ATM-dependent Phosphorylation of Nemo SQ Motifs Is Dispensable for Nemo-mediated Gene Expression Changes in Response to DNA Double-Strand Breaks.

In response to DNA double-strand breaks (DSBs), the ATM kinase activates NF-κB factors to stimulate gene expression changes that promote survival and allow time for cells to repair damage. In cell lines, ATM can activate NF-κB transcription factors via two independent, convergent mechanisms. One is ATM-mediated phosphorylation of nuclear NF-κB essential modulator (Nemo) protein, which leads to monoubiquitylation and export of Nemo to the cytoplasm where it engages the IκB kinase (IKK) complex to activate NF-κB. Another is DSB-triggered migration of ATM into the cytoplasm, where it promotes monoubiquitylation of Nemo and the resulting IKK-mediated activation of NF-κB. ATM has many other functions in the DSB response beyond activation of NF-κB, and Nemo activates NF-κB downstream of diverse stimuli, including developmental or proinflammatory stimuli such as LPSs. To elucidate the in vivo role of DSB-induced, ATM-dependent changes in expression of NF-κB-responsive genes, we generated mice expressing phosphomutant Nemo protein lacking consensus SQ sites for phosphorylation by ATM or related kinases. We demonstrate that these mice are viable/healthy and fertile and exhibit overall normal B and T lymphocyte development. Moreover, treatment of their B lineage cells with LPS induces normal NF-κB-regulated gene expression changes. Furthermore, in marked contrast to results from a pre-B cell line, primary B lineage cells expressing phosphomutant Nemo treated with the genotoxic drug etoposide induce normal ATM- and Nemo-dependent changes in expression of NF-κB-regulated genes. Our data demonstrate that ATM-dependent phosphorylation of Nemo SQ motifs in vivo is dispensable for DSB-signaled changes in expression of NF-κB-regulated genes.

The Cyclin D3 Protein Enforces Monogenic TCRß Expression by Mediating TCRß Protein-Signaled Feedback Inhibition of Vß Recombination.

In jawed vertebrates, adaptive immunity depends on the process of V(D)J recombination creating vast numbers of T and B lymphocytes that each expresses unique Ag receptors of uniform specificity. The asynchronous initiation of V-to-(D)J rearrangement between alleles and the resulting protein from one allele signaling feedback inhibition of V recombination on the other allele ensures homogeneous receptor specificity of individual cells. Upon productive Vß-to-DßJß rearrangements in noncycling double-negative thymocytes, TCRß protein signals induction of the cyclin D3 protein to accelerate cell cycle entry, thereby driving proliferative expansion of developing αß T cells. Through undetermined mechanisms, the inactivation of cyclin D3 in mice causes an increased frequency of αß T cells that express TCRß proteins from both alleles, producing lymphocytes of heterogeneous specificities. To determine how cyclin D3 enforces monogenic TCRß expression, we used our mouse lines with enhanced rearrangement of specific Vß segments due to replacement of their poor-quality recombination signal sequence (RSS) DNA elements with a better RSS. We show that cyclin D3 inactivation in these mice elevates the frequencies of αß T cells that display proteins from RSS-augmented Vß segments on both alleles. By assaying mature αß T cells, we find that cyclin D3 deficiency increases the levels of Vß rearrangements that occur within developing thymocytes. Our data demonstrate that a component of the cell cycle machinery mediates TCRß protein-signaled feedback inhibition in thymocytes to achieve monogenic TCRß expression and resulting uniform specificity of individual αß T cells.

Menin Maintains Cholesterol Content in Colorectal Cancer via Repression of LXR-Mediated Transcription.

