Abstract 08: Soluble agonists in the in vivo macroenvironment generate phenotypic de novo resistance to BTK/Bcl-2 targeted therapies in diverse B-cell malignancies
Authors: Jayappa Kallesh D. , Portell Craig A. , Gordon Vicki L. , Bender Timothy P. , Williams Michael E. , Weber Michael J. 
Published: August 2018

Abstract
The Bruton's tyrosine kinase (BTK) inhibitor ibrutinib (IBR) is FDA approved for chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL). However, IBR responses are incomplete in majority of cases, and not durable in MCL and high-risk CLL. We hypothesize that de novo resistance, incomplete responses, and rapid recurrence can be due to adaptive signaling that should be cotargeted with BTK to achieve deeper and durable responses. We previously showed that venetoclax (VEN), an inhibitor of Bcl-2, generates synergistic cytotoxicity with IBR in MCL lines (Axelrod et al. Leukemia 2014;28:407-10). Recently, we extended these findings to circulating leukemic B cells in CLL, MCL (Jayappa et al. ASH 2016), and follicular lymphoma (FL) patient PBMCs treated ex vivo. Based on these results we initiated a phase I/Ib trial for IBR+VEN combination in MCL.
Our ex vivo cytotoxicity studies showed a surprisingly high frequency of de novo resistance to IBR+VEN. Resistance did not correlate with previous therapy or clinical characteristics of patients. However, FACS analysis of primary CLL cells showed that cells displaying an “activation” phenotype (CD5+/CD19+/CD69+) were less sensitive to cytotoxicity of IBR+VEN, suggesting that cellular activation by extrinsic factors could generate drug resistance. To test this, we cocultured or preincubated CLL patient PBMCs with various stromal cells, cytokines, and other agonists and found that soluble CD40L, IL10, and CpG oligodeoxynucleotides (ODN) generated variable resistance to IBR+VEN in CLL samples, with CpG ODN, a specific TLR9 agonist, being the most effective. The combination of CpG ODN, sCD40L, and IL10 (“agonist mix”) generated near-complete resistance to IBR+VEN in most CLL(N=22)/MCL(N=7)/FL(N=4) patient PBMCs. The agonist mix treatment significantly upregulated nuclear localization of RelA and RelB proteins in CLL/MCL/FL cells analyzed by ImageStream, suggesting activation of NF-kB signaling. Agonist mix treatment also induced the NF-kB dependent overexpression of anti-apoptotic proteins Mcl-1, Bcl-xL, and survivin, and inhibitors of NF-kB or of upregulated anti-apoptotic proteins overcame this drug resistance (Jayappa et al. ASH 2016).
To further explore the role of TLR9 and other innate immune receptors in resistance, we performed IBR+VEN cytotoxicity analysis in CLL/MCL patient PBMCs cocultured with agonists of various TLRs and NOD1/2 receptors. Preliminary results showed that TLR9 agonist CpG ODN generated significant resistance to IBR+VEN in most CLL/MCL samples, and agonists of TLR1/2, TLR7, and NOD1/2 induced a moderate level of resistance in a MCL sample. The CpG ODN treatment induced a significant increase in NF-kB signaling measured by increased nuclear localization of RelB in CLL patient samples. To determine if soluble agonists in the extranodal macroenvironment of patients could generate NF-kB dependent resistance to IBR+VEN, we analyzed drug cytotoxicity in CLL(N=6) and MCL(N=1) patient PBMCs cultured with autologous plasma. We found that preincubation with autologous plasma induced variable levels of resistance to IBR+VEN in CLL/MCL samples, but did not induce resistance to a NF-kB inhibitor BMS-345541. In conclusion, soluble factors in the patient nodal/extranodal macroenvironment, particularly a TLR9 agonist, generate resistance to IBR+VEN by activating NF-kB signaling and overexpression of multiple anti-apoptotic proteins. The same resistance mechanism, based on innate immune signaling, occurs in three mature B-cell malignancies, CLL, MCL and FL, that differ in etiology and clinical course. The signaling pathway responsible for resistance contains new targets for therapeutic intervention with potential to prevent or reverse resistance to IBR and VEN in these and perhaps other cancers as well.
Citation Format: Kallesh D. Jayappa, Craig A. Portell, Vicki L. Gordon, Timothy P. Bender, Michael E. Williams, Michael J. Weber. Soluble agonists in the in vivo macroenvironment generate phenotypic de novo resistance to BTK/Bcl-2 targeted therapies in diverse B-cell malignancies [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr 08.