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Supplementary Table 1 lists oligonucleotide sequences and cell line information

Supplementary Table 2 details known and de novo motif analysis for distal acetylation clusters

Supplementary table 4 lists ETV6-regulated gene-enhancer linkages

Supplementary Table 6 details GGAA repeat overlap analysis of new and previously published ETS factor datasets.

Contains Supplementary Figures 1-12 and additional information about Supplementary tables

Distal enhancers play critical roles in sustaining oncogenic gene-expression programs. We identify aberrant enhancer-like activation of GGAA tandem repeats as a characteristic feature of B-cell acute lymphoblastic leukemia (B-ALL) with genetic defects of the ETV6 transcriptional repressor, including ETV6–RUNX1+ and ETV6-null B-ALL. We show that GGAA repeat enhancers are direct activators of previously identified ETV6–RUNX1+/− like B-ALL “signature” genes, including the likely leukemogenic driver EPOR. When restored to ETV6-deficient B-ALL cells, ETV6 directly binds to GGAA repeat enhancers, represses their acetylation, downregulates adjacent genes, and inhibits B-ALL growth. In ETV6-deficient B-ALL cells, we find that the ETS transcription factor ERG directly binds to GGAA microsatellite enhancers and is required for sustained activation of repeat enhancer-activated genes. Together, our findings reveal an epigenetic gatekeeper function of the ETV6 tumor suppressor gene and establish microsatellite enhancers as a key mechanism underlying the unique gene-expression program of ETV6–RUNX1+/− like B-ALL.Significance:We find a unifying mechanism underlying a leukemia subtype-defining gene-expression signature that relies on repetitive elements with poor conservation between humans and rodents. The ability of ETV6 to antagonize promiscuous, nonphysiologic ERG activity may shed light on other roles of these key regulators in hematolymphoid development and human disease.See related commentary by Mercher, p. 2.This article is highlighted in the In This Issue feature, p. 1

Supplementary Table 5 annotates genome-wide candidate ETV6-repressed enhancers

Supplementary Table 3 summarizes TF ChIP-Seq results and motif enrichment statistics

Fibrous dysplasia of the mandible typically begins during toddler years and culminates during puberty. These lesions may cause jaw disfigurement and may not be amendable to surgery. Imatinib successfully has treated cherubism associated with SH3BP2 mutations, but similar lesions can present in other conditions such as Noonan syndrome. We report diagnosis of Noonan syndrome caused by a PTPN11 activating mutation and successful treatment of the fibrous dysplasia of the mandible with imatinib.

Distal enhancers play critical roles in sustaining oncogenic gene-expression programs. We identify aberrant enhancer-like activation of GGAA tandem repeats as a characteristic feature of B-cell acute lymphoblastic leukemia (B-ALL) with genetic defects of the ETV6 transcriptional repressor, including ETV6–RUNX1+ and ETV6-null B-ALL. We show that GGAA repeat enhancers are direct activators of previously identified ETV6–RUNX1+/− like B-ALL “signature” genes, including the likely leukemogenic driver EPOR. When restored to ETV6-deficient B-ALL cells, ETV6 directly binds to GGAA repeat enhancers, represses their acetylation, downregulates adjacent genes, and inhibits B-ALL growth. In ETV6-deficient B-ALL cells, we find that the ETS transcription factor ERG directly binds to GGAA microsatellite enhancers and is required for sustained activation of repeat enhancer-activated genes. Together, our findings reveal an epigenetic gatekeeper function of the ETV6 tumor suppressor gene and establish microsatellite enhancers as a key mechanism underlying the unique gene-expression program of ETV6–RUNX1+/− like B-ALL. Significance: We find a unifying mechanism underlying a leukemia subtype-defining gene-expression signature that relies on repetitive elements with poor conservation between humans and rodents. The ability of ETV6 to antagonize promiscuous, nonphysiologic ERG activity may shed light on other roles of these key regulators in hematolymphoid development and human disease. See related commentary by Mercher, p. 2. This article is highlighted in the In This Issue feature, p. 1

Distal enhancers play critical roles in sustaining oncogenic gene expression programs. We identify aberrant enhancer-like activation of GGAA tandem repeats as a characteristic feature of B-cell acute lymphoblastic leukemia (B-ALL) with genetic defects of the ETV6 transcriptional repressor, including ETV6-RUNX1+ and ETV6-null B-ALL. We show that GGAA repeat enhancers are direct activators of previously identified ETV6-RUNX1+ B-ALL “signature” genes, including likely oncogenic drivers. When restored to ETV6-deficient B-ALL cells, ETV6 directly binds to GGAA repeat enhancers, represses their acetylation, downregulates adjacent genes, and inhibits B-ALL growth. In ETV6-deficient B-ALL cells, we find that the ETS transcription factor ERG directly binds to GGAA microsatellite enhancers and is required for sustained activation of many repeat enhancer-activated genes. Together, our findings reveal a novel epigenetic gatekeeper function of the ETV6 tumor suppressor gene and establish microsatellite enhancers as a key mechanism underlying the unique gene expression program of ETV6-RUNX1+ B-ALL.SignificanceWe show that the oncogenic gene expression program of a common pediatric leukemia relies on repetitive noncoding elements that are not conserved between humans and rodents, placing important limitations on animal models for this disease. Our findings may present new opportunities for targeting cancer-specific chromatin dysregulation in leukemia.

