miR-10b is silenced in normal neuroglial cells of the brain but commonly activated in glioma, where it assumes an essential tumor-promoting role. We demonstrate that the entire miR-10b-hosting HOXD locus is activated in glioma via the cis -acting mechanism involving 3D chromatin reorganization and CTCF-cohesin-mediated looping. This mechanism requires two interacting lncRNAs, HOXD-AS2 and LINC01116, one associated with HOXD3/HOXD4/miR-10b promoter and another with the remote enhancer. Knockdown of either lncRNA in glioma cells alters CTCF and cohesin binding, abolishes chromatin looping, inhibits the expression of all genes within HOXD locus, and leads to glioma cell death. Conversely, in cortical astrocytes, enhancer activation is sufficient for HOXD/miR-10b locus reorganization, gene derepression, and neoplastic cell transformation. LINC01116 RNA is essential for this process. Our results demonstrate the interplay of two lncRNAs in the chromatin folding and concordant regulation of miR-10b and multiple HOXD genes normally silenced in astrocytes and triggering the neoplastic glial transformation. [Display omitted] • Chromatin topology of the HOXD locus is altered between the brain and glioblastoma • lncRNAs regulate CTCF/cohesin-dependent loop formation and HOXD gene expression • Activation of LINC01116 enhancer RNA leads to astrocyte transformation • The transformation depends on the loop formation and miR-10b derepression Deforzh et al. investigated a common mechanism of HOXD/miR-10b genes' derepression in glioblastoma and revealed the coordinated activity of two lncRNAs, HOXD-embedded HOXD-AS2 and distant enhancer-associated LINC01116, in CTCF/cohesin binding, chromatin topology, and astrocyte transformation. The work shed light on the molecular mechanisms of gliomagenesis. [ABSTRACT FROM AUTHOR]