Dereje D. Jima

Genetic heterogeneity of diffuse large B-cell lymphoma

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.

The genetic landscape of mutations in Burkitt lymphoma

Burkitt lymphoma is characterized by deregulation of MYC, but the contribution of other genetic mutations to the disease is largely unknown. Here, we describe the first completely sequenced genome from a Burkitt lymphoma tumor and germline DNA from the same affected individual. We further sequenced the exomes of 59 Burkitt lymphoma tumors and compared them to sequenced exomes from 94 diffuse large B-cell lymphoma (DLBCL) tumors. We identified 70 genes that were recurrently mutated in Burkitt lymphomas, including ID3, GNA13, RET, PIK3R1 and the SWI/SNF genes ARID1A and SMARCA4. Our data implicate a number of genes in cancer for the first time, including CCT6B, SALL3, FTCD and PC. ID3 mutations occurred in 34% of Burkitt lymphomas and not in DLBCLs. We show experimentally that ID3 mutations promote cell cycle progression and proliferation. Our work thus elucidates commonly occurring gene-coding mutations in Burkitt lymphoma and implicates ID3 as a new tumor suppressor gene.

Patterns of microRNA expression characterize stages of human B-cell differentiation

Mature B-cell differentiation provides an important mechanism for the acquisition of adaptive immunity. Malignancies derived from mature B cells constitute the majority of leukemias and lymphomas. These malignancies often maintain the characteristics of the normal B cells that they are derived from, a feature that is frequently used in their diagnosis. The role of microRNAs in mature B cells is largely unknown. Through concomitant microRNA and mRNA profiling, we demonstrate a potential regulatory role for microRNAs at every stage of the mature B-cell differentiation process. In addition, we have experimentally identified a direct role for the microRNA regulation of key transcription factors in B-cell differentiation: LMO2 and PRDM1 (Blimp1). We also profiled the microRNA of B-cell tumors derived from diffuse large B-cell lymphoma, Burkitt lymphoma, and chronic lymphocytic leukemia. We found that, in contrast to many other malignancies, common B-cell malignancies do not down-regulate microRNA expression. Although these tumors could be distinguished from each other with use of microRNA expression, each tumor type maintained the expression of the lineage-specific microRNAs. Expression of these lineage-specific microRNAs could correctly predict the lineage of B-cell malignancies in more than 95% of the cases. Thus, our data demonstrate that microRNAs may be important in maintaining the mature B-cell phenotype in normal and malignant B cells.

Deep sequencing of the small RNA transcriptome of normal and malignant human B cells identifies hundreds of novel microRNAs.

A role for microRNA (miRNA) has been recognized in nearly every biologic system examined thus far. A complete delineation of their role must be preceded by the identification of all miRNAs present in any system. We elucidated the complete small RNA transcriptome of normal and malignant B cells through deep sequencing of 31 normal and malignant human B-cell samples that comprise the spectrum of B-cell differentiation and common malignant phenotypes. We identified the expression of 333 known miRNAs, which is more than twice the number previously recognized in any tissue type. We further identified the expression of 286 candidate novel miRNAs in normal and malignant B cells. These miRNAs were validated at a high rate (92%) using quantitative polymerase chain reaction, and we demonstrated their application in the distinction of clinically relevant subgroups of lymphoma. We further demonstrated that a novel miRNA cluster, previously annotated as a hypothetical gene LOC100130622, contains 6 novel miRNAs that regulate the transforming growth factor-β pathway. Thus, our work suggests that more than a third of the miRNAs present in most cellular types are currently unknown and that these miRNAs may regulate important cellular functions.

