- Letter to the Editor
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WDR26 and MTF2 are therapeutic targets in multiple myeloma
Journal of Hematology & Oncology volume 14, Article number: 203 (2021)
Abstract
Unbiased genetic forward screening using retroviral insertional mutagenesis in a genetically engineered mouse model of human multiple myeloma may further our understanding of the genetic pathways that govern neoplastic plasma cell development. To evaluate this hypothesis, we performed a tumor induction study in MYC-transgenic mice infected as neonates with the Moloney-derived murine leukemia virus, MOL4070LTR. Next-generation DNA sequencing of proviral genomic integration sites yielded rank-ordered candidate tumor progression genes that accelerated plasma cell neoplasia in mice. Rigorous clinical and biological validation of these genes led to the discovery of two novel myeloma genes: WDR26 (WD repeat-containing protein 26) and MTF2 (metal response element binding transcription factor 2). WDR26, a core component of the carboxy-terminal to LisH (CTLH) complex, is overexpressed or mutated in solid cancers. MTF2, an ancillary subunit of the polycomb repressive complex 2 (PRC2), is a close functional relative of PHD finger protein 19 (PHF19) which is currently emerging as an important driver of myeloma. These findings underline the utility of genetic forward screens in mice for uncovering novel blood cancer genes and suggest that WDR26-CTLH and MTF2-PRC2 are promising molecular targets for new approaches to myeloma treatment and prevention.
To the Editor,
Multiple myeloma (MM) is a common blood cancer derived from terminally differentiated B-lymphocytes called plasma cells (PCs). Despite recent advancements in treatment options, MM remains incurable in the great majority of cases, with no more than half of patients surviving past 5 years [1]. Reasons for poor outcome include tumor heterogeneity and severe limitations in our knowledge base on genetic pathways that drive neoplastic PC development from an early progenitor stage to frank malignancy. Unbiased genetic forward screening using proviral insertional mutagenesis [2] in a dedicated mouse model of human myeloma may lend itself to attacking this knowledge gap. Here, we employ this approach, for the first time, to discover two candidate genes that may yield new opportunities for molecularly targeted myeloma treatments: WDR26 (WD repeat-containing protein 26) and MTF2 (metal response element binding transcription factor 2).
Our experimental strategy for detecting presumptive therapeutic targets in MM is depicted in Fig. 1a. The first step was a tumor induction study in iMycΔEµ mice, a gene-insertion model of the chromosomal T(12;15) translocation that results in deregulated expression of Myc in B-lineage cells [3]. Because T(12;15) is a tumor-initiating event in mouse plasmacytoma [4] and upregulation of MYC is a well-established mechanism of tumor progression in human myeloma [5], the iMycΔEµ transgene served as an ideal “sensitizer” for skewing the oncogenic potency of the murine leukemia virus (MuLV), MOL4070LTR, to plasmablasts and PCs. MOL4070LTR is a modified Moloney-MuLV that contains the LTR U3 enhancer region from the amphotropic MuLV, 4070A [6]. Infection of newborn iMycΔEµ mice with MOL4070LTR resulted in accelerated tumor development (Fig. 1b): 51 of 68 (75%) virus-treated mice developed tumors by 210 days of age, whereas less than a quarter of untreated mice demonstrated malignant growth by 505 days. Histopathological tumor classification relied on immunostaining for T cell (CD3), B cell (Pax5, B220) and PC (CD138) markers to assign tumor-bearing mice with virus-accelerated neoplasms to the B-lineage (31%) or T-lineage (44%). A quarter of mice (25%) contained both B- and T-cell tumors (Fig. 1c). From all mice carrying B-lineage tumors (n = 21), eight individual tumor samples (spleen plus peripheral and deep lymph nodes) were collected on average. Most tumors were categorized as plasmacytoma (Fig. 1d, left) or plasmablastic lymphoma (Fig. 1d, right) in accordance with the Bethesda proposal of lymphoid tumors in mice [7]. A total of 168 tumor specimens were analyzed for common retroviral insertion sites (CIS) as depicted in Additional file 1: Fig. S1. From nearly half a million mapped sequence reads, approximately 45 thousand proviral integration events were extracted. To unequivocally identify CIS, we used a biocomputational algorithm based on Monte Carlo statistics that considered both the number of independent integration sites in a given DNA window and the distance between the sites. We defined a CIS as the minimum genomic region in which 5 to 7 unique insertions were found to be significant at p < 0.05, provided that no more than two insertions were derived from the same tumor. CIS windows ranged from 10 to 40 kb, corresponding to the size of the transcriptional unit of the average mouse gene (~ 30 kb). A total of 171 CIS-tagged candidate genes were identified and rank ordered according to proviral insertion frequency. The top 100 genes are shown in Fig. 1e. Included are many genes one might have expected in a forward genetic screen of neoplastic PC development; e.g., Ccnd2 on Chr 6, Hras on Chr 7 and Myc on Chr 15.
