Associating drug sensitivity with differentiation status identifies effective combinations for acute myeloid leukemia

Abstract

Using ex vivo drug screening of primary patient specimens, we identified the combination of the p38 MAPK inhibitor doramapimod (DORA) with the BCL2 inhibitor venetoclax (VEN) as demonstrating broad, enhanced efficacy compared with each single agent across 335 acute myeloid leukemia (AML) patient samples while sparing primary stromal cells. Single-agent DORA and VEN sensitivity was associated with distinct, nonoverlapping tumor cell differentiation states. In particular, increased monocytes, M4/M5 French-American-British classification, and CD14+ immunophenotype tracked with sensitivity to DORA and resistance to VEN but were mitigated with the combination. Increased expression of MAPK14 and BCL2, the respective primary targets of DORA and VEN, were observed in monocytic and undifferentiated leukemias, respectively. Enrichment for DORA and VEN sensitivities was observed in AML with monocyte-like and progenitor-like transcriptomic signatures, respectively, and these associations diminished with the combination. The mechanism underlying the combination's enhanced efficacy may result from inhibition of p38 MAPK-mediated phosphorylation of BCL2, which in turn enhances sensitivity to VEN. These findings suggest exploiting complementary drug sensitivity profiles with respect to leukemic differentiation state, such as dual targeting of p38 MAPK and BCL2, offers opportunity for broad, enhanced efficacy across the clinically challenging heterogeneous landscape of AML.

Figure 1.

VEN+DORA demonstrate enhanced efficacy and synergy toward AML leukemic cells. (A) Scatter and boxplot of ex vivo sensitivities for matched samples from 335 unique patients with AML displayed as area under the dose-response curve as a percentage of maximum (AUC % of max) for DORA, VEN, and the combination. n.s., not significant. (B) Highest single-agent synergy surface plot for a 7 × 7 dose matrix of VEN and DORA combinations on primary AML cells from a representative sample (21-00779). Red coloring denotes a positive synergy score. (C) Viability response curves for VEN upon increasing concentrations of DORA in MOLM14 AML cells. Cells were cultured with drug for 72 hours and analyzed by MTS-based assay, normalizing to untreated wells. Mean percent viability ± standard error of the mean for 4 replicates is shown. (D) HSA synergy surface plot for a 7 × 7 dose matrix of VEN and DORA combinations on MOLM14 AML cells from panel B. Red coloring denotes a positive synergy score. (E) Comparison of single-agent and combination sensitivity for matched mononuclear cell (MNCs) and stromal cell populations from a representative patient sample (20-00494). (F) Viability response curves for single agents and the combination in HS-5 stromal cells. A control kill cocktail of flavopiridol + staurosporine + velcade (FSV) was also tested. Mean percent viability for 3 replicates is shown. HSA, highest single-agent.

Figure 2.

Cell state gene expression signatures distinguish VEN and DORA sensitivities. (A) Spearman correlation of differential cell counts at the time of specimen collection with AUC for DORA, VEN, and the combination. Red-colored bars indicate no statistical significance. (B) Scatter and boxplots of sensitivities with respect to CD14 surface expression for matched samples from 105 AML unique patient samples displayed as AUC (% of maximum) for DORA, VEN, and the combination. (C) Normalized RNAseq-based gene expression levels for BCL2 and MAPK14 relative to surface expression of the monocytic marker CD14. Boxplots indicate the interquartile range. (D) Bar plot depicting log2 mean fold-change in expression for cell differentiation gene sets comparing sensitive (lowest quintile) and resistant (upper quintile) patient samples according to AUC distributions for DORA, VEN, and VEN+DORA. Significant false discovery rate–corrected P values for gene set enrichment are shown. BM, bone marrow; cDC, conventional dendritic cell; GMP, granulocyte-monocyte progenitor; HSC, hematopoietic stem cell; PB, peripheral blood; RBCs, red blood cells.