RUNX1 Activity Research Articles

Identification of Regulatory circRNAs Involved in the Pathogenesis of Acute Myocardial Infarction.

BackgroundAcute myocardial infarction (AMI) has high morbidity and mortality worldwide. However, the pathogenesis of AMI is still unclear, and the impact of circular RNAs (circRNAs) on AMI has rarely been recognized and needs to be explored.Materials and MethodsThe circRNA array was applied to investigate the expression level of circRNAs in the blood samples of coronary arteries of three AMI patients and three normal persons. Principal component analysis (PCA) and unsupervised clustering analysis were performed to reveal the distinguished expression patterns of circRNAs. The miRNA expression profiles of AMI patients were identified from a public dataset from the Gene Expression Omnibus (GEO) database (GSE31568). The miRNA binding sites on the circRNAs were predicted by miRanda. The miRNA enrichment analysis and annotation tool were used to explore the pathways that the dysregulated circRNAs may participate in.ResultsIn total, 142 differentially expressed circRNAs, including 89 upregulated and 53 downregulated in AMI samples, were identified by the differential expression analysis. AMI patients had quite different circRNA expression profiles to those of normal controls. Functional characterization revealed that circRNAs that had the potential to regulate miRNAs were mainly involved in seven pathways, such as the Runt-related transcription factor-1 (RUNX1) expression and activity-related pathway. Specifically, hsa_circRNA_001654, hsa_circRNA_091761, hsa_circRNA_405624, and hsa_circRNA_406698 were predicted to sponge four miRNAs including hsa-miR-491-3p, hsa-miR-646, hsa-miR-603, and hsa-miR-922, thereby regulating RUNX1 expression or activity.ConclusionWe identified dysregulated blood circRNAs in the coronary arteries of AMI patients and predicted that four upregulated circRNAs were involved in the regulation of RUNX1 expression or activity through sponging four miRNAs.

Restoring RUNX1 deficiency in RUNX1 familial platelet disorder by inhibiting its degradation

AbstractRUNX1 familial platelet disorder (RUNX1-FPD) is an autosomal dominant disorder caused by a monoallelic mutation of RUNX1, initially resulting in approximately half-normal RUNX1 activity. Clinical features include thrombocytopenia, platelet functional defects, and a predisposition to leukemia. RUNX1 is rapidly degraded through the ubiquitin-proteasome pathway. Moreover, it may autoregulate its expression. A predicted kinetic property of autoregulatory circuits is that transient perturbations of steady-state levels result in continued maintenance of expression at adjusted levels, even after inhibitors of degradation or inducers of transcription are withdrawn, suggesting that transient inhibition of RUNX1 degradation may have prolonged effects. We hypothesized that pharmacological inhibition of RUNX1 protein degradation could normalize RUNX1 protein levels, restore the number of platelets and their function, and potentially delay or prevent malignant transformation. In this study, we evaluated cell lines, induced pluripotent stem cells derived from patients with RUNX1-FPD, RUNX1-FPD primary bone marrow cells, and acute myeloid leukemia blood cells from patients with RUNX1 mutations. The results showed that, in some circumstances, transient expression of exogenous RUNX1 or inhibition of steps leading to RUNX1 ubiquitylation and proteasomal degradation restored RUNX1 levels, thereby advancing megakaryocytic differentiation in vitro. Thus, drugs retarding RUNX1 proteolytic degradation may represent a therapeutic avenue for treating bleeding complications and preventing leukemia in RUNX1-FPD.

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.

Suppression of malignant rhabdoid tumors through Chb-M'-mediated RUNX1 inhibition.

Malignant rhabdoid tumor (MRT) is a rare and highly aggressive pediatric malignancy primarily affecting infants and young children. Intensive multimodal therapies currently given to MRT patients are not sufficiently potent to control this highly malignant tumor. Therefore, additive or alternative therapy for these patients with a poor prognosis is necessary. We herein demonstrated that the inhibition of runt-related transcription factor 1 (RUNX1) by novel alkylating conjugated pyrrole-imidazole (PI) polyamides, which specifically recognize and bind to RUNX-binding DNA sequences, was highly effective in the treatment of rhabdoid tumor cell lines in vitro as well as in an in vivo mouse model. Therefore, suppression of RUNX1 activity may be a novel strategy for MRT therapy.

Hematopoietic stem cells acquire survival advantage by loss of RUNX1 methylation identified in familial leukemia

AbstractRUNX1 is among the most frequently mutated genes in human leukemia, and the loss or dominant-negative suppression of RUNX1 function is found in myelodysplastic syndrome and acute myeloid leukemia (AML). How posttranslational modifications (PTMs) of RUNX1 affect its in vivo function, however, and whether PTM dysregulation of RUNX1 can cause leukemia are largely unknown. We performed targeted deep sequencing on a family with 3 occurrences of AML and identified a novel RUNX1 mutation, R237K. The mutated R237 residue is a methylation site by protein arginine methyltransferase 1, and loss of methylation reportedly impairs the transcriptional activity of RUNX1 in vitro. To explore the biologic significance of RUNX1 methylation in vivo, we used RUNX1 R233K/R237K double-mutant mice, in which 2 arginine-to-lysine mutations precluded RUNX1 methylation. Genetic ablation of RUNX1 methylation led to loss of quiescence and expansion of hematopoietic stem cells (HSCs), and it changed the genomic and epigenomic signatures of phenotypic HSCs to a poised progenitor state. Furthermore, loss of RUNX1 R233/R237 methylation suppressed endoplasmic reticulum stress–induced unfolded protein response genes, including Atf4, Ddit3, and Gadd34; the radiation-induced p53 downstream genes Bbc3, Pmaip1, and Cdkn1a; and subsequent apoptosis in HSCs. Mechanistically, activating transcription factor 4 was identified as a direct transcriptional target of RUNX1. Collectively, defects in RUNX1 methylation in HSCs confer resistance to apoptosis and survival advantage under stress conditions, a hallmark of a preleukemic clone that may predispose affected individuals to leukemia. Our study will lead to a better understanding of how dysregulation of PTMs can contribute to leukemogenesis.

