Global RNA Synthesis Research Articles

CCG-1423-derived compounds reduce global RNA synthesis and inhibit transcriptional responses.

Myocardin-related transcription factors (MRTFs) are coactivators of serum response factor (SRF), and thereby regulate cytoskeletal gene expression in response to actin dynamics. MRTFs have also been implicated in transcription of heat shock protein (HSP)-encoding genes in fly ovaries, but the mechanisms remain unclear. Here, we demonstrate that, in mammalian cells, MRTFs are dispensable for gene induction of HSP-encoding genes. However, the widely used small-molecule inhibitors of the MRTF-SRF transcription pathway, derived from CCG-1423, also efficiently inhibit gene transcription of HSP-encoding genes in both fly and mammalian cells in the absence of MRTFs. Quantifying RNA synthesis and RNA polymerase distribution demonstrates that CCG-1423-derived compounds have a genome-wide effect on transcription. Indeed, tracking nascent transcription at nucleotide resolution reveals that CCG-1423-derived compounds reduce RNA polymerase II elongation, and severely dampen the transcriptional response to heat shock. The effects of CCG-1423-derived compounds therefore extend beyond the MRTF-SRF pathway into nascent transcription, opening novel opportunities for their use in transcription research.

First person – Bina Prajapati

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping researchers promote themselves alongside their papers. Bina Prajapati is co-first author on ‘ CCG-1423-derived compounds reduce global RNA synthesis and inhibit transcriptional responses’, published in JCS. Bina conducted the research described in this article while a PhD Student in Maria Vartiainen's lab at the Institute of Biotechnology, University of Helsinki. She is now also a lab coordinator at Finnish Genome Editing Centre (FINGEEC) with Topi Tervonen (FINGEEC PI) at the University of Helsinki. Bina is interested in anything and everything related to cellular health, stress response and disease targets.

MiR-330-5p and miR-1270 target essential components of RNA polymerase I transcription and exhibit a novel tumor suppressor role in lung adenocarcinoma.

Upregulation of RNA polymerase I (Pol I) transcription and the overexpression of Pol I transcriptional machinery are crucial molecular alterations favoring malignant transformation. However, the causal molecular mechanism(s) of this aberration remain largely unknown. Here, we found that Pol I transcription and its core machinery are upregulated in lung adenocarcinoma (LUAD). We show that the loss of miRNAs (miR)-330-5p and miR-1270 expression contributes to the upregulation of Pol I transcription in LUAD. Constitutive overexpression of these miRs in LUAD cell lines suppressed the expression of core components of Pol I transcription, and reduced global ribosomal RNA synthesis. Importantly, miR-330-5p/miR-1270-mediated repression of Pol I transcription exerted multiple tumor suppressive functions including reduced proliferation, cell cycle arrest, enhanced apoptosis, reduced migration, increased drug sensitivity, and reduced tumor burden in a mouse xenograft model. Mechanistically, the downregulation of miR-330-5p and miR-1270 is regulated by Pol I subunit-derived circular RNA circ_0055467 and DNA hypermethylation, respectively. This study uncovers a novel miR-330-5p/miR-1270 mediated post-transcriptional regulation of Pol I transcription, and establish tumor suppressor properties of these miRs in LUAD. Ultimately, our findings provide a rationale for the therapeutic targeting of Pol I transcriptional machinery for LUAD.

A transcriptionally repressed quiescence program is associated with paused RNA polymerase II and is poised for cell cycle re-entry.

