BRAF inhibitor-associated ERK activation drives development of chronic lymphocytic leukemia - PubMed (original) (raw)

Case Reports

. 2014 Nov;124(11):5074-84.

doi: 10.1172/JCI76539. Epub 2014 Oct 20.

Frank Meiss, Justin Mastroianni, Thorsten Zenz, Hana Andrlova, Nimitha R Mathew, Rainer Claus, Barbara Hutter, Stefan Fröhling, Benedikt Brors, Dietmar Pfeifer, Milena Pantic, Ingrid Bartsch, Timo S Spehl, Philipp T Meyer, Justus Duyster, Katja Zirlik, Tilman Brummer, Robert Zeiser

Case Reports

BRAF inhibitor-associated ERK activation drives development of chronic lymphocytic leukemia

Niuscha Yaktapour et al. J Clin Invest. 2014 Nov.

Abstract

Patients with BRAFV600E/K-driven melanoma respond to the BRAF inhibitor vemurafenib due to subsequent deactivation of the proliferative RAS/RAF/MEK/ERK pathway. In BRAF WT cells and those with mutations that activate or result in high levels of the BRAF activator RAS, BRAF inhibition can lead to ERK activation, resulting in tumorigenic transformation. We describe a patient with malignant melanoma who developed chronic lymphocytic leukemia (CLL) in the absence of RAS mutations during vemurafenib treatment. BRAF inhibition promoted patient CLL proliferation in culture and in murine xenografts and activated MEK/ERK in primary CLL cells from additional patients. BRAF inhibitor-driven ERK activity and CLL proliferation required B cell antigen receptor (BCR) activation, as inhibition of the BCR-proximal spleen tyrosine kinase (SYK) reversed ERK hyperactivation and proliferation of CLL cells from multiple patients, while inhibition of the BCR-distal Bruton tyrosine kinase had no effect. Additionally, the RAS-GTP/RAS ratio in primary CLL cells exposed to vemurafenib was reduced upon SYK inhibition. BRAF inhibition increased mortality and CLL expansion in mice harboring CLL xenografts; however, SYK or MEK inhibition prevented CLL proliferation and increased animal survival. Together, these results suggest that BRAF inhibitors promote B cell malignancies in the absence of obvious mutations in RAS or other receptor tyrosine kinases and provide a rationale for combined BRAF/MEK or BRAF/SYK inhibition.

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Figures

Figure 6

Figure 6. Impact of SYK inhibition on BRAF inhibitor–induced primary CLL proliferation.

Primary CLL cells of different patients were exposed to DMSO or vemurafenib (1 μM), and the percentage of living cells relative to all cells (patients n = 7) when the MEK inhibitor (A) or the Syk inhibitor (B) was included, was determined for different time points. **P < 0.01; ***P < 0.001. (C) The amount of CLL cells in the peripheral blood of Rag2–/– γC–/– mice relative to start of treatment. * P < 0.05 when the group was compared with the vemurafenib-only group. (D) The survival of the Rag2–/– γC–/– mice treated with vemurafenib (24 mg/kg/d) alone or in combination with fostamatinib (60 mg/kg/d) or trametinib (1 mg/kg/every other day for 12 days) is shown (Vem vs. Vem/Fosta, P < 0.001; Vem vs. Vem/Tram, P < 0.001) (E) Proposed mechanism illustrating how BRAF inhibition could cooperate with SYK in paradoxical ERK activation. Vemurafenib binds to 1 protomer, e.g., BRAF, and stimulates a drug-free RAF molecule, e.g., RAF1, in an allosteric and RAS-dependent manner (yellow arrow). Active and RAF-binding competent RAS is supplied via SYK, which is hyperactivated due to the autonomous signaling capacity of the CLL-specific BCR.

Figure 5

Figure 5. Impact of BRAF inhibition on viability, proliferation, and in vivo expansion of primary CLL cells.

(A–C) Primary CLL cells from different patients were highly purified (>97% CD19+CD5+) and exposed to DMSO or vemurafenib. (A) The percentage of living cells relative to all cells (patients n = 3) or the absolute number of CLL cells (patients n = 4) (B) was determined for different time points. (C) Metabolic activity measured by MTT on day 52 of culture was higher in primary CLL cells (patients n = 3) when exposed to vemurafenib compared with DMSO. One of 3 independent experiments with similar results is shown. *P < 0.05; **P < 0.01. (D and E) On day 0, the amount of 2.5 × 107 patient-derived CD19+CD5+ cells was injected i.p. and i.v. (D) The amount of CLL cells in the peripheral blood of Rag2–/– γC–/– mice on days 0 and 7 relative to start of treatment. (E) The survival of the Rag2–/– γC–/– mice treated with DMSO or vemurafenib (24 mg/kg/d) is shown for the CLL cells derived from 3 different patients.

Figure 4

Figure 4. Impact of BRAF and SYK/MEK inhibition on ERK activation in primary CLL cells.