CHD7 and Runx1 interaction provides a braking mechanism for hematopoietic differentiation

Hematopoietic stem and progenitor cell (HSPC) formation and lineage differentiation involve gene expression programs orchestrated by transcription factors and epigenetic regulators. Genetic disruption of the chromatin remodeler chromodomain-helicase-DNA-binding protein 7 (CHD7) expanded phenotypic HSPCs, erythroid, and myeloid lineages in zebrafish and mouse embryos. CHD7 acts to suppress hematopoietic differentiation. Binding motifs for RUNX and other hematopoietic transcription factors are enriched at sites occupied by CHD7, and decreased RUNX1 occupancy correlated with loss of CHD7 localization. CHD7 physically interacts with RUNX1 and suppresses RUNX1-induced expansion of HSPCs during development through modulation of RUNX1 activity. Consequently, the RUNX1:CHD7 axis provides proper timing and function of HSPCs as they emerge during hematopoietic development or mature in adults, representing a distinct and evolutionarily conserved control mechanism to ensure accurate hematopoietic lineage differentiation.

Abstract 5041: Inhibition of RUNX-CBFβ binding reduces RSPO3 expression and EMT features in breast cancer cells

Abstract We have recently identified R-spondin3 (RSPO3) as a novel key modulator of breast cancer development that may become a potential target for treatment of the basal subtype, which lacks efficient therapeutic options to date. RSPO3 is expressed in the basal stem cell-enriched compartment of mouse mammary glands whereas it is absent in the mature luminal cells. RSPO3 knockdown in mouse mammary tumor cells reduces canonical Wnt signaling pathway, epithelial-to-mesenchymal transition (EMT)-like features, migration capacity, and tumor formation in vivo. To evaluate the relevance of RSPO3 in human breast cancer, we analyzed samples from 75 patients with ductal infiltrating breast carcinomas, where we found a high prevalence of positive immunoreactivity for RSPO3 (70%). Expression data of The Cancer Genome Atlas Network from 1985 human breast carcinoma samples showed that RSPO3 is primarily expressed in Claudin-low breast cancer subtype, which is characterized for having stem cells features. In culture assays revealed that knockdown of RSPO3 expression reduced SOX2 expression and impaired mammary tumor cell ability to form mammospheres. Previous reports suggested the potential involvement of RUNX1-CBFβ on RSPO3 transcription in mammary tumor cells. Therefore, we evaluated if pharmacologic inhibition of RUNX1 activity would reduce RSPO3 expression and activity in the cancer stem cell compartment. Treatment of breast cancer cells with AI-10-104, a small molecule that inhibits CBFβ-RUNX interaction, reduced RSPO3 mRNA and protein levels, and reduced the migration ability of MDA-MB231 breast cancer cells, ability that was recovered upon treatment with recombinant RSPO3. In addition, AI-10-104 inhibited the expression of canonical Wnt pathway targets, EMT and stem-cell markers. Therefore, our results demonstrate that inhibition of RUNX-CBFβ transcriptional activity leads to a down-regulation of RSPO3 expression, constituting a new molecular target for therapeutic modulation in breast cancer treatment. Citation Format: Carla M. Felcher, Johanna M. Tocci, Martin E. Garcia Sola, John H. Bushweller, Lucio H. Castilla, Edith C. Kordon. Inhibition of RUNX-CBFβ binding reduces RSPO3 expression and EMT features in breast cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5041.

LncRNA Hoxaas3 promotes lung fibroblast activation and fibrosis by targeting miR-450b-5p to regulate Runx1

Long noncoding RNAs (lncRNAs) participate in organ fibrosis and various pulmonary diseases, but its role in idiopathic pulmonary fibrosis (IPF) is not fully understood. In this study, we found lncRNA Hoxaas3 (Hoxaas3) was up-regulated in the mice model of BLM-induced PF and TGF-β1-induced fibrogenesis in lung fibroblasts (LF). Overexpression of Hoxaas3 promoted fibrogenesis, whereas Hoxaas3 inhibition attenuated lung fibrosis both in vitro and in vivo, through regulation of miR-450b-5p. Furthermore, miR-450b-5p inhibition stimulated fibrogenesis by regulating runt-related transcription factor 1 (Runx1), whereas up-regulation of miR-450b-5p alleviated fibrogenesis in LF. Mechanistically, our study showed that Hoxaas3 regulated lung fibroblast activation and fibrogenesis by acting as a competing endogenous RNA for miR-450b-5p: Hoxaas3 decreased the expression of miR-450b-5p to stimulate level and activity of Runx1 and induced fibrotic LF, whereas Runx1 inhibition alleviated the pro-fibrotic effect of Hoxaas3. In addition, Hoxaas3 was regulated by TGF-β1/Smad4 pathway as its transcriptional target. In conclusion, our study showed the role and mechanism of the TGF-β1/Smad4- Hoxaas3–miR-450b-5p–Runx1 axis for a better understanding of PF, demonstrated Hoxaas3 maybe a new diagnostic biomarker or potential therapeutic target for IPF.

