The HSP90 inhibitor XL888 overcomes BRAF inhibitor resistance mediated through diverse mechanisms

Abstract

Purpose: The clinical use of BRAF inhibitors is being hampered by the acquisition of drug resistance. This study shows the potential therapeutic use of the HSP90 inhibitor (XL888) in six different models of vemurafenib resistance.

Experimental design: The ability of XL888 to inhibit growth and to induce apoptosis and tumor regression of vemurafenib-resistant melanoma cell lines was shown in vitro and in vivo. A novel mass spectrometry-based pharmacodynamic assay was developed to measure intratumoral HSP70 levels following HSP90 inhibition in melanoma cell lines, xenografts, and melanoma biopsies. Mechanistic studies were carried out to determine the mechanism of XL888-induced apoptosis.

Results: XL888 potently inhibited cell growth, induced apoptosis, and prevented the growth of vemurafenib-resistant melanoma cell lines in 3-dimensional cell culture, long-term colony formation assays, and human melanoma mouse xenografts. The reversal of the resistance phenotype was associated with the degradation of PDGFRβ, COT, IGFR1, CRAF, ARAF, S6, cyclin D1, and AKT, which in turn led to the nuclear accumulation of FOXO3a, an increase in BIM (Bcl-2 interacting mediator of cell death) expression, and the downregulation of Mcl-1. In most resistance models, XL888 treatment increased BIM expression, decreased Mcl-1 expression, and induced apoptosis more effectively than dual mitogen-activated protein-extracellular signal-regulated kinase/phosphoinositide 3-kinase (MEK/PI3K) inhibition.

Conclusions: HSP90 inhibition may be a highly effective strategy at managing the diverse array of resistance mechanisms being reported to BRAF inhibitors and appears to be more effective at restoring BIM expression and downregulating Mcl-1 expression than combined MEK/PI3K inhibitor therapy.

Figure 1. The HSP90 inhibitor XL888 blocks the growth and survival of melanoma cell lines with diverse mechanisms of vemurafenib resistance

A: Growth assay showing the response of matched pairs of vemurafenib naïve and resistant melanoma cell lines and melanoma cell lines with intrinsic resistance. (Left) Cells were treated with increasing concentrations of vemurafenib (1nM – 10 µM: 72 hrs) before being subject to the MTT assay. (Right) Cell growth assay showing the response of the cell line panel from (A) to the HSP90 inhibitor (1nM-10 µM: 72 hrs). B: Cell cycle effects of XL888 (300 nM: 24 hrs) upon vemurafenib sensitive and naïve cell lines. Cells were fixed, stained with propidium iodide and distributions analyzed by flow cytometry. C: XL888 induces apoptosis in every model of acquired vemurafenib resistance tested. Cells were treated for either 72 or 144 hrs with XL888 (300 nM) followed by Annexin-V. Apoptosis was measured by flow cytometry. D: (Upper) Colony formation assay showing the long-term effectiveness of XL888. Cell lines were treated with 300nM XL888 for 4 weeks before being fixed and stained with crystal violet. (Lower) Quantification of absorbance after 4 weeks of drug treatment.

Figure 2. XL888 degrades proteins involved in BRAF inhibitor resistance leading to apoptosis induction

A: (Left) XL888 degrades IGF1R, PDGFRβ, ARAF, CRAF and cyclin D1 and inhibits pAKT, pERK and pS6 signaling in 4 melanoma cell lines with acquired BRAF inhibitor resistance. XL888 degrades the expression of COT and cyclin D1 in melanoma cell lines with intrinsic resistance to vemurafenib. (Right) Time-dependency of the XL888 mediated effects upon pAKT, pERK, pS6, COT and BIM. RPMI7951 cells were treated with XL888 for 0–48 hrs. B: XL888 (1 µM, 144 hrs) is effective at blocking the growth and survival of vemurafenib resistant melanoma cell lines grown as 3D collagen implanted spheroids. Staining shows cell viability, where green corresponds to live cells and red: dead cells. Magnification X4. C: Combining XL888 with vemurafenib leads to enhanced levels of apoptosis in melanoma cell lines with COT overexpression (RPMI7951), or loss of PTEN expression (WM793 and 1205Lu). Cells were treated with vemurafenib (3 µM), XL888 (300nM), or the two in combination for 48 hr. Apoptosis was measured by Annexin-V staining and flow cytometry. D: XL888 in combination with vemurafenib reduces the survival of intrinsically resistant melanoma cell lines grown as 3D collagen-implanted spheroids. RPMI7951 cells (COT amplified) and WM793 cells (PTEN deficient) were treated with either XL888 (1 µM), vemurafenib (3 µM), or a combination of the two for 48 hrs. Viability was measured as described above.

