Combined Pan-RAF and MEK Inhibition Overcomes Multiple Resistance Mechanisms to Selective RAF Inhibitors

Abstract

RAF and MEK inhibitors are effective in BRAF-mutant melanoma but not in BRAF-mutant colorectal cancer. To gain additional insights into this difference, we performed a genome-scale pooled shRNA enhancer screen in a BRAF-mutant, RAF inhibitor-resistant colorectal cancer cell line exposed to the selective RAF inhibitor PLX4720. We identified multiple genes along the receptor tyrosine kinase (RTK)/mitogen-activated protein kinase (MAPK) signaling axis that, when suppressed, either genetically or pharmacologically, sensitized cells to the selective RAF inhibitor through sustained inhibition of MAPK signaling. Strikingly, CRAF was a key mediator of resistance that could be overcome by the use of pan-RAF inhibitors in combination with a MEK inhibitor. Furthermore, the combination of pan-RAF and MEK inhibitors displayed strong synergy in melanoma and colorectal cancer cell lines with RAS-activating events such as RTK activation, KRAS mutation, or NF1 loss-of-function mutations. Combinations of selective RAF inhibitors, such as PLX4720 or dabrafenib, with MEK inhibitors did not incur such profound synergy, suggesting that inhibition of CRAF by pan-RAF inhibitors plays a key role in determining cellular response. Importantly, in contrast to the modest activity seen with single-agent treatment, dual pan-RAF and MEK inhibition results in the induction of apoptosis, greatly enhancing efficacy. Notably, combined pan-RAF and MEK inhibition can overcome intrinsic and acquired resistance to single-agent RAF/MEK inhibition, supporting dual pan-RAF and MEK inhibition as a novel therapeutic strategy for BRAF- and KRAS-mutant cancers.

Figure 1. A synthetic lethal shRNA screen identifies enhancers of BRAF inhibition in colorectal cancer

(A) BRAF-mutant melanoma and colorectal cancer cell lines were profiled for sensitivity to PLX4720 following a 4 d exposure. GI₅₀ values were determined using non-linear regression analysis in GraphPad Prism. Box and whisker plots represent the median values, 25^th and 75^th percentiles and min/max. (B) Cell lines were treated with PLX4720 and ERK phosphorylation was determined by in-cell Western. Results are presented as a heat map with PLX4720 GI₅₀ values plotted for comparison. (C) Protocol for a synthetic lethal RNAi screen in RKO colorectal cancer cells for modifiers of sensitivity to PLX4720. (D) Proliferation of RKO cells infected with the shRNA library and cultured in the presence of either DMSO or 3 μM PLX4720. (E) Pearson correlation was determined between experimental replicates for each condition in the screen to assess reproducibility. (F) Following 16 population doublings, the abundance of each shRNA was determined and used to generate a ranked list of shRNAs based upon the log-fold change between the DMSO- and PLX4720-treated replicates. RIGER analysis was employed to rank synthetic lethal genes according to the second best hairpin metric. The top ten ranking genes are indicated.

Figure 2. Validation of candidate synthetic lethal genes

(A) RKO cells were infected with individual lentiviral shRNA expression vectors targeting high-ranking genes from the primary screen or a control shRNA targeting luciferase. Cells were treated with increasing concentrations of PLX4720 for 4 d. Cell proliferation was determined using the CellTiter-Glo assay. GI₅₀ values were determined using GraphPad Prism. (B) RKO cells were infected as in (A) then, 72 h after infection, were treated with 3 μM PLX4720 for 18 h and protein lysates were analysed by Western blotting for the indicated proteins.

Figure 3. PLX4720-resistant cells are exquisitely sensitive to combined pan-RAF and MEK inhibition

(A) The sensitivity of BRAF mutant melanoma and colorectal cancer cell lines treated with either AZ628 or AZD6244 was assessed following a 96 h treatment with a 10-point titration of the drugs. CellTiter-glo data was used to generate GI₅₀ values. (B) RKO and LOXIMVI cells were treated with a titration of AZD6244 in the presence of either DMSO vehicle, 10 nM AZ628 or 100 nM AZ628 for 96 h. Cell proliferation was assessed by CellTiter-glo. (C) RKO and LOXIMVI cells were treated with 300 nM AZD6244, 30 nM AZ628 alone or in combination. Cells were cultured for 7–10 days and then stained with crystal violet. Cell proliferation was quantified by measuring the absorbance of solubilized crystal violet stain.

Figure 4. Combinatorial inhibition of BRAF, CRAF and MEK is synergistic in cell lines displaying intrinsic resistance to PLX4720

(A) RKO, LOXIMVI, WiDr, A375 and COLO205 cancer cell lines were treated with 1 μM AZD6244, 0.1 μM AZ628, 10 μM PLX4720 or a combination of AZD6244 with either AZ628 or PLX4720 for 18 h. Cell lysates were analyzed by Western blotting for the indicated proteins. (B) Cell lines were treated as in (A) for 96 h and cell proliferation was assessed by CellTiter-Glo assay. (C) Cells were treated with a titration of AZD6244 versus a titration of either AZ628 or PLX4720 in combination for 96 h. Cell proliferation was assessed using CellTiter-Glo and presented in the blue-red heat maps. The green-red heat maps present the degree of synergistic interaction between the compounds, as determined using the ‘Bliss Independence model’ where values greater than 0 indicate an effect greater than the combined fractional inhibition of the single agents, indicative of synergy.

Figure 5. RAS activation is associated with sensitivity to combined pan-RAF and MEK inhibition

(A) Synergy between AZD6244 and AZ628 was determined using the Bliss independence model in colorectal and melanoma cell lines with genetic lesions in BRAF, NF1, KRAS or NRAS (red dots indicate mutation, black indicate wild type). The sum of excess over Bliss was calculated across the matrix of concentrations tested. Values in excess of 2, indicated by the dotted red line, indicated a synergistic response. (B) A375 cells were infected with lentiviral expression vectors encoding ORFs for LacZ and KRAS^G12V or shRNAs for luciferase and NF1. After 72 h, cells were treated with 1 μM AZD6244, 0.1 μM AZ628 or a combination of both compounds for 96 h. Cell proliferation was assessed using CellTiter-Glo. (C) A375 cells were infected with lentiviral expression vectors as in (B). After 72 h, cells were treated with 1 μM AZD6244, 0.1 μM AZ628 or a combination of both compounds for 18 h. Cell lysates were analyzed for the indicated proteins.

Figure 6. Pan-RAF and MEK inhibition is cytotoxic and induces apoptosis

(A) RKO, WIDR and HCT116 cells were treated with 1 μM AZD6244, 0.1 μM AZ628 or a combination of the two for 96 h. Cell proliferation was determined relative to the starting number of cells prior to treatment. Genes known to play a role in resistance to BRAF inhibition are indicated in red for each cell line. (B) RKO, WIDR and HCT116 cells were treated as in (A) and after 72 h cytotoxicity was determined relative to the proportion of viable cells using the Cytotox-Glo assay. (C) Cells were treated as in (B) and after 72 h cell lysates were generated and analyzed by Western blotting for the stated proteins.