Venetoclax-resistant CLL cells show an aggressive phenotype

CORDINI, GREGORIO; FERNANDEZ GRECCO, HORACIO; SANCHEZ AVALOS, JULIO CESAR; BORGE, MERCEDES; ELÍAS, ESTEBAN E; VERGARA-RUBIO, MARICEF; BEZARES, FERNANDO R.; VICENTE, ÁNGELES; GIORDANO, MIRTA N.; COLADO, ANA; CABREJO, MARÍA; CUSTIDIANO, MARÍA DEL ROSARIO; GARATE, GONZALO MARTÍN; GAMBERALE, ROMINA

Edimburgo

Workshop; XVIII International Workshop on Chronic Lymphocytic Leukemia; 2019

It is well known that tumor microenvironment is crucial for survival and proliferation of CLL cells and also for generating drug resistance. We have previously reported that in vitro activation of autologous T cells favors the activation of the leukemic clone and the upregulation of MCL-1 and/or BCL-XL, fostering venetoclax resistance in CLL cells (Elías EE, Haematologica, 2018). Our results and those obtained by others, showing that signaling through the BCR or signals provided by fibroblast CD40L+ and by stromal cell lines were shown to reduce the sensitivity of CLL cells to the drug, suggest that tissue-resident leukemic cells might not be properly targeted by venetoclax alone. We here aimed to characterize venetoclax-resistant CLL cells. To this aim peripheral blood mononuclear cells (PBMC) from CLL patients were cultured for 48hs without (control) or with anti-CD3 (aCD3) to activate T cells, and then venetoclax was added to the cultures. Leukemic cell survival, activation and proliferation capacity were evaluated by flow cytometry. While control CLL cells treated with venetoclax showed more than 97% of cell death after 24hs, CLL cells from aCD3 cultures are still alive with venetoclax 0.1µM at 120hs (mean±SEM of %CD19+ viable cells: 2.0±0.5 vs 15.0±5.0 for venetoclax vs aCD3+venetoclax n=16, * p˂0.05). Interestingly, resistant CLL cells from aCD3+venetoclax cultures showed an increased size (Figure 1A), higher expression of CD86 (Figure 1B) and Ki67 (Figure 1C) compared to CLL cells from aCD3 cultures. Moreover, we found that CLL cells previously cultured with aCD3+venetoclax for 120hs, were more resistant to a second treatment with venetoclax (Figure 1D). To overcome venetoclax resistance induced by autologous T cell activation we previously used the BCR kinase inhibitor (BCR-KI) entospletinib (Elías EE, Haematologica, 2018). Here we showed that entospletinib did not directly affect CLL cell survival or modify venetoclax-induced cell death (n=10) (Figure 2), corroborating that its effects were through impairment of T cell activation (Colado A, Cancer.Immunol.Immunother, 2017). In conclusion, our results highlight the importance of the tumor microenvironment in the generation of venetoclax-resistant CLL cells, which showed a more aggressive phenotype: increased expression of activation and proliferation markers and more resistance to a second treatment with the drug. They also encourage the combination of venetoclax with BCR-KIs, such as entospletinib, which impairs activation signals provided by the tumor microenvironment.