Runx1 and Runx3 drive progenitor to T-lineage transcriptome conversion in mouse T cell commitment via dynamic genomic site switching.

Runt domain-related (Runx) transcription factors are essential for early T cell development in mice from uncommitted to committed stages. Single and double Runx knockouts via Cas9 show that target genes responding to Runx activity are not solely controlled by the dominant factor, Runx1. Instead, Runx1 and Runx3 are coexpressed in single cells; bind to highly overlapping genomic sites; and have redundant, collaborative functions regulating genes pivotal for T cell development. Despite stable combined expression levels across pro-T cell development, Runx1 and Runx3 preferentially activate and repress genes that change expression dynamically during lineage commitment, mostly activating T-lineage genes and repressing multipotent progenitor genes. Furthermore, most Runx target genes are sensitive to Runx perturbation only at one stage and often respond to Runx more for expression transitions than for maintenance. Contributing to this highly stage-dependent gene regulation function, Runx1 and Runx3 extensively shift their binding sites during commitment. Functionally distinct Runx occupancy sites associated with stage-specific activation or repression are also distinguished by different patterns of partner factor cobinding. Finally, Runx occupancies change coordinately at numerous clustered sites around positively or negatively regulated targets during commitment. This multisite binding behavior may contribute to a developmental "ratchet" mechanism making commitment irreversible.

Dasatinib response in acute myeloid leukemia is correlated with FLT3/ITD, PTPN11 mutations and a unique gene expression signature.

Novel targeted therapies demonstrate improved survival in specific subgroups (defined by genetic variants) of acute myeloid leukemia (AML) patients, validating the paradigm of molecularly targeted therapy. However, identifying correlations between AML molecular attributes and effective therapies is challenging. Recent advances in high-throughput in vitro drug sensitivity screening applied to primary AML blasts were used to uncover such correlations; however, these methods cannot predict the response of leukemic stem cells (LSCs). Our study aimed to predict in vitro response to targeted therapies, based on molecular markers, with subsequent validation in LSCs. We performed ex vivo sensitivity screening to 46 drugs on 29 primary AML samples at diagnosis or relapse. Using unsupervised hierarchical clustering analysis we identified group with sensitivity to several tyrosine kinase inhibitors (TKIs), including the multi-TKI, dasatinib, and searched for correlations between dasatinib response, exome sequencing and gene expression from our dataset and from the Beat AML dataset. Unsupervised hierarchical clustering analysis of gene expression resulted in clustering of dasatinib responders and non-responders. In vitro response to dasatinib could be predicted based on gene expression (AUC=0.78). Furthermore, mutations in FLT3/ITD and PTPN11 were enriched in the dasatinib sensitive samples as opposed to mutations in TP53 which were enriched in resistant samples. Based on these results, we selected FLT3/ITD AML samples and injected them to NSG-SGM3 mice. Our results demonstrate that in a subgroup of FLT3/ITD AML (4 out of 9) dasatinib significantly inhibits LSC engraftment. In summary we show that dasatinib has an anti-leukemic effect both on bulk blasts and, more importantly, LSCs from a subset of AML patients that can be identified based on mutational and expression profiles. Our data provide a rational basis for clinical trials of dasatinib in a molecularly selected subset of AML patients.

Runx3 prevents spontaneous colitis by directing the differentiation of anti-inflammatory mononuclear phagocytes.

Mice deficient in the transcription factor Runx3 develop a multitude of immune system defects, including early onset colitis. This paper demonstrates that Runx3 is expressed in colonic mononuclear phagocytes (MNP), including resident macrophages (RM) and dendritic cell subsets (cDC2). Runx3 deletion in MNP causes early onset colitis due to their impaired maturation. Mechanistically, the resulting MNP subset imbalance leads to up-regulation of pro-inflammatory genes as occurs in IL10R-deficient RM. In addition, RM and cDC2 display a marked decrease in expression of anti-inflammatory/TGF ß-regulated genes and ß-catenin signaling associated genes, respectively. MNP transcriptome and ChIP-seq data analysis suggest that a significant fraction of genes affected by Runx3 loss are direct Runx3 targets. Collectively, Runx3 imposes intestinal immune tolerance by regulating maturation of colonic anti-inflammatory MNP, befitting the identification of RUNX3 as a genome-wide associated risk gene for various immune-related diseases in humans, including gastrointestinal tract diseases such as Crohn's disease and celiac.

