Genome-Wide Analysis of Menin Binding Provides Insights into MEN1 Tumorigenesis

Multiple endocrine neoplasia type I (MEN1) is a familial cancer syndrome characterized primarily by tumors of multiple endocrine glands. The gene for MEN1 encodes a ubiquitously expressed tumor suppressor protein called menin. Menin was recently shown to interact with several components of a trithorax family histone methyltransferase complex including ASH2, Rbbp5, WDR5, and the leukemia proto-oncoprotein MLL. To elucidate menin's role as a tumor suppressor and gain insights into the endocrine-specific tumor phenotype in MEN1, we mapped the genomic binding sites of menin, MLL1, and Rbbp5, to approximately 20,000 promoters in HeLa S3, HepG2, and pancreatic islet cells using the strategy of chromatin-immunoprecipitation coupled with microarray analysis. We found that menin, MLL1, and Rbbp5 localize to the promoters of thousands of human genes but do not always bind together. These data suggest that menin functions as a general regulator of transcription. We also found that factor occupancy generally correlates with high gene expression but that the loss of menin does not result in significant changes in most transcript levels. One exception is the developmentally programmed transcription factor, HLXB9, which is overexpressed in islets in the absence of menin. Our findings expand the realm of menin-targeted genes several hundred-fold beyond that previously described and provide potential insights to the endocrine tumor bias observed in MEN1 patients

Symptomatic benefit of momelotinib in patients with myelofibrosis: results from the SIMPLIFY phase III studies

Background: Myelofibrosis (MF)-associated constitutional symptoms can severely impact health-related quality of life. Clinical trials in MF traditionally measure symptom response to treatment as a landmark endpoint of total symptom score (TSS) reduction ≥50% from baseline. However, this dichotomous assessment provides a limited view of clinically relevant symptomatic changes. Herein we evaluated longitudinal change from baseline in TSS over the continuous 24-week period and individual symptom scores to obtain a more comprehensive understanding of symptom benefits experienced by patients with MF receiving therapy. Methods: Longitudinal symptom change was evaluated using mixed-effect model repeated measure (MMRM) methodology with individual item-level analyses to complement the interpretation of the landmark symptom results in the completed phase III SIMPLIFY studies of momelotinib in MF. MMRM compared mean change in TSS from baseline with Week 24 using data from all patient visits. Generalized estimating equations were used to estimate item-level odds ratios using multiple predictive imputations for missing data. Results: Momelotinib and ruxolitinib groups reported similar overall symptom improvements, with a TSS difference of <1.5 points between groups for each post-baseline visit in SIMPLIFY-1. In SIMPLIFY-2, the improvement in TSS observed in momelotinib-treated patients was consistent with that observed in SIMPLIFY-1, whereas progressive TSS deterioration was observed with control. Item-level scores were heterogeneous in both studies. A similar and greater proportion of momelotinib-treated patients were categorized as “improved” or “stable” compared with control in SIMPLIFY-1 and SIMPLIFY-2, respectively. Odds ratios for between-group comparison ranged from 0.75 to 1.21 in SIMPLIFY-1, demonstrating similarity in likelihood of symptom improvement. In SIMPLIFY-2, the likelihood of symptom improvement in each item was higher in the momelotinib arm. Conclusions: These findings suggest that momelotinib provides clinically relevant symptom benefits in the JAK inhibitor-naïve and JAK inhibitor-exposed settings

A Multifunctional Mesothelin Antibody-tagged Microparticle Targets Human Mesotheliomas

Pleural and peritoneal mesotheliomas (MMs) are chemoresistant tumors with no effective therapeutic strategies. The authors first injected multifunctional, acid-prepared mesoporous spheres (APMS), microparticles functionalized with tetraethylene glycol oligomers, intraperitoneally into rodents. Biodistribution of APMS was observed in major organs, peritoneal lavage fluid (PLF), and urine of normal mice and rats. After verification of increased mesothelin in human mesotheliomas injected into severe combined immunodeficient (SCID) mice, APMS were then functionalized with an antibody to mesothelin (APMS-MB) or bovine serum albumin (BSA), a nonspecific protein control, and tumor targeting was evaluated by inductively coupled plasma mass spectrometry and multifluorescence confocal microscopy. Some APMS were initially cleared via the urine over a 24 hr period, and small amounts were observed in liver, spleen, and kidneys at 24 hr and 6 days. Targeting with APMS-MB increased APMS uptake in mesenteric tumors at 6 days. Approximately 10% to 12% of the initially injected amount was observed in both spheroid and mesenteric MM at this time point. The data suggest that localized delivery of APMS-MB into the peritoneal cavity after encapsulation of drugs, DNA, or macromolecules is a novel therapeutic approach for MM and other tumors (ovarian and pancreatic) that overexpress mesothelin

