Targeting B-Raf inhibitor resistant melanoma with novel cell penetrating peptide disrupters of PDE8A - C-Raf

Abstract

Background: Recent advances in the treatment of melanoma that involve immunotherapy and B-Raf inhibition have revolutionised cancer care for this disease. However, an un-met clinical need remains in B-Raf inhibitor resistant patients where first-generation B-Raf inhibitors provide only short-term disease control. In these cases, B-Raf inhibition leads to paradoxical activation of the C-Raf - MEK - ERK signalling pathway, followed by metastasis. PDE8A has been shown to directly interact with and modulate the cAMP microdomain in the vicinity of C-Raf. This interaction promotes C-Raf activation by attenuating the PKA-mediated inhibitory phosphorylation of the kinase.

Methods: We have used a novel cell-penetrating peptide agent (PPL-008) that inhibits the PDE8A - C-Raf complex in a human malignant MM415 melanoma cell line and MM415 melanoma xenograft mouse model to investigate ERK MAP kinase signalling.

Results: We have demonstrated that the PDE8A - C-Raf complex disruptor PPL-008 increased inhibitory C-Raf-S259 phosphorylation and significantly reduced phospho-ERK signalling. We have also discovered that the ability of PPL-008 to dampen ERK signalling can be used to counter B-Raf inhibitor-driven paradoxical activation of phospho-ERK in MM415 cells treated with PLX4032 (Vemurafenib). PPL-008 treatment also significantly retarded the growth of these cells. When applied to a MM415 melanoma xenograft mouse model, PPL-008C penetrated tumour tissue and significantly reduced phospho-ERK signalling in that domain.

Conclusion: Our data suggests that the PDE8A-C-Raf complex is a promising therapeutic treatment for B-Raf inhibitor resistant melanoma.

Keywords: B-Raf; C-Raf; CellPorter®; Disruption; Melanoma; NRAS; PDE8A; PPL-008.

Fig. 1

Effects of PPL008 conjugates on pERK levels and rate of cell proliferation. Normalised phospho-ERK (mean ± SEM) following treatment with DMSO (lane 1), PLX4032 B-Raf inhibitor (lane 2), PPL008 conjugates (lanes 11–14) or PLX co-treatments with PDE8A – C-Raf peptide disrupters (original stearylated ‘disrupter’ lane 3, or scrambled control, lane 4) or PPL-008 conjugates (lanes 7–10) in: a MM415 (NRAS Q61L) and (b) A375 (BRAF V600E) human malignant melanoma cell lines. Respective pERK and GAPDH immunoblot examples shown below (N ≥ 3, * P < 0.05, ** P < 0.01). c Real-time cell analyses (xCELLigence platform) of MM415 cell proliferation following treatments described above. Treatments occurred at 21 h and the slope of normalised cell index (mean ± STDEV) was measured between 21 and 39 h, with (ii) representing DMSO vs. peptide disrupter treatments only (10 μM) and (iii) representing PLX (1 μM) vs. co-treatments of peptide disrupters and PLX4032 (n = 3, *** P < 0.001). D, stearylated disrupter; S, stearylated scrambled; N, PPL-008 N; C, PPL-008C; NSS, PPL-008NSS; CSS, PPL-008CSS

Fig. 2

PPL008-C/N dose response in MM415 (NRAS Q61L) human malignant melanoma cell line. a & b: Normalised pERK expression (mean ± SEM) in MM415 cells following dose response co-treatment with PLX4032 (1 μM) and PPL-008 N or PPL-008C (1 nM – 10 μM). Respective pERK and GAPDH immunoblot examples shown below. (N = 3, * P < 0.05, ** P < 0.01, *** P < 0.001), DMSO vs. PPL008 only, PLX vs. PLX + PPL008). c & d (i) Real-time cell analyses (xCELLigence platform) of MM415 cell proliferation following dose response co-treatments with PPL-008 N or PPL-008C (1 nM - 10 μM) and PLX4032 (1 μM). Treatments occurred at 32 h and the slope of normalised cell index (mean ± STDEV) was measured between 32 and 48 h (n = 3, *** P < 0.001). c & d (ii) Representative traces of normalised cell index of each treatment shown below. N, PPL-008 N; C, PPL-008C

Fig. 3

In vivo suppression of phospho-ERK signalling in an MM415 murine xenograft model. a Normalised total pERK1/2, b pERK1 (T202, 44 kDa) and (c) pERK2 (Y204, 42 kDa) levels (mean ± SEM) in MM415 (Q61L) tumour xenografts from NSG immuno-deficient mice following PPL-008C treatment, at multiple time points, with either 25 mg/kg or 100 mg/kg doses (control N = 3, treated N = 4, * P < 0.05). Control mice were treated with a 5% dextrose in dH₂O solution and PPL-008C was administered via subcutaneous injection at the site of tumour. Representative pERK1/2 and GAPDH immunoblot examples shown below (a)

Fig. 4

Dual inhibition of B-Raf and C-Raf inhibits melanoma tumour progression. a B-Raf inhibition leads to the Ras negative feedback mechanism switching to C-Raf driven tumourigenesis via potentiation of the Raf/MEK/ERK signalling axis. b PPL-008 (PDE8A – C-Raf disrupter peptide) binds to C-Raf, preventing PDE8A localisation within the C-Raf cAMP microdomain and exposing serine 259 – C-Raf to inhibitory phosphorylation by PKA. Co-treatment with B-Raf inhibitor and PPL-008 blocks onco-Ras driven tumour progression via inhibition of the Raf/MEK/ERK axis