Abstract 3031: Diverse microenvironmental agonists promote a multi drug tolerant persister phenotype CLL and MCL
Authors: Jayappa Kallesh D. , Gordon Vicki L. , Cios Konrad J. , Morris Christopher G. , Arora Puja C. , Bender Timothy P. , Williams Michael E. , Portell Craig A. , Weber Michael J. 
Published: August 2019

Abstract
Responses to ibrutinib (IBR) or venetoclax (VEN) in Chronic Lymphocytic Leukemia (CLL) or Mantle Cell Lymphoma (MCL) are often partial. We reported synergistic toxicity for the IBR+VEN combination in CLL/MCL ex vivo and initiated an IBR+VEN trial in MCL (NCT02419560). However, we noted variable de novo resistance even to IBR+VEN ex vivo, and this has been found in patients (Tam et al., 2018). We also reported that microenvironmental agonists (CpG-ODN, sCD40L, and IL10; “agonist mix”) rapidly induce de novo resistance to IBR+VEN in CLL/MCL (Jayappa et al., 2017).
Here we show that microenvironmental agonists induce multi-drug tolerance to diverse pro-apoptotic drugs (bendamustine, fludarabine, vincristine, inhibitors of Mcl-1, Bcl-xL, and Bcl-2, and BAX activator) including IBR+VEN in CLL PBMCs (N=10). This is mediated by a pre-mitochondrial apoptosis blockade, as BAX activation failed in these cells. CLL cells with an activation phenotype (CD5+/19+/69+) in treatment naive patient PBMCs (N=3) also showed tolerance to several drugs, indicating that multi-drug tolerant cells pre-exist in vivo.
To identify extrinsic inducers of drug tolerance, we tested stromal cells, and agonists for TLRs, NOD1/2, CD40, and IL10R for induction of IBR+VEN tolerance. Variable levels of tolerance were noted in most CLL/MCL PBMCs (N=15) cultured with the TLR9 agonist CpG-ODN, sCD40L, or Jurkat cells expressing CD40L. IL10 and HUVEC induced modest levels of drug tolerance in a few CLL/MCL PBMCs, and TLR1/2, TLR7, and NOD1/2 agonists were effective only in MCL. Prior exposure to CpG-ODN enhanced the drug tolerance/proliferation response to sCD40L and vice versa in CLL/MCL (N=8), predicting mutually reinforcing interactions of agonists in vivo. CLL cells exposed to CpG-ODN/CD40L upregulated cognate receptors and/or NFkB proteins downstream, suggesting a mechanism for mutual reinforcement.
The agonist mix induced NFkB-dependent over-expression of anti-apoptotic proteins Mcl-1 and Bcl-xL. Consistently, multi-drug tolerance was rescued with NFkB inhibitors (BMS345541, MI-2, bortezomib) or drug combinations that inhibit multiple anti-apoptotic proteins in CLL cells exposed to agonist mix or CD5+/19+/69+ cells without agonists. By analyzing non-cancer (CD5+/19-) cells in patient PBMCs, bortezomib and MALT1 inhibitor MI-2 were selectively toxic to cancer cells, showing their value for clinical testing.
In summary, diverse microenvironmental agonists, mainly agonists of TLR9 and CD40, induce multi-drug tolerance in CLL/MCL, which is mediated by NFkB dependent over-expression of multiple anti-apoptotic proteins. Inhibitors of NFkB or drug combinations targeting apoptotic proteins overcame multi-drug tolerance. Thus, durable responses may require drug combinations targeting multiple apoptotic proteins, convergent pathways that induce multi-drug tolerance, and/or agents activating non-apoptotic death.
Citation Format: Kallesh D. Jayappa, Vicki L. Gordon, Konrad J. Cios, Christopher G. Morris, Puja C. Arora, Timothy P. Bender, Michael E. Williams, Craig A. Portell, Michael J. Weber. Diverse microenvironmental agonists promote a multi drug tolerant persister phenotype CLL and MCL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3031.