BRAF targets in melanoma. Biological mechanisms, resistance, and drug discovery. Cancer drug discovery and development. Volume 82. Ed. Ryan J. Sullivan. Springer (2015)
Although the activating BRAF mutation is the most frequent somatic event in melanoma, and a valuable therapeutic target, several lines of evidence support that other pathways likely play a critical role in this disease. For example, benign nevi have a rate of BRAF V600 mutations that is similar to or higher than the rate observed in melanomas . As benign nevi have an extremely low rate of malignant transformation, this finding demonstrates that other events must complement the BRAF mutation to fully explain the aggressive biology of this cancer. Of note, mutations in NRAS are also common in benign nevi . In addition to this observation, functional studies in zebrafish, mice, and human cells have demonstrated that introducing expression of V600-mutant BRAF proteins alone in normal melanocytes fails to induce malignant transformation [88–90]. These model systems have provided a way to functionally interrogate candidates that may contribute to melanomagenesis. Finally, while many clinical specimens and cell lines with acquired resistance to selective BRAF inhibitor exhibit re-activation of the MAPK pathway, this has not been a universal finding [69, 77, 91]. While many pathways remain to be interrogated, a number of studies support that the PI3K-AKT pathway can play an important role in this disease.
The PI3K-AKT pathway is a critical regulator of many cellular processes, including growth, survival, anchorage independence, motility/invasion, angiogenesis and metabolism, among others. The significance of the PI3K-AKT pathway in cancer is supported by the finding of a high rate of somatic alterations, including mutations, amplifications, and deletions, in multiple components of this pathway in many tumor types [92–94]. As described previously, activation of the PI3K-AKT pathway was initially implicated in melanoma by the identification of activating NRAS mutations and loss of function of the PTEN tumor suppressor. Interestingly, similar to previous comparison of PTEN loss and PI3K mutations, quantitative analysis demonstrated that PTEN loss correlates with significantly greater activation of AKT than NRAS mutations, as measured by expression of phosphorylated (activated) AKT protein, in melanoma cell lines and clinical specimens [95, 96]. While mutations in the catalytic subunit of PI3K, PIK3CA, are common in several tumor types, they are detected in only 1–2% of melanomas . Point mutations in the regulatory pleckstrin homology (PH) domain of AKT1 have been identified as rare events in several tumor types, including melanoma (~ 1%) . In addition, the analagous mutation in AKT3 has been identified uniquely in melanoma . This finding builds upon several other studies specifically implicating AKT3 as an important AKT isoform in this disease, whereas most research in other cancers implicates AKT1 and/or AKT2 [99–101]. Finally, mutations and amplifications of oncogenic receptor tyrosine kinases (RTKs) that activate signaling through the PI3K-AKT pathway in other cancers, such as HER2/neu and the epidermal growth factor receptor (EGFR), have not been detected as significant events in cutaneous melanomas, although aberrations of the KIT RTK have been implicated in other subtypes . One report indicated that mutations throughout the sequence of the ERBB4 (HER4) gene were detected in ~ 20% of melanomas . Although this pattern of mutations was curious for a proposed oncogene, functional studies in cell lines with enforced expression of several of the variants detected in patients did suggest that the mutations were activating. However, recent whole exome sequencing efforts have not identified somatic mutations of ERBB4 as a significant event [42, 43].
A role for activation of the PI3K-AKT pathway in the transformation of melanocytes has been suggested primarily in preclinical models. In a genetically engineered mouse model (GEMM) in which inducible loss of PTEN in melanocytes was achieved with topical treatment with 4-hydroxytamoxifen, no melanocytic lesions were observed. In the same model, induction of the BRAF V600E mutation in newborn mice resulted in melanocyte hyperplasia, but no invasive lesions (melanomas) were observed. However, crossing of the two strains of mice to generate targeted expression of the BRAF V600E mutation with loss of PTEN expression in melanocytes resulted in invasive melanomas in all mice within 7–10 days of 4-hydroxtamoxifen treatment. In addition to being 100% penetrant, the tumors formed spontaneous metastases in all of the mice. All mice required euthanasia within 25–50 days of induction . Expression of an activated form of AKT3 (myr-AKT3) also transforms human melanocytes that express the BRAF V600E protein . Interestingly, although NRAS mutations and genetic loss of PTEN are mutually exclusive in patients, loss of PTEN increased the metastatic potential and invasive behavior of NRAS-mutant melanomas in another GEMM .