Subtypes of B-lymphoblastic leukemia (B-ALL) are defined by unique genetic aberrations and gene expression programs. ETV6-RUNX1+ (E-R+) B-ALL, a common pediatric subtype, shares a gene activation signature with “ETV6-RUNX1-like” B-ALL that lacks the E-R fusion gene. Both E-R+ and E-R-like B-ALL show frequent inactivating mutations or deletions of ETV6, which encodes an ETS family transcriptional repressor (core binding motif GGAA/T). Signature genes overexpressed in E-R+ B-ALL, such as EPOR and PIK3C3, were shown to sustain the malignant phenotype. We performed H3K27ac ChIP-Seq and ATAC-Seq on 26 B-cell cancer cell lines to identify subtype-specific active enhancer-like elements. Unbiased K-means clustering identified distal elements that were selectively acetylated in four E-R+ B-ALL cell lines (all with inactivation of the second ETV6 allele) and in a fifth B-ALL cell line with biallelic ETV6 deletion. Motif analysis of this cluster showed strong enrichment for tandem repeats of the sequence “GGAA” (p Doxycycline-inducible expression of ETV6 in ETV6-deficient, E-R+ B-ALL cells led to significant growth inhibition. Restored ETV6 bound extensively to GGAA repeats (80% of ETV6 binding sites), repressed repeat enhancer acetylation, and repressed expression of adjacent E-R+ signature genes (n=40). Repression of a published E-R+ signature gene set was significant by GSEA analysis (p GGAA repeat enhancers are a known driver of Ewing sarcoma, where they are activated by oncogenic fusions of the ETS activators FLI1 or ERG. Active GGAA repeat enhancers and repeat-regulated genes in E-R+ B-ALL show only partial overlap with repeat-regulated genes in Ewings, possibly due to tissue-specific factors. Wild-type ERG and FLI1 are highly expressed in B-ALL. We found that knockdown of ERG, but not FLI1, led to significant downregulation of repeat enhancer-regulated E-R+ signature genes in B-ALL. ChIP-Seq showed that the ERG binds nearly all active GGAA repeat enhancers in three ETV6-deficient B-ALL cell lines, but not in ETV6-intact B-ALL. We identify GGAA repeat enhancers, sustained by ETV6 deficiency and strong ERG expression, as a unifying mechanism for a subtype-specific oncogenic gene expression program in B-ALL. Our findings may have crucial implications for E-R+ B-ALL modeling, as most GGAA repeats are not conserved between mice and humans. Mechanisms that activate leukemia-specific repeat enhancers may represent appealing targets for future therapeutic development. Citation Format: Joshua W. Goldman, Rohan Kodgule, Alexander C. Monovich, Travis Saari, Cody N. Hall, Niharika Rajesh, Juhi Gupta, Athalee Aguilar, Noah Brown, Mark Y. Chiang, Marcin P. Cieslik, Russell J. Ryan. ETV6 deficiency and microsatellite enhancers drive transcriptional dysregulation in B-lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1461.

Background Knock-in mice provide useful models of congenital and age-related cataracts caused by α-crystallin mutations. R49C αA-crystallin and R120G αB-crystallin mutations are linked with hereditary cataracts. Knock-in αA-R49C +/− heterozygotes develop cataracts by 1–2 months, whereas homozygote mice have cataracts at birth. The R49C mutation drastically reduces lens protein water solubility and causes cell death in knock-in mouse lenses. Mutant crystallin cannot function as a chaperone, which leads to protein aggregation and lens opacity. Protein aggregation disrupts the lens fiber cell structure and normal development and causes cell death in epithelial and fiber cells. We determined what aspects of the wild-type phenotype are age-dependently altered in the mutant lens. Methods Wild-type, heterozygote (αA-R49C +/−), and homozygote (αA-R49C +/+) mouse lenses were assessed pre- and postnatally for lens morphology (electron microscopy, immunohistochemistry), and autophagy or unfolded protein response markers (immunoblotting). Results Morphology was altered by embryonic day 17 in R49C +/+ lenses; R49C +/− lens morphology was unaffected at this stage. Active autophagy in the lens epithelium of mutant lenses was indicated by the presence of autophagosomes using electron microscopy. Protein p62 levels, which are degraded specifically by autophagy, increased in αA-R49C mutant versus wild-type lenses, suggesting autophagy inhibition in the mutant lenses. The unfolded protein response marker XBP-1 was upregulated in adult lenses of αB-R120G +/+ mice, suggesting its role in lens opacification. Conclusions Mutated crystallins alter lens morphology, autophagy, and stress responses. General significance Therapeutic modulation of autophagic pathways may improve protein degradation in cataractous lenses and reduce lens opacity. This article is part of a Special Issue entitled Crystallin Biochemistry in Health and Disease.