Runt-related transcription factor 1 is required for murine osteoblast differentiation and bone formation

Despite years of research investigating osteoblast differentiation, the mechanisms by which transcription factors regulate osteoblast maturation, bone formation, and bone homeostasis is still unclear. It has been reported that runt-related transcription factor 1 (Runx1) is expressed in osteoblast progenitors, pre-osteoblasts, and mature osteoblasts; yet, surprisingly, the exact function of RUNX1 in osteoblast maturation and bone formation remains unknown. Here, we generated and characterized a pre-osteoblast and differentiating chondrocyte-specific Runx1 conditional knockout mouse model to study RUNX1's function in bone formation. Runx1 ablation in osteoblast precursors and differentiating chondrocytes via osterix-Cre (Osx-Cre) resulted in an osteoporotic phenotype and decreased bone density in the long bones and skulls of Runx1f/fOsx-Cre mice compared with Runx1f/f and Osx-Cre mice. RUNX1 deficiency reduced the expression of SRY-box transcription factor 9 (SOX9), Indian hedgehog signaling molecule (IHH), Patched (PTC), and cyclin D1 in the growth plate, and also reduced the expression of osteocalcin (OCN), OSX, activating transcription factor 4 (ATF4), and RUNX2 in osteoblasts. ChIP assays and promoter activity mapping revealed that RUNX1 directly associates with the Runx2 gene promoter and up-regulates Runx2 expression. Furthermore, the ChIP data also showed that RUNX1 associates with the Ocn promoter. In conclusion, RUNX1 up-regulates the expression of Runx2 and multiple bone-specific genes, and plays an indispensable role in bone formation and homeostasis in both trabecular and cortical bone. We propose that stimulating Runx1 activity may be useful in therapeutic approaches for managing some bone diseases such as osteoporosis.

Identification of tRNA-derived small RNA (tsRNA) responsive to the tumor suppressor, RUNX1, in breast cancer.

Despite recent advances in targeted therapies, the molecular mechanisms driving breast cancer initiation, progression, and metastasis are minimally understood. Growing evidence indicate that transfer RNA (tRNA)-derived small RNAs (tsRNA) contribute to biological control and aberrations associated with cancer development and progression. The runt-related transcription factor 1 (RUNX1) transcription factor is a tumor suppressor in the mammary epithelium whereas RUNX1 downregulation is functionally associated with breast cancer initiation and progression. We identified four tsRNA (ts-19, ts-29, ts-46, and ts-112) that are selectively responsive to expression of the RUNX1 tumor suppressor. Our finding that ts-112 and RUNX1 anticorrelate in normal-like mammary epithelial and breast cancer lines is consistent with tumor-related activity of ts-112 and tumor suppressor activity of RUNX1. Inhibition of ts-112 in MCF10CA1a aggressive breast cancer cells significantly reduced proliferation. Ectopic expression of a ts-112 mimic in normal-like mammary epithelial MCF10A cells significantly increased proliferation. These findings support an oncogenic potential for ts-112. Moreover, RUNX1 may repress ts-112 to prevent overactive proliferation in breast epithelial cells to augment its established roles in maintaining the mammary epithelium.

Characterisation of transcription factor profiles in polycystic kidney disease (PKD): identification and validation of STAT3 and RUNX1 in the injury/repair response and PKD progression

Autosomal dominant polycystic kidney disease (ADPKD) is the most common genetic renal disease, caused in the majority of the cases by a mutation in either the PKD1 or the PKD2 gene. ADPKD is characterised by a progressive increase in the number and size of cysts, together with fibrosis and distortion of the renal architecture, over the years. This is accompanied by alterations in a complex network of signalling pathways. However, the underlying molecular mechanisms are not well characterised. Previously, we defined the PKD Signature, a set of genes typically dysregulated in PKD across different disease models from a meta-analysis of expression profiles. Given the importance of transcription factors (TFs) in modulating disease, we focused in this paper on characterising TFs from the PKD Signature. Our results revealed that out of the 1515 genes in the PKD Signature, 92 were TFs with altered expression in PKD, and 32 of those were also implicated in tissue injury/repair mechanisms. Validating the dysregulation of these TFs by qPCR in independent PKD and injury models largely confirmed these findings. STAT3 and RUNX1 displayed the strongest activation in cystic kidneys, as demonstrated by chromatin immunoprecipitation (ChIP) followed by qPCR. Using immunohistochemistry, we showed a dramatic increase of expression after renal injury in mice and cystic renal tissue of mice and humans. Our results suggest a role for STAT3 and RUNX1 and their downstream targets in the aetiology of ADPKD and indicate that the meta-analysis approach is a viable strategy for new target discovery in PKD.Key messagesWe identified a list of transcription factors (TFs) commonly dysregulated in ADPKD.Out of the 92 TFs identified in the PKD Signature, 35% are also involved in injury/repair processes.STAT3 and RUNX1 are the most significantly dysregulated TFs after injury and during PKD progression.STAT3 and RUNX1 activity is increased in cystic compared to non-cystic mouse kidneys.Increased expression of STAT3 and RUNX1 is observed in the nuclei of renal epithelial cells, also in human ADPKD samples.