Adult stem cells persist in mammalian tissues by entering a state of reversible quiescence, referred to as G0, which is associated with low levels of transcription. Using cultured myoblasts and muscle stem cells, we report that in G0, global RNA content and synthesis are substantially repressed, correlating with decreased RNA polymerase II (RNAPII) expression and activation. Integrating RNAPII occupancy and transcriptome profiling, we identify repressed networks and a role for promoter-proximal RNAPII pausing in G0. Strikingly, RNAPII shows enhanced pausing in G0 on repressed genes encoding regulators of RNA biogenesis (such as Ncl, Rps24, Ctdp1), and release of pausing is associated with increased expression of these genes in G1. Knockdown of these transcripts in proliferating cells leads to induction of G0 markers, confirming the importance of their repression in establishment of G0. A targeted screen of RNAPII regulators revealed that knockdown of Aff4 (a positive regulator of elongation) unexpectedly enhances expression of G0-stalled genes and hastens S phase; however, the negative elongation factor (NELF) complex, a regulator of pausing, appears to be dispensable. We propose that RNAPII pausing contributes to transcriptional control of a subset of G0-repressed genes to maintain quiescence and impacts the timing of the G0-G1 transition. This article has an associated First Person interview with the first authors of the paper.

Symmetrically dimethylated histone H3R2 promotes global transcription during minor zygotic genome activation in mouse pronuclei

Paternal genome reprogramming, such as protamine–histone exchange and global DNA demethylation, is crucial for the development of fertilised embryos. Previously, our study showed that one of histone arginine methylation, asymmetrically dimethylated histone H3R17 (H3R17me2a), is necessary for epigenetic reprogramming in the mouse paternal genome. However, roles of histone arginine methylation in reprogramming after fertilisation are still poorly understood. Here, we report that H3R2me2s promotes global transcription at the 1-cell stage, referred to as minor zygotic genome activation (ZGA). The inhibition of H3R2me2s by expressing a histone H3.3 mutant H3.3R2A prevented embryonic development from the 2-cell to 4-cell stages and significantly reduced global RNA synthesis and RNA polymerase II (Pol II) activity. Consistent with this result, the expression levels of MuERV-L as minor ZGA transcripts were decreased by forced expression of H3.3R2A. Furthermore, treatment with an inhibitor and co-injection of siRNA to PRMT5 and PRMT7 also resulted in the attenuation of transcriptional activities with reduction of H3R2me2s in the pronuclei of zygotes. Interestingly, impairment of H3K4 methylation by expression of H3.3K4M resulted in a decrease of H3R2me2s in male pronuclei. Our findings suggest that H3R2me2s together with H3K4 methylation is involved in global transcription during minor ZGA in mice.

Transcriptional Heterogeneity of Neural Stem Cells during Axolotl Salamander Spinal Cord Regeneration

Unlike humans, axolotl salamanders are capable of restoring sensory and motor function after spinal cord injury. Upon tail amputation or spinal cord transection, endogenous neural stem cells (NSCs) increase proliferation and differentiate into multiple neural and glial cell types of the central and peripheral nervous system. How NSCs transition from a resting state to a regenerative state and coordinate proper neural differentiation is poorly understood. Here, we study the global changes that occur in NSCs after spinal cord injury as well as specific changes in gene transcription as the NSC regenerative program unfolds. We first studied global changes in NSCs after injury by measuring global RNA synthesis temporally and spatially through incorporation of 5‐ethynyl‐uridine into nascent RNA. Substantial upward shifts in overall transcriptional output occurred in specific NSC populations during regeneration, which was correlated with loss of facultative repressive chromatin marks, and was dependent on Hippo‐YAP signaling. We also found an evolutionarily conserved upregulation of nascent RNA production in active neural progenitors in axolotls and mice. In order to dig deeper into neural stem cell dynamics during regeneration, we adopted multiplexed fluorescence in situ hybridization (FISH) in tissue section and whole mount tissues. We produced a user‐friendly, sharable, Docker Container for automated high‐throughput design of Third Generation Hybridization Chain Reaction (HCR) FISH probe sets. An advantage of HCR‐FISH is that it utilizes non‐enzymatic signal amplification, while maintaining low background signal, leading to high signal‐to‐noise detection of mRNAs. We demonstrate that the HCR‐FISH method is amenable to multiplexing through five sequential rounds of hybridization (SeqFISH). Using our probe design pipeline and HCR‐FISH, we imaged NSC activation and differentiation in tissue sections collected after injury using 15 simultaneously‐detected probe sets indicative to CNS and PNS cell types. Whole mount HCR‐FISH for three neural markers were also imaged simultaneously to assess NSC and neuron responses after injury. Overall, the genetic neuroanatomical mapping performed here using molecular classification of NSCs provides novel information on how NSCs respond to injury to repair a CNS injury.Support or Funding InformationThis project was funded by grants NSF 1656429, NSF 1558017, and NIH OD024909

