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.
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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 2

Interaction between BLM (aa 181–212) and RAD54 enhanced chemoresistance in cells.

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Interaction between BLM (aa 181–212) and RAD54 enhanced chemoresistance ...
(A) Cellular uptake of BLM (aa 181–212) cell-permeable peptide (BLM_CPP) and scrambled cell–permeable peptide (SCM_CPP). The intake of the TAMRA tagged peptides was monitored by live-cell imaging. (B and C) Levels of RAD51, RAD54, and γH2AX were altered after treatment with BLM_CPP. GM03509 GFP cells were grown in presence of HU for 16 hours or 6 more hours after washing away HU, in presence of (B) 180 nM BLM_CPP or SCM_CPP or (C) BLM_NP or SCM_NP. Lysates made were probed with indicated antibodies. Experiment was repeated 4 times, and representative blots are presented. (D) RAD51 and RAD54 foci numbers were increased after treatment with BLM (aa 181–212) peptide. As in B and C, except HCT116 BLM–/– cells were fixed and processed for immunofluorescence with indicated antibodies. (D and H) Experiment was repeated 3 times, and representative images and quantitation (foci/cell) are presented. Number of cells analyzed = 45. (E) Presence of BLM_CPP allowed GM03509 GFP cells to proliferate. As in B and C, except cells released after HU treatment were grown for 4 hours with 180 nM BLM_CPP or SCM_CPP, washed, and allowed to grow for the indicated time intervals. Cells were analyzed by flow cytometry. (F and G) Presence of BLM_CPP or BLM_NP decreased the levels of the CDK inhibitors. GM03509 GFP-BLM and GM03509 GFP cells released after HU treatment were grown for 6 hours with (F) 180 nM BLM_CPP or SCM_CPP or (G) BLM_NP or SCM_NP. Lysates made were probed with indicated antibodies. Experiment was repeated 4 times, and representative blots are presented. The asterisk represents a cross-reactive band in G. (H) γH2AX foci numbers were decreased after treatment with BLM_CPP. As in B and C, except HCT116 BLM–/– cells were processed for immunofluorescence with γH2AX antibody. (I) BLM_CPP decreased the levels of cellular DNA damage. Cells treated with HU (16 hours) were grown for 6 hours with 180 nM BLM_CPP or SCM_CPP, after which Comet assays were carried out. (J and K) BLM_CPP increased cellular resistance to cisplatin and camptothecin. Cells were treated with 180 nM BLM_CPP or SCM_CPP in presence of (J) 1, 2, 3, 4, or 5 nM CDDP or (K) 10 nM, 50 nM, 100 nM, 150 nM, 200 nM of CPT. The percentage of viable cells was determined by MTT assays. The data are from (J) 4 and (I and K) 3 independent experiments. Data are shown as the mean ± SD. *P < 0.05, **P < 0.01, ****P < 0.0001, (I) 1-way ANOVA; (D, H, J, and K) 2-way ANOVA. Scale bar: 5 μM.