ALBERT

Description: Betula platyphylla bark has been evaluated for the treatment of dermatitis, inflammatory conditions, and cancer. Diarylheptanoids are the major constituents of the B. platyphylla bark and possess various pharmacological effects. Our previous study confirmed the selective antiproliferative effect of platyphylloside (BPP) isolated from B. platyphylla on colon cancer and leukemic cells using 60 different cancer cell lines from thr National Cancer Institution (NCI). In line with previous reports, this study focuses on the apoptotic pathway of BPP, a phenolic glycoside composed of two aromatic rings joined by a seven-carbon chain. Cytotoxicity assays in solid tumor and blood cancer cell models demonstrated that BPP possesses potent antiproliferative activity. The level of apoptosis increased with BPP treatment, causing cell cycle arrest at the G1 phase along with the downregulation of IκBα phosphorylation and BCL-2, as well as upregulation of cleaved caspase 3 and BAX proteins. In addition, BPP displayed potent mitochondrial depolarization effects in Jurkat cells. The combined findings revealed that the cytotoxic effects of BPP were mediated by intracellular signaling, possibly through a mechanism involving the upregulation of mitochondrial reactive oxygen species (ROS). Thus, BPP could be a potential multitarget therapeutic agent in leukemia and colon cancer.

Description: Cancer genome and transcriptome sequencing can identify novel oncogenes and tumor suppressors, discover distinct cancer subtypes, and predict therapeutic responses. Analysis of the coding genome of diffuse large B cell lymphoma (DLBCL) has identified various genetic mutations in the ABC and GCB molecular subtypes. Notably, ABC DLBCLs have recurrent activating mutations involving the canonical NF-kB pathway. Although an oncogenic role for constitutive NF-kB activity has been demonstrated in ABC DLBCL, the molecular mechanisms of various oncogenic mutations are still elusive. To understand the pathogenesis of DLBCL, we used high-throughput exome and transcriptome sequencing of more than 500 DLBCL biopsies. To enable functional screening of mutant alleles identified by exome-seq and RNA-seq, we are constructing an inducible retroviral expression library of over 200 open reading frames (ORFs) of genes mutated in DLBCL. We have cloned the mutant and wildtype forms of these genes together with a unique 26-base-pair 'bar code' to facilitate screening with high-throughput sequencing. We have transduced a subset of this ORF library into DLBCL cell lines, induced ORF expression by doxycycline, and FACS sorted into high and low populations based on expression the known NF-κB target gene CD83. We then performed barcode sequencing to assay relative enrichment of ORFs that confer higher or lower NF-κB activity. ORF screening revealed that MYD88L265P, CARD11L232LI, both well-characterized gain-of-function mutants, were among the highest ranking genes for induction of NF-κB activity. Interestingly, we observed enrichment for USP7D271E in NF-κB high populations, though these genes have not been shown to have a tumorigenic role in DLBCL. Ubiquitin Specific Protease-7 (USP7) is a regulator of NF-κB transcriptional activity, in part by de-ubiquitinating p65. To test how USP7 mutations contribute to NF-κB activity, we engineered the ABC DLBCL cell line TMD8 to express an NF-κB reporter consisting of the NF-κB transcriptional response element (TRE) fused to GFP. NF-κB activity upon overexpression of wildtype or mutant USP7 was monitored by flow cytometry (FACS). We observed that the mutated form of USP7 significantly increased NF-κB activity over that seen with wildtype, suggesting this is a gain-of-function mutant. We next used the CRISPR gene-targeting system to probe the function of USP7 genetically. Single guide RNAs (sgRNAs) targeting the USP7 coding regions were coexpressed in TMD8 cells with the endonuclease Cas9. SgRNA-expressing, GFP+ cells were monitored over time among total live cells by flow cytometry, and a relative reduction of the GFP+ population was observed in the sgUSP7 population. These results suggest that USP7 may function as an oncogene in DLBCL. This ongoing work demonstrates the efficacy of a high-throughput ORF expression screen to characterize mutations found in the genomic landscape of the DLBCL. Disclosures No relevant conflicts of interest to declare.

