDK4/6 and MEK inhibition significantly decreased tumor growth in two BRAF/MEK inhibitor-resistant patient-derived xenograft models. In vitro, we observed that the combination of CDK4/6 and MEK inhibition resulted in synergy and significantly reduced cellular growth, promoted cell-cycle arrest, and effectively inhibited downstream signaling of MAPK and cell-cycle pathways in BRAF inhibitor-resistant cell lines. Knockdown of CDKN2A in BRAF inhibitor-resistant cells increased sensitivity to CDK4/6 inhibition alone and in combination with MEK inhibition. A key implication of our study is that the combination of CDK4/6 and MEK inhibitors overcomes acquired resistance to BRAF/MEK inhibitors, and loss of CDKN2A may represent a biomarker of response to the combination. Inhibition of the cell-cycle and MAPK pathway represents a promising strategy for patients with metastatic melanoma who are refractory to BRAF/MEK inhibitor therapy.

Trial registration: ClinicalTrials.gov NCT02159066 NCT00246506 NCT02645149.

©2021 American Association for Cancer Research.

Figures

Figure 1.

Patients with BRAF-V600E–mutant melanoma refractory to BRAFi/MEKi therapy harbor mutations in NRAS and CDKN2A and dual inhibition of MEK and CDK4/6 overcomes BRAFi/MEKi acquired resistance in PDX models. A, Schematic representation of experimental design and work flow. B and C, Whole-exome sequencing (WES) was performed on tumor samples from two patients were collected at the indicated time points (before, during and upon resistance to BRAF/MEK inhibition; PD, progressive disease). Two PDX models were established. D and E, Mice bearing BRAFi/MEKi-resistant PDX tumors were treated with vehicle, MEK inhibitor (trametinib, 0.5 mg/kg daily dose), CDK4/6 inhibitor (palbociclib, 75 mg/kg daily dose) alone or in combination (error bars, ± SEM). F, Western blotting of PDX tumors harvested at end of study (30 days), representative images are shown. G, Western blot quantifications of the band densities of phospho-RB-S807 and phospho-ERK were quantitated and normalized to those of the corresponding loading control, β-tubulin, from Fig. 1F of PDX tumors harvested at the end of study (30 days). Representative images are shown and the data expressed as the mean ± SEM, n = 3. H, Quantification of IHC of phospho-ERK from MB1998 PDX tumors harvested at the end of study (error bars, ± SEM). I, Quantification of IHC of Ki-67 from MB1998 PDX tumors harvested at the end of study (error bars, ± SEM). J, Quantification of IHC of phospho-ERK from MB2132 PDX tumors harvested at the end of study (error bars, ± SEM). K, Quantification of immunohistochemistry of Ki-67 from MB2132 PDX tumors harvested at the end of study (error bars, ± SEM). Asterisks (*) indicate *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001; n.s., not significant.

Figure 2.

In vitro cell line models of acquired resistance to vemurafenib are more sensitive to CDK4/6i alone or in combination with MEKi. A and B, A375 and A375-VR cell proliferation (nuclear count) was measured every 6 hours using an IncuCyte for 7 days, where cells were treated with MEKi (trametinib; A) or CDK4/6i (palbociclib; B). Results shown are mean ± SEM of three independent experiments performed in triplicate. C, IC50 values we calculated for A375 and A375-VR cells treated with CDK4/6i single agent, analyzed by three independent IncuCyte experiments performed in triplicate (from Fig. 3A). IC50 values are normalized to SKMEL28, with IC50 values represented in the legend, and fold change is noted above the bars. D and E, A375 (2D) and A375-VR (2E) cell proliferation (nuclear count) was measured every 6 hours using an IncuCyte for 7 days, where cells were treated with single agent and the combination of CDK4/6i and MEKi. Results shown are mean ± SEM of three independent experiments performed in triplicate. F, Cell cycle was analyzed after treating cells with 5 nmol/L MEKi (trametinib), 0.5 μmol/L CDK4/6i (palbociclib) or the combination for 24 hours and analyzed by propidium iodide (PI) staining. The relative distribution of cells in the G1, S, and G2–M phases of the cell cycle are shown (n = 3, error bars, ± SEM). Black stars indicate a significant increase in G1 phase together with a significant decrease in S-phase compared with vehicle (DMSO). *, P ≤ 0.05. G, Cells were treated with 5 nmol/L MEKi (trametinib), 0.5 μmol/L CDK4/6i (palbociclib), or the combination 24 hours prior to collection. Western blot analysis was performed on cell lysates as indicated. Blots shown are representative images from three independent experiments.

Figure 3.

