Ibrutinib Inhibits Concomitant TLR and BCR- Driven Proliferation of Chronic Lymphocytic Leukemia Cells and Overrides the Supportive Survival-Promoting Effects of Microenvironmental Signals
Authors: Temburni Sonal , Barrientos Jacqueline C. , Kolitz Jonathan E. , Allen Steven L. , Rai Kanti R. , Chang Betty Y. , Chiorazzi Nicholas , Damle Rajendra N 
Published: October 2019

Abstract
Chronic lymphocytic leukemia (CLL) cells interact with external stimuli via several receptors, including the B cell receptor (BCR) and cytokine/chemokine receptors. CLL cells also express varying levels of functional Toll-like receptors (TLR), including TLR7 and TLR9. In vivo, the interaction of CLL cells via BCR or TLR occurs within a supportive microenvironment that provides them with survival and proliferative signals. CLL clonal turnover, occurring as an outcome of these and other interactions might lead to the release of naked nuclear material into the microenvironment and permit an interaction with these remnants of dying/dead cells. This could lead to repeated low level T cell-independent activation, possibly resulting in disease progression. To this effect, concomitant engagement of BCR and TLR leads to differential Erk phosphorylation and apoptosis rescue in CLL cases segregated by IgV gene mutational status.
Here, we have studied the impact of stromal cells on TLR-mediated activation of negatively selected CLL cells from 24 untreated cases. Marked increases in percentages of CD69+ CLL cells and density of HLA-DR expression were observed within 18 hrs after exposure to TLR7 or TLR9 agonists (Imiquimod or ODN2006, respectively). However, although CLL cells activated via their TLRs in the presence of irradiated stromal cells (HS-5) did not show further changes in CD69 and HLA-DR expression, they exhibited significantly elevated levels (p<0.01) of anti-apoptotic molecules, Survivin and Mcl-1, compared to CLL cells activated in the absence of HS-5 cells. B cells from 16 CLL cases stimulated with Imiquimod or ODN2006 in the absence/presence of HS-5 cells were assayed for rescue from apoptosis using Annexin/PI (on day 1) and cell proliferation using 3H- thymidine incorporation (on day 3). Stromal cells alone induced ~12.8% (range: 8-31%) rescue from spontaneous CLL apoptosis in culture, whereas TLR7- and TLR9-mediated activation induced ~12.2% (range: 9-44%) and ~20.1% (range: 7-41%) rescue from cell death, respectively. TLR7 and TLR9 signaling upregulated proliferation of CLL cells by 1.83 fold (range: 1.3-11.6) and 9.9 fold (range: 1.3-43.2), respectively. Co-culture with stromal cells further enhanced cell proliferation by an average of 3.54 fold (range: 1.4-7.8) for TLR7 or 2.45 fold (range: 1.1-5.6) for TLR9.
Ibrutinib covalently binds and inhibits Bruton tyrosine kinase, BTK, a molecule critical for BCR signaling. In addition, ibrutinib inhibits cell signaling occurring when CXCR4 binds its ligand, stromal derived factor-1/CXCL12. However, the effects of ibrutinib on concomitant TLR+ BCR-mediated signaling in CLL have not yet been reported. We evaluated the impact of ibrutinib over a dose range of 0.1 - 25mg/ml on TLR-mediated CLL cell apoptosis and proliferation in presence/absence of stromal help, as mentioned above. Pre-incubation of CLL cells with ibrutinib for 2 hrs significantly lowered TLR-mediated induction of Survivin and Mcl-1 within 36 hrs of culture. Exposure to ibrutinib also inhibited 3H-thymidine incorporation in non-stimulated (average: 22%; range: 7-43%) and TLR7- (average: 21%; range: 7-49%) or TLR9- (average: 43%; range: 11-98%) activated CLL cells in 14/14 cases. Ibrutinib treatment resulted in an increase in TLR7-induced apoptosis in 3 of 4 CLL cases and in TLR9-induced apoptosis in 2 of 4 CLL cases. Even when CLL cells from 6 patients were cocultured with stromal cells, ibrutinib reduced cell proliferation to 45-65% of that observed in untreated cells induced via TLR7 or TLR9 alone or when combined with an anti-BCR signal.
These findings reiterate the pivotal role of the microenvironment in nurturing CLL cells activated via several types of receptors. Ibrutinib, known to be efficacious in inhibiting BCR signaling, can also abrogate events downstream of TLR7 and TLR9 signaling in CLL. Importantly, inhibition of TLR 7 and 9 signaling by ibrutinib can override the protective effects from stroma-derived signals. These anti-proliferative effects of ibrutinib may be especially relevant and important for curbing concomitant activation via the BCR, TLR, and chemokine receptor pathways that likely synergize and thereby contribute to the aggressiveness of the disease.

Disclosures
Barrientos: Pharmacyclics, Inc: Membership on an entity's Board of Directors or advisory committees, Research Funding. Rai:Pharmacyclics, Inc: Membership on an entity's Board of Directors or advisory committees. Chang:Pharmacyclics, Inc: Employment, Equity Ownership. Chiorazzi:Pharmacyclics, Inc: Research Funding.