Bioinformatics analysis of the top 100 genes using STRING (string-db.org) demonstrated their tight association with the oncogenic MYC network (Fig. 1f). KEGG analysis (www.kegg.jp) revealed significant enrichment in cancer-relevant pathways including blood cancers such as AML and CML (Fig. 1g). GO analysis of biological processes (geneontology.org) demonstrated strong enrichment in pathways of hematopoiesis, hematopoietic or lymphoid organ development, and regulation of leukocyte differentiation (Fig. 1h). These results underscored the relevance of the top 100 genes for MM and encouraged us to narrow them down to the most promising candidates. This process began with two steps denoted “Filter 1” in Fig. 1a, top right. The first step asked the question whether upregulation of the human orthologs of the top 100 mouse genes predicted to be upregulated by proviral insertion might be associated with inferior survival in human myeloma. We chose the MMRF CoMMpass study to test for associations of gene expression and survival because this study evaluates outcomes in over one thousand patients in a publicly accessible fashion (https://research.themmrf.org). The second filtering step relied on the DepMap data explorer tool (depmap.org/portal), which provides CRISPR and RNAi dependency scores that indicate whether a gene of interest is important and functionally non-redundant in myeloma in vitro. Twenty-two of the top 100 mouse genes (labeled with colored dots in Fig. 1e) passed Filter 1. Next, we performed a rigorous PubMed analysis of the 22 genes for published evidence on their involvement in MM and related diseases (“Filter 2” in Fig. 1a). Only eight genes (green and red in Fig. 1e) promised novelty for myeloma. These candidates proceeded to “Filter 3” in Fig. 1a, which assessed whether shRNA-mediated knockdown (KD) of gene expression inhibited myeloma in cell culture. Three HMCLs were transfected with eight different Mission EHU esiRNAs, and gene KD was verified by qPCR in 8 of 8 cases (not shown). However, significant (p < 0.01) and consistent inhibition (in 3 of 3 cell lines) was only seen in two cases: WDR26 and MTF2 (Fig. 1i–k).
To validate WDR26 and MTF2 in greater depth, we gathered additional clinical and biological data (Fig. 2). Clinical results in support of the contention that WDR26 and MTF2 are important in MM include the circumstance that gene expression was upregulated in smoldering and frank myeloma (Fig. 2a), that message levels in the DREAM Challenge study [8] were elevated in high-risk compared to standard-risk myeloma (Fig. 2b) and that high amounts of mRNA in myeloma cells of CoMMpass patients predicted inferior overall survival (Fig. 2c). To strengthen the biological evidence on the impact of WDR26 and MTF2 in MM, we complemented the KD data shown in Fig. 1i–k with loss-of-function studies using CRISPR-Cas9 engineered gene knockouts (KO) in myeloma cells (Fig. 2d). WDR26 or MTF2 deficiency compromised the growth of myeloma in both bulk suspension (Fig. 2e) and clonogenic soft-agar culture (Fig. 2f). KO led to a significant increase in apoptotic cell death measured with the help of annexin V immunoreactivity (Fig. 2g, h). In vivo studies using HMCL-in-mouse xenografts added further confidence to these results: WDR26 or MTF2 deficient tumors grew more slowly than their normal counterparts (Fig. 2i, j) and thus permitted longer survival of host mice (Fig. 2k). Employment of GFP as reporter of malignant growth produced similar results; e.g., the abundance of tumor cells in the bone marrow of mice harboring WDR26 or MTF2 deficient myeloma was cut in half compared to controls (Fig. 2l, m).
In conclusion, this study used a sensitized forward genetic screen in laboratory mice to nominate WDR26 and MTF2 as candidate myeloma genes. WDR26 is a component of the CTLH complex that is mutated or upregulated in many solid cancers [9]. WDR26 has not been implicated in blood cancers, yet its significance as therapeutic target in carcinomas has been recognized [10]. MTF2, an accessory unit of the PRC2 complex involved in gene repression and growth promotion of various cancers [11], is a validated molecular target in AML [12]. MTF2 is new in myeloma, but PHF19, another accessory unit of PRC2, has emerged as a major player in MM [8, 13,14,15]. Both MTF2 and PHF19 are preferentially overexpressed in high-risk myeloma. Additional research is warranted to elucidate the oncogenic networks of WDR26 and MTF2 in myeloma because this may point to new avenues for molecularly targeted treatments and preventions.
Availability of data and materials
Detailed information on materials and methods used, including KD and KO primer sequences, are available from the corresponding author upon request.
