FoxO1/Rictor axis induces a nongenetic adaptation to ibrutinib via Akt activation in chronic lymphocytic leukemia
Laura Ondrisova, … , Jiri Mayer, Marek Mraz
Laura Ondrisova, … , Jiri Mayer, Marek Mraz
Published October 22, 2024
Citation Information: J Clin Invest. 2024;134(23):e173770. https://doi.org/10.1172/JCI173770.
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FoxO1/Rictor axis induces a nongenetic adaptation to ibrutinib via Akt activation in chronic lymphocytic leukemia

Abstract

Bruton tyrosine kinase (BTK) inhibitor therapy induces peripheral blood lymphocytosis in chronic lymphocytic leukemia (CLL), which lasts for several months. It remains unclear whether nongenetic adaptation mechanisms exist, allowing CLL cells’ survival during BTK inhibitor–induced lymphocytosis and/or playing a role in therapy resistance. We show that in approximately 70% of CLL cases, ibrutinib treatment in vivo increases Akt activity above pretherapy levels within several weeks, leading to compensatory CLL cell survival and a more prominent lymphocytosis on therapy. Ibrutinib-induced Akt phosphorylation (pAktS473) is caused by the upregulation of Forkhead box protein O1 (FoxO1) transcription factor, which induces expression of Rictor, an assembly protein for the mTORC2 protein complex that directly phosphorylates Akt at serine 473 (S473). Knockout or inhibition of FoxO1 or Rictor led to a dramatic decrease in Akt phosphorylation and growth disadvantage for malignant B cells in the presence of ibrutinib (or PI3K inhibitor idelalisib) in vitro and in vivo. The FoxO1/Rictor/pAktS473 axis represents an early nongenetic adaptation to B cell receptor (BCR) inhibitor therapy not requiring PI3Kδ or BTK kinase activity. We further demonstrate that FoxO1 can be targeted therapeutically and its inhibition induces CLL cells’ apoptosis alone or in combination with BTK inhibitors (ibrutinib, acalabrutinib, pirtobrutinib) and blocks their proliferation triggered by T cell factors (CD40L, IL-4, and IL-21).

Authors

Laura Ondrisova, Vaclav Seda, Krystof Hlavac, Petra Pavelkova, Eva Hoferkova, Giorgia Chiodin, Lenka Kostalova, Gabriela Mladonicka Pavlasova, Daniel Filip, Josef Vecera, Pedro Faria Zeni, Jan Oppelt, Zuzana Kahounova, Rachel Vichova, Karel Soucek, Anna Panovska, Karla Plevova, Sarka Pospisilova, Martin Simkovic, Filip Vrbacky, Daniel Lysak, Stacey M. Fernandes, Matthew S. Davids, Alba Maiques-Diaz, Stella Charalampopoulou, Jose I. Martin-Subero, Jennifer R. Brown, Michael Doubek, Francesco Forconi, Jiri Mayer, Marek Mraz

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Figure 4

FoxO1-KO or inhibition leads to decrease of Rictor and pAktS473 levels.

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FoxO1-KO or inhibition leads to decrease of Rictor and pAktS473 levels. ...
(A) Representative immunoblot of FoxO1-KO MEC1 clones and densitometric quantification of relative Rictor and pAktS473 protein levels in all obtained FoxO1-KO MEC1 clones with complete FoxO1 KO (n = 15). P values were calculated using unpaired t test. (B) Representative immunoblot of WT and FoxO1-KO MEC1 clones (n = 4). Cells were treated with ibrutinib (1 μM) for 7 days. Fresh ibrutinib was added to culture media every other day. (C) Competitive growth of WT MEC1 cells versus FoxO1-KO MEC1 clones in medium with ibrutinib (2 μM, 4 weeks) relative to growth in medium with vehicle (DMSO). WT and FoxO1-KO cells marked with GFP or AZURIT (and vice versa) and mixed in 1:1 ratio (n = 4 repetitions for each of the 3 clones, WT clones are marked by numbers corresponding to the specific KO clone that was used in the corresponding competitive growth experiment). Cells were treated with ibrutinib (fresh ibrutinib was added 3 times a week) or DMSO. Graph represents the percentage of KO versus WT ibrutinib-treated cells, and this is plotted relatively to vehicle-treated (DMSO) KO or WT cells, respectively, to correct for any effect of the KO on ibrutinib-unrelated cell fitness. Statistical difference was tested using 2-way ANOVA with Geisser-Greenhouse correction. (D) Representative immunoblot of MEC1 cells treated with various FoxO1 inhibitor concentrations (72 hours). (E) Representative immunoblot of MEC1 cells treated with FoxO1 inhibitor (inh) or mTOR inhibitor (both 0.5 μM, 24 hours). (F) Representative immunoblot of primary CLL cells treated with FoxO1 inhibitor or mTOR inhibitor for 48 hours (both 0.5 μM) and then stimulated with anti-IgM (20 μg/ml, 10 minutes). (G) Representative immunoblot of primary CLL cells treated with FoxO1 inhibitor (0.5 μM) for 48 hours and then stimulated with bead-bound anti-IgM (3 hours). (H) Relative protein levels of pAktS473 and cMYC obtained by densitometric quantification of immunoblots from experiment in G (n = 6). P values were calculated using paired t test.