The Ubiquitin Ligase DTX2 Promotes Nuclear Export of RUNX1 and Inhibits RUNX1-Dependent Leukemogenesis

Transcription factor RUNX1 plays important roles in hematopoiesis and leukemogenesis. As a master regulator for hematopoiesis, RUNX1function is tightly controlled through posttranslational modifications, including ubiquitination. We and others have identified multiple E3 ubiquitin ligases that promote RUNX1 ubiquitination, such as STUB1, RNF38 and APC complex. However, mechanisms for ubiquitination-mediated regulation of RUNX1 function, especially the proteasome-independent roles of ubiquitination, have not been fully understood. We previously established a high-throughput AlphaScreen assayto measure the interaction between RUNX1 and E3 ubiquitin ligases.In this assay, biotinylated full-length RUNX1 was captured by streptavidin-coated donor beads and FLAG-tagged E3 ligases were bound by protein A-conjugated acceptor beads. The binding of RUNX1 to each E3 ligase brings the donor and acceptor beads in close proximity, enabling energy transfer between them, resulting in a chemiluminescence signal. This cell-free AlphaScreen assay allows for the efficient detection of RUNX1-interacting E3 ligases with high sensitivity, and have revealed several E3 ligases, including DTX2, that interact with RUNX1. DTX2 is a mammalian homolog of Drosophila deltex and functions as a RING-finger E3 ubiquitin ligase. Interestingly, a RUNX1 fusion gene connected to reversed DTX2 was identified in acute myeloid leukemia with t(7;21). Given that the reversed sequence of DTX2could inhibit activities of wild-type DTX2, the presence of RUNX1-DTX2 (reversed-sequence) chimera in AML indicates the functional association between RUNX1 and DTX2 in hematopoietic cells. To test this possibility, we first examined whether DTX2 physically interacts with RUNX1 to promote its ubiquitination. Immunoprecipitation assays revealed that DTX2 in fact interacted with RUNX1 in 293T cells. We also found that DTX2, but not a DTX2 mutant lacking Ring finger domain (DTX2-DR), induced ubiquitination of RUNX1 in both nucleus and cytoplasm. However, DTX2-induced RUNX1 ubiquitination did not result in degradation of RUNX1 protein, suggesting that DTX2 modifies RUNX1 function mainly through a proteasome-independent mechanism. We therefore examined if DTX2 alters subcellular localization of RUNX1 using immunofluorescence analysis. Strikingly, ectopic expression of DTX2 altered RUNX1 distribution from nuclear towards cytoplasm in 293T cells (Figure 1). Furthermore, we observed similar cytoplasmic localization of endogenous RUNX1 in DTX2-transduced K562 cells. We then assessed whether DTX2 is involved in RUNX1-mediated transcriptional regulation using the MCSFR luciferase reporter, and found that RUNX1-induced reporter activation was inhibited by ectopic expression of DTX2. Taken together, these data suggest that DTX2 promotes nuclear export of RUNX1, thereby inhibits its transcriptional activity. Next, we assessed the effect of DTX2 on RUNX1 function in primary human cord blood cells. In agreement with previous reports, RUNX1 overexpression promoted myeloid differentiation and downregulated a stem cell marker CD34 in cord blood cells. This RUNX1-induced CD34 downregulation was attenuated by DTX2 coexpression, suggesting the inhibitory effect of DTX2 on RUNX1 activity. We then examined the effect of DTX2 expression in several leukemia cell lines: Jurkat, TF-1, Kasumi-1, SKNO-1 and K562 cells. Previous studies have shown that Jurkat, TF-1, Kasumi-1 and SKNO-1 cells are dependent on RUNX1 activity for their growth. In contrast, K562 cells were shown to be relatively resistant to RUNX1 depletion. Consistent with our earlier results, expression of DTX2 promoted cytoplasmic localization of RUNX1 in all these leukemia cell lines. Functionally, DTX2 overexpression inhibited the growth of RUNX1-dependent leukemia cell lines (Jurkat, TF-1, Kasumi-1, and SKNO-1) by inducing apoptosis and cell-cycle arrest, while it showed only modest effect in K562 cells. Thus, these results indicate again that DTX2 inhibit RUNX1 function by promoting its cytoplasmic localization in hematopoietic and leukemia cells. In summary, we identified DTX2 as an E3 ligase to promote RUNX1 ubiquitination. DTX2 does not induce RUNX1 degradation, but instead promotes nuclear export of RUNX1 to inhibit its function. Activation of DTX2 could be a therapeutic approach to treat RUNX1-dependent leukemias. Disclosures No relevant conflicts of interest to declare.