Transcriptional Modulation by Idelalisib Synergizes with Bendamustine in Chronic Lymphocytic Leukemia

The phosphatidyl-inositol 3 kinase (PI3K) δ inhibitor, idelalisib (IDE), is a potent inhibitor of the B-cell receptor pathway and a novel and highly effective agent for the treatment of chronic lymphocytic leukemia (CLL). We evaluated the activities of IDE in comparison to bendamusine (BEN), a commonly used alkylating agent, in primary CLL cells ex vivo. In contrast to BEN, IDE was cytotoxic to cells from extensively-treated patients, including those with a deletion (del)17p. Cross-resistance was not observed between BEN and IDE, confirming their different modes of cytotoxicity. Marked synergy was seen between BEN and IDE, even in cases that were resistant to BEN or IDE individually, and those with deletion (del) 17p. CD40L/interleukin 4 (IL4) co-treatment mimicking the CLL microenvironment increased resistance to IDE, but synergy was retained. PI3Kδ-deficient murine splenic B cells were more resistant to IDE and showed reduced synergy with BEN, thus confirming the importance of functional PI3Kδ protein. Although IDE was observed to induce γH2AX, IDE did not enhance activation of the DNA damage response nor DNA repair activity. Interestingly, IDE decreased global RNA synthesis and was antagonistic with 5,6-Dichlorobenzimidazole 1-b-D-ribofuranoside (DRB), an inhibitor of transcription. These findings add to the increasingly complex cellular effects of IDE, and B cell receptor (BCR) inhibitors in general, in CLL.

CDK4/6 inhibition mitigates stem cell damage in a novel model for taxane‐induced alopecia

Taxanes are a leading cause of severe and often permanent chemotherapy‐induced alopecia. As the underlying pathobiology of taxane chemotherapy‐induced alopecia remains poorly understood, we investigated how paclitaxel and docetaxel damage human scalp hair follicles in a clinically relevant ex vivo organ culture model. Paclitaxel and docetaxel induced massive mitotic defects and apoptosis in transit amplifying hair matrix keratinocytes and within epithelial stem/progenitor cell‐rich outer root sheath compartments, including within Keratin 15+ cell populations, thus implicating direct damage to stem/progenitor cells as an explanation for the severity and permanence of taxane chemotherapy‐induced alopecia. Moreover, by administering the CDK4/6 inhibitor palbociclib, we show that transit amplifying and stem/progenitor cells can be protected from paclitaxel cytotoxicity through G1 arrest, without premature catagen induction and additional hair follicle damage. Thus, the current study elucidates the pathobiology of taxane chemotherapy‐induced alopecia, highlights the paramount importance of epithelial stem/progenitor cell‐protective therapy in taxane‐based oncotherapy, and provides preclinical proof‐of‐principle in a healthy human (mini‐) organ that G1 arrest therapy can limit taxane‐induced tissue damage.

Visualization of global RNA synthesis in a human (mini-) organ in situ by click chemistry.