Cell type-specific actions of Bcl11b in early T-lineage and group 2 innate lymphoid cells.

The zinc finger transcription factor, Bcl11b, is expressed in T cells and group 2 innate lymphoid cells (ILC2s) among hematopoietic cells. In early T-lineage cells, Bcl11b directly binds and represses the gene encoding the E protein antagonist, Id2, preventing pro-T cells from adopting innate-like fates. In contrast, ILC2s co-express both Bcl11b and Id2. To address this contradiction, we have directly compared Bcl11b action mechanisms in pro-T cells and ILC2s. We found that Bcl11b binding to regions across the genome shows distinct cell type-specific motif preferences. Bcl11b occupies functionally different sites in lineage-specific patterns and controls totally different sets of target genes in these cell types. In addition, Bcl11b bears cell type-specific post-translational modifications and organizes different cell type-specific protein complexes. However, both cell types use the same distal enhancer region to control timing of Bcl11b activation. Therefore, although pro-T cells and ILC2s both need Bcl11b for optimal development and function, Bcl11b works substantially differently in these two cell types.

RUNX represses Pmp22 to drive neurofibromagenesis.

Patients with neurofibromatosis type 1 (NF1) are predisposed to develop neurofibromas, but the underlying molecular mechanisms of neurofibromagenesis are not fully understood. We showed dual genetic deletion of Runx1 and Runx3 in Schwann cells (SCs) and SC precursors delayed neurofibromagenesis and prolonged mouse survival. We identified peripheral myelin protein 22 (Pmp22/Gas3) related to neurofibroma initiation. Knockdown of Pmp22 with short hairpin RNAs increased Runx1fl/fl;Runx3fl/fl;Nf1fl/fl;DhhCre tumor-derived sphere numbers and enabled significantly more neurofibroma-like microlesions on transplantation. Conversely, overexpression of Pmp22 in mouse neurofibroma SCs decreased cell proliferation. Mechanistically, RUNX1/3 regulated alternative promoter usage and induced levels of protein expression of Pmp22 to control SC growth. Last, pharmacological inhibition of RUNX/core-binding factor ß (CBFB) activity significantly reduced neurofibroma volume in vivo. Thus, we identified a signaling pathway involving RUNX1/3 suppression of Pmp22 in neurofibroma initiation and/or maintenance. Targeting disruption of RUNX/CBFB interaction might provide a novel therapy for patients with neurofibroma.

The Proprioceptive System Masterminds Spinal Alignment: Insight into the Mechanism of Scoliosis.

Maintaining posture requires tight regulation of the position and orientation of numerous spinal components. Yet, surprisingly little is known about this regulatory mechanism, whose failure may result in spinal deformity as in adolescent idiopathic scoliosis. Here, we use genetic mouse models to demonstrate the involvement of proprioception in regulating spine alignment. Null mutants for Runx3 transcription factor, which lack TrkC neurons connecting between proprioceptive mechanoreceptors and spinal cord, developed peripubertal scoliosis not preceded by vertebral dysplasia or muscle asymmetry. Deletion of Runx3 in the peripheral nervous system or specifically in peripheral sensory neurons, or of enhancer elements driving Runx3 expression in proprioceptive neurons, induced a similar phenotype. Egr3 knockout mice, lacking muscle spindles, but not Golgi tendon organs, displayed a less severe phenotype, suggesting that both receptor types may be required for this regulatory mechanism. These findings uncover a central role for the proprioceptive system in maintaining spinal alignment.

