Rituximab and obinutuzumab differentially hijack the B cell receptor and NOTCH1 signaling pathways

Abstract

Summary The anti-CD20 monoclonal antibodies rituximab and obinutuzumab differ in their mechanisms of action, with obinutuzumab evoking greater direct B cell death. To characterize the signaling processes responsible for improved B cell killing by obinutuzumab, we undertook a phosphoproteomics approach and demonstrate that rituximab and obinutuzumab differentially activate pathways downstream of the B cell receptor. Although both antibodies induce strong ERK and MYC activation sufficient to promote cell-cycle arrest and B cell death, obinutuzumab exceeds rituximab in supporting apoptosis induction by means of aberrant SYK phosphorylation. In contrast, rituximab elicits stronger anti-apoptotic signals by activating AKT, by impairing pro-apoptotic BAD, and by releasing membrane-bound NOTCH1 to up-regulate pro-survival target genes. As a consequence, rituximab appears to reinforce BCL2-mediated apoptosis resistance. The unexpected complexity and differences by which rituximab and obinutuzumab interfere with signaling pathways essential for lymphoma pathogenesis and treatment provide important impetus to optimize and personalize the application of different anti-CD20 treatments.

NOTCH1 Signalling: A key pathway for the development of high-risk chronic lymphocytic leukaemia

“…Modulation of NOTCH1 signalling through BCR signalling, as suggested above, was supported by in-vitro and in-vivo data obtained under BTK inhibition, since ibrutinib treatment was shown to suppress NOTCH1 signalling ( 39 , 40 ). In keeping with this notion, the presence of NOTCH1 mutation had no negative impact on response to ibrutinib treatment in the RESONATE trial ( 13 ).…”

“…In keeping with this notion, the presence of NOTCH1 mutation had no negative impact on response to ibrutinib treatment in the RESONATE trial ( 13 ). Suppressive effects on NOTCH1 signalling were also found for the Pi3K-inhibitor idelalisib in an in-vitro setting ( 39 ), whereas clinical data based on nine patients suggested poor response of NOTCH1 mutant CLL patients to idelalisib ( 41 ).…”

“…However, this explanation remained contradictory since associations between low CD20 expression and NOTCH1 mutation were not reproducible in the GCLLSG CLL8 nor in the COMPLEMENT-1 trial ( 9 , 11 ). As another explanation, in-vitro studies on SU-DHL4 revealed strong activation of NOTCH1 signalling by rituximab, but not obinutuzumab, possibly explained by distinct intracellular signalling events that both anti-CD20 mAbs induce in the B-cell receptor (BCR) signalling cascade ( Figure 2 ) ( 39 ). Exceedingly strong transcriptional changes induced after release of mutant NICD1 in concert with pro-survival signalling changes following rituximab binding to CD20 could thus also be responsible for a higher resistance of NOTCH1 mutant CLL cells towards rituximab-based chemo-immunotherapy.…”

“…This mode is less well understood, likely executed by ADAM17 and possibly occurring in the intracellular compartment where NOTCH1 is expressed on endosomal membranes ( 47 , 48 ). Importantly, ligand-independent NOTCH1 cleavage was described to occur shortly after activation of T-cell as well as B-cell receptors ( 39 , 49 – 54 ), a finding that associates NOTCH1 signalling with the response of T- and B-cells to antigen recognition and is interesting against the notion that high frequencies of NOTCH1 mutation could be associated with aggressive stereotyped B-cell receptor subsets (subsets #1, #6, #8, and #59) ( 55 ).…”

Multiple Mechanisms of NOTCH1 Activation in Chronic Lymphocytic Leukemia: NOTCH1 Mutations and Beyond

“…In contrast, CLL is strongly characterized by the other class of NOTCH1 mutations, which are restricted to the TAD and PEST domains; therefore, NOTCH1 signaling in CLL still requires external receptor engagement for cleavage to be triggered. Because of this mechanism of action, NOTCH1 mutations in CLL are mostly passenger events, acquired during the course of the disease and are rarely found within precursor populations [ 116 , 117 , 118 ].…”