Mosaic BRAF Fusions Are a Recurrent Cause of Congenital Melanocytic Nevi Targetable by MAPK Pathway Inhibition.

Among children with multiple congenital melanocytic nevi, 25% have no established genetic cause, of whom many develop a hyperproliferative and severely pruritic phenotype resistant to treatment. Gene fusions have been reported in individual cases of congenital melanocytic nevi. We studied 169 patients with congenital melanocytic nevi in this study, 38 of whom were double wild type for pathogenic NRAS/BRAF variants. Nineteen of these 38 patients had sufficient tissue to undergo RNA sequencing, which revealed mosaic BRAF fusions in 11 of 19 patients and mosaic RAF1 fusions in 1 of 19. Recurrently, fusions involved the loss of the 5´ regulatory domain of BRAF or RAF1 but preserved the kinase domain. We validated all cases and detected the fusions in two separate nevi in 5 of 12 patients, confirming clonality. The absence of the fusion in blood in 8 of 12 patients indicated mosaicism. Primary culture of BRAF-fusion nevus cells from 3 of 12 patients demonstrated highly increased MAPK activation, despite only mildly increased BRAF expression, suggesting additional mechanisms of kinase activation. Trametinib quenched MAPK hyperactivation in vitro, and treatment of two patients caused rapid improvement in bulk tissue, improving bodily movement and reducing inflammation and severe pruritus. These findings offer a genetic diagnosis to an additional group of patients and trametinib as a treatment option for the severe associated phenotypes.

GNAQ/GNA11 Mosaicism Causes Aberrant Calcium Signaling Susceptible to Targeted Therapeutics.

Mosaic variants in genes GNAQ or GNA11 lead to a spectrum of vascular and pigmentary diseases including Sturge-Weber syndrome, in which progressive postnatal neurological deterioration led us to seek biologically targeted therapeutics. Using two cellular models, we find that disease-causing GNAQ/11 variants hyperactivate constitutive and G-protein coupled receptor ligand-induced intracellular calcium signaling in endothelial cells. We go on to show that the aberrant ligand-activated intracellular calcium signal is fueled by extracellular calcium influx through calcium-release-activated channels. Treatment with targeted small interfering RNAs designed to silence the variant allele preferentially corrects both the constitutive and ligand-activated calcium signaling, whereas treatment with a calcium-release-activated channel inhibitor rescues the ligand-activated signal. This work identifies hyperactivated calcium signaling as the primary biological abnormality in GNAQ/11 mosaicism and paves the way for clinical trials with genetic or small molecule therapies.

GNAQ/GNA11 Mosaicism Is Associated with Abnormal Serum Calcium Indices and Microvascular Neurocalcification.

Mosaic mutations in genes GNAQ or GNA11 lead to a spectrum of diseases including Sturge-Weber syndrome and phakomatosis pigmentovascularis with dermal melanocytosis. The pathognomonic finding of localized "tramlining" on plain skull radiography, representing medium-sized neurovascular calcification and associated with postnatal neurological deterioration, led us to study calcium metabolism in a cohort of 42 children. In this study, we find that 74% of patients had at least one abnormal measurement of calcium metabolism, the commonest being moderately low serum ionized calcium (41%) or high parathyroid hormone (17%). Lower levels of ionized calcium even within the normal range were significantly associated with seizures, and with specific antiepileptics despite normal vitamin D levels. Successive measurements documented substantial intrapersonal fluctuation in indices over time, and DEXA scans were normal in patients with hypocalcemia. Neurohistology from epilepsy surgery in five patients revealed not only intravascular, but perivascular and intraparenchymal mineral deposition and intraparenchymal microvascular disease in addition to previously reported findings. Neuroradiology review clearly demonstrated progressive calcium deposition in individuals over time. These findings and those of the adjoining paper suggest that calcium deposition in the brain of patients with GNAQ/GNA11 mosaicism may not be a nonspecific sign of damage as was previously thought, but may instead reflect the central postnatal pathological process in this disease spectrum.

PTPN11 Mosaicism Causes a Spectrum of Pigmentary and Vascular Neurocutaneous Disorders and Predisposes to Melanoma.