The Leo Sachs' legacy: a pioneer's journey through hematopoiesis.

Leo Sachs spent almost his entire scientific career in Israel, at the Weizmann Institute of Science, and became a worldwide renowned scientist for his pioneering studies in normal hematopoiesis, its breakdown in leukemia and the suppression of malignancy by inducing differentiation, thereby bypassing genetic defects that give rise to malignancy. The cell culture system he established in the early 1960s for the clonal development of normal hematopoietic cells, made it possible to discover the proteins that regulate the viability, proliferation and differentiation of different blood cell lineages, the molecular basis of normal hematopoiesis and the changes that drive leukemia. His studies established significant general concepts including: a) the value of a multi-gene cytokine network in regulating the viability, number and development of different cell types; b) the existence of alternative pathways that give flexibility to development in both normal and cancer cells; c) the response of some cancer cells to normal regulators of development; d) suppression of myeloid leukemia by inducing differentiation, bypassing malignancy-driving genetic defects; e) identification of chromosomes that control tumor suppression; f) discovering apoptosis as a major mechanism by which WT-p53 suppresses malignancy and g) the ability of hematopoietic cytokines to suppress apoptosis in both normal and leukemic cells. It is gratifying that Leo had the good fortune to witness his pioneering discoveries and ideas move from the basic science stage to effective clinical applications, augmenting normal hematopoiesis in patients with various hematopoietic deficiencies, in patients requiring hematopoietic stem cell transplantation and in the suppression of malignancy by inducing differentiation and apoptosis.

Runx3 in Immunity, Inflammation and Cancer.

In this chapter we summarize the pros and cons of the notion that Runx3 is a major tumor suppressor gene (TSG). Inactivation of TSGs in normal cells provides a viability/growth advantage that contributes cell-autonomously to cancer. More than a decade ago it was suggested that RUNX3 is involved in gastric cancer development, a postulate extended later to other epithelial cancers portraying RUNX3 as a major TSG. However, evidence that Runx3 is not expressed in normal gastric and other epithelia has challenged the RUNX3-TSG paradigm. In contrast, RUNX3 is overexpressed in a significant fraction of tumor cells in various human epithelial cancers and its overexpression in pancreatic cancer cells promotes their migration, anchorage-independent growth and metastatic potential. Moreover, recent high-throughput quantitative genome-wide studies on thousands of human samples of various tumors and new investigations of the role of Runx3 in mouse cancer models have unequivocally demonstrated that RUNX3 is not a bona fide cell-autonomous TSG. Importantly, accumulating data demonstrated that RUNX3 functions in control of immunity and inflammation, thereby indirectly influencing epithelial tumor development.

An ensemble of regulatory elements controls Runx3 spatiotemporal expression in subsets of dorsal root ganglia proprioceptive neurons.

The Runx3 transcription factor is essential for development and diversification of the dorsal root ganglia (DRGs) TrkC sensory neurons. In Runx3-deficient mice, developing TrkC neurons fail to extend central and peripheral afferents, leading to cell death and disruption of the stretch reflex circuit, resulting in severe limb ataxia. Despite its central role, the mechanisms underlying the spatiotemporal expression specificities of Runx3 in TrkC neurons were largely unknown. Here we first defined the genomic transcription unit encompassing regulatory elements (REs) that mediate the tissue-specific expression of Runx3. Using transgenic mice expressing BAC reporters spanning the Runx3 locus, we discovered three REs-dubbed R1, R2, and R3-that cross-talk with promoter-2 (P2) to drive TrkC neuron-specific Runx3 transcription. Deletion of single or multiple elements either in the BAC transgenics or by CRISPR/Cas9-mediated endogenous ablation established the REs' ability to promote and/or repress Runx3 expression in developing sensory neurons. Our analysis reveals that an intricate combinatorial interplay among the three REs governs Runx3 expression in distinct subtypes of TrkC neurons while concomitantly extinguishing its expression in non-TrkC neurons. These findings provide insights into the mechanism regulating cell type-specific expression and subtype diversification of TrkC neurons in developing DRGs.