The genomic landscape of mantle cell lymphoma is related to the epigenetically determined chromatin state of normal B cells

In this study, we define the genetic landscape of mantle cell lymphoma (MCL) through exome sequencing of 56 cases of MCL. We identified recurrent mutations in ATM, CCND1, MLL2, and TP53. We further identified a number of novel genes recurrently mutated in patients with MCL including RB1, WHSC1, POT1, and SMARCA4. We noted that MCLs have a distinct mutational profile compared with lymphomas from other B-cell stages. The ENCODE project has defined the chromatin structure of many cell types. However, a similar characterization of primary human mature B cells has been lacking. We defined, for the first time, the chromatin structure of primary human naïve, germinal center, and memory B cells through chromatin immunoprecipitation and sequencing for H3K4me1, H3K4me3, H3Ac, H3K36me3, H3K27me3, and PolII. We found that somatic mutations that occur more frequently in either MCLs or Burkitt lymphomas were associated with open chromatin in their respective B cells of origin, naïve B cells, and germinal center B cells. Our work thus elucidates the landscape of gene-coding mutations in MCL and the critical interplay between epigenetic alterations associated with B-cell differentiation and the acquisition of somatic mutations in cancer.

The Epstein-Barr Virus (EBV)-Induced Tumor Suppressor MicroRNA MiR-34a Is Growth Promoting in EBV-Infected B Cells

ABSTRACT Epstein-Barr virus (EBV) infection of primary human B cells drives their indefinite proliferation into lymphoblastoid cell lines (LCLs). B cell immortalization depends on expression of viral latency genes, as well as the regulation of host genes. Given the important role of microRNAs (miRNAs) in regulating fundamental cellular processes, in this study, we assayed changes in host miRNA expression during primary B cell infection by EBV. We observed and validated dynamic changes in several miRNAs from early proliferation through immortalization; oncogenic miRNAs were induced, and tumor suppressor miRNAs were largely repressed. However, one miRNA described as a p53-targeted tumor suppressor, miR-34a, was strongly induced by EBV infection and expressed in many EBV and Kaposis sarcoma-associated herpesvirus (KSHV)-infected lymphoma cell lines. EBV latent membrane protein 1 (LMP1) was sufficient to induce miR-34a requiring downstream NF-κB activation but independent of functional p53. Furthermore, overexpression of miR-34a was not toxic in several B lymphoma cell lines, and inhibition of miR-34a impaired the growth of EBV-transformed cells. This study identifies a progrowth role for a tumor-suppressive miRNA in oncogenic-virus-mediated transformation, highlighting the importance of studying miRNA function in different cellular contexts.

Analysis of somatic hypermutation in X-linked hyper-IgM syndrome shows specific deficiencies in mutational targeting

Subjects with X-linked hyper-IgM syndrome (X-HIgM) have a markedly reduced frequency of CD27(+) memory B cells, and their Ig genes have a low level of somatic hypermutation (SHM). To analyze the nature of SHM in X-HIgM, we sequenced 209 nonproductive and 926 productive Ig heavy chain genes. In nonproductive rearrangements that were not subjected to selection, as well as productive rearrangements, most of the mutations were within targeted RGYW, WRCY, WA, or TW motifs (R = purine, Y = pyrimidine, and W = A or T). However, there was significantly decreased targeting of the hypermutable G in RGYW motifs. Moreover, the ratio of transitions to transversions was markedly increased compared with normal. Microarray analysis documented that specific genes involved in SHM, including activation-induced cytidine deaminase (AICDA) and uracil-DNA glycosylase (UNG2), were up-regulated in normal germinal center (GC) B cells, but not induced by CD40 ligation. Similar results were obtained from light chain rearrangements. These results indicate that in the absence of CD40-CD154 interactions, there is a marked reduction in SHM and, specifically, mutations of AICDA-targeted G residues in RGYW motifs along with a decrease in transversions normally related to UNG2 activity.

PAK1 Mediates Resistance to PI3K Inhibition in Lymphomas

Purpose: The phosphoinositide 3-kinase (PI3K) pathway is known to play an active role in many malignancies. The role of PI3K inhibition in the treatment of lymphomas has not been fully delineated. We sought to identify a role for therapeutic PI3K inhibition across a range of B-cell lymphomas. Experimental Design: We selected three small molecule inhibitors to test in a panel of 60 cell lines that comprised diverse lymphoma types. We tested the selective PI3K inhibitor BKM120 and the dual PI3K/mTOR inhibitors BEZ235 and BGT226 in these cell lines. We applied gene expression profiling to better understand the molecular mechanisms associated with responsiveness to these drugs. Results: We found that higher expression of the PAK1 gene was significantly associated with resistance to all three PI3K inhibitors. Through RNA–interference-mediated knockdown of the PAK1 gene, we showed a dramatic increase in the sensitivity to PI3K inhibition. We further tested a small-molecule inhibitor of PAK1 and found significant synergy between PI3K and PAK1 inhibition. Conclusion: Thus, we show that PI3K inhibition is broadly effective in lymphomas and PAK1 is a key modulator of resistance to PI3K inhibition. Clin Cancer Res; 19(5); 1106–15. ©2012 AACR.