BRAFi-resistant CDKN2A-WT SKMEL28-VR cells are sensitive to CDK4/6i alone and in combination with MEKi. A, Cell growth of SKMEL28 and SKMEL28-VR cells treated with single-agent MEKi (trametinib) or CDK4/6i (palbociclib) for 5 days, measured by BrdU. Results shown are the mean ± SEM of three independent experiments performed in triplicate. B, IC50 values for SKMEL28 and SKMEL28-VR cells treated with CDK4/6i single agent (from Fig. 3A), graphed normalized to SKMEL28, with fold change values denoted above the black arrows and with IC50 values represented in the legend. IC50 values calculated in GraphPad. C and D, Top, cell growth of SKMEL28 (3C) or SKMEL28-VR (3D) cells treated with the combination of CDK4/6i and MEKi for 5 days. Results shown are mean ± SEM of three independent experiments performed in triplicate. Bottom, combination index (CI) values for the combination or CDK4/6i and MEKi, calculated using CalcuSyn software from 5-day BrdU growth assays for SKMEL28 (C) or SKMEL28-VR (D). White boxes correspond to combination concentrations that are synergistic, between 0 and 0.3 (CI values: synergism: 0–0.3; slight synergism: 0.3–0.9; additive: 0.9–1.10; slight antagonism: 1.10–1.45; antagonism: 1.45–>10.). E, Cell cycle was analyzed after treating cells with 5 nmol/L MEKi (trametinib), 0.5 μmol/L CDK4/6i (palbociclib) or the combination for 24 hours and analyzed by propidium iodide (PI) staining. The relative distribution of cells in the G1, S, and G2–M phases of the cell cycle are shown (n = 3, error bars, ± SEM). Black stars indicate a significant increase in G1 phase together with a significant decrease in S-phase compared with vehicle (DMSO), *, P ≤ 0.05. F, Cells were treated with 5 nmol/L MEKi (trametinib), 0.5 μmol/L CDK4/6i (palbociclib) or the combination 24 hours prior to collection. Western blot analysis was performed on cell lysates as indicated. Blots shown are representative images from three independent experiments.

Figure 4.

Loss of CDKN2A/p16INK4A expression increases sensitivity of BRAFi-resistant cells to single-agent CDK4/6i. A, Knockdown of CDKN2A using two shRNAs targeting CDKN2A (shCDKN2A_15 and shCDKN2A_49) and a negative control nontargeting shRNA (shScr) in SKMEL28-VR cells. CDK2NA knockdown mRNA validation measured by qRT-PCR. CDKN2A mRNA expression levels normalized to β-actin, graphed as the relative fold change normalized to SKMEL28-VR shScr control cells. B, Analysis of p16INK4A and RB expression via Western blot analysis in SKMEL28-VR shScr and shCDKN2A cells (left). Western blot quantifications of the band densities of phospho-RBS807, phospho-ERK and cyclin D1, were quantitated and normalized to those of the corresponding loading control β-tubulin. The data expressed as the mean ± SEM, n = 3. C, Cell growth of SKMEL28-VR-shScr and VR-shCDKN2A cells treated with single-agent CDK4/6i (palbociclib) for 5 days, measured by BrdU. Results shown are the mean ± SEM of three independent experiments performed in triplicate. D, IC50 values for SKMEL28-VR-shCDKN2A cells treated with CDK4/6i (palbociclib) single agent (from C), graphed normalized to SKMEL28-VR-shScr, with fold change values denoted below the black arrows and with IC50 values represented in the legend. IC50 values calculated in GraphPad. E, Cell growth of SKMEL28-VR shScr, shCDKN2A cells treated with single-agent MEKi (trametinib) for 5 days, measured by BrdU. Results shown are the mean ± SEM of three independent experiments performed in triplicate. F, IC50 values for SKMEL28-VR shCDKN2A cells treated with MEKi single agent (from E), graphed normalized to SKMEL28-VR shScr, with fold change values denoted below the black arrows, with IC50 values represented in the legend. IC50 values calculated in GraphPad.

Figure 5.

Loss of CDKN2A/p16 expression results in a synergistic growth response to the combination of CDK4/6i and MEKi in BRAFi-resistant cells. A–C, Top, cell growth of SKMEL28-VR shScr (A), SKMEL28-VR shCDKN2A_15 (B), or SKMEL28-VR shCDKN2A_49 (C) cells treated with the combination of CDK4/6i and MEKi for 5 days. Results shown are mean ± SEM of three independent experiments performed in triplicate. Bottom, combination index (CI) values for the combination or CDK4/6i and MEKi, calculated using CalcuSyn software from 5-day BrdU growth assays for SKMEL28-VR shScr (A), SKMEL28-VR shCDKN2A_15 (B) or SKMEL28-VR shCDKN2A_49 (C; CI values: synergism: 0–0.3; slight synergism: 0.3–0.9; additive: 0.9–1.10; slight antagonism: 1.10–1.45; antagonism: 1.45–>10.). D, Cell cycle was analyzed after treating SKMEL28-VR shCDKN2A cells with 5 nmol/L MEKi (trametinib), 0.5 μmol/L CDK4/6i (palbociclib), or the combination for 24 hours and analyzed by propidium iodide (PI) staining. The relative distribution of cells in the G1, S, and G2–M phases of the cell cycle are shown. (n = 3, error bars, ± SEM). Black stars indicate a significant increase in G1 phase together with a significant decrease in S-phase compared with vehicle (DMSO). *, P ≤ 0.05. E, SKMEL28-VR shCDKN2A cells were treated with 5 nmol/L MEKi (trametinib), 0.5 μmol/L CDK4/6i (palbociclib), or the combination 24 hours prior to collection. Western blot analysis was performed on cell lysates as indicated. Blots shown are representative images from two independent experiments.