Combinatorial genetics reveals the Dock1-Rac2 axis as a potential target for the treatment of NPM1;Cohesin mutated AML.

Acute myeloid leukemia (AML) is driven by mutations that occur in numerous combinations. A better understanding of how mutations interact with one another to cause disease is critical to developing targeted therapies. Approximately 50% of patients that harbor a common mutation in NPM1 (NPM1cA) also have a mutation in the cohesin complex. As cohesin and Npm1 are known to regulate gene expression, we sought to determine how cohesin mutation alters the transcriptome in the context of NPM1cA. We utilized inducible Npm1cAflox/+ and core cohesin subunit Smc3flox/+ mice to examine AML development. While Npm1cA/+;Smc3Δ/+ mice developed AML with a similar latency and penetrance as Npm1cA/+ mice, RNA-seq suggests that the Npm1cA/+; Smc3Δ/+ mutational combination uniquely alters the transcriptome. We found that the Rac1/2 nucleotide exchange factor Dock1 was specifically upregulated in Npm1cA/+;Smc3Δ/+ HSPCs. Knockdown of Dock1 resulted in decreased growth and adhesion and increased apoptosis only in Npm1cA/+;Smc3Δ/+ AML. Higher Rac activity was also observed in Npm1cA/+;Smc3Δ/+ vs. Npm1cA/+ AMLs. Importantly, the Dock1/Rac pathway is targetable in Npm1cA/+;Smc3Δ/+ AMLs. Our results suggest that Dock1/Rac represents a potential target for the treatment of patients harboring NPM1cA and cohesin mutations and supports the use of combinatorial genetics to identify novel precision oncology targets.

Decitabine and vorinostat with FLAG chemotherapy in pediatric relapsed/refractory AML: Report from the therapeutic advances in childhood leukemia and lymphoma (TACL) consortium.

Survival outcomes for relapsed/refractory pediatric acute myeloid leukemia (R/R AML) remain dismal. Epigenetic changes can result in gene expression alterations which are thought to contribute to both leukemogenesis and chemotherapy resistance. We report results from a phase I trial with a dose expansion cohort investigating decitabine and vorinostat in combination with fludarabine, cytarabine, and G-CSF (FLAG) in pediatric patients with R/R AML [NCT02412475]. Thirty-seven patients enrolled with a median age at enrollment of 8.4 (range, 1-20) years. There were no dose limiting toxicities among the enrolled patients, including two patients with Down syndrome. The recommended phase 2 dose of decitabine in combination with vorinostat and FLAG was 10 mg/m2 . The expanded cohort design allowed for an efficacy evaluation and the overall response rate among 35 evaluable patients was 54% (16 complete response (CR) and 3 complete response with incomplete hematologic recovery (CRi)). Ninety percent of responders achieved minimal residual disease (MRD) negativity (<0.1%) by centralized flow cytometry and 84% (n = 16) successfully proceeded to hematopoietic stem cell transplant. Two-year overall survival was 75.6% [95%CI: 47.3%, 90.1%] for MRD-negative patients vs. 17.9% [95%CI: 4.4%, 38.8%] for those with residual disease (p < .001). Twelve subjects (34%) had known epigenetic alterations with 8 (67%) achieving a CR, 7 (88%) of whom were MRD negative. Correlative pharmacodynamics demonstrated the biologic activity of decitabine and vorinostat and identified specific gene enrichment signatures in nonresponding patients. Overall, this therapy was well-tolerated, biologically active, and effective in pediatric patients with R/R AML, particularly those with epigenetic alterations.

DOT1L inhibitors block abnormal self-renewal induced by cohesin loss.

Acute myeloid leukemia (AML) is a high-risk malignancy characterized by a diverse spectrum of somatic genetic alterations. The mechanisms by which these mutations contribute to leukemia development and how this informs the use of targeted therapies is critical to improving outcomes for patients. Importantly, how to target loss-of-function mutations has been a critical challenge in precision medicine. Heterozygous inactivating mutations in cohesin complex genes contribute to AML in adults by increasing the self-renewal capacity of hematopoietic stem and progenitor cells (HSPCs) by altering PRC2 targeting to induce HOXA9 expression, a key self-renewal transcription factor. Here we sought to delineate the epigenetic mechanism underpinning the enhanced self-renewal conferred by cohesin-haploinsufficiency. First, given the substantial difference in the mutational spectrum between pediatric and adult AML patients, we first sought to identify if HOXA9 was also elevated in children. Next, using primary HSPCs as a model we demonstrate that abnormal self-renewal due to cohesin loss is blocked by DOT1L inhibition. In cohesin-depleted cells, DOT1L inhibition is associated with H3K79me2 depletion and a concomitant increase in H3K27me3. Importantly, we find that there are cohesin-dependent gene expression changes that promote a leukemic profile, including HoxA overexpression, that are preferentially reversed by DOT1L inhibition. Our data further characterize how cohesin mutations contribute to AML development, identifying DOT1L as a potential therapeutic target for adult and pediatric AML patients harboring cohesin mutations.

