Fig 2: BKT300 downregulates CDC25C without affecting CDC2, upregulates p21 and arrests PRC1 in a phosphorylated state.(A) 7-AAD cell cycle analysis of U937 cells after incubation with 9 mM RO-3306, for 20 h (green), incubated with RO-3306 for 20 h followed by a 4-h washout period (red), or incubated with RO-3306 followed by a 4-h washout period and subsequent addition of 1000 nM BKT300 (yellow). Control cells were left untreated (purple). (B) Western blots for CDC25C, CDC2, cyclin B1 and p21 in U937 and MV4–11 cells following 24 h treatment with 50, 100, or 1000 nM BKT300. (C) Western blots for CDC25C and CDC2 in U937, NB4, Jurkat and K562 cells following 24 h treatment with 1000 nM BKT300. (D) Western blots for pPRC1 (T481) in U937 cells after 24 h incubation with 100, 500, or 1000 nM BKT300. (E) Western blots for pPRC1 (T481) in U937 cells after 4 h or 24 h incubation with 1000 nM BKT300. (F) U937 cells were treated for 24 h with 31.25, 125, 250, or 500 nM BKT300 and then stained with a fluorescent antibody targeting pPRC1 (T481). Images were captured using the IncuCyte system. (G) Mean fluorescence intensity (MFI) of pPRC1 in U937 cells and normal peripheral blood mononuclear cells (PBMCs) following 24 h treatment with 31.25, 62.5, 125, or 500 nM BKT300, as measured by flow cytometry. (H-I) Representative fluorescence-activated cell sorting histograms showing the expression of pPRC1 in control untreated U937 cells and PBMCs (H), and after a 24 h treatment with 125nM BKT300 (I). (J) Effect of 24 h treatment with 125, 250 and 500 nM BKT300 on the expression of total PRC1 in U937 cells and normal PBMCs, shown as the change from control levels, as measured by flow cytometry. Analyses were performed in triplicates. Data represent mean ± SD of two independent experiments. * p <0.05 Student’s t-test.

Fig 3: BKT300 efficiently and specifically binds PRC1(A) Sensorgram showing the interaction between BKT300 and recombinant PRC1, as measured by surface plasmon resonance (SPR). (B) Binding curve determined using the MicroScale Thermophoresis (MST) assay, showing the interaction between BKT300 and PRC1. (C-D) ELISA results, presented as optical density (OD) values, illustrating the binding of the active BKT300–3-C5–4 analog (C) and the inactive BKT300-3-c5-1b analog (D) to PRC1, with nonspecific binding to BSA used as a control.

Fig 4: Bioinformatic analysis of PRC1 and CDC25C expression, mutation correlations, and prognostic significance in AML(A, B) Distribution of PRC1 (A) and CDC25C (B) gene expression levels across 14 cancer types. Batch-normalized RSEM values are plotted on the Y-axis and displayed as box plots. Cancer types on the X-axis are abbreviated as follows: BCLA - bladder urothelial carcinoma, BRCA - breast invasive carcinoma, COADREAD - colon and rectum adenocarcinoma, GBM - glioblastoma multiforme, KIRC - kidney renal clear cell carcinoma, LAML - acute myeloid leukemia, LGG - low-grade glioma, LIHC - liver hepatocellular carcinoma, LUAD - lung adenocarcinoma, OV - ovarian serous cystadenocarcinoma, SKCM - skin cutaneous melanoma, STAD - stomach adenocarcinoma, THCA - thyroid carcinoma, UCEC - uterine corpus endometrial carcinoma. (C) Scatter plot illustrating the correlation between CDC25C expression levels and PRC1 expression levels of 3172 AML patients. Data were downloaded from GDC TARGET-AML project. Each dot represents one sample (N=3172). The calculated Pearson correlation is 0.704 (p-value < 1E-200). This same expression data was also employed in (D, E). Box plots demonstrating expression level distribution of PRC1 (D) and CDC25C (E) for primary and for recurrent cancer samples derived from bone marrow (BM) or peripheral blood (PB). (F) A summary table for figures D and E indicating the number of patients in each category and p value assessing the similarity between BM and PB groups. p value was computed using the Kruskal-Wallis test. (G-J) Gene expression levels of PRC1 (G, I) and CDC25C (H, J) in patients carrying common AML-associated gene mutations. Gene expression levels are presented as batch-normalized RNA-seq by expectation maximization (RSEM) values (G-H), or as log2-transformed reads per kilobase per million mapped reads (RPKM) values (I-J). (K-P) Kaplan-Meier survival curves of patients with varying expression levels of PRC1 (K-M) or of CDC25C (N-P). Results are shown for three sample sources: peripheral blood from primary cancers (K, N), recurrent cancers (L, O), and BM from primary cancers (M, P).

Fig 5: BKT300 inhibits tumor growth and induces tumor regression in xenograft mouse models of AML(A) Average tumor volume over time in NSG mice bearing a subcutaneous human AML U937 cell tumor and treated with 2.5 mg BKT300, once daily, on days 3–7 (n=7). (B) Tumor volume in NSG mice bearing a subcutaneous human AML U937 cell tumor and treated once daily with 1, 1.5, 2, or 2.5 mg/day BKT300, on days 3–7. Tumor weight was measured 24 h after the last treatment (n=7). (C) Representative images of U937 tumor tissue extracted from untreated animals and from animals treated with BKT300, as described in panel A, showing DAPI/TUNEL staining for apoptotic cell death and immunohistochemical stains for total PRC1, CDC25C and phosphorylated PRC1 (pPRC1). Scale bar: 200 μm. (D) Average tumor volume over time in NSG mice bearing a subcutaneous human AML U937 cell tumor and treated with 2.5 mg BKT300, twice a day on days 10–12 (n=7). (E-F) Complete blood count (E) and blood chemistry (F) of untreated (n=2), vehicle-treated (n=4), or BKT300-treated mice (n=6) (2.5 mg once daily on days 3–7 and days 10–14) bearing subcutaneous U937 tumors. Blood samples were collected 72 hours after the last treatment. (G) Experimental schema for in vivo efficacy assessment of BKT300 in an intravenously established human MV4–11 xenograft model in NSG mice. (H) Average percentage of human MV4–11 AML cells in the blood of untreated control mice and mice treated with BKT300 (n=10) as measured on day 21 by flowcytometry following staining with anti-human CD45 antibody. (I) Relative number of hematopoietic mouse cells in the blood of untreated control mice and mice treated with BKT300. (J) Average percentage of human MV4–11 AML cells in the bone marrow (BM) of untreated control mice and BKT300-treated mice. (K) Relative number of mouse cells in the BM of untreated control mice and BKT300-treated mice. (L) Representative flow cytometry data of BM cells following staining with anti-human CD45 antibody, from untreated control mice and mice treated with BKT300, as described in panel J. Error bars represent mean ± SEM. Data are from two independent experiments. * p < 0.05, Student’s t-test.