Non-Hotspot DIS3 Mutations in Multiple Myeloma Result in Loss of Function Accompanied By Transcriptional and Proteomic Dysregulation

Introduction

DIS3 mutations in multiple myeloma (MM) are associated with a poor prognosis, therefore impacting the clinical risk of patients. Mutations in DIS3 recurrently occur at three amino acid residues, known as hotspot mutations, and are found to be heterozygous. The remaining DIS3 mutations—non-hotspot mutations—are typically homozygous and span the DIS3 protein. However, the functional difference between these DIS3 mutation groups and their cellular effects is yet to be investigated. This knowledge is critical to understanding the biological role of DIS3 mutations in the progression of multiple myeloma.

Methods

Patient samples (SMM, n=14; NDMM, n=36; RRMM, n=54) underwent whole genome sequencing to determine DIS3 mutation rates. Endogenous copies of DIS3 were mutated in the KMS11 cell line using CRISPR-Cas9 homology directed repair and individual colonies isolated. The mutations were individually generated and included 4 non-hotspot homozygous mutations (Y106C (PIN domain), T262P, L544P, and H788Y (all RNB catalytic domain)), 3 hotspot heterozygous mutations (D479G, D488N, and R780K (all RNB domain)), and synonymous control mutations. To examine the impact of these DIS3 mutations, we performed total RNA-sequencing for differential gene expression and pathway analysis, R-loop detection, cell proliferation analysis (Incucyte S3 live cell imaging), tandem mass spectrometry, and Western blot analysis.

Results

We identified DIS3 mutations in SMM (7%), newly diagnosed MM (7%) and relapsed refractory MM (19%) patient samples with increasing clonal fraction, indicating an ongoing role in disease progression and relapse. To investigate their effect, mutations were engineered in the endogenous copies of DIS3 in KMS11 cells. Differential expression analysis of mutated cell lines showed that non-hotspot mutations had a greater effect on transcription factors than hotspot mutations, resulting in 49 unique differentially expressed transcription factors which were mostly downregulated. In turn, most differentially expressed genes were downregulated in non-hotspot mutations and were involved in RNA metabolic processing, transcription, and protein processing pathways; which was not seen in the hotspot mutations. Across all mutations, lncRNAs were significantly upregulated (p<0.0001), except for Y106C, possibly due to the mutation being in a different domain from the other non-hotspot mutations.

Although DIS3 is an essential gene, Western blot analysis revealed that non-hotspot mutations had a 25-fold decrease (p<0.0001) in DIS3 protein expression while hotspot mutations had no change in expression compared to wild-type DIS3. This difference in protein abundance could not be accounted for by changes in RNA expression, implying a post-transcriptional loss. The same loss of DIS3 protein was also observed in the KMS26 cell line which harbors a V171G non-hotspot mutation in DIS3. Loss of DIS3 expression at the protein level was not due to proteasomal degradation, as seen by MG-132 inhibition. However, we observed an increase in the expression of no-go mRNA decay proteins—such as XRN1 and SKIV2L—in the non-hotspot mutations which was not seen in the hotspot mutations.

Mass spectrometry also showed a decrease in DIS3 protein in non-hotspot mutated cells that resulted in a vast downstream difference (|log2FC|>0.49, p<0.05) in the proteome with abundance changes in a median of 1,129 proteins (range=1,112-1,219); however, this was not found in hotspot mutated cell lines (median=3; range=0-42). Non-hotspot mutations resulted in the upregulation of RNA processing genes and histone linkers, consistent with a role in RNA processing and chromatin compaction. Despite these differences we discovered, cell proliferation analysis exhibited no difference in proliferation rates between the hotspot and non-hotspot mutation groups, as well as no difference when compared to wild-type DIS3 cells.

Conclusions

Our findings provide insight into the cellular impact of DIS3 mutations in multiple myeloma, highlighting key differences between hotspot and non-hotspot mutations. These results suggest distinct functional roles for DIS3 mutations which may depend on the location of the mutation. Overall, we observed the molecular effects of DIS3 mutations to further understand the mechanism of pathogenesis.