B cell receptor (BCR) signalling has emerged as a therapeutic target in B cell lymphomas, but inhibiting this pathway in diffuse large B cell lymphoma (DLBCL) has benefited only a subset of patients. Gene expression profiling identified two major subtypes of DLBCL, known as germinal centre B cell-like and activated B cell-like (ABC), that show poor outcomes after immunochemotherapy in ABC. Autoantigens drive BCR-dependent activation of NF-κB in ABC DLBCL through a kinase signalling cascade of SYK, BTK and PKCβ to promote the assembly of the CARD11-BCL10-MALT1 adaptor complex, which recruits and activates IκB kinase. Genome sequencing revealed gain-of-function mutations that target the CD79A and CD79B BCR subunits and the Toll-like receptor signalling adaptor MYD88, with MYD88(L265P) being the most prevalent isoform. In a clinical trial, the BTK inhibitor ibrutinib produced responses in 37% of cases of ABC. The most striking response rate (80%) was observed in tumours with both CD79B and MYD88(L265P) mutations, but how these mutations cooperate to promote dependence on BCR signalling remains unclear. Here we used genome-wide CRISPR-Cas9 screening and functional proteomics to determine the molecular basis of exceptional clinical responses to ibrutinib. We discovered a new mode of oncogenic BCR signalling in ibrutinib-responsive cell lines and biopsies, coordinated by a multiprotein supercomplex formed by MYD88, TLR9 and the BCR (hereafter termed the My-T-BCR supercomplex). The My-T-BCR supercomplex co-localizes with mTOR on endolysosomes, where it drives pro-survival NF-κB and mTOR signalling. Inhibitors of BCR and mTOR signalling cooperatively decreased the formation and function of the My-T-BCR supercomplex, providing mechanistic insight into their synergistic toxicity for My-T-BCR DLBCL cells. My-T-BCR supercomplexes characterized ibrutinib-responsive malignancies and distinguished ibrutinib responders from non-responders. Our data provide a framework for the rational design of oncogenic signalling inhibitors in molecularly defined subsets of DLBCL.

Hematotoxicity represents a frequent chimeric antigen receptor (CAR) T-cell related adverse event and remains poorly understood. In this multicenter analysis, we studied patterns of hematopoietic reconstitution and evaluated potential predictive markers in 258 patients receiving Axicabtagene ciloleucel (Axi-cel) or Tisagenlecleucel (Tisa-cel) for relapsed/refractory large B-cell lymphoma. We observed profound (ANC<100/µl) and prolonged (≥day 21) neutropenia in 72 and 64% of patients respectively. The median duration of severe neutropenia (ANC<500/µl) was 9 days. We aimed to identify predictive biomarkers of hematotoxicity using the duration of severe neutropenia until day +60 as the primary endpoint. In the training cohort (n=58), we observed a significant correlation with baseline thrombocytopenia (r= -0.43, P=0.001) and hyperferritinemia (r=0.54, P<0.0001) on uni- and multivariate analysis. Incidence and severity of CRS, ICANS and peak cytokine levels were not associated with the primary endpoint. We calculated the CAR-HEMATOTOX model, which included markers associated with hematopoietic reserve (e.g. platelet count, hemoglobin and ANC) and baseline inflammation (e.g. C-reactive-protein, ferritin). This model was validated in two independent cohorts from Europe (n=91) and the USA (n=109), and discriminated patients with severe neutropenia ≥/<14 days (pooled validation: AUC=0.89, Sensitivity 89%, Specificity 68%). A high CAR-HEMATOTOX score resulted in a longer duration of neutropenia (12 vs. 5.5 days, P<0.001), and a higher incidence of severe thrombocytopenia (87% vs. 34%, P<0.001) and anemia (96% vs. 40%, P<0.001). The score implicates pre-CART bone marrow reserve and inflammatory state as key features associated with delayed cytopenia and will be useful for risk-adapted management of hematotoxicity.

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