Diffuse large B-cell lymphoma (DLBCL) represents a heterogeneous diagnostic category with distinct molecular subtypes that can be defined by gene expression profiling. However, even within these defined subtypes, heterogeneity prevails. To further elucidate the pathogenesis of these entities, we determined the expression of the tumor suppressor phosphatase and tensin homolog (PTEN) in 248 primary DLBCL patient samples. These analyses revealed that loss of PTEN was detectable in 55% of germinal center B-cell-like (GCB) DLBCLs, whereas this abnormality was found in only 14% of non-GCB DLBCL patient samples. In GCB DLBCL, the PTEN status was inversely correlated with activation of the oncogenic PI3K/ protein kinase B (AKT) pathway in both DLBCL cell lines and primary patient samples. Reexpression of PTEN induced cytotoxicity in PTEN-deficient GCB DLBCL cell line models by inhibiting PI3K/AKT signaling, indicating an addiction to this pathway in this subset of GCB DLBCLs. PI3K/AKT inhibition induced down-regulation of the transcription factor MYC. Reexpression of MYC rescued GCB DLBCL cells from PTEN-induced toxicity, identifying a regulatory mechanism of MYC expression in DLBCL. Finally, pharmacologic PI3K inhibition resulted in toxicity selectively in PTEN-deficient GCB DLBCL lines. Collectively, our results indicate that PTEN loss defines a PI3K/ AKT-dependent GCB DLBCL subtype that is addicted to PI3K and MYC signaling and suggest that pharmacologic inhibition of PI3K might represent a promising therapeutic approach in these lymphomas. Diffuse large B-cell lymphoma (DLBCL) represents the most frequent lymphoma subtype and is considered a heterogeneous diagnostic category (1). Using gene expression profiling, two major molecular subtypes can be distinguished termed germinal center B-cell-like (GCB) DLBCL and activated B-cell-like (ABC) DLBCL (2). GCB DLBCLs are derived from germinal center B cells, whereas ABC DLBCLs originate from postgerminal center B cells that are in the transition of being differentiated into plasma cells. However, full plasma cell maturation is blocked in ABC DLBCL by different genetic abnormalities inhibiting the function of BLIMP1 that regulates plasmacytic differentiation (3-5).Recent work suggested constitutive activation of the PI3K/ protein kinase B (AKT) pathway that plays a crucial role in mediating growth, proliferation, and cell survival in a substantial number of DLBCL patient samples determined by immunohistochemical staining for phospho-AKT (p-AKT) (6, 7). However, these studies did not investigate the molecular mechanisms leading to constitutive PI3K/AKT signaling. The tumor suppressor PTEN is the major negative regulator of PI3K/AKT. PTEN functions as a lipid phosphatase dephosphorylating the 3′ position of phosphatidyl-inositol-3,-4,-5-trisphosphate, which serves as a trigger for AKT activation (8, 9). However, recent studies showed that PTEN has additional PI3K/AKT-independent tumor suppressor functions. Nuclear PTEN, for example, acts as guardian of genome integrity by up-...

Key Points• IkB-z is essential for nuclear NF-kB activity in ABC DLBCL.• ABC DLBCL survival depends on IkB-z signaling.Constitutive activation of the nuclear factor-k B (NF-kB) pathway is a hallmark of the activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL). Recurrent mutations of NF-kB regulators that cause constitutive activity of this oncogenic pathway have been identified. However, it remains unclear how specific target genes are regulated. We identified the atypical nuclear IkB protein IkB-z to be upregulated in ABC compared with germinal center B-cell-like (GCB) DLBCL primary patient samples. Knockdown of IkB-z by RNA interference was toxic to ABC but not to GCB DLBCL cell lines. Gene expression profiling after IkB-z knockdown demonstrated a significant downregulation of a large number of known NF-kB target genes, indicating an essential role of IkB-z in regulating a specific set of NF-kB target genes. To further investigate how IkB-z mediates NF-kB activity, we performed immunoprecipitations and detected a physical interaction of IkB-z with both p50 and p52 NFkB subunits, indicating that IkB-z interacts with components of both the canonical and the noncanonical NF-kB pathway in ABC DLBCL. Collectively, our data demonstrate that IkB-z is essential for nuclear NF-kB activity in ABC DLBCL, and thus might represent a promising molecular target for future therapies. (Blood. 2013;122(13):2242-2250

