Recent studies have demonstrated that myelodysplastic syndromes (MDSs) arise from a small population of disease-initiating hematopoietic stem cells (HSCs) that persist and expand through conventional ...therapies and are major contributors to disease progression and relapse. MDS stem and progenitor cells are characterized by key founder and driver mutations and are enriched for cytogenetic alterations. Quantitative alterations in hematopoietic stem and progenitor cell (HSPC) numbers are also seen in a stage-specific manner in human MDS samples as well as in murine models of the disease. Overexpression of several markers such as interleukin-1 (IL-1) receptor accessory protein (IL1RAP), CD99, T-cell immunoglobulin mucin-3, and CD123 have begun to differentiate MDS HSPCs from healthy counterparts. Overactivation of innate immune components such as Toll-like receptors, IL-1 receptor–associated kinase/tumor necrosis factor receptor–associated factor-6, IL8/CXCR2, and IL1RAP signaling pathways has been demonstrated in MDS HSPCs and is being targeted therapeutically in preclinical and early clinical studies. Other dysregulated pathways such as signal transducer and activator of transcription 3, tyrosine kinase with immunoglobulinlike and EGF-like domains 1/angiopoietin-1, p21-activated kinase, microRNA 21, and transforming growth factor β are also being explored as therapeutic targets against MDS HSPCs. Taken together, these studies have demonstrated that MDS stem cells are functionally critical for the initiation, transformation, and relapse of disease and need to be targeted therapeutically for future curative strategies in MDSs.
Emerging clonal complexity has brought into question the way in which we perceive and, in turn, treat disorders of the hematopoietic system. Former models of cell-intrinsic clonal dominance driven by ...acquisition of driver genes in a stereotypic sequence are often insufficient in explaining observations such as clonal hematopoiesis, and new paradigms are in order. Here, we review the evidence both within the hematologic malignancy field and also borrow from perspectives rooted in evolutionary biology to reframe pathogenesis of hematologic disorders as dynamic processes involving complex interplays of genetic and non-genetic subclones and the tissue microenvironment in which they reside.
Haematopoietic stem cells (HSCs) maintain lifelong blood production and increase blood cell numbers in response to chronic and acute injury. However, the mechanism(s) by which inflammatory insults ...are communicated to HSCs and their consequences for HSC activity remain largely unknown. Here, we demonstrate that interleukin-1 (IL-1), which functions as a key pro-inflammatory 'emergency' signal, directly accelerates cell division and myeloid differentiation of HSCs through precocious activation of a PU.1-dependent gene program. Although this effect is essential for rapid myeloid recovery following acute injury to the bone marrow, chronic IL-1 exposure restricts HSC lineage output, severely erodes HSC self-renewal capacity, and primes IL-1-exposed HSCs to fail massive replicative challenges such as transplantation. Importantly, these damaging effects are transient and fully reversible on IL-1 withdrawal. Our results identify a critical regulatory circuit that tailors HSC responses to acute needs, and is likely to underlie deregulated blood homeostasis in chronic inflammation conditions.
Nongenetic heterogeneity, or gene expression stochasticity, is an important source of variability in biological systems. With the advent and improvement of single molecule resolution technologies, it ...has been shown that transcription dynamics and resultant transcript number fluctuations generate significant cell-to-cell variability that has important biological effects and may contribute substantially to both tissue homeostasis and disease. In this respect, the pathophysiology of stem cell-derived malignancies such as acute myeloid leukemia and myelodysplastic syndromes, which has historically been studied at the ensemble level, may require reevaluation. To that end, it is our aim in this review to highlight the results of recent single-molecule, biophysical, and systems studies of gene expression dynamics, with the explicit purpose of demonstrating how the insights from these basic science studies may help inform and progress the field of leukemia biology and, ultimately, research into novel therapies.
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Spliceosome mutations are common in myelodysplastic syndromes (MDS) and acute myeloid leukaemia (AML), but the oncogenic changes due to these mutations have not been identified. Here a global ...analysis of exon usage in AML samples revealed distinct molecular subsets containing alternative spliced isoforms of inflammatory and immune genes. Interleukin-1 receptor-associated kinase 4 (IRAK4) was the dominant alternatively spliced isoform in MDS and AML and is characterized by a longer isoform that retains exon 4, which encodes IRAK4-long (IRAK4-L), a protein that assembles with the myddosome, results in maximal activation of nuclear factor kappa-light-chain-enhancer of B cells (NF-κB) and is essential for leukaemic cell function. Expression of IRAK4-L is mediated by mutant U2 small nuclear RNA auxiliary factor 1 (U2AF1) and is associated with oncogenic signalling in MDS and AML. Inhibition of IRAK4-L abrogates leukaemic growth, particularly in AML cells with higher expression of the IRAK4-L isoform. Collectively, mutations in U2AF1 induce expression of therapeutically targetable 'active' IRAK4 isoforms and provide a genetic link to activation of chronic innate immune signalling in MDS and AML.
