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Key Points• Analysis of coding genomes of FL tumor subpopulations reveals striking clonal diversity at diagnosis and progression.• Within a hierarchy of somatic evolution of FL coding genomes, many recurrent mutations are subclonal at diagnosis.Follicular lymphoma (FL) is currently incurable using conventional chemotherapy or immunotherapy regimes, compelling new strategies. Advances in high-throughput sequencing technologies that can reveal oncogenic pathways have stimulated interest in tailoring therapies toward actionable somatic mutations. However, for mutation-directed therapies to be most effective, the mutations must be uniformly present in evolved tumor cells as well as in the self-renewing tumor-cell precursors. Here, we show striking intratumoral clonal diversity within FL tumors in the representation of mutations in the majority of genes as revealed by whole exome sequencing of subpopulations. This diversity captures a clonal hierarchy, resolved using immunoglobulin somatic mutations and IGH-BCL2 translocations as a frame of reference and by comparing diagnosis and relapse tumor pairs, allowing us to distinguish early versus late genetic eventsduring lymphomagenesis. We provide evidence that IGH-BCL2 translocations and CREBBP mutations are early events, whereas MLL2 and TNFRSF14 mutations probably represent late events during disease evolution. These observations provide insight into which of the genetic lesions represent suitable candidates for targeted therapies. (Blood. 2013;121(9):1604-1611) IntroductionFollicular lymphoma (FL) is a common form of non-Hodgkin lymphoma (NHL) arising from mature B cells. FL tumor cells share identical immunoglobulin (Ig) gene rearrangements, indicating that the transforming founder mutation(s) occurs subsequent to VDJ recombination. These cells express markers of mature B-lineage including surface-Ig and CD19, and germinal center B-cell markers such as LMO2, CD10, and BCL6. 1 The B-cell marker CD20, which is also the target of the anti-lymphoma therapy Rituximab, 2,3 is expressed to a variable degree on FL tumors. 4,5 CD20 levels can predict primary responsiveness of diverse lymphomas to Rituximab,6 and changes in CD20 expression contribute to Rituximab resistance and relapse. 7,8 For instance, CD20 expression levels are associated with survival in patients with aggressive lymphomas treated with or without Rituximab. 9 These studies each found CD20 to be variably expressed on cells within the same tumor, suggesting that this may be a marker of underlying clonal diversity.The genetic hallmark of FL is the t(14;18)(q32;q21) translocation that places the antiapoptotic BCL2 oncogene under control of the Ig heavy-chain enhancer. 10 This lesion is present in Ͼ 90% of FL cases, 11 but is also detectable in the majority of older healthy adults suggesting that it is not sufficient to induce clinical disease. 12 Recently, mutation of a histone methyltransferase gene, MLL2, was identified in 89% of FL cases, indicating that this may also be a founder mutation. 13 Genes encodin...

The transcriptional program regulated by the tumor suppressor p53 was analysed using oligonucleotide microarrays. A human lung cancer cell line that expresses the temperature sensitive murine p53 was utilized to quantitate mRNA levels of various genes at dierent time points after shifting the temperature to 328C. Inhibition of protein synthesis by cycloheximide (CHX) was used to distinguish between primary and secondary target genes regulated by p53. In the absence of CHX, 259 and 125 genes were up or down-regulated respectively; only 38 and 24 of these genes were up and down-regulated by p53 also in the presence of CHX and are considered primary targets in this cell line. Cluster analysis of these data using the super paramagnetic clustering (SPC) algorithm demonstrate that the primary genes can be distinguished as a single cluster among a large pool of p53 regulated genes. This procedure identi®ed additional genes that co-cluster with the primary targets and can also be classi®ed as such genes. In addition to cell cycle (e.g. p21, TGF-b, Cyclin E) and apoptosis (e.g. Fas, Bak, IAP) related genes, the primary targets of p53 include genes involved in many aspects of cell function, including cell adhesion (e.g. Thymosin, Smoothelin), signaling (e.g. H-Ras, Diacylglycerol kinase), transcription (e.g. ATF3, LISCH7), neuronal growth (e.g. Ninjurin, NSCL2) and DNA repair (e.g. BTG2, DDB2). The results suggest that p53 activates concerted opposing signals and exerts its eect through a diverse network of transcriptional changes that collectively alter the cell phenotype in response to stress.

The newly discovered p53 family member, p73, has a striking homology to p53 in both sequence and modular structure. Ectopic expression of p73 promotes transcription of p53 target genes and recapitulates the most characterized p53 biological effects such as growth arrest, apoptosis, and differentiation. Unlike p53-deficient mice that develop normally but are subject to spontaneous tumor formation, p73-deficient mice exhibit severe defects in the development of central nervous system and suffer from inflammation but are not prone to tumor development. These phenotypes suggest different biological activities mediated by p53 and p73 that might reflect activation of specific sets of target genes. Here, we have analyzed the gene expression profile of H1299 cells after p73␣ or p53 activation using oligonucleotide microarrays capable of detecting ϳ11,000 mRNA species. Our results indicate that p73␣ and p53 activate both common and distinct groups of genes. We found 141 and 320 genes whose expression is modulated by p73␣ and p53, respectively. p73␣ up-regulates 85 genes, whereas p53 induces 153 genes, of which 27 are in common with p73␣. Functional classification of these genes reveals that they are involved in many aspects of cell function ranging from cell cycle and apoptosis to DNA repair. Furthermore, we report that some of the upregulated genes are directly activated by p73␣ or p53.

Flexible maturation rates underlie part of the diversity of leaf shape, and tomato (Solanum lycopersicum) leaves are compound due to prolonged organogenic activity of the leaf margin. The CINCINNATA -TEOSINTE BRANCHED1, CYCLOIDEA, PCF (CIN-TCP) transcription factor LANCEOLATE (LA) restricts this organogenic activity and promotes maturation. Here, we show that tomato APETALA1/FRUITFULL (AP1/FUL) MADS box genes are involved in tomato leaf development and are repressed by LA. AP1/FUL expression is correlated negatively with LA activity and positively with the organogenic activity of the leaf margin. LA binds to the promoters of the AP1/FUL genes MBP20 and TM4. Overexpression of MBP20 suppressed the simple-leaf phenotype resulting from upregulation of LA activity or from downregulation of class I knotted like homeobox (KNOXI) activity. Overexpression of a dominant-negative form of MBP20 led to leaf simplification and partly suppressed the increased leaf complexity of plants with reduced LA activity or increased KNOXI activity. Tomato plants overexpressing miR319, a negative regulator of several CIN-TCP genes including LA, flower with fewer leaves via an SFTdependent pathway, suggesting that miR319-sensitive CIN-TCPs delay flowering in tomato. These results identify a role for AP1/FUL genes in vegetative development and show that leaf and plant maturation are regulated via partially independent mechanisms.