Suppression of RAC1-driven malignant melanoma by group A PAK inhibitors

Abstract

Activating mutations in the RAC1 gene have recently been discovered as driver events in malignant melanoma. Expression of this gene is associated with melanocyte proliferation, and melanoma cells bearing this mutation are insensitive to BRAF inhibitors such as vemurafenib and dabrafenib, and also may evade immune surveillance due to enhanced expression of PD-L1. Activating mutations in RAC1 are of special interest, as small-molecule inhibitors for the RAC effector p21-activated kinase (PAK) are in late-stage clinical development and might impede oncogenic signaling from mutant RAC1. In this work, we explore the effects of PAK inhibition on RAC1^P29S signaling in zebrafish embryonic development, in the proliferation, survival and motility of RAC1^P29S-mutant human melanoma cells, and on tumor formation and progression from such cells in mice. We report that RAC1^P29S evokes a Rasopathy-like phenotype on zebrafish development that can be blocked by inhibitors of PAK or MEK. We also found and that RAC1-mutant human melanoma cells are resistant to clinical inhibitors of BRAF but are uniquely sensitive to PAK inhibitors. These data suggest that suppressing the PAK pathway might be of therapeutic benefit in this type of melanoma.

Figure 1. RAC1^P29S yields a Rasopathy-like phenotype in zebrafish that requires PAK1 activation

One-cell embryos were injected with mRNA encoding the indicated genes. A) RNA expression of BRAF^V600E, KRAS 4A^G12V and Rac1^P29S produce phenotypes similar to Rasopathies at 12hpf in zebrafish embryos. B) Overexpression of proteins increases the activity of p-Erk and p-Pak 1/2; β-actin was used as a loading control. Transgenic fish were injected with Rac1 mRNA, C) and D) Tg (CMLC:GFP) embryos injected with Rac1^P29S were treated with 1µM inhibitors of RAC1 (NSC23766), BRAF (Vemurafenib), PAK1 (Frax-1036) or MEK (PD325901) at 72 hpf. Phase contrast and GFP images are shown in panel C, and quantitated in panel D. Embryo phenotypes were scored as mild abnormal if elongated (body axis ratio >1.1 but <1.3) and/or small or absent eye development was noted, and abnormal if boxy axis ration was >1.3 and/or pericardial edema was also noted.

Figure 2. PAK and MEK inhibitors block effects of RAC1 on zebrafish embryonic development

One-cell embryos were injected with mRNA encoding the indicated genes. 1 µM inhibitors were added to water between 4.5–5.5hpf, then removed and replaced with fresh water. Embryo morphology was scored at 12hpf by a blinded observer. A) Morphology at 12hpf. B) Quantitation of developmental abnormalities.

Figure 3. Effects of targeted inhibitors on proliferation of BRAF, NRAS, and RAC1-mutant melanoma cell lines

A) Western blots for activated PAK in the indicated cell lines. B) Cells were grown under standard conditions or treated with C) the MEK inhibitor PD325901 (100 nM), D) the PAK inhibitor Frax-1036 (100 nM), or E) the BRAF inhibitor vemurafenib (100 nM). Cell number was measured using an XCELLigence device.

Figure 4. Effects of targeted inhibitors on viability of BRAF, NRAS, and RAC1-mutant melanoma cell lines

Cells were grown under standard conditions and treated with either A) PAK inhibitor Frax-1036 (100 nM), B) the BRAF inhibitor vemurafenib (100 nM), C) the MEK inhibitor PD325901 (100 nM), or D) the RAC inhibitor NSC23766 (1 µM). Cell viability was determined by a redox indicator (Alamar Blue).

Figure 5. Effects of inhibitors on cell migration

Cell Culture Wound Closure Assay was developed in 6-well confluent plates. After the scratch the different cell lines were treated with 100 nM BRAF inhibitor (vemurafenib), Pak1 inhibitor (Frax-1036), or MEK inhibitor (PD325901). Snapshot images in an inverted microscope were taken after 24h. Migration was quantified by measuring the size of the cell-free area. % migration for each cell line was measured against control (untreated cells), with the latter defined as 100% migration for each cell line. Where significant, p values are shown comparing responses of a RAC1-mutant cell line (YUHEF) to a BRAF-mutant cell line (501mel).

Figure 6. Consequences of small molecule inhibitors on melanoma cell signaling

Cells were grown under standard conditions or treated with vehicle (control) or 100 nM of the indicated inhibitors for 24h. Lysates were analyzed by Western blot for PAK, ERK, and AKT activity.

Figure 7. Inhibition of xenograft growth by small molecule inhibitors

501mel (BRAF), YURIF (BRAF+RAC1) and YUHEF (RAC1) cells were subcutaneously injected into the flanks of Nude mice (cohorts of ten mice/cell line). Ten days post-inoculation, when the tumors reached a volume of at least 25 mm³, the animals were treated by oral gavage with inhibitors for 2 weeks. A) Volumetric changes in tumor size between untreated mice (vehicle) and mice treated with inhibitor (vemurafenib or Frax-1036). B) Tumor weight was measured after mice were sacrificed. p<0.01 and p<0.05 values were determined by Student’s t-test. C) Immunblots for markers of proliferation (PCNA and MCM2) and apoptosis (cleaved caspase 3).