Hmga2 is a direct target gene of RUNX1 and regulates expansion of myeloid progenitors in mice.

RUNX1 is a master transcription factor in hematopoiesis and mediates the specification and homeostasis of hematopoietic stem and progenitor cells (HSPCs). Disruptions in RUNX1 are well known to lead to hematologic disease. In this study, we sought to identify and characterize RUNX1 target genes in HSPCs by performing RUNX1 chromatin immunoprecipitation with high-throughput sequencing (ChIP-seq) using a murine HSPC line and complementing this data with our previously described gene expression profiling of primary wild-type and RUNX1-deficient HSPCs (Lineage(-)/cKit(+)/Sca1(+)). From this analysis, we identified and confirmed that Hmga2, a known oncogene, as a direct target of RUNX1. Hmga2 was strongly upregulated in RUNX1-deficient HSPCs, and the promoter of Hmga2 was responsive in a cell-type dependent manner upon coexpression of RUNX1. Conditional Runx1 knockout mice exhibit expansion of their HSPCs and myeloid progenitors as hallmark phenotypes. To further validate and establish that Hmga2 plays a role in inducing HSPC expansion, we generated mouse models of HMGA2 and RUNX1 deficiency. Although mice lacking both factors continued to display higher frequencies of HSPCs, the expansion of myeloid progenitors was effectively rescued. The data presented here establish Hmga2 as a transcriptional target of RUNX1 and a critical regulator of myeloid progenitor expansion.

Runx1 exon 6-related alternative splicing isoforms differentially regulate hematopoiesis in mice.

RUNX1 is an important transcription factor for hematopoiesis. There are multiple alternatively spliced isoforms of RUNX1. The best known isoforms are RUNX1a from use of exon 7A and RUNX1b and c from use of exon 7B. RUNX1a has unique functions due to its lack of C-terminal regions common to RUNX1b and c. Here, we report that the ortholog of human RUNX1a was only found in primates. Furthermore, we characterized 3 Runx1 isoforms generated by exon 6 alternative splicing. Runx1bEx6(-) (Runx1b without exon 6) and a unique mouse Runx1bEx6e showed higher colony-forming activity than the full-length Runx1b (Runx1bEx6(+)). They also facilitated the transactivation of Runx1bEx6(+). To gain insight into in vivo functions, we analyzed a knock-in (KI) mouse model that lacks isoforms Runx1b/cEx6(-) and Runx1bEx6e. KI mice had significantly fewer lineage-Sca1(+)c-Kit(+) cells, short-term hematopoietic stem cells (HSCs) and multipotent progenitors than controls. In vivo competitive repopulation assays demonstrated a sevenfold difference of functional HSCs between wild-type and KI mice. Together, our results show that Runx1 isoforms involving exon 6 support high self-renewal capacity in vitro, and their loss results in reduction of the HSC pool in vivo, which underscore the importance of fine-tuning RNA splicing in hematopoiesis.

Cooperation between RUNX1-ETO9a and novel transcriptional partner KLF6 in upregulation of Alox5 in acute myeloid leukemia.

Fusion protein RUNX1-ETO (AML1-ETO, RUNX1-RUNX1T1) is expressed as the result of the 8q22;21q22 translocation [t(8;21)], which is one of the most common chromosomal abnormalities found in acute myeloid leukemia. RUNX1-ETO is thought to promote leukemia development through the aberrant regulation of RUNX1 (AML1) target genes. Repression of these genes occurs via the recruitment of the corepressors N-COR and SMRT due to their interaction with ETO. Mechanisms of RUNX1-ETO target gene upregulation remain less well understood. Here we show that RUNX1-ETO9a, the leukemogenic alternatively spliced transcript expressed from t(8;21), upregulates target gene Alox5, which is a gene critically required for the promotion of chronic myeloid leukemia development by BCR-ABL. Loss of Alox5 expression reduces activity of RUNX1-ETO9a, MLL-AF9 and PML-RARα in vitro. However, Alox5 is not essential for the induction of leukemia by RUNX1-ETO9a in vivo. Finally, we demonstrate that the upregulation of Alox5 by RUNX1-ETO9a occurs via the C2H2 zinc finger transcription factor KLF6, a protein required for early hematopoiesis and yolk sac development. Furthermore, KLF6 is specifically upregulated by RUNX1-ETO in human leukemia cells. This identifies KLF6 as a novel mediator of t(8;21) target gene regulation, providing a new mechanism for RUNX1-ETO transcriptional control.

