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

Abstract

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.T ranscription factors RUNX1 and CBFβ form a heterodimer for DNA binding and regulation of gene expression. Genes encoding both proteins play key roles in hematopoiesis (1) and are involved in leukemogenesis through recurrent chromosome abnormalities (2), such as a chromosome 16 inversion [(inv)16] that generates a fusion gene between CBFB and MYH11 (encoding the smooth muscle myosin heavy chain, SMMHC) in acute myeloid leukemia (AML) subtype M4Eo (3, 4), a translocation between chromosomes 8 and 21 that generates a fusion gene between RUNX1 and ETO in AML subtype M2 (5), and a translocation between chromosomes 12 and 21 that generates a fusion gene called TEL-RUNX1 in pediatric precursor B-cell acute lymphocytic leukemia (ALL) (6). All together, the CBF leukemias, which contain translocations involving RUNX1 or CBFB, account for 24% of adult AML cases (7) and 25% of pediatric ALL cases (8). Although core binding factor (CBF) leukemias are generally associated with relatively favorable prognoses, long-term survival for adult patients with CBF AML is only about 50% (9). Although children with CBF leukemias have survival rates of >80% (8, 10), standard therapy takes years to complete. Moreover, the current standard of care for all patients is frequently associated with significant morbidity and mortality. Therefore, targeted treatments for CBF leukemia with high efficacy and low toxicity are clearly desirable.Previous studies suggest that the physical interactions between RUNX1 fusion proteins (RUNX1-ETO and TEL-RUNX1) and CBFβ, and between the CBFβ fusion protein (CBFβ-SMMHC) and RUNX1 are critical for the pathogenesis of CBF leukemias (11-13). We therefore hypothesize that inhibitors of the RUNX1-CBFβ interaction will be therapeutic for all...

CBFβ Enhances De Novo Protein Biosynthesis of Its Binding Partners HIV-1 Vif and RUNX1 and Potentiates the Vif-Induced Degradation of APOBEC3G

“…CBF␤ was previously known as a transcriptional cofactor regulating the DNA binding capacity of RUNX1 (61). In addition, RUNX1 and CBF␤ were found to synergistically inhibit Tat-mediated HIV-1 long terminal repeat (LTR) transactivation (52). However, our data rule out a role of CBF␤ in the transcriptional regulation of Vif expression.…”

Transcription factor RUNX1 promotes survival of acute myeloid leukemia cells

“…Next, we assessed the effect of a recently reported RUNX1 inhibitor, Ro5-3335, on the growth of MLL-AF9 cells (31). We confirmed an inhibitory effect of Ro5-3335 on human RUNX1 and RUNX2 using a CB cell culture assay (Supplemental Figure 6, B and C).…”

“…Recent efforts to target this transcription factor complex in CBF leukemia (31) should be extended to AML associated with MLL rearrangements and potentially to other myeloid neoplasms with RUNX1 dysfunction.…”

Role of RUNX1 in hematological malignancies

“…Moreover, inhibition of RUNX1, either through short hairpin RNA knockdown or a RUNX1 chemical inhibitor, Ro5-3335, 184 suppressed leukemia development by MLL-AF9 in a mouse model. They also found that additional MLL fusion genes require RUNX1 for leukemogenesis.…”