Fig 2: The combination of MI-238 and venetoclax potently inhibited the development of AML in murine model. A Schematic diagram of the experimental design showing the timeline for the treatment and imaging. B Representative bioluminescent images of the Molm13 tumor burden in mice treated with vehicle, MI-238 (70 mg/kg), venetoclax (50 mg/kg) or their combination. C, D The percentage of human CD45 (hCD45) and human CD33 (hCD33) positive cells were analyzed by flow cytometry to measure the Molm13 tumor burden. The representative flow cytometry plots (C) and quantification of hCD45 and hCD33 double positive (hCD45+/hCD33+) cells (D) were shown. Data are represented as mean ± SD from three independent replicates. ***P < 0.001 and ****P < 0.0001 by two-tailed t-test. E, F Immunochemistry (IHC) analysis of hCD45 expression in bone marrow from experimental mice. Representative staining (E) and quantification (F) were shown. Data represent mean ± SD from three independent replicates, *P < 0.05 and ****P < 0.0001 by two-tailed t-test. G Kaplan–Meier analysis showed MI-238 in combination with venetoclax resulted in a survival benefit in Molm13 AML xenograft mice. **P < 0.01 by log-rank (Mantel–Cox) test (n = 5)

Fig 3: Gating strategies for identifying MDSC subsets and ARG1 expression(A) The identification of viable murine tumor-associated M-MDSCs (CD11b+ CD11c- Ly6Chi) and PMN-MDSCs (CD11b+ Ly6Clow Ly6G+).(B) The identification of viable human tumor-associated M-MDSCs (CD11b+ HLA-DRlow CD33+ CD14+) and PMN-MDSCs (CD11b+ HLA-DRlow CD33+ CD15+). ARG1 expression in each MDSC subset is detected in comparison to isotype IgG staining control.

Fig 4: Large expansion of phenotypically defined sx-MDSCs comprised mainly of M-MDSC and PMN-MDSCs immediately after surgery (POD1). (A) Representative flow plots showing CD33 versus Lineage (CD3/CD56/CD19) for baseline (left) and POD1 (right) PBMCs. (B) Representative flow plots gated down on CD33+Lin− cells showing CD15 versus CD14 expression. (C) Healthy controls (n=15), Baseline, and POD1 patients (n=44) proportion of CD33+Lin− cells (left) and MDSC subsets (right). (D) The MFI of HLA-DR gated on M-MDSCs (left) and the proportion of M-MDSCs that are HLA-DRlo (right) in healthy controls, baseline and POD1 patients with cancer. (E) Representative histograms of common M-MDSC and PMN-MDSC markers, before and after surgery. Statistical analysis was performed using Wilcoxon matched pairs signed-rank test or Kruskal-Wallis with Dunn’s post test. **** p≤0.0001. FMO, fluorescence minus one control; HLA-DR, human leukocyte antigen - DR isotype; E-MDSC, early stage-MDSC; M-MDSC, monocytic-MDSC; MDSC, myeloid derived suppressor cell; sx-MDSC, surgery-induced MDSC, MFI; mean fluorescence instensity; PBMC, peripheral blood mononuclear cell; PMN-MDSC, polymorphonuclear-MDSC; POD1, postoperative day 1.

Fig 5: Sx-MDSCs suppress NK92-MI cytotoxicity. (A) Schematic of MDSC-NK92 co-culture suppression assay with CP450-labeled K562 target cells. (B) NK92-MIs were co-cultured at increasing ratios with CD33+ cells for 6 or 24 hours (in the absence of K562s) to assess CD33+ cell death by NK92-MI (left). Sx-MDSCs from POD1 patient samples (n=10) were co-cultured with NK92-MI cells and NK cytotoxicity was measured as % dead K562 (right). (C) % suppression was calculated as the reduction of NK cytotoxicity, normalized to NK cells alone (ratio=0). CD33+ cells isolated from baseline and patient-matched POD1 blood were co-cultured with NK92-MI and K562 targets to measure their suppressive capacity (n=5). (D) Representative images of sorted cells stained with Giemsa Wright. (E) Bulk Sx-MDSCs, M-MDSCs, PMN-MDSCs, and HDN from POD1 patients (n=3) co-cultured with NK92-MI to measure effect on NK cytotoxicity. The NK92-MI alone control group is indicated by the broken horizontal line. Statistical analysis used Kruskal-Wallis with Dunn’s post-test. ****p≤0.0001. HDN, high-density neutrophils; M-MDSC, monocytic-MDSC; MDSC, myeloid-derived suppressor cell; NK, natural killer; sx-MDSC, surgery-induced MDSC, PMN-MDSC, polymorphonuclear-MDSC; POD1, postoperative day 1.