Genomic-wide transcriptional profiling in primary myoblasts reveals Runx1-regulated genes in muscle regeneration.

In response to muscle damage the muscle adult stem cells are activated and differentiate into myoblasts that regenerate the damaged tissue. We have recently showed that following myopathic damage the level of the Runx1 transcription factor (TF) is elevated and that during muscle regeneration this TF regulates the balance between myoblast proliferation and differentiation (Umansky et al.). We employed Runx1-dependent gene expression, Chromatin Immunoprecipitation sequencing (ChIP-seq), Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) and histone H3K4me1/H3K27ac modification analyses to identify a subset of Runx1-regulated genes that are co-occupied by the TFs MyoD and c-Jun and are involved in muscle regeneration (Umansky et al.). The data is available at the GEO database under the superseries accession number GSE56131.

Runx3 specifies lineage commitment of innate lymphoid cells.

Subsets of innate lymphoid cells (ILCs) reside in the mucosa and regulate immune responses to external pathogens. While ILCs can be phenotypically classified into ILC1, ILC2 and ILC3 subsets, the transcriptional control of commitment to each ILC lineage is incompletely understood. Here we report that the transcription factor Runx3 was essential for the normal development of ILC1 and ILC3 cells but not of ILC2 cells. Runx3 controlled the survival of ILC1 cells but not of ILC3 cells. Runx3 was required for expression of the transcription factor RORγt and its downstream target, the transcription factor AHR, in ILC3 cells. The absence of Runx3 in ILCs exacerbated infection with Citrobacter rodentium. Therefore, our data establish Runx3 as a key transcription factor in the lineage-specific differentiation of ILC1 and ILC3 cells.

Runx1 Transcription Factor Is Required for Myoblasts Proliferation during Muscle Regeneration.

Following myonecrosis, muscle satellite cells proliferate, differentiate and fuse, creating new myofibers. The Runx1 transcription factor is not expressed in naïve developing muscle or in adult muscle tissue. However, it is highly expressed in muscles exposed to myopathic damage yet, the role of Runx1 in muscle regeneration is completely unknown. Our study of Runx1 function in the muscle's response to myonecrosis reveals that this transcription factor is activated and cooperates with the MyoD and AP-1/c-Jun transcription factors to drive the transcription program of muscle regeneration. Mice lacking dystrophin and muscle Runx1 (mdx-/Runx1f/f), exhibit impaired muscle regeneration leading to age-dependent muscle waste, gradual decrease in motor capabilities and a shortened lifespan. Runx1-deficient primary myoblasts are arrested at cell cycle G1 and consequently differentiate. Such premature differentiation disrupts the myoblasts' normal proliferation/differentiation balance, reduces the number and size of regenerating myofibers and impairs muscle regeneration. Our combined Runx1-dependent gene expression, ChIP-seq, ATAC-seq and histone H3K4me1/H3K27ac modification analyses revealed a subset of Runx1-regulated genes that are co-occupied by MyoD and c-Jun in mdx-/Runx1f/f muscle. The data provide unique insights into the transcriptional program driving muscle regeneration and implicate Runx1 as an important participant in the pathology of muscle wasting diseases.

Runx3 at the interface of immunity, inflammation and cancer.

