TET2 Down-Regulation Confers Bortezomib Resistance in Multiple Myeloma

Introduction: TET2, one of the ten-eleven-translocation proteins (TET1-3), is an epigenetic modifying enzyme that catalyzes the oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) to promote DNA demethylation. Acquired somatic deletions or loss-of-function mutations have been found in various myeloid and lymphoid malignancies, including acute myeloid leukemia (16%), myelodysplastic syndromes (19%), chronic myelomonocytic leukemia (44%), etc. These genetic abnormalities are associated to aggressive disease and poor outcome, suggesting its function as a tumor suppressor. Interestingly, vitamin C mimics TET2 restoration in TET2-deficient mouse hematopoietic stem and progenitor cells and thus suppresses leukemia progression. While TET2 mutations have also been observed in patients with multiple myeloma (MM; ~7%), the role of TET2 remains unknown in MM cells.

Materials and Methods: DNAseq and RNAseq were conducted to examine genetic aberrations and transcriptional dysregulation of TET2 and its related genes in primary patient samples and drug-naive vs. -resistant MM cells. Flow cytometry, qPCR, Western blot analysis, and immunofluorescence were utilized to monitor apoptosis or mRNA and protein levels of target genes. Dot blot was performed to examine 5hmC and 5mC. TET2 knockdown by shRNA was carried out to evaluate its function. Analysis of the GEP databases (R2: Genomics Analysis and Visualization Platform) was performed to validate the clinical significance of TET2 and its related genes in MM patients.

Results: GEP analysis revealed that expression of TET2 was increased during myelomagenesis while decreased with increasing 1q21 copy number in MM patients. In contrast to TET1 and TET3, of which low mRNA levels were associated with favorable prognosis, TET2 down-regulation significantly correlated with poor outcome. Interestingly, TET2 negatively correlated with expression of a majority of 1q21.1-23.3 genes. KEGG analysis showed that TET2-related down-regulated genes were enriched for oxidative phosphorylation, proteasome, and protein processing in ER, largely overlapping with those for TET1 and TET3. DNAseq unveiled multiple TET2 mutations (e.g., I1762V) in MM patients, which were less frequent than TET1 mutations. RNAseq demonstrated down-regulation of TET2 and TET3 but up-regulation of TET1 in CD138+ cells of MM patients. Notably, MM cells acquired bortezomib (Btz) resistance exhibited a sharp down-regulation of TET2 at both mRNA and protein levels, together with a reduction in 5hmC methylation. Unlike its nuclear localization in drug-naive cells, strong TET2 signal was observed in cytoplasm of Btz-resistant cells. Whereas vitamin C treatment resulted in an increase in 5hmC, it clearly down-regulated TET2 expression, resulting in a marked reduction in the lethality of Btz and ixazomib, but not carfilzomib. Similar phenomena were observed when metformin was used, but multiple TET2 inhibitors failed to do so. Consistent with these findings, TET2 knockdown (KD) by shRNA in drug-naive cells resulted in resistance to Btz and and ixazomib, but not carfilzomib. In contrast, overexpression of wild-type TET2 in Btz-resistant cells significantly increased Btz sensitivity, but its I1762V mutant was less active, both of which could be prevented by administration of vitamin C. Interestingly, blockade of autophagy by chloroquine largely restored TET2 protein level, sensitizing MM cells to Btz. RNAseq showed that TET2 knockdown mainly up-regulated the expression of numerous genes, including a number of 1q21 genes. qPCR and Western blot analysis confirmed up-regulation of the 1q21 genes, including CKS1B, MCL1, PSMD4, and ARNT (encoding HIF-1β), in TET2 KD drug-naive cells, while overexpression of wild-type TET2 or its I1762V mutant moderately but clearly down-regulated these genes in Btz-resistant cells. Similar results were obtained when TET2 was down-regulated by vitamin C. DNA methylation sequencing showed robust genome-wide alterations in TET2 KD cells, mostly enriched for metabolism. Seahorse analysis revealed that TET2 KD increased oxidative phosphorylation via mitochondrial respiration, but reduced glycolysis. Last, TET2 KD in MM cells promoted tumor growth in vivo in a xenograft model.

Conclusion: TET2 is a tumor suppressor in MM, while loss of TET2 confers acquired Btz resistance via up-regulation of 1q21 genes.

Kumar:AbbVie,: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen,: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda,: Membership on an entity's Board of Directors or advisory committees, Research Funding; Adaptive,: Membership on an entity's Board of Directors or advisory committees, Research Funding; KITE,: Research Funding; MedImmune/Astra Zeneca,: Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck,: Research Funding; Novartis,: Research Funding; Roche: Research Funding; Sanofi: Research Funding; Oncopeptides: Other: Independent review committee.