The Inherited Platelet Disorder of RUNX1 Haploinsufficiency (Familial Platelet Disorder with Associated Myeloid Malignancy (FPDMM)) Has a Marked Deficiency of Megakaryocyte-Biased Hematopoietic Progenitor Cells: Mechanistic Studies and Drug Correction

FPDMM is an autosomal dominant disorder associated with qualitative and quantitative platelet defects and an elevated risk of developing acute myeloid leukemia (AML). FPDMM is due to haploinsufficiency of RUNX1, a transcription factor critical for definitive hematopoiesis and megakaryopoiesis. Currently, there are no drugs available for preventing either the bleeding diathesis or AML predisposition in FPDMM. We hypothesize that drugs that correct RUNX1 expression and/or activity in megakaryopoiesis would not only correct the platelet defect, but prevent leukemia progression. In our studies, we established two cell lines with isogenic controls from induced pluripotent stem cells (iPSC) to study the effects of RUNX1 haploinsufficiency in hematopoietic progenitor cells (iHPCs) and megakaryocytes (iMKs). The first iPSC line (FPD) was derived from patient cells harboring a splice mutation in RUNX1 and its isogenic control was created using the CRISPR/Cas9 technology (FPD-cor). The second line was a normal control iPSC line (WT6) in which the FPD splicing mutation was created by CRISPR/Cas9 gene editing (WT6-mut). When differentiated to iMKs, both lines recapitulated a disease phenotype showing decreased iMK yield per iHPC compared to their respective isogenic controls (29% and 42%, respectively). This decreased yield appeared to be due to depletion of a novel CD42+-iHPC subpopulation in both mutant lines compared to their isogenic controls (5% of isogenic controls (Fig. 1A)). Not shown, these HPCs are von Willibrand factor-positive and almost exclusively gives rise to Mks, thus we termed them Mk-biased. To investigate global and local gene expression changes underlying this depletion, single cell RNA sequencing was performed on sorted CD42+-iHPCs derived from the FPD and FPD-cor lines. In both, we defined 7 identical separate subpopulations, several of which were markedly deficient in the FPD CD42+-HPCs (Fig. 1B). Gene set enrichment analysis (GSEA) of genes upregulated in FPD CD42+-iHPCs from the Gene Ontology and Reactome databases indicates that regulation of response to stress and positive regulation of gene expression gene sets are enriched in FPD CD42+-iHPCs (Fig. 1C, shaded in red). Previously, RepSox, a TGFβ receptor 1 inhibitor, was described by others to enhance megakaryopoiesis in humans and mice, and to increase RUNX1 levels. We now show that RepSox can correct the deficient Mk-biased-HPC population (Fig. 1A) as well as return final iMK yield (not shown) to near FPD-cor levels. GSEA of genes downregulated in RepSox-treated FPD CD42+-iHPCs revealed enrichment of stress-response genes(Fig. 1C, shaded in blue). The stress-activated protein kinase c-Jun N-terminal kinase (JNK) is known to be activated by TGFβ1, various cellular stressors, and pro-inflammatory cytokines. Immunoblotting studies of FPD iMKs showed an elevation in baseline JNK2 phosphorylation (p-JNK) levels (Fig. 1D, left), which was not due to alteration in total JNK2 protein compared to isogenic control iMKs. Immunofluorescence microscopy studies supported these findings (Fig. 1D, right). In summary, we describe a novel defect in the inherited platelet disorder FPDMM by studying iPSC-derived hematopoietic cells. We note a marked deficiency of an MK-biased HPC population that may be related to a proinflammatory state in these HPCs potentially involving TGFβ1-related pathways. These findings need to be confirmed in other FPDMM patient iPSCs and show that it also applies to adult hematopoiesis. Whether the described defect is solely related to the observed thrombocytopenia or also is related to the observed AML defect needs also to be addressed. Disclosures No relevant conflicts of interest to declare.

Runx1 and RORγt Cooperate to Upregulate IL-22 Expression in Th Cells through Its Distal Enhancer.

IL-22 is a cytokine that plays a pivotal role in regulating tissue homeostasis at barrier surfaces and is produced by activated CD4+ Th cells. Currently, the molecular mechanisms regulating Il22 gene expression are still unclear. In this study, we have identified a crucial cis-regulatory element located 32 kb upstream of the mouse Il22 promoter, termed conserved noncoding sequence (CNS)-32. We demonstrated that CNS-32 acts as an enhancer in reporter assays and contains binding motifs for Runt-related transcription factor (Runx)1 and retinoic acid-related orphan receptor γt (RORγt). Mutation of these motifs significantly abrogated the reporter activity, suggesting a role for both factors in the control of enhancer-mediated Il22 expression. Runx1 and RORγt occupancy and elevated histone H4 acetylation at CNS-32 were evident, as naive T cells differentiated into IL-22-producing Th22 cells. Overexpression of Runx1 promoted IL-22 production by inducing RORγt and IL-23 receptor, all critical to Th22 cell induction. Although Runx1 alone enhanced IL-22 production in Th22 cells, it was further enhanced in the presence of RORγt. Conversely, short hairpin RNA-mediated knockdown of core-binding factor β, a cofactor essential for Runx1 activity, was effective in limiting IL-22 production. Collectively, our results suggest that IL-22 production is controlled by a regulatory circuit in which Runx1 induces RORγt and then partners with RORγt to direct Il22 expression through their targeting of the Il22 enhancer.

Thermodynamic investigation of DNA-binding affinity of wild-type and mutant transcription factor RUNX1.