BACKGROUND AND AIMS: Menin is a nuclear scaffold protein that regulates gene transcription in an oftentimes tissue-specific manner. Our previous work showed that menin is over-expressed in colorectal cancer (CRC); however, the full spectrum of menin function in colonic neoplasia remains unclear. Herein, we aimed to uncover novel menin-regulated pathways important for colorectal carcinogenesis. METHODS: RNA-Seq analysis identified that menin regulates LXR-target gene expressions in CRC cell lines. Isolated colonic epithelium from Men1f/f;Vil1-Cre and Men1f/f mice was used to validate the results in vivo. Cholesterol content was quantified via an enzymatic assay. RESULTS: RNA-Seq analysis in the HT-29 CRC cell line identified that menin inhibition upregulated LXR-target genes, specifically ABCG1 and ABCA1, with protein products that promote cellular cholesterol efflux. Similar results were noted across other CRC cell lines and with different methods of menin inhibition. Consistent with ABCG1 and ABCA1 upregulation, and similarly to LXR agonists, menin inhibition reduced the total cellular cholesterol in both HT-29 and HCT-15 cells. To confirm the effects of menin inhibition in vivo, we assessed Men1f/f;Vil1-Cre mice lacking menin expression in the colonic epithelium. Men1f/f;Vil1-Cre mice were found to have no distinct baseline phenotype compared to control Men1f/f mice. However, similarly to CRC cell lines, Men1f/f;Vil1-Cre mice showed an upregulation of Abcg1 and a reduction in total cellular cholesterol. Promoting cholesterol efflux, either via menin inhibition or LXR activation, was found to synergistically suppress CRC cell growth under cholesterol-depleted conditions and when administered concomitantly with small molecule EGFR inhibitors. CONCLUSIONS: Menin represses the transcription of LXR-target genes, including ABCA1 and ABCG1 in the colonic epithelium and CRC. Menin inhibition conversely upregulates LXR-target genes and reduces total cellular cholesterol, demonstrating that menin inhibition may be an important mechanism for targeting cholesterol-dependent pathways in colorectal carcinogenesis.

Nemo-Dependent, ATM-Mediated Signals from RAG DNA Breaks at Igk Feedback Inhibit V κ Recombination to Enforce Igκ Allelic Exclusion.

Monoallelic AgR gene expression underlies specific adaptive immune responses. AgR allelic exclusion is achieved by sequential initiation of V(D)J recombination between alleles and resultant protein from one allele signaling to prevent recombination of the other. The ATM kinase, a regulator of the DNA double-strand break (DSB) response, helps enforce allelic exclusion through undetermined mechanisms. ATM promotes repair of RAG1/RAG2 (RAG) endonuclease-induced DSBs and transduces signals from RAG DSBs during Igk gene rearrangement on one allele to transiently inhibit RAG1 protein expression, Igk accessibility, and RAG cleavage of the other allele. Yet, the relative contributions of ATM functions in DSB repair versus signaling to enforce AgR allelic exclusion remain undetermined. In this study, we demonstrate that inactivation in mouse pre-B cells of the NF-κB essential modulator (Nemo) protein, an effector of ATM signaling, diminishes RAG DSB-triggered repression of Rag1/Rag2 transcription and Igk accessibility but does not result in aberrant repair of RAG DSBs like ATM inactivation. We show that Nemo deficiency increases simultaneous biallelic Igk cleavage in pre-B cells and raises the frequency of B cells expressing Igκ proteins from both alleles. In contrast, the incidence of biallelic Igκ expression is not elevated by inactivation of the SpiC transcriptional repressor, which is induced by RAG DSBs in an ATM-dependent manner and suppresses Igk accessibility. Thus, we conclude that Nemo-dependent, ATM-mediated DNA damage signals enforce Igκ allelic exclusion by orchestrating transient repression of RAG expression and feedback inhibition of additional Igk rearrangements in response to RAG cleavage on one Igk allele.

The RAG1 N-terminal region regulates the efficiency and pathways of synapsis for V(D)J recombination.

Immunoglobulin and T cell receptor gene assembly depends on V(D)J recombination initiated by the RAG1-RAG2 recombinase. The RAG1 N-terminal region (NTR; aa 1-383) has been implicated in regulatory functions whose influence on V(D)J recombination and lymphocyte development in vivo is poorly understood. We generated mice in which RAG1 lacks ubiquitin ligase activity (P326G), the major site of autoubiquitination (K233R), or its first 215 residues (Δ215). While few abnormalities were detected in R1.K233R mice, R1.P326G mice exhibit multiple features indicative of reduced recombination efficiency, including an increased Igκ+:Igλ+ B cell ratio and decreased recombination of Igh, Igκ, Igλ, and Tcrb loci. Previous studies indicate that synapsis of recombining partners during Igh recombination occurs through two pathways: long-range scanning and short-range collision. We find that R1Δ215 mice exhibit reduced short-range Igh and Tcrb D-to-J recombination. Our findings indicate that the RAG1 NTR regulates V(D)J recombination and lymphocyte development by multiple pathways, including control of the balance between short- and long-range recombination.