A cohesive look at leukemogenesis: The cohesin complex and other driving mutations in AML.

Acute myeloid leukemia (AML) affects tens of thousands of patients a year, yet survival rates are as low as 25% in certain populations. This poor survival rate is partially due to the vast genetic diversity of the disease. Rarely do 2 patients with AML have the same mutational profile, which makes the development of targeted therapies particularly challenging. However, a set of recurrent mutations in chromatin modifiers have been identified in many patients, including mutations in the cohesin complex, which have been identified in up to 20% of cases. Interestingly, the canonical function of the cohesin complex in establishing sister chromatid cohesin during mitosis is unlikely to be the affected role in leukemogenesis. Instead, the cohesin complex's role in DNA looping and gene regulation likely facilitates disease. The epigenetic mechanisms by which cohesin complex mutations promote leukemia are not completely elucidated, but alterations of enhancer-promoter interactions and differential histone modifications have been shown to drive oncogenic gene expression changes. Such changes commonly include HoxA upregulation, which may represent a common pathway that could be therapeutically targeted. As cohesin mutations rarely occur alone, examining the impact of common co-occurring mutations, including those in NPM1, the core-binding factor complex, FLT3, and ASXL1, will yield additional insight. While further study of these mutational interactions is required, current research suggests that the use of combinatorial genetics could be the key to uncovering new targets, allowing for the treatment of AML patients based on their individual genetic profiles.

Genome-Wide Maps of Transcription Regulatory Elements and Transcription Enhancers in Development and Disease.

Gene expression is regulated by numerous elements including enhancers, insulators, transcription factors, and architectural proteins. Regions of DNA distal to the transcriptional start site, called enhancers, play a central role in the temporal and tissue-specific regulation of gene expression through RNA polymerase II. The identification of enhancers and other cis regulatory elements has largely been possible due to advances in next generation sequencing technologies. Enhancers regulate gene expression through chromatin loops mediated by architectural proteins such as YY1, CTCF, the cohesin complex, and LDB1. Additionally, enhancers can be transcribed to produce noncoding RNAs termed enhancer RNAs that likely participate in transcriptional regulation. The central role of enhancers in regulating gene expression implicates them in both normal physiology but also many disease states. The importance of enhancers is evident by the suggested role of SNPs, duplications, and other alterations of enhancer function in many diseases, ranging from cancer to atherosclerosis to chronic kidney disease. Although much progress has been made in recent years, the field of enhancer biology and our knowledge of the cis regulome remains a work in progress. This review will highlight recent seminal studies which demonstrate the role of enhancers in normal physiology and disease pathogenesis. © 2019 American Physiological Society. Compr Physiol 9:439-455, 2019.

Restricted MHC class I A locus diversity in olive and hybrid olive/yellow baboons from the Southwest National Primate Research Center.

Baboons are valuable models for complex human diseases due to their genetic and physiologic similarities to humans. Deep sequencing methods to characterize full-length major histocompatibility complex (MHC) class I (MHC-I) alleles in different nonhuman primate populations were used to identify novel MHC-I alleles in baboons. We combined data from Illumina MiSeq sequencing and Roche/454 sequencing to characterize novel full-length MHC-I transcripts in a cohort of olive and hybrid olive/yellow baboons from the Southwest National Primate Research Center (SNPRC). We characterized 57 novel full-length alleles from 24 baboons and found limited genetic diversity at the MHC-I A locus, with significant sharing of two MHC-I A lineages between 22 out of the 24 animals characterized. These shared alleles provide the basis for development of tools such as MHC:peptide tetramers for studying cellular immune responses in this important animal model.

Major histocompatibility complex haplotyping and long-amplicon allele discovery in cynomolgus macaques from Chinese breeding facilities.

