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Unlabelled: The identification of genes maintaining cancer growth is critical to our understanding of tumorigenesis. We report the first in vivo genetic screen of patient-derived tumors, using metastatic melanomas and targeting 236 chromatin genes by expression of specific shRNA libraries. Our screens revealed unprecedented numerosity of genes indispensable for tumor growth (∼50% of tested genes) and unexpected functional heterogeneity among patients (<15% in common). Notably, these genes were not activated by somatic mutations in the same patients and are therefore distinguished from mutated cancer driver genes. We analyzed underlying molecular mechanisms of one of the identified genes, the Histone-lysine N-methyltransferase KMT2D, and showed that it promotes tumorigenesis by dysregulating a subset of transcriptional enhancers and target genes involved in cell migration. The assembly of enhancer genomic patterns by activated KMT2D was highly patient-specific, regardless of the identity of transcriptional targets, suggesting that KMT2D might be activated by distinct upstream signaling pathways., Significance: Drug targeting of biologically relevant cancer-associated mutations is considered a critical strategy to control cancer growth. Our functional in vivo genetic screens of patient-derived tumors showed unprecedented numerosity and interpatient heterogeneity of genes that are essential for tumor growth, but not mutated, suggesting that multiple, patient-specific signaling pathways are activated in tumors. Cancer Discov; 6(6); 650-63. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 561., (©2016 American Association for Cancer Research.)

The T-box transcription factors Tbx2 and Tbx3 are overexpressed in many cancers and in melanoma promote proliferation by actively suppressing senescence. Whether they also contribute to tumor progression via other mechanisms is not known. Here, we identify a novel role for these factors, providing evidence that Tbx3, and potentially Tbx2, directly repress the expression of E-cadherin, a keratinocyte-melanoma adhesion molecule whose loss is required for the acquisition of an invasive phenotype. Overexpression of Tbx2 and Tbx3 in melanoma cells down-regulates endogenous E-cadherin expression, whereas depletion of Tbx3, but not Tbx2, increases E-cadherin mRNA and protein levels and decreases melanoma invasiveness in vitro. Consistent with these observations, in melanoma tissue, Tbx3 and E-cadherin expression are inversely correlated. Depletion of Tbx3 also leads to substantial up-regulation of Tbx2. The results suggest that Tbx2 and Tbx3 may play a dual role during the radial to vertical growth phase transition by both inhibiting senescence via repression of p21(CIP1) expression, and enhancing melanoma invasiveness by decreasing E-cadherin levels.

The Src homology and collagen (Src) family of adaptor proteins comprises six Shc-like proteins encoded by three loci in mammals (Shc, Rai, and Sli). Shc-like proteins are tyrosine kinase substrates, which regulate diverse signaling pathways and cellular functions, including Ras and proliferation (p52/p46Shc), phosphatidylinositol 3-kinase and survival (p54Rai), and mitochondrial permeability transition and apoptosis (p66Shc). Here, we report the identification, cloning, and sequence characterization of a new member of the Shc family that we termed RaLP. RaLP encodes a 69-kDa protein characterized by the CH2-PTB-CH1-SH2 modularity, typical of the Shc protein family, and expressed, among adult tissues, only in melanomas. Analysis of RaLP expression during the melanoma progression revealed low expression in normal melanocytes and benign nevi, whereas high levels of RaLP protein were found at the transition from radial growth phase to vertical growth phase and metastatic melanomas, when tumor cells acquire migratory competence and invasive potential. Notably, silencing of RaLP expression in metastatic melanomas by RNA interference reduced tumorigenesis in vivo. Analysis of RaLP in melanoma signal transduction pathways revealed that (a) when ectopically expressed in RaLP-negative melanocytes and nonmetastatic melanoma cells, it functions as a substrate of activated insulin-like growth factor-1 and epidermal growth factor receptors and increases Ras/mitogen-activated protein kinase (MAPK) signaling and cell migration, whereas (b) its silencing in RaLP-positive melanoma cells abrogates cell migration in vitro, without affecting MAPK signaling, suggesting that RaLP activates both Ras-dependent and Ras-independent migratory pathways in melanomas. These findings indicate that RaLP is a specific marker of metastatic melanomas, a critical determinant in the acquisition of the migratory phenotype by melanoma cells, and a potential target for novel anti-melanoma therapeutic strategies.

Anaplastic lymphoma kinase (ALK)-positive lymphomas ("ALKomas") constitute a distinct molecular and clinicopathological entity within the heterogeneous group of CD30-positive large cell lymphomas. In 80-85% of cases tumor cells express a Mr 80,000 hybrid protein comprising the nucleolar phosphoprotein nucleophosmin (NPM) and the ALK. We report here the cloning and expression of a novel ALK-fusion protein from an ALK-positive lymphoma. This case was selected for molecular investigation because of (a) the absence of NPM-ALK transcripts; (b) the atypical staining patterns for ALK (cytoplasm-restricted) and for NPM (nucleus-restricted); and (c) the presence of a Mr 96,000 ALK-protein differing in size from NPM-ALK. Nucleotide sequence analysis of ALK transcripts isolated by 5'-rapid amplification of cDNA ends revealed a chimeric mRNA corresponding to an ATIC-ALK in-frame fusion. ATIC is a bifunctional enzyme (5-aminoimidazole-4-carboxamide ribonucleotide transformylase and IMP cyclohydrolase enzymatic activities) that catalyzes the penultimate and final enzymatic activities of the purine nucleotide synthesis pathway. Expression of full-length ATIC-ALK cDNA in mouse fibroblasts revealed that the fusion protein (a) possesses constitutive tyrosine kinase activity; (b) forms stable complexes with the signaling proteins Grb2 and Shc; (c) induces tyrosine-phosphorylation of Shc; and (d) provokes oncogenic transformation. These findings point to fusion with ATIC as an alternative mechanism of ALK activation.

We report a retroviral expression vector (PINCO) that allows high-efficiency gene transfer and selection of hemopoietic progenitor cells (HPCs). The main characteristics of this vector are the presence outside the two long terminal repeats of the EBV origin of replication and the EBNA-1 gene and the presence in the retrovirus of the cDNA that encodes for the enhanced green fluorescence protein (GFP), controlled by a cytomegalovirus promoter. Transient transfection of PINCO in Phoenix packaging cells results in episomal propagation of the plasmid and generates viral titers as high as 10(7) colony-forming units/ml. Infection of established cell lines with the PINCO retrovirus yields more than 95% GFP-expressing cells. GFP expression remains stable for months in infected cell cultures and can easily be monitored by fluorescent microscopy or fluorescence-activated cell-sorting (FACS) analysis of living cells. The PINCO vector allows efficient expression of a second gene (thymidine kinase, Shc, and PML), and there is strict correlation between GFP and second gene expression levels in the infected cells. PINCO was used to infect human HPCs; infection efficiency was about 50%. GFP-positive cells can be FACS sorted to yield a homogeneous population of infected cells. FACS-sorted GFP-positive HPC cells have, with respect to unfractionated HPC cells, the same frequency of long-term culture initiating cells and an identical capacity to undergo multilineage and unilineage differentiation. The entire gene transfer procedure, from the transfection of the packaging cell line to the infection of target cells, requires less than a week. The high viral titer and the easy obtainment of homogeneously infected cell populations without drug selection procedures make PINCO an ideal vector for gene transfer of human primary hemopoietic cells.