R. Katherine Hyde

Accelerated leukemogenesis by truncated CBFβ-SMMHC defective in high-affinity binding with RUNX1

Dominant RUNX1 inhibition has been proposed as a common pathway for CBF leukemia. CBF beta-SMMHC, a fusion protein in human acute myeloid leukemia (AML), dominantly inhibits RUNX1 largely through its RUNX1 high-affinity binding domain (HABD). However, the type I CBF beta-SMMHC fusion in AML patients lacks HABD. Here, we report that the type I CBF beta-SMMHC protein binds RUNX1 inefficiently. Knockin mice expressing CBF beta-SMMHC with a HABD deletion developed leukemia quickly, even though hematopoietic defects associated with Runx1-inhibition were partially rescued. A larger pool of leukemia-initiating cells, increased MN1 expression, and retention of RUNX1 phosphorylation are potential mechanisms for accelerated leukemia development in these mice. Our data suggest that RUNX1 dominant inhibition may not be a critical step for leukemogenesis by CBF beta-SMMHC.

Identification of benzodiazepine Ro5-3335 as an inhibitor of CBF leukemia through quantitative high throughput screen against RUNX1–CBFβ interaction

Core binding factor (CBF) leukemias, those with translocations or inversions that affect transcription factor genes RUNX1 or CBFB, account for ∼24% of adult acute myeloid leukemia (AML) and 25% of pediatric acute lymphocytic leukemia (ALL). Current treatments for CBF leukemias are associated with significant morbidity and mortality, with a 5-y survival rate of ∼50%. We hypothesize that the interaction between RUNX1 and CBFβ is critical for CBF leukemia and can be targeted for drug development. We developed high-throughput AlphaScreen and time-resolved fluorescence resonance energy transfer (TR-FRET) methods to quantify the RUNX1–CBFβ interaction and screen a library collection of 243,398 compounds. Ro5-3335, a benzodiazepine identified from the screen, was able to interact with RUNX1 and CBFβ directly, repress RUNX1/CBFB-dependent transactivation in reporter assays, and repress runx1-dependent hematopoiesis in zebrafish embryos. Ro5-3335 preferentially killed human CBF leukemia cell lines, rescued preleukemic phenotype in a RUNX1–ETO transgenic zebrafish, and reduced leukemia burden in a mouse CBFB–MYH11 leukemia model. Our data thus confirmed that RUNX1–CBFβ interaction can be targeted for leukemia treatment and we have identified a promising lead compound for this purpose.

GATA2 mutations lead to MDS and AML

Several new studies report mutations in the gene GATA2 in three different familial syndromes characterized by predisposition to myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Before the onset of MDS and AML, patients with similar GATA2 mutations had distinct hematological abnormalities.

Cbfb/Runx1-repression independent blockage of differentiation and accumulation of Csf2rb expressing cells by Cbfb-MYH11

It is known that CBFB-MYH11, the fusion gene generated by inversion of chromosome 16 in human acute myeloid leukemia, is causative for oncogenic transformation. However, the mechanism by which CBFB-MYH11 initiates leukemogenesis is not clear. Previously published reports showed that CBFB-MYH11 dominantly inhibits RUNX1 and CBFB, and such inhibition has been suggested as the mechanism for leukemogenesis. Here we show that Cbfb-MYH11 caused Cbfb/Runx1 repression-independent defects in both primitive and definitive hematopoiesis. During primitive hematopoiesis, Cbfb-MYH11 delayed differentiation characterized by sustained expression of Gata2, Il1rl1, and Csf2rb, a phenotype not found in Cbfb and Runx1 knockout mice. Expression of Cbfb-MYH11 in the bone marrow induced the accumulation of abnormal progenitor-like cells expressing Csf2rb in preleukemic mice. The expression of all 3 genes was detected in most human and murine CBFB-MYH11(+) leukemia samples. Interestingly, Cbfb-MYH11(+) preleukemic progenitors and leukemia-initiating cells did not express Csf2rb, although the majority of leukemia cells in our Cbfb-MYH11 knockin mice were Csf2rb(+). Therefore Csf2rb can be used as a negative selection marker to enrich preleukemic progenitor cells and leukemia-initiating cells from Cbfb-MYH11 mice. These results suggest that Cbfb/Runx1 repression-independent activities contribute to leukemogenesis by Cbfb-MYH11.

Core binding factor beta (CBFB) haploinsufficiency due to an interstitial deletion at 16q21q22 resulting in delayed cranial ossification, cleft palate, congenital heart anomalies, and feeding difficulties but favorable outcome

The core binding factor beta gene (CBFB), essential to bone morphogenesis, is located at 16q22.1. Homozygous deficiency of CBFB leads to ossification defects in mice. CBFB forms a heterodimer with RUNX2 (CBFA1) during embryonic bone development. RUNX2 mutations lead to cleidocranial dysplasia in humans. We describe an infant boy with an interstitial deletion of 16q21q22, delayed skull ossification, cleft palate, and heart anomalies who had a difficult course in infancy but eventually improved and is healthy. He was found to have CBFB haploinsufficiency, but did not have mutations in RUNX2. We suggest that 16q21q22 deletion be considered when there are antenatal or postnatal findings of enlarged cranial sutures with or without cleft palate. The finding of CBFB haploinsufficiency in our case and the similarity of cranial ossification defects with a mouse model of CBFB deletion suggest a role for CBFB in cranial bone development in humans.

RUNX1 repression-independent mechanisms of leukemogenesis by fusion genes CBFB-MYH11 and AML1-ETO (RUNX1-RUNX1T1).

