Non-Hodgkin’s lymphoma (NHL) is the 7th most common cancer in adults in most countries worldwide. Although there have been significant advances in treatments for patients with NHL, relapse can occur within the first two years after remission. Therefore, novel targets and treatment modalities are urgently needed.

PRMT5 is a type II protein arginine methyltransferase (PRMT) that catalyzes symmetric dimethylation of arginine residues on histones and non-histone proteins. During embryonic development, PRMT5 is key for the maintenance of hematopoietic stem cells and B-cell development, by regulating transcription and splicing fidelity¹.

Overexpression of PRMT5 is associated with worse a prognosis across multiple cancer types, including lymphomas, where it has been found especially upregulated in germinal center-derived lymphomas and mantle cell lymphomas (MCL)^{2, 3, 4}. PRMT5 plays an important role in lymphomagenesis through the transcriptional downregulation of tumor suppressors, such as P53⁵, while promoting the expression and/or activity of the lymphoma-drivers, c-MYC, CYCLIN D1 and BCL-6^{6, 7}.

What did we discover?

 To uncover the mechanism of action and predictors of response to the potent selective PRMT5 inhibitor, GSK3203591 (GSK-591), we performed a genome-wide CRISPR/Cas9 screen in an MCL cell line and a validation CRISPR /Cas9 screen in a DLBCL cell line. CRISPR screens identified TP53 as the top sensitizer to GSK-591, which validated our screens, as it is a well characterized target of PRMT5. Furthermore, we demonstrate that TP53 deletion and the hot spot TP53^R248W mutation are biomarkers of resistance to PRMT5 inhibition.

RNA-binding protein MUSASHI-2 (MSI2) was identified as the top driver of resistance to GSK-591. We found a strong correlation of PRMT5 and MSI2 mRNA levels in DLBCL patients. We detected both PRMT5 and MSI2 in relapsed MCL and DLBCL patient samples.  Moreover, MSI2 depletion decreased cell viability and sensitized lymphoma cells to PRMT5 inhibition both in vitro and in vivo. Additionally, MSI2 overexpression rescued the reduced growth effects from PRMT5 inhibition. Consistent with our genetic studies, inhibition of MSI2 using Ro 08-2750 (Ro)⁸, conferred sensitivity to GSK-591, and the combination therapy was synergistic, inducing cell cycle arrest.

To identify the molecular mechanism responsible for the synergy of PRMT5 and MSI2 inhibitors, we performed RNA-Seq analysis of cells treated with GSK-591, Ro, or combination. Gene set enrichment analysis (GSEA) showed dual treatment results in synergistic gene expression changes including cell cycle, P53, and c-MYC signatures.

We then focused validating c-MYC as a target of GSK-591 and Ro combination. MSI2 RNA-IP assays demonstrated that MSI2 binds c-MYC mRNA and this interaction is disrupted by the drug combination. We observed that the combination of MSI2 and PRMT5 inhibitors does not affect c-MYC mRNA levels but, rather, controls its translation. These findings provide new insights on the potential mechanism involved in PRMT5’s regulation of c-MYC.  We find MSI2 binds c-MYC 3’UTR and enhances its translation.  

 To understand the landscape of MSI2 mRNA targets, we performed MSI2-HyperTRIBE⁹. By using a fusion of MSI2 and the RNA editing enzyme ADAR, we could quantitatively assess MSI2 binding across its targets for the first time in B-cell lymphoma cells. Importantly, we find  Ro significantly reduces MSI2 binding across its targets, while GSK591 had no effect. These data suggest that PRMT5 does not affect MSI2’s ability to bind its targets.

Using a multi-omic analysis with the genome-wide CRISPR screen, RNA-Seq and MSI2-HyperTRIBE, we identified the anti-apoptotic protein BCL-2, as a target of the PRMT5-MSI2 axis. Genetic depletion or inhibition of BCL-2 using venetoclax synergized with the PRMT5 inhibitor by inducing apoptosis of lymphoma cells.

Our study uncovers a novel oncogenic axis, PRMT5/MSI2/c-MYC/BCL-2 that drives resistance to PRMT5-targeted therapies in lymphoma.

Translational relevance

There are currently six ongoing or actively recruiting clinical trials involving PRMT5 inhibitors, which include: GSK, Prelude, Amgen, Tango, Mirati Therapeutics, and IDEAYA Therapeutics.  

We found that of TP53 LOF and MSI2 expression could be used as biomarkers for patient stratification. Second, we propose two drug combination modalities, to overcome the resistance to PRMT5-targeted therapy with the BCL-2 inhibitor, venetoclax, and the MSI2 inhibitor, Ro. Both drug combinations induced potent anti-proliferative activity across different lymphoma subtypes.

It is important to note that Ro is a micromolar inhibitor with poor drug like properties thus prohibiting its clinical use. Our study suggests the need for the development of more potent and clinically relevant MSI2 inhibitors. Although venetoclax is a clinically approved drug, its toxicity profile in combination with PRMT5 inhibitors may preclude any future therapeutic benefit. Additional pre-clinical studies and development of a careful combination treatment regimen may dictate the feasibility of our strategy. Overall, we performed an unbiased screen which identified novel pathways and targets that enhance PRMT5 inhibition.

References

1. Litzler LC, et al. PRMT5 is essential for B cell development and germinal center dynamics. Nat Commun 10, 22 (2019).
2. Chung J, et al. Protein arginine methyltransferase 5 (PRMT5) inhibition induces lymphoma cell death through reactivation of the retinoblastoma tumor suppressor pathway and polycomb repressor complex 2 (PRC2) silencing. J Biol Chem 288, 35534-35547 (2013).
3. Alinari L, et al. Selective inhibition of protein arginine methyltransferase 5 blocks initiation and maintenance of B-cell transformation. Blood 125, 2530-2543 (2015).
4. Li Y, et al. PRMT5 is required for lymphomagenesis triggered by multiple oncogenic drivers. Cancer Discov 5, 288-303 (2015). 

5. Jansson M, et al. Arginine methylation regulates the p53 response. Nat Cell Biol 10, 1431-1439 (2008).
6. Karkhanis V, et al. Protein arginine methyltransferase 5 represses tumor suppressor miRNAs that down-regulate CYCLIN D1 and c-MYC expression in aggressive B-cell lymphoma. J Biol Chem 295, 1165-1180 (2020).
7. Lu X, et al. PRMT5 interacts with the BCL6 oncoprotein and is required for germinal center formation and lymphoma cell survival. Blood 132, 2026-2039 (2018).
8. Minuesa G, et al. Small-molecule targeting of MUSASHI RNA-binding activity in acute myeloid leukemia. Nat Commun 10, 2691 (2019).
9. Nguyen DTT, et al. HyperTRIBE uncovers increased MUSASHI-2 RNA binding activity and differential regulation in leukemic stem cells. Nat Commun 11, 2026 (2020).