Phakomatosis pigmentovascularis is a diagnosis that denotes the coexistence of pigmentary and vascular birthmarks of specific types, accompanied by variable multisystem involvement, including CNS disease, asymmetrical growth, and a predisposition to malignancy. Using a tight phenotypic group and high-depth next-generation sequencing of affected tissues, we discover here clonal mosaic variants in gene PTPN11 encoding SHP2 phosphatase as a cause of phakomatosis pigmentovascularis type III or spilorosea. Within an individual, the same variant is found in distinct pigmentary and vascular birthmarks and is undetectable in blood. We go on to show that the same variants can cause either the pigmentary or vascular phenotypes alone, and drive melanoma development within pigmentary lesions. Protein structure modeling highlights that although variants lead to loss of function at the level of the phosphatase domain, resultant conformational changes promote longer ligand binding. In vitro modeling of the missense variants confirms downstream MAPK pathway overactivation and widespread disruption of human endothelial cell angiogenesis. Importantly, patients with PTPN11 mosaicism theoretically risk passing on the variant to their children as the germline RASopathy Noonan syndrome with lentigines. These findings improve our understanding of the pathogenesis and biology of nevus spilus and capillary malformation syndromes, paving the way for better clinical management.

Inherited duplications of PPP2R3B predispose to nevi and melanoma via a C21orf91-driven proliferative phenotype.

PURPOSE: Much of the heredity of melanoma remains unexplained. We sought predisposing germline copy-number variants using a rare disease approach. METHODS: Whole-genome copy-number findings in patients with melanoma predisposition syndrome congenital melanocytic nevus were extrapolated to a sporadic melanoma cohort. Functional effects of duplications in PPP2R3B were investigated using immunohistochemistry, transcriptomics, and stable inducible cellular models, themselves characterized using RNAseq, quantitative real-time polymerase chain reaction (qRT-PCR), reverse phase protein arrays, immunoblotting, RNA interference, immunocytochemistry, proliferation, and migration assays. RESULTS: We identify here a previously unreported genetic susceptibility to melanoma and melanocytic nevi, familial duplications of gene PPP2R3B. This encodes PR70, a regulatory unit of critical phosphatase PP2A. Duplications increase expression of PR70 in human nevus, and increased expression in melanoma tissue correlates with survival via a nonimmunological mechanism. PPP2R3B overexpression induces pigment cell switching toward proliferation and away from migration. Importantly, this is independent of the known microphthalmia-associated transcription factor (MITF)-controlled switch, instead driven by C21orf91. Finally, C21orf91 is demonstrated to be downstream of MITF as well as PR70. CONCLUSION: This work confirms the power of a rare disease approach, identifying a previously unreported copy-number change predisposing to melanocytic neoplasia, and discovers C21orf91 as a potentially targetable hub in the control of phenotype switching.

Combined targeting of G protein-coupled receptor and EGF receptor signaling overcomes resistance to PI3K pathway inhibitors in PTEN-null triple negative breast cancer.

Triple-negative breast cancer (TNBC) has poorer prognosis compared to other types of breast cancers due to the lack of effective therapies and markers for patient stratification. Loss of PTEN tumor suppressor gene expression is a frequent event in TNBC, resulting in over-activation of the PI 3-kinase (PI3K) pathway and sensitivity to its inhibition. However, PI3K pathway inhibitors show limited efficacy as monotherapies on these tumors. We report a whole-genome screen to identify targets whose inhibition enhanced the effects of different PI3K pathway inhibitors on PTEN-null TNBC. This identified a signaling network that relies on both the G protein-coupled receptor for thrombin (PAR1/F2R) and downstream G protein ßγ subunits and also epidermal growth factor receptor (EGFR) for the activation of the PI3K isoform p110ß and AKT. Compensation mechanisms involving these two branches of the pathway could bypass PI3K blockade, but combination targeting of both EGFR and PI3Kß suppressed ribosomal protein S6 phosphorylation and exerted anti-tumor activity both in vitro and in vivo, suggesting a new potential therapeutic strategy for PTEN-null TNBC.

Oncogenic RAS Signaling Promotes Tumor Immunoresistance by Stabilizing PD-L1 mRNA.

The immunosuppressive protein PD-L1 is upregulated in many cancers and contributes to evasion of the host immune system. The relative importance of the tumor microenvironment and cancer cell-intrinsic signaling in the regulation of PD-L1 expression remains unclear. We report that oncogenic RAS signaling can upregulate tumor cell PD-L1 expression through a mechanism involving increases in PD-L1 mRNA stability via modulation of the AU-rich element-binding protein tristetraprolin (TTP). TTP negatively regulates PD-L1 expression through AU-rich elements in the 3' UTR of PD-L1 mRNA. MEK signaling downstream of RAS leads to phosphorylation and inhibition of TTP by the kinase MK2. In human lung and colorectal tumors, RAS pathway activation is associated with elevated PD-L1 expression. In vivo, restoration of TTP expression enhances anti-tumor immunity dependent on degradation of PD-L1 mRNA. We demonstrate that RAS can drive cell-intrinsic PD-L1 expression, thus presenting therapeutic opportunities to reverse the innately immunoresistant phenotype of RAS mutant cancers.

