Loss of MYSM1 inhibits the oncogenic activity of cMYC in B cell lymphoma

Abstract MYSM1 is a chromatin‐binding protein, widely investigated for its functions in haematopoiesis in human and mouse; however, its role in haematologic malignancies remains unexplored. Here, we investigate the cross‐talk between MYSM1 and oncogenic cMYC in the transcriptional regulation of genes encoding ribosomal proteins, and the implications of these mechanisms for cMYC‐driven carcinogenesis. We demonstrate that in cMYC‐driven B cell lymphoma in mouse models, MYSM1‐loss represses ribosomal protein gene expression and protein synthesis. Importantly, the loss of MYSM1 also strongly inhibits cMYC oncogenic activity and protects against B cell lymphoma onset and progression in the mouse models. This advances the understanding of the molecular and transcriptional mechanisms of lymphomagenesis, and suggests MYSM1 as a possible drug target for cMYC‐driven malignancies.

Intracellular staining for flow cytometry was performed as previously described 5,8 . Briefly, the cells were fixed in 2% paraformaldehyde (PFA) in PBS with 2% FCS at 37 C for 10 minutes, and permeabilized in 90% methanol for 30 minutes on ice. The cells were stained with intracellular antibodies: Alexa Fluor 488 anti-p53 (clone 1C12, Cell Signaling), or unconjugated anti-cMYC (clone D84C12, Cell Signaling) or anti-eEF1G (EPR7200, Abcam) with Alexa Fluor 488 antirabbit IgG highly cross-adsorbed secondary antibody (Life Technologies), or appropriate isotype controls. All data were acquired on FACS Canto II flow cytometer (BD Biosciences) and analyzed with FACS Diva (BD Biosciences) or FlowJo (Tree Star) software.

Protein Synthesis Rate Measurements
Analysis of protein synthesis rates was performed using the O-propargyl-puromycin (OPP) incorporation method. Briefly, cells were cultured in the presence of 20 μM OPP for 30 minutes, stained with Fixable Viability Dye eFluor506 (eBioscience), fixed in 2% paraformaldehyde (PFA) in PBS with 2% FCS at 37 C for 10 minutes, and permeabilized in 90% methanol for 30 minutes on ice. The cells were then washed with PBS, and staining for OPP incorporation using the Click-iT™ Plus OPP Alexa Fluor 488 Protein Synthesis Assay Kit (Life Technologies, Thermo Fisher Scientific) according to the manufacturer's protocols. Samples were analyzed by flow cytometry on FACS Canto II with FACS Diva software (BD Biosciences).

RNA Isolation and qPCR
RNA isolation from cell lines was carried out using the MagMAX total RNA kit (Ambion, Life Technologies) according to the manufacturer's protocol. RNA quality was assessed on Bioanalyzer RNA Pico chips (Agilent), and cDNA was prepared using the qScript XLT cDNA Supermix (Quanta Biosciences) with 2-5ng RNA input per reaction. qPCRs were performed on a StepOnePlus instrument with Power SYBR Mastermix (Applied Biosystems, Life Technologies). The primers were purchased from IDT Technologies, and the sequences are provided in Supplemental Table S2.

ChIP-Seq data analyses
The following ChIP-Seq datasets were included in the analyses: (I) cMYC ChIP-Seq from HPC7 murine hematopoeitic progenitor cells from Wilson NK et. al. 10,11 ; (II) MYSM1 ChIP-Seq from Ba/F3 murine B cell progenitors stably expressing 3xFLAG-tagged MYSM1 12 . The reads were mapped to the UCSC mouse mm9 reference genome with Bowtie 1.0.0 13 , and chromatin binding sites identified using peak detection algorithm MACS1.4.1 14 , with comparisons for read enrichment against control input DNA from the same cells. Normalized sequence read density profiles (bigwig) were generated with Homer tool 15 and visualized with IGV 16 .

Statistical analyses
Statistical comparisons were performed with Prism 7.01 (GraphPad), using Student's t-test for two groups, ANOVA for multiple comparisons, and Log-rank (Mantel-Cox) test for survival data.  Figure S1. Supplemental cMYC and MYSM1 ChIP-qPCR analysis. The data demonstrates the binding of cMYC at known MYC target genes Ncl and Cdk7, the binding of MYSM1 at MYSM1 target genes Npas4 and Mgmt, and the binding of both transcriptional regulators at Rpl11, consistent with previous data and confirming the specificity of the ChIP analyses. Data was acquired in Ba/F3 cells and is from one experiment. All Ct values were normalized to those of the pro-opiomelanocortin (Pomc) gene, which serves as a negative binding region. Enrichment was calculated relative to input DNA.