RUNX1 or runt-related transcription factor 1 is a transcription factor in the RUNX family of proteins. This family is composed of RUNX1, RUNX2, and RUNX3--all of which contain the runt homology domain, which mediates interaction with DNA and other transcriptional co-factors. RUNX1 has garnered much attention over the past decades primarily because its disruption has been associated with a variety of human diseases and malignancies. Several chromosomal translocations in leukemias involve RUNX1. The most famous one is between chromosomes 8 and 21 resulting in the RUNX1-ETO fusion and leads to acute myeloid leukemia. Since then, RUNX1 mutations have been found in acute lymphoid leukemia, myelodysplastic syndrome, myeloproliferative neoplasm, familial platelet disease, and others. Although RUNX1 has been a major focus in the field of hematology, many of the molecular and cellular mechanisms of how RUNX1 disruption leads to disease remain unknown. Uncovering these mechanisms will result in a better understanding of these diseases and may lead to meaningful therapies. As a transcription factor, RUNX1 primarily exerts its functions by regulating target genes. This dissertation explores how these target genes ultimately facilitate RUNX1 functions in hematopoiesis and in hematopoietic stem and progenitor cells (HSPCs). We first focus on Hmga2 or high mobility group AT-hook 2, which was found to be up-regulated in RUNX1 loss-of- function HSPCs. Hmga2 is an established oncogene and has roles in inducing cellular proliferation. Notably, Hmga2 was found to contribute to myeloid progenitor cell expansion, which is a main characteristic of RUNX1 loss-of -function. Next, we analyzed differential gene expression data from wildtype and RUNX1 loss-of-function HSPCs and determined that these genes are involved in pathways associated with cell-to-cell interaction and signaling. Upon further examination, we discovered that RUNX1 loss-of -function mice exhibit hypersensitivity to HSPC mobilization regimens. Finally, we focus on another RUNX1 target gene known as Gimap4 or GTPase of the immunity- associated protein 4 and confirm that up-regulation of Gimap4 contributes to HSPC expansion, a finding also found in RUNX1 loss-of-function mice. Together, these studies provide critical insight in the role of RUNX1 in hematopoiesis and blood-related diseases, and offer additional avenues for therapies for these diseases

Protein homeostasis preserves stem cell function, but underlying mechanisms are largely unknown. A study reveals that protein quality control mediated by the endoplasmic reticulum-associated degradation pathway ensures proper expression of MPL, a key cell surface receptor that promotes haematopoietic stem cell function through niche interaction.

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.

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.

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.

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.