RESISTANCE TO B-RAF INHIBITORS IN MELANOMA

B-RAF is a serine/threonine kinase that activates the mitogen-activated protein kinase (MAPK) signaling pathway. Hanahan and Weinberg (2000) describe six hallmarks of cancer:

• Independence from growth signals
• Evasion of cell death (apoptosis)
• Resistance to anti-growth signals
• Unlimited replication
• The ability to invade and metastasize
• The ability to trigger blood vessel growth (angiogenesis)

Abnormal MAPK signaling affects most of these processes, making it central to how cancer develops and progresses. Activating mutations in B-RAF kinase (mostly V600E B-RAF) appear in about 50% of melanomas, locking the MAPK pathway in the “on” position.

As researchers learned more about V600E B-RAF‘s role in melanoma, they developed drugs to block this kinase and its downstream signaling. Two ATP-competitive type I B-RAF inhibitors — vemurafenib and dabrafenib (GSK2118436) — show strong anti-cancer activity in patients with V600E B-RAF mutant melanoma. But almost all patients treated with B-RAF inhibitors eventually develop drug resistance. In clinical trials, most patients’ disease progressed again after just 6-7 months of initial response.

How Resistance Develops

Resistance can develop when cells pick up secondary mutations in the kinase’s ATP-binding site. These mutations block the drug from binding to the hydrophobic pocket at the so-called “gatekeeper” residue. In a preclinical study, Whittaker et al. (2010) identified a gatekeeper mutation in BRAF at the Threonine-259 (T259) residue. This mutation caused resistance to the B-RAF inhibitors SB590885 and PLX4720.

However, researchers didn’t find this mutation in patients whose disease progressed after vemurafenib treatment. This suggests a different mechanism — one that bypasses V600E B-RAF entirely — drives acquired resistance to B-RAF inhibitors in patients instead.

Researchers found active MAPK signaling and higher C-RAF (a B-RAF isoform) expression in B-RAF-inhibitor-resistant melanoma cells. Villanueva et al. (2010) also found constant activation of insulin-like growth factor receptor 1 (IGFR1) in these resistant cells. IGFR1 is a receptor tyrosine kinase (RTK). Because IGFR1 activates PI3K/Akt signaling, combining PI3K and MEK inhibitors reversed the resistance.

Researchers also found higher IGFR1 levels in melanoma patients who failed vemurafenib treatment. This suggests IGFR1-driven PI3K/Akt signaling can limit how well B-RAF inhibitors work. Other RTKs were also linked to acquired vemurafenib resistance. Tumor biopsies from melanoma patients failing vemurafenib showed overexpression of platelet-derived growth factor receptor-β (Nazarian et al., 2010).

Genetic Alterations Behind Resistance

Beyond increased RTK activity, genetic changes in the MAPK signaling pathway itself can also drive resistance to B-RAF inhibitors. Genetic analysis detected an activating mutation in NRAS at codon 61 in tumor biopsies from patients treated with vemurafenib (Nazarian et al., 2010).

Researchers have also found evidence of B-RAF alterations in tumor samples from patients whose cancer progressed after initially responding to B-RAF inhibitors. A study by Shi et al. (2012) found that 20% of vemurafenib-resistant melanoma patients had extra copies of V600E B-RAF (genomic copy-number gains). These extra copies caused its overexpression. In an in vitro study, Shi et al. restored sensitivity to vemurafenib in these B-RAF amplification-driven, resistant cells. They did this by treating the cells with the MEK inhibitor selumetinib (AZD6244). This result shows that MAPK pathway reactivation drives this resistance mechanism.

Structural changes in B-RAF could also confer resistance, as Poulikakos et al. (2011) discovered. Their study identified a splice variant of B-RAF (V600E). This 61-kDa B-RAF variant lacks the RAS-binding domain. This variant turned up in the tumors of 32% of patients with acquired vemurafenib resistance.

What This Means for Treatment

One of melanoma treatment’s biggest challenges is developing effective strategies to overcome intrinsic and acquired drug resistance to small molecule B-RAF inhibitors. The resistance mechanisms identified so far are diverse. Most, however, seem to rely on reactivating the MAPK signaling pathway and boosting signaling output through the PI3K/Akt pathway. This suggests that dual inhibition of the BRAF and PI3K/Akt signaling pathways may prevent or delay resistance from developing in melanoma.

References:

1. Hanahan D, Weinberg RA. The hallmarks of cancer. Cell. Vol. 100. United States; 2000:57-70.

2. Whittaker S, Kirk R, Hayward R, et al. Gatekeeper mutations mediate resistance to BRAF-targeted therapies. Sci Transl Med. Vol. 2. United States; 2010:35ra41.

3. Villanueva J, Vultur A, Lee JT, et al. Acquired resistance to BRAF inhibitors mediated by a RAF kinase switch in melanoma can be overcome by cotargeting MEK and IGF-1R/PI3K. Cancer Cell. Vol. 18. United States: 2010 Elsevier Inc; 2010:683-695.

4. Nazarian R, Shi H, Wang Q, et al. Melanomas acquire resistance to B-RAF(V600E) inhibition by RTK or N-RAS upregulation. Nature. Vol. 468. England; 2010:973-977.

5. Shi H, Moriceau G, Kong X, et al. Melanoma whole-exome sequencing identifies (V600E)B-RAF amplification-mediated acquired B-RAF inhibitor resistance. Nat Commun. Vol. 3. England; 2012:724.

6. Alas S, Bonavida B. Inhibition of constitutive STAT3 activity sensitizes resistant non-Hodgkin’s lymphoma and multiple myeloma to chemotherapeutic drug-mediated apoptosis. Clin Cancer Res. 2003;9(1):316-326.