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 2

Induction in Akt activity during ibrutinib treatment is caused by Rictor upregulation.

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Induction in Akt activity during ibrutinib treatment is caused by Rictor...
(A) Heatmap of differentially expressed genes from samples obtained before and during ibrutinib treatment in vivo (P adj < 0.05; base mean > 100; n = 11 pairs). List includes genes overlapping with genes involved in PI3K/Akt signaling (gene sets: no. M27162, reactome_PI3K_AKT_signalling _in_cancer and has04151, KEGG: PI3K-Akt signaling pathway). (B) PI3K/Akt pathway with visualization of genes differentially expressed in patients treated with ibrutinib in vivo. (C) Representative immunoblots of Rictor in primary CLL samples obtained before and during ibrutinib treatment in vivo (n = 2). (D) Densitometric quantification of Rictor protein levels in primary CLL samples obtained before and during ibrutinib treatment in vivo for 2–8 weeks (n = 39). (E) Relative Rictor protein levels analyzed by densitometric quantification of immunoblots in MEC1 cells treated with ibrutinib (2 μM) for 5 days (n = 5; representative immunoblot in Figure 1C). (F) Representative immunoblot of WT and Rictor-KO MEC1 clones (Rictor-KO). Cells treated with ibrutinib (2 μM) for 1 or 9 days. (G) Competitive growth of WT versus Rictor-KO MEC1 cells. (n = 4 repetitions for each of the 2 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 (2 μM, fresh ibrutinib was added 3 times a week) or vehicle (DMSO). Graph represents percentage of KO versus WT ibrutinib-treated cells, and this is plotted relative 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. (H) Representative immunoblot of MEC1 cells treated with mTOR inhibitor AZD8055 (0.1–10 μM, 24 hours). (I) Representative immunoblot of primary CLL cells treated with mTOR inhibitor AZD8055 (0.5 μM) for 24 hours and then stimulated with anti-IgM (20 μg/ml) for 10 minutes. (J) Representative immunoblot of primary CLL cells treated with mTOR inhibitor AZD8055 (0.5 μM) for 24 hours and then stimulated with bead-bound anti-IgM for 3 hours. (K) Relative protein levels of pAktS473 and cMYC obtained by densitometric quantification of immunoblots from the experiment described in J (n = 5). (L) Relative viability (WST-1 absorbance) in CLL cells (n = 6) treated with ibrutinib (ibr, 1 μM), idelalisib (idela, 1 μM), AZD8055 (mTOR inh, 0.5 μM), or their combination (48 hours). P values in D, E, K, and L were calculated using paired t test. For patient characteristics, see Supplemental Table 8.