Haider Ahsan

The bar graphs represent the quantification of the effect of BMH-21 on nucleolar proteins in PanCa cells by Image J. (A) Immunofluorescence (IF) of RPA194 decreases by several folds in Capan-2, MIA PaCa-2 and HPAF-II cells treated with 2µM BMH-21 as compared to untreated cells. (B) Immunofluorescence (IF) of UBTF decreases in Capan-2, MIA PaCa-2 and HPAF-II cells treated with 2µM BMH-21 as compared to untreated cells. The p values were expressed as: *p ≤ 0.05, **p ≤ 0.01, and ****p ≤ 0.0001.

(A): Ex vivo luminescence imaging of pancreatic tumors excised from orthotopically xenografted with MIA PaCa-2 luciferase cells. (B): Detection of metastasis through ex vivo luminescence imaging of excised organs. Various organs are denoted by different numbers i.e., 1: Liver; 2: heart; 3: kidneys; 4: lungs, and 5: spleen.

Dysregulated ribosome biogenesis and p53 mutations are known to play oncogenic roles in various cancers, including pancreatic cancer. In this study, we demonstrated the therapeutic potential of BMH-21, a pharmacologic inhibitor of RNA polymerase I, against pancreatic cancer by uncovering a novel molecular mechanism involving RPA194-mediated ubiquitination of mutant p53 without affecting the ubiquitination of wild-type p53. Our key findings are that (i) BMH-21 selectively induces apoptosis and cell growth inhibition of pancreatic cancer cells with no effect on normal human pancreatic ductal epithelial cells; (ii) BMH-21 degrades RPA194; (iii) BMH-21 inhibits recruitment of both RPA194 and RPA135 on rDNA to suppress pre-rRNA synthesis; (iv) RPA194 physically interacts with p53 and BMH-21–induced degradation of RPA194 selectively exposes truncated and mutated p53 for ubiquitination with no effect on ubiquitination of wild-type p53 in pancreatic cancer cells; and (v) BMH-21 treatment significantly reduces the growth of orthotopic xenograft pancreatic tumors in athymic nude mice with no observed toxicity. Altogether, these findings suggest that BMH-21 is a promising, nontoxic therapeutic agent for patients with pancreatic cancer with aberrant ribosome biogenesis and mutant p53, offering a potential new avenue for targeted treatment.

Representative pictures of hematoxylin & eosin (H & E) staining showing the effect of BMH-21 treatment does not pose any cytotoxic effect on the histopathological features of different organs in a mice model of pancreatic cancer, orthotopically xenografted with MIA PaCa-2-Luciferase cells. Magnification: 20X; Scale bar: 50 µm.

(A) Effect of BMH-21 treatment on the body weight of mice in orthotopic xenograft experiment with MIA PaCa-2-Luciferase cells. (B) Effect of BMH-21 treatment on the body weight of mice in orthotopic xenografted with AsPC-1 luciferase cells.

IC50 values of HPDEC and PanCa cells expressing different WT and mutant p53. BMH-21 was found to be least effective against normal HPDEC (IC50 = 33.4 µM).

Contains supplementary tables.

The status of p53 of the various PanCa cells used in the study. Western blotting for the screening of p53 expression using antibodies from different vendors in normal and PanCa cells. Ab-1: Cell Signaling Technologies, Cat. No. 2524 (1:1000); Ab-2: Abcam, Cat. No. ab-32049 (1:1000); Ab-3: p53 (DO-1), Santacruz, Cat. No. Sc-126 (1:1000). Based on the binding profile, we used p53 (DO-1) for our experiments.

Contains methodology, supplementary figures, and references.

The bar graph shows the frequency of variation in p53 in different cancer samples. The data for this graph was obtained from TP3 database (https://tp53.cancer.gov/prevalence_somatic_stats).