|Home | About | Journals | Submit | Contact Us | Français|
Identifying the spectrum of genetic alterations that cooperate with critical oncogenes to promote transformation provides a foundation for understanding the diversity of clinical phenotypes observed in human cancers. Here, we performed integrated analyses to identify genomic alterations that co-occur with oncogenic BRAF in melanoma and abrogate cellular dependence upon this oncogene. We identified concurrent mutational inactivation of the PTEN and RB1 tumor suppressors as a mechanism for loss of BRAF/MEK dependence in melanomas harboring V600EBRAF mutations. RB1 alterations were mutually exclusive with loss of p16INK4A, suggesting that whereas p16INK4A and RB1 may have overlapping roles in preventing tumor formation, tumors with loss of RB1 exhibit diminished dependence upon BRAF signaling for cell proliferation. These findings provide a genetic basis for the heterogeneity of clinical outcomes in patients treated with targeted inhibitors of the mitogen-activated protein kinase pathway. Our results also suggest a need for comprehensive screening for RB1 and PTEN inactivation in patients treated with RAF and MEK-selective inhibitors to determine whether these alterations are associated with diminished clinical benefit in patients whose cancers harbor mutant BRAF.
Mitogen-activated protein (MAP) kinase pathway activation is among the most frequent findings in human cancer and is often the result of activating mutations in BRAF (Davies et al., 2002). BRAF mutations are found in ~8% of human tumors, most commonly in melanoma where they are observed in ~50% of patients. These mutations cluster in the glycine-rich loop and activation segments of the kinase domain, with a single missense substitution (V600E), identified in over 90% of cases. Most cell lines with V600EBRAF mutations are dependent upon ERK signaling for cyclin D1 expression, G1 progression, and proliferation and are extremely sensitive to inhibition of the pathway with selective inhibitors of MEK or RAF (Solit et al., 2006; Tsai et al., 2008).
The high frequency of BRAF kinase domain mutations in human cancer and the dependence of such tumors on ERK signaling identify mutant BRAF as a driver oncogene in the tumors in which it occurs (Hingorani et al., 2003; Wellbrock et al., 2004). Transfection of V600EBRAF into primary human melanocytes induces senescence and not transformation, however, and BRAF mutations are observed frequently in non-invasive colonic polyps and melanocytic nevi (Michaloglou et al., 2005). These findings suggest that, in at least some lineages, additional genetic events are required for BRAF-mediated transformation (Patton et al., 2005; Dankort et al., 2007, 2009).
To identify genetic alterations that co-occur with V600EBRAF in human melanoma and diminish BRAF dependence, we performed a multifaceted genomic analysis of 149 melanoma cell lines and short-term cultures. Analysis of the V600EBRAF-mutant cohort demonstrated a large degree of genomic heterogeneity. Despite the large mutational burden of tumors in this class, cells harboring V600EBRAF mutations were pre-dominantly dependent upon MEK activation for proliferation and survival. Subsequent analyses of MEK-independent cells that expressed V600EBRAF indicated that the requirement for BRAF and MEK activity was abrogated by concurrent mutational inactivation of the retinoblastoma (RB1) and PTEN tumor suppressor genes. Notably, MEK-independent, V600EBRAF cells with concurrent RB1/PTEN loss were wild type for p16INK4A, whereas those without RB1 and PTEN mutations generally inactivated the RB pathway through p16INK4A alterations. These findings suggest that the complement of oncogenic mutations associated with the evolution of mutant BRAF melanoma condition the biologic function of ERK signaling in melanomas and thus sensitivity to selective MAP kinase pathway inhibition.
To systematically explore the complement of mutational changes that co-occur with V600EBRAF, and condition dependence on this oncogene, we performed an integrated genomic and proteomic analysis on a large panel of melanoma cell lines and short-term cultures. To identify cells harboring activating BRAF alleles, we profiled 149 melanoma cell lines for alterations in BRAF and NRAS using a mass spectrometry-based genotyping assay (Janakiraman et al., 2010). In total, 78 of the 149 (52%) melanoma cell lines examined harbored BRAF mutations (72 V600E, 3 V600K, and 3 within exon 11; See Figure 1a and Supplementary Table 1). An additional 31 cell lines (21%) were NRAS mutant with mutations in BRAF and NRAS being mutually exclusive in all but two cases (P-value=1.9×10−10). All mutations were confirmed by sequencing with an orthogonal platform (Supplementary Figure 1).