Studies in advanced melanomas also support that PTEN loss is important functionally. In particular, a number of studies have compared BRAF-mutant human melanoma cell lines that lack PTEN to those that have normal PTEN function. Loss of PTEN correlates with increased activation of AKT in BRAF-mutant cell lines and tumors, and is also observed after knockdown of PTEN expression with RNAi . Treatment of BRAF-mutant, PTEN-null human melanoma cell lines with BRAF or MEK inhibitors generally results in cytostatic effects, although one study identify a subset of resistant lines that also had loss of Rb . In contrast to other BRAF-mutant cell lines, most of the cell lines with loss of PTEN fail to undergo apoptosis following treatment with BRAF or MEK inhibitors [104–107]. Resistance to apoptosis can also be induced in BRAF-mutant cell lines by inhibiting PTEN expression with RNAi . These findings support that BRAF-mutant melanomas with loss of PTEN may exhibit at least some degree of de novo resistance to MAPK pathway inhibitors. Sequencing and copy number analysis of 34 patients enrolled in the phase I and phase II studies of dabrafenib detected aberrations in the PTEN gene in 11 (32%) of the patients . Patients with PTEN loss had a similar rate of clinical response (36%) as those with genetically intact PTEN (43%). However, PTEN loss showed a very strong trend, even in this relatively small set of patients, for shorter PFS (18 weeks versus 32 weeks, p = 0.06). Overall, analysis of samples collected at the time of disease progression found that homozygous deletion of PTEN was observed more frequently (4/10) than in the pre-treatment samples (2/34, p = 0.017). A previous analysis of 5 patient that had matching pre-treatment and disease progression samples found discordance in 1 sample, which exhibited homozygous loss of PTEN at disease progression .
In addition to constitutive activation in melanomas with PTEN loss, it appears that activation of the PI3K-AKT pathway through growth factor receptors can mediate resistance to BRAF and MEK inhibitors. Characterization of two BRAFmutant, PTEN-expressing human melanoma cell lines with de novo cell resistance to apoptosis induction demonstrated that these cell lines had similar degree and duration of inhibition of the MAPK pathway as cell lines destined to undergo apoptosis, but they were unique in that they developed marked activation of AKT after MEK inhibitor treatment . Similar results were also observed subsequently with selective BRAF inhibitors . Inhibition of the insulin-like growth factor 1 receptor (IGF1R), which both of the resistant cell lines expressed at high levels, abrogated the compensatory activation of AKT. Inhibition of IGF1R alone did not induce apoptosis in the cells, but marked cell death was observed when that was combined with MEK inhibition. This synergistic effect on apoptosis induction was recapitulated by knocking down AKT, or by inhibiting AKT activation with a dual TORC1/2 inhibitor, demonstrating that PI3K-AKT activation was mediating IGF1R-induced resistance.
Overexpression of IGF1R was also observed independently by investigators characterizing cell lines selected in vitro for secondary resistance to selective BRAF inhibitors . These cell lines also demonstrated resistance to MEK inhibition by BRAF inhibitors through utilization of multiple RAF isoforms. While the MAPK pathway activation could be blocked in these cells by treatment with a MEK inhibitor, this failed to induce apoptosis in the resistant clones. Apoptosis was only seen with the MEK inhibitor when it was combined with a small molecule inhibitor of either IGF1R or PI3K. Analysis of matching samples from 5 patients treated with a selective BRAF inhibitor detected increased IGF1R expression in 2 patients at the time of disease progression (a third tumor had loss of PTEN). Resistant cell lines developed and characterized by another group of investigators also identified multiple RTKs that were upregulated at the time of resistance . Although multiple RTKs were overexpressed (i.e. KIT, MET, EGFR), only the PDGFRβ was found to be activated by antibody array analysis. Increased activation of PDGFRβ was also identified in 4 (36%) of 11 patients with matching pretreatment and progression samples following BRAF inhibitors. Functional testing demonstrated that the cell lines did not undergo apoptosis with MEK inhibitors alone, but did when MEK inhibitors were combined with either AKT or dual PI3KmTOR inhibitors . It is interesting to note that the two groups of investigators found completely non-overlapping RTKs mediating resistance in their different experimental systems. In addition, investigations by both groups failed to identify any mutations or amplifications of the genes encoding the implicated receptors [69, 74]. Thus, the induction of the RTKs appears to reflect an epigenetically-mediated mechanism of resistance.
