Extrinsic interactions in the microenvironment in vivo activate an antiapoptotic multidrug-resistant phenotype in CLL

Abstract

The Bcl-2 inhibitor venetoclax (VEN) has yielded exceptional clinical responses in chronic lymphocytic leukemia (CLL). However, de novo resistance can result in failure to achieve negative minimal residual disease and predicts poor treatment outcomes. Consequently, additional pro-apoptotic drugs, such as inhibitors of Mcl-1 and Bcl-xL, are in development. By profiling anti-apoptotic proteins using flow cytometry, we find that leukemic B-cells recently emigrated from the lymph node (LN) (CD69Pos/CXCR4Low) in vivo are enriched for cell clusters simultaneously overexpressing multiple anti-apoptotic proteins (Mcl-1high/Bcl-xLhigh/Bcl-2high), in both treated and treatment naïve CLL patients. These cells exhibited anti-apoptotic resistance to multiple BH-domain antagonists, including inhibitors of Bcl-2, Mcl-1, and Bcl-xL, when tested as single agents in a flow cytometry-based functional assay. Anti-apoptotic multi-drug resistance declines ex vivo, consistent with resistance being generated in vivo by extrinsic microenvironmental interactions. Surviving "persister" cells in patients undergoing venetoclax treatment are enriched for CLL cells displaying the functional and molecular properties of microenvironmentally-induced multi-drug resistance. Overcoming this resistance required simultaneous inhibition of multiple anti-apoptotic proteins, with potential for unwanted toxicities. Using a drug screen performed using patient PBMCs cultured in an ex vivo microenvironment model we identify novel venetoclax drug combinations that induce selective cytotoxicity in multi-drug resistant CLL cells. Thus, we demonstrate that anti-apoptotic multi-drug resistant CLL cells exist in patients de novo, and show that these cells persist during pro-apoptotic treatment such as venetoclax. We validate clinically actionable approaches to selectively deplete this reservoir in patients.

Notch2 Increases the Resistance to Venetoclax-Induced Apoptosis in Chronic Lymphocytic Leukemia B Cells by Inducing Mcl-1

“…Of relevance, tumor microenvironment may determine an additional support in maintaining the expression of Notch2 and Mcl-1, especially at the lymph node level. This is in line with emerging clinical data that suggest an involvement of lymph node niches to induce resistance to proapoptotic treatments ( 38 ).…”

Targeting Mcl-1 by AMG-176 During Ibrutinib and Venetoclax Therapy in Chronic Lymphocytic Leukemia

“…Mcl-1 was recognized among the top molecule in cancers for somatic copy number alterations (34). Mcl-1 protein levels have been shown to be important in the survival of CLL cells (20,21,(35)(36)(37)(38)(39)(40)(41). Recently, direct inhibitors of Mcl-1 have been designed by Astra Zeneca [AZD-5991 (42)], Amgen [AMG-176 (43,44)] and Novartis [S63845 and S64315 (45,46)].…”

Venetoclax resistance induced by activated T cells can be counteracted by sphingosine kinase inhibitors in chronic lymphocytic leukemia

“…While the first one is enriched in recently born/divided cells mostly present in lymphoid tissues that can be also found in peripheral blood as a small proportion of recently emigrant CLL cells, the “quiescent fraction” enriched in older, less vital cells, are mostly present in peripheral blood and need to immigrate to lymphoid tissue or die ( 42 ). Two independent groups recently demonstrated that in treatment naïve patients the small proportion of recently emigrant CLL cells overexpress anti-apoptotic proteins including MCL-1 and BCL-XL ( 9 , 10 ). Of note, this subpopulation survives and increases upon in vivo venetoclax treatment of the patients showing that resistant CLL cells already exist, even in untreated CLL patients and these cells persist during proapoptotic treatment with venetoclax ( 10 ).…”

“…Multiple independent mechanisms contributed to venetoclax resistance, including the acquisition of various mutations in BCL-2 and/or the upregulation of other anti-apoptotic proteins which are not targeted by venetoclax, such as BCL-XL and MCL-1 ( 7 , 8 ). Different groups reported that malignant cells that recently interacted in vivo with the supportive microenvironment of lymphoid tissues ( 9 , 10 ), or those that were cultured in vitro with different signals that mimic microenvironment stimuli ( 11 – 15 ), show an increased expression of BCL-XL and MCL-1 and are less sensitive to venetoclax compared to quiescent or unstimulated CLL cells. In line with this, we previously reported that when peripheral blood mononuclear cells (PBMC) from CLL patients were incubated on immobilized anti-CD3 monoclonal antibodies (aCD3) to induce T cell activation, autologous activated T lymphocytes induced the activation of CLL cells, and in vitro venetoclax resistance due to the upregulation of BCL-XL and MCL-1 ( 11 , 12 ).…”