To identify alterations that co-occur with V600EBRAF in cutaneous melanomas, we performed genome-wide DNA copy-number profiling on 31 V600EBRAF-mutant cutaneous melanoma cell lines (Figures 1b and c). Global analysis of the V600EBRAF cell line data revealed significant variability in the levels of both broad and focal copy-number alterations (median of 88 alterations per sample (±50 median absolute deviation; range of 16–276), Figure 1b). To identify recurrent, statistically significant candidate copy-number alterations for further biological characterization, we used the statistical method RAE (Taylor et al., 2008). Consistent with previous studies, deletions encompassing the CDKN2A and PTEN loci were common, as was focal amplification of the MITF gene (Figure 1c), among other events (See Supplementary Table 2).
As loss of the 10q23 locus encompassing the PTEN gene was common in the V600EBRAF melanoma cell lines, we characterized 40 of the BRAF-mutant samples for loss of PTEN expression and activation of AKT (Supplementary Figure 2). In this analysis, we identified nine (22.5%) that lacked detectable PTEN expression (Figure 2a). Consistent with its role as a negative regulator of AKT activity, all nine V600EBRAF, PTEN-null models exhibited high levels of phosphorylated AKT (serine 473 and threonine 308). Loss of PTEN function was not, however, the only mechanism of AKT pathway activation in the melanoma cell line panel as elevated expression of phosphorylated AKT was identified in a subset of the PTEN-expressing cells lines (Gopal et al., 2010). The loss of PTEN expression in all nine PTEN-null cell lines could be directly attributed to genetic events. Six of the nine V600EBRAF, PTEN-null models had detectable PTEN mRNA expression whereas the other three (SKMEL-133, SKMEL-178 and SKMEL-190) lacked demonstrable PTEN mRNA expression by reverse transcription–PCR (Figure 2b). In cell lines that expressed PTEN mRNA, we sequenced all PTEN coding exons and performed cDNA sequencing of the reverse transcription–PCR products (Supplementary Figure 3a and Supplementary Table 3). In all six of the PTEN-null models that expressed PTEN mRNA, mutations in PTEN were identified including three cell lines harboring small homozygous insertion or deletion events (indels) causing frameshift and subsequent early truncation (Supplementary Table 3).
In the C136RPTEN-mutant SKMEL-269 cell line, PTEN was biallelically inactivated by heterozygous loss of the remaining allele (Figure 2c and data not shown). In the broader V600EBRAF panel, heterozygous loss of PTEN was common, and was often a result of large-scale chromosome 10q loss (Figure 2c). In two of the three models lacking PTEN mRNA expression (SKMEL-190 and SKMEL-133), we detected focal homozygous deletions affecting the PTEN gene using the Agilent 244K aCGH platform (Figure 2c and Supplementary Figure 3). To determine whether intragenic microdeletions in PTEN existed beyond the resolution of the 244K array, all nine of the PTEN-null cell lines were profiled using a 1M-probe array. We detected in these data a micro-scale deletion of a single PTEN exon in SKMEL-178 (Figures 2c and d), and an even smaller deletion in SKMEL-105 (Figure 2c), neither event being visible with the lower-resolution aCGH platform. In summary, complete loss of PTEN expression in BRAF-mutant melanoma cell lines was the result of focal genomic events.
Given the high frequency of PTEN inactivation in V600EBRAF melanomas, we examined the MEK dependence of a large panel of V600EBRAF melanomas as a function of PTEN status. Cells were treated with PD0325901, a highly selective, allosteric inhibitor of MEK that inhibits the kinase by locking the enzyme in a closed, but catalytically inactive conformation (Ohren et al., 2004; Sebolt-Leopold and Herrera, 2004; Sebolt-Leopold and English, 2006). All eight V600EBRAF, PTEN-expressing cell lines examined were sensitive to PD0325901 (as defined by an IC50<200 nM), as were seven of the nine V600EBRAF PTEN-null lines (Figure 3a). In fact, only two V600EBRAF cell lines (SKMEL-207 and A2058), both lacking detectable PTEN, were resistant to PD0325901. In summary, loss of PTEN expression alone, mediated by either gene deletion or missense mutation, was insufficient to confer MEK independence in cells expressing V600EBRAF.
