Chemical biology. A small-molecule inhibitor of the aberrant transcription factor CBFβ-SMMHC delays leukemia in mice

Abstract

Acute myeloid leukemia (AML) is the most common form of adult leukemia. The transcription factor fusion CBFβ-SMMHC (core binding factor β and the smooth-muscle myosin heavy chain), expressed in AML with the chromosome inversion inv(16)(p13q22), outcompetes wild-type CBFβ for binding to the transcription factor RUNX1, deregulates RUNX1 activity in hematopoiesis, and induces AML. Current inv(16) AML treatment with nonselective cytotoxic chemotherapy results in a good initial response but limited long-term survival. Here, we report the development of a protein-protein interaction inhibitor, AI-10-49, that selectively binds to CBFβ-SMMHC and disrupts its binding to RUNX1. AI-10-49 restores RUNX1 transcriptional activity, displays favorable pharmacokinetics, and delays leukemia progression in mice. Treatment of primary inv(16) AML patient blasts with AI-10-49 triggers selective cell death. These data suggest that direct inhibition of the oncogenic CBFβ-SMMHC fusion protein may be an effective therapeutic approach for inv(16) AML, and they provide support for transcription factor targeted therapy in other cancers.

Fig. 1. Development of potent selective inhibitor of CBFβ-SMMHC-RUNX binding

(A) ¹⁵N-¹H HSQC spectrum (peaks correspond to all NH moieties of protein) of CBFβ alone (blue) and CBFβ + AI-4-57 (red). (B) Schematic diagram for the application of polyvalency to develop a specific and potent inhibitor of CBFβ-SMMHC-RUNX binding. Equations refer to predicted KD values for a bivalent inhibitor binding to CBFβ and CBFβ-SMMHC. Ceff is the effective local concentration, which depends on the distance between CBFβ domains in the oligomeric CBFβ-SMMHC. (C) FRET assay measurements for bivalent inhibitors with varying linker lengths with 10 nM Cerulean-Runt domain and 10 nM Venus-CBFβ-SMMHC.The y axis is the ratio of emission intensities at 525 and 474 nm. Three independent measurements were performed, and their average and standard deviation were used for IC50 data fitting. (D) FRETassay measurements of inhibition of CBFβ-SMMHC- RUNX binding for AI-4-57 and AI-4-83 with 10 nM Cerulean-Runt domain and 10 nM Venus-CBFβ-SMMHC. Data for AI-4-83 are the same as presented in (C). Data for these two compounds are presented separately for clarity of comparison to one another. Left y axis is the ratio of emission intensities at 525 and 474 nm. Right y axis indicates the FRET ratios observed with addition of 100 nM and 1000 nM untagged CBFβ, corresponding to roughly 1-fold and 10-fold dissociation of CBFβ-SMMHC and Runt domain [CBFβ-SMMHC binds with 7-fold the affinity of CBFβ ()]. Three independent measurements were performed, and their average and standard deviation were used for IC50 data fitting. (E) Dose-dependent effect of a 24-hour treatment of ME-1 cells with bivalent inhibitors with varying linker lengths measured by MTT assay and normalized to the DMSO-treated group. Each symbol represents the mean of triplicate experiments; error bars represent the SD. (F and G) Dose-dependent effect of AI-10-47 (red) and AI-10-49 (black) treatment for 48 hours; (F) ME-1 cells were assessed by annexin V and 7-amino-actinomycin (7AAD) viability staining, and (G) human bone marrow cells were assessed by MTT assay. The data were normalized to the DMSOtreated group. Each data point represents the mean of triplicate experiments; error bars represent the SD.

Fig. 2. Specificity of AI-10-49 activity on CBFβ-SMMHC-RUNX1 binding

(A) Effect of 1 μM AI-10-49 on CBFβ-RUNX1 and CBFβ-SMMHC-RUNX1 binding at 3 and 6 hours in ME-1 cells, measured by coimmunoprecipitation (quantification of three experiments is shown on the right). (B) Schematic of the effect of CBFβ-SMMHC on RUNX1 occupancy and target gene expression and the effect of AI-10-49 on occupancy and expression. (C) Chromatin immunoprecipitation assay showing RUNX1 occupancy on RUNX3, CSF1R, and CEBPA in ME-1 and U937 cells treated with 1 μM AI-10-49 for 6 hours and represented as fold enrichment relative to DMSOtreated cells. Each symbol represents the mean of triplicate experiments; error bars represent the SD. (D) Relative expression (qRT-PCR) of RUNX3, CSF1R, and CEBPA in ME-1 and U937 cells treated with 1 μM AI-10-49 for 6 and 12 hours, and normalized to the DMSO control group. Each symbol represents the mean of triplicate experiments; error bars represent the SD. For all panels, significance was calculated as unpaired t-test, *P < 0.05, or ***P < 0.001.

Fig. 3. Activity of AI-10-49 in inv(16)mousemodel and inv(16) AML patient samples

(A) Kaplan-Meier survival curve of mice (n = 11 per group) transplanted with 2 × 10³ cbfb^+/MYH11;Ras^+/G12D leukemic cells and treated between days 5 and 15 posttransplantation (blue arrows)with DMSO(black line) or 200 mg/kg of body weight per day AI-10-49 (red line); Statistics described in the statistical methods section. (B) Percent viability (annexin V and 7AAD assay) relative to vehicle control (DMSO) for CD34+ purified primary human inv(16) AML samples treated for 48 hours with either AI-10-49 or AI-10-47 at the indicated concentrations. Each symbol represents the average for an individual sample from duplicate treatments. The line represents the mean; error bars represent the SD. (C) Percent viability (annexin V and 7AAD assay) relative to vehicle control (DMSO) for primary human AML samples with normal karyotype treated for 48 hours with either AI-10-49 or AI-10-47 at the indicated concentrations. Each symbol represents the average for an individual sample from duplicate treatments.The line represents the mean of all biological replicates; error bars represent the SD. (D) Percentage of colony-forming units (CFUs) after treatment with AI-10-49 relative to vehicle control (DMSO) for primary human AML cells. Each symbol represents the average for an individual sample from duplicate treatments; error bars represent the SD. (E) Percent CFUs to vehicle control (DMSO) for CD34+ purified primary human inv(16) AML samples treated with either AI-10-49 or AI-10-47 at the indicated concentrations. CFU assays were performed in triplicate. Error bars represent the SD. (F) Percent CFUs to vehicle control (DMSO) for CD34+ purified primary human AML samples with normal karyotype treated with either AI-10-49 or AI-10-47 at the indicated concentrations. CFU assays were performed in triplicate. Error bars represent the SD. (G) Percent CFUs to vehicle control (DMSO) for CD34+ purified primary CD34+ cord blood cells treated with either AI-10-49 or AI-10-47 at the indicated concentrations. Significance calculated as unpaired t test, *P < 0.05 or ***P < 0.001.