RNA synthesis can be detected by 5-ethynyl uridine (EU) incorporation and click chemistry. Despite identifying a fundamental functional process, this technique has yet to be widely applied to complex human tissue systems. By incorporating EU into human hair follicle (HF) organs cultured ex vivo, nascent RNA synthesis was detected in situ. EU differentially incorporated across the HF epithelium.Interestingly, RNA synthesis did not correlate with protein synthesis, proliferation or epithelial progenitor cell marker expression. By treating human HFs with the cytotoxic cell cycle inhibitor (R)-CR8, which inhibits transcriptional regulators CDK7 and CDK9, it was further shown that this technique can be used to sensitively detect changes in global RNA synthesis in situ. Together, this work delineates new insights into nascent RNA synthesis within a human (mini)- organ and describes a novel read-out parameter that will enrich future ex vivo human tissue research studies.

BET Inhibition Induces HEXIM1- and RAD51-Dependent Conflicts between Transcription and Replication

BET bromodomain proteins are epigenetic readers required for oncogenic transcription activities, and BET inhibitors have been rapidly advanced into clinical trials. Understanding the effects of BET inhibition on other nuclear processes such as DNA replication will be important for future clinical applications. Here we show that BET inhibition causes replication stress in cancer and non-cancer cells due to a rapid burst in global RNA synthesis and interference of transcription with replication. We identify BRD4 as the main BET inhibitor target in this process and provide evidence that BRD4 inhibition causes transcription-replication interference through release of P-TEFb from its inhibitor HEXIM1, promoting RNA Polymerase II phosphorylation. Unusually, BET inhibitor-induced transcription-replication interference does not activate the classic ATM/ATR-dependent DNA damage response. We show however that they promote foci formation of the homologous recombination factor RAD51. Both HEXIM1 and RAD51 are required for BET inhibitor-induced fork slowing, but rescuing fork slowing by HEXIM1 or RAD51 depletion activate a DNA damage response. Our data support a new mechanism where BRD4 inhibition slows replication and suppresses DNA damage through concerted action of transcription and homologous recombination machineries. They shed new light on the roles of DNA replication and recombination in the action of this new class of cancer drugs.

Idelalisib and bendamustine combination is synergistic and increases DNA damage response in chronic lymphocytic leukemia cells.

Idelalisib is a targeted agent that potently inhibits PI3Kδ which is exclusively expressed in hematological cells. Bendamustine is a well-tolerated cytotoxic alkylating agent which has been extensively used for treatment of chronic lymphocytic leukemia (CLL). Both these agents are FDA-approved for CLL. To increase the potency of idelalisib and bendamustine, we tested their combination in primary CLL lymphocytes. While each compound alone produced a moderate response, combination at several concentrations resulted in synergistic cytotoxicity. Idelalisib enhanced the bendamustine-mediated DNA damage/repair response, indicated by the phosphorylation of ATM, Chk2, and p53. Each drug alone activated γH2AX but combination treatment further increased the expression of this DNA damage marker. Compared with the control, idelalisib treatment decreased global RNA synthesis, resulting in a decline of early-response and short-lived MCL1 transcripts. In concert, there was a decline in total Mcl-1 protein in CLL lymphocytes. Isogenic mouse embryonic fibroblasts lacking MCL1 had higher sensitivity to bendamustine alone or in combination compared to MCL1 proficient cells. Collectively, these data indicate that bendamustine and idelalisib combination therapy should be investigated for treating patients with CLL.

Regulation of Mcl-1 Expression in Context to Bone Marrow Stromal Microenvironment in Chronic Lymphocytic Leukemia

