FoxO1/Rictor axis induces a nongenetic adaptation to ibrutinib via Akt activation in chronic lymphocytic leukemia
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
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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Research Article Hematology Oncology

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 5

FoxO1/Rictor/pAktS473 is responsible for adaptation to idelalisib.

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FoxO1/Rictor/pAktS473 is responsible for adaptation to idelalisib. (A) H...
(A) Heatmap of differentially expressed genes from samples obtained before and during idelalisib treatment in vivo (P adj < 0.05; base mean > 100; n = 11 pairs) and overlapped with 2 databases of genes involved in PI3K/Akt signaling (gene sets: no. M27162, reactome_PI3K_AKT_signalling_in_cancer and has04151, KEGG: PI3K-Akt signaling pathway). Lower expression indicated in blue, higher in yellow. Genes marked with asterisks were also differentially expressed after ibrutinib treatment in vivo (see Figure 2A). List of top 500 differentially expressed genes is included in Supplemental Table 5. (B) Representative immunoblot of Rictor, FoxO1, and pAktS473 in primary CLL sample treated with idelalisib in vivo. (C) Densitometric quantification of relative pAktS473, Rictor, and FoxO1 protein levels analyzed by immunoblot in primary samples of CLL patients treated with idelalisib in vivo for 2–4 weeks (n = 11 for pAktS473 and FoxO1; n = 9 for Rictor). P values were calculated using Wilcoxon’s test. For patient characteristics, see Supplemental Table 8. (D) Competitive growth of WT versus FoxO1-KO MEC1 cells in medium with idelalisib (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). Graph represents the percentage of KO versus WT idelalisib-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 idelalisib-unrelated cell fitness. Statistical difference was tested using 2-way ANOVA with Geisser-Greenhouse correction.