Editor’s Highlight: Comparative Dose-Response Analysis of Liver and Kidney Transcriptomic Effects of Trichloroethylene and Tetrachloroethylene in B6C3F1 Mouse

Trichloroethylene (TCE) and tetrachloroethylene (PCE) are ubiquitous environmental contaminants and occupational health hazards. Recent health assessments of these agents identified several critical data gaps, including lack of comparative analysis of their effects. This study examined liver and kidney effects of TCE and PCE in a dose-response study design. Equimolar doses of TCE (24, 80, 240, and 800 mg/kg) or PCE (30, 100, 300, and 1000 mg/kg) were administered by gavage in aqueous vehicle to male B6C3F1/J mice. Tissues were collected 24 h after exposure. Trichloroacetic acid (TCA), a major oxidative metabolite of both compounds, was measured and RNA sequencing was performed. PCE had a stronger effect on liver and kidney transcriptomes, as well as greater concentrations of TCA. Most dose-responsive pathways were common among chemicals/tissues, with the strongest effect on peroxisomal β-oxidation. Effects on liver and kidney mitochondria-related pathways were notably unique to PCE. We performed dose-response modeling of the transcriptomic data and compared the resulting points of departure (PODs) to those for apical endpoints derived from long-term studies with these chemicals in rats, mice, and humans, converting to human equivalent doses using tissue-specific dosimetry models. Tissue-specific acute transcriptional effects of TCE and PCE occurred at human equivalent doses comparable to those for apical effects. These data are relevant for human health assessments of TCE and PCE as they provide data for dose-response analysis of the toxicity mechanisms. Additionally, they provide further evidence that transcriptomic data can be useful surrogates for in vivo PODs, especially when toxicokinetic differences are taken into account.

Nuclear localized LSR, a novel regulator of breast cancer behavior and tumorigenesis

Lipolysis-stimulated lipoprotein receptor (LSR) has been found in the plasma membrane and is believed to function in lipoprotein endocytosis and tight junctions. Given the impact of cellular metabolism and junction signaling pathways on tumor phenotypes and patient outcome, it is important to understand how LSR cellular localization mediates its functions. We conducted localization studies, evaluated DNA binding, and examined the effects of nuclear LSR in cells, xenografts, and clinical specimens. We found LSR within the membrane, cytoplasm, and the nucleus of breast cancer cells representing multiple intrinsic subtypes. Chromatin immunoprecipitation (ChIP) showed direct binding of LSR to DNA, and sequence analysis identified putative functional motifs and post-translational modifications of the LSR protein. While neither overexpression of transcript variants, nor pharmacologic manipulation of post-translational modification significantly altered localization, inhibition of nuclear export enhanced nuclear localization, suggesting a mechanism for nuclear retention. Coimmunoprecipitation and proximal ligation assays indicated LSR–pericentrin interactions, presenting potential mechanisms for nuclear-localized LSR. The clinical significance of LSR was evaluated using data from over 1,100 primary breast tumors, which showed high LSR levels in basal-like tumors and tumors from African-Americans. In tumors histosections, nuclear localization was significantly associated with poor outcomes. Finally, in vivo xenograft studies revealed that basal-like breast cancer cells that overexpress LSR exhibited both membrane and nuclear localization, and developed tumors with 100% penetrance, while control cells lacking LSR developed no tumors. These results show that nuclear LSR alters gene expression and may promote aggressive cancer phenotypes. Implications: LSR functions in the promotion of aggressive breast cancer phenotypes and poor patient outcome via differential subcellular localization to alter cell signaling, bioenergetics, and gene expression. Mol Cancer Res; 15(2); 165–78. ©2016 AACR.