The role of the 5′ HoxA genes in the development of the hindgut, vent, and a novel sphincter in a derived teleost (bluebanded goby, Lythrypnus dalli )

Abstract Several HOX loci associated long noncoding RNAs (lncRNAs) have been shown to regulate transcription of HOX genes through influencing epigenetic landscape. Especially, the posterior HOXA domain associated lncRNA HOTTIP acts as an epigenetic regulator that recruits WDR5/MLL complex to coordinate active chromatin modifications and HOXA genes expression in the development of animal digits. Despite HOX genes, especially HOXA genes, are highly expressed in many acute myeloid leukemia (AML) patients, it remains largely unknown whether and how HOTTIP lncRNA regulates hematopoietic stem cell (HSC) function and contributes to leukemogenesis. We showed previously that disruption of the CTCF boundary located between HOXA7 and HOXA9 genes (CBS7/9) resulted in reduced lncRNA HOTTIP and HOXA genes expression in MLL rearranged AML suggesting that HOTTIP may play a role in ectopic expression of the posterior HOXA gene. We employed a pooled CRISPR-Cas9 KO library to specifically screen lncRNAs in four HOX gene loci and identify HOTTIP as acritical regulator in controlling oncogenic HOX chromatin signature and associated gene expression patterns in AML by collaborating with posterior HOXA chromatin boundary. HOTTIP is upregulated in AML patients with MLL-rearrangement or NPM1 mutation. AML patients with high HOTTIP expression exhibits a significantly shortened survival compared to low HOTTIP expressing patients. To test whether HOTTIP acts to coordinate posterior chromatin domain and HOXA genes activation in AML, we manipulated HOTTIP lncRNA expression levels in the MLL-AF9 rearranged MOLM13 by loss-of-function KO and gain-of function rescue, as well as carried out genome wide chromatin and transcriptomic analysis to intterrogate the role of HOTTIP in control of AML specific posterior HOXA chromatin domain. We found that knock-out of HOTTIP lncRNA led to a loss of active chromatin structure and invasion of repressive H3K27me3 mark over the posterior HOXA domain. HOTTIP KO attenuated progression of AML in the transplanted AML mouse model resembling the effect of CBS7/9 boundary disruption, while transcriptional activation of HOTTIP lncRNA in the CBS7/9 boundary-disrupted AML cells restored HOXA locus chromatin signature and gene expression as well as reversed the CBS7/9-mediated anti-leukemic effects. To further determine the role of HOTTIP lncRNA in regulating HSC function and leukemogenesis, we generated transgenic mice that expresses Hottip lncRNA under the control of the hematopoietic specific Vav1 enhancer and promoter. The Hottip transgenic (Tg) mice exhibited increased WBC and neutrophil counts and developed splenomegaly indicating that enforced expression of Hottip lncRNA resulted in perturbation of hematopoiesis. Furthermore, overexpression of Hottip lncRNA in mice bone marrow hematopoietic compartment strongly perturbed hematopoietic stem and progenitor cell (HSC/HPC) function by altering self-renewal and differentiation property of HSC/HPCs through affecting homeotic gene associated oncogenic transcription program. Approximately 20% of Hottip lncRNA transgenic mice developed abnormal hematopoietic phenotypes resembling AML-like disease. RNA-seq and ATAC-seq analysis indicated that overexpression of Hottip enhanced promoter chromatin accessibility and stimulates transcription of genes and pathways involved in HSC function and leukemogenesis, including WNT signaling, hematopoietic cell lineage, cell cycle, Hoxa9, Hoxa13, and Meis1, Runx1, and Twist1 genes. Thus, Hottip lncRNA overexpression acts as an oncogenic event to promote HSC self-renewal and HPC proliferation by reprograming leukemic associated chromatin signature and transcription programs. Disclosures No relevant conflicts of interest to declare.

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SOX17 Is Essential for Integration of Arterial and HOXA Programs in Hemogenic Endothelium

Blood ◽

10.1182/blood-2019-130789 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2476-2476

Author(s):

Ho Sun Jung ◽

Gene I. Uenishi ◽

Mi Ae Park ◽

Peng Liu ◽

Matthew Raymond ◽

...

Keyword(s):