The stress phenotype makes cancer cells addicted to CDT2, a substrate receptor of the CRL4 ubiquitin ligase.

CDT2/L2DTL/RAMP is one of the substrate receptors of the Cullin Ring Ubiquitin Ligase 4 that targets for ubiquitin mediated degradation a number of substrates, such as CDT1, p21 and CHK1, involved in the regulation of cell cycle and survival. Here we show that CDT2 depletion was alone able to induce the apoptotic death in 12/12 human cancer cell lines from different tissues, regardless of the mutation profile and CDT2 expression level. Cell death was associated to rereplication and to loss of CDT1 degradation. Conversely, CDT2 depletion did not affect non-transformed human cells, such as immortalized kidney, lung and breast cell lines, and primary cultures of endothelial cells and osteoblasts. The ectopic over-expression of an activated oncogene, such as the mutation-activated RAS or the amplified MET in non-transformed immortalized breast cell lines and primary human osteoblasts, respectively, made cells transformed in vitro, tumorigenic in vivo, and susceptible to CDT2 loss. The widespread effect of CDT2 depletion in different cancer cells suggests that CDT2 is not in a synthetic lethal interaction to a single specific pathway. CDT2 likely is a non-oncogene to which transformed cells become addicted because of their enhanced cellular stress, such as replicative stress and DNA damage.

Intrinsic resistance to MEK inhibition in KRAS mutant lung and colon cancer through transcriptional induction of ERBB3.

There are no effective therapies for the ~30% of human malignancies with mutant RAS oncogenes. Using a kinome-centered synthetic lethality screen, we find that suppression of the ERBB3 receptor tyrosine kinase sensitizes KRAS mutant lung and colon cancer cells to MEK inhibitors. We show that MEK inhibition results in MYC-dependent transcriptional upregulation of ERBB3, which is responsible for intrinsic drug resistance. Drugs targeting both EGFR and ERBB2, each capable of forming heterodimers with ERBB3, can reverse unresponsiveness to MEK inhibition by decreasing inhibitory phosphorylation of the proapoptotic proteins BAD and BIM. Moreover, ERBB3 protein level is a biomarker of response to combinatorial treatment. These data suggest a combination strategy for treating KRAS mutant colon and lung cancers and a way to identify the tumors that are most likely to benefit from such combinatorial treatment.

Reversible and adaptive resistance to BRAF(V600E) inhibition in melanoma.

Treatment of BRAF(V600E) mutant melanoma by small molecule drugs that target the BRAF or MEK kinases can be effective, but resistance develops invariably. In contrast, colon cancers that harbour the same BRAF(V600E) mutation are intrinsically resistant to BRAF inhibitors, due to feedback activation of the epidermal growth factor receptor (EGFR). Here we show that 6 out of 16 melanoma tumours analysed acquired EGFR expression after the development of resistance to BRAF or MEK inhibitors. Using a chromatin-regulator-focused short hairpin RNA (shRNA) library, we find that suppression of sex determining region Y-box 10 (SOX10) in melanoma causes activation of TGF-ß signalling, thus leading to upregulation of EGFR and platelet-derived growth factor receptor-ß (PDGFRB), which confer resistance to BRAF and MEK inhibitors. Expression of EGFR in melanoma or treatment with TGF-ß results in a slow-growth phenotype with cells displaying hallmarks of oncogene-induced senescence. However, EGFR expression or exposure to TGF-ß becomes beneficial for proliferation in the presence of BRAF or MEK inhibitors. In a heterogeneous population of melanoma cells having varying levels of SOX10 suppression, cells with low SOX10 and consequently high EGFR expression are rapidly enriched in the presence of drug, but this is reversed when the drug treatment is discontinued. We find evidence for SOX10 loss and/or activation of TGF-ß signalling in 4 of the 6 EGFR-positive drug-resistant melanoma patient samples. Our findings provide a rationale for why some BRAF or MEK inhibitor-resistant melanoma patients may regain sensitivity to these drugs after a 'drug holiday' and identify patients with EGFR-positive melanoma as a group that may benefit from re-treatment after a drug holiday.

BRAF V600E is a determinant of sensitivity to proteasome inhibitors.