Inactivation of tumor suppressor genes (TSG) in normal cells provides a viability/growth advantage that contributes cell-autonomously to cancer. More than a decade ago claims arose that the RUNX3 member of the RUNX transcription factor family is a major TSG inactivated in gastric cancer, a postulate extended later to other cancers. However, evidence that Runx3 is not expressed in normal gastric and other epithelia has challenged the RUNX3-TSG paradigm. Here we critically re-appraise this paradigm in light of recent high-throughput, quantitative genome-wide studies on thousands of human samples of various tumors and new investigations of the role of Runx3 in mouse cancer models. Collectively, these studies unequivocally demonstrate that RUNX3 is not a bona fide cell-autonomous TSG. Accordingly, RUNX3 is not recognized as a TSG and is not included among the 2000 cancer genes listed in the "Cancer Gene Census" or "Network for Cancer Genes" repositories. In contrast, RUNX3 does play important functions in immunity and inflammation and may thereby indirectly influence epithelial tumor development.

Loss of osteoblast Runx3 produces severe congenital osteopenia.

Congenital osteopenia is a bone demineralization condition that is associated with elevated fracture risk in human infants. Here we show that Runx3, like Runx2, is expressed in precommitted embryonic osteoblasts and that Runx3-deficient mice develop severe congenital osteopenia. Runx3-deficient osteoblast-specific (Runx3(fl/fl)/Col1α1-cre), but not chondrocyte-specific (Runx3(fl/fl)/Col1α2-cre), mice are osteopenic. This demonstrates that an osteoblastic cell-autonomous function of Runx3 is required for proper osteogenesis. Bone histomorphometry revealed that decreased osteoblast numbers and reduced mineral deposition capacity in Runx3-deficient mice cause this bone formation deficiency. Neonatal bone and cultured primary osteoblast analyses revealed a Runx3-deficiency-associated decrease in the number of active osteoblasts resulting from diminished proliferation and not from enhanced osteoblast apoptosis. These findings are supported by Runx3-null culture transcriptome analyses showing significant decreases in the levels of osteoblastic markers and increases in the levels of Notch signaling components. Thus, while Runx2 is mandatory for the osteoblastic lineage commitment, Runx3 is nonredundantly required for the proliferation of these precommitted cells, to generate adequate numbers of active osteoblasts. Human RUNX3 resides on chromosome 1p36, a region that is associated with osteoporosis. Therefore, RUNX3 might also be involved in human bone mineralization.

Carcinogen-induced skin tumor development requires leukocytic expression of the transcription factor Runx3.

Carcinogen-induced skin tumorigenesis depends heavily on proinflammatory tumor-promoting processes. Here, we show that leukocytic Runx3 expression is central to the two-stage DMBA/TPA-induced skin tumorigenesis. Runx3-null mice were highly resistant to this process and concomitant ablation of Runx3 in dendritic and T cells fully recapitulated this resistance. Mechanistically, this resistance was associated with a shift in the skin cytokine milieu toward a tumor nonpermissive microenvironment. Specifically, leukocytic Runx3 loss substantially increased the antitumorigenic cytokine thymic stromal lymphopoietin (TSLP) and profoundly decreased two protumorigenic cytokines, interleukin-17a and osteopontin. Therefore, inflammation-mediated tumor promotion requires leukocytic Runx3 expression, as its loss creates a unique cytokine composition that polarizes the tumor microenvironment to a potent antitumorigenic state.

Transcription factor Runx3 regulates interleukin-15-dependent natural killer cell activation.

Natural killer cells belong to the family of innate lymphoid cells comprising the frontline defense against infected and transformed cells. Development and activation of natural killer cells is highly dependent on interleukin-15 signaling. However, very little is known about the transcription program driving this process. The transcription factor Runx3 is highly expressed in natural killer cells, but its function in these cells is largely unknown. We show that loss of Runx3 impaired interleukin-15-dependent accumulation of mature natural killer cells in vivo and under culture conditions and pregnant Runx3(-/-) mice completely lack the unique population of interleukin-15-dependent uterine natural killer cells. Combined chromatin immunoprecipitation sequencing and differential gene expression analysis of wild-type versus Runx3-deficient in vivo activated splenic natural killer cells revealed that Runx3 cooperates with ETS and T-box transcription factors to drive the interleukin-15-mediated transcription program during activation of these cells. Runx3 functions as a nuclear regulator during interleukin-15-dependent activation of natural killer cells by regulating the expression of genes involved in proliferation, maturation, and migration. Similar studies with additional transcription factors will allow the construction of a more detailed transcriptional network that controls natural killer cell development and function.