A growing body of evidence suggests that the resistance of CLL cells to apoptosis is partly mediated through the interactions between leukemia cells and adjacent stromal cells residing in the lymphatic tissue or bone marrow microenvironment. Mcl-1, an anti-apoptotic protein that is associated with failure to treatment is up-regulated in CLL lymphocytes after interaction with microenvironment. However, the regulation of its expression in context to microenvironment is unclear. We evaluated and compared changes in Mcl-1 in CLL B-cells in suspension culture and when co-cultured on stromal cells. The blockade of apoptosis in co-cultured CLL cells is associated with diminution in caspase-3 and PARP cleavage and is not dependent on cytogenetic profile or prognostic factors of the disease. Stroma-derived resistance to apoptosis is associated with a cascade of transcriptional events such as increase in levels of total RNA Pol II and its phosphorylation at Ser2 and Ser5, increase in the rate of global RNA synthesis, and amplification of Mcl-1 transcript levels. The latter is associated with increase in Mcl-1 protein level without an impact on the levels of Bcl-2 and Bcl-xL. Post-translational modifications of protein kinases show increased phosphorylation of Akt at Ser473, Erk at Thr202/Tyr204 and Gsk-3β at Ser9 and augmentation of total Mcl-1 accumulation along with phosphorylation at Ser159/Thr163 sites. Collectively, stroma-induced apoptosis resistance is mediated through signaling proteins that regulate transcriptional and translational expression and post-translational modification of Mcl-1 in CLL cells in context to bone marrow stromal microenvironment.

Abstract 4529: The PI3Kδ inhibitor, idelalisib, inhibits transcription and translation through PI3K/Akt pathway in mantle cell lymphoma

Abstract PI3Kδ is the major PI3K family class I isoform expressed in B-cells that is known to promote cancer cell growth and survival. PI3Kδ pathway is activated through cell surface receptors, e.g. B-cell receptor signaling. This suggests that PI3Kδ is a therapeutic target for B-cell hematological malignancies. Idelalisib is a potent and selective PI3Kδ inhibitor (IC50=19nM) that is currently under investigation in Phase II/III trials in B-cell neoplasms. In solid tumors, it has been established that PI3K/Akt axis regulates transcription and translation processes. Based on the overexpression and hyperactivity of PI3Kδ in B-cell lymphoma such as mantle cell lymphoma (MCL), and PI3Kδ-specific inhibition by idelalisib, we hypothesized that idelalisib treatment will 1) impact the transcription and translation processes that are PI3K/Akt-dependent, 2) disrupt RNA and protein synthetic capacity, and 3) decrease short-lived oncogenic mRNA and proteins levels that are important for MCL cell survival. Idelalisib (0.5 to 5μM) induced modest levels of apoptosis (<15%) in MCL cell lines and primary cells. A reverse-phase protein array (RPPA) study suggested that idelalisib treatment resulted in a decrease in phosphorylation of transcription regulators (GSK-3αβ and c-Myc), and protein synthesis regulators (S6, p70S6K, 4E-BP1, eIF4 and mTOR) in MCL cell lines and primary cells. Immunoblot analysis suggests that a 4hr treatment with idelalisib (0.5 or 1μM) resulted in a decrease of phosphorylated Akt (T308 and S473) levels in both JeKo-1 and Mino cells following IgM stimulation. In parallel, phospho-levels of GSK-3β (S9), 4E-BP1 (S65) and S6 (S235/236) were also decreased. In IgM-stimulated MCL cell lines, moderate to significant decrease in global RNA synthesis (10-15% decrease in JeKo-1, 50% decrease in Mino) and protein synthesis (15% decrease in JeKo-1, >20% decrease in Mino) were detected with 72hr incubation of idelalisib (0.5 to 1μM). Consistent with cell line data, in B-cell lymphoma primary cells, transcription levels were decreased by 20% or >50% following a 24hr treatment with 1 or 5µM idelalisib, respectively; and a 40% decrease of translation level was detected with 5µM idelalisib. In Jeko-1, early response genes MCL1 mRNA level was reduced following 24hr idelalisib treatment, whereas the other mRNAs (PIM2, MYC, CCND1) were decreased to a lesser extent. Mcl-1 and cyclin D1 protein levels were decreased in JeKo-1 and Mino cells following short incubation of idelalisib (0.5 or 1µM). In summary, idelalisib was effective in reducing phosphorylation of Akt and its downstream effectors in MCL cell lines (GSK-3β, 4E-BP1 and S6). Idelalisib inhibits global RNA and protein synthesis, and decreases the levels of short-lived survival proteins. Collectively, these data imply that similar to solid tumors, PI3K regulates key factors involved in gene transcription and protein translation in MCL. Citation Format: Qingshan Yang, Lisa S. Chen, Sattva S. Neelapu, Varsha Gandhi. The PI3Kδ inhibitor, idelalisib, inhibits transcription and translation through PI3K/Akt pathway in mantle cell lymphoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4529. doi:10.1158/1538-7445.AM2014-4529

