Hamid Dolatshad

Combining gene mutation with gene expression data improves outcome prediction in myelodysplastic syndromes

Cancer is a genetic disease, but two patients rarely have identical genotypes. Similarly, patients differ in their clinicopathological parameters, but how genotypic and phenotypic heterogeneity are interconnected is not well understood. Here we build statistical models to disentangle the effect of 12 recurrently mutated genes and 4 cytogenetic alterations on gene expression, diagnostic clinical variables and outcome in 124 patients with myelodysplastic syndromes. Overall, one or more genetic lesions correlate with expression levels of ~20% of all genes, explaining 20–65% of observed expression variability. Differential expression patterns vary between mutations and reflect the underlying biology, such as aberrant polycomb repression for ASXL1 and EZH2 mutations or perturbed gene dosage for copy-number changes. In predicting survival, genomic, transcriptomic and diagnostic clinical variables all have utility, with the largest contribution from the transcriptome. Similar observations are made on the TCGA acute myeloid leukaemia cohort, confirming the general trends reported here.

Disruption of SF3B1 results in deregulated expression and splicing of key genes and pathways in myelodysplastic syndrome hematopoietic stem and progenitor cells.

The splicing factor SF3B1 is the most commonly mutated gene in the myelodysplastic syndrome (MDS), particularly in patients with refractory anemia with ring sideroblasts (RARS). We investigated the functional effects of SF3B1 disruption in myeloid cell lines: SF3B1 knockdown resulted in growth inhibition, cell cycle arrest and impaired erythroid differentiation and deregulation of many genes and pathways, including cell cycle regulation and RNA processing. MDS is a disorder of the hematopoietic stem cell and we thus studied the transcriptome of CD34+ cells from MDS patients with SF3B1 mutations using RNA sequencing. Genes significantly differentially expressed at the transcript and/or exon level in SF3B1 mutant compared with wild-type cases include genes that are involved in MDS pathogenesis (ASXL1 and CBL), iron homeostasis and mitochondrial metabolism (ALAS2, ABCB7 and SLC25A37) and RNA splicing/processing (PRPF8 and HNRNPD). Many genes regulated by a DNA damage-induced BRCA1–BCLAF1–SF3B1 protein complex showed differential expression/splicing in SF3B1 mutant cases. This is the first study to determine the target genes of SF3B1 mutation in MDS CD34+ cells. Our data indicate that SF3B1 has a critical role in MDS by affecting the expression and splicing of genes involved in specific cellular processes/pathways, many of which are relevant to the known RARS pathophysiology, suggesting a causal link.

Differential Expression of the Circadian Clock in Maternal and Embryonic Tissues of Mice

Background Molecular feedback loops involving transcription and translation and several key genes are at the core of circadian regulatory cycles affecting cellular pathways and metabolism. These cycles are active in most adult animal cells but little is known about their expression or influence during development. Methodology/Principal Findings To determine if circadian cycles are active during mammalian development we measured the expression of key circadian genes during embryogenesis in mice using quantitative real-time RT-PCR. All of the genes examined were expressed in whole embryos beginning at the earliest age examined, embryonic day 10. In contrast to adult tissues, circadian variation was absent for all genes at all of the embryonic ages examined in either whole embryos or individual tissues. Using a bioluminescent fusion protein that tracks translation of the circadian gene, per2, we also analyzed protein levels. Similar to mRNA, a protein rhythm was observed in adult tissue but not in embryonic tissues collected in-vivo. In contrast, when tissues were placed in culture for the continuous assay of bioluminescence, rhythms were observed in embryonic (E18) tissues. We found that placing embryonic tissues in culture set the timing (phase) of these rhythms, suggesting the importance of a synchronizing signal for the expression of circadian cycles in developing tissues. Conclusions/Significance These results show that embryonic tissues express key circadian genes and have the capacity to express active circadian regulatory cycles. In vivo, circadian cycles are not expressed in embryonic tissues as they are in adult tissues. Individual cells might express oscillations, but are not synchronized until later in development.

