Synergistic Targeting of BTK and E-Selectin/CXCR4 in the Microenvironment of Mantle Cell Lymphomas
Authors: Basyal Mahesh , Zhang Weiguo , Patel Nalini B , Piya Sujan , Zhang Hongying , Fogler William E. , Rice William G , Magnani John L. , Borthakur Gautam M. , Andreeff Michael 
Published: November 2019

Mantle cell lymphoma (MCL) is a rare subtype of aggressive B-cell non-Hodgkin lymphoma that remains incurable with standard therapy. Overexpression of B-cell receptor signaling through Bruton tyrosine kinase (BTK) is a hallmark of MCL (Pal Singh et al., 2018). Inactivation of BTK signaling with the small molecule inhibitor ibrutinib is currently the most broadly used treatment of B cell lymphoma. However, it induces only low rates of apoptosis in vitro at clinically achievable concentrations. Frequently, primary and acquired resistance is observed (Chiron et al., 2014; Wang et al., 2013). One of the molecular mechanisms of acquired resistance is the development of BTKC481S mutations (Martin et al., 2016). In addition, the tumor microenvironment (TME), in which mesenchymal stroma cells (MSC) and vascular endothelial cells (ECs) are specialized components, has increasingly been recognized as a central determinant of drug resistance, subclonal evolution and late progression/transformation of B-cell lymphomas (Balsas et al., 2017; Weis and Cheresh, 2011).
Although the pro-tumoral ecosystem that supports MCL is still poorly understood, it has been reported that MCL cells express high levels of functional CXCR4 and CXCR5 chemokine receptors and VLA-4 adhesion molecules (Kurtova et al., 2009) . Lymphoma cells also display high levels of CD44, one of E-selectin ligands, in co-culture with ECs (Cao et al., 2014). These findings strongly suggest the association of acquired BTK mutations and the TME with resistance to BTK-targeted therapy in MCL. Therefore, we hypothesize that disrupting the crosstalk of MCL cells and TME by blocking CXCR4/CXCL12 or E-selectin/CD44 might benefit BTK-targeted therapy against MCL.
In this study, we investigated the anti-lymphoma effect of a novel small-molecule multi-kinase inhibitor CG-806 which exerts promising enzymatic inhibitory activity against the C481S mutation and wild type BTK at extremely low doses (IC50s were 2.52 and 5 nM, respectively). CG-806 demonstrated impressive anti-lymphoma effects in MCL cell lines Z138, MINO, Jeko-1 and JVM2 (IC50s of 2.7, 3.87, 3.79 and 8.27 nM, respectively), all of which were much less sensitive to ibrutinib (IC50s ≈ 10,000 nM). Mechanistically, CG-806 not only suppressed BTK activation, but also its downstream signaling targets phospho-Stat3,-AKT,-ERK and -Src, as well as NF-κB and c-Myc, and surprisingly upregulated p53 in MCL cells but exerted no suppression of phospho-FLT3 and aurora kinase at tested doses in MCL cells, two of the other potential CG-806 targets. Interestingly, CG-806 triggered profound apoptosis in Z138 and MINO cells as evidenced by increased cleavage of caspase-3 and PARP and expression of annexin V (EC50s 4.91 and 6.35 nM, respectively), but showed resistance in Jeko-1 and JVM2 cells (EC50s 7,800 and 3260 nM, respectively), which was accompanied with marked upregulation of autophagy, implicating autophagy as a novel resistance mechanism to BTK inhibition. Interestedly, our previous studies demonstrated that TME components MSC/hypoxia mediated autophagy upregulation which was associated with resistance in AML cells (Zhang et al., 2018), and, on the other hand, upregulation of autophagy was also observed in FLT3 wild type AML cells after CG-806 treatment, which resulted in resistance to CG-806-triggered apoptosis induction (Zhang et al., unpublished). Nevertheless, suppression of autophagy with the ULK1 inhibitor SBI-0206965 (Egan et al., 2015) or a putative autophagy inhibitor Chloroquine (CQ) (Mauthe et al., 2018) partially enhanced CG-806-induced apoptosis in the resistant Jeko-1 cells, confirming a role for autophagy in resistance to BTK inhibitors in MCL.
Furthermore, CXCR4 and E-selectin ligand levels were upregulated by exposing MCL cells in either ibrutinib or CG-806, and co-culture of MCL cells Z138 with MSC or HUVEC cells partially protected MCL cells from CG-806-triggered apoptosis. Of note, blockade of CXCR4 or E-selectin with their antagonist plerixafor or GMI-1271, respectively, re-sensitized to CG-806-induced apoptosis in MCL cells, suggesting potential benefit of disrupting the crosstalk of TME and lymphoma cells in MCL therapy. Taken together, our findings may provide the basis for a new therapeutic strategy co-targeting TME, autophagy and BTK with the goal of overcoming the resistance to BTK-targeted therapy in MCL.

Disclosures
Zhang: Aptose Biosciences, Inc: Employment. Fogler:GlycoMimetics Inc: Employment, Equity Ownership. Rice:Aptose Biosciences, Inc: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Magnani:GlycoMimetics Inc: Employment, Equity Ownership. Borthakur:BMS: Research Funding; Tetralogic Pharmaceuticals: Research Funding; AstraZeneca: Research Funding; Eisai: Research Funding; Xbiotech USA: Research Funding; Argenx: Membership on an entity's Board of Directors or advisory committees; AbbVie: Research Funding; Cyclacel: Research Funding; Janssen: Research Funding; NKarta: Consultancy; BioLine Rx: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; PTC Therapeutics: Consultancy; Cantargia AB: Research Funding; FTC Therapeutics: Membership on an entity's Board of Directors or advisory committees; Eli Lilly and Co.: Research Funding; Strategia Therapeutics: Research Funding; Bayer Healthcare AG: Research Funding; Oncoceutics: Research Funding; Novartis: Research Funding; Arvinas: Research Funding; Merck: Research Funding; Oncoceutics, Inc.: Research Funding; Agensys: Research Funding; GSK: Research Funding; Incyte: Research Funding; Polaris: Research Funding; BioTheryX: Membership on an entity's Board of Directors or advisory committees. Andreeff:CLL Foundation: Membership on an entity's Board of Directors or advisory committees; BiolineRx: Membership on an entity's Board of Directors or advisory committees; Eutropics: Equity Ownership; Aptose: Equity Ownership; Reata: Equity Ownership; 6 Dimensions Capital: Consultancy; AstaZeneca: Consultancy; Amgen: Consultancy; Celgene: Consultancy; Daiichi Sankyo, Inc.: Consultancy, Patents & Royalties: Patents licensed, royalty bearing, Research Funding; Jazz Pharmaceuticals: Consultancy; Center for Drug Research & Development: Membership on an entity's Board of Directors or advisory committees; NIH/NCI: Research Funding; CPRIT: Research Funding; Breast Cancer Research Foundation: Research Funding; Oncolyze: Equity Ownership; NCI-RDCRN (Rare Disease Cliln Network): Membership on an entity's Board of Directors or advisory committees; Leukemia Lymphoma Society: Membership on an entity's Board of Directors or advisory committees; German Research Council: Membership on an entity's Board of Directors or advisory committees; NCI-CTEP: Membership on an entity's Board of Directors or advisory committees; Cancer UK: Membership on an entity's Board of Directors or advisory committees; Oncoceutics: Equity Ownership; Senti Bio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.