n4-(2-2-dimethyl-3-oxo-4h-pyrid(1-4)oxazin-6-yl)-5-fluoro-n2-(3-4-5-trimethoxyphenyl)-2-4-pyrimidinediamine and Leukemia--Lymphocytic--Chronic--B-Cell

Small molecules targeting kinases involved in B cell receptor signaling are showing encouraging clinical activity in chronic lymphocytic leukemia (CLL) patients. Fostamatinib (R406) and entospletinib (GS-9973) are ATP-competitive inhibitors designed to target spleen tyrosine kinase (Syk) that have shown clinical activity with acceptable toxicity in trials with CLL patients. Preclinical studies with these inhibitors in CLL have focused on their effect in patient-derived leukemic B cells. In this work we show that clinically relevant doses of R406 and GS-9973 impaired the activation and proliferation of T cells from CLL patients. This effect could not be ascribed to Syk-inhibition given that we show that T cells from CLL patients do not express Syk protein. Interestingly, ζ-chain-associated protein kinase (ZAP)-70 phosphorylation was diminished by both inhibitors upon TCR stimulation on T cells. In addition, we found that both agents reduced macrophage-mediated phagocytosis of rituximab-coated CLL cells. Overall, these results suggest that in CLL patients treated with R406 or GS-9973 T cell functions, as well as macrophage-mediated anti-tumor activity of rituximab, might be impaired. The potential consequences for CLL-treated patients are discussed.

Topics: Aged; Aged, 80 and over; Cell Proliferation; Cells, Cultured; Female; Humans; Indazoles; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphocyte Activation; Macrophages; Male; Middle Aged; Oxazines; Phagocytosis; Phosphorylation; Pyrazines; Pyridines; Receptors, Antigen, T-Cell; Rituximab; Syk Kinase; T-Lymphocytes; ZAP-70 Protein-Tyrosine Kinase

Topics: Adult; Aged; Cell Line, Tumor; Cell Movement; Cell Survival; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Middle Aged; Multiple Myeloma; Oxazines; Protein Kinase Inhibitors; Pyridines; Syk Kinase

Topics: Antigens, CD19; Antineoplastic Agents; B-Lymphocytes; CD40 Ligand; Cell Proliferation; Clinical Trials as Topic; Cyclohexylamines; Gene Expression Regulation, Leukemic; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphocyte Activation; Organ Specificity; Oxazines; p38 Mitogen-Activated Protein Kinases; Primary Cell Culture; Protein Kinase Inhibitors; Pyridines; Pyrimidines; Signal Transduction; Spleen; Syk Kinase; Transcription Factor RelA

The Bcl-2 antagonist ABT-199 (venetoclax) has demonstrated promising clinical activity in patients with chronic lymphocytic leukemia (CLL). ABT-199 is strongly cytotoxic against unstimulated peripheral blood CLL cells in vitro but is much less effective against CLL cells that have received survival signals from the microenvironment. In particular, stimulation of CLL cells with CD40L results in substantial resistance mediated by induction of the antiapoptotic Bcl-2 family proteins Bcl-xL and Bfl-1. In this study, we investigated whether resistance to ABT-199 can be conferred by B-cell receptor (BCR) stimulation, which is another important survival signal from the leukemic microenvironment. We show that sustained BCR stimulation results in significant ABT-199 resistance, which correlates with induction of the antiapoptotic protein Mcl-1 and less consistently with downregulation of proapoptotic Bmf, Hrk, and BimEL A major role for Mcl-1 in conferring ABT-199 resistance is additionally supported by knockdown and enforced expression experiments with primary CLL cells. We further show that SYK, BTK, and phosphatidylinositol 3-kinase δ (PI3Kδ) inhibitors significantly downregulate Mcl-1, but with different efficacy. Complete Mcl-1 downregulation was consistently achieved only with SYK inhibitors R406 and GS-9973 (entospletinib), whereas the BTK inhibitor ibrutinib and the PI3Kδ inhibitor idelalisib in more than half of the cases had only a partial effect. The greater ability of SYK inhibitors to downregulate Mcl-1 correlated with their greater capacity to block BCR-mