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).

• Keywords
• Drug therapy, Hematology, Leukemias, Oncology, Signal transduction,
• MeSH
• Adenine * analogs & derivatives   pharmacology   MeSH
• Leukemia, Lymphocytic, Chronic, B-Cell * drug therapy   metabolism   genetics   pathology   MeSH
• Forkhead Box Protein O1 * metabolism   genetics   MeSH
• Phosphorylation MeSH
• Humans MeSH
• Mice MeSH
• Cell Line, Tumor MeSH
• Neoplasm Proteins metabolism   genetics   MeSH
• Piperidines * pharmacology   MeSH
• Rapamycin-Insensitive Companion of mTOR Protein * genetics   metabolism   MeSH
• Agammaglobulinaemia Tyrosine Kinase metabolism   genetics   antagonists & inhibitors   MeSH
• Proto-Oncogene Proteins c-akt * metabolism   genetics   MeSH
• Pyrazoles * pharmacology   MeSH
• Pyrimidines * pharmacology   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Adenine * MeSH
• BTK protein, human MeSH Browser
• Forkhead Box Protein O1 * MeSH
• FOXO1 protein, human MeSH Browser
• ibrutinib MeSH Browser
• Neoplasm Proteins MeSH
• Piperidines * MeSH
• Rapamycin-Insensitive Companion of mTOR Protein * MeSH
• Agammaglobulinaemia Tyrosine Kinase MeSH
• Proto-Oncogene Proteins c-akt * MeSH
• Pyrazoles * MeSH
• Pyrimidines * MeSH

Several in vitro models have been developed to mimic chronic lymphocytic leukemia (CLL) proliferation in immune niches; however, they typically do not induce robust proliferation. We prepared a novel model based on mimicking T-cell signals in vitro and in patient-derived xenografts (PDXs). Six supportive cell lines were prepared by engineering HS5 stromal cells with stable expression of human CD40L, IL4, IL21, and their combinations. Co-culture with HS5 expressing CD40L and IL4 in combination led to mild CLL cell proliferation (median 7% at day 7), while the HS5 expressing CD40L, IL4, and IL21 led to unprecedented proliferation rate (median 44%). The co-cultures mimicked the gene expression fingerprint of lymph node CLL cells (MYC, NFκB, and E2F signatures) and revealed novel vulnerabilities in CLL-T-cell-induced proliferation. Drug testing in co-cultures revealed for the first time that pan-RAF inhibitors fully block CLL proliferation. The co-culture model can be downscaled to five microliter volume for large drug screening purposes or upscaled to CLL PDXs by HS5-CD40L-IL4 ± IL21 co-transplantation. Co-transplanting NSG mice with purified CLL cells and HS5-CD40L-IL4 or HS5-CD40L-IL4-IL21 cells on collagen-based scaffold led to 47% or 82% engraftment efficacy, respectively, with ~20% of PDXs being clonally related to CLL, potentially overcoming the need to co-transplant autologous T-cells in PDXs.

• MeSH
• Stromal Cells * metabolism   pathology   MeSH
• Leukemia, Lymphocytic, Chronic, B-Cell * pathology   genetics   drug therapy   MeSH
• Protein Kinase Inhibitors pharmacology   MeSH
• Interleukin-21 MeSH
• Interleukins genetics   metabolism   MeSH
• Coculture Techniques * MeSH
• Humans MeSH
• CD40 Ligand * metabolism   genetics   MeSH
• Mice MeSH
• Cell Proliferation * MeSH
• T-Lymphocytes immunology   metabolism   MeSH
• Xenograft Model Antitumor Assays MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Protein Kinase Inhibitors MeSH
• Interleukin-21 MeSH
• Interleukins MeSH
• CD40 Ligand * MeSH