(A–F) Primary CLL cells from multiple patients were highly purified (>97% CD19+CD5+) and exposed to the indicated inhibitors. (A) Representative Western blot analysis for pERK and tERK of the protein lysate at the indicated concentrations of vemurafenib and dabrafenib for patients 1 and 2. The experiment was performed 2 times with similar results. (B) Quantification of the pERK/14-3-3 ratios and pERK/tERK ratios of 10 different patients are displayed (Vem, 1 μM). (C) Addition of the SYK inhibitor R406 decreased the ERK phosphorylation as shown for the protein lysates for patient 3. The experiment was performed 2 times with similar results. (D) Quantification of the pERK/14-3-3 ratios and pERK/tERK ratios of 9 and 5 different patients, respectively, are displayed (Vem, 2.5 μM). (E) Western blot analysis for pERK and tERK of the protein lysate at the indicated concentrations of vemurafenib and trametinib (1 μM) for patient 4. The experiment was performed 3 times with similar results. (F) Quantification of the pERK/tERK ratios and pERK/14-3-3 ratios of all analyzed patients (n = 4) are displayed (Vem, 2.5 μM).

Figure 3

Figure 3. BRAF inhibition increases ERK phosphorylation and CLL proliferation in vivo, which can be reverted by SYK inhibition.

Highly purified (>97%) CD19+CD5+ cells (CLL) obtained from the patient were exposed to dabrafenib (6 μM) and R406 at increasing concentrations, as indicated, or DMSO as control. (A) A representative Western blot is shown. The experiment was performed twice with similar results. The pERK/tERK ratios (B) and the pSYK/tSYK ratios (C) are shown for the indicated conditions. (D) The patient’s CLL cells were exposed to dabrafenib, vemurafenib, R406, or DMSO as control at the indicated concentrations, and the resulting Western blot and the RAS-GTP/tRAS ratios are shown. One of 3 independent experiments with similar results is shown. (E) The amounts of CLL cells in the peripheral blood of Rag2–/– γC–/– mice on day 7 relative to treatment starting with vemurafenib (24 mg/kg/d) alone or vemurafenib and fostamatinib (60 mg/kg/d) are shown. On day 0, 2.5 × 107 patient-derived CD19+CD5+ cells were injected i.p. and i.v. (F) The survival of the Rag2–/– γC–/– mice treated as described in E is shown (P = 0.0001). Data from 2 experiments were pooled.

Figure 2

Figure 2. Impact of BRAF and MEK inhibitor treatment on signaling and survival of CLL cells.

Highly purified (>97%) CD19+CD5+ cells or CD14+ myeloid cells obtained from the patient were cultured in the presence of the BRAF inhibitors (vemurafenib, dabrafenib), the MEK inhibitor trametinib, BRAF inhibitor combined with MEK inhibitor, or DMSO only at the indicated concentrations. (A) OD as an indicator for viability and metabolic activity of CLL cells under different vemurafenib concentrations in an MTT assay. The experiment was performed twice using in dependent samples from the patient with similar results. Levels of pERK and tERK in the CLL cells (B) or CD14+ cells (C) derived from PBMC were measured by Western blot. We used vemurafenib, dabrafenib, and trametinib (0.07 μM) as indicated. One of 3 independent experiments with similar results is shown. (D) Western blot analysis for pERK and tERK of the protein lysate of highly purified (>97%) patient-derived CD19+CD5+ cells at the indicated concentrations of dabrafenib (Dab; 6 μM) and trametinib (MEK-i; 0.03, 0.07, 0.14 μM). Quantification of the protein amount of the described groups shown as a bar diagram. The experiment was performed 3 times with similar results. (E) The amount of CLL cells in the peripheral blood of Rag2–/– γC–/– mice relative to start of treatment with vehicle or vemurafenib (24 mg/kg/d). (F) The survival of the Rag2–/– γC–/– mice treated as described in panel E is shown (P = 0.0004). Data from 3 independent experiments were pooled.

Figure 1

Figure 1. Clinical course of a melanoma patient with CLL progressing during treatment with vemurafenib.

Displayed are the white-cell count (A) and the lymphocyte count (B) at multiple time points prior to and after vemurafenib treatment (gray area). (C) A representative blood smear of the patient during vemurafenib treatment is shown. Original magnification, ×100; ×200 (inset). The dominant population has a mature lymphocyte phenotype. (D) Immunophenotyping of the white blood cells during vemurafenib treatment revealed a CD19+CD200+ population that could also be seen as CD19+CD5+ cells. (E) Combined CT and FDG PET scans obtained 1 month before the patient started taking vemurafenib and 6 weeks after vemurafenib was discontinued showed a partial response to treatment. The maximal standardized uptake value (SUVmax; semi-quantitative measure of tumor glucose metabolism) decreased from 9.5 to 4.6 g/ml (–52%) in the parailiacal LN (red arrow) and from 6.6 g/ml to 1.9 g/ml (–71%) in the right inguinal LN (not shown). Arrows indicate melanoma metastasis. (F) Lambda light-chain restriction in CD19+ B lymphocytes on day 732 relative to treatment initiation is shown.

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