Analysis of global RNA synthesis at the single cell level following hypoxia.

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification. Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification. Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

The yeast and human FACT chromatin-reorganizing complexes solve R-loop-mediated transcription–replication conflicts

FACT (facilitates chromatin transcription) is a chromatin-reorganizing complex that swaps nucleosomes around the RNA polymerase during transcription elongation and has a role in replication that is not fully understood yet. Here we show that recombination factors are required for the survival of yeast FACT mutants, consistent with an accumulation of DNA breaks that we detected by Rad52 foci and transcription-dependent hyperrecombination. Breaks also accumulate in FACT-depleted human cells, as shown by γH2AX foci and single-cell electrophoresis. Furthermore, FACT-deficient yeast and human cells show replication impairment, which in yeast we demonstrate by ChIP-chip (chromatin immunoprecipitation [ChIP] coupled with microarray analysis) of Rrm3 to occur genome-wide but preferentially at highly transcribed regions. Strikingly, in yeast FACT mutants, high levels of Rad52 foci are suppressed by RNH1 overexpression; R loops accumulate at high levels, and replication becomes normal when global RNA synthesis is inhibited in FACT-depleted human cells. The results demonstrate a key function of FACT in the resolution of R-loop-mediated transcription-replication conflicts, likely associated with a specific chromatin organization.

Combination of Pim kinase inhibitor SGI-1776 and bendamustine in B-cell lymphoma.

SGI-1776 is a small-molecule Pim kinase inhibitor that primarily targets c-MYC-driven transcription and cap-dependent translation in mantle cell lymphoma (MCL) cells. Bendamustine is an alkylating chemotherapeutic agent approved for use in B-cell lymphoma that is known to induce DNA damage and initiate response to repair. Our studies were conducted in MCL cell lines JeKo-1 and Mino, as well as primary B-cell lymphoma samples of MCL and splenic marginal zone lymphoma (SMZL), where we treated cells with SGI-1776 and bendamustine. We measured levels of cellular apoptosis, macromolecule synthesis inhibition, and DNA damage induced by drug treatments. Both SGI-1776 and bendamustine effectively induced apoptosis as single agents, and when used in combination, an additive effect in cell killing was observed in MCL cell lines JeKo-1 and Mino, as well as in MCL and SMZL primary cells. As expected, SGI-1776 was effective in inducing a decrease of global RNA and protein synthesis, and bendamustine significantly inhibited DNA synthesis and generated a DNA damage response. When used in combination, the effects were intensified in DNA, RNA, and protein synthesis inhibition compared with single-agent treatments. These data provide a foundation and suggest the feasibility of using Pim kinase inhibitors in combination with chemotherapeutic agents such as bendamustine in B-cell lymphoma.

Abstract 3265: PI3Kδ inhibitor, GS-1101, impacts transcription and translation in mantle cell lymphoma.