Very little is currently known about the major histocompatibility complex (MHC) region of cynomolgus macaques (Macaca fascicularis; Mafa) from Chinese breeding centers. We performed comprehensive MHC class I haplotype analysis of 100 cynomolgus macaques from two different centers, with animals from different reported original geographic origins (Vietnamese, Cambodian, and Cambodian/Indonesian mixed-origin). Many of the samples were of known relation to each other (sire, dam, and progeny sets), making it possible to characterize lineage-level haplotypes in these animals. We identified 52 Mafa-A and 74 Mafa-B haplotypes in this cohort, many of which were restricted to specific sample origins. We also characterized full-length MHC class I transcripts using Pacific Biosciences (PacBio) RS II single-molecule real-time (SMRT) sequencing. This technology allows for complete read-through of unfragmented MHC class I transcripts (~1100 bp in length), so no assembly is required to unambiguously resolve novel full-length sequences. Overall, we identified 311 total full-length transcripts in a subset of 72 cynomolgus macaques from these Chinese breeding facilities; 130 of these sequences were novel and an additional 115 extended existing short database sequences to span the complete open reading frame. This significantly expands the number of Mafa-A, Mafa-B, and Mafa-I full-length alleles in the official cynomolgus macaque MHC class I database. The PacBio technique described here represents a general method for full-length allele discovery and genotyping that can be extended to other complex immune loci such as MHC class II, killer immunoglobulin-like receptors, and Fc gamma receptors.

Arteriviruses, Pegiviruses, and Lentiviruses Are Common among Wild African Monkeys.

UNLABELLED: Nonhuman primates (NHPs) are a historically important source of zoonotic viruses and are a gold-standard model for research on many human pathogens. However, with the exception of simian immunodeficiency virus (SIV) (family Retroviridae), the blood-borne viruses harbored by these animals in the wild remain incompletely characterized. Here, we report the discovery and characterization of two novel simian pegiviruses (family Flaviviridae) and two novel simian arteriviruses (family Arteriviridae) in wild African green monkeys from Zambia (malbroucks [Chlorocebus cynosuros]) and South Africa (vervet monkeys [Chlorocebus pygerythrus]). We examine several aspects of infection, including viral load, genetic diversity, evolution, and geographic distribution, as well as host factors such as age, sex, and plasma cytokines. In combination with previous efforts to characterize blood-borne RNA viruses in wild primates across sub-Saharan Africa, these discoveries demonstrate that in addition to SIV, simian pegiviruses and simian arteriviruses are widespread and prevalent among many African cercopithecoid (i.e., Old World) monkeys. IMPORTANCE: Primates are an important source of viruses that infect humans and serve as an important laboratory model of human virus infection. Here, we discover two new viruses in African green monkeys from Zambia and South Africa. In combination with previous virus discovery efforts, this finding suggests that these virus types are widespread among African monkeys. Our analysis suggests that one of these virus types, the simian arteriviruses, may have the potential to jump between different primate species and cause disease. In contrast, the other virus type, the pegiviruses, are thought to reduce the disease caused by human immunodeficiency virus (HIV) in humans. However, we did not observe a similar protective effect in SIV-infected African monkeys coinfected with pegiviruses, possibly because SIV causes little to no disease in these hosts.

Novel MHC class I full-length allele and haplotype characterization in sooty mangabeys.

Sooty mangabeys (Cercocebus atys) are natural SIV hosts and the presumed source of HIV-2 and SIVmac, which makes them a valuable model for HIV/SIV research. However, like other African primates, little is known about their major histocompatibility complex (MHC) genetics. In this study, we used Roche/454 and Illumina MiSeq deep sequencing in order to determine the MHC class I transcripts in a cohort of 165 sooty mangabeys from the Yerkes National Primate Research Center (YNPRC). We have characterized 121 functionally full-length classical (Ceat-A and Ceat-B) and non-classical (Ceat-F and Ceat-I) alleles and have also identified 22 Ceat-A/Ceat-B haplotype chromosomal combinations. We correlated these Ceat-A/Ceat-B haplotype combinations to recently described microsatellite haplotypes from the YNPRC colony. These newly identified alleles and haplotypes establish a resource for studying cellular immunity in sooty mangabeys and provide a framework for rapidly cataloging MHC class I sequences in an understudied, yet important, nonhuman primate species.

Survey of major histocompatibility complex class II diversity in pig-tailed macaques.

Pig-tailed macaques (Macaca nemestrina) serve as important models for human infectious disease research. Major histocompatibility complex (MHC) class II molecules are important to this research since they present peptides to CD4+ T cells. Despite the importance of characterizing the MHC-II alleles expressed in model species like pig-tailed macaques, to date, less than 150 MHC-II alleles have been named for the six most common classical class II loci (DRA, DRB, DQA, DQB, DPA, and DPB) in this population. Additionally, only a small percentage of these alleles are full-length, making it impossible to use the known sequence for reagent development. To address this, we developed a fast, high-throughput method to discover full-length MHC-II alleles and used it to characterize alleles in 32 pig-tailed macaques. By this method, we identified 128 total alleles across all six loci. We also performed an exon 2-based genotyping assay to validate the full-length sequencing results; this genotyping assay could be optimized for use in determining MHC-II allele frequencies in large cohorts of pig-tailed macaques.