Description: After neonatal HTLV-I infection through breast feeding, approximately 5% of HTLV-I carriers eventually develop Adult T-Cell Leukemia/Lymphoma (ATLL) with a latency of ~50 years, suggesting that acquired genetic and epigenetic changes in cellular genes act in concert with HTLV-I to initiate and maintain oncogenic transformation. We and others have recently utilized next generation sequencing technology to identify mutated genes that could be pivotal in the pathogenesis of ATLL. However, due to the complexity of genomic/epigenetic alteration in the ATLL genome, the identification of indispensable genes for proliferation and/or survival of ATLL cells remains a formidable challenge. To discover essential regulatory networks that are required for the proliferation and survival of ATLL cells, we performed a pooled shRNA screen in 8 ATLL cell lines using a library enriched for shRNAs targeting lymphoid regulatory factors and discovered that two BATF3 shRNAs and one IRF4 shRNA were highly toxic for all ATLL lines, but had little if any effect in other T cell and B cell lines. It is recently shown that a transcriptional complex of Irf4 and Batf binds to AP1-IRF composite (AICE) DNA motifs and plays key roles in the differentiation and function of certain mouse helper T cell subsets. A close paralogue of Batf, Batf3, is an indispensable transcription factor in a mouse dendritic cell subset, but also appears to play a redundant role with Batf in the differentiation of TH2 cells and can substitute for Batf in Batf knockout T cells. Our observations from shRNA screening suggested that IRF4 and BATF3 may cooperate to drive a transcriptional program that is essential for ATLL viability. We next used genome-wide chromatin precipitation (ChIP-seq) to identify the loci that are bound by BATF3 and IRF4. The set of binding peaks and the associated genes in IRF4 and BATF3 ChIP-seq intersected significantly. By integrating the ChIP-seq and gene expression profiling data of shBATF3- and shIRF4-ATLL cells, we defined a set of 68 BATF3-IRF4 direct target genes. Gene set enrichment analysis using gene expression profiling data from primary T cell lymphomas demonstrated that BATF3-IRF4 direct target genes were significantly enriched among genes that are more highly expressed in ATLL than in peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS), suggesting that the BATF3 and IRF4 cooperatively regulate transcription in primary ATLL cells. HBZ is unique among HTLV-I viral proteins in being maintained in expression in all ATLL cases, suggesting that it may help maintain the malignant phenotype. Given that BATF3 and IRF4 are essential regulators in ATLL, we hypothesized possible relationship between HBZ and BATF3-IRF4 complex. We defined HBZ direct target genes by integrating the ChIP-seq and gene expression profiling data of HBZ-knockout ATLL cell lines by CRISPR/Cas9. Notably we discovered that BATF3 was among these. BATF3 mRNA and protein expression decreased following HBZ inactivation. The above considerations suggested that pharmacologic inhibition of the BATF3-IRF4 regulatory network might be a means to attack the HBZ oncogenic program therapeutically. ChIP-seq analysis of two enhancer marks, H3K27ac and BRD4, identified super-enhancers at the BATF3 locus in two ATLL cell lines. The small molecule JQ1 prevents the BET-protein BRD4 from interacting with chromatin, which is required for the function of super-enhancers. JQ1 treatment reduced BATF3 mRNA and protein levels in all ATLL lines tested, correlating with the eviction of BRD4 from the BATF3 super-enhancer. MYC mRNA and protein expression was also broadly downmodulated by JQ1. JQ1 treatment was consistently toxic for all ATLL cell lines tested at dose ranges that killed cell line models of T-ALL and DLBCL, which are known to rely on BET-proteins. In a dose-dependent manner, JQ1 also reduced the viability of primary ATLL samples and downregulated their expression BATF3 and MYC mRNA. Finally, we treated mouse xenograft models of ATLL with the BET-protein inhibitor CPI-203, a JQ1 analog with superior bioavailability in mice. In two different xenograft models, we observed significant tumor regression or growth inhibition, without evidence of systemic toxicity. Our study demonstrates that the HTLV-I virus exploits a regulatory module that can potentially be attacked therapeutically with BET protein inhibitors. Disclosures Yu: Celgene Corporation: Employment.