In six of eight PTEN wild-type models, PD0325901-induced cell cycle arrest was accompanied by induction of cell death as shown by an increase in the sub G1 fraction (Figures 3b and c). In contrast, although PTEN loss did not confer resistance to the anti-proliferative effects of MEK inhibition, all nine V600EBRAF, PTEN-null cell lines exhibited at best a cytostatic response (Figures 3b and c). Differences in PD0325901 sensitivity among the cell lines was not attributable to differences in the ability of the drug to inhibit ERK activity as for all cell lines tested, PD0325901 treatment resulted in a rapid and sustained loss of phosphorylated ERK expression (Figure 3d and additional data not shown).
As PTEN loss was associated with a cytostatic response to MEK inhibition in melanomas with concurrent BRAF mutation, we hypothesized that combined inhibition of MEK and phosphoinositide 3-kinase (PI3-kinase) may synergistically induce apoptosis in cell lines with mutational activation of both pathways. We therefore assessed the effects of PI3-kinase pathway inhibition using PI-103, a selective inhibitor of the p110α subunit of PI3 kinase and mTOR kinase, alone and in combination with inhibition of MEK (Figure 4 and Supplementary Figure 4). Concurrent inhibition of PI3-kinase and MEK was associated with synergistic induction of apoptosis in V600EBRAF/PTEN-null SKMEL-190 and SKMEL-133 cells (Figure 4b and Supplementary Figure 4). Nevertheless, we observed variable synergy with this dual inhibitor combination in V600EBRAF, PTEN-null cells as the attenuated response of the SKMEL-178 (V600EBRAF/PTEN-null) cell line highlights (Figure 4b). These data support combined targeting of the MAP kinase and PI3-kinase pathways in melanoma patients with concurrent mutational activation of both pathways, but also suggest that additional heterogeneity within the V600EBRAF class will limit the effectiveness of this combination in some patients.
Given the high level of resistance of the SKMEL-207 and A2058 cell lines to MEK inhibition, we further examined these two models for alterations downstream of MEK that could account for their lack of dependence on ERK. We have previously shown that cyclin D1 expression is MEK-dependent in tumors that harbor BRAF mutations (Solit et al., 2006). In MEK inhibitor sensitive, BRAF-mutant cells, inhibition of MAP kinase signaling results in decreased expression of cyclin D1, induction of p27, RB1 hypophosphorylation and thus accumulation of cells in G1 (Figures 5a and b). As discussed above, growth arrest following treatment with PD0325901 is followed by induction of cell death as indicated by an increase in the sub G1 fraction (Figures 3b and c) in a subset of the PTEN wild-type cell lines but not in the PTEN-null cohort. Notably, in MEK-inhibitor resistant SKMEL-207 cells, cyclin D1 expression was still sensitive to PD0325901 and thus ERK-dependent (Figure 5a), but p27 was not induced and G1 arrest did not occur following drug treatment (Figure 5b). Proteomic profiling of the V600EBRAF-mutant cell lines revealed that both SKMEL-207 and A2058 cells were devoid of RB1 expression and in contrast to most melanomas both of these cell lines expressed wild-type p16INK4A (Figure 2a, Supplementary Figure 5 and Supplementary Table 3) (Gonzalgo et al., 1997).
Loss of RB1 expression in SKMEL-207 was attributable to a focal intragenic homozygous deletion within the RB1 coding locus (Figure 5c) whereas A2058 was copy-number neutral at the RB1 locus but harbored a truncating RB1 Q93* mutation (data not shown). While deletion of the 9p21.3 locus encoding CDKN2A is common in melanomas including those in our panel (Figure 1b), SKMEL-207 was copy-number neutral for the CDKN2A locus while a focal homozygous deletion affected this region in A2058. The 9p21.3 locus contains two genes CDKN2A and CDKN2B, the former of which encodes two proteins, p16INK4A and the tumor suppressor p14ARF (Sherr, 2006). A2058 has a 10.3-kb homozygous deletion that disrupts exon 1β of ARF (Figure 5d). This event disrupts p14ARF expression but leaves intact the p16INK4A encoding portion of the gene (Figure 2a).