While these studies identified resistance mechanisms that are intrinsic to the tumor cells, there is also evidence that activation of the PI3K-AKT pathway may be mediated in part by extrinsic factors. Two groups independently demonstrated that production of the growth factor HGF by stromal cells was capable of mediating resistance to BRAF inhibitors in BRAF-mutant human melanoma cells in co-culture systems [110, 111]. Supporting the clinical relevance of this finding, analysis of pre-treatment samples of patients treated with BRAF inhibitors demonstrated that increased expression of HGF in stromal cells correlated with a decreased chance of achieving a clinical response . While not evaluated in patients, analysis of BRAF-mutant human melanoma cell lines showed that HGF did not rescue the cells from inhibition of MAPK signaling by BRAF inhibitors, but it induced PI3K-AKT pathway activation. The resistance mediated by exogenous HGF could be overcome by treating the cells with inhibitors of c-MET, the receptor for HGF, or with PI3K inhibitors.
The data implicating PTEN loss, RTK overexpression, and stromal growth factors together provide a strong rationale targeting the PI3K-AKT pathway in BRAFmutant melanomas. Of note, data from these preclinical models suggests that only inhibiting the PI3K-AKT pathway is unlikely to be effective, due to both constitutive and compensatory activation of MAPK pathway signaling. In contrast, multiple studies have demonstrated that inhibition of the PI3K-AKT pathway can specifically sensitize cells to apoptosis induction by BRAF or MEK inhibitors [104–107, 109, 112]. In addition to increasing the degree of apoptosis, it appears that the timing of apoptosis induction is also shorter than what is observed with MAPK pathway inhibition alone. This suggests that intermittent dosing of PI3K-AKT pathway inhibitors may be an effective therapeutic strategy, which is supported by xenograft studies . Examination of various dosing schedules may be critical to clinical development in this area, as the important role of the PI3K-AKT pathway in many basic physiological processes will likely make achieving an acceptable therapeutic index challenging. In contrast to the opportunity to target a tumor-specific activating mutation afforded by the BRAF V600 mutations in the MAPK pathway, activating mutations in the PI3K-AKT pathway are rare in melanoma. One possible route to improved therapeutic indices may be the use of isoform-specific inhibitors. For example, data in melanoma supports that the AKT3 isoform may be selectively important in melanoma progression, whereas its expression and function in most normal tissues appears to be rather limited [99, 114]. While inactivating mutations in PTEN are not directly targetable, two different studies have shown that PTEN loss may result in selective dependence on the β-isoform of the catalytic unit of PI3K (P110β, or PIK3CB) [115, 116]. As P110β appears to have a much more limited role in normal physiology, this may again allow for selective targeting of PTEN-null tumor cells, and thus an acceptable therapeutic index.
The clinical development of combinatorial strategies against the PI3K-AKT pathway is also complicated by several other factors [117, 118]. First, there are multiple different classes of inhibitors available to target the pathway, and generally multiple agents in each class undergoing clinical evaluation (Table 6.2). These classes include PI3K inhibitors (pan-PI3K and isoform-specific), dual PI3K-mTOR inhibitors, AKT inhibitors, TORC1 inhibitors (rapamycin-like agents), and dual TORC1/2 inhibitors. Previous studies support that different mechanisms of PI3KAKT pathway activation can result in functional dependence on different effectors . Thus, optimal clinical testing of the pathway may need to match the choice of therapeutic agent to the mechanism of pathway activation that is present in the patient. As the development of vemurafenib demonstrated, the rational testing and assessment of PI3K-AKT pathway inhibitors for melanoma would be facilitated by the identification of a reliable biomarker that correlates with efficacy/clinical benefit. However, while pharmacodynamic markers that do exist to determine if targets in the pathway have been inhibited, it still is unclear which targets, and what degree of target inhibition, are required for efficacy/synergy. Finally, studies in both patients and cell lines have demonstrated that the PI3K-AKT pathway is regulated by multiple feedback loops . As a result, inhibition of a single target in the pathway may rapidly lead to a compensatory signaling mechanism that reactivates itself and/or other pathway effectors. Such feedback compensatory mechanisms have been observed with AKT, TORC1, and dual TORC1/2 inhibitors [120–122]. Thus, meaningful analysis of the effects of PI3K-AKT pathway inhibitors will likely require broad analysis of pathway markers in additional to pharmacodynamic evaluation of on-target effects.
Table 6.2. Classes of PI3K-AKT pathway inhibitors. GSK = GlaxoSmithKline.
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