Menin Sites Can Be Bound in the Presence or Absence of HMT Complex Members

<div><p>(A) Comparison of promoters bound by menin and Rbbp5 showing the broad range in signal intensity for each factor. The −log10 <i>p</i>-value for each site is plotted on the <i>x</i> and <i>y</i> axes. Vertical and horizontal lines represent confidence thresholds at <i>p</i> < 0.0001. Points in the lower left quadrant represent promoters that are bound by neither menin nor Rbbp5. Points in the upper right quadrant represent promoters occupied by both menin and Rbbp5. Plots comparing menin to MLL and H3 K4 appeared similar (unpublished data).</p><p>(B and C) Heat maps illustrating the overlap of factor-occupied promoters. Promoters bound by either menin (B) or MLL (C) at the <i>p</i> < 0.0001 confidence threshold in HeLa S3 cells were plotted with corresponding <i>p</i>-values for each indicated factor. The heat maps reveal subsets of genes that are bound by all factors (blue brackets), in addition to subsets that are bound by menin or MLL but not the other factors (green brackets).</p><p>(D) Real-time PCR validation of randomly selected promoters determined by ChIP-chip to be bound by menin and not Rbbp5 (left), and vice versa (right). Each pair of bars corresponds to a unique promoter region.</p></div

Factor Occupancy Correlates with High Gene Expression but Absence of Menin Does Not Generally Affect Gene Expression Levels

<div><p>(A) Overall expression of genes for which we had both expression and binding data in HeLa S3 cells was plotted (lane 1, “All expression”) and compared to the expression of genes whose promoter regions were bound (<i>p</i> < 0.0001) by each of the factors indicated at the top. Asterisks denote significance (<i>p</i> < 0.0001) as determined by two-tailed <i>t</i>-test analyses between each set of factor-bound genes compared to all genes. Box plots comparing ChIP-chip and expression data from HepG2 and pancreatic islets revealed similar correlations between expression and factor occupancy (unpublished data).</p><p>(B) Overall expression of genes in islets isolated from wild-type control mice (lane 1, “All Expression”) compared to those normally bound by menin in wild-type islets (lane 2) and those normally bound by menin in islets from 15-wk (lane 3) and 25-wk (lane 4) mice that are conditionally null for <i>Men1</i> (genotype: <i>RIP-cre; Men1</i>^{−<i>/</i>−}).</p></div

Hb9 Expression Is Elevated in Islets from Conventional and Conditional <i>Men1</i> Knockout Mice

<div><p>(A) Pancreatic sections from an 18-mo conventional <i>Men1</i> knockout mouse <i>(Men1^+/</i>⁻<i>)</i> were analyzed for Hb9 expression by immunohistochemistry. Brown stain corresponds to positive Hb9 signal. Compared to normal-size islets (left), an atypical hyperplastic islet (middle) and tumor (right) show increased Hb9 expression. All pictures were taken from different regions of the same section, all at the same exposure. Top panels: ×100 magnification, bottom panels: ×200 to ×400.</p><p>(B) Real-time PCR analysis of <i>Men1</i> and <i>Hlxb9</i> expression in <i>Men1^+/+</i> and <i>Men1</i>^{−<i>/</i>−} islets from conditional animals. Blue bars indicate expression relative to <i>Gapdh;</i> red bars, expression relative to beta-actin; +/+, average expression of five control mice (three <i>RIP-Cre; Men1^+/+</i>, two <i>Men1^+/+</i>); 15–25 wk <i>Men1</i>^{−<i>/</i>−}, average of one pool of conditional islets at 15 wk (five) and three individual preps from 25-wk <i>Men1</i>^{−<i>/</i>−} islets <i>(RIP-cre; Men1^loxP/loxP);</i> and tumor, average expression of five tumors from conditional <i>Men1</i> knockout mice <i>(RIP-cre; Men1^loxP/loxP)</i>. Asterisks denote statistical significance (<i>p</i> < 0.02) for both blue and red bars as determined by two-tailed <i>t</i>-tests.</p></div