COT drives resistance to RAF inhibition through MAP kinase pathway reactivation

Abstract

Oncogenic mutations in the serine/threonine kinase B-RAF (also known as BRAF) are found in 50-70% of malignant melanomas. Pre-clinical studies have demonstrated that the B-RAF(V600E) mutation predicts a dependency on the mitogen-activated protein kinase (MAPK) signalling cascade in melanoma-an observation that has been validated by the success of RAF and MEK inhibitors in clinical trials. However, clinical responses to targeted anticancer therapeutics are frequently confounded by de novo or acquired resistance. Identification of resistance mechanisms in a manner that elucidates alternative 'druggable' targets may inform effective long-term treatment strategies. Here we expressed ∼600 kinase and kinase-related open reading frames (ORFs) in parallel to interrogate resistance to a selective RAF kinase inhibitor. We identified MAP3K8 (the gene encoding COT/Tpl2) as a MAPK pathway agonist that drives resistance to RAF inhibition in B-RAF(V600E) cell lines. COT activates ERK primarily through MEK-dependent mechanisms that do not require RAF signalling. Moreover, COT expression is associated with de novo resistance in B-RAF(V600E) cultured cell lines and acquired resistance in melanoma cells and tissue obtained from relapsing patients following treatment with MEK or RAF inhibitors. We further identify combinatorial MAPK pathway inhibition or targeting of COT kinase activity as possible therapeutic strategies for reducing MAPK pathway activation in this setting. Together, these results provide new insights into resistance mechanisms involving the MAPK pathway and articulate an integrative approach through which high-throughput functional screens may inform the development of novel therapeutic strategies.

Figures

Figure 1. An ORF-based functional screen identifies COT and C-RAF kinases as drivers of resistance to B-RAF inhibition

1. Overview of the CCSB/Broad Institute Kinase ORF collection. Kinase classification and number of kinases per classification are noted.

2. A375 expressing the CCSB/Broad Institute Kinase ORF collection were assayed for relative viability in 1 µM PLX4720 and normalized to constitutively active MEK1 (MEK1DD). Nine ORFs (orange circles) scored 2 standard deviations (red dashed line, 58.64%) from the mean of all ORFs (green dashed line, 44.26%).

3. Indicated ORFs were expressed in 5 B-RAFV600E cell lines and treated with DMSO or 1 µM PLX4720. Viability (relative to DMSO) was quantified after 4 days. Error bars represent standard deviation between replicates (n=6).

Figure 2. Resistance to B-RAF inhibition via MAPK pathway activation

1. Indicated ORFs were expressed in A375. Levels of phosphorylated MEK and ERK were assayed following 18 h. treatment with DMSO (−) or PLX4720 (concentration noted).

2. Proliferation of A375 expressing indicated ORFs. Error bars represent standard deviation between replicates (n=6).

3. C-RAF (S338) and ERK phosphorylation in lysates from A375 expressing indicated ORFs.

4. COT expression in lysates from immortalized primary melanocytes expressing BRAFV600E or empty vector. COT mRNA has an internal start codon (30M) resulting in two protein products of different lengths; amino acids 1–467 or 30–467, noted with arrows.

5. COT and ERK phosphorylation in lysates from A375 expressing indicated ORFs following shRNA-mediated B-RAF depletion (shBRAF) relative to control shRNA (shLuc).

6. ERK phosphorylation in lysates from A375 expressing indicated ORFs following shRNA-mediated C-RAF depletion (shCRAF) or control shRNA (shLuc), following 18 h. treatment with DMSO (−) or 1 µM PLX4720 (+).

Figure 3. COT expression predicts resistance to B-RAF inhibition in cancer cell lines

1. MAP3K8/COT copy number; red bars: COT amplification, blue bars: non-amplified COT.

2. COT expression in B-RAFV600E cell lines and

3. short-term cultures.

4. PLX4720 GI50 in B-RAFV600E cell lines. Colors as in (a).

5. MEK and ERK phosphorylation following treatment with DMSO or PLX4720 (concentration indicated).

6. ERK phosphorylation in M307 lysates (AZD-R; AZD6244-resistant) treated with DMSO or 1 µM PLX4720 (PLX) or CI-1040 (CI).

7. COT mRNA expression (QRT/PCR) in patient/lesion-matched PLX4032-treated metastatic melanoma tissue samples. Pts. 1 and 3 had multiple biopsies from the same lesion. Error bars represent SEM (n=3). U; undetermined/undetectable.

8. ERK and MEK phosphorylation in RPMI-7951 following shRNA-mediated COT depletion (shCOT) versus control (shLuc) and treatment with DMSO (−) or 1 µM PLX4720 (+). ERK and MEK phosphorylation are quantified.

9. ERK and MEK phosphorylation in RPMI-7951 following 1 h. treatment with a small molecule COT kinase inhibitor. ERK and MEK phosphorylation are quantified.

Figure 4. COT-expressing B-RAFV600E cell lines exhibit resistance to allosteric MEK inhibitors

1. CI-1040 GI50 in a panel of B-RAFV600E cell lines; red bars: COT expression/amplification, blue bars: undetectable/non-amplified COT.

2. MEK and ERK phosphorylation in lysates from indicated cell lines treated with DMSO or CI-1040 (concentration noted).

3. Fold change (relative to MEK1) GI50 of A375 ectopically expressing the indicated ORFs for PLX4720, RAF265, CI-1040 and AZD6244.

4. ERK phosphorylation in A375 expressing indicated ORFs following treatment with DMSO or 1 µM of PLX4720, RAF265, CI-1040 or AZD6244.

5. Viability of A375 expressing the indicated ORFs and treated with DMSO, PLX4720 (concentration indicated) and PLX4720 in combination with CI-1040 or AZD6244 (all 1 µM). Error bars represent the standard deviation (n=6).

6. ERK phosphorylation in A375 expressing indicated ORFs following treatment with DMSO, PLX4720 (1 µM) or PLX4720 in combination with CI-1040 or AZD6244 (all 1 µM).