30. Venetoclax-resistant CLL cells show an aggressive phenotype.

ELIAS ESTEBAN ENRIQUE; GAMBERALE ROMINA

Edimburgo

Workshop; XVIII International Workshop on Chronic Lymphocytic Leukemia,; 2019

It iswell known that tumor microenvironment is crucial for survival and proliferationof CLL cells and also for generating drug resistance. We have previouslyreported that in vitro stimulation ofautologous T cells favors the activation of the leukemic clone and theupregulation of MCL-1 and/or BCL-XL, fostering venetoclax resistance in CLLcells (Elías EE, Haematologica, 2018).Our results and those obtained by others, showing that signaling through theBCR or signals provided by fibroblast CD40L+ and by stromal celllines reduce the sensitivity of CLL cells to the drug, suggest thattissue-resident leukemic cells might not be properly targeted by venetoclaxalone. We here aimed to characterize venetoclax-resistant CLL cells. To thisaim, peripheral blood mononuclear cells from CLL patientswere cultured for 48hs without (control) or with anti-CD3 (aCD3) to activate Tcells, and then venetoclax was added to the cultures. Leukemic cellsurvival, activation and proliferation capacity was evaluatedby flow cytometry. While control CLL cells treated with venetoclax 0.1µM showedmore than 97% of cell death after 24hs, the percentage of viable CLL cellstreated with venetoclax was significantly higher in cultures with activated Tcells, even at 120 hs (mean±SEM of %CD19+ viable cells: 2.0±0.5 vs 15.0±5.0for venetoclax 0.1µM vs aCD3+venetoclax 0.1µM, n=16, * p˂0.05). Interestingly, resistantCLL 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 aCD3cultures. Moreover, we found that CLL cells previously cultured withaCD3+venetoclax for 120hs, became more resistant to a second treatment withvenetoclax (Figure1D). To overcome venetoclax resistance induced by autologous T cellactivation we previously used the BCR kinase inhibitor (BCR-KI) entospletinib (Elías EE, Haematologica, 2018). Here weshowed that entospletinib did not directly affect CLL cell survival or modify venetoclax-inducedcell 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 theimportance of the tumor microenvironment in the generation of venetoclax-resistantCLL cells, which showed a more aggressive phenotype: increased expression ofactivation and proliferation markers andmore resistance to a second treatmentwith the drug. They also encourage the combination of venetoclax with BCR-KIs,such as entospletinib, which impairs activation signals provided by the tumor microenvironment.