IMT+venetoclax combination treatment synergistically inhibits cell viability/growth of AML cell lines in vitro.

(A) Chemical structure of IMT (LDC204857) (B) AML cell lines were treated with single agents IMT or venetoclax at a range of concentrations (0.1–1000 nM and 0.01–100 nM, respectively), and assessed by cell viability tests after three and six days. (C) Synergism analysis of AML cell lines treated with a combination of IMT (0.05, 0.1, 0.2 μM for MV4-11 and MOLM-13, and 0.1, 0.5, and 1 μM for THP-1 and KG-1) and venetoclax (Vex; 1, 2, 4 nM for MV4-11 and MOLM-13, and 1, 5, 10 nM for THP-1 and KG-1) for six days. ‘S’ represents synergy of the combination treatment, evaluated using the Bliss independent model. Proliferation percentages are normalized to DMSO treated. All results are from three independent experiments.

Combination treatment significantly enhances apoptosis induction in AML cell lines.

AML cell lines were treated with IMT (500 nM) and venetoclax (5 nM) for 72–96 h. (A) Apoptosis was determined by Annexin V/PI staining of the cells and analyzed by flow cytometry. (B) Loss of mitochondrial membrane potential was calculated as the percentage of depolarized (apoptotic) cells out of the total cell population. Error bars represent standard error of the mean where * represents P < 0.05, **P < 0.01, and ***p < 0.001.

Combination of IMT and venetoclax dramatically decreases OXPHOS (in a glycolysis-independent manner) in sensitive cell lines.

(A) Representative graph of an OXPHOS measurement using mitostress compounds: oligomycin (OMY), carbonyl cyanide m-chlorophenyl hydrazone (CCCP), and antimycin (AMA). (B) AML cell lines (MV4-11, MOLM-13, and THP-1) were treated with either IMT (500 nM), venetoclax (5 nM), or a combination of both for four days. DMSO was added to control wells. Oxygen consumption rate (OCR) was measured with an Oroboros O2K instrument at physiological glucose levels (5 mM) using mitostress compounds. (C) AML cell lines were cultured in growth medium supplemented with either 5 mM glucose or galactose, and cell viability tests (ATP measurements) were performed after 24 and 48 h. All results are from at least three independent experiments. Error bars show ± SEM. Statistical tests on all comparisons in (C) showed non-significant differences.

Combination of IMT and venetoclax significantly prolongs the survival of mice with leukemia.

(A) Outline of CDX efficacy experiment: NBSGW mice were transplanted with MV4-11 human leukemia cells, then treated with either vehicle, IMT, venetoclax, or a combination of IMT and venetoclax (n=10 per treatment group). Peripheral blood was taken before treatment (week 2) and after treatment at regular intervals until disease progression was observed. (B) Analysis of proportion of human CD45+ cells in mice peripheral blood in all treatment groups at indicated time points, determined by flow cytometry. (C) Kaplan-Meier analysis of survival. Statistical significance was calculated using log-rank (Mantle-Cox) test. ****p < 0.0001, Error bars represent ± SEM. (D) Analysis of proportion of human CD45+ cells in the bone marrow of all treatment groups (four mice per group compared to two surviving mice) at time of euthanization.

Synergistic effect of (IMT and venetoclax) combination treatment on tumor burden and survival of AML-PDX transplanted mice.

(A) Experimental outline: NSGS mice were transplanted with primary AML splenocytes from PDX mice and were then treated with either vehicle, IMT, venetoclax, or a combination of IMT and venetoclax (n=10 per treatment group). After the final dosing, three or four mice were euthanized (end-of-treatment group) and the rest (n=6) were maintained for survival analysis. (B) Percentage of viable human CD45-positive (hCD45+) cells in peripheral blood, BM, and spleen of treated mice determined by flow cytometry. (C) Immunohistochemistry analysis of mouse BM and spleen sections stained with anti-human CD45 (brown) and counterstained with DAB (blue). (D) Quantitative analysis of hCD45+ cells on immunohistochemistry sections of BM and spleen sections of treated mice. (E) Proliferating tumor cells (CD45) are shown by quantification of Ki67 positive cells in immunohistochemistry-stained BM sections at end-of-treatment (results from at least three technical replicates and two biological replicates in each treatment group).

Synergistic effect of (IMT and venetoclax) combination treatment on gene transcription and survival of AML-PDX transplanted mice.

The experimental outline was shown in figure 5A, but n=10 per treatment group. After the final dosing, three or four mice were euthanized (end-of-treatment group) and the rest (n=6) were maintained for survival analysis. (A) Gene expression profiling by RNA-seq of purified AML blasts isolated from PDX models treated with venetoclax, IMT, or a combination thereof. Indicated expression changes are relative to a vehicle treated group. Significantly enriched genes are indicated in red (q < 0.05). (B) Peripheral blood engraftment analysis of survival group. (C) Survival analysis of treated mice shown by Kaplan-Meier analysis (n=6/treatment group). *P < 0.05, **P < 0.01, and ***p < 0.001.

Leukemia progression induced by primary AML cells is inhibited by venetoclax treatment.

As in Fig. 5A, efficacy experiment outline: NSGS mice were transplanted with primary AML splenocytes from PDX mice and were then treated with either vehicle, IMT, venetoclax, or a combination of IMT and venetoclax (n=12 per treatment group). 8–12 h after the last dosing, half of the mice (n=6/treatment group) were euthanized (end-of-treatment group) and the other half were maintained for survival analysis. (A) Percentage of viable human CD45-positive (hCD45+) cells in peripheral blood and bone marrow of treated mice (n=6 for each treatment group), determined by flow cytometry. (B) Immunohistochemistry analysis of hCD45+ cells on bone marrow and spleen tissue sections of treated mice (images are representative from at least two mice per group). Scale bars represent 100 μM. (C) Peripheral blood engraftment analysis of survival group. (D) Mice treated with single or a combination of compounds showed prolonged survival in comparison to the vehicle-treated group. Kaplan-Meier analysis shows the relative survival curve. *P < 0.05, **P < 0.01, and ***p < 0.001.