Utx Insufficiency Cooperates with Braf V600E in the Induction of Myeloma in Mice
Authors: Rizq Ola , Mimura Naoya , Oshima Motohiko , Momose Shuji , Nakajima-Takagi Yaeko , Aoyama Kazumasa , Saraya Atsunori , Iseki Tohru , Sakaida Emiko , Nakaseko Chiaki , Okuno Yutaka , Honda Hiroaki , Tamaru Jun-ichi , Iwama Atsushi 
Published: October 2019

Abstract
Introduction:
Dysfunction of epigenetic pathways has been frequently implicated in hematological malignancies. In multiple myeloma (MM), EZH2, a methyltransferase that induces histone H3 lysine 27 trimethylation (H3K27me3), acts as an oncogene as evidenced by its overexpression, which was found to be positively correlated with disease progression (Kalushkova et al. PloS One. 2010). We have shown that inhibition of both EZH2 and its homolog EZH1 is effective in eradicating MM cells in vitro and in vivo (Rizq et al. Clin Cancer Res. 2017). In addition, inactivating somatic mutations in UTX/KDM6A, an X-linked histone demethylase that removes di- and tri-methyl groups from H3K27, are found in 3 - 10% of MM patients (van Haaften et al. Nat Genet. 2009 and Pawlyn et al. Clin Cancer Res. 2016), indicating a tumor suppressive role for UTX, which has yet to be delineated. Up till now, no mouse model has been generated to test Utx insufficiency in post germinal center (GC) B cells and plasma cells. On the other hand, an activating mutation V600E in the BRAF kinase gene is closely associated with aggressive disease features such as extramedullary disease and shorter overall survival in MM patients (Andrulis et al. Cancer Discov. 2013) and could accelerate induction of myeloma in mice.
Methods:
To investigate whether loss of Utx cooperates with Braf V600E in myelomagenesis in mice, we generated and analyzed mice with conditional knock-out allele of Utx and/or knock-in allele of Braf V600E combined with Cγ1-Cre allele, in which Cre is activated by immunization in post GC B cells.
Results:
Loss of Utx and Braf V600E synergistically induced post GC B-cell lymphoma and plasma cell neoplasms in mice and significantly shortened the survival of mice compared with control mice and either allele alone. Utx-/-Braf V600E females succumbed to death earlier than Utx-/YBraf V600Emales and Utx-/+Braf V600Efemales. Of note, plasma cell neoplasms developed at a high frequency in Utx-/YBraf V600Emales and Utx-/+Braf V600Efemales and, less frequently, in Utx-/-Braf V600E females. Mice with plasma cell neoplasms showed expansion of CD138+ plasma cells in bone marrow as well as spleen and/or lymph nodes, exhibiting extramedullary disease. Loss of Utx alleles and expression of Braf V600E were confirmed by genomic PCR of plasma cells. Importantly, the clonality of plasma cells was demonstrated by genomic PCR detecting rearrangements of immunoglobulin heavy and light chain genes. In addition, M protein was detected by serum protein electrophoresis (SPEP) at a high frequency. Notably, we were able to establish murine myeloma cell lines from moribund compound mice. These cells readily engrafted in the bone marrow of NOG mice after transplantation and caused myeloma-associated phenotypes including paraplegia in recipient mice. Interestingly, Utx-/-Braf V600E cells were sensitive to dual inhibition of EZH2 and EZH1 but not to specific inhibition of EZH2 in culture. They also showed decreased susceptibility to proteasome inhibitors when compared with human MM cell lines. To gain insight into the changes in the transcriptional landscape following Utx loss, we performed RNA sequencing (RNA seq) and then gene set enrichment analysis (GSEA). We found positive enrichment of gene sets related to Myc, implying that Myc is one of the main drivers of myelomagenesis in our mouse model. In addition, gene sets related to MM were significantly enriched following Utx loss. We are now working on ChIP sequencing (ChIP seq) of UTX-related histone modifications to evaluate the epigenetic impact of Utx loss on myelomagenesis.
Conclusion:
Utx insufficiency cooperates with Braf V600E in the induction of myeloma in mice. Our mouse model is a promising tool for understanding the role of epigenetic dysregulation in the pathogenesis of MM and evaluating novel anti-myeloma agents.

Disclosures
Okuno: Celgene: Research Funding. Tamaru:Nichirei Bioscience INC.: Research Funding; Takeda Pharmaceutical Company Limited: Speakers Bureau.