Transcription factor RUNX1 and its binding partner CBFβ play a critical role in gene regulation for hematopoiesis. Mutations of RUNX1 cause ~10% of acute myeloid leukemia (AML) with a particularly poor prognosis. The current paradigm for the leukemogenesis is that insufficient activity of wild-type (WT) RUNX1 impairs hematopoietic differentiation. The majority of mutant RUNX1 proteins lose the DNA-binding affinity and inhibit WT RUNX1 by depletion of CBFβ. Here, isothermal titration calorimetry (ITC) was used to quantitatively study the interactions of WT and three clinical mutant RUNX1, CBFβ and DNA. Our data show that the binding of RUNX1 to DNA is enthalpy-driven, and the affinity decreases in the order of WT > S114L > R139Q >> K83E, which support previous observations and conclusion. To find potentially beneficial RUNX1 mutations that could increase the overall RUNX1 activity, K83R and H179K mutations of RUNX1 were designed, using structure-based computational modeling, and found to possess significantly higher DNA-binding affinities than does WT RUNX1. K83R and H179K mutant RUNX1 could therefore be protein-based RUNX1 activators.

Exploiting altered enhancer landscapes to decode pathogenic changes in gene expression of diverse hepatic macrophages

Abstract Kupffer cells have specialized roles supporting the environment of the liver during homeostasis and disease. However, the key regulatory elements governing these behaviors are unknown. Using scRNA-seq, we found diversification of Kupffer cells and recruitment of additional macrophage subtypes during nonalcoholic steatohepatitis (NASH). A significant source of macrophage heterogeneity during NASH was traced to Cx3cr1 expressing monocytes. Further, macrophage subsets were localized in distinct niches, suggesting environmental specification as a key determinant of macrophage heterogeneity. We profiled chromatin accessibility of the major NASH associated macrophage populations to identify transcription factors governing their environmental specification. These results predict greater NFκB, RUNX, and AP1 activity in recruited hepatic macrophages compared to Kupffer cells. Surprisingly, we found minimal significant chromatin accessibility changes comparing Kupffer cells from healthy mice to mice with NASH, even though several thousand genes were differentially expressed. Instead, NASH led to altered chromatin activity, as measured by H3K27ac ChIP-seq, at Kupffer cell enhancer regions. A binding element for LXR (liver X receptor) was the top transcription factor motif identified in Kupffer cell enhancers with reduced activity during NASH. Furthermore, LXRα was required to maintain expression of a unique gene signature defining healthy Kupffer cells. Thus, our studies establish for the first time gene regulatory events controlling diverse hepatic macrophages during homeostasis and NASH.

Alternative translation initiation generates the N-terminal truncated form of RUNX1 that retains hematopoietic activity

Transcription factor RUNX1 plays a crucial role in hematopoiesis and its activity is tightly regulated at both the transcriptional and posttranslational levels. However, translational control of RUNX1 expression has not been fully understood. In this study, we demonstrated that RUNX1b mRNA is translated from two alternative initiation sites, Met-1 and Met-25, giving full-length RUNX1b and a shorter protein lacking the first 24 amino acids (RUNX1ΔN24). Presence/absence of strong Kozak consensus sequences around Met-1 determines which initiation site is mainly used in RUNX1b cDNA. Selective disruption of either Met-1 or Met-25 abrogates expression of the corresponding protein while facilitating the production of another protein. The RUNX1b cDNA containing 65bp natural promoter sequences mainly produces full-length RUNX1b in human cord blood cells, but disruption of Met-1 in this cDNA also induced translation from Met-25. Consistent with these data, disruption of endogenous RUNX1b around Met-1 using CRISPR/Cas9 induced selective expression of RUNX1ΔΝ24 in several leukemia cell lines. RUNX1ΔN24 protein is more stable than full-length RUNX1b and retains hematopoietic activity. We also found that FLAG-tagged full-length RUNX1 showed altered activity, indicating the influence of N-terminal FLAG-tag on RUNX1 function. The alternative translation initiation of RUNX1b may participate in fine tuning RUNX1 activity.

The VWRPY Domain Is Essential for RUNX1 Function in Hematopoietic Progenitor Cell Maturation and Megakaryocyte Differentiation