To confirm that loss of RB1 function is sufficient to confer MEK-independence in a V600EBRAF, PTEN-null context, we stably infected human papillomavirus E7 DNA and a mutant E7 construct (E7Δ21–24) (Psyrri et al., 2004) incapable of binding to RB1 into three MEK-dependent, BRAF-mutant, PTEN-null models. E7 inhibits RB1 function by binding to RB1 and disrupting the RB1/E2F complex (Dyson et al., 1989; Munger et al., 1989; Chellappan et al., 1992; Knudsen and Knudsen, 2008). Viral E7 has also been shown to promote transformation by enhancing RB1 degradation. Infection of E7 into SKMEL-39, SKMEL-105 and SKMEL-133 cells (all V600EBRAF, PTEN-null and wild-type RB1) resulted in resistance to PD0325901 concentrations of up to 3000 nM in all three models (Figure 5e). As was observed in the RB1-null SKMEL-207 and A2058 cell lines, resistance to MEK inhibition in E7-transfected cell lines was associated with failure of the cells to arrest in G1 (Figure 5f). In contrast, transfection of the E7Δ21–24 mutant incapable of binding to RB1 or the vector alone had no effect on MEK inhibitor sensitivity or cell cycle distribution upon treatment with the MEK inhibitor.
Although MEK kinases are the primary downstream effectors of RAF, other RAF substrates have been identified that may promote proliferation or survival in a MEK-independent manner. To confirm that in a PTEN-null context, loss of RB1 reduced dependence on mutant BRAF and not only BRAF-dependent MEK activation, we treated cells representing each genetically distinct class with the RAF inhibitor PLX4720 (Tsai et al., 2006). PLX4720 is an ATP competitive, selective inhibitor of RAF that effectively inhibits ERK signaling in tumor cells with V600EBRAF. Treatment of V600EBRAF-expressing cells with PLX4720 inhibited BRAF-mediated MEK activation in cell lines harboring V600EBRAF as shown by a rapid and sustained decrease in the expression of phosphorylated MEK1/2 (Figure 6a). In contrast and as previously reported, PLX4720 caused a paradoxical induction of phosphorylated MEK expression in cells wild type for BRAF including those harboring NRAS mutation (SKMEL-30) (Hatzivassiliou et al., 2010; Heidorn et al., 2010; Poulikakos et al., 2010). As was observed with MEK inhibition, V600EBRAF cell lines with concurrent loss of both RB1 and PTEN were resistant to RAF inhibition whereas PLX4720 had a purely cytostatic effect in cell lines with PTEN loss and wild-type RB1 (Figures 6b–d). Indeed, ERK pathway inhibition was accompanied by G1 arrest and induction of apoptosis only in cells with intact PTEN and RB1 (Figures 6c and d). Furthermore, analogous to the result described above, stable infection of SKMEL-39 (V600EBRAF, PTEN-null, wild-type RB1) cells with viral E7 but not the E7Δ21–24 mutant incapable of binding to RB1 resulted in resistance to the anti-proliferative effects of PLX4720 (Supplementary Figure 6).
Our results suggest that tumors with concurrent V600EBRAF and PTEN/RB1 loss will be resistant to inhibitors of ERK signaling and that, in these cells, the ERK pathway does not regulate the transformed phenotype. To address this possibility, we generated mice bearing LOX (V600EBRAF/WTPTEN/WTRB1) and A2058 (V600EBRAF/PTEN-null/RB1-null) xenografts. SKMEL-207 cells (V600EBRAF/PTEN-null/RB1-null) were not used, as this cell line was not consistently tumorigenic in mice. Both PD0325901 and PLX4720, each administered by oral gavage, caused significant tumor regression in LOX-bearing mice (Figures 7a and b). In contrast, and consistent with our in vitro data, PD0325901 and PLX4720 had no effect on the growth of established A2058 xenografts (Figures 7c and d). Also, consistent with our in vitro data, treatment of mice with established SKMEL-39 (V600EBRAF, PTEN null, WTRB1) and SKMEL-133 (V600EBRAF, PTEN null, WTRB1) xenografts resulted in growth delay but no tumor regression (Supplementary Figure 7). Thus, loss of RB1 expression is likely to confer clinical resistance to inhibitors of ERK-signaling in V600EBRAF melanomas.
One of the hallmarks of cancer treatment is the heterogeneity of clinical outcomes. Such heterogeneity has been observed with both classical cytotoxics and newer targeted approaches that inhibit the pathways dysregulated by alterations in the oncogenes or tumor suppressors responsible for cancer initiation and progression. With inhibitors of activated kinases such as BRAF, variability in treatment response has been hypothesized to be the result of differences in the complement of genetic and epigenetic changes present within individual tumors.
