Two lymphoid cell lines potently silence unintegrated HIV-1 DNAs.

Mammalian cells mount a variety of defense mechanisms against invading viruses to prevent or reduce infection. One such defense is the transcriptional silencing of incoming viral DNA, including the silencing of unintegrated retroviral DNA in most cells. Here, we report that the lymphoid cell lines K562 and Jurkat cells reveal a dramatically higher efficiency of silencing of viral expression from unintegrated HIV-1 DNAs as compared to HeLa cells. We found K562 cells in particular to exhibit an extreme silencing phenotype. Infection of K562 cells with a non-integrating viral vector encoding a green fluorescent protein reporter resulted in a striking decrease in the number of fluorescence-positive cells and in their mean fluorescence intensity as compared to integration-competent controls, even though the levels of viral DNA in the nucleus were equal or in the case of 2-LTR circles even higher. The silencing in K562 cells was functionally distinctive. Histones loaded on unintegrated HIV-1 DNA in K562 cells revealed high levels of the silencing mark H3K9 trimethylation and low levels of the active mark H3 acetylation, as detected in HeLa cells. But infection of K562 cells resulted in low H3K27 trimethylation levels on unintegrated viral DNA as compared to higher levels in HeLa cells, corresponding to low H3K27 trimethylation levels of silent host globin genes in K562 cells as compared to HeLa cells. Most surprisingly, treatment with the HDAC inhibitor trichostatin A, which led to a highly efficient relief of silencing in HeLa cells, only weakly relieved silencing in K562 cells. In summary, we found that the capacity for silencing viral DNAs differs between cell lines in its extent, and likely in its mechanism.

Inhibition of Karyopherin ß1-Mediated Nuclear Import Disrupts Oncogenic Lineage-Defining Transcription Factor Activity in Small Cell Lung Cancer.

Genomic studies support the classification of small cell lung cancer (SCLC) into subtypes based on the expression of lineage-defining transcription factors ASCL1 and NEUROD1, which together are expressed in ∼86% of SCLC. ASCL1 and NEUROD1 activate SCLC oncogene expression, drive distinct transcriptional programs, and maintain the in vitro growth and oncogenic properties of ASCL1 or NEUROD1-expressing SCLC. ASCL1 is also required for tumor formation in SCLC mouse models. A strategy to inhibit the activity of these oncogenic drivers may therefore provide both a targeted therapy for the predominant SCLC subtypes and a tool to investigate the underlying lineage plasticity of established SCLC tumors. However, there are no known agents that inhibit ASCL1 or NEUROD1 function. In this study, we identify a novel strategy to pharmacologically target ASCL1 and NEUROD1 activity in SCLC by exploiting the nuclear localization required for the function of these transcription factors. Karyopherin ß1 (KPNB1) was identified as a nuclear import receptor for both ASCL1 and NEUROD1 in SCLC, and inhibition of KPNB1 led to impaired ASCL1 and NEUROD1 nuclear accumulation and transcriptional activity. Pharmacologic targeting of KPNB1 preferentially disrupted the growth of ASCL1+ and NEUROD1+ SCLC cells in vitro and suppressed ASCL1+ tumor growth in vivo, an effect mediated by a combination of impaired ASCL1 downstream target expression, cell-cycle activity, and proteostasis. These findings broaden the support for targeting nuclear transport as an anticancer therapeutic strategy and have implications for targeting lineage-transcription factors in tumors beyond SCLC. SIGNIFICANCE: The identification of KPNB1 as a nuclear import receptor for lineage-defining transcription factors in SCLC reveals a viable therapeutic strategy for cancer treatment.

ASCL1, NKX2-1, and PROX1 co-regulate subtype-specific genes in small-cell lung cancer.

Lineage-defining transcription factors (LTFs) play key roles in small-cell lung cancer (SCLC) pathophysiology. Delineating the LTF-regulated genes operative in SCLC could provide a road map to identify SCLC dependencies. We integrated chromatin landscape and transcriptome analyses of patient-derived SCLC preclinical models to identify super-enhancers (SEs) and their associated genes in the ASCL1-, NEUROD1-, and POU2F3-high SCLC subtypes. We find SE signatures predict LTF-based classification of SCLC, and the SE-associated genes are enriched with those defined as common essential genes in DepMap. In addition, in ASCL1-high SCLC, we show ASCL1 complexes with NKX2-1 and PROX1 to co-regulate genes functioning in NOTCH signaling, catecholamine biosynthesis, and cell-cycle processes. Depletion of ASCL1 demonstrates it is a key dependency factor in preclinical SCLC models and directly regulates multiple DepMap-defined essential genes. We provide LTF/SE-based subtype-specific gene sets for SCLC for further therapeutic investigation.

ASCL1 represses a SOX9+ neural crest stem-like state in small cell lung cancer.