RUNX1 is a transcription factor essential during definitive hematopoiesis. Germline mutations in RUNX1 results in a disorder called Familial Platelet Disorder with Associated Myeloid Malignancy (FPDMM). FPDMM patients have abnormal bleeding due to reduced platelet count and/or function. Importantly, 20-60% of the FPDMM patients develop hematological malignancies, which are mainly myeloid. Reported RUNX1 mutations in FPDMM families are mostly clustered in the N-terminal runt domain and the C-terminal transactivation domain. Recently, three mutations have been reported at or near the end of the C-terminal repression domain, the VWRPY motif. However, the mechanism behind the VWRPY motif involvement in the FPDMM pathogenesis has not been studied. Interestingly, these VWRPY-mutated RUNX1 proteins still have intact runt and transactivation domains, but patients still show FPDMM phenotype. Here, we evaluate the functional defects of a RUNX1 mutation, L472fsX, which was reported in a FPDMM family, using three different experimental models. Our study is aimed to unravel the significance of the VWRPY motif in FPDMM pathogenesis.The RUNX1 L472fsX mutation is caused by a GC insertion upstream of the VWRPY motif. The mutation results in a frameshift and a run on protein for an additional 123 amino acids. The frameshift abolishes the VWRPY motif, which is responsible for the binding between RUNX1 and a co-repressor protein, TLE1. As expected, from both FRET and co-IP assays, the mutated RUNX1 lost binding with TLE1. Interestingly, we observed increased binding between the mutated RUNX1 and its co-factor CBFβ in the FRET assay, as compared to the wildtype RUNX1. Furthermore, in reporter assays we found that TLE1 failed to repress the expression of a RUNX1 target, M-CSFR promoter, when co-transfected with the mutated RUNX1, which is contrary to what has been seen with wildtype RUNX1. Consistent with increased binding between the mutated RUNX1 and CBFβ in the FRET assay, co-transfecting mutant RUNX1 and CBFβ resulted in a significant increase of M-CSFR promoter expression as compared to wildtype RUNX1 with CBFβ. For another RUNX1 target, Hmga2, wildtype RUNX1 and CBFβ decreased Hmga2 expression, which could be restored by adding TLE1. Transfected mutant RUNX1 and CBFβ also decreased Hmga2 expression, but TLE1 could not restore Hmga2 expression when co-transfected with the mutant RUNX1. These findings suggest that the VWRPY-disrupting L472fsX mutation leads to the loss of binding between mutant RUNX1 and TLE1, which in turn resulted in defective repression of the RUNX1 activity by TLE1.To assess for hematopoietic defects in the FPDMM patients with the L472fsX mutation, blood cells from two family members were reprogrammed to induced pluripotent stem cells (iPSCs). Similar to previous studies, iPSCs from these patients gave rise to fewer megakaryocyte progenitors and mature megakaryocytes during in vitro differentiation. In addition, these FPDMM iPSCs showed decrease in hematopoietic stem cell (HSCs) maturation and differentiation to progenitors. The L472fsX mutation in the iPSCs was then corrected by genome editing using zinc finger nuclease. Importantly, the hematopoietic defects of the FPDMM iPSCs mentioned above were rescued after mutation correction. Overall, the findings in these iPSCs differentiation assays showed that the VWRPY motif is essential for RUNX1 activity in megakaryocytes differentiation. In addition, the VWRPY motif is important for HSCs maturation and differentiation to progenitors.To evaluate the impact of this VWRPY-deletion mutation on hematopoiesis in an in vivo model, CRISPR-mediated genome editing was used to generate mice with frameshift mutations that remove the VWRPY domain. Preliminary observations showed that the mutant mice have minor defects in the peripheral blood. More data on this mouse model will be presented at the meeting.In conclusion, we present a novel RUNX1 mutation (L472fsX) with unique hematopoietic defect that has not been reported previously in FPDMM. Our findings imply the significance of the VWRPY motif in megakaryopoiesis, as well as HSCs maturation and differentiation. DisclosuresNo relevant conflicts of interest to declare.

Resistance to Lenalidomide in Del(5q) MDS Is Mediated By Inhibition of Drug-Induced Megakaryocytic Differentiation

The immunomodulatory drug lenalidomide (LEN) is the treatment of choice for del(5q) MDS patients. LEN has been shown to trigger the specific degradation of CSNK1A1 and IKZF1 proteins after binding the E3-ligase substrate adaptor CRBN. When brought below a certain expression threshold, CSNK1A1 deficiency activates a p53-dependent apoptotic response. Thus, the unique sensitivity of del(5q) cells to LEN is explained by CSNK1A1 haploinsufficiency in del(5q) MDS patients. Despite its efficacy, 50% of LEN-treated patients eventually relapse within an interval of 2-3 years after treatment. Treatment failure is associated to low platelet counts and occurrence of additional mutations, such as TP53. To identify novel genetic determinants of LEN resistance, we have compared whole genome sequencing data of paired samples from six del(5q) patients who have been treated with LEN and eventually became resistant to the treatment. We identified 2 patients with mutations in TP53. The remaining four presented RUNX1 alterations: two patients had protein coding mutations in RUNX1 and two had a significant reduction in RUNX1, but not TP53, transcript levels.As a model of sensitivity, we studied the response to LEN in two human del(5q) cell lines, MDS-L and KG-1a. RUNX1 protein levels are postranscriptionally upregulated upon exposure to LEN, accompanied by increased levels of RUNX1 activity. Deletion of CRBN expression cancelled these effects. RUNX1 overexpression inhibited clonogenic growth and induced apoptosis. We then generated RUNX1 knock-out (KO) clones derived from MDS-L cells using CRISPR/Cas9 system. RUNX1 KO cells presented increased proliferation, increased colony growth and reduced apoptosis in the presence of LEN compared to wild-type (WT) control clones. These results were validated with different shRNAs against RUNX1. Genetic rescue experiments showed that RUNX1 mutants were unable to restore sensitivity to the drug compared to RUNX1 WT. Finally, modeling RUNX1 loss-of-function (LOF) in CSNK1A-depleted human CD34+ cells abrogated the effects of LEN on colony forming cell assays. Thus, RUNX1 function is required for the elimination of del(5q) cells by LEN.To understand the molecular mechanisms underlying the resistant phenotype, we performed RNA-seq on MDS-L cells treated with LEN for 24h. We observed a significant upregulation of Platelet specific genes (ITGB3, ITGA2B, VWF, THBD, SELP, TREML1, GATA1) coupled to downregulation of Cell Cycle genes (E2F2, E2F1, MCM5, CDKN1A), suggesting that LEN induces differentiation in to the Megakaryocytic (Meg) lineage. We found a significant upregulation of CD41+/CD61+ double positive cells after LEN exposure in vitro and in vivo, associated to the appearance of multinucleated cells. Importantly, the apoptotic response was associated to the emergence of the differentiating population. At the molecular level, CRBN is required for LEN-induced differentiation. Further downstream we identified IKZF1 degradation as key trigger, as IKZF1 overexpression restrained Meg differentiation and a IKZF1 dominant negative isoform enhanced it. In contrast, CSNK1A overexpression did not alter differentiation after LEN, but did reduce apoptotic induction. Moreover, we identified GATA2 targets enriched in LEN-regulated genes and showed that GATA2 overexpression or downregulation using shRNAs significantly increased or reduced LEN induced differentiation respectively.Finally, gene expression analysis after LEN exposure showed that Meg signatures were not enriched in resistant RUNX1 KO cells compared to WT control. Accordingly, RUNX1 KO cells did not undergo differentiation upon LEN exposure. RUNX1 LOF in CSNK1A-depleted primary human CD34+ cells blocked CFU-Mk growth in LEN treated cells. GATA2 overexpression was unable to restore LEN effects in RUNX1 deficient cells, suggesting a cooperative mechanism between both transcription factors. Luciferase assays using the human CD41 promoter showed that RUNX1 mutants reduced promoter transactivation compared to RUNX1 WT. Remarkably, we observed a similar phenotype for LEN-resistant TP53 KO cells.As a conclusion, our results suggest that GATA2, RUNX1 and TP53 cooperate to drive Meg differentiation after LEN-mediated degradation of IKZF1 protein. Loss of function mutations affecting RUNX1 or TP53 alter the activity of GATA2 transcriptional complex, rendering del(5q) cells unresponsive to LEN. DisclosuresPlatzbecker:Celgene: Research Funding.

