Rapid activation of ARF6 after RAF inhibition augments BRAFV600E and promotes therapy resistance

Abstract

The intrinsic ability of cancer cells to evade death underpins tumorigenesis, progression, metastasis and the survival of drug-tolerant persister (DTP) cells. Herein, we discovered that when activated, the small GTPase ARF6 plays a central role in tumor survival by facilitating expression of the BRAF^V600E oncoprotein. Tumor-specific Arf6 deletion caused a significant reduction in BRAF^V600E protein and MAPK signaling and prevented rapid tumor progression. In the context of targeted therapy, BRAF inhibition induced swift activation of ARF6, driving a positive feedback loop that restored MAPK-driven anti-apoptotic signaling, facilitated DTP cell survival during the early phases of treatment and contributed to drug-tolerant growth. In patient-derived melanoma cells with innate or clinically acquired resistance to MAPK inhibitors, ARF6 inhibition enhanced sensitivity to combined BRAF + MEK inhibition. Collectively, these findings elucidate an ARF6-dependent mechanism of BRAF oncoprotein synthesis that may be exploited in BRAF^V600E driven cancers as a therapeutic vulnerability.

Figure 1. ARF6 is sufficient to control oncogenic BRAF protein levels through protein translation.

a, Relative amount of MAPK signaling proteins in tumor cells derived from Braf^V600E/ Cdkn2a^f/f mice detected by Reverse Phase Protein Array, two-tailed t-test. n= 3 replicates per cell line. b-h, Western Blot for indicated proteins. b, murine melanoma cells derived from Braf^V600E/ Cdkn2a^f/f mice. n=3 biological independent experiments c, dox-inducible ectopic expressed ARF6^Q67L. n=3 biological independent experiments d, Western blot for indicated proteins in UACC.62 and A375 cells with or without adenoviral-mediated ectopic expression of ARF6^Q67L, control= empty vector. e, 4μM QS11 for 48h in A2058, HT-29, and DBTRG-05MG. 2μM QS11 for 24h other cell lines f, 2μM QS11. g, 17-AAG and QS11 for 24h in A375 with doxycycline-inducible ectopic expressed ARF6^Q67L. h, 20μg/ml cycloheximide (CHX) in A375 with doxycycline-inducible ectopic expressed ARF6^Q67L. BRAF^V600E protein quantification at 48h. n=3 biological independent experiments. i, Quantitative RT-PCR for BRAF mRNA in A375 with doxycycline-inducible ectopic expressed ARF6^Q67L, n=3 biological independent experiments. b, c, h, Two-tailed ratio paired t-test.

Figure 2. ARF6 is necessary to control oncogenic BRAF protein levels.

a, Representative immunofluorescence images of cryo-embedded frozen tumor tissues from Braf^V600E/Cdkn2a^f/f mice, 1200X magnification. b-e, Western blot for indicated proteins. b, murine melanoma cells derived from Braf^V600E/ Cdkn2a^f/f mice, n=3 biological independent experiments. c, 10μM SecinH3, BRAF^V600E protein quantification at 48h, n=3 biological independent experiments. d, 5μM NAV-2729. e, A375 cells with or without adenoviral-mediated ectopic expression of ARF6^T27N, control= empty vector. a, Two-tailed unpaired t-test. b, e, Two-tailed ratio paired t-test.

Figure 3. ARF6 promotes tumor survival and accelerated disease progression.

a, Apoptotic protein profile of tumor cells derived from Braf^V600E/ Cdkn2a^f/f mice detected by Reverse Phase Protein Array, two-tailed t-test. n= 3 replicates per cell line. b, Western Blot for indicated proteins in murine melanoma cells derived from Braf^V600E/ Cdkn2a^f/f mice, n=3 biological independent experiments. Two-tailed ratio paired t-test. c, Apoptosis detection, measured at 48h, dox-inducible ectopic expressed ARF6^WT and ARF6^Q67L in A375 cells, n=4 replicates per condition, One-way ANOVA with multiple comparisons. d, Apoptosis detection, 4μM QS11, measured at 48h, n=5 for A375 and n=3 for A2058 replicates per condition, Two-tailed unpaired t-test. e, Cell viability detection, measured at 72h, n=5 replicates per condition. Two-tailed unpaired t-test. f, Rate of tumor growth measured from the time of initial detection in Dct::TVA;Braf^V600E;Cdkn2a^f/f mice, n= 24 Pten^WT, n=14 Pten^f/f mice, two-tailed t-test with Welch’s correction. g, Rate of tumor growth measured from the time of initial detection in Dct::TVA;Braf^V600E;Cdkn2a^f/f;Pten^f/f mice, n = 14 Arf6^WT, n = 22 Arf6^f/f mice, two-tailed t-test with Welch’s correction. h, Survival of mice (before primary tumor reached 2 cm) after Cre injection (day 0) within 130 days, n = 14 Arf6^WT, n = 18 Arf6^f/f mice, Log-rank (Mantle-Cox) test. Solid line withing data points = mean. i, Apoptotic protein profile of tumors from Dct::TVA;Braf^V600E;Cdkn2a^f/f;Pten^f/f mice (n=6 mice per group) detected by Reverse Phase Protein Array, two-tailed t-test.

