BACKGROUND Ibrutinib is an irreversible inhibitor of Bruton’s tyrosine kinase (BTK) and is effective in chronic lymphocytic leukemia (CLL). Resistance to irreversible kinase inhibitors and resistance associated with BTK inhibition have not been characterized. Although only a small proportion of patients have had a relapse during ibrutinib therapy, an understanding of resistance mechanisms is important. We evaluated patients with relapsed disease to identify mutations that may mediate ibrutinib resistance. METHODS We performed whole-exome sequencing at baseline and the time of relapse on samples from six patients with acquired resistance to ibrutinib therapy. We then performed functional analysis of identified mutations. In addition, we performed Ion Torrent sequencing for identified resistance mutations on samples from nine patients with prolonged lymphocytosis. RESULTS We identified a cysteine-to-serine mutation in BTK at the binding site of ibrutinib in five patients and identified three distinct mutations in PLCγ2 in two patients. Functional analysis showed that the C481S mutation of BTK results in a protein that is only reversibly inhibited by ibrutinib. The R665W and L845F mutations in PLCγ2 are both potentially gain-of-function mutations that lead to autonomous B-cell–receptor activity. These mutations were not found in any of the patients with prolonged lymphocytosis who were taking ibrutinib. CONCLUSIONS Resistance to the irreversible BTK inhibitor ibrutinib often involves mutation of a cysteine residue where ibrutinib binding occurs. This finding, combined with two additional mutations in PLCγ2 that are immediately downstream of BTK, underscores the importance of the B-cell–receptor pathway in the mechanism of action of ibrutinib in CLL. (Funded by the National Cancer Institute and others.)

BackgroundMyeloid cells are an abundant leukocyte in many types of tumors and contribute to immune evasion. Expression of the enzyme arginase 1 (Arg1) is a defining feature of immunosuppressive myeloid cells and leads to depletion of L-arginine, a nutrient required for T cell and natural killer (NK) cell proliferation. Here we use CB-1158, a potent and orally-bioavailable small-molecule inhibitor of arginase, to investigate the role of Arg1 in regulating anti-tumor immunity.MethodsCB-1158 was tested for the ability to block myeloid cell-mediated inhibition of T cell proliferation in vitro, and for tumor growth inhibition in syngeneic mouse models of cancer as a single agent and in combination with other therapies. Tumors from animals treated with CB-1158 were profiled for changes in immune cell subsets, expression of immune-related genes, and cytokines. Human tumor tissue microarrays were probed for Arg1 expression by immunohistochemistry and immunofluorescence. Cancer patient plasma samples were assessed for Arg1 protein and L-arginine by ELISA and mass spectrometry, respectively.ResultsCB-1158 blocked myeloid cell-mediated suppression of T cell proliferation in vitro and reduced tumor growth in multiple mouse models of cancer, as a single agent and in combination with checkpoint blockade, adoptive T cell therapy, adoptive NK cell therapy, and the chemotherapy agent gemcitabine. Profiling of the tumor microenvironment revealed that CB-1158 increased tumor-infiltrating CD8+ T cells and NK cells, inflammatory cytokines, and expression of interferon-inducible genes. Patient tumor samples from multiple histologies expressed an abundance of tumor-infiltrating Arg1+ myeloid cells. Plasma samples from cancer patients exhibited elevated Arg1 and reduced L-arginine compared to healthy volunteers.ConclusionsThese results demonstrate that Arg1 is a key mediator of immune suppression and that inhibiting Arg1 with CB-1158 shifts the immune landscape toward a pro-inflammatory environment, blunting myeloid cell-mediated immune evasion and reducing tumor growth. Furthermore, our results suggest that arginase blockade by CB-1158 may be an effective therapy in multiple types of cancer and combining CB-1158 with standard-of-care chemotherapy or other immunotherapies may yield improved clinical responses.

No abstract

Positive regulation of gene expression by the yeast Saccharomyces cerevisiae transcription factor Yap1p is required for normal tolerance of oxidative stress elicited by the redox-active agents diamide and H 2 O 2 . Several groups have provided evidence that a cluster of cysteine residues in the extreme C terminus of the factor are required for normal modulation of Yap1p by oxidant challenge. Deletion of this C-terminal cysteine-rich domain (c-CRD) produces a protein that is highly active under both stressed and nonstressed conditions and is constitutively located in the nucleus. We have found that a variety of different c-CRD mutant proteins are hyperactive in terms of their ability to confer diamide tolerance to cells but fail to provide even normal levels of H 2 O 2 resistance. Although the c-CRD mutant forms of Yap1p activate an artificial Yap1p-responsive gene to the same high level in the presence of either diamide or H 2 O 2 , these mutant factors confer hyperresistance to diamide but hypersensitivity to H 2 O 2 . To address this discrepancy, we have examined the ability of c-CRD mutant forms of Yap1p to activate expression of an authentic target gene required for H 2 O 2 tolerance, TRX2. When assayed in the presence of c-CRD mutant forms of Yap1p, a TRX2-lacZ fusion gene fails to induce in response to H 2 O 2 . We have also identified a second cysteine-rich domain, in the N terminus (n-CRD), that is required for H 2 O 2 but not diamide resistance and influences the localization of the protein. These data are consistent with the idea that the function of Yap1p is different at promoters of loci involved in H 2 O 2 tolerance from promoters of genes involved in diamide resistance.To grow in the presence of oxygen, cells must be able to deal with reactive oxygen species (ROS) that are produced during metabolism. Aerobes have the ability both to detoxify ROS and to repair macromolecules that are damaged by these highly reactive compounds (26). Owing to the potentially lethal action of ROS, cells maintain constant surveillance of intracellular ROS levels and rapidly activate the expression of loci involved in oxidative stress tolerance (7).The yeast Saccharomyces cerevisiae has been a useful model for studies of the eukaryotic response to oxidant challenge (15). S. cerevisiae produces a variety of enzymes, such as catalase, superoxide dismutase, and glutathione peroxidase, and small molecules and peptides (glutathione and thioredoxins) that detoxify ROS (reviewed in reference 10). Recent data have provided insight into the regulation of the biosynthesis of these enzymatic activities.One of the key regulators of oxidative stress tolerance in S. cerevisiae is the Yap1p transcription factor. The YAP1 gene is required for normal tolerance to a wide variety of oxidants and is essential for normal synthesis of a variety of antioxidant activities, including glutathione and glutathione reductase (4,5,19,27). Later studies established that Yap1p-dependent transactivation was markedly enhanced when cells were challenged w...

Key Points MCL cells are mobilized into the peripheral blood of patients treated with the BTK inhibitor ibrutinib. Ibrutinib dose-dependently inhibits BCR- and chemokine-mediated adhesion and migration of MCL cells.