Deregulated transcription factor (TF) activities are commonly observed in hematopoietic malignancies. Understanding tumorigenesis therefore requires determining the function and hierarchical role of individual TFs. To identify TFs central to lymphomagenesis, we identified lymphoma type-specific accessible chromatin by global mapping of DNaseI hypersensitive sites and analyzed enriched TFbinding motifs in these regions. Applying this unbiased approach to classical Hodgkin lymphoma (HL), a common B-cell-derived lymphoma with a complex pattern of deregulated TFs, we discovered interferon regulatory factor (IRF) sites among the top enriched motifs. High-level expression of the proinflammatory TF IRF5 was specific to HL cells and crucial for their survival. Furthermore, IRF5 initiated a regulatory cascade in human non-Hodgkin B-cell lines and primary murine B cells by inducing the TF AP-1 and cooperating with NF-κB to activate essential characteristic features of HL. Our strategy efficiently identified a lymphoma type-specific key regulator and uncovered a tumor promoting role of IRF5.T ranscription factor (TF) activities have to be tightly controlled because their aberrant regulation alters tissue-specific gene expression programs and contributes to cancer pathogenesis. Therefore, the identification of altered TF activities in malignancies is of crucial importance to understand malignant transformation and to develop new treatment strategies. Deregulated TF activities are commonly observed in hematopoietic malignancies including human lymphomas and leukemias, and the link between structural or functional alterations in TFs and malignant transformation has been documented in various in vitro and in vivo studies (1-3). Apart from the direct modulation of cellular processes like cellular growth or cell death, alterations of the activity of even single TFs might enforce malignant transformation by switching differentiation programs and consequently altering the cellular fate of the respective cells, as exemplarily demonstrated for the B-lymphoid TF PAX5 (4, 5).Among lymphoid malignancies, one of the most prominent examples for complex patterns of deregulated TFs is classical Hodgkin lymphoma (HL), a common B cell-derived malignancy (6). Pathogenic hallmarks of the malignant Hodgkin/Reed-Sternberg (HRS) cells of HL include the constitutive activation of TFs that are only transiently activated in normal B cells, such as nuclear factor kappa B (NF-κB) or activator protein-1 (AP-1), and a profound deregulation of lineage-specific TFs such as E2A (6-8). Thus, although originating from B-lymphoid cells, HRS cells have lost their B cell-specific gene expression pattern and instead upregulate expression of genes characteristic for other hematopoietic lineages. However, the nature of the TFs initiating and maintaining HRS-specific gene expression remains poorly understood.As an unbiased approach for the identification of deregulated TF activities central to lymphoma biology, we identified HL-specific accessible chromatin regions...

Constitutional mismatch repair deficiency (CMMRD) is caused by germline pathogenic variants in both alleles of a mismatch repair gene. Patients have an exceptionally high risk of numerous pediatric malignancies and benefit from surveillance and adjusted treatment. The diversity of its manifestation, and ambiguous genotyping results, particularly from PMS2, can complicate diagnosis and preclude timely patient management. Assessment of low‐level microsatellite instability in nonneoplastic tissues can detect CMMRD, but current techniques are laborious or of limited sensitivity. Here, we present a simple, scalable CMMRD diagnostic assay. It uses sequencing and molecular barcodes to detect low‐frequency microsatellite variants in peripheral blood leukocytes and classifies samples using variant frequencies. We tested 30 samples from 26 genetically‐confirmed CMMRD patients, and samples from 94 controls and 40 Lynch syndrome patients. All samples were correctly classified, except one from a CMMRD patient recovering from aplasia. However, additional samples from this same patient tested positive for CMMRD. The assay also confirmed CMMRD in six suspected patients. The assay is suitable for both rapid CMMRD diagnosis within clinical decision windows and scalable screening of at‐risk populations. Its deployment will improve patient care, and better define the prevalence and phenotype of this likely underreported cancer syndrome.