Transcriptomic Analysis in Mantle Cell Lymphoma Patients Identified MALAT1 Long-Non-Coding RNA to Regulate Cell Cycle Progression By Modulating EZH2/p57/Sox11 Axis

Introduction: Mantle Cell Lymphoma (MCL), a rare and incurable form of B-cell lymphoma, presents significant challenges in treatment, with limited median progression-free survival. MCL is characterized by the translocation t(11;14) leading to overexpression of the cyclinD1 and deregulation of the cell cycle processes and genomic instability. Molecular heterogeneity in MCL patients reveals that MCL patients with high expression of SOX11 unmutated IGHV correlate poorly with clinical outcomes. The targeted therapies targeting cell cycle and DNA Damage response pathways have shown efficacy. However, the outcomes are dismal. Recent studies have suggested that lncRNAs regulate the cancer cell cycle, and identifying the mechanistic insights of lncRNAs in regulating cell cycle factors will provide new insights into the regulation of fundamental cellular processes to develop rational therapies.

Methods: To identify the differential expression of lncRNA in MCL patients, we conducted RNA-Sequencing (RNA-Seq) analysis on MCL patients (n=18), comparing them to CD19+ B-cells sorted from the peripheral blood of healthy donors (n=10). We trimmed the reads for quality, removed adapters using Trim Galore, and mapped them to the human genome (HG38 assembly) using HISAT 2. All the reads mapped within the genomic coordinates of lncRNAs were counted using featureCounts. We used LIMMA to analyze differential expression between the MCL and healthy donor samples. To validate our findings from the MCL cohort, we obtained publicly available RNA-Seq data from 16 MCL patients and analyzed them using the same pipeline. Finally, the top candidates were validated in the third cohort of 48 MCL patients and 10 normal CD19+ B-cells. We performed loss of Function, RNA-protein interaction studies, and complementation studies to confirm the role of identified lncRNA in activating critical molecular events in MCL using human MCL cell lines (n=3).

Results: Our analyses revealed dysregulations of lncRNA in MCL. Specifically, we identified 385 upregulated and 387 downregulated lncRNAs (|log2FC| > 0.58 and adjusted p-value < 0.05) in our internal MCL cohort (n=18), and over 60% of them were also confirmed in the external cohort (n=16). Notably, the lncRNA called MALAT1 was among the top ten upregulated in both cohorts, further validated in our third cohort (n=48). To investigate the potential role of MALAT1 in MCL, we analyzed MCL samples with high MALAT1 expression (upper quartile) compared to those with low expression (lower quartile) across two cohorts. Differentially expressed genes identified from this comparison were used for functional network-based topological pathway analysis via MITHrIL. This analysis highlighted the KEGG pathway "Cell Cycle" as one of the top upregulated pathways in MCL samples with high MALAT1 expression. We further demonstrate that the loss of MALAT1 in MCL cell lines (n=3) downregulates the expression of Sox11, an important transcription factor in MCL. Further studies illustrated that loss of MALAT1 downregulates SOX11 expression by inhibiting the Rb/E2F1 mediated transactivation and halting MCL progression at the G1/S stage. Mechanistically, MALAT1 loss downregulates EZH2, a polycomb repressive complex protein, and activates the cell cycle-dependent kinase inhibitor p57 (CDKNIC). In MCL Patients (n=43) the expression of MALAT1 negatively correlates with p57 (r²=-0.55; pValue=1.19e-4) and positively correlate with SOX11 (r²=.38;pValue=0.0124). Reactivation of p57 inhibits the Rb/E2F1 transactivation of SOX11 by binding to E2F1 and inhibiting its transactivation function. Interestingly, the SOX11 direct targets (SMAD3, CACYBP, and MAPK8) are also downregulated upon Loss of MALAT1 in MCL.

Conclusion: Our study is the first to describe the novel dysregulation signature of lncRNAs in MCL across two independent cohorts and dissect the role of MALAT1 in regulating the cell cycle progression by EZH2/p57/-Sox11 axis in MCL. Our study also demonstrates that loss of MALAT1 lncRNA in MCL upregulates p57 and regulates the Rb/E2F1 transactivation to downregulate SOX11 and further alter the expression of SOX11 direct target genes controlling cell cycle and proliferation in MCL. We are currently optimizing the novel LNA-Gapmers targeting MALAT1 to target SOX11-positive MCL and identify novel drug combinations to target MCL progression.