Epigenetic loss of m¹A RNA demethylase ALKBH3 in Hodgkin lymphoma targets collagen, conferring poor clinical outcome

TO THE EDITOR:

The dysregulation of RNA and protein levels, in comparison with the counterpart normal tissues, is a constant molecular feature of all human malignancies. Many causes can be invoked as culprits of the altered transcriptome and proteome of cancer cells, but recently, the emergence of disrupted chemical modifications of the RNA molecule (that can affect its stability, targeting, or translation) is gaining momentum as a primary cause. In this regard, the emergence of a so-termed altered epitranscriptome,^1,2  including malignant hematopoiesis, is starting to be described in tumorigenesis.³  More than 100 distinctly modified nucleotides have been described in RNA molecules.^4,5  Although some of these, such as cytosine methylation, have also been described in DNA, DNA has a smaller repertoire of modified nucleotides, whereas RNA molecules can show a more diverse spectrum of modifications, including, among others, pseudouridine or queuosine. However, the most abundant modification of RNA is the methylation of adenosine (A) in the form of m⁶A and to a lesser extent of m¹A. m⁶A is not only the most common internal modification of messenger RNA (mRNA), affecting various facets of RNA metabolism,^5,6  but it is also relevant to microRNAs, controlling their maturation and expression levels (as has been described for the tumor-suppressor microRNA let-7).⁷  The other methylated adenosine present in RNA, m¹A, is typically found in abundant noncoding RNAs, and is also observed around the start codon upstream of the first splice site and in the 5′ untranslated regions (UTRs) of mRNAs.^8-10  The m¹A location is opposed to m⁶A, which is enriched in 3′ UTRs and near stop codons.^11,12 

The discovery of an m⁶A eraser, FTO,¹³  provided the first evidence of reversible posttranscriptional modifications in mRNAs, further generating interest in the epitranscriptome and its possible impact in cancer biology. Since then, complex pathways of highly regulated enzymes that add (writers) or remove (erasers) the RNA-modification marks have been dissected at the physiological level, but little is still know about their possible alterations in the cancer arena. Focusing on the methylation of adenosine, the most prevalent type of RNA modification, and knowing that promoter cytosine guanine dinucleotide (CpG) island hypermethylation-associated transcriptional silencing is a common mechanism of gene inactivation in cancer cells,^14,15  we interrogated the presence of this type of epigenetic inactivation in the genes encoding the writers and erasers of the m⁶A and m¹A marks. Thus, herein, we have investigated cancer-specific DNA-methylation changes at the 5′-end promoter regions of the METTL3 and METTL14 core subunits of the m⁶A writer complex; the m⁶A erasers ALKBH5 and FTO; the m¹A writers TRMT6, TRMT61A, TRMT10C, and TRMT61B; and the m¹A eraser ALKBH3.⁵  To uncover candidate epigenetic changes in these enzymes, we first data-mined a collection of close to 1000 human cancer cell lines in which we have characterized the DNA-methylation profiles.¹⁶  The promoter-associated CpG islands of METTL3, METTL14, ALKBH5, FTO, TRMT6, TRMT61A, TRMT10C, and TRMT61B were unmethylated in all of the assessed cancer cell lines (supplemental Data Set 1, available on the Blood Web site). However, the ALKBH3 promoter CpG island was commonly methylated in lymphoma cell lines (35%; 23 of 66) among different cancer cell line types (Figure 1A; supplemental Data Set 1). The second tumor type with the highest percentage of ALKBH3 hypermethylation was breast cancer (10.20%; 5 of 49), where this epigenetic alteration has recently been described.¹⁷  Although ALKBH3 has been reported to be upregulated in head and neck cancer,¹⁸  data-mining of the available transcriptional patterns in the studied cancer cell line set¹⁶  showed that ALKBH3 hypermethylation was associated with mRNA downregulation (Figure 1B). This genomic locus was found unmethylated in all of the different normal tissue samples analyzed from the TCGA data set (n = 730; supplemental Data Set 2). Further detailed in silico analyses of the hematological malignancies according to their subtype identified that ALKBH3 promoter CpG island hypermethylation was most frequent in Hodgkin lymphoma cell lines (44%; 4 of 9), followed by Burkitt lymphoma (38%; 5 of 13), other non-Hodgkin lymphoma cell lines (37%; 12 of 32), and anaplastic large cell lymphoma (33%; 1 of 3) (Figure 1C). Data-mining of the available microarray-expression results¹⁶  demonstrated that ALKBH3 hypermethylation was linked to transcript downregulation in the cell lines derived from hematological malignancies (Figure 1D). Further in silico analyses showed that the 5′ end of of ALKBH3 was unmethylated in all tested samples of peripheral blood mononuclear cells, naive B lymphocytes, and naive T lymphocytes obtained from healthy donors (supplemental Data Set 3). Thus, the cancer-specific DNA-methylation event at the ALKBH3 promoter became our focus of interest and was herein further studied in the context of Hodgkin lymphoma, the lymphoma type exhibiting the highest hypermethylation frequency.