Figure 3. Development of a quantitative pharmacodynamic assay for HSP90 inhibition

A: Work flow of the LC-MRM experiment to measure HSP chaperone levels. After reversed-phase HPLC separation, peptides are selected by their mass-to-charge ratio and dissociated by collisions with background gas before the fragment ions are mass selected to enable specific detection and quantification of individual peptides in complex mixtures. B: Heatmap showing XL888-induced (0–48 hrs, 300 nM) HSP70 expression in all of the melanoma cell lines irrespective of vemurafenib resistance mechanism. C: Western Blot confirmation of HSP70 upregulation following XL888 treatment (300 nM, 48 hr). D: Quantification of absolute (fmol/µg) expression of the HSP chaperone protein expression in fine needle aspirates from two melanoma specimens.

Figure 4. XL888 induces the regression of established M229R xenografts and is associated with increased intratumoral HSP70 expression

A: XL888 leads to regression of M229R melanoma xenografts. M229R cells were grown until a palpable tumor had formed before being treated with XL888 thrice per week (100 mg/kg) by oral gavage. Data shows growth curves normalized to starting volumes, bar graph shows mean tumor volumes after 15 days of XL888 treatment. XL888 treatment led to significant levels of tumor regression (P=0.003). B: Heatmap showing the increase in HSP70 isoform 1 (HSP71) expression in XL888 treated (15 days, 100mg per kg) xenograft samples compared to vehicle controls. C: XL888 treatment (10 days) led to the induction of apoptosis in established M229R xenografts as measured by increased TUNEL staining (green), and was associated with the induction of BIM expression and the suppression of Mcl-1 expression.

Figure 5. HSP90 inhibition increased BIM, decreases Mcl-1 and restores apoptosis in vemurafenib-resistant melanoma cell lines

A: (Left) Western blot showing that XL888 (48 hrs, 300nM) decreases BIM phosphorylation (Ser69) and increases BIM expression. (Right) Quantitative RT-PCR experiment showing that treatment with XL888 (300 nM, 48 hrs) increases the expression of BIM at the mRNA level. B: siRNA knockdown of BIM significantly decreases XL888 (300 nM, 48hrs) mediated apoptosis in two vemurafenib resistant melanoma cell lines (M229R and 1205LuR). C: (Left) Western blot of Mcl-1 expression in vemurafenib resistant melanoma cell lines treated with XL888 (300 nM) for 48 hrs. (Right) Quantitative RT-PCR showing that XL888 (300 nM, 48hrs) treatment downregulates Mcl-1 expression at the mRNA level. D: Induction of Mcl-1 reduces the magnitude of XL888 induced apoptosis. Western blot shows the induction of Mcl-1 following doxycycline treatment. Induction of Mcl-1 (DOX+XL) significantly reduces the level of XL888-induced apoptosis compared to XL888 (XL: 300nM, 72 hrs) alone. *P<0.05

Figure 6. HSP90 inhibition is more effective at restoring the apoptotic response than combined MEK+PI3K inhibition

A: Immunofluorescence staining of 1205LuR and M229R cells for BIM and FOXO3a following treatment with either vehicle, XL888 (300nM), AZD6244 (3µM), GDC-0941 (3µM) and the combination of AZD6244 + GDC-0941 (each at 3µM). B: XL888 is more effective than MEK+PI3K inhibitors at increasing BIM and decreasing Mcl-1 mRNA expression in 1205LuR and M229R cell lines. Cells were treated with vehicle, XL888, AZD6244, GDC-0941 and the combination of AZD6244 + GDC-0941 (as above) and Quantitative RT-PCR was performed on BIM and Mcl-1. C: XL888 is more effective than MEK+PI3K inhibitors at increasing BIM and decreasing Mcl-1 protein expression in 1205LuR and M229R cell lines. Cells were treated with vehicle, XL888, AZD6244, GDC-0941 and the combination of AZD6244 + GDC-0941 as above and Western blotting was performed for BIM and Mcl-1. D: XL888 is more effective at inducing apoptosis of melanoma cell lines where resistance is mediated through COT and PDGFRβ expression and in 2 models where the resistance mechanism is unknown. Cells were treated with XL888, AZD6244, GDC-0941 and AZD6244 + GDC-0941 as described above for 72 or 144 hrs. Apoptosis was measured by Annexin-V staining and flow cytometry.