Expression of the runt homology domain of RUNX1 disrupts homeostasis of hematopoietic stem cells and induces progression to myelodysplastic syndrome.

Mutations of RUNX1 are detected in patients with myelodysplastic syndrome (MDS). In particular, C-terminal truncation mutations lack a transcription regulatory domain and have increased DNA binding through the runt homology domain. The expression of the runt homology domain, RUNX1(41-214), in mouse hematopoietic cells induced progression to MDS and acute myeloid leukemia. Analysis of premyelodysplastic animals found expansion of c-Kit(+)Sca-1(+)Lin(-) cells and skewed differentiation to myeloid at the expense of the lymphoid lineage. These abnormalities correlate with the phenotype of Runx1-deficient animals, as expected given the reported dominant-negative role of C-terminal mutations over the full-length RUNX1. However, MDS is not observed in Runx1-deficient animals. Gene expression profiling found that RUNX1(41-214) c-Kit(+)Sca-1(+)Lin(-) cells have an overlapping yet distinct gene expression profile from Runx1-deficient animals. Moreover, an unexpected parallel was observed between the hematopoietic phenotype of RUNX1(41-214) and aged animals. Genes deregulated in RUNX1(41-214), but not in Runx1-deficient animals, were inversely correlated with the aging gene signature of HSCs, suggesting that disruption of the expression of genes related to normal aging by RUNX1 mutations contributes to development of MDS. The data presented here provide insights into the mechanisms of development of MDS in HSCs by C-terminal mutations of RUNX1.

Assessment of the Efficacy of Therapies Following Venetoclax Discontinuation in CLL Reveals BTK Inhibition as an Effective Strategy.

PURPOSE: Venetoclax-based therapy is a standard-of-care option in first-line and relapsed/refractory chronic lymphocytic leukemia (CLL). Patient management following venetoclax discontinuation remains nonstandard and poorly understood. EXPERIMENTAL DESIGN: To address this, we conducted a large international study to identify a cohort of 326 patients who discontinued venetoclax and have been subsequently treated. Coprimary endpoints were overall response rate (ORR) and progression-free survival for the post-venetoclax treatments stratified by treatment type [Bruton's tyrosine kinase inhibitor (BTKi), PI3K inhibitor (PI3Ki), and cellular therapies]. RESULTS: We identified patients with CLL who discontinued venetoclax in the first-line (4%) and relapsed/refractory settings (96%). Patients received a median of three therapies prior to venetoclax; 40% were BTKi naïve (n = 130), and 81% were idelalisib naïve (n = 263). ORR to BTKi was 84% (n = 44) in BTKi-naïve patients versus 54% (n = 30) in BTKi-exposed patients. We demonstrate therapy selection following venetoclax requires prior novel agent exposure consideration and discontinuation reasons. CONCLUSIONS: For BTKi-naïve patients, selection of covalently binding BTKis results in high ORR and durable remissions. For BTKi-exposed patients, covalent BTK inhibition is not effective in the setting of BTKi resistance. PI3Kis following venetoclax do not appear to result in durable remissions. We conclude that BTKi in naïve or previously responsive patients and cellular therapies following venetoclax may be the most effective strategies.See related commentary by Rogers, p. 3501.

Bone regeneration is regulated by wnt signaling.

UNLABELLED: Tissue regeneration is increasingly viewed as reactivation of a developmental process that, when misappropriated, can lead to malignant growth. Therefore, understanding the molecular and cellular pathways that govern tissue regeneration provides a glimpse into normal development as well as insights into pathological conditions such as cancer. Herein, we studied the role of Wnt signaling in skeletal tissue regeneration. INTRODUCTION: Some adult tissues have the ability to regenerate, and among these, bone is one of the most remarkable. Bone exhibits a persistent, lifelong capacity to reform after injury, and continual bone regeneration is a prerequisite to maintaining bone mass and density. Even slight perturbations in bone regeneration can have profound consequences, as exemplified by conditions such as osteoporosis and delayed skeletal repair. Here, our goal was to determine the role of Wnts in adult bone regeneration. MATERIALS AND METHODS: Using TOPgal reporter mice, we found that damage to the skeleton instigated Wnt reporter activity, specifically at the site of injury. We used a skeletal injury model to show that Wnt inhibition, achieved through adenoviral expression of Dkk1 in the adult skeleton, prevented the differentiation of osteoprogenitor cells. RESULTS: As a result, injury-induced bone regeneration was reduced by 84% compared with controls. Constitutive activation of the Wnt pathway resulting from a mutation in the Lrp5 Wnt co-receptor results in high bone mass, but our experiments showed that this same point mutation caused a delay in bone regeneration. In these transgenic mice, osteoprogenitor cells in the injury site were maintained in a proliferative state and differentiation into osteoblasts was delayed. CONCLUSIONS: When considered together, these data provide a framework for understanding the roles of Wnt signaling in adult bone regeneration and suggest a feasible approach to treating clinical conditions where enhanced bone formation is desired.