Having found the ALKBH3 CpG island–methylation patterns shown herein, we assessed in detail the possible association of the hypermethylation event with the loss of ALKBH3 gene expression at the RNA and protein levels. We performed bisulfite genomic sequencing of multiple clones in the Hodgkin lymphoma cell lines KM-H2, L540, HS445, and HD-MY-Z using primers that encompassed the transcription start site–linked CpG island (supplemental Methods). We observed that the 5′-end CpG island of ALKBH3 in the L540 and KMH2 cell lines was hypermethylated (Figure 1E), whereas the HD-MY-Z and HS445 cells were unmethylated at these sites (Figure 1E). Naive B cells were also found unmethylated (Figure 1E). These data corroborated the DNA-methylation profiles obtained by the DNA-methylation microarray platform (Figure 1F). The methylated ALKBH3 cell lines KM-2 and L540 did not express the ALKBH3 RNA transcript and protein, as determined by quantitative reverse transcription polymerase chain reaction (PCR) and western blot, respectively (Figure 1G). Expression of ALKBH3 RNA and protein was found in the unmethylated cell lines HD-MY-Z and HS445 (Figure 1G). Treatment of the ALKBH3-hypermethylated cell lines with the DNA-demethylating agent 5′-aza-2′-deoxycytidine restored ALKBH3 RNA and protein expression (Figure 1H). The association between ALKBH3 promoter hypermethylation and gene silencing was further validated in 2 additional Hodgkin cell lines not included in our original screening, SUP-HD1 and HS611-T (supplemental Figure 1). Overall, these results indicate the presence of cancer-specific promoter CpG island hypermethylation–associated loss of ALKBH3 gene expression in Hodgkin lymphoma cells. DNA methylation–associated silencing of ALKBH3 was also validated in 13 cell lines derived from Burkitt lymphoma and other non-Hodgkin lymphomas (supplemental Figure 2).

Once we had shown the presence of ALKBH3 CpG island hypermethylation–linked transcriptional inactivation in human Hodgkin lymphoma cell lines, we then wondered about the molecular targets of ALKBH3 loss in these cells. Beyond its role in DNA repair,^19-21  ALKBH3 exhibits an RNA-demethylase activity for m¹A mRNA.⁹  Thus, the epigenetic inactivation of ALKBH3 in Hodgkin lymphoma cells hinders a downstream m¹A RNA-demethylating event and this can contribute to the biology of these malignancies. To prove this idea, we performed RNA high-throughput sequencing using an m¹A antibody to enrich m¹A-modified mRNA fragments⁸  in HD-MY-Z cells, unmethylated at the ALKBH3 promoter CpG island (Figure 1E-F), and expressing the transcript and protein (Figure 1G), transfected with a doxycycline-inducible short harpin RNA (shRNA) against ALKBH3 (Figure 2A). Read-mapping statistics from the m¹A-sequencing samples are shown in supplemental Table 1.