Abstract PI3Kα, β, γ and δ are the most ubiquitous and important PI3K family class I member isoforms that contribute to cell growth and survival processes in cancer. Among these isoforms, PI3Kα and β are broadly expressed, where PI3Kγ and δ are uniquely expressed in T- and B-cells, respectively, making them ideal candidates for targeted therapies in hematological malignancies. GS-1101, also known as CAL-101, is a potent and selective PI3Kδ inhibitor (IC50=2.5 nM) that is currently in Phase II/III clinical trials in B-cell diseases. Previous studies have elucidated disruption of survival factors and cytokine signaling mediated by PI3K/Akt pathways as major mechanism of activity in B-cell malignancies. It has been established that oncoprotein PI3Kα isoform deregulates transcription and translation processes in solid tumors, and based on the knowledge of PI3Kδ and GS-1101, we hypothesized that 1) GS-1101 treatment will inhibit gene transcription and protein translation, 2) will impact transcription and translation regulators that are downstream of PI3K/Akt pathways and 3) will reduce short-lived mRNA and proteins such as Mcl-1, c-Myc and cyclin D1, which are important in mantle cell lymphoma (MCL), a B-cell lymphoma. In JeKo-1 and Mino MCL cell lines, PI3Kδ is the most prominent isoform. In these cell lines and in primary MCL cells, the level of apoptosis was modest (less than 15%) when cells were treated with 5 μM GS-1101 for 24 hr. Consistent with Annexin/PI data, slight levels of PARP cleavage was detected. Of the two Akt phosphorylation sites, Thr308 and Ser473, phosphorylation of Akt (Thr308) showed dose-dependent decrease in both cell lines when treated by GS-1101 for 1 hr following serum starvation; however, there was no change in Akt (Ser347) levels following 24 hr of GS-1101 treatment in full serum conditions. Global RNA synthesis was substantially reduced in MCL primary samples, reaching 80% of control at 1 μM and 50% or less at 5 μM for 24 hr treatment, while only minor changes (80-90% of control) were observed in JeKo-1 and Mino. Global translation inhibition measured by leucine incorporation was observed in JeKo-1, Mino and primary MCL cells. Phosphorylation levels of translation regulators p70S6K (Ser9) and S6 (Ser235/236) showed dose-dependent decrease when treated with GS-1101 for 24 hr in JeKo-1. Meanwhile, minor decrease of 4E-BP1 (Ser65) phosphorylation level was observed in JeKo-1 under the same conditions. In Jeko-1 cells, early response genes such as short-lived MCL1 mRNA level was reduced following 24 hr of GS-1101 treatment, however, other (PIM2, MYC, CCND1) mRNA levels decreased to a lesser extent. At the protein level, both Mcl-1 and cyclin D1 expression decreased in JeKo-1 cells. In conclusion, similar to inhibition of other PI3K isoforms, PI3Kδ inhibition impacted transcription and translation, and reduced cyclin D1 and Mcl-1 levels which are important for MCL cell survival and proliferation. Citation Format: Qingshan Yang, Lisa S. Chen, Sattva S. Neelapu, Brian J. Lanutti, Varsha Gandhi. PI3Kδ inhibitor, GS-1101, impacts transcription and translation in mantle cell lymphoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3265. doi:10.1158/1538-7445.AM2013-3265

Endothelin-1­induced cardiomyocyte hypertrophy is partly regulated by transcription factor II-F interacting C-terminal domain phosphatase of RNA polymerase II

AimsCardiac hypertrophy is associated with the increase of total amount of RNA, which is in accordance with RNA polymerase II (RNAPII) activation via C-terminal domain (CTD) phosphorylation of the largest subunit of RNAPII. It has been demonstrated that endothelin-1 (ET-1) phosphorylates CTD at the hypertrophic response in cardiomyocytes. However, it is unclear whether ET-1-induced hypertrophy is affected by the CTD phosphatase, transcription factor IIF-interacting CTD phosphatase1 (FCP1). Main methodsWe analyzed whether ET-1-induced cardiomyocyte hypertrophy was affected by overexpression of FCP1 or dominant-negative form of FCP1 (dnFCP1) in neonatal rat cardiomyocytes. Key findingsThe level of ET-1-induced RNAPII CTD phosphorylation was decreased by FCP1 overexpression, whereas it was sustained by dnFCP1. Global RNA synthesis evaluated by [3H]-uridine incorporation showed that the ET-1-induced increase in RNA synthesis was suppressed by FCP1 and was augmented by dnFCP1. ET-1-induced increase in cell surface area was suppressed by FCP1 and was preserved by dnFCP1. Furthermore, the ET-1-induced increase in molecular markers of cardiac hypertrophy, expression of ANP and β-MHC gene, was suppressed by FCP1 and was not inhibited by dnFCP1. SignificanceET-1-induced cardiac hypertrophy and CTD phosphorylation level are functionally regulated by FCP1. These findings suggest that FCP1 plays an important role in ET-1-induced cardiac hypertrophy via controlling phosphorylation level of the RNAPII CTD.