Cryptic splicing events in the iron transporter ABCB7 and other key target genes in SF3B1 -mutant myelodysplastic syndromes

The splicing factor SF3B1 is the most frequently mutated gene in myelodysplastic syndromes (MDS), and is strongly associated with the presence of ring sideroblasts (RS). We have performed a systematic analysis of cryptic splicing abnormalities from RNA sequencing data on hematopoietic stem cells (HSCs) of SF3B1-mutant MDS cases with RS. Aberrant splicing events in many downstream target genes were identified and cryptic 3′ splice site usage was a frequent event in SF3B1-mutant MDS. The iron transporter ABCB7 is a well-recognized candidate gene showing marked downregulation in MDS with RS. Our analysis unveiled aberrant ABCB7 splicing, due to usage of an alternative 3′ splice site in MDS patient samples, giving rise to a premature termination codon in the ABCB7 mRNA. Treatment of cultured SF3B1-mutant MDS erythroblasts and a CRISPR/Cas9-generated SF3B1-mutant cell line with the nonsense-mediated decay (NMD) inhibitor cycloheximide showed that the aberrantly spliced ABCB7 transcript is targeted by NMD. We describe cryptic splicing events in the HSCs of SF3B1-mutant MDS, and our data support a model in which NMD-induced downregulation of the iron exporter ABCB7 mRNA transcript resulting from aberrant splicing caused by mutant SF3B1 underlies the increased mitochondrial iron accumulation found in MDS patients with RS.

A Fear-Inducing Odor Alters PER2 and c-Fos Expression in Brain Regions Involved in Fear Memory

Evidence demonstrates that rodents learn to associate a foot shock with time of day, indicating the formation of a fear related time-stamp memory, even in the absence of a functioning SCN. In addition, mice acquire and retain fear memory better during the early day compared to the early night. This type of memory may be regulated by circadian pacemakers outside of the SCN. As a first step in testing the hypothesis that clock genes are involved in the formation of a time-stamp fear memory, we exposed one group of mice to fox feces derived odor (TMT) at ZT 0 and one group at ZT 12 for 4 successive days. A separate group with no exposure to TMT was also included as a control. Animals were sacrificed one day after the last exposure to TMT, and PER2 and c-Fos protein were quantified in the SCN, amygdala, hippocampus, and piriform cortex. Exposure to TMT had a strong effect at ZT 0, decreasing PER2 expression at this time point in most regions except the SCN, and reversing the normal rhythm of PER2 expression in the amygdala and piriform cortex. These changes were accompanied by increased c-Fos expression at ZT0. In contrast, exposure to TMT at ZT 12 abolished the rhythm of PER2 expression in the amygdala. In addition, increased c-Fos expression at ZT 12 was only detected in the central nucleus of the amygdala in the TMT12 group. TMT exposure at either time point did not affect PER2 or c-Fos in the SCN, indicating that under a light-dark cycle, the SCN rhythm is stable in the presence of repeated exposure to a fear-inducing stimulus. Taken together, these results indicate that entrainment to a fear-inducing stimulus leads to changes in PER2 and c-Fos expression that are detected 24 hours following the last exposure to TMT, indicating entrainment of endogenous oscillators in these regions. The observed effects on PER2 expression and c-Fos were stronger during the early day than during the early night, possibly to prepare appropriate systems at ZT 0 to respond to a fear-inducing stimulus.

Circadian clock genes in reproductive tissues and the developing conceptus.

The circadian (near 24-h) clock is involved in the temporal organisation of physiological and biochemical activities of many organisms, including humans. The clock functions through the rhythmic transcription and translation of several genes, forming an oscillatory feedback loop. Genetic analysis has shown that the circadian clock exists in both a central circadian pacemaker (i.e. the suprachiasmatic nucleus of the hypothalamus), as well as in most peripheral tissues. In particular, the circadian clockwork genes are expressed in all female and male reproductive tissues studied so far, as well as in the conceptus itself. The current data clearly show a robust rhythm in female reproductive tissues, but whether rhythmicity also exists in male reproductive tissues remains uncertain. Although the conceptus also expresses most of the canonical circadian genes, the rhythmicity of their expression is still under investigation. Published data indicate that environmental and genetic manipulations influence reproductive function and fecundity, suggesting an important role for the circadian clock in reproduction, and possibly early development.

ASXL1 mutation correction by CRISPR/Cas9 restores gene function in leukemia cells and increases survival in mouse xenografts

Recurrent somatic mutations of the epigenetic modifier and tumor suppressor ASXL1 are common in myeloid malignancies, including chronic myeloid leukemia (CML), and are associated with poor clinical outcome. CRISPR/Cas9 has recently emerged as a powerful and versatile genome editing tool for genome engineering in various species. We have used the CRISPR/Cas9 system to correct the ASXL1 homozygous nonsense mutation present in the CML cell line KBM5, which lacks ASXL1 protein expression. CRISPR/Cas9-mediated ASXL1 homozygous correction resulted in protein re-expression with restored normal function, including down-regulation of Polycomb repressive complex 2 target genes. Significantly reduced cell growth and increased myeloid differentiation were observed in ASXL1 mutation-corrected cells, providing new insights into the role of ASXL1 in human myeloid cell differentiation. Mice xenografted with mutation-corrected KBM5 cells showed significantly longer survival than uncorrected xenografts. These results show that the sole correction of a driver mutation in leukemia cells increases survival in vivo in mice. This study provides proof-of-concept for driver gene mutation correction via CRISPR/Cas9 technology in human leukemia cells and presents a strategy to illuminate the impact of oncogenic mutations on cellular function and survival.

