A Multi-Drug Tolerant Phenotype Is Induced By Diverse Microenvironmental Agonists in Chronic Lymphocytic Leukemia and Mantle Cell Lymphoma

Responses to single-agent ibrutinib (IBR) or venetoclax (VEN) in Chronic Lymphocytic Leukemia (CLL) or Mantle Cell Lymphoma (MCL) are often incomplete suggesting drug combinations are needed to overcome de novo and acquired resistance. We previously reported synergistic cytotoxicity for the IBR+VEN combination in CLL/MCL cells ex vivo and have initiated an IBR+VEN clinical trial in MCL (NCT02419560). However, ex vivo analysis of patient samples showed de novo resistance even to the IBR+VEN combination in some samples, consistent with recent clinical experience (Tam et al. N Engl J Med, 2018). We noted that sensitivity to IBR+VEN was lower in CLL cells showing an "activation" phenotype (CD5⁺/19⁺/69⁺) that can be induced by extracellular factors in vivo, suggesting that the microenvironment could induce drug tolerance in the cancer cells.This was supported by our finding that ex vivo exposure of CLL/MCL samples to a mixture of microenvironmental agonists (CpG-ODN, sCD40L, and IL10; "agonist mix") induced significant loss of sensitivity to IBR+VEN (Jayappa et al. Blood Adv., 2017). Here we show that various microenvironmental agonists, including of innate immunity, are able to reduce sensitivity to IBR+VEN as well as other pro-apoptotic drugs, generating a multi-drug tolerant phenotype.

To explore drug tolerance, we performed flow cytometric analysis of apoptosis induced by IBR+VEN in CD5⁺/19⁺cancer cells in CLL/MCL PBMCs cultured with agonists of TLRs, NOD1/2, CD40, and IL10R and various stromal cells. Tolerance to IBR+VEN as determined by resistance to apoptosis was noted in most samples cultured with TLR9 agonist CpG-ODN, sCD40L, or Jurkat cells expressing CD40L. IL10 and HUVEC induced modest levels of drug tolerance in a few CLL/MCL samples, and TLR1/2, TLR7, and NOD1/2 agonists were effective only in MCL samples.

Prior exposure to CpG-ODN enhanced the ability of sCD40L to induce proliferation and drug tolerance and vice versa in CLL/MCL, predicting mutually reinforcing interactions in vivo. We noted that CLL cells exposed to CpG-ODN displayed higher levels of CD40 and downstream signaling proteins (TRAF2, RelB, and p100/52). Conversely, CD40L induced a modest increase in NF-kB transcription factors, providing a possible mechanism for mutual reinforcement.

Using flow cytometric analysis of apoptosis, we noted tolerance to several pro-apoptotic agents (bendamustine, fludarabine, and inhibitors of Mcl-1, Bcl-xL, Bcl-2, and Bcl-2/Bcl-xL) including IBR+VEN in CD5⁺/19⁺cells from CLL samples treated with agonist mix, showing development of multi-drug tolerance in these cells. CpG-ODN or agonist mix induced NFkB-dependent over-expression of pro-survival proteins (Mcl-1 and Bcl-xL), and increased ratio of these to cognate pro-apoptotic proteins. This increased expression of pro-survival proteins underlies the multi-drug tolerant phenotype. Consistently, multi-drug tolerance was rescued with inhibitors of NFkB (BMS345541 or Bortezomib) or drug combinations simultaneously inhibiting multiple anti-apoptotic proteins (inhibition of Mcl-1 with Bcl-2, Bcl-xL, or Bcl-2 and Bcl-xL).

CLL cells with activation phenotype (CD5⁺/19⁺/69⁺) in patient PBMCs cultured without the agonist mix also showed tolerance to several apoptotic protein inhibitors ex vivo, and this was also effectively rescued with NF-kB inhibition or combination of apoptotic protein inhibitors ex vivo. These results suggest that the cancer cells with activation phenotype exist in vivo anddisplay multi-drug tolerant phenotype consistent with the drug tolerance induced by agonist mix ex vivo.

In conclusion, several microenvironmental factors, particularly agonists of TLR9 and CD40, induce tolerance to IBR+VEN and cell proliferation in CLL/MCL, and response to these agonists is enhanced by combinatorial exposure. These agonists generate tolerance to several apoptosis-inducing agents beyond IBR+VEN. This microenvironment-induced multi-drug tolerance is mediated by NFkB dependent over-expression of multiple pro-survival proteins. Inhibitors of NFkB, or drug combinations targeting multiple pro-survival proteins, overcame multi-drug tolerance in agonist mix-treated samples and in CD69⁺CLL cells in patient PBMCs that represent multi-drug tolerant cells in vivo. Thus, more complete and durable responses might be achieved in MCL/CLL with therapies that target multi-drug tolerant persister cells.