A critical step toward defining tailored therapy in patients with cancer is the identification of genetic interactions that may impair-or boost-the efficacy of selected therapeutic approaches. Cell models able to recapitulate combinations of genetic aberrations are important to find drug-genotype interactions poorly affected by the heterogeneous genetics of human tumors. In order to identify novel pharmacogenomic relationships, we employed an isogenic cell panel that reconstructs cancer genetic scenarios. We screened a library of 43 compounds in human hTERT-HME1 epithelial cells in which PTEN or RB1 were silenced in combination with the targeted knockin of cancer-associated mutations in EGFR, KRAS, BRAF, or PIK3CA oncogenes. Statistical analysis and clustering algorithms were applied to display similar drug response profiles and mutation-specific patterns of activity. From the screen, we discovered that proteasome inhibitors show selectivity toward BRAF V600E-mutant cells, irrespective of PTEN or RB1 expression. Preferential targeting of BRAF-mutant cells by proteasome inhibitors was corroborated in a second BRAF V600E isogenic model, as well as in a panel of colorectal cancer cell lines by the use of the proteasome inhibitor carfilzomib. Notably, carfilzomib also showed striking in vivo activity in a BRAF-mutant human colorectal cancer xenograft model. Vulnerability to proteasome inhibitors is dependent on persistent BRAF signaling, because BRAF V600E blockade by PLX4720 reversed sensitivity to carfilzomib in BRAF-mutant colorectal cancer cells. Our findings indicated that proteasome inhibition might represent a valuable targeting strategy in BRAF V600E-mutant colorectal tumors.

Amplification of the MET receptor drives resistance to anti-EGFR therapies in colorectal cancer.

EGF receptor (EGFR)-targeted monoclonal antibodies are effective in a subset of metastatic colorectal cancers. Inevitably, all patients develop resistance, which occurs through emergence of KRAS mutations in approximately 50% of the cases. We show that amplification of the MET proto-oncogene is associated with acquired resistance in tumors that do not develop KRAS mutations during anti-EGFR therapy. Amplification of the MET locus was present in circulating tumor DNA before relapse was clinically evident. Functional studies show that MET activation confers resistance to anti-EGFR therapy both in vitro and in vivo. Notably, in patient-derived colorectal cancer xenografts, MET amplification correlated with resistance to EGFR blockade, which could be overcome by MET kinase inhibitors. These results highlight the role of MET in mediating primary and secondary resistance to anti-EGFR therapies in colorectal cancer and encourage the use of MET inhibitors in patients displaying resistance as a result of MET amplification.

Modeling tumor progression by the sequential introduction of genetic alterations into the genome of human normal cells.

Cancer genomes display a complex blend of genetic lesions affecting oncogenes and tumor suppressor genes. Multiple modeling approaches indicate that 5-15 driver oncogenic events are required to achieve tumor progression in common epithelial cancers. In vitro, a lower number (2-3) of events is typically sufficient to achieve full transformation. We developed cellular models that closely resemble the occurrence of multiple genetic lesions to understand their role in tumor progression. Homologous recombination and transcriptional downregulation were used to recapitulate the co-occurrence of driver mutations targeting oncogenes and inactivation of tumor suppressor genes in human nontransformed epithelial cells. Knockdown of the tumor suppressor genes PTEN or RB1 was combined with mutagenic activation of individual oncogenes (EGFR, KRAS, BRAF, or PIK3CA), thus generating a combinatorial model. The simultaneous presence of oncogenic and tumor suppressive events resulted in distinct biochemical properties and anchorage-independent growth abilities. Notably, however, we found that even when up to four individual alterations were concomitantly present they were not sufficient to fully transform the target cells. Our results suggest that the close recapitulation of cancer lesions in not-transformed cells is essential to unveil their oncogenic potential and raise questions concerning the minimal requirements for neoplastic transformation of epithelial cells.

Unresponsiveness of colon cancer to BRAF(V600E) inhibition through feedback activation of EGFR.