Introduction Engraftment of mesenchymal stem cells (MSCs) has emerged as a powerful candidate for mediating myocardial repair. In this study, we genetically modified MSCs with an adenovector encoding thioredoxin-1 (Ad.Trx1). Trx1 has been described as a growth regulator, a transcription factor regulator, a cofactor, and a powerful antioxidant. We explored whether engineered MSCs, when transplanted, are capable of improving cardiac function and angiogenesis in a rat model of myocardial infarction (MI). Methods Rat MSCs were cultured and divided into MSC, MSC+Ad.LacZ, and MSC+Ad.Trx1 groups. The cells were assayed for proliferation, and differentiation potential. In addition, rats were divided into control-sham (CS), control-MI (CMI), MSC+Ad.LacZ-MI (MLZMI), and MSC+Ad.Trx1-MI (MTrxMI) groups. MI was induced by left anterior descending coronary artery (LAD) ligation, and MSCs preconditioned with either Ad.LacZ or Ad.Trx1 were immediately administered to four sites in the peri-infarct zone. Results The MSC+Ad.Trx1 cells increased the proliferation capacity and maintained pluripotency, allowing them to divide into cardiomyocytes, smooth muscle, and endothelial cells. Western blot analysis, 4days after treatment showed increased vascular endothelial growth factor (VEGF), heme oxygenase-1 (HO-1), and C-X-C chemokine receptor type 4 (CXCR4). Also capillary density along with myocardial function as examined by echocardiography was found to be increased. Fibrosis was reduced in the MTrxMI group compared to MLZMI and CMI. Visualization of Connexin-43 by immunohistochemistry confirmed increased intercellular connections in the MTrxMI rats compared to MLZMI. Conclusion Engineering MSCs to express Trx1 may prove to be a strategic therapeutic modality in the treatment of cardiac failure.

A visionary approach to transparency Cataracts are the most common cause of vision loss, especially in our ever-increasing elderly population. Cataracts arise when crystallin, a major protein component of the eye lens, begins to aggregate, which causes the lens to become cloudy. Makley et al. explored whether small molecules that reverse this aggregation might have therapeutic potential for treating cataracts, which normally require surgery (see the Perspective by Quinlan). They used a screening method that monitors the effect of ligands on temperature-dependent protein unfolding and identified several compounds that bind and stabilize the soluble form of crystallin. In proof-of-concept studies, one of these compounds improved lens transparency in mice. Science , this issue p. 674 ; see also p. 636

Significance: In this review, we have discussed the efficacy and effect of small molecules that act as prolyl hydroxylase domain inhibitors (PHDIs). The use of these compounds causes upregulation of the pro-angiogenic factors and hypoxia inducible factor-1α and -2α (HIF-1α and HIF-2α) to enhance angiogenic, glycolytic, erythropoietic, and anti-apoptotic pathways in the treatment of various ischemic diseases responsible for significant morbidity and mortality in humans. Recent Advances: Sprouting of new blood vessels from the existing vasculature and surgical intervention, such as coronary bypass and stent insertion, have been shown to be effective in attenuating ischemia. However, the initial reentry of oxygen leads to the formation of reactive oxygen species that cause oxidative stress and result in ischemia/reperfusion (IR) injury. This apparent “oxygen paradox” must be resolved to combat IR injury. During hypoxia, decreased activity of PHDs initiates the accumulation and activation of HIF-1α, wherein the modulation of both PHD and HIF-1α appears as promising therapeutic targets for the pharmacological treatment of ischemic diseases. Critical Issues: Research on PHDs and HIFs has shown that these molecules can serve as therapeutic targets for ischemic diseases by modulating glycolysis, erythropoiesis, apoptosis, and angiogenesis. Efforts are underway to identify and synthesize safer small-molecule inhibitors of PHDs that can be administered in vivo as therapy against ischemic diseases. Future Directions: This review presents a comprehensive and current account of the existing small-molecule PHDIs and their use in the treatment of ischemic diseases with a focus on the molecular mechanisms of therapeutic action in animal models. Antioxid. Redox Signal. 20, 2631–2665.