Figure 4. ARF6 activation protects against MAPKi-induced apoptosis and promotes the development of MAPKi-resistance cells.

a-b, Total ARF6 and ARF6-GTP pulldown in A375, 5μM vemurafenib for 4h, Dabrafenib treatment for 4h,PF-07799933 treatment for 2h in 0%FBS media. b, 5μM vemurafenib. c-f, Apoptosis detection. c, 1μM Vemurafenib, dox-inducible ectopic expressed ARF6^WT and ARF6^Q67L in A375, apoptosis measured at 48h, Ctrl= no doxycycline, n=5 replicates per condition. d, 1μM Vemurafenib, 4μM QS11 for A375, n=4 replicates per condition, apoptosis measured at 48h, 2μM Vemurafenib, 4μM QS11 for UACC.62, n=3 replicates per condition, apoptosis measured at 24h. e, 1.25μM Dabrafenib, 0.0625μM Trametinib, doxinducible ectopic expressed ARF6^WT and ARF6^Q67L in A375, apoptosis measured at 48h, Ctrl= no doxycycline, n=3 replicates per condition. f, 1.25μM Dabrafenib, 0.0625μM Trametinib, 4μM QS11, apoptosis measured at 48h, n=3 replicates per condition. g, Western Blot for indicated proteins. 1μM Vemurafenib, 4μM QS11 in A375. 2μM Vemurafenib, 4μM QS11 in UACC.62. h-i, Colony outgrowth assay in A375. h, 1μM Vemurafenib, 4μM QS11, for 30 days. i, 250nM Dabrafenib, 12.5nM Trametinib, 2μM QS11, 4μM QS11, for 30 days. c-f, One-way ANOVA with multiple comparisons. h-i, Two-tailed unpaired t-test. n=4 biological independent experiments.

Figure 5. ARF6 inhibition sensitizes MAPKi-resistant cells.

a, Schematics of in vivo and in vitro experiments with patient-derived xenograft cell lines. b,Rate of tumor growth measurements started six days after initial engraftment of MTG013 cells [stably transduced with doxycycline-induced short hairpin RNA (shRNA) for ARF6] in NRG mice, n=10 controls, n=10 fed doxycycline chow (shARF6), two-tailed t-test with Welch’s correction. c, e, g, h, k, l, Cell viability detection measured at 48 hours patient-derived cell lines (see Supplementary Table 1). c, Dose response to Dabrafenib plus Trametinib (Dab+Tram) in MTG013, n= 5 replicates per condition. d, Schematic of cell viability assay. e-f, Doxycycline-induced shARF6. e, n=4 replicates per condition. f, Apoptosis detection. n= 3 replicates per condition. g, h, i, k, l, pharmacologic inhibition of ARF6. g, n= 5 replicates per condition. h, n= 5 replicates per condition. j,Dose response of Dab+Tram in MTG030. n= 5 replicates per condition. k, n=4 replicates per condition. l, n=4 replicates per condition. i, m, Colony outgrowth assay in MTG013 and MTG030 for 14 days. i, MTG013 treated with 5 μM Dabrafenib and 0.25μM Trametinib and/or 1.25 μM NAV-2729. n=4 biological independent experiments. m, MTG030 cells treated with 5 μM Dabrafenib and 0.25μM Trametinib, Ctrl= no doxycycline. n=4 biological independent experiments. e, f, g, h, j, k, l, One-way ANOVA with multiple comparisons. i, m, Two-tailed unpaired t-test.

Figure 6. Proposed model of ARF6-dependent drug tolerant persister cell survival.

Pharmacologic inhibition of BRAF^V600E induces ARF6 activation, triggering an adaptive stress response pathway that fortifies BRAF oncoprotein synthesis, reactivation of the MAPK pathway and DTP cell survival. Combined inhibition of ARF6 and MAPK signaling limits drug tolerance and enhances tumor cell death.