Menin-mediated Repression of Glycolysis in Combination with Autophagy Protects Colon Cancer Against Small-molecule EGFR Inhibitors.

Menin serves both tumor suppressor and promoter roles in a highly tumor-specific manner. In colorectal cancer, menin is overexpressed and plays a critical role in regulating transcription of SKP2, and combined treatment with a menin inhibitor and small-molecule EGFR inhibitor (EGFRi) leads to synergistic killing of colorectal cancer cells. However, the full spectrum of menin function in colorectal cancer remains uncertain. Herein, we demonstrate that menin inhibition increases glycolysis in colorectal cancer cells. This menin inhibitor-induced increase in glycolysis occurs in an mTOR-independent manner and enhances the sensitivity of colorectal cancer cells to EGFRis. In addition, we show that EGFRis induce autophagy in colorectal cancer cells, which is important for cell survival in the setting of combined treatment with an EGFRi and menin inhibitor. Inhibition of autophagy with chloroquine further sensitizes colorectal cancers to treatment with the combination of an EGFRi and menin inhibitor. Together, these findings uncover a novel role for menin in colorectal cancer as a repressor of glycolysis and demonstrate that menin inhibitor-induced increases in glycolysis sensitize colorectal cancer cells to EGFRis. In addition, these findings illustrate the importance of autophagy as a protective mechanism against EGFRis, especially in the presence of menin inhibition. Ultimately, these data open the possibility of using menin-mediated regulation of glycolysis to potentially improve treatment modalities for colorectal cancer.

Combined Menin and EGFR Inhibitors Synergize to Suppress Colorectal Cancer via EGFR-Independent and Calcium-Mediated Repression of SKP2 Transcription.

Menin is a nuclear epigenetic regulator that can both promote and suppress tumor growth in a highly tissue-specific manner. The role of menin in colorectal cancer, however, remains unclear. Here, we demonstrate that menin was overexpressed in colorectal cancer and that inhibition of menin synergized with small-molecule inhibitors of EGFR (iEGFR) to suppress colorectal cancer cells and tumor xenografts in vivo in an EGFR-independent manner. Mechanistically, menin bound the promoter of SKP2, a pro-oncogenic gene crucial for colorectal cancer growth, and promoted its expression. Moreover, the iEGFR gefitinib activated endoplasmic reticulum calcium channel inositol trisphosphate receptor 3 (IP3R3)-mediated release of calcium, which directly bound menin. Combined inhibition of menin and iEGFR-induced calcium release synergistically suppressed menin-mediated expression of SKP2 and growth of colorectal cancer. Together, these findings uncover a molecular convergence of menin and the iEGFR-induced, IP3R3-mediated calcium release on SKP2 transcription and reveal opportunities to enhance iEGFR efficacy to improve treatments for colorectal cancer. SIGNIFICANCE: Menin acts as a calcium-responsive regulator of SKP2 expression, and small molecule EGFR inhibitors, which induce calcium release, synergize with Menin inhibition to reduce SKP2 expression and suppress colorectal cancer.

Efficacy of Peptide Receptor Radionuclide Therapy in a United States-Based Cohort of Metastatic Neuroendocrine Tumor Patients: Single-Institution Retrospective Analysis.

OBJECTIVES: The aim of this study was to analyze in a retrospective cohort study the outcomes of a United States-based group of metastatic neuroendocrine tumor (NET) patients who underwent peptide receptor radionuclide therapy (PRRT). METHODS: Twenty-eight patients from a single US NET Center were treated with PRRT. Toxicities were assessed using Common Terminology Criteria for Adverse Events version 4.03. Progression was determined by the Response Evaluation Criteria in Solid Tumors version 1.1. Univariate and multivariate Cox regression was performed to identify potential predictors of progression-free survival (PFS) and overall survival (OS). RESULTS: The median age at NET diagnosis was 56 years, 50% of the patients were male, 46% of NET primaries were located in the pancreas, 71% of tumors were nonfunctional, 25% were World Health Organization (WHO) grade III, and 20% had at least a 25% hepatic tumor burden. Anemia (36%) was the most common post-PRRT toxicity, followed by leukopenia (31%), nephrotoxicity (27%), and thrombocytopenia (24%). Median PFS was 18 months, and median OS was 38 months. Having a WHO grade III NET and receiving systemic chemotherapy prior to PRRT were found to be to independent predictors of shorter PFS and OS. CONCLUSIONS: Peptide receptor radionuclide therapy is an effective therapy in a US population. Progression-free survival and OS were better in WHO grade I/II NETs and when PRRT was sequenced prior to systemic chemotherapy.