Abstract 5419: HDAC1 regulates RUNX1 activity in inv(16) acute myeloid leukemia

Abstract RUNX1 and CBFβ form a transcription factor dimer that regulates normal hematopoiesis and leukemogenesis. Inversion of chromosome 16 (inv(16)) is one of the most common mutations in acute myeloid leukemia (AML), fusing CBFβ with the gene encoding smooth muscle myosin heavy chain (MYH11). The fusion protein encoded by CBFB-MYH11 (CM), retains the ability to bind to RUNX1, and together they cause changes in gene expression leading to leukemogenesis. Recently, we found that Histone Deacetylase 1 (HDAC1) is part of the RUNX1:CM complex, that all three proteins co-localize on the promoters of target genes, and that HDAC1 is required for target gene expression. By deacetylating histones, HDAC1 can act as a transcriptional repressor. However, HDAC1 can also deacetylate non-histone proteins, which may explain its unexpected role in gene activation. We hypothesized that HDAC1 acts by deacetylating proteins in the RUNX1:CM complex. To test this, we immunoprecipitated the RUNX1:CM complex and probed for acetyl-lysine. The only visible band was at the expected size for RUNX1, suggesting that RUNX1 is the target of HDAC1 activity. Indeed, cells transfected with RUNX1 and HDAC1 showed significantly less acetylation than cells with RUNX1 alone. To test if HDAC1 deacetylates endogenous RUNX1, we treated leukemia cells from CM-expressing knockin mice with the HDAC1 selective inhibitor entinostat. The level of RUNX1 acetylation was significantly increased in treated cells compared to control, providing further support that HDAC1 deacetylates RUNX1. Acetylation at lysines 24 and 43 of RUNX1 was previously shown to increase its activity. To determine the effect of deacetylation on RUNX1 activity, we used the M-CSFR promoter fused to luciferase. Cells transfected with RUNX1, CBFβ, and HDAC1 showed significantly decreased promoter activity compared to cells with RUNX1 and CBFβ. Importantly, in cells expressing RUNX1 acetylation and deacetylation mimetics, promoter activity was unchanged with the addition of HDAC1, indicating that lysines 24 and 43 are the relevant HDAC1 targets. To test if RUNX1 acetylation status affects the activity of the CM complex, we transfected a CM+ cell line with WT or mutated RUNX1 and examined target gene expression. In CM+ cells over-expressing WT RUNX1, we found similar target gene expression as untransfected CM+ cells. However, expression of either of RUNX1 mutant blocked the CM-induced changes in gene expression. This implies that regulation of RUNX1 acetylation is important for the activity of the RUNX1:CM complex. The observation that both RUNX1 mimetics had the same effect on CM activity may imply that alternate rounds of RUNX1 acetylation and deacetylation is required for continuous RUNX1:CM transcriptional activity. This work provides a possible explanation for HDAC1's role in gene activation in inv(16) AML, and may have implications for other RUNX1 dependent leukemias as well. Citation Format: Lisa Richter, Yiqian Wang, Michelle Becker, Jake Williams, R. Katherine Hyde. HDAC1 regulates RUNX1 activity in inv(16) acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5419.