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).

4th Department of Internal Medicine Haematology University Hospital Hradec Kralove and Faculty of Medicine Hradec Kralove Charles University Prague Czechia

Cancer Sciences Faculty of Medicine University of Southampton Southampton United Kingdom

Department of Cytokinetics Institute of Biophysics of the Czech Academy of Sciences Brno Czechia

Department of Haematology and Oncology University Hospital Pilsen Pilsen Czechia

Department of Internal Medicine Hematology and Oncology University Hospital Brno and Faculty of Medicine Masaryk University Brno Czechia

Department of Medical Oncology Dana Farber Cancer Institute Boston Massachusetts USA

Haematology Department Cancer Care Directorate University Hospital Southampton NHS Trust Southampton United Kingdom

Institut d'Investigacions Biomèdiques August Pi 1 Sunyer University of Barcelona Barcelona Spain

Molecular Medicine CEITEC Masaryk University Brno Czechia

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