The formation of cataracts is associated with the accumulation of protein aggregates in the ocular lens, suggesting that defective protein degradation plays a role in cataract pathogenesis. Accumulation of the p62 protein has recently been identified as a marker for impaired autophagy in a variety of tissues; however, little information exists on its expression in the ocular lens and in cataracts. In the present study we examined the expression of p62 in the mouse lens and compared its expression in wild-type lenses with that in lenses from knock-in mice with an arginine to glycine mutation in αB-crystallin (αB-R120G) that is known to cause human hereditary cataract. Immunohistochemical, immunoblotting, and transmission electron microscopic analyses of wild type and αB-R120G mutant mice were performed. To assess the effect of increased protein aggregation on autophagy, immunohistochemical staining was performed with an anti-p62 antibody, revealing the presence of p62-positive punctate staining in a band of denucleated cortical fiber cells. The number and size of p62 puncta were significantly greater in αB-R120G homozygous mutant lenses than in wild type and heterozygous mutant lenses. p62 staining was also abundant in lens epithelial cells and was concentrated around the nuclear membrane. Double-membraned structures similar to autophagosomes containing cellular cytoplasmic content were detected in lens epithelial cells by transmission electron microscopy. The autophagosomes in lens epithelial cells from αB-R120G homozygous mutant mice were larger than those in wild type mice. Double-membraned structures that are probably autophagosomes were also detected in cortical fiber cells and were more abundant in the αB-R120G homozygous mutant lens than the wild type lens. This study demonstrates p62 distribution as speckles in the lens fiber cells, altered levels of p62 expression, and the presence of autophagosomes in the ocular lens of αB-R120G mutant mice. We propose that autophagy is inhibited in the αB-R120G mutant lenses because of a defect in protein degradation after autophagosome formation. Further work is necessary to determine the relationship between αB-crystallin function, autophagy, and cataract formation.

Although chronobiology is of growing interest to scientists, physicians, and the general public, access to recent discoveries and historical perspectives is limited. Wikipedia is an online, user-written encyclopedia that could enhance public access to current understanding in chronobiology. However, Wikipedia is lacking important information and is not universally trusted. Here, 46 students in a university course edited Wikipedia to enhance public access to important discoveries in chronobiology. Students worked for an average of 9 h each to evaluate the primary literature and available Wikipedia information, nominated sites for editing, and, after voting, edited the 15 Wikipedia pages they determined to be highest priorities. This assignment ( http://www.nslc.wustl.edu/courses/Bio4030/wikipedia_project.html ) was easy to implement, required relatively short time commitments from the professor and students, and had measurable impacts on Wikipedia and the students. Students created 3 new Wikipedia sites, edited 12 additional sites, and cited 347 peer-reviewed articles. The targeted sites all became top hits in online search engines. Because their writing was and will be read by a worldwide audience, students found the experience rewarding. Students reported significantly increased comfort with reading, critiquing, and summarizing primary literature and benefited from seeing their work edited by other scientists and editors of Wikipedia. We conclude that, in a short project, students can assist in making chronobiology widely accessible and learn from the editorial process.

To the Editor.— The review article by Zarrabi et al entitled "Chronic Lymphocytic Leukemia Terminating in Acute Leukemia," 1 questions the origin of the acute leukemic termination of chronic lymphocytic leukemia (CLL). The issue raised is whether the occurrence of acute leukemia (AL) with CLL is a second malignancy or a "blast crisis (transformation)," which occurs as a natural evolution of CLL. We would like to demonstrate that the number of cases with CLL and AL reported, and the number predicted, could occur by chance. Since 1936, 2 19 cases of CLL and an associated AL have been reported 1 in the American medical literature. Demographic and morphologic characteristics of the patients with AL have not been consistently reported. The average death rate in 1967 for all ages and sexes was 1.6 per 100,000 for CLL, and 2.4 per 100,000 for AL. 3 The average annual population in the United

A patient with hypercalcemia, hypophosphatemia, and an undifferentiated lymphoma involving only the bone marrow was treated by us. The finding of an elevated serum parathyroid hormone (PTH) level in the presence of normal parathyroid glands and increased metabolic bone activity on bone biopsy suggests an ectopic PTH syndrome. To our knowledge, no previous case of ectopic PTH syndrome associated with a lymphoma with bone marrow involvement only has been reported. (Arch Intern Med138:1290-1291, 1978)

Excerpt To the editor: With reference to the article of Dr. Shenhman and Dr. Bottone (1) on the origin of subacute thyroiditis, we wish to report a case of subacute thyroiditis observed during a re...