Application of CRISPR/Cas9 genome editing to the study and treatment of disease

CRISPR/Cas is a microbial adaptive immune system that uses RNA-guided nucleases to cleave foreign genetic elements. The CRISPR/Cas9 method has been engineered from the type II prokaryotic CRISPR system and uses a single-guide RNA to target the Cas9 nuclease to a specific genomic sequence. Cas9 induces double-stranded DNA breaks which are repaired either by imperfect non-homologous end joining to generate insertions or deletions (indels) or, if a repair template is provided, by homology-directed repair. Due to its specificity, simplicity and versatility, the CRISPR/Cas9 system has recently emerged as a powerful tool for genome engineering in various species. This technology can be used to investigate the function of a gene of interest or to correct gene mutations in cells via genome editing, paving the way for future gene therapy approaches. Improvements to the efficiency of CRISPR repair, in particular to increase the rate of gene correction and to reduce undesired off-target effects, and the development of more effective delivery methods will be required for its broad therapeutic application.

The role of splicing factor mutations in the pathogenesis of the myelodysplastic syndromes.

Accurate pre-mRNA splicing by the spliceosome is a fundamental cellular mechanism required to remove introns that are present in most protein-coding transcripts. The recent discovery of a variety of somatic spliceosomal mutations in the myelodysplastic syndromes (MDS), a heterogeneous group of myeloid malignancies, has revealed a new leukemogenic pathway involving spliceosomal dysfunction. Spliceosome mutations are found in over half of all MDS patients and are likely founder mutations. The spliceosome mutations are highly specific to MDS and closely related conditions and, to some extent, appear to define distinct clinical phenotypes in MDS. The high frequency of mutations in different components of the RNA splicing machinery in MDS suggests that abnormal RNA splicing is the common consequence of these mutations. The identification of the downstream targets of the spliceosome mutations is an active area of research. Emerging data from the study of the MDS transcriptome suggests that spliceosomal mutations have effects on specific genes, including some previously shown to play a role in MDS pathogenesis. The effects of the spliceosomal mutations on RNA splicing and cell growth have been evaluated only in a limited context to date, however, and the determination of the impact of these mutations in primary human hematopoietic cells is essential in order to elucidate fully the molecular mechanism by which they contribute to MDS pathogenesis.

Chronic stimulation of the hypothalamic vasoactive intestinal peptide receptor lengthens circadian period in mice and hamsters.

Evidence suggests that circadian rhythms are regulated through diffusible signals generated by the suprachiasmatic nucleus (SCN). Vasoactive intestinal peptide (VIP) is located in SCN neurons positioned to receive photic input from the retinohypothalamic tract and transmit information to other SCN cells and adjacent hypothalamic areas. Studies using knockout mice indicate that VIP is essential for synchrony among SCN cells and for the expression of normal circadian rhythms. To test the hypothesis that VIP is also an SCN output signal, we recorded wheel-running activity rhythms in hamsters and continuously infused the VIP receptor agonist BAY 55-9837 in the third ventricle for 28 days. Unlike other candidate output signals, infusion of BAY 55-9837 did not affect activity levels. Instead, BAY 55-9837 lengthened the circadian period by 0.69 +/- 0.04 h (P < 0.0002 compared with controls). Period returned to baseline after infusions. We analyzed the effect of BAY 55-9837 on cultured SCN from PER2::LUC mice to determine if lengthening of the period by BAY 55-9837 is a direct effect on the SCN. Application of 10 muM BAY 55-9837 to SCN in culture lengthened the period of PER2 luciferase expression (24.73 +/- 0.24 h) compared with control SCN (23.57 +/- 0.26, P = 0.01). In addition, rhythm amplitude was significantly increased, consistent with increased synchronization of SCN neurons. The effect of BAY 55-9837 in vivo on period is similar to the effect of constant light. The present results suggest that VIP-VPAC2 signaling in the SCN may play two roles, synchronizing SCN neurons and setting the period of the SCN as a whole.