ACTIVATION OF BCR SIGNALING REWIRES THE RESPONSE OF BCR-ASSOCIATED KINASES TO IBRUTINIB IN MANTLE CELL LYMPHOMA CELLS

Mantle cell lymphoma (MCL) is a rare B-cell malignancy with sig- nificant clinical heterogeneity. We have recently shown that a deeper re- sponse of MCL cells to B-cell receptor (BCR) stimulation identified a subset of patients with a higher risk of progression. Although therapies targeting the BCR, such as the BTK inhibitor ibrutinib (Ibr), have a high initial response rate in MCL, relapse remains a challenge. To investigate the ability of Ibr to modulate the BCR signaling network, we analyzed the activating phosphorylation status of BCR-associated kinases (BAK), i.e., SYK, PLCγ2, STAT5, ERK1/2, NF-κB p65, AKT, BTK, STAT3, in cells from peripheral blood of 29 MCL patients at diagnosis following Ibr treatment in vitro, in the basal condition and after BCR stimulation with anti-IgM, using phospho-specific flow cytometry, a multiparametric assay allowing functional signaling analysis at a single-cell level, com- bined with fluorescent cell barcoding. In the basal condition, Ibr induced a significant average reduction of phosphorylation level for all BAKs but NF-κB p65 and STAT3. In the BCR-stimulated condition, we de- tected a significant average reduction of phosphorylation for all BAKs, including NF-κB p65, but STAT3. Comparison of the phosphorylation responses to Ibr for each BAK between the basal and BCR-stimulated conditions showed that the phosphorylation reduction in response to Ibr was significantly deeper in the BCR-stimulated condition compared with the basal one for SYK, ERK1/2, NF-κB p65, and AKT. In contrast, re- duction in BTK phosphorylation resulted attenuated, although not sig- nificantly, in the BCR-stimulated condition compared with the basal one (Figure 1). In conclusion, our data show that in the basal condition most BAKs are highly connected in response to Ibr, thus suggesting that these proteins belong to the same signaling subnetwork. Importantly, the acti- vation of the BCR quantitatively and qualitatively rewires the network of BAKs sensible to Ibr, thus suggesting that the microenvironment may influence the response of tumor cells to therapies. Moreover, identifying novel BAKs that are inhibited by Ibr may form the rationale to target multiple signaling nodes to overcome resistance in MCL. Thank to Excellence Project 2023-2027 of the DNBM, University of Verona funded by MUR; European Union, MUR and MSAL (PNRR- PNC-E3-2022-23683266 PNC-HLS-DA-INNOVA and PNRR-TR1-2023- 12378287) for funding support.