Runx3-mediated transcriptional program in cytotoxic lymphocytes.

The transcription factor Runx3 is highly expressed in CD8(+) T and NK cytotoxic lymphocytes and is required for their effective activation and proliferation but molecular insights into the transcription program regulated by Runx3 in these cells are still missing. Using Runx3-ChIP-seq and transcriptome analysis of wild type vs. Runx3(-/-) primary cells we have now identified Runx3-regulated genes in the two cell types at both resting and IL-2-activated states. Runx3-bound genomic regions in both cell types were distantly located relative to gene transcription start sites and were enriched for RUNX and ETS motifs. Bound genomic regions significantly overlapped T-bet and p300-bound enhancer regions in Runx3-expressing Th1 helper cells. Compared to resting cells, IL-2-activated CD8(+) T and NK cells contain three times more Runx3-regulated genes that are common to both cell types. Functional annotation of shared CD8(+) T and NK Runx3-regulated genes revealed enrichment for immune-associated terms including lymphocyte activation, proliferation, cytotoxicity, migration and cytokine production, highlighting the role of Runx3 in CD8(+) T and NK activated cells.

Transcriptional reprogramming of CD11b+Esam(hi) dendritic cell identity and function by loss of Runx3.

Classical dendritic cells (cDC) are specialized antigen-presenting cells mediating immunity and tolerance. cDC cell-lineage decisions are largely controlled by transcriptional factor regulatory cascades. Using an in vivo cell-specific targeting of Runx3 at various stages of DC lineage development we show that Runx3 is required for cell-identity, homeostasis and function of splenic Esam(hi) DC. Ablation of Runx3 in DC progenitors led to a substantial decrease in splenic CD4(+)/CD11b(+) DC. Combined chromatin immunoprecipitation sequencing and gene expression analysis of purified DC-subsets revealed that Runx3 is a key gene expression regulator that facilitates specification and homeostasis of CD11b(+)Esam(hi) DC. Mechanistically, loss of Runx3 alters Esam(hi) DC gene expression to a signature characteristic of WT Esam(low) DC. This transcriptional reprogramming caused a cellular change that diminished phagocytosis and hampered Runx3(-/-) Esam(hi) DC capacity to prime CD4(+) T cells, attesting to the significant role of Runx3 in specifying Esam(hi) DC identity and function.

Addiction of t(8;21) and inv(16) acute myeloid leukemia to native RUNX1.

The t(8;21) and inv(16) chromosomal aberrations generate the oncoproteins AML1-ETO (A-E) and CBFß-SMMHC (C-S). The role of these oncoproteins in acute myeloid leukemia (AML) etiology has been well studied. Conversely, the function of native RUNX1 in promoting A-E- and C-S-mediated leukemias has remained elusive. We show that wild-type RUNX1 is required for the survival of t(8;21)-Kasumi-1 and inv(16)-ME-1 leukemic cells. RUNX1 knockdown in Kasumi-1 cells (Kasumi-1(RX1-KD)) attenuates the cell-cycle mitotic checkpoint, leading to apoptosis, whereas knockdown of A-E in Kasumi-1(RX1-KD) rescues these cells. Mechanistically, a delicate RUNX1/A-E balance involving competition for common genomic sites that regulate RUNX1/A-E targets sustains the malignant cell phenotype. The broad medical significance of this leukemic cell addiction to native RUNX1 is underscored by clinical data showing that an active RUNX1 allele is usually preserved in both t(8;21) or inv(16) AML patients, whereas RUNX1 is frequently inactivated in other forms of leukemia. Thus, RUNX1 and its mitotic control targets are potential candidates for new therapeutic approaches.