Epigenetically Programmed Defenseless Signaling in Malignant Lymphoma
Authors: Yamagishi Makoto , Katano Harutaka , Hishima Tsunekazu , Ota Yasunori , Okada Seiji , Watanabe Toshiki 
Published: October 2019

Abstract
B-cell receptor (BCR) signaling is a major source of gene expression signature important for B cell survival, functions, and development. The integrated signaling amplitude should be equilibrated; when chronically activated by genetic perturbations or other mechanisms, BCR signaling has been accepted as a stem in the pathogenesis of malignant lymphoma/leukemia. The promiscuous signaling network without appropriate alleviation is an essential property of cancers.
Diffuse large B-cell lymphoma (DLBCL) with aggressive phenotypes often associates with BCR signaling activation and epigenetic abnormalities, both of which are well correlated with lymphoma subtypes and disease progression. However, the relationship of the two fundamental characteristics is unknown. It is also still unclear the mechanism by which the expression of functionally important genes is continuously deregulated.
microRNAs (miRNAs) are an emerging class of intrinsic buffering molecules that have diverse functions in mainly post-transcriptional regulation through miRNA-RISC (miRISC) formation. Using optimized RISC-capture assay, we revealed that multiple BCR signaling factors were persistently regulated by miRNA in human B cells. Clinical samples from newly diagnosed patients with DLBCL (n=83) showed epigenetic loss of an essential miRNA set (miR-200c, miR-203, miR-31). Conventional screening and RISC profiling identified multiple targets (CD79B, SYK, PKCbII, PLCg1, IKKb, NIK, MYD88, PI3K class I (a/b/d/g), RasGRP3); the miRNA-orchestrated overlapping interactome suggested multiple regulatory windows in signaling pathways, which implied their compensatory roles. The shared components within the interconnected signaling pathways were under constant surveillance by the miRNAs. We demonstrated that simultaneous depletion of the key miRNAs enhanced translation of the multiple targets and caused seamless signaling crosstalk among canonical/noncanonical NF-kB pathways, PI3K-Akt-mTOR and Ras-Erk cascades, and downstream of BCR and BAFF receptors. In addition to the miRNA loss, mTOR activation was also positively correlated with signaling amplitude, offering the possibility of selective therapy targeting the translation machinery. The defenseless signaling crosstalk appeared to be a prerequisite for lymphoma development because the essential miRNA set were functionally lost in all tested lymphoma samples.
Surprisingly, a common epigenetic mechanism was identified. Polycomb-mediated H3K27me3 accumulation and reciprocal H3K4me3 loss were frequently observed at the miRNAs loci in primary DLBCL samples. We found that lymphoma-associated deregulation (mutation and/or expression change) of EZH2 and MLL2 cooperated to cause the sustained signaling activities of NF-kB, Ras-Erk, PI3K-Akt and mTOR pathways through miRNA silencing en masse. Of note, the perturbation of histone-modifying polycomb and trithorax groups resulted from variations of pooled miRNAs and signaling integrity. The identified coherent circuit may be a source of pathological robustness and phenotypic convergence.
Most malignant B cell clones have some kind of genetic lesions. In particular, recurrent variations have been identified within signaling cascades, where miRNA-formed cooperative gene interference (CGI) could buffer the signaling noise. We re-established some experimental models that mimicked the signaling cascade observed in clinically established malignant clones. We experimentally confirmed the buffering roles of the key miRNAs against genetic mutation/alteration of CARD11, A20, MYD88 and PTEN. Reciprocally, coordinated reduction of the functional miRNAs promoted biological processes caused by genetic perturbations. It is therefore conceivable that systematic redundancy of miRNA is inherently required for robustness against genetic alterations that are acquired in lymphoma evolution.
We illustrate an example of a biological masterplan comprised of signaling pathways, compensatory actions of multi-layered miRNAs, translation regulation and epigenetic mechanisms. Malignant cell opts for epigenetic reprogramming to stabilize mutation-driven processes and to surmount the demarcation threshold of transformation. Reversing the plastic characteristics of epigenetic alterations is expected in future clinical settings.

Disclosures
Yamagishi: Daiichi Sankyo Co., Ltd.: Research Funding. Watanabe:Daiichi Sankyo Co., Ltd.: Research Funding.