DLBCL associated NOTCH2 mutations escape ubiquitin-dependent degradation and promote chemo-resistance.

Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin's lymphoma. Up to 40% of DLBCL patients display refractory disease or relapse after standard chemotherapy treatment (R-CHOP; rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone), leading to significant morbidity and mortality. The molecular mechanisms of chemo-resistance in DLBCL remain incompletely understood. Utilizing a CULLIN-RING ligases based CRISPR-Cas9 library, we identify that inactivation of the E3 ubiquitin ligase KLHL6 promotes DLBCL chemo-resistance. Furthermore, proteomic approaches identified KLHL6 as a novel master regulator of plasma membrane-associated NOTCH2 via proteasome-dependent degradation. In CHOP-resistant DLBCL tumors, mutations of NOTCH2 result in a protein that escapes the mechanism of ubiquitin-dependent proteolysis, leading to protein stabilization and activation of the oncogenic RAS signaling pathway. Targeting CHOP-resistant DLBCL tumors with the Phase 3 clinical trial molecules nirogacestat, a selective g-secretase inhibitor, and ipatasertib, a pan-AKT inhibitor, synergistically promotes DLBCL death. These findings establish the rationale for therapeutic strategies aimed at targeting the oncogenic pathway activated in KLHL6- or NOTCH2-mutated DLBCL.