Critical Role ◽

Lymphoid Cells ◽

Hemogenic Endothelium ◽

Contributing Factors ◽

Functional Deficits ◽

Stage Of Development ◽

Hoxa Genes ◽

Transcriptional Start Sites ◽

Upregulated Genes

Recent advances in understanding the major bottlenecks in derivation of engraftable HSCs and lymphoid cells from pluripotent stem cells (PSC), have identified deficiencies in NOTCH and HOXA signaling as contributing factors to the observed functional deficits of PSC-derived hematopoietic progenitors. However, little is known about the mechanisms that are essential for establishing these pathways during PSC differentiation. Here, we revealed the critical role of SOX17 in linking HOXA and NOTCH-mediated arterial programs in hemogenic endothelium (HE) and specification of definitive lympho-myeloid hematopoiesis. Using SOX17-knockout (SOX17-/-) and SOX17 DOX-inducible (iSOX17) hESCs, we found that SOX17-deficiency substantially reduces formation of CD144+CD43-CD73-DLL4+CXCR4+/- arterial HE and definitive lympho-myeloid hematopoiesis, while SOX17 upregulation at mesodermal stage of development causes the opposite effect. Molecular profiling of HE generated from iSOX17 hESCs in DOX+ and DOX- conditions using RNAseq, SOX17 ChIPseq and ATACseq, revealed that SOX17 overexpression upregulates 522 genes enriched in NOTCH, TGFb, HEDGEHOG and WNT signaling, including DLL1, DLL4, NOTCH4, LFNG, WNT5a, WNT5b, GLI3, and genes associated with HSC development, CXCR4,KITLG and ALDH1A2. In addition, we noted significant upregulation of HOXA7,HOXA9, HOXA10, HOXB8, HOXC4 and CDX2 homeobox genes in SOX17-induced cultures, with no expression of HOXA genes observed in HE from SOX17-/- cells. ChIPSeq analysis revealed DOX+ specific SOX17 binding at transcriptional start sites (TSS) of 316 significantly upregulated genes, including ALDH1A2, CDX2, DLL1, DLL4, HEY1, HOXA7, HOXB8, HOXC4 and KITLG, suggesting that upregulation of these genes could be explained by their direct activation by SOX17. Since ALDH1A2 and CDX2 are known to play a role in the activation of HOXA genes, we investigated whether SOX17's effect on HOXA expression could also be mediated by ALDH1A2 and CDX2. We found that adding ALDH1 inhibitor to DOX+ cultures had no effect on arterial HE development and HOXA expression. In contrast, transfection of iSOX17 hPSCs cultures with CDX2 shRNA significantly decreased arterial HE formation and downregulated HOXA7, HOXA9, and HOXA10 expression. Overall, our studies indicate that SOX17 plays a critical role in the activation and integration of arterial and HOXA programs in HE, which is mediated by CDX2. These findings will be important for designing a strategy for direct HSC fate programming from hPSCs. Disclosures Uenishi: Casebia Therapeutics: Employment. Slukvin:Cynata Therapeutics: Consultancy, Other: Founder and Stockholder.

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The SIX1 homeobox Gene Is a Novel Therapeutic Target in CALM-AF10 Leukemogenesis

Blood ◽

10.1182/blood-2020-140754 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 38-39

Author(s):

Waitman K. Aumann ◽

Catherine P. Lavau ◽

Amanda Harrington ◽

Donald Tope ◽

Amanda E. Conway ◽

...

Keyword(s):

Therapeutic Target ◽

Nuclear Export ◽

Homeobox Gene ◽

Leukemia Cells ◽

Hematopoietic Progenitors ◽

Self Renewal ◽

Downstream Targets ◽

Hoxa Genes ◽

Novel Therapeutic

Background : The CALM-AF10 translocation is found 5-10% of T-cell acute lymphoblastic leukemias (T-ALL), and a subset of acute myeloid leukemias (AML). CALM-AF10 leukemias are characterized by elevated expression of proleukemic HOXA genes. Since HOXA genes are difficult to target, we hypothesized that identification of non-HOXA CALM-AF10 effector genes could potentially yield novel therapeutic targets. To discover novel CALM-AF10-regulated genes, we took advantage of our prior observation that the nuclear export factor CRM1/XPO1 tethers CALM-AF10 to HOXA genes by interacting with a nuclear export signal within CALM. Using microarrays, we identified a set of genes that showed decreased expression in response to the CRM1 inhibitor, Leptomycin B (LMB), similar to Hoxa genes, in murine CALM-AF10 leukemia cells. Then using RNA-sequencing, we discovered a set of genes increased in murine hematopoietic stem cells transduced with CALM-AF10. There were 11 genes that were both decreased in response to LMB and increased in response to CALM-AF10, which included the Hoxa gene cluster, as well as Six1. Similar to HOXA genes, SIX1 is a homeobox gene that is associated with embryogenesis and is quiescent post-embryologically. Additionally, SIX1 and its cofactor EYA2 have been found to be overexpressed in numerous solid tumors, and inhibitor of the SIX1/EYA2 complex has recently been described. While there is evidence of a role for SIX1 in solid tumors, its role in leukemias has not been explored. Objective: To evaluate the role of SIX1 in CALM-AF10 leukemias. Design/Methods: RT-qPCR and Chromatin Immunoprecipitation (ChIP) were performed using bone marrow progenitors transduced with CALM-AF10 or an empty vector, with and without LMB. Methylcellulose colony assays assessed the ability of SIX1 to enhance self-renewal of hematopoietic progenitors. An inhibitor of the Six1/Eya2 interaction (compound 8430) was used to evaluate cell proliferation. Downstream targets of Six1 were evaluated using RT-qPCR in CALM-AF10 cells treated with Six1/Eya2 inhibitor (8430). Results: RT-qPCR confirmed overexpression of SIX1 in CALM-AF10 leukemia cells, and showed decreased SIX1 expression in the presence of LMB. Furthermore, ChIP revealed that CALM-AF10 binds to the SIX1 gene locus. Overexpression of SIX1 in fetal liver progenitors was sufficient to increase self-renewal potential. The 8430 Six1/Eya2 inhibitor slowed cell growth in CALM-AF10 cells compared to cells treated with DMSO alone. Finally, downstream targets such as Slc2a1, Cdk2, and Cyclina2 were decreased in 8430-treated CALM-AF10 leukemia cells. Conclusions: The SIX1 homeobox gene is highly expressed during embryogenesis, and its expression is silenced post-embryogenesis. Through an initial unbiased screen, we discovered that Six1 may play a role in CALM-AF10 leukemogenesis. We have determined that Six1 expression is upregulated in the presence of CALM-AF10. Further, we have shown a potential oncogenic role for Six1, as it was able to increase the self-renewal potential of hematopoietic progenitors. The role of Six1 in CALM-AF10 leukemia is further supported by the ability of a SIX1/EYA2 inhibitor to slow the growth of CALM-AF10 leukemia cells and decrease the expression of downstream targets of SIX1. These observations suggest that Six1 plays a pathogenic role in leukemogenesis, and may be a novel therapeutic target in CALM-AF10 leukemias. Disclosures No relevant conflicts of interest to declare.

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