Small molecules that disrupt RAD54-BLM interaction hamper tumor proliferation in colon cancer chemoresistance models
Ekjot Kaur, … , Avinash Bajaj, Sagar Sengupta
Ekjot Kaur, … , Avinash Bajaj, Sagar Sengupta
Published February 29, 2024
Citation Information: J Clin Invest. 2024;134(8):e161941. https://doi.org/10.1172/JCI161941.
View: Text | PDF
Research Article Oncology Article has an altmetric score of 33

Small molecules that disrupt RAD54-BLM interaction hamper tumor proliferation in colon cancer chemoresistance models

Abstract

RAD54 and BLM helicase play pivotal roles during homologous recombination repair (HRR) to ensure genome maintenance. BLM amino acids (aa 181–212) interact with RAD54 and enhance its chromatin remodeling activity. Functionally, this interaction heightens HRR, leading to a decrease in residual DNA damage in colon cancer cells. This contributes to chemoresistance in colon cancer cells against cisplatin, camptothecin, and oxaliplatin, eventually promoting tumorigenesis in preclinical colon cancer mouse models. ChIP-Seq analysis and validation revealed increased BLM and RAD54 corecruitment on the MRP2 promoter in camptothecin-resistant colon cancer cells, leading to BLM-dependent enhancement of RAD54-mediated chromatin remodeling. We screened the Prestwick small-molecule library, with the intent to revert camptothecin- and oxaliplatin-induced chemoresistance by disrupting the RAD54-BLM interaction. Three FDA/European Medicines Agency–approved candidates were identified that could disrupt this interaction. These drugs bound to RAD54, altered its conformation, and abrogated RAD54-BLM–dependent chromatin remodeling on G5E4 and MRP2 arrays. Notably, the small molecules also reduced HRR efficiency in resistant lines, diminished anchorage-independent growth, and hampered the proliferation of tumors generated using camptothecin- and oxaliplatin-resistant colon cancer cells in both xenograft and syngeneic mouse models in BLM-dependent manner. Therefore, the 3 identified small molecules can serve as possible viable candidates for adjunct therapy in colon cancer treatment.

Authors

Ekjot Kaur, Ritu Agrawal, Rimpy Arun, Vinoth Madhavan, Vivek Srivastava, Dilip Kumar, Pragyan Parimita Rath, Nitin Kumar, Sreekanth Vedagopuram, Nishant Pandey, Swati Priya, Patrick Legembre, Samudrala Gourinath, Avinash Bajaj, Sagar Sengupta

×

Figure 1

BLM (aa 181–212) enhanced RAD54-mediated chromatin remodeling.

Options: View larger image (or click on image) Download as PowerPoint
BLM (aa 181–212) enhanced RAD54-mediated chromatin remodeling. (A) The N...
(A) The N-terminal region of RAD54 interacted with BLM (aa 181–212) in cells. N-RAD54-F1, C-RAD54-F1, BLM-F1, and BLM-F2 were transfected in HEK293T cells. Then, Renilla luciferase–based PCAs were carried out with the indicated combination of expressed proteins. (B) Lack of amino acids 181–212 in BLM abrogates its interaction with RAD54 in cells. HCT116 BLM–/– cells were transfected with GFP BLM WT or GFP BLM (aa Δ181–212), and lysates were made. Immunoprecipitations were carried out with anti-GFP antibodies and probed for RAD54. One representative experiment is shown. (C) BLM (aa 181–212) interacts with endogenous RAD54 in cells. As in B, except HCT116 BLM–/– cells were transfected with p3XFlag-Myc-CMV24 BLM (aa 181–212) or the empty vector. (D and E) BLM (aa 181–212) enhanced ATP-dependent RAD54-mediated chromatin remodeling. (D) REA assays were carried out with chromatinized G5E4 array using indicated experimental conditions in presence of ATP. The reactions were stopped after 0, 2, 4, 6, 8, and 10 minutes. (E) Quantitation of D. (F) BLM (aa 181–212) enhanced the ATP binding capacity of RAD54. Quantitation of the ATP binding assays carried out as indicated. (G) BLM (aa 181–212) peptide increased the ATPase activity of RAD54. Quantitation of the ATPase activity carried out as indicated. (H) BLM (aa 181–212) peptide altered the conformation of RAD54. Tryptophan fluorescence assays were carried out with RAD54 WT or RAD54 WT in presence of concentrations of BLM_peptide or SCM_peptide. Experiment was repeated 3 times. One representative experiment is shown. (A and E–G) Data are shown as the mean ± SD. Data are from 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, (E) 2-way ANOVA, (F) 1-way ANOVA; (G) paired t test.