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 3

Chromatin remodeling by RAD54-BLM complex on MRP2 promoter enhances chemoresistance.

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Chromatin remodeling by RAD54-BLM complex on MRP2 promoter enhances chem...
(A) Circos plot obtained from BLM Chip-Seq analysis carried out on GM03509 BLM Clone 9.6 cells. (B and C) Both BLM and RAD54 were corecruited to MRP2 promoter. Chromatin isolated from HCT116 WT and HCT116 IC60 CPTR cells was used for (B) ChIP or (C) Re-ChIP. DNA obtained was used to determine the enrichment on (B) MRP2, MRP3, MDR1, and GAPDH promoters and (C) MRP2, MDR1, and GAPDH promoters by qPCR. Data are from 3 independent experiments. (D and E) BLM (aa 1–212) enhanced ATP-dependent RAD54-mediated chromatin remodeling. (D) REA assays were carried out with chromatinized MRP2 array. Reactions were stopped after 1, 5, and 10 minutes. (E) Quantitation of D. Data are from 4 independent experiments. (F) Enhanced transcription of MDR genes occurred in HCT116 IC60 CPTR cells. RNA isolated from HCT116 WT and HCT116 IC60 CPTR cells was used for RT-qPCR. The levels of MRP1, MRP2, MRP3, MRP4, MRP5, MXR, MDR1, BSEP, ABCA2, and ABCB5 were quantitated from 3 independent experiments. (G) HCT116 WT IC60 CPTR cells have enhanced MRP2 efflux activity. HCT116 WT and HCT116 WT IC60 CPTR cells were incubated with MRP2 substrate (CDF) for 30 minutes at 37°C. The accumulation of fluorescent product CDF was determined as a measure of MRP2 activity. The experiment was carried out 9 times. (H) BLM_CPP enhanced the anchorage-independent growth of HCT116 BLM–/– cells. Soft agar assay was carried out in HCT116 BLM–/– cells by treating them with 180 nM BLM_CPP or SCM_CPP in absence or presence of CPT (120 nM). The number of soft agar colonies in each condition was counted. Data are from 3 independent experiments. (I) Treatment with CPT-BLM-Gel enhanced tumor growth in a xenograft mice model. HCT116 BLM–/– cells were injected into SCID mice (n = 7 in each group). On day 1, when the tumors were 50 mm3, CPT-Gel was injected at the base of the tumors alone or along with the injection of CPT-BLM-Gel or CPT-SCM-Gel. The volume of the tumors was estimated for the indicated days. (J) BLM (aa 181–212) region enhanced tumor growth in xenograft mice model. HCT116 BLM–/– cells stably expressing EGFP or EGFP-BLM (aa 181–212) were injected into SCID mice (n = 7 in each group). The volume of the tumors was estimated for the indicated days. Data are shown as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, (B, C, E, F, I) 2-way ANOVA; (G) Mann-Whitney test; (H) 1-way ANOVA; (J) Wilcoxon’s test.