Novel Insights into SOX11 Modulation of BCR Signaling and Targeting of BTK Resistance

Background: Mantle cell lymphoma (MCL) is a highly aggressive and currently incurable subtype of B-cell non-Hodgkin lymphoma, accounting for about 7% of all non-Hodgkin lymphoma (NHL) cases. Bruton tyrosine kinase inhibitors (BTKi), have been extremely effective in treating MCL; however, patients often experience relapse following these treatments. The transcription factor SOX11, a protein containing an SRY-related high-mobility group (HMG) box, is expressed in the majority of MCL cases. Its presence is associated with a more aggressive disease course and a poorer clinical prognosis. Our previous studies (Kuo et al., Blood 2018) demonstrated that SOX11 overexpression contributes to MCL pathogenesis by enhancing BCR signaling in transgenic mouse models of MCL. The present study reveals the mechanism by which SOX11 modulates the BCR pathway employing a combination of functional and pharmacological approaches to overcome resistance to ibrutinib.

Methods: To explore the alterations in signaling pathways regulated by SOX11, we utilized CRISPR/Cas9 gene editing to create stable knockouts of SOX11 in two MCL cell lines. For SOX11 overexpression, we used a lentiviral vector containing a 3xFLAG-tagged SOX11 construct. We conducted single-cell RNA sequencing (scRNA-seq) on normal and tumor B cells in BTKi-sensitive and BTKi-resistant patients. Additionally, we employed a small molecule inhibitor of SOX11 (Jatiani et al., CCR 2021), SOX11 degraders, and performed apoptosis assay, western blot, and flow cytometry experiments, along with in vivo studies.

Results: Our previous studies (Kuo et al., Oncogene 2015) and others (Vegliante et al., Blood 2013) have shown that PAX5 is a direct target of SOX11. The CD19 receptor is a co-stimulatory molecule downstream of PAX5 that amplifies BCR signaling. Our scRNA-seq results (Zhang et al., Nat. Commun. 2021) showed SOX11 is overexpressed in tumor B cells from Ibrutinib-resistant patients as compared to Ibrutinib-sensitive patients (p<0.05). Moreover, SOX11 mRNA expression correlated with PAX5 (R=0.62, P<0.05) and CD19 expression (R=0.55, p<0.05) in Ibrutinib resistant samples. CRISPR-cas9 mediated depletion of SOX11 in MCL cell lines Z-138 and JeKo-1 resulted in decreased expression of PAX5 and CD19 resulting in the concurrent decrease of BCR signaling (p-BTK, p-SYK, p-AKT, and p-PLCγ). Consistently, overexpression of SOX11 could restore PAX5 and CD19 expression. We also confirmed that depleting PAX5 in JeKo-1 and MAVER-1 cells overexpressing SOX11 reduces CD19 expression. These findings were further supported by observations in Eμ-SOX11 overexpressing transgenic mice, which showed higher PAX5 expression in splenic B1a cells compared to wild-type mice. Treatment with a SOX11 DNA binding inhibitor (SOX11i) in MCL cells significantly decreased the expression of PAX5, and CD19 and reduced the p-BTK, p-SYK, p-AKT, and p-PLCγ in both Ibrutinib sensitive Z-138, JeKo-1, and Ibrutinib resistant JeKo-1_IBNR cell line with increased PI3K-AKT signaling modeling BCR bypass (Zhao et al., Nat. Commun. 2017). Importantly, SOX11 inhibitor was able to demonstrate cytotoxicity in Ibrutinib-resistant PDX models as well as synergize with Ibrutinib in JeKo-1_IBNR cells. The SOX11i treatment reduced the tumor growth in vivo in a Z-138 MCL xenograft model without any weight loss or organ toxicity. Furthermore, we developed a novel SOX11-specific degrader using PROTAC technology, consisting of a warhead ligand for SOX11, an E3 ubiquitin ligase-binding ligand, and a linker. The degrader treatment in MCL cells (JeKo-1, Z-138) successfully replicated the effects observed with SOX11 genetic knockdown.

Conclusion: Our findings highlight a novel pathway for BCR signaling modulated via the SOX11-PAX5-CD19 axis. We demonstrate the expression of SOX11 in Ibrutinib-resistant primary tumors, and the ability to inhibit SOX11-driven BCR signaling in Ibrutinib-sensitive as well as Ibrutinib-resistant models. Developing SOX11 inhibitors and SOX11-targeting degraders offers significant potential for MCL patients, especially those resistant to ibrutinib, by targeting upstream resistance mechanisms.