Mutated RAS-associating proteins and ERK activation in relapse/refractory diffuse large B cell lymphoma

Alexandre Benoit,Sarit Assouline,Vincent Mingyi Luo,Hsiang Chou,Koren K Mann,Elisabeth Bou-Petit,Svetlana Dmitrienko,Melissa Lu,Cynthia Guilbert,Roger Estrada-Tejedor,Ryan D Morin,Nathalie A Johnson

doi:10.1038/s41598-021-04736-0

Abstract

Diffuse large B cell lymphoma (DLBCL) is successfully treated with combination immuno-chemotherapy, but relapse with resistant disease occurs in ~ 40% of patients. However, little is known regarding relapsed/refractory DLBCL (rrDLBCL) genetics and alternative therapies. Based on findings from other tumors, we hypothesized that RAS-MEK-ERK signaling would be upregulated in resistant tumors, potentially correlating with mutations in RAS, RAF, or associated proteins. We analyzed mutations and phospho-ERK levels in tumor samples from rrDLBCL patients. Unlike other tumor types, rrDLBCL is not mutated in any Ras or Raf family members, despite having increased expression of p-ERK. In paired biopsies comparing diagnostic and relapsed specimens, 33% of tumors gained p-ERK expression, suggesting a role in promoting survival. We did find mutations in several Ras-associating proteins, including GEFs, GAPs, and downstream effectors that could account for increased ERK activation. We further investigated mutations in one such protein, RASGRP4. In silico modeling indicated an increased interaction between H-Ras and mutant RASGRP4. In cell lines, mutant RASGRP4 increased basal p-ERK expression and lead to a growth advantage in colony forming assays when challenged with doxorubicin. Relapsed/refractory DLBCL is often associated with increased survival signals downstream of ERK, potentially corresponding with mutations in protein controlling RAS/MEK/ERK signaling.

Highlights

Abbreviations activated B-cell (ABC) Activated B-cell COO Cell of origin CFU Colony forming unit DLBCL Diffuse large B cell lymphoma GTPase activating proteins (GAP) GTPase-activating protein GCB Germinal center B-cell guanyl exchange factors (GEF) Guanyl exchange factor Molecular Dynamics (MD) Molecular dynamics Molecular Operating Environment (MOE) Molecular operating environment phorbol 12-myristate 13-acetate (PMA) Phorbol 12-myristate 13-acetate pMIG PMSCV-IRES-GFP PTM Post-translational modification RAS Guanyl Releasing Protein 4 (RASGRP4) RAS guanyl releasing protein 4
We hypothesized that mutations in RAS-associating proteins give a gain of function to GEFs and effectors or a loss of function to GAPs, which will result in enhanced downstream signaling and resistance to R-CHOP
We further explored one candidate GEF, RAS Guanyl Releasing Protein 4 (RASGRP4), which was previously shown to play an oncogenic role in DLBCL21

Summary

Introduction

Abbreviations ABC Activated B-cell COO Cell of origin CFU Colony forming unit DLBCL Diffuse large B cell lymphoma GAP GTPase-activating protein GCB Germinal center B-cell GEF Guanyl exchange factor MD Molecular dynamics MOE Molecular operating environment PMA Phorbol 12-myristate 13-acetate pMIG PMSCV-IRES-GFP PTM Post-translational modification RASGRP4 RAS guanyl releasing protein 4. R-CHOP resistance mechanisms in vivo are unclear, several means have been postulated[5] These include downregulation of the target for rituximab, CD20, co-expression of MYC and BCL-2, overexpression of other anti-apoptotic proteins, and sustained survival signals from NF-κB and E RK6–8. Based on the importance of RAS/MEK/ERK activation in other resistant tumors, we predicted activation of this pathway in relapsed/refractory DLBCL (rrDLBCL). We hypothesized that mutations in RAS-associating proteins give a gain of function to GEFs and effectors or a loss of function to GAPs, which will result in enhanced downstream signaling and resistance to R-CHOP. While no RASGRP4 mutations were identified in this study, we show that RASGRP4 mutations observed in rrDLBCL patient samples enhanced p-ERK activation, as well as increased resistance to doxorubicin

Methods

Results

Conclusion