The core binding factor (CBF) acute myeloid leukemias (AMLs) are a prognostically distinct subgroup that includes patients with the inv(16) and t(8:21) chromosomal rearrangements. Both of these rearrangements result in the formation of fusion proteins, CBFB–MYH11 and AML1–ETO, respectively, that involve members of the CBF family of transcription factors. It has been proposed that both of these fusion proteins function primarily by dominantly repressing normal CBF transcription. However, recent reports have indicted that additional, CBF‐repression independent activities may be equally important during leukemogenesis. This article will focus on these recent advances. J. Cell. Biochem. 110: 1039–1045, 2010. Published 2010 Wiley‐Liss, Inc.

Germline PAX5 mutations and B cell leukemia

The transcription factor PAX5 is required for normal B cell development and is frequently mutated or deleted in B cell precursor acute lymphoblastic leukemia (B-ALL). A new study demonstrates that germline hypomorphic mutations of PAX5 are associated with susceptibility to B-ALL, implicating PAX5 in a growing list of hematopoietic transcription factors mutated in familial leukemia predisposition syndromes.

The C-terminus of CBFβ-SMMHC is required to induce embryonic hematopoietic defects and leukemogenesis

The C-terminus of CBFβ-SMMHC, the fusion protein produced by a chromosome 16 inversion in acute myeloid leukemia subtype M4Eo, contains domains for self-multimerization and transcriptional repression, both of which have been proposed to be important for leukemogenesis by CBFβ-SMMHC. To test the role of the fusion proteins C-terminus in vivo, we generated knock-in mice expressing a C-terminally truncated CBFβ-SMMHC (CBFβ-SMMHCΔC95). Embryos with a single copy of CBFβ-SMMHCΔC95 were viable and showed no defects in hematopoiesis, whereas embryos homozygous for the CBFβ-SMMHCΔC95 allele had hematopoietic defects and died in mid-gestation, similar to embryos with a single-copy of the full-length CBFβ-SMMHC. Importantly, unlike mice expressing full-length CBFβ-SMMHC, none of the mice expressing CBFβ-SMMHCΔC95 developed leukemia, even after treatment with a mutagen, although some of the older mice developed a nontransplantable myeloproliferative disease. Our data indicate that the CBFβ-SMMHCs C-terminus is essential to induce embryonic hematopoietic defects and leukemogenesis.

The role of microRNAs in acute myeloid leukemia.

MicroRNAs (miRs) are short (18-22 nucleotides) non-coding RNAs that are important in regulating gene expression. MiR expression is deregulated in many types of cancers, including leukemias. In acute myeloid leukemia (AML), the expression of specific miRs has been linked with both prognostically and cytogenetically defined subgroups. Recent studies have shown that deregulation of miR expression is not simply a consequence of AML but a potential contributer to leukemogenesis. This commentary will focus on select findings that describe the different mechanistic roles for miRs in the development of leukemia.

Abstract 3850: The interaction of RUNX1 with CBFβ-SMMHC during leukemogenesis.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Chromosome 16 inversion is associated with acute myeloid leukemia subtype M4Eo and produces a fusion gene CBFB-MYH11 that contains part of the core binding factor (CBF) β gene CBFB, and part of the smooth muscle myosin heavy chain (SMMHC) gene MYH11. This fusion gene encodes a fusion protein CBFβ-SMMHC, which is oncogenic and binds to the runt domain (RD) of RUNX1, another member of the CBF transcription factor family, resulting in repression of RUNX1 transactivation. We have generated mouse models by conventional and conditional knock-in of the Cbfb-MYH11 fusion gene and demonstrated that Cbfb-MYH11 represses Runx1 function in hematopoiesis and predisposes mice to myeloid leukemia (Castilla et. al., Cell 1996; Nat Genet, 1999). RUNX1 binding and repression was previously considered a key step in leukemogenesis by CBFβ-SMMHC. In order to dissect the molecular mechanism of RUNX1 and CBFβ-SMMHC interaction during leukemogenesis, we generated a knock-in mouse model with deleted high affinity binding site of Cbfb-MYH11. We found accelerated leukemia development in these mice (Kamikubo et.al., Cancer cell, 2010) suggesting that Cbfb-MYH11 play an independent role apart from Runx1 binding and repression. To test if Runx1 is involved in the leukemia development and progression, we crossed Cbfb-MYH11 knock-in mice with mice harboring one of the two mutant alleles of Runx1 - Runx1+/- and Runx1+/Lzd. Runx1+/- contains a null allele while Runx1+/Lzd contains a knocked-in fusion between the RD of Runx1 and the LacZ gene, which is partially dominant-negative in reporter assays. We have determined the rate and percentage of leukemia development in these mice. We found that the Cbfb-MYH11 mice that were Runx1+/- had a similar rate of leukemogenesis when compared with Cbfb-MYH11 mice that were Runx1+/+. However, the Cbfb-MYH11 mice that were Runx1+/Lzd had significantly delayed leukemogenesis as compared to Cbfb-MYH11 mice that were Runx1+/+. Moreover, some of the Cbfb-MYH11; Runx1+/Lzd mice did not develop leukemia at the end of the one-year observation. We detected a decrease of BrdU incorporation in the bone marrow cells in mice with the Runx1+/Lzd allele, suggesting that the delayed leukemia development resulted, at least in part, from decreased proliferation. These data demonstrated that Runx1 is likely required for leukemogenesis by CBFβ-SMMHC. Citation Format: Ling Zhao, R Katherine Hyde, Lemlem Alemu, P Paul Liu. The interaction of RUNX1 with CBFβ-SMMHC during leukemogenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3850. doi:10.1158/1538-7445.AM2013-3850