Monoclonal antibodies (mAb) are key therapeutic agents in cancer immunotherapy and exert their effects through Fc receptor-dependent and -independent mechanisms. However, the nanoscale receptor reorganization resulting from mAb binding and its implications for the therapeutic mode of action remain poorly understood. Here, we present a multi-target 3D RESI super-resolution microscopy technique that directly visualizes the structural organization of CD20 receptors and the Type I (e.g., Rituximab) and Type II (e.g., Obinutuzumab) anti-CD20 therapeutic antibodies and quantitatively analyze these interactions at single-protein resolution in situ. We discover that, while Type I mAbs promote higher-order CD20 oligomerization, Type II mAbs induce limited clustering, leading to differences in therapeutic function. Correlating RESI with functional studies for Type II antibodies with different hinge region flexibilities, we show that the oligomeric CD20 arrangement determines the Type I or Type II function. Thus, the nanoscale characterization of CD20-mAb complexes enhances our understanding of the structure-function relationships of therapeutic antibodies and offers insights into the design of next-generation mAb therapies.

• MeSH
• Antigens, CD20 * immunology   chemistry   metabolism   MeSH
• Antibodies, Monoclonal, Humanized * therapeutic use   chemistry   MeSH
• Immunotherapy * methods   MeSH
• Humans MeSH
• Antibodies, Monoclonal * chemistry   therapeutic use   MeSH
• Protein Multimerization MeSH
• Cell Line, Tumor MeSH
• Neoplasms * therapy   immunology   MeSH
• Rituximab * therapeutic use   chemistry   immunology   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Antigens, CD20 * MeSH
• Antibodies, Monoclonal, Humanized * MeSH
• Antibodies, Monoclonal * MeSH
• obinutuzumab MeSH Browser
• Rituximab * MeSH

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

• MeSH
• Antigens, CD20 metabolism   MeSH
• Quinazolinones pharmacology   therapeutic use   MeSH
• Leukemia, Lymphocytic, Chronic, B-Cell * drug therapy   MeSH
• Interleukin-4 MeSH
• Humans MeSH
• Antineoplastic Agents * therapeutic use   MeSH
• Purines pharmacology   therapeutic use   MeSH
• STAT6 Transcription Factor MeSH
• Check Tag
• Humans MeSH
• Publication type
• Letter MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Antigens, CD20 MeSH
• Quinazolinones MeSH
• idelalisib MeSH Browser
• Interleukin-4 MeSH
• Antineoplastic Agents * MeSH
• Purines MeSH
• STAT6 protein, human MeSH Browser
• STAT6 Transcription Factor MeSH

The approval of BTK and PI3K inhibitors (ibrutinib, idelalisib) represents a revolution in the therapy of B cell malignancies such as chronic lymphocytic leukemia (CLL), mantle-cell lymphoma (MCL), diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL), or Waldenström's macroglobulinemia (WM). However, these "BCR inhibitors" function by interfering with B cell pathophysiology in a more complex way than anticipated, and resistance develops through multiple mechanisms. In ibrutinib treated patients, the most commonly described resistance-mechanism is a mutation in BTK itself, which prevents the covalent binding of ibrutinib, or a mutation in PLCG2, which acts to bypass the dependency on BTK at the BCR signalosome. However, additional genetic aberrations leading to resistance are being described (such as mutations in the CARD11, CCND1, BIRC3, TRAF2, TRAF3, TNFAIP3, loss of chromosomal region 6q or 8p, a gain of Toll-like receptor (TLR)/MYD88 signaling or gain of 2p chromosomal region). Furthermore, relative resistance to BTK inhibitors can be caused by non-genetic adaptive mechanisms leading to compensatory pro-survival pathway activation. For instance, PI3K/mTOR/Akt, NFkB and MAPK activation, BCL2, MYC, and XPO1 upregulation or PTEN downregulation lead to B cell survival despite BTK inhibition. Resistance could also arise from activating microenvironmental pathways such as chemokine or integrin signaling via CXCR4 or VLA4 upregulation, respectively. Defining these compensatory pro-survival mechanisms can help to develop novel therapeutic combinations of BTK inhibitors with other inhibitors (such as BH3-mimetic venetoclax, XPO1 inhibitor selinexor, mTOR, or MEK inhibitors). The mechanisms of resistance to PI3K inhibitors remain relatively unclear, but some studies point to MAPK signaling upregulation via both genetic and non-genetic changes, which could be co-targeted therapeutically. Alternatively, drugs mimicking the BTK/PI3K inhibition effect can be used to prevent adhesion and/or malignant B cell migration (chemokine and integrin inhibitors) or to block the pro-proliferative T cell signals in the microenvironment (such as IL4/STAT signaling inhibitors). Here we review the genetic and non-genetic mechanisms of resistance and adaptation to the first generation of BTK and PI3K inhibitors (ibrutinib and idelalisib, respectively), and discuss possible combinatorial therapeutic strategies to overcome resistance or to increase clinical efficacy.

• Keywords
• B cell malignancies, B cell receptor, BCR inhibitor, adaptation, ibrutinib, resistance, targeted therapy,
• Publication type
• Journal Article MeSH