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

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.

Figure 1

(A) Representative images of p-ERK in matched pairs of diagnostic and rrDLBCL samples. (B) Gain of phosphorylation of ERK in rrDLBCL tissues compared to diagnostic DLBCL tissues, including matched paired biopsies.

Figure 2

(A) Proportion of non-synonymous, synonymous and truncated mutations in each type of RAS-associating proteins. (B) RAS mRNA expression level is higher in rrGCB-DLBCL samples compared to rrABC-DLBCL samples. GEF (C), GAP (D), and Effector (E) mRNA expression level is higher in rrGCB-DLBCL samples compared to rrABC-DLBCL samples. (unpaired t-test; * = p < 0.05, ** = p < 0.01, *** = p < 0.001).

Figure 3

(A) Diagram on relevant domains within RASGRP4 with mutations highlighted. (B) Graphical representation of the three HRAS-RASGRP4 complexes predicted after applying molecular docking and molecular dynamics simulations. Mutations under study are highlighted. (C) Comparison of the intramolecular H-bond pattern established with the wild-type protein (left) and D404N and R280K mutated amino acids (right). (D) Changes on the intermolecular H-bond pattern between HRAS and RASGRP4 that could explain the different stabilities of the complexes. The first residue refers to HRAS and the second to RASGRP4.

Figure 4

Mutant RASGRP4^D404N Phoenix AMPHO cells (A) and RASGRP4^D404N GCB-DLBCL (B) have a higher level of p-ERK than RASGRP4^WT cells in normal serum condition (10% FBS). AKT and p-AKT (Ser473) were not changed by mutational status.

Figure 5

(A) Growth curves of stable cell lines show no difference between mutant and wild-type RASGRP4 GCB-DLBCL cell lines. Viable cell number was assessed by trypan blue staining over 3 days in culture. (B) SU-DHL-8 cells overexpressing mutant RASGRP4^D404N are more resistant than RASGRP4^WT cells or parental cells expressing empty vector when treated with 2.5 ng/mL doxorubicin for 12 days. A representative plot of one biological replicate is shown.