Routine Multiplex Mutational Profiling of Melanomas Enables Enrollment in Genotype-Driven Therapeutic Trials

Purpose: Knowledge of tumor mutation status is becoming increasingly important for the treatment of cancer, as mutation-specific inhibitors are being developed for clinical use that target only sub-populations of patients with particular tumor genotypes. Melanoma provides a recent example of this paradigm. We report here development, validation, and implementation of an assay designed to simultaneously detect 43 common somatic point mutations in 6 genes (BRAF, NRAS, KIT, GNAQ, GNA11, and CTNNB1) potentially relevant to existing and emerging targeted therapies specifically in melanoma. Methods: The test utilizes the SNaPshot method (multiplex PCR, multiplex primer extension, and capillary electrophoresis) and can be performed rapidly with high sensitivity (requiring 5–10% mutant allele frequency) and minimal amounts of DNA (10–20 nanograms). The assay was validated using cell lines, fresh-frozen tissue, and formalin-fixed paraffin embedded tissue. Clinical characteristics and the impact on clinical trial enrollment were then assessed for the first 150 melanoma patients whose tumors were genotyped in the Vanderbilt molecular diagnostics lab. Results: Directing this test to a single disease, 90 of 150 (60%) melanomas from sites throughout the body harbored a mutation tested, including 57, 23, 6, 3, and 2 mutations in BRAF, NRAS, GNAQ, KIT, and CTNNB1, respectively. Among BRAF V600 mutations, 79%, 12%, 5%, and 4% were V600E, V600K, V600R, and V600M, respectively. 23 of 54 (43%) patients with mutation harboring metastatic disease were subsequently enrolled in genotype-driven trials. Conclusion: We present development of a simple mutational profiling screen for clinically relevant mutations in melanoma. Adoption of this genetically-informed approach to the treatment of melanoma has already had an impact on clinical trial enrollment and prioritization of therapy for patients with the disease

Acquired Resistance to BRAF Inhibitors Mediated by a RAF Kinase Switch in Melanoma Can Be Overcome by Cotargeting MEK and IGF-1R/PI3K

SummaryBRAF is an attractive target for melanoma drug development. However, resistance to BRAF inhibitors is a significant clinical challenge. We describe a model of resistance to BRAF inhibitors developed by chronic treatment of BRAFV600E melanoma cells with the BRAF inhibitor SB-590885; these cells are cross-resistant to other BRAF-selective inhibitors. Resistance involves flexible switching among the three RAF isoforms, underscoring the ability of melanoma cells to adapt to pharmacological challenges. IGF-1R/PI3K signaling was enhanced in resistant melanomas, and combined treatment with IGF-1R/PI3K and MEK inhibitors induced death of BRAF inhibitor-resistant cells. Increased IGF-1R and pAKT levels in a post-relapse human tumor sample are consistent with a role for IGF-1R/PI3K-dependent survival in the development of resistance to BRAF inhibitors

Modulators of Prostate Cancer Cell Proliferation and Viability Identified by Short-Hairpin RNA Library Screening

There is significant need to identify novel prostate cancer drug targets because current hormone therapies eventually fail, leading to a drug-resistant and fatal disease termed castration-resistant prostate cancer. To functionally identify genes that, when silenced, decrease prostate cancer cell proliferation or induce cell death in combination with antiandrogens, we employed an RNA interference-based short hairpin RNA barcode screen in LNCaP human prostate cancer cells. We identified and validated four candidate genes (AKT1, PSMC1, STRADA, and TTK) that impaired growth when silenced in androgen receptor positive prostate cancer cells and enhanced the antiproliferative effects of antiandrogens. Inhibition of AKT with a pharmacologic inhibitor also induced apoptosis when combined with antiandrogens, consistent with recent evidence for PI3K and AR pathway crosstalk in prostate cancer cells. Recovery of hairpins targeting a known prostate cancer pathway validates the utility of shRNA library screening in prostate cancer as a broad strategy to identify new candidate drug targets

Probes depleted in bicalutamide-treated LNCaP cells.

*<p>Number of probes that had a log2 value ≤−0.58.</p>**<p>Bic, bicalutamide; Veh, vehicle.</p

shRNA probes depleted or enriched in bicalutamide-treated LNCaP cells.

<p>(A) Schematic of shRNA screen. Details can be found in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034414#s4" target="_blank">Materials and Methods</a> section. (B) A heatmap was generated by clustering based on probes that were depleted or enriched (log2 bicalutamide/vehicle ≤+/−0.58 and a p-value≤0.01) in the bicalutamide (0.4 uM and 1.0 uM)-treated LNCaP cells at T = 1 and T = 2 compared to the vehicle-treated cells at the same timepoints. The shRNA target gene associated with each probe is indicated to the right of the heatmap. Target genes that appear more than one time on the heatmap indicate that more than one probe scored for that gene.</p

Silencing of a subset of genes inhibited VCaP proliferation and induced apoptosis.

<p>Candidate target genes from LNCaP screen probes that were depleted in the presence of bicalutamide were selectively targeted using siRNAs. (A, left panel), VCaP cells were transfected with siRNAs that scored in the LNCaP drug screen and were incubated with 1 uM MDV3100 or vehicle, and the number of viable cells was measured 6 days post-treatment. (A, right panel), VCaP cells were transfected and treated as in (A, left panel) except Caspase 3/7 activity was measured after 3 days of treatment. <i>PLK1</i> siRNA transfection was used as a positive control. Dashed lines indicate the level of growth inhibition or apoptosis induced by MDV3100, for comparison. NT, non-targeting siRNA. (B) Gene silencing (10–50%) was confirmed by RT-qPCR 6 days post-transfection of VCaP cells with the siRNA SMARTpools. Reactions were done in triplicate and normalized to RPL27 for each cDNA and then normalized to vehicle-treated NT. Standard error of the mean was calculated. Bic, bicalutamide.</p

Higher <i>TTK</i> expression is associated with biochemical recurrence.

<p>Kaplan Meier plot of the risk of biochemical recurrence in patients (n = 131) with TTK overexpressing prostate tumors (n = 20; green line) versus those without TTK overexpressing (n = 111; blue line) tumors (p = 0.003, log-rank test).</p

Candidate gene expression and alterations in human prostate tumors.

*<p>4 of the samples with upregulated AR mRNA also had <i>AR</i> amplification.</p