Paradox-breaking RAF inhibitors that also target SRC are effective in drug-resistant BRAF mutant melanoma

Abstract

BRAF and MEK inhibitors are effective in BRAF mutant melanoma, but most patients eventually relapse with acquired resistance, and others present intrinsic resistance to these drugs. Resistance is often mediated by pathway reactivation through receptor tyrosine kinase (RTK)/SRC-family kinase (SFK) signaling or mutant NRAS, which drive paradoxical reactivation of the pathway. We describe pan-RAF inhibitors (CCT196969, CCT241161) that also inhibit SFKs. These compounds do not drive paradoxical pathway activation and inhibit MEK/ERK in BRAF and NRAS mutant melanoma. They inhibit melanoma cells and patient-derived xenografts that are resistant to BRAF and BRAF/MEK inhibitors. Thus, paradox-breaking pan-RAF inhibitors that also inhibit SFKs could provide first-line treatment for BRAF and NRAS mutant melanomas and second-line treatment for patients who develop resistance.

Graphical abstract

Figure 1

CCT196969 and CCT241161 Are BRAF Inhibitors (A) Chemical structures of CCT196969 and CCT241161. (B) Efficacy of PLX4720, CCT196969, and CCT241161 (1 μM) against a panel of 63 protein kinases. Color bar shows percent activity compared to DMSO. (C) Phospho-MEK (pMEK), phospho-ERK (pERK), and ERK2 in WM266.4 and D35 cells treated for 24 hr with DMSO (D), CCT196969, or CCT241161. (D) BRAF^V600E A375 cell proliferation assay (CellTiter Glo) with PLX4720, CCT196969, or CCT241161. (E) BRAF^V600E A375 xenograft growth in nude mice treated with vehicle, CCT196969, or CCT241161 14 days after cell injection. ^∗∗∗p ≤ 0.001 (t test, two-tailed). (F) BRAF^V600E and BRAF^T529N,V600E kinase inhibition by CCT196969 and CCT241161. (G) Growth of Ba/F3 cells ([³H]-thymidine incorporation) expressing BRAF^V600E or BRAF^T529N,V600E treated with CCT196969 or CCT241161. Bars represent SEM. See also Figure S1 and Tables S1–S3.

Figure 2

CCT196969 and CCT241161 Inhibit RAS Mutant Cells (A) Cell growth inhibition by CCT196969 or CCT241161 (expressed as log₂ GI₅₀ in micromolar) in cells carrying BRAF (red), RAS (blue), or neither (green) mutation. ^∗, melanoma cell line; ˆ, colorectal carcinoma cell line; >, breast cancer cell line. WT, wild-type. (B) Heat map showing sensitivity of cancer cell lines bearing mutations in BRAF, NRAS, or KRAS (shown in the grid below the heat map) presented as GI₅₀ values determined after a 5-day exposure to each compound (BRAF inhibitors PLX4720 and SB590885, MEK inhibitor PD184352, and our compounds CCT241161 and CCT196969) and analysis by sulphorhodamine B staining. Values were log₂-transformed, and hierarchical clustering was performed with “one minus Pearson correlation” using Gene E (www.broadinstitute.org/cancer/software/GENE-E/). (C) Phospho-MEK (pMEK), phospho-ERK (pERK), and ERK2 in D04 cells treated for 24 hr with DMSO (D), CCT196969, or CCT241161. (D) NRAS mutant D04 cell proliferation assay (CellTiter Glo) with PLX4720, CCT196969, or CCT241161. (E) NRAS mutant D04 xenograft growth in nude mice treated with vehicle, PLX4720, CCT196969, or CCT241161 10 days after cell injection. ^∗∗∗p ≤ 0.001 (t test, two-tailed). Bars represent SEM. See also Figure S2.