Identification of mutants in inbred Xenopus tropicalis.

Xenopus tropicalis offers the potential for genetic analysis in an amphibian. In order to take advantage of this potential, we have been inbreeding strains of frogs for future mutagenesis. While inbreeding a population of Nigerian frogs, we identified three mutations in the genetic background of this strain. These mutations are all recessive embryonic lethals. We show that multigenerational mutant analysis is feasible and demonstrate that mutations can be identified, propagated, and readily characterized using hybrid, dihybrid, and even trihybrid crosses. In addition, we are optimizing conditions to raise frogs rapidly and present our protocols for X. tropicalis husbandry. We find that males mature faster than females (currently 4 versus 6 months to sexual maturity). Here we document our progress in developing X. tropicalis as a genetic model organism and demonstrate the utility of the frog to study the genetics of early vertebrate development.

RUNX1 and RUNX1-ETO: roles in hematopoiesis and leukemogenesis.

RUNX1 is a transcription factor that regulates critical processes in many aspects of hematopoiesis. RUNX1 is also integral in defining the definitive hematopoietic stem cell. In addition, many hematological diseases like myelodysplastic syndrome and myeloproliferative neoplasms have been associated with mutations in RUNX1. Located on chromosomal 21, the RUNX1 gene is involved in many forms of chromosomal translocations in leukemia. t(8;21) is one of the most common chromosomal translocations found in acute myeloid leukemia (AML), where it results in a fusion protein between RUNX1 and ETO. The RUNX1-ETO fusion protein is found in approximately 12% of all AML patients. In this review, we detail the structural features, functions, and models used to study both RUNX1 and RUNX1-ETO in hematopoiesis over the past two decades.

Accelerated bone repair after plasma laser corticotomies.

OBJECTIVE: To reveal, on a cellular and molecular level, how skeletal regeneration of a corticotomy is enhanced when using laser-plasma mediated ablation compared with conventional mechanical tissue removal. SUMMARY BACKGROUND DATA: Osteotomies are well-known for their most detrimental side effect: thermal damage. This thermal and mechanical trauma to adjacent bone tissue can result in the untoward consequences of cell death and eventually in a delay in healing. METHODS: Murine tibial corticotomies were performed using a conventional saw and a Ti:Sapphire plasma-generated laser that removes tissue with minimal thermal damage. Our analyses began 24 hours after injury and proceeded to postsurgical day 6. We investigated aspects of wound repair ranging from vascularization, inflammation, cell proliferation, differentiation, and bone remodeling. RESULTS: Histology of mouse corticotomy sites uncovered a significant difference in the onset of bone healing; whereas laser corticotomies showed abundant bone matrix deposition at postsurgical day 6, saw corticotomies only exhibited undifferentiated tissue. Our analyses uncovered that cutting bone with a saw caused denaturation of the collagen matrix due to thermal effects. This denatured collagen represented an unfavorable scaffold for subsequent osteoblast attachment, which in turn impeded deposition of a new bony matrix. The matrix degradation induced a prolonged inflammatory reaction at the cut edge to create a surface favorable for osteochondroprogenitor cell attachment. Laser corticotomies were absent of collagen denaturation, therefore osteochondroprogenitor cell attachment was enabled shortly after surgery. CONCLUSION: In summary, these data demonstrate that corticotomies performed with Ti:Sapphire lasers are associated with a reduced initial inflammatory response at the injury site leading to accelerated osteochondroprogenitor cell migration, attachment, differentiation, and eventually matrix deposition.