In contrast to many solid tumors, cell line models can be readily derived from the majority of patients with invasive melanomas. Furthermore, a single missense mutation in BRAF (V600E) is found in over 90% of BRAF-mutant melanomas. These two factors allowed the generation of a large number of V600EBRAF-mutant melanoma models for use in studying the genomic diversity of this disease. To identify the V600EBRAF-mutant cohort, we first screened 149 melanoma cell lines and short-term cultures for mutations in BRAF. Genome-wide copy-number profiling was then performed to identify genomic alterations that co-occur with and potential diminish dependence on V600EBRAF. In our analysis, we noted a large number of both broad and focal copy-number alterations in cells expressing V600EBRAF. This high mutational burden in melanoma raised concern that inhibition of a single oncogenic driver such as BRAF would be unlikely to result in meaningful anti-tumor activity. Despite such concerns, we observed that V600EBRAF melanoma cell lines were dependent upon MAP kinase pathway activation for cell proliferation with only rare exception. Thus, the existence of multiple genetic lesions in melanoma does not, in general, eliminate dependence on the V600EBRAF mutation. This conclusion is consistent with a recent clinical trial of the RAF inhibitor PLX4032 in which an 81% partial response rate was observed in patients with advanced melanoma whose tumor harbored V600EBRAF (Flaherty et al., 2010).
However, our data and that of others do suggest that the complement of mutations that are found to co-occur with mutant BRAF does impact the response of such cells to selective BRAF and more generally ERK pathway inhibition (Gopal et al., 2010; Sondergaard et al., 2010). This contention is consistent with the clinical data, in which the responses to PLX4032 varied widely in both durability and the extent of tumor regression. In the current study, we identify PTEN loss as one genetic event that abrogates the effects of ERK pathway inhibition in V600EBRAF tumors. Loss of PTEN expression was observed in approximately one quarter (9/40) of the V600EBRAF melanoma cell lines examined. We observed that cells with concurrent BRAF mutation and PTEN loss were typically dependent upon ERK signaling, but the nature of the dependence was altered by concurrent loss of PTEN. Although, ERK was required for the proliferation of 17 of the 19 V600EBRAF cell lines examined in detail, it only promoted survival in the subset wild type for PTEN. It thus appears that secondary mutation of PTEN in a tumor with mutant BRAF renders the survival function of the latter redundant. These data suggest that tumor regressions are likely to be less frequent and of decreased magnitude in such tumors. Furthermore, our data provide a rationale for combined targeting of the RAF/MEK and PI3-kinase pathways in the cohort of patients with concurrent BRAF mutation and PTEN loss.
Resistance to the anti-proliferative effects of RAF and MEK inhibition was observed in two of the 19 V600E BRAF-mutant melanoma cell lines, a fraction consistent with the ~20% of patients who fail to derive any clinical benefit from the RAF inhibitor PLX4032 (Flaherty et al., 2010). In each case, both PTEN and RB1-loss coexisted with V600EBRAF. Furthermore, inactivation of RB1 in MEK-dependent, V600EBRAF-mutant, PTEN-null, RB1 wild-type cell lines upon infection with viral E7 conferred high-level resistance to MEK inhibition. These results suggest that loss of RB1 function in the setting of PTEN loss is sufficient to render the proliferation of V600EBRAF-mutant cell lines independent of oncogenic BRAF.
