Drugging the lncRNA MALAT1 via LNA gapmeR ASO inhibits gene expression of proteasome subunits and triggers anti-multiple myeloma activity

Abstract

The biological role and therapeutic potential of long non-coding RNAs (lncRNAs) in multiple myeloma (MM) are still to be investigated. Here, we studied the functional significance and the druggability of the oncogenic lncRNA MALAT1 in MM. Targeting MALAT1 by novel LNA-gapmeR antisense oligonucleotide antagonized MM cell proliferation and triggered apoptosis both in vitro and in vivo in a murine xenograft model of human MM. Of note, antagonism of MALAT1 downmodulated the two major transcriptional activators of proteasome subunit genes, namely NRF1 and NRF2, and resulted in reduced trypsin, chymotrypsin and caspase-like proteasome activities and in accumulation of polyubiquitinated proteins. NRF1 and NRF2 decrease upon MALAT1 targeting was due to transcriptional activation of their negative regulator KEAP1, and resulted in reduced expression of anti-oxidant genes and increased ROS levels. In turn, NRF1 promoted MALAT1 expression thus establishing a positive feedback loop. Our findings demonstrate a crucial role of MALAT1 in the regulation of the proteasome machinery, and provide proof-of-concept that its targeting is a novel powerful option for the treatment of MM.

Fig. 1

Effects of MALAT1 manipulation on in vitro and in vivo MM growth. a MALAT1 relative levels (left panel) and cell viability of MM cells (right panel) were determined by qRT-PCR and CTG assay respectively, 4 days after treatment with 5 μM g CNT or g#5. b CTG assay in MM cell lines and primary PCs co-cultured on HS-5 stromal cells, and treated for 4 days with 5 μM g CNT or g#5. c Colony formation assay performed on MM cell lines treated for 14 days with 5 μM g CNT or g#5; representative pictures of colonies at day 14 are also shown. d Annexin V staining of MM cell lines, 5 days after treatment with naked g CNT or g#5. Data are representative of at least three independent experiments. e Caliper measurements of tumor volumes in mice bearing AMO-ABZB-luc xenografts IP-treated with g CNT (25 mg/kg) or g#5. *p < 0.05

Fig. 2

MALAT1 depletion reduces proteasome gene expression in MM cells. a GSEA performed 48 h after transfection with 50 nM g CNT or g#5 (nominal p-values for RPMI-8226 and MM1s were 0.00 and 0.028, respectively). b Caspase-like (C-L), Trypsin-like (T-L), Chimotrypsin-like (CT-L) activities evaluated by proteasome assay (Promega), 4 days after delivery of naked gCNT or g#5; WB of polyubiquitinated (Ub) proteins is reported on the right. c WB of ER stress markers 4 days after delivery of naked gCNT or g#5. * = p < 0.05

Fig. 3

MALAT1 regulates NRF1 and NRF2 expression and activity. a WB of NRF1, NRF2, and PSMβ5 in MM cell lines or patient-derived MM cells, 5 days after delivery of naked g CNT or g#5. b CTG viability assay was performed in AMO-BZB cells transfected with 2.5 μg of NRF1-flag or NRF2-flag expression vectors, then treated for 4 days with 2.5 μM naked g#5. c Luciferase assay performed in cells transfected with an ARE reporter (Promega), and then treated for 4 days with 5.0 μM g CNT or naked g#5. d ROS measurement by luminescent assay, 4 days after delivery of 5.0 μM naked g CNT or g#5. e Representative immunofluorescence of γH2AX foci, 4 days after delivery of g CNT (5.0 μM) or g#5 (×63 magnification). Data are representative of at least three independent experiments. *p < 0.05

Fig. 4

MALAT1 epigenetically regulates KEAP1. a qRT-PCR of KEAP1 mRNA relative expression, 5 days after delivery of naked g CNT or g#5; right panel shows WB of KEAP1. b qRT-PCR of KEAP1 and EZH2 mRNA levels in AMO-BZB cells, 24 h after transfection with 100 nM of the corresponding siRNAs. c qPCR for KEAP1 promoter performed after ChIP with H3K27Me3 antibody, in AMO-BZB cells treated for 72 h with 2.5 μM naked g#5 or 2 μM DZNep. Data are representative of at least three independent experiments. *p < 0.05

Fig. 5

NRF1 induces MALAT1 expression in a clinically relevant feedback loop. a qPCR for MALAT1 promoter performed after ChIP with an NRF1 antibody or IgG control. b Luciferase assay carried out in AMO-BZB cells, 48 h after transfection with MALAT1 promoter reporter (2.5 μg), together with 2.5 μg of NRF1 (V-NRF1), NRF2 (V-NRF2), or empty vector (V-CNT). c Cartoon illustrating the regulatory loop identified in this study. d qRT-PCR of MALAT1 and NRF1, 24 h after bortezomib treatment. CTG viability assay performed in AMO-1 cells (e) and in AMO-BZB cells (f), 4 days after treatment with g#5 and bortezomib. *p < 0.05; °combination index < 1.0 (calculated using Calcusyn)