Abbreviations
- AAD:
-
7-Aminoactinomycin D
- AML:
-
Acute myeloid leukemia
- CML:
-
Chronic myeloid leukemia
- CIS:
-
Common retroviral insertion site
- CTLH:
-
Carboxy-terminal to LisH
- GFP:
-
Green fluorescent protein
- HMCL(s):
-
Human myeloma cell line(s)
- LTR:
-
Long terminal repeat
- KD:
-
Knockdown
- KO:
-
Knockout
- LTR:
-
Long terminal repeat
- MM:
-
Multiple myeloma
- MMRF:
-
Multiple Myeloma Research Foundation
- MOL4070LTR:
-
Moloney derived MuLV
- MTF2:
-
Metal response element binding transcription factor 2
- MuLV:
-
Murine leukemia virus
- NGS:
-
Next-generation sequencing
- PC(s):
-
Plasma cell(s)
- PRC2:
-
Polycomb repressive complex 2
- WDR26:
-
WD repeat-containing protein 26
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Acknowledgements
We wish to thank Dr. Linda Wolff, NCI, NIH for providing the batch WV523 of MOL4070LTR used in this study. We are grateful to Drs. Herbert C. Morse III, NIAID, NIH, and Alicia Olivier, University of Iowa, for help with tumor diagnosis and classification. We wish to acknowledge expert technical assistance from MCW Cancer Center core facility staff, especially Galina Petrova, Flow Cytometry Shared Resource, and Donna McAllister, Biomedical Imaging Shared Resource.
Funding
This work was supported by NCI R01CA151354 and the William G. Schuett, Jr., Multiple Myeloma Research Endowment to SJ. Additional support was provided by the Intramural Research Program of the National Institutes of Health, National Cancer Institute, Center for Cancer Research to BM and WD; by the Advancing a Healthier Wisconsin Endowment and Medical College of Wisconsin Cancer Center to JD; and by NCI R01CA236814, DoD CA180190 and the Riney Family Multiple Myeloma Research Program to FZ.
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Contributions
FS and YC performed in vitro and in vivo studies, carried out bioinformatic and statistical analyses, designed the figures, and wrote the manuscript. AD and JDR performed NGS studies and determined CIS. WD performed the tumor induction study, harvested and shipped tissue specimens, and took care of animal husbandry. MP performed Pubmed searches and edited the manuscript. JD contributed to experimental procedures and data analytical approaches. BM and FZ provided infrastructure support and understanding of genetic and biological pathways of mouse plasmacytoma and human myeloma. PH provided infrastructure support and insights into myeloma treatment and prevention. SJ conceived and supervised the study and edited the figures and the manuscript. All authors contributed to reading and approved the final version of the manuscript.
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Studies involving laboratory mice were approved by the institutional IACUC and performed in accordance with prevailing guidelines for the welfare and use of animals in cancer research. Because analysis of clinical data did not contain any personally identifiable information from any sources and did not require approval of the Institutional Review Board, informed consent is not applicable.
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Supplementary Information
Additional file 1
. Fig. S1. Identification of proviral integration sites and candidate driver genes. Genomic DNA was extracted from malignant tissues harvested from MOL4070LTR-infected mice. Approximately 1 μg of genomic DNA was then digested using either MseI or NlaIII. Next, 200 ng of digested DNA was ligated to double-stranded adaptors (NlaIII linker: 5’-GTA ATA CGA CTC ACT ATA GGG CTC CGC TTA AGG GAC CAT G-3’ and 5’-Phos-GTC CCT TAA GCG GAG-C3spacer-3’, MseI linker: 5’-GTA ATA CGA CTC ACT ATA GGG CTC CGC TTA AGG GAC-3’ and 5’- Phos-TAG TCC CTT AAG CGG AG-C3spacer-3’). Following adaptor ligation, DNA was digested with EcoRV to eliminate the internal proviral fragment (indicated by red cross). EcoRV-digested DNA was then amplified (primary PCR) using primers annealing to the adaptor (5’-GTA ATA CGA CTC ACT ATA GGG CTC CG-3’) and the proviral LTR (5’-GCT AGC TTG CCA AAC CTA CAG GTG G-3’). PCR products were diluted 1:50 in sterile water. Two microliters of diluted PCR product was re-amplified (secondary PCR) using nested primers annealing to the adaptor (5’-AGG GCT CCG CTT AAG GGA C-3’) and proviral LTR (5’-CCA AAC CTA CAG GTG GGG TCT TTC-3’). Amplicons from the second round of PCR were purified to remove unincorporated primers and nucleotides and directly sequenced on an Illumina platform. Raw sequences were trimmed to remove adaptors and viral sequences and mapped to the mouse reference genome. Candidate driver genes were identified using Monte Carlo simulation as previously described (PMID: 21931803).
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Sun, F., Cheng, Y., Riordan, J.D. et al. WDR26 and MTF2 are therapeutic targets in multiple myeloma. J Hematol Oncol 14, 203 (2021). https://doi.org/10.1186/s13045-021-01217-9
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DOI: https://doi.org/10.1186/s13045-021-01217-9