Effect of second generation BTK inhibitors on macrophage and neutrophil functions.

COLADO, ANA; VERGARA RUBIO, MARICEF; CRANCO, SANTIAGO; ZERGA, MARTA; BEZARES, FERNANDO; BORGE, MERCEDES; ELÍAS, ESTEBAN E.; FERNÁNDEZ GRECCO, HORACIO; AIZPURUA, FLORENCIA; MORANDE, PABLO; GAMBERALE, ROMINA; MARÍN FRANCO, JOSÉ L.; CORDINI, GREGORIO; MAHUAD, CAROLINA; BALBOA, LUCIANA; GIORDANO, MIRTA

Edimburgo

Workshop; XVIII International workshop on chronic lymphocytic leukemia; 2019

The development of BTK inhibitors (BTKi) has changed the treatment landscape in CLL.To date, ibrutinib is the only BTKi approved for CLL treatment, while second generationBTKi with greater selectivity for BKT, such as acalabrutinib, approved for MCL, andspebrutinib, are being evaluated in clinical trials. Besides its effects on leukemic B-cells,ibrutinib also affects key innate immune cells such as macrophages and neutrophils. In thisregard we and others have reported that ibrutinib impairs macrophage-M1 polarization andmacrophage response to TLR`s ligands, ​Mycobacterium tuberculosis (Mtb) and Aspergillusfumigatus ​(​Colado A, et al. Blood Cancer Journal. 2018, Fiorcari, S et al. Oncotarget.2016, Bercusson A, et al. Blood, 2018)​ , while others have reported that ibrutinib affectsneutrophil functionality (​Stadler, N. et al. Haematologica. 2017​).Infections are a main cause of morbidity and mortality in CLL, and while rates of infectionsduring ibrutinib treatment are similar or lower compared to other treatments (​Ball S, et al.European journal of haematology. 2018 and O'Brien S, Clinical Lymphoma, Myeloma &Leukemia. 2018​), they are still one of the most frequent adverse events (​O'Brien S, et al.Blood. 2018)​, and they are among the most common toxicities leading todrug-discontinuation in relapsed/refractory patients (​Mato A, et al. Haematologica. 2018​).Therefore, to better understand the impact of new agents on immune cells may help toimprove CLL patients´ care. Our aim was to investigate how second generation BTKi affectmacrophage and neutrophil functions.First we evaluated the effect of acalabrutinib and spebrutinib on ​macrophagepolarization towards the IFN-γ-induced M1 profile, which is associated with aneffective ​anti-microbial immune response. To this aim, human monocytes were culturedwith GM-CSF and IFN-​γ ​with or without BTKi at clinically relevant doses. We found that,similar to ibrutinib, acalabrutinib impaired M1 polarization as shown by the down-regulationin the expression of the M1-associated marker CD86, the decrease in ​TNF-α secretion​,the up-regulation of the M2-associated markers CD16, CD14, CD163, CD206 and theincrease in IL-10 secretion. In contrast, we found that spebrutinib did not modify M1/M2markers or cytokine secretion (Figure 1A-G).When we analyzed the effect of the BTKi on macrophage response towards microbialstimulation we found that acalabrutinib, as reported for ibrutinib, ​significantly decreasedTNF-α secretion by macrophages ​stimulated with irradiated-​Mtb​, heat inactivatedAspergillus fumigatus ​conidia or heat-inactivated Candida albicans ​yeast (Figure 1H-J). Inline with this, we found that ibrutinib ​and acalabrutinib impaired TNF-α secretion whenmacrophages were stimulated with Pam3CSK4, a TLR2 ligand, or zymosan a TLR2 andDectin-1 ligand, which are receptors involved in macrophage-recognition of Mtb and fungi(n=8, p<0.05). On the other hand, we found that spebrutinib only affected ​TNF-αsecretion by macrophages in response to ​heat-inactivated ​C. albicans ​(Figure 1J).Similar results were found with macrophages from CLL patients (n=6, p<0.05). In contrastto ​TNF-α ​secretion, we found that BTKi did not affect zymosan or ​C. albicansphagocytosis by macrophages (Figure 1K and L).Then we evaluated the effect of second generation BTKi on neutrophil response tomicrobial stimulation. We found that the three BTKi impaired neutrophil activationevaluated as CD62L downregulation, increase in CD11b expression and oxidative burst inresponse to zymosan and ​C. albicans (Figure 2A-H). In contrast to what we observed inmacrophages, BTKi decreased zymosan and ​C. albicans ​phagocytosis by neutrophils(Figure 3A-D). On the other hand, we found that upregulation of CD11b anddownregulation of CD62L on neutrophils activated with ​A. fumigatus ​conidia were notaffected by the BTKi (Figure 3E-F). Same results were obtained with neutrophils from CLLpatients (n=6, p<0.05).In conclusion our results show that BTKi have inhibitory effects on key innate immunecells. The diverse effects they have on macrophages and neutrophils could be due todifferences in kinase-selectivity and kinase expression on each cell type. Macrophagepolarization and activation in response to microbial stimulation were mostly affected byacalabrutinib and ibrutinib, and not by spebrutinib, while neutrophil activation was similarlyaffected by the three BTKi. We speculate that this inhibition may impact on theanti-microbial immune response ​in vivo and should be taken into account especially in thepresence of other risk factors or in heavily pre-treated patients.