Inhibition of the BRAF(V600E) oncoprotein by the small-molecule drug PLX4032 (vemurafenib) is highly effective in the treatment of melanoma. However, colon cancer patients harbouring the same BRAF(V600E) oncogenic lesion have poor prognosis and show only a very limited response to this drug. To investigate the cause of the limited therapeutic effect of PLX4032 in BRAF(V600E) mutant colon tumours, here we performed an RNA-interference-based genetic screen in human cells to search for kinases whose knockdown synergizes with BRAF(V600E) inhibition. We report that blockade of the epidermal growth factor receptor (EGFR) shows strong synergy with BRAF(V600E) inhibition. We find in multiple BRAF(V600E) mutant colon cancers that inhibition of EGFR by the antibody drug cetuximab or the small-molecule drugs gefitinib or erlotinib is strongly synergistic with BRAF(V600E) inhibition, both in vitro and in vivo. Mechanistically, we find that BRAF(V600E) inhibition causes a rapid feedback activation of EGFR, which supports continued proliferation in the presence of BRAF(V600E) inhibition. Melanoma cells express low levels of EGFR and are therefore not subject to this feedback activation. Consistent with this, we find that ectopic expression of EGFR in melanoma cells is sufficient to cause resistance to PLX4032. Our data suggest that BRAF(V600E) mutant colon cancers (approximately 8-10% of all colon cancers), for which there are currently no targeted treatment options available, might benefit from combination therapy consisting of BRAF and EGFR inhibitors.

Transfection and DNA-mediated gene transfer.

The advent of recent technologies such as gene expression microarrays and high-throughput sequencing methods has allowed for unveiling the molecular complexity of cancer. However, compared to the genomic discovery stage, the functional characterization of genes that have been found altered (by somatic mutations, rearrangements, or copy number variations) or differentially regulated at the expression level is still lagging behind. In the future, it is anticipated that efforts would be aimed at addressing the impact of such genes on several cancer traits, including tumor formation, dissemination, and response to therapies. These studies would likely have to rely on introducing the gene(s) of interest (in its -wild-type or altered version) in cellular models. We describe here a number of techniques to introduce nucleic acids into eukaryotic cells, ranging from conventional plasmid transfection to lentiviral -transduction and adeno-associated viral (AAV)-mediated DNA transfer.

Deregulation of the PI3K and KRAS signaling pathways in human cancer cells determines their response to everolimus.

Personalized cancer medicine is based on the concept that targeted therapies are effective on subsets of patients whose tumors carry specific molecular alterations. Several mammalian target of rapamycin (mTOR) inhibitors are in preclinical or clinical trials for cancers, but the molecular basis of sensitivity or resistance to these inhibitors among patients is largely unknown. Here we have identified oncogenic variants of phosphoinositide-3-kinase, catalytic, alpha polypeptide (PIK3CA) and KRAS as determinants of response to the mTOR inhibitor everolimus. Human cancer cells carrying alterations in the PI3K pathway were responsive to everolimus, both in vitro and in vivo, except when KRAS mutations occurred concomitantly or were exogenously introduced. In human cancer cells with mutations in both PIK3CA and KRAS, genetic ablation of mutant KRAS reinstated response to the drug. Consistent with these data, PIK3CA mutant cells, but not KRAS mutant cells, displayed everolimus-sensitive translation. Importantly, in a cohort of metastatic cancer patients, the presence of oncogenic KRAS mutations was associated with lack of benefit after everolimus therapy. Thus, our results demonstrate that alterations in the KRAS and PIK3CA genes may represent biomarkers to optimize treatment of patients with mTOR inhibitors.

Tracking the genomic evolution of breast cancer metastasis.

Therapeutic choices for metastatic tumors are, in most cases, based upon the histological and molecular analysis of the corresponding primary tumor. Understanding whether and to what extent the genomic landscape of metastasis differs from the tumors from which they originated is critical yet largely unknown. A recent report tackled this key issue by comparing the genomic and transcriptional profile of a metastatic lobular breast tumor with that of the primary tumor surgically removed 9 years earlier. The extent of the differences suggests a high degree of mutational heterogeneity between primary and metastatic lesions and indicates that significant evolution occurs during breast cancer progression.

Replacement of normal with mutant alleles in the genome of normal human cells unveils mutation-specific drug responses.

Mutations in oncogenes and tumor suppressor genes are responsible for tumorigenesis and represent favored therapeutic targets in oncology. We exploited homologous recombination to knock-in individual cancer mutations in the genome of nontransformed human cells. Sequential introduction of multiple mutations was also achieved, demonstrating the potential of this strategy to construct tumor progression models. Knock-in cells displayed allele-specific activation of signaling pathways and mutation-specific phenotypes different from those obtainable by ectopic oncogene expression. Profiling of a library of pharmacological agents on the mutated cells showed striking sensitivity or resistance phenotypes to pathway-targeted drugs, often matching those of tumor cells carrying equivalent cancer mutations. Thus, knock-in of single or multiple cancer alleles provides a pharmacogenomic platform for the rational design of targeted therapies.