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<p>Chronic myeloid leukemia (CML) is a hematological malignancy that has its cause as Philadelphia chromosome and BCR: ABL fusion gene. Although specific drugs such as imatinib have been used to a great effect to enhance the outcome of treatment, resistance is still a challenge especially in later stages of the disease. This work has explored the phenotypic role of eight single nucleotide variants (SNVs) in the downstream sequence of MIR5195 gene, which are determined in real-life sample of patients. The initial experiment involved whole-exome sequencing (WES) to identify genetic variants in advanced phase CML patients and Sanger sequencing to validate the variants. The results indicate the possible importance of the established mutations as well as mutations that are yet to be characterized in the regulation of MIR5195 and their potential impact on the development of CML. Comprehensive bioinformatics analyses (MEME, TOMTOM, and RegulomeDB) revealed that these mutations profoundly reconfigure local cis-regulatory architecture, resulting in the loss of conserved transcription factor binding motifs and the emergence of a mutation-specific motif enriched for hematopoietic and cancer-associated transcription factors, including RXRB, FOXP1, GATA-1, and GATA-2. Three-dimensional structural modeling using AlphaFold3 showed that both wild-type and mutant downstream MIR5195 sequences retain canonical B-form DNA, but the mutant exhibits localized changes in backbone curvature and groove geometry within the mutation-enriched motif. Molecular docking analyses revealed transcription factor–specific redistribution of binding affinities rather than uniform loss of interaction, with increased binding observed for GATA1 (− 242.20 vs. − 228.26) and RXRB (− 223.57 vs. − 221.44), and reduced binding for FOXP1 (− 213.63 vs. − 219.93) and GATA2 (− 242.15 vs. − 281.57). These findings indicate selective modulation of transcription factor engagement driven by downstream non-coding mutations. Based on the emergence of a mutation-specific regulatory motif, we designed a transcription factor decoy oligonucleotide targeting the altered binding site as a potential mutation-directed regulatory strategy. Overall, this integrative computational and experimental framework demonstrates that downstream MIR5195 mutations quantitatively reprogram transcription factor binding networks in CML and highlights non-coding regulatory variation as a biologically meaningful contributor to leukemia pathogenesis.</p>

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Experimental Validation and Computational Deconstruction of Downstream Mutations in MIR5195: Integrating Whole-Exome Sequencing, Structural Dynamics, and Molecular Docking to Decipher Transcriptional Reprogramming in CML

  • 期刊论文

  • 发表日期：

    2026年4月11日

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  • Sameen Shahid,
  • Muhammad Farooq Sabar,
  • Zafar Iqbal

摘要

Chronic myeloid leukemia (CML) is a hematological malignancy that has its cause as Philadelphia chromosome and BCR: ABL fusion gene. Although specific drugs such as imatinib have been used to a great effect to enhance the outcome of treatment, resistance is still a challenge especially in later stages of the disease. This work has explored the phenotypic role of eight single nucleotide variants (SNVs) in the downstream sequence of MIR5195 gene, which are determined in real-life sample of patients. The initial experiment involved whole-exome sequencing (WES) to identify genetic variants in advanced phase CML patients and Sanger sequencing to validate the variants. The results indicate the possible importance of the established mutations as well as mutations that are yet to be characterized in the regulation of MIR5195 and their potential impact on the development of CML. Comprehensive bioinformatics analyses (MEME, TOMTOM, and RegulomeDB) revealed that these mutations profoundly reconfigure local cis-regulatory architecture, resulting in the loss of conserved transcription factor binding motifs and the emergence of a mutation-specific motif enriched for hematopoietic and cancer-associated transcription factors, including RXRB, FOXP1, GATA-1, and GATA-2. Three-dimensional structural modeling using AlphaFold3 showed that both wild-type and mutant downstream MIR5195 sequences retain canonical B-form DNA, but the mutant exhibits localized changes in backbone curvature and groove geometry within the mutation-enriched motif. Molecular docking analyses revealed transcription factor–specific redistribution of binding affinities rather than uniform loss of interaction, with increased binding observed for GATA1 (− 242.20 vs. − 228.26) and RXRB (− 223.57 vs. − 221.44), and reduced binding for FOXP1 (− 213.63 vs. − 219.93) and GATA2 (− 242.15 vs. − 281.57). These findings indicate selective modulation of transcription factor engagement driven by downstream non-coding mutations. Based on the emergence of a mutation-specific regulatory motif, we designed a transcription factor decoy oligonucleotide targeting the altered binding site as a potential mutation-directed regulatory strategy. Overall, this integrative computational and experimental framework demonstrates that downstream MIR5195 mutations quantitatively reprogram transcription factor binding networks in CML and highlights non-coding regulatory variation as a biologically meaningful contributor to leukemia pathogenesis.