ASCL1 is a neuroendocrine lineage-specific oncogenic driver of small cell lung cancer (SCLC), highly expressed in a significant fraction of tumors. However, ∼25% of human SCLC are ASCL1-low and associated with low neuroendocrine fate and high MYC expression. Using genetically engineered mouse models (GEMMs), we show that alterations in Rb1/Trp53/Myc in the mouse lung induce an ASCL1+ state of SCLC in multiple cells of origin. Genetic depletion of ASCL1 in MYC-driven SCLC dramatically inhibits tumor initiation and progression to the NEUROD1+ subtype of SCLC. Surprisingly, ASCL1 loss promotes a SOX9+ mesenchymal/neural crest stem-like state and the emergence of osteosarcoma and chondroid tumors, whose propensity is impacted by cell of origin. ASCL1 is critical for expression of key lineage-related transcription factors NKX2-1, FOXA2, and INSM1 and represses genes involved in the Hippo/Wnt/Notch developmental pathways in vivo. Importantly, ASCL1 represses a SOX9/RUNX1/RUNX2 program in vivo and SOX9 expression in human SCLC cells, suggesting a conserved function for ASCL1. Together, in a MYC-driven SCLC model, ASCL1 promotes neuroendocrine fate and represses the emergence of a SOX9+ nonendodermal stem-like fate that resembles neural crest.

Different Originating Cells Underlie Intertumoral Heterogeneity in Lung Neuroendocrine Tumors.

Studies in genetically engineered mouse models of neuroendocrine lung cancer suggest that differences in cells of origin underlie subtype variations in this class of cancers. These findings highlight the concept that the same driver mutations introduced into different cells of origin lead to tumors with the same histology but dramatically different metastatic programs and potentially different therapeutic responses. Cancer Discov; 8(10); 1216-8. ©2018 AACR See related article by Yang et al., p. 1316.

ASCL1 regulates proliferation of NG2-glia in the embryonic and adult spinal cord.

NG2-glia are highly proliferative oligodendrocyte precursor cells (OPCs) that are widely distributed throughout the central nervous system (CNS). During development, NG2-glia predominantly differentiate into oligodendrocytes (OLs) to myelinate axon fibers, but they can also remain as OPCs persisting into the mature CNS. Interestingly, NG2-glia in the gray matter (GM) are intrinsically different from those in the white matter (WM) in terms of proliferation, differentiation, gene expression, and electrophysiological properties. Here we investigate the role of the transcriptional regulator, ASCL1, in controlling NG2-glia distribution and development in the GM and WM. In the spinal cord, ASCL1 levels are higher in WM NG2-glia than those in the GM. This differential level of ASCL1 in WM and GM NG2-glia is maintained into adult stages. Long-term clonal lineage analysis reveals that the progeny of single ASCL1+ oligodendrocyte progenitors (OLPs) and NG2-glia are primarily restricted to the GM or WM, even though they undergo extensive proliferation to give rise to large clusters of OLs in the postnatal spinal cord. Conditional deletion of Ascl1 specifically in NG2-glia in the embryonic or adult spinal cord resulted in a significant reduction in the proliferation but not differentiation of these cells. These findings illustrate that ASCL1 is an intrinsic regulator of the proliferative property of NG2-glia in the CNS.

A central role for the primary microRNA stem in guiding the position and efficiency of Drosha processing of a viral pri-miRNA.

Processing of primary microRNA (pri-miRNA) stem-loops by the Drosha-DGCR8 complex is the initial step in miRNA maturation and crucial for miRNA function. Nonetheless, the underlying mechanism that determines the Drosha cleavage site of pri-miRNAs has remained unclear. Two prevalent but seemingly conflicting models propose that Drosha-DGCR8 anchors to and directs cleavage a fixed distance from either the basal single-stranded (ssRNA) or the terminal loop. However, recent studies suggest that the basal ssRNA and/or the terminal loop may influence the Drosha cleavage site dependent upon the sequence/structure of individual pri-miRNAs. Here, using a panel of closely related pri-miRNA variants, we further examine the role of pri-miRNA structures on Drosha cleavage site selection in cells. Our data reveal that both the basal ssRNA and terminal loop influence the Drosha cleavage site within three pri-miRNAs, the Simian Virus 40 (SV40) pri-miRNA, pri-miR-30a, and pri-miR-16. In addition to the flanking ssRNA regions, we show that an internal loop within the SV40 pri-miRNA stem strongly influences Drosha cleavage position and efficiency. We further demonstrate that the positions of the internal loop, basal ssRNA, and the terminal loop of the SV40 pri-miRNA cooperatively coordinate Drosha cleavage position and efficiency. Based on these observations, we propose that the pri-miRNA stem, defined by internal and flanking structural elements, guides the binding position of Drosha-DGCR8, which consequently determines the cleavage site. This study provides mechanistic insight into pri-miRNA processing in cells that has numerous biological implications and will assist in refining Drosha-dependent shRNA design.