Prenatal stress and newborn telomere length.

BACKGROUND: The developmental origin of the health and disease hypothesis is based on the premise that many chronic diseases have their roots in fetal development. Specifically, maternal stress during pregnancy is associated with altered fetal development and many adverse long-term health outcomes. Although the mechanisms underlying this effect are currently unclear, at the cellular level 1 possible mediator is the regulation of telomere length. Telomere dynamics appear to play a role in disease progression, and an adverse intrauterine environment may contribute in the establishment of short telomeres in newborns. In accordance with this, it was recently reported that prenatal stress is significantly associated with shorter mean newborn telomere length. However, this finding has yet to be replicated, and currently we know nothing about whether different size classes of telomeres within the telomere length distribution are differentially affected by prenatal stress. Examining telomere length frequency distributions is important, because the shortest telomeres in the distribution appear to be the most indicative of telomere dysfunction and thus the best predictors of mortality and morbidity in humans. OBJECTIVE: We investigated the effects of intrauterine exposure to maternal stress over the whole course of gestation on newborn mean telomere length and telomere length frequency distributions. STUDY DESIGN: We conducted a prospective cohort study of 24 mother-newborn dyads at an urban teaching hospital. Pregnant women with nonanomalous, uncomplicated pregnancies were recruited and assessed in the third trimester of gestation. Maternal psychosocial stress was quantified using the Holmes and Rahe Stress Scale and categorized as high stress (≥300 points) or low stress (≤299 points) exposure. Newborn telomere length was measured from cord blood at delivery using the Telomere Restriction Fragment assay. RESULTS: We found a significant negative association between maternal stress and newborn telomere length (ß = -0.463, P = 0.04). Newborns whose mothers experienced a high level of stress during pregnancy had significantly shorter telomere length (6.98 ± 0.41 kb) compared to newborns of mothers with low stress (8.74 ± 0.24 kb; t = -3.99, P = .003). Moreover, the difference in newborn telomere length between high-stress and low-stress mothers was due to a shift in the telomere length distribution, with the high-stress group showing an underrepresentation of longer telomeres and an over-representation of shorter telomeres. CONCLUSION: Our findings replicate those of other recent studies and also show, for the first time, that the prenatal stress-associated difference in newborn mean telomere length is due to a shift in the overall telomere distribution.

A sensitive luminescent assay for the histone methyltransferase NSD1 and other SAM-dependent enzymes.

A major focus of our pediatric cancer research is the discovery of chemical probes to further our understanding of the biology of leukemia harboring fusion proteins arising from chromosomal rearrangements, and to develop novel specifically targeted therapies. The NUP98-NSD1 fusion protein occurs in a highly aggressive subtype of acute myeloid leukemia after rearrangement of the genes NUP98 and NSD1. The methyltransferase activity of NSD1 is retained in the fusion, and it gives rise to abnormally high levels of methylation at lysine 36 on histone 3, enforcing oncogene activation. Therefore, inhibition of the methyltransferase activity of NUP98-NSD1 may be considered a viable therapeutic strategy. Here, we report the development and validation of a highly sensitive and robust luminescence-based assay for NSD1 and other methyltransferases that use S-adenosylmethionine (SAM) as a methyl donor. The assay quantifies S-adenosylhomocysteine (SAH), which is produced during methyl transfer from SAM. SAH is converted enzymatically to adenosine monophosphate (AMP); in the process, adenosine triphosphate (ATP) is consumed and the amount of ATP remaining is measured using a luminescent assay kit. The assay was validated by pilot high-throughput screening (HTS), dose-response confirmation of hits, and elimination of artifacts through counterscreening against SAH detection in the absence of NSD1. The known methyltransferase inhibitor suramin was identified, and profiled for selectivity against the histone methyltransferases EZH2, SETD7, and PRMT1. HTS using the luminescent NSD1 assay described here has the potential to deliver selective NSD1 inhibitors that may serve as leads in the development of targeted therapies for NUP98-NSD1-driven leukemias.