Molecular noise is a natural phenomenon that is inherent to all biological systems
. How stochastic processes give rise to the robust outcomes that support tissue homeostasis remains unclear. Here we ...use single-molecule RNA fluorescent in situ hybridization (smFISH) on mouse stem cells derived from haematopoietic tissue to measure the transcription dynamics of three key genes that encode transcription factors: PU.1 (also known as Spi1), Gata1 and Gata2. We find that infrequent, stochastic bursts of transcription result in the co-expression of these antagonistic transcription factors in the majority of haematopoietic stem and progenitor cells. Moreover, by pairing smFISH with time-lapse microscopy and the analysis of pedigrees, we find that although individual stem-cell clones produce descendants that are in transcriptionally related states-akin to a transcriptional priming phenomenon-the underlying transition dynamics between states are best captured by stochastic and reversible models. As such, a stochastic process can produce cellular behaviours that may be incorrectly inferred to have arisen from deterministic dynamics. We propose a model whereby the intrinsic stochasticity of gene expression facilitates, rather than impedes, the concomitant maintenance of transcriptional plasticity and stem cell robustness.
Chromosomal translocations are critically involved in the molecular pathogenesis of B-cell lymphomas, and highly recurrent and specific rearrangements have defined distinct molecular subtypes linked ...to unique clinicopathological features. In contrast, several well-characterized lymphoma entities still lack disease-defining translocation events. To identify novel fusion transcripts resulting from translocations, we investigated two Hodgkin lymphoma cell lines by whole-transcriptome paired-end sequencing (RNA-seq). Here we show a highly expressed gene fusion involving the major histocompatibility complex (MHC) class II transactivator CIITA (MHC2TA) in KM-H2 cells. In a subsequent evaluation of 263 B-cell lymphomas, we also demonstrate that genomic CIITA breaks are highly recurrent in primary mediastinal B-cell lymphoma (38%) and classical Hodgkin lymphoma (cHL) (15%). Furthermore, we find that CIITA is a promiscuous partner of various in-frame gene fusions, and we report that CIITA gene alterations impact survival in primary mediastinal B-cell lymphoma (PMBCL). As functional consequences of CIITA gene fusions, we identify downregulation of surface HLA class II expression and overexpression of ligands of the receptor molecule programmed cell death 1 (CD274/PDL1 and CD273/PDL2). These receptor-ligand interactions have been shown to impact anti-tumour immune responses in several cancers, whereas decreased MHC class II expression has been linked to reduced tumour cell immunogenicity. Thus, our findings suggest that recurrent rearrangements of CIITA may represent a novel genetic mechanism underlying tumour-microenvironment interactions across a spectrum of lymphoid cancers.
Abstract
Tet enzymes (Tet1/2/3) oxidize 5-methylcytosine to promote DNA demethylation and partner with chromatin modifiers to regulate gene expression. Tet1 is highly expressed in embryonic stem ...cells (ESCs), but its enzymatic and non-enzymatic roles in gene regulation are not dissected. We have generated Tet1 catalytically inactive (Tet1m/m) and knockout (Tet1−/−) ESCs and mice to study these functions. Loss of Tet1, but not loss of its catalytic activity, caused aberrant upregulation of bivalent (H3K4me3+; H3K27me3+) developmental genes, leading to defects in differentiation. Wild-type and catalytic-mutant Tet1 occupied similar genomic loci which overlapped with H3K27 tri-methyltransferase PRC2 and the deacetylase complex Sin3a at promoters of bivalent genes and with the helicase Chd4 at active genes. Loss of Tet1, but not loss of its catalytic activity, impaired enrichment of PRC2 and Sin3a at bivalent promoters leading to reduced H3K27 trimethylation and deacetylation, respectively, in absence of any changes in DNA methylation. Tet1−/−, but not Tet1m/m, embryos expressed higher levels of Gata6 and were developmentally delayed. Thus, the critical functions of Tet1 in ESCs and early development are mediated through its non-catalytic roles in regulating H3K27 modifications to silence developmental genes, and are more important than its catalytic functions in DNA demethylation.
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