Figure 3

CCT196969 and CCT241161 Inhibit SFK in Patient-Derived Resistant Cells (A) A375 cell colony formation with DMSO, PLX4720, CCT196969, or CCT241161 (0.5 μM) after 7 or 20 days. (B) A375/R cell proliferation assay (CellTiter Glo) with PLX4720, CCT196969, and CCT241161. (C) A375/R xenograft growth in nude mice treated with vehicle, PLX4720, CCT196969, or CCT241161. ^∗∗∗p ≤ 0.001 (t test, two-tailed). (D) Patient #1 PDX growth in NSG mice treated with PLX4720. (E) Growth of PLX4720-resistant PDX from patient #1, from (D), in mice treated with PLX4720, CCT196969, or CCT241161 7 days after cell injection. ^∗∗∗p ≤ 0.001 (t test, two-tailed). (F) Patient #2 cell proliferation assay (CellTiter Glo) with PLX4720, CCT196969, or CCT241161. (G) RPPA quantification for pMEK, pERK, and pSFK in three vemurafenib-resistant patient-derived cell lines treated with DMSO (vehicle), PLX4720, CCT196969, and CCT241161 (1 μM; 4 hr). (H) pMEK, pERK, ERK2, pSFK, and SRC in patient #2 cells treated with DMSO, PLX4720, CCT196969, or CCT241161 (1 μM; 4 hr). (I) PARP and caspase 3 in patient #2 cells treated with DMSO, PLX4720, CCT196969, or CCT241161 (1 μM; 4 hr). (J) Patient #2 cell xenograft growth in nude mice treated with vehicle, PLX4720, CCT196969, or CCT241161 16 days after tumor implant. ^∗∗∗p ≤ 0.001 (t test, two-tailed). Bars represent SEM. See also Figure S3 and Tables S4 and S5.

Figure 4

CCT196969 and CCT241161 Inhibit Tumors with Acquired Resistance to Vemurafenib (A) pERK and pSFK in pre- and postvemurafenib treatment tumors from patient #3. Scale bars, 50 μm. (B) Patient #3 tumor cell proliferation assay (CellTiter Glo) with PLX4720, CCT196969, and CCT241161. (C) pERK, ERK2, and pSFK in patient #3 PDX in mice treated with PLX4720, CCT196969, or CCT241161. (D) Growth of patient #3 PDX in NSG mice treated with PLX4720, CCT196969, or CCT241161. (E) pERK and pSFK in pre- and post-vemurafenib treatment tumors from patient #4. Scale bars, 100 μm. (F) Growth of patient #4 PDX in NSG mice treated with PLX4720, CCT196969, or CCT241161 21 days after tumor implant. ^∗∗∗p ≤ 0.001 (t test, two-tailed). Bars represent SEM. See also Figure S4.

Figure 5

CCT196969 and CCT241161 Inhibit PDX from a Patient with Intrinsic Resistance to Vemurafenib (A) pERK and pSFK in pre- and post-vemurafenib treatment tumors from patient #5. Scale bars, 50 μm. (B) Growth of cell lines from patient #5 tumor (CellTiter Glo) with PLX4720, CCT196969, and CCT241161. (C) Growth of PDX from patient #5 in NSG mice treated with PLX4720, CCT196969, or CCT241161 21 days after tumor implant. ^∗∗∗p ≤ 0.001 (t test, two-tailed). Bars represent SEM. See also Figure S5.

Figure 6

CCT196969 and CCT241161 Inhibit Tumors with Acquired Resistance to Dabrafenib plus Trametinib (A) pERK and pSFK in PDX from patient #13 before and after treatment with dabrafenib plus trametinib. Scale bars, 100 μm. (B) Sequencing electropherograms confirming BRAF^V600E and NRAS^Q61R mutations in PDX from patient #13. (C) Growth of PDX from patient #13 in NSG mice treated with dabrafenib plus trametinib (dab + tram), CCT196969, or CCT241161 21 days after tumor implant. ^∗∗∗p ≤ 0.001 (t test, two-tailed). Bars represent SEM. See also Figure S6.

Figure 7

Model Showing Targets of Vemurafenib, Dabrafenib, Trametinib, CCT196969, and CCT241161