Members of the INK4 protein family inhibit cdk4 (cyclin-dependent kinase 4) and cdk6-mediated phosphorylation of the retinoblastoma susceptibility gene product (RB1). p16INK4A, a prototypic INK4 protein, functions as a tumor suppressor in many human cancers and is mutated or deleted in most but not all melanomas. Patients with germline mutations in CDK4 and those with hereditary retinoblastoma demonstrate an increased risk of developing melanoma, and in patients with CDK4 mutations, somatic alterations in the INK4A gene locus are not observed (Draper et al., 1986; Eng et al., 1993; Ohta et al., 1994). Consistent with these reports, the SKMEL-1 and SKMEL-28 cell lines, which harbor cdk4 amplification and mutation, respectively, express p16INK4A (Supplementary Figure 5). We observed that loss of RB1 and p16INK4A were also mutually exclusive events in our cohort of V600EBRAF melanoma cell lines. Detailed characterization of the two V600EBRAF, RB1-null models, demonstrated that in both cases, p16INK4A protein was expressed (Figure 2a). Furthermore, direct sequencing indicated that both RB1-null models were CDKN2A wild type within the p16 coding region of the gene (Supplementary Table 3). In the case of A2058, a small focal deletion was detected within the CDKN2A locus as previously reported (Kumar et al., 1998). Close inspection of this deletion confirmed that it encompassed only exon 1β of the ARF gene, which encodes the N-terminal portion of p14ARF but is outside of the coding region of INK4A (Figure 5d). These findings are consistent with the notion that p14ARF and p16INK4A have distinct tumor suppressive functions in melanoma. They also suggest that somatic loss of RB1 function may represent an alternative pathogenic event in melanomagenesis, which is mutually exclusive with INK4A inactivation.
Recent data demonstrating that RB1, like p16INK4A, can abrogate oncogene-induced senescence, provides a potential basis for the co-selection for RB1-loss in V600EBRAF tumors and its mutual exclusivity with CDKN2A (Chicas et al., 2010). However, whereas loss of both p16INK4A and RB1 may have overlapping roles in promoting tumor formation by preventing oncogene-induced senescence, our data suggest that loss of RB1, in contrast to loss of p16INK4A, leads to a diminished dependence upon BRAF.
Our work has translational implications for the ongoing clinical trials of RAF and MEK-selective inhibitors. Specifically, the data suggest that despite the high level of genomic complexity in melanoma, the vast majority of V600EBRAF melanoma models exhibit BRAF dependence and therefore inhibitors of BRAF or MEK may be effective in this setting. Early clinical trials of the MEK inhibitors PD0325901 and AZD6244 demonstrated low, but reproducible response rates (Adjei et al., 2008; Dummer et al., 2008). Although these early trials were not stratified for BRAF mutational status, the majority of responders were patients with melanoma. Additionally, in those cases where tissue was available for study, most patients with objective responses had tumors with BRAF mutations (Dummer et al., 2008). Nevertheless, the low response rate with MEK inhibitors even in patients whose tumors harbored BRAF mutation, raised the possibility that the high level of genomic complexity observed in melanoma was the basis for the limited efficacy of this approach in patients. The high response rate of PLX4032 along with our current data, however, imply that the lower response rate of MEK inhibitors in BRAF-mutant patients was unlikely to be due to a lack of BRAF dependence in the majority of patients harboring the oncogene.
One explanation for the significantly greater clinical efficacy of the RAF versus the MEK inhibitors in patients with BRAF-mutant melanoma is that oncogenic BRAF may promote tumor progression in part through MEK-independent effectors. The data we present here suggest that this possibility is unlikely to be the major determinant of individual variability, as BRAF-mutant tumors with concurrent PTEN and RB1 loss that exhibited resistance to MEK inhibition were also resistant to RAF inhibition. Rather, the greater efficacy of the RAF inhibitor observed in clinical trials is more likely attributable to the more potent inhibition of MAP kinase pathway activity achievable with PLX4032 in patients (Bollag et al., 2010). The MEK inhibitor downregulates MAP kinase activity in all cells including normal tissues whereas the RAF inhibitor PLX4032 inhibits MAP kinase activity only in cells expressing mutant BRAF (Figure 6a; Joseph et al., 2010). This appears to confer a broader therapeutic index, which allows for greater pathway inhibition with PLX4032 and, thus, increased anti-tumor efficacy in patients with BRAF-mutant tumors.
In summary, our data suggest that the complement of mutations associated with the evolution of mutant BRAF melanoma condition the biologic function of ERK signaling and thus sensitivity to selective MAP kinase pathway inhibitors. The prevalence of functional inactivation of RB1 in melanoma remains unknown. Given the higher frequency of CDKN2A deletions in cultured melanoma cells versus tumors, it is possible that the prevalence of RB1 alterations will also prove to be lower in primary and metastatic melanomas than that observed in our cell line panel (Gonzalgo et al., 1997). It is anticipated that the ongoing Tumor Cancer Genome Atlas project in melanoma will define the frequency of RB1 alterations in previously untreated melanomas and their co-occurrence with BRAF mutation and PTEN loss. The data presented here support screening for RB1 and PTEN inactivation in patients with advanced metastatic melanoma to determine whether RB1 and PTEN loss are associated with diminished clinical benefit with selective inhibitors of RAF and MEK.