Upon efficient shRNA-mediated depletion of ALKBH3 in the HDMYZ cells (Figure 2A; supplemental Figure 3), our epitranscriptomic approach identified that, whereas 2511 m¹A peaks (corresponding to 1522 transcripts) were unchanged in both conditions (supplemental Table 2), the shRNA-mediated depletion of ALKBH3 in HD-MY-Z cells caused a change of 165 m¹A peaks (corresponding to 147 transcripts; supplemental Table 3). Most importantly, the induced downregulation of ALKBH3 caused a gain of m¹A, being observed in 159 m¹A sites (96% of the total 165 sites with distinct m¹A content) corresponding to 141 transcripts (supplemental Table 3), a finding that it is consistent with the described role of ALKBH3 as an m¹A RNA demethylase.⁹  The most frequent location of the differential m¹A peaks was the 5′ UTR of the transcripts (Figure 2B), in agreement with previous reports for this epitranscriptomic mark,^8,9,22  although they were also observed in other transcript regions such as exons, 3′ UTR, and introns. To better characterize the identified set of 147 genes with significantly distinct m¹A content upon ALKBH3 depletion, we performed a gene functional annotation by gene-set enrichment analysis using gene ontology (GO) signature collections. We observed an overrepresentation of GO biological processes, molecular function, cellular component, and reactome pathways related to cell migration (such as ”locomotion” and “cell motility”), the cytoskeleton (such as “cytoskeleton organization” and “actin filament based process”), and the microenvironment (such as “extracellular matrix structural component”, “extracellular matrix organization,” and collagen formation, degradation, and trimerization) (Figure 2C; supplemental Figure 4). These processes are highly relevant in Hodgkin lymphoma, in which the malignant cells are surrounded by a large tumor microenvironment that exerts a critical role in the disease²³  and the cytoskeleton, through association with extracellular connective tissues, acts as a “guardian” for tissue stabilization and the prevention of cell migration. In this regard, the 2 transcripts with the highest number of gained m¹A peaks upon ALKBH3 depletion corresponded to 2 collagens, type I α 2 (COL1A2) and type I α 1 (COL1A1) (supplemental Table 4), critical noncellular components of the Hodgkin lymphoma microenvironment.^24,25  Genome browser screenshots for differential m¹A peaks in COL1A2 and COL1A1 are shown in supplemental Figure 5. Presence of m¹A in 5′ UTRs correlates with increased protein expression,^8,9  and we indeed observed that the shRNA-mediated depletion of ALKBH3 in HD-MY-Z cells induced high protein-expression levels of both collagens (Figure 2D). We also performed the reverse experiment, in which we restored ALKBH3 expression by transduction in the hypermethylated/silenced cell line L540 (supplemental Figure 6). Herein, we found the opposite scenario compared with the shRNA model (Figure 2D): the recovery of ALKBH3 expression reduced m¹A content in the COL1A2 mRNA and decreased COL1A2 protein levels (supplemental Figure 6). The link between ALKBH3 epigenetic silencing and collagen expression was further reinforced by showing how restoration of ALKBH3 expression in hypermethylated KM-H2 and L540 cells, upon the use of the DNA-methylating agent, decreased COL1A2 levels (supplemental Figure 7).

To further improve the real representation of the disease beyond the limitations of established cell lines, we also studied the relevance of ALKBH3 aberrant CpG island methylation in human primary Hodgkin lymphomas. To achieve this goal, we determined the ALKBH3 promoter-methylation status by bisulfite-coupled pyrosequencing (supplemental Methods) in a collection of 80 well-characterized primary Hodgkin lymphoma cases whose clinicopathological features are shown in supplemental Table 5. We detected ALKBH3 promoter hypermethylation in 18% (14 of 80) of the primary Hodgkin lymphoma cases. The presence of ALKBH3 aberrant methylation was not associated with any of the studied clinicopathological parameters (supplemental Table 5). We also analyzed ALKBH3 expression by western blot in 4 samples of the studied primary Hodgkin lymphoma cases, observing that ALKBH3 CpG island hypermethylation was associated with protein loss (supplemental Figure 8). Importantly, ALKBH3 epigenetic silencing was also associated with COL1A2 and COL1A1 overexpression, whereas an unmethylated ALKBH3 CpG island was linked to the absence of collagen expression (supplemental Figure 8), mimicking the results found in the cell line models (Figure 2D). Most importantly, we wondered whether ALKBH3 hypermethylation also conferred any clinical outcome value. We observed that ALKBH3 hypermethylation was associated with shorter overall survival (OS) (log-rank; P = .010; hazard ratio [HR] = 4.231; 95% confidence interval [CI] = 1.290 - 13.872) (Figure 2E) in the studied Hodgkin lymphoma cohort. In the multivariate analysis, ALKBH3 promoter hypermethylation was shown to be an independent prognostic factor for reduced OS (HR = 4.752; 95% CI = 1.277-17.683; P = .020) (Figure 2F).

Overall, the described research provides an illustrative example that aberrations of the so-called epitranscriptome occur in human lymphomas, herein shown by the epigenetic silencing of the m¹A RNA demethylase ALKBH3 in Hodgkin lymphoma. The epigenetic event we describe shifts the m¹A targeting of migration and cytoskeleton-related genes, and provides an independent biomarker of poor OS in this disease of otherwise good prognosis.