Longitudinal Assessment of CLL Patients Under Ibrutinib Treatment Reveals Maintained Capacity to Respond to Microenvironmental Stimuli through the Toll-like Receptors
Authors: Gounari Maria , Ntoufa Stavroula , Kotta Konstantia , Vilia Maria Giovanna , Nenou Athanasia , Gerousi Marina , Scarfò Lydia , Xochelli Aliki , Fonte Eleonora , Coscia Marta , Tedeschi Alessandra , Stavroyianni Niki , Muzio Marta , Stamatopoulos Kostas , Ghia Paolo 
Published: October 2019

Abstract
Despite the remarkable clinical results obtained with the novel kinase inhibitors i.e. the BTK inhibitor Ibrutinib and the PI3Kδ inhibitor idelalisib in both relapsed/refractory and treatment-naïve patients, most patients achieve only partial responses underscoring the existence of resistance mechanisms that warrant further investigation. Here we explored two major mechanisms that may underlie less than optimal responses to BcR inhibition by ibrutinib, namely resistance to apoptosis due to a decreased dependence on proximal BcR signaling as it might be occurring in the context of BcR anergy; and, "bypass" activation from other non-BcR immune pathways, in particular the Toll-like receptors (TLRs).
The study group included 33 CLL patients who received ibrutinib as monotherapy in 1st (n=4) or subsequent lines (n=29) of treatment. CLL cells were isolated by negative selection from peripheral blood samples collected prior to treatment initiation and, thereafter, at fixed sampling times throughout the 1st year of treatment. In keeping with the literature, we observed decreased ERK phosphorylation after 1 and 3 months of treatment as assessed by flow cytometry (p=0.0002 and <0.0001 respectively; n=25). We also report for the first time a significant reduction in basal intracellular Ca2+ levels at 1 and 3 months of Ibrutinib treatment compared to the pre-treatment paired samples (p=0.04 and 0.006 respectively; n=27). These were accompanied by attenuated Ca2+ fluxes after BcR cross-linking compared to the pre-treatment paired samples (p=0.0022 and 0.0004 respectively; n=23), implying a significant decrease in BCR signaling capacity. Using chemiluminesence-based protein arrays and Western Blotting, we assessed the activation of key molecules participating in immune signaling pathways and found that the phosphorylation status of critical MAP kinases (pERK, pJNK, pp38, pAKT), pIKB, and STATs (pSTAT1, pSTAT3) decreased at 1 month of treatment (n=13). Interestingly, only molecules proximal to BTK remained inhibited after 6 months of therapy (pBTK, pPLCγ2), while the phosphorylation of the downstream MAPks (pERK, pp38) increased above baseline levels at 6 months (n=13).
We then studied the effects of Ibrutinib on the capacity of CLL cells to respond to additional immune pathways such as TLR. We stimulated primary cells from 13 CLL cases with specific TLR ligands for TLR1/2, TLR2/6, TLR7 and TLR9 and assessed the functional outcome after 24 hours by flow cytometric determination of CD25 and CD86 expression as a measure of cell activation. Based on the pattern of responses observed in cells collected at +1 month under treatment in comparison to the pre-treatment sample, cases were subdivided in two subsets: the first (8/13 cases, 61%) displays significantly augmented functional responses to TLR triggering ('TLR responders') while the second (5/13 cases, 39%) shows an opposite pattern i.e. attenuated responses ('TLR non-responders'). No significant differences in TLR1, TLR2, TLR6, TLR7, TLR9 expression (flow cytometry) were identified between the two subsets either pre-treatment or at +1 month under ibrutinib. Probing into the mechanisms implicated in the observed responses, we found that at +1 month under Ibrutinib TLR9 stimulation with CpG in 'TLR responders' resulted in higher activation of several TLR-pathway signaling molecules, including the MAPKs, STATs and SRC kinases. The exact opposite i.e. dampened activation of these molecules was observed in 'TLR non-responders'. Interestingly, in 2/2 'TLR non-responders' for whom data was available, upregulation of basal Ca2+ levels was noted at +1 month under ibrutinib, while, in contrast, 5/5 TLR responders did not show any such upregulation.
In conclusion, we confirm and significantly extend previous observations that CLL cells under ibrutinib treatment display a molecular profile of B cells anergized through the BcR. Importantly, we show for the first time a dichotomous pattern of TLR pathway signaling capacity under ibrutinib whereby one subset of case exhibits augmented while the other exhibits dampened responses to TLR triggering. In the latter subset, longitudinally elevated basal calcium levels and constitutive activation of MAPK signaling allude to severely diminished immune receptor signaling capacity. Studies are underway to correlate these findings with in vivo clinical response and patient outcome.

Disclosures
Coscia: Mundipharma: Honoraria; ROCHE: Honoraria, Other: Advisory board; Janssen: Honoraria; Gilead: Honoraria; Karyopharm: Research Funding. Stamatopoulos:Abbvie: Honoraria, Other: Travel expenses; Novartis: Honoraria, Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Janssen: Honoraria, Other: Travel expenses, Research Funding. Ghia:Roche: Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Speakers Bureau; Gilead: Consultancy, Honoraria, Research Funding, Speakers Bureau; Adaptive: Consultancy; Abbvie: Consultancy, Honoraria.