Comprehensive DNA Methylation Analysis Indicates That Pancreatic Intraepithelial Neoplasia Lesions Are Acinar-Derived and Epigenetically Primed for Carcinogenesis.

Pancreatic ductal adenocarcinoma (PDAC) is believed to arise from the accumulation of a series of somatic mutations and is also frequently associated with pancreatic intraepithelial neoplasia (PanIN) lesions. However, there is still debate as to whether the cell type-of-origin of PanINs and PDACs in humans is acinar or ductal. As cell type identity is maintained epigenetically, DNA methylation changes during pancreatic neoplasia can provide a compelling perspective to examine this question. Here, we performed laser-capture microdissection on surgically resected specimens from 18 patients to isolate, with high purity, DNA for whole-genome bisulfite sequencing from four relevant cell types: acini, nonneoplastic ducts, PanIN lesions, and PDAC lesions. Differentially methylated regions (DMR) were identified using two complementary analytical approaches: bsseq, which identifies any DMRs but is particularly useful for large block-like DMRs, and informME, which profiles the potential energy landscape across the genome and is particularly useful for identifying differential methylation entropy. Both global methylation profiles and block DMRs clearly implicated an acinar origin for PanINs. At the gene level, PanIN lesions exhibited an intermediate acinar-ductal phenotype resembling acinar-to-ductal metaplasia. In 97.6% of PanIN-specific DMRs, PanIN lesions had an intermediate methylation level between normal and PDAC, which suggests from an information theory perspective that PanIN lesions are epigenetically primed to progress to PDAC. Thus, epigenomic analysis complements histopathology to define molecular progression toward PDAC. The shared epigenetic lineage between PanIN and PDAC lesions could provide an opportunity for prevention by targeting aberrantly methylated progression-related genes. SIGNIFICANCE: Analysis of DNA methylation landscapes provides insights into the cell-of-origin of PanIN lesions, clarifies the role of PanIN lesions as metaplastic precursors to human PDAC, and suggests potential targets for chemoprevention.

Mutant Cas9-transcriptional activator activates HIV-1 in U1 cells in the presence and absence of LTR-specific guide RNAs.

CRISPR/Cas9 systems have been advanced as promising tools in the HIV eradication armamentarium for sequence-specific disruption or latency reversal. Enthusiasm is balanced by concerns about off-target host genome modification and effects on HIV evolution. In the chronically HIV-1-infected U1 promonocytic latency model, we have confirmed stimulation of HIV-1 production by a mutant Cas9-transcriptional activator and guide RNAs with two guide RNAs apparently more potent than one. However, significant increases were also observed in the absence of guide RNAs. We encourage continued careful evaluation of non-sequence-specific and off-target effects of Cas9-mediated approaches.

Replication-competent simian immunodeficiency virus (SIV) Gag escape mutations archived in latent reservoirs during antiretroviral treatment of SIV-infected macaques.

In response to pressure exerted by major histocompatibility complex (MHC) class I-mediated CD8(+) T cell control, human immunodeficiency virus (HIV) escape mutations often arise in immunodominant epitopes recognized by MHC class I alleles. While the current standard of care for HIV-infected patients is treatment with highly active antiretroviral therapy (HAART), suppression of viral replication in these patients is not absolute and latently infected cells persist as lifelong reservoirs. To determine whether HIV escape from MHC class I-restricted CD8(+) T cell control develops during HAART treatment and then enters latent reservoirs in the periphery and central nervous system (CNS), with the potential to emerge as replication-competent virus, we tracked the longitudinal development of the simian immunodeficiency virus (SIV) Gag escape mutation K165R in HAART-treated SIV-infected pigtailed macaques. Key findings of these studies included: (i) SIV Gag K165R escape mutations emerged in both plasma and cerebrospinal fluid (CSF) during the decaying phase of viremia after HAART initiation before suppression of viral replication, (ii) SIV K165R Gag escape mutations were archived in latent proviral DNA reservoirs, including the brain in animals receiving HAART that suppressed viral replication, and (iii) replication-competent SIV Gag K165R escape mutations were present in the resting CD4(+) T cell reservoir in HAART-treated SIV-infected macaques. Despite early administration of aggressive antiretroviral treatment, HIV immune escape from CD8(+) T cell control can still develop during the decaying phases of viremia and then persist in latent reservoirs, including the brain, with the potential to emerge if HAART therapy is interrupted.