Abstract 1490: Impact of bone marrow microenvironment on mRNA expression of genes in the apoptotic pathway in CLL cells

Abstract Chronic lymphocytic leukemia (CLL) is thought to arise due to dysregulated apoptosis rather than uncontrolled proliferation. Recent reports revealed that CLL cells accumulate due to both an intrinsic defect in apoptosis and extrinsic signals delivered by the microenvironment. Although several entities of bone marrow stroma cell (MSC) have been unveiled, the critical components involved in CLL survival remain obscure. In the present study, we used low density microarray analysis to identify the essential apoptotic genes (n=93) that are involved in cell survival. Primary cells obtained from CLL patients (n=12) were co-cultured with MSC (Nktert, human stromal cell line that mimics bone marrow microenvironment) in a ratio of 100:1. At the end of the incubation (12hr, d1, and d3), the expression profile of 93 genes as well as the percent apoptosis were evaluated. When CLL lymphocytes were co-cultured on MSC for 6 days, the viability of CLL cells, measured by Annexin/PI staining, increased by 6-27% at d1-d6 (n=9, p<0.001) compared to time matched controls. Next, we investigated the effect of MSC on global transcription. There was a 1.7+0.5 (d1, n=13) and 2.5+1.0 (d3, n=7) fold increase in global RNA synthesis as measured by [3H]uridine incorporation (p<0.001). Examination of mRNA profile of 93 genes showed that among the BCL2 family, the mRNA levels of the pro-apoptotic members such as BCL2L13, BAK1 and BIK decreased, while mRNA levels of anti-apoptotic, BCL2, BFL-1, BCL-XL, increased at d1 (1.46+0.56, p=0.015; 1.68+0.67, p=0.005; 2.17+1.46, p=0.018; respectively). Among caspase recruitment domain targets, pro-apoptotic APAF1 mRNA level significantly decreased at d1 but NALP1 mRNA level increased significantly (1.53+0.42, p=0.001), suggesting this gene may have alternate function in apoptosis pathway. In addition, NFKB-REL complex family members demonstrated decrease with MSC; in addition, member of IKB family, inhibitor of NFKB complex, such as NFKBIA increased significantly (1.56+0.7, p=0.018) at d1. Furthermore, in tumor necrosis factor receptor super family, TNFRSF1B expression significantly decreased (d1,0.74+0.21, p=0.001; d3,0.75+0.18, p=0.005), while TNFRSF10A and TNFRSF21 mRNA expression levels significantly increased (1.85+0.72, p=0.002; 2.81+2.08, p=0.016) at d1 but returned to baseline at d3. Additionally, 4 out of 6 DED domain containing pro-apoptotic proteins, (FADD, TRADD-adaptor proteins interacting with TNFRSF proteins, DEDD, DEDD2) demonstrated increase in mRNA levels compared to control. Moreover, mRNA levels of anti-apoptotic FLIP, which inhibits Caspase8 and Caspase10, increased significantly (1.50+0.59, p=0.013) after incubation with MSC. In summary, our data shows that MSC promoted survival to CLL lymphocytes is associated with transcriptional regulation of several genes including TNFRSF and BCL2 anti-apoptotic family members. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1490. doi:1538-7445.AM2012-1490