T cells are key components in environments that support chronic lymphocytic leukemia (CLL), activating CLL-cell proliferation and survival. Here, we review in vitro and in vivo model systems that mimic CLL-T-cell interactions, since these are critical for CLL-cell division and resistance to some types of therapy (such as DNA-damaging drugs or BH3-mimetic venetoclax). We discuss approaches for direct CLL-cell co-culture with autologous T cells, models utilizing supportive cell lines engineered to express T-cell factors (such as CD40L) or stimulating CLL cells with combinations of recombinant factors (CD40L, interleukins IL4 or IL21, INFγ) and additional B-cell receptor (BCR) activation with anti-IgM antibody. We also summarize strategies for CLL co-transplantation with autologous T cells into immunodeficient mice (NOD/SCID, NSG, NOG) to generate patient-derived xenografts (PDX) and the role of T cells in transgenic CLL mouse models based on TCL1 overexpression (Eµ-TCL1). We further discuss how these in vitro and in vivo models could be used to test drugs to uncover the effects of targeted therapies (such as inhibitors of BTK, PI3K, SYK, AKT, MEK, CDKs, BCL2, and proteasome) or chemotherapy (fludarabine and bendamustine) on CLL-T-cell interactions and CLL proliferation.

• Keywords
• B cells, CD40L, Eμ-TCL1, IL-21, IL-4, T cells, chronic lymphocytic leukemia, co-culture, fludarabine, ibrutinib, interactions, interleukin, microenvironment, models, therapy resistance, venetoclax, xenograft,
• Publication type
• Journal Article MeSH
• Review MeSH

Recirculation of chronic lymphocytic leukemia (CLL) cells between the peripheral blood and lymphoid niches plays a critical role in disease pathophysiology, and inhibiting this process is one of the major mechanisms of action for B-cell receptor (BCR) inhibitors such as ibrutinib and idelalisib. Migration is a complex process guided by chemokine receptors and integrins. However, it remains largely unknown how CLL cells integrate multiple migratory signals while balancing survival in the peripheral blood and the decision to return to immune niches. Our study provided evidence that CXCR4/CD5 intraclonal subpopulations can be used to study the regulation of migration of CLL cells. We performed RNA profiling of CXCR4dimCD5bright vs CXCR4brightCD5dim CLL cells and identified differential expression of dozens of molecules with a putative function in cell migration. GRB2-associated binding protein 1 (GAB1) positively regulated CLL cell homing capacity of CXCR4brightCD5dim cells. Gradual GAB1 accumulation in CLL cells outside immune niches was mediated by FoxO1-induced transcriptional GAB1 activation. Upregulation of GAB1 also played an important role in maintaining basal phosphatidylinositol 3-kinase (PI3K) activity and the "tonic" AKT phosphorylation required to sustain the survival of resting CLL B cells. This finding is important during ibrutinib therapy, because CLL cells induce the FoxO1-GAB1-pAKT axis, which represents an adaptation mechanism to the inability to home to immune niches. We have demonstrated that GAB1 can be targeted therapeutically by novel GAB1 inhibitors, alone or in combination with BTK inhibition. GAB1 inhibitors induce CLL cell apoptosis, impair cell migration, inhibit tonic or BCR-induced AKT phosphorylation, and block compensatory AKT activity during ibrutinib therapy.

• MeSH
• Adaptor Proteins, Signal Transducing biosynthesis   MeSH
• Adenine analogs & derivatives   pharmacology   MeSH
• Leukemia, Lymphocytic, Chronic, B-Cell drug therapy   metabolism   pathology   MeSH
• Forkhead Box Protein O1 metabolism   MeSH
• Humans MeSH
• Cell Line, Tumor MeSH
• Piperidines pharmacology   MeSH
• Cell Movement * MeSH
• Proto-Oncogene Proteins c-akt metabolism   MeSH
• Gene Expression Regulation, Leukemic * MeSH
• Signal Transduction * MeSH
• Up-Regulation * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Adaptor Proteins, Signal Transducing MeSH
• Adenine MeSH
• Forkhead Box Protein O1 MeSH
• FOXO1 protein, human MeSH Browser
• GAB1 protein, human MeSH Browser
• ibrutinib MeSH Browser
• Piperidines MeSH
• Proto-Oncogene Proteins c-akt MeSH