Ligands That Mimic the Tissue Microenvironment of Replicating Chronic Lymphocytic Leukemia (CLL) and Mantle Cell Lymphoma (MCL) Protect Ex Vivo Patient Cell Samples from the Cytotoxicity of Combined Treatment with Ibrutinib and Venetoclax (ABT-199)

Ibrutinib (IBR) is a small molecule inhibitor of Bruton's Tyrosine Kinase, a key component of the BCR pathway. In phase II studies of single-agent IBR in CLL and MCL, the overall response rate was 89% and 68% respectively. However, only a minority of responses were complete and few are durable, suggesting that drug combinations will be required to achieve broader and more durable responses. In a previous report from our laboratory, Venetoclax (VEN) (GDC-0199; ABT-199), an inhibitor of the anti-apoptotic protein BCL2, showed synergistic cytotoxicity with IBR in MCL cell lines (Axelrod M et al, Leukemia 2014). A clinical trial testing this combination for MCL patients is now open (NCT02419560). Here, we sought to investigate the cytotoxic effects of the IBR and VEN combination in CLL and MCL patient samples and the mechanisms of resistance to drug treatment that might be expected to occur in the tissue microenvironment of these malignancies in patients.

Peripheral blood mononuclear cells (PBMCs) from CLL/MCL patients containing circulating neoplastic B cells were incubated with IBR (0.1 μM), VEN (25 nM), and the combination of IBR and VEN for different time intervals. Cytotoxicity following drug treatment was determined by FACS analysis of cleaved PARP in CD5/CD19 double positive cells (Portell CA et al ASH 2014) and loss of CD5/CD19 double positive cells (i.e., neoplastic B cells). Cytotoxicity induced by IBR was variable, but was invariably enhanced by the addition of VEN. In a cohort of CLL and MCL patient samples that were treated (n=14), eleven patients showed synergistic cytotoxicity following treatment with IBR and VEN as evaluated by the Bliss model of independence.

Recognizing that CLL and MCL cells populate replication centers (pseudofollicles) of the tissue microenvironment such as lymph node and bone marrow, we attempted to emulate a pseudofollicle in vitro by co-culturing or pre-incubating PBMC with agonistic components of that microenvironment.  We screened several cytokines, other agonistic proteins, and supporting cells, including stromal cells (HS-5 cell line), follicular dendritic cells (HK cell line), soluble CD40L, CxCL13, IL-10, IL-2, CpG oligodeoxynucleotides (CpG ODN), BAFF, or IgM. A 12 h pre-incubation with soluble CD40L, IL-10, or CpG ODN generated significant levels of resistance to IBR and VEN. Moreover, the combination of soluble CD40L, IL-10, and CpG ODN resulted in nearly complete resistance to the IBR-VEN combination in cells from three CLL patient samples. The resistance was not overcome by increasing concentrations of IBR up to 10 μM, suggesting that resistance is not due to upregulation of BTK.  

To gain insight into the mechanism(s) of resistance and ways to overcome it, CLL patient PBMC pre-treated with soluble CD40L, IL-10, and CpG ODN were treated in three-way combination with IBR, VEN and inhibitors of potential resistance pathways known to be downstream from the tested ligands. These included small molecule inhibitors of IKKα and IKKβ, JAK1, 2, and 3, MAP Kinase, or PI3Kδ as well as the anti-apoptotic proteins MCL1, BCL-xL, or Survivin.  Cytotoxicity was evaluated with an alamarBlue^®assay. The NF-kB, JAK-STAT, PI3Kδ, and anti-apoptotic protein inhibitors all exhibited synergistic interactions with the IBR-VEN combination, as well as substantial cytotoxicity as single agents at higher doses. Inhibitors of the MAP Kinase pathway were ineffective. Taken together our data are consistent with the hypothesis that resistance to IBR, VEN and the combination in patients with CLL or MCL could arise by NF-kB, JAK-STAT, or PI3K signaling from CD40, IL10R and Toll-like receptors.  

In conclusion, IBR and VEN induced synergistic cytotoxicity in a majority of CLL and MCL patient samples. An in vitro model of the pseudofollicle microenvironment provided protection against cytotoxicity of IBR and VEN, suggesting that the tissue microenvironment provides a niche that supports drug resistance. This resistance was overcome by inhibition of NF-kB, JAK-STAT, or PI3Kδ, or the anti-apoptotic proteins MCL1, BCL-xL, or Survivin.