PD0325901 was synthesized based upon the reported structure. PLX4720 was obtained from Plexxikon (Berkeley, CA, USA) and PI-103 from CalBiochem (Gibbstown, NJ, USA). Cell lines were checked for cross-contamination and misidentification using a multiplexed PCR/MS-based genetic finger-printing assay (Janakiraman et al., 2010). Cell growth inhibition was measured by Alamar Blue assay as previously described (Solit et al., 2006). To measure apoptosis, both adherent and floating cells were harvested and stained with ethidium bromide using the method of Nusse (Nusse et al., 1990).
Cell lysates were prepared as previously described (Solit et al., 2006). Anti–p42/44 MAPK, phospho–p42/44 MAPK, Akt, phospho-Akt (ser473), phospho-AKT (thr308), and RB1 antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-PTEN, cyclin D1, cyclin D2, cyclin D3, p16, p21, and p27 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-actin (42 KDa; rabbit; Sigma-Aldrich, St Louis, MO, USA) antibody was used to check for total protein loading.
Retroviral E7, E7Δ21–24, and MSCV-neo constructs (Demers et al., 1996) were first packaged and produced in NIH 293FT cells by co-transfection with pVSVG and pCL-Ampho constructs using FuGene transfection reagent (Roche Applied Sciences, Indianapolis, IN, USA). Two days after transfection, virus production media was collected, centrifuged and filtered through 0.45-μm sterile cellulose acetate filters to remove 293FT cells. Upon infection under established conditions, the retroviruses were removed 1 day after incubation with the recipient cells. Infected cells were selected with neomycin (G418) at established concentrations for the individual cell lines.
The iPLEX assay (Sequenom, Inc., San Diego, CA, USA) is based on a single-base primer extension assay (Thomas et al., 2007; Pratilas et al., 2008). The assay method was described in detail in Janakiraman et al., 2010. All primer sequences are available upon request.
Three micrograms of DNA was digested and labeled by random priming using Cy3 or Cy5-dUTP labeled primers (Invitrogen, Carlsbad, CA, USA). Labeled tumor DNA was co-hybridized to Agilent aCGH microarrays with a pool of reference normal DNA for 40 h at 60 °C. After washing, the slides were scanned and images were quantified using Feature Extraction 9.1 (Agilent Technologies, Wilmington, DE, USA). Raw copy-number estimates were normalized and segmented with Circular Binary Segmentation (Venkatraman and Olshen, 2007), both as previously described and additionally analyzed using the RAE algorithm (Taylor et al., 2008). Alteration counts were determined from segments exceeding either A0 or D0>0.99, respectively, per the multicomponent model in RAE (Taylor et al., 2008) excluding regions of either known or presumed germline copy-number polymorphism (as in The Cancer Genome Atlas Research Network, 2008). Segmented copy-number data was visualized in the Integrative Genomics Viewer (http://www.broadinstitute.org/igv) and all genome coordinates were standardized to NCBI build 36.1 (hg18) of the reference human genome.
Studies were performed under an IACUC-approved protocol, and guidelines for the proper and humane use of animals in research were followed. Xenografts were generated by injecting 1×107 tumor cells in matrigel (BD Biosciences, San Jose, CA, USA) into nu/nu athymic mice. Xenograft tumor-bearing mice were randomized to receive PD0325901 at a dose of 25 mg/kg, q.d., 5 days/week, PLX4720 at a dose of 12.5 mg/kg, b.i.d., 5 days/week, or vehicle only as control. PD0325901 and PLX4032 were formulated in 0.5% hydroxypropyl methylcellulose plus 0.2% Tween 80, and administered by oral gavage.
We thank Drs Meenhard Herlyn and Kate Nathanson (University of Pennsylvania) and Dr Øystein Fodstad (Department of Tumor Biology of The Norwegian Radium Hospital, Norway) for kindly providing cell lines. This study was supported by grants from the National Institutes of Health (DBS), the Kimmel Foundation (DBS), Golfers-Against-Cancer (DBS), the Melanoma Research Alliance (DBS) and STARR Foundation (DBS). BST is the David H Koch Fellow in cancer genomics.