Diffuse large B-cell lymphoma (DLBCL) is the most common form of lymphoma in adults. The disease exhibits a striking heterogeneity in gene expression profiles and clinical outcomes, but its genetic causes remain to be fully defined. Through whole genome and exome sequencing, we characterized the genetic diversity of DLBCL. In all, we sequenced 73 DLBCL primary tumors (34 with matched normal DNA). Separately, we sequenced the exomes of 21 DLBCL cell lines. We identified 322 DLBCL cancer genes that were recurrently mutated in primary DLBCLs. We identified recurrent mutations implicating a number of known and not previously identified genes and pathways in DLBCL including those related to chromatin modification (ARID1A and MEF2B), NF-κB (CARD11 and TNFAIP3), PI3 kinase (PIK3CD, PIK3R1, and MTOR), B-cell lineage (IRF8, POU2F2, and GNA13), and WNT signaling (WIF1). We also experimentally validated a mutation in PIK3CD, a gene not previously implicated in lymphomas. The patterns of mutation demonstrated a classic long tail distribution with substantial variation of mutated genes from patient to patient and also between published studies. Thus, our study reveals the tremendous genetic heterogeneity that underlies lymphomas and highlights the need for personalized medicine approaches to treating these patients.next-generation sequencing | cancer genetics | cancer heterogeneity D iffuse large B-cell lymphoma (DLBCL) is the most common form of lymphoma in adults (1). Although nearly half the patients can be cured with standard regimens, the majority of relapsed patients succumb. Thus, there is an urgent need to identify the genetic underpinnings of the disease and to identify novel treatment strategies. Gene expression profiling (2, 3) has uncovered distinct molecular signatures for DLBCL subtypes that have unique biology and prognoses. High-throughput sequencing has provided rich opportunities for the comprehensive identification of the genetic causes of cancer (4-6). Whereas exhaustive portraits of individual cancer genomes are emerging, the degree to which these genomes represent the disease is unclear.We generated a detailed analysis of a DLBCL genome by sequencing a primary human tumor and paired normal tissue (Dataset S1). We further characterized the genetic diversity of DLBCL by sequencing the exomes of 73 DLBCL primary tumors (34 with matched normal DNA) and 21 DLBCL cell lines for comparative purposes. This in-depth sequencing identified 322 DLBCL cancer genes that were recurrently mutated in DLBCLs. We also experimentally validated the effects of genetic alteration of PIK3CD, an oncogene that we identified in DLBCL. Our work provides one of the largest genetic portraits yet of human DLBCLs and offers insights into the molecular heterogeneity of the disease, especially in the context of other recently published studies in DLBCL (7, 8).

Oncogene-induced senescence is an important mechanism by which normal cells are restrained from malignant transformation. Here we report that the suppression of the c-Myc (MYC) oncogene induces cellular senescence in diverse tumor types including lymphoma, osteosarcoma, and hepatocellular carcinoma. MYC inactivation was associated with prototypical markers of senescence, including acidic ␤-gal staining, induction of p16INK4a, and p15INK4b expression. Moreover, MYC inactivation induced global changes in chromatin structure associated with the marked reduction of histone H4 acetylation and increased histone H3 K9 methylation. Osteosarcomas engineered to be deficient in p16INK4a or Rb exhibited impaired senescence and failed to exhibit sustained tumor regression upon MYC inactivation. Similarly, only after lymphomas were repaired for p53 expression did MYC inactivation induce robust senescence and sustained tumor regression. The pharmacologic inhibition of signaling pathways implicated in oncogene-induced senescence including ATM/ATR and MAPK did not prevent senescence associated with MYC inactivation. Our results suggest that cellular senescence programs remain latently functional, even in established tumors, and can become reactivated, serving as a critical mechanism of oncogene addiction associated with MYC inactivation.oncogene addiction ͉ tumorigenesis ͉ tumor maintenance

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Tyrosine kinase inhibitors (TKIs), despite efficacy as anti-cancer therapeutics, are associated with cardiovascular side effects ranging from induced arrhythmias to heart failure. We used patient-specific human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), generated from 11 healthy individuals and 2 patients receiving cancer treatment, to screen FDA-approved TKIs for cardiotoxicities by measuring alterations in cardiomyocyte viability, contractility, electrophysiology, calcium handling, and signaling. With these data, we generated a “cardiac safety index” to assess cardiotoxicities of existing TKIs. TKIs with low cardiac safety indices exhibit cardiotoxicity in patients. We also derived endothelial cells (hiPSC-ECs) and cardiac fibroblasts (hiPSC-CFs) to examine cell type-specific cardiotoxicities. Using high-throughput screening, we determined that VEGFR2/PDGFR-inhibiting TKIs caused cardiotoxicity in hiPSC-CMs, hiPSC-ECs, and hiPSC-CFs. Using phosphoprotein analysis, we determined that VEGFR2/PDGFR-inhibiting TKIs led to a compensatory increase in cardioprotective insulin and insulin-like growth factor (IGF) signaling in hiPSC-CMs. Upregulating cardioprotective signaling with exogenous insulin or IGF1 improved hiPSC-CM viability during co-treatment with cardiotoxic VEGFR2/PDGFR-inhibiting TKIs. Thus, hiPSC-CMs can be used to screen for cardiovascular toxicities associated with anti-cancer TKIs, correlating with clinical phenotypes. This approach provides unexpected insights, as illustrated by our finding that toxicity can be alleviated via cardioprotective insulin/IGF signaling.

Summary Oncogene addiction is thought to occur cell autonomously. Immune effectors are implicated in the induction and restraint of tumorigenesis, but their role in oncogene inactivation mediated tumor regression is unclear. Here, we show that an intact immune system, specifically CD4+ T-cells, is required for the induction of cellular senescence, shut down of angiogenesis and chemokine expression resulting in sustained tumor regression upon inactivation of the MYC or BCR-ABL oncogenes in mouse models of T-cell acute lymphoblastic lymphoma and pro-B-cell leukemia, respectively. Moreover, immune effectors knocked out for thrombospondins failed to induce sustained tumor regression. Hence, CD4+ T-cells are required for the remodeling of the tumor microenvironment through the expression of chemokines, such as thrombospondins, in order to elicit oncogene addiction.