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

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

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Figure 4

Disruption of RAD54-BLM interaction by small molecules decreased chromatin remodeling.

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Disruption of RAD54-BLM interaction by small molecules decreased chromat...
(A) Disruption of RAD54-BLM interaction by small molecules was done by screening the Prestwick chemical library using Renilla luciferase–based PCA. Percentage disruption of the interaction between BLM F2 and N-RAD54 F1 was plotted in form of a heatmap. (B) C3, C7, and C17 disrupted RAD54-BLM interaction in vitro. In vitro interactions were carried out between bound GST-BLM WT and soluble His-RAD54 WT in the absence or presence of 10 μM C3, C7, or C17. Levels of bound RAD54 were determined by immunoblotting. (C and D) C3, C7, and C17 decreased the efficiency of BLM-dependent enhancement of RAD54 chromatin remodeling activity. (C) REA assays were carried out as indicated using MRP2 array. The reactions were stopped after 1, 5, and 10 minutes. (D) Quantitation of C. The data are from 3 independent experiments. (E) C3, C7, and C17 decreased BLM-dependent enhancement of the binding of ATP by RAD54. Quantitation of the ATP binding assays was carried out. Data are from 3 independent experiments. (F) C3, C7, and C17 decreased BLM-dependent enhancement of the ATPase activity of RAD54. Quantitation of the ATPase activity was carried out. Data are from 4 independent experiments. (G) C17 altered the conformation of RAD54. Tryptophan fluorescence assays were carried out with His-RAD54 WT, alone or in presence of the indicated concentrations of C17. RAD54 fluorescence was measured in a fluorometer. Experiment was repeated 3 times, and a representative experiment is shown. (H) The affinity of RAD54 to C17 was similar to that of biotinylated BLM (aa 181–212) peptide. Octet BLI-based studies were performed to determine the dissociation constant of the interaction of different concentrations of biotin BLM_peptide and C17 with His-RAD54 WT immobilized onto Ni-NTA-sensor. The affinity constant (KD) ± SD is shown. The experiment was repeated 3 times, and 1 representative experiment is shown. Data are shown as the mean ± SD. *P < 0.05, ***P < 0.001, ****P < 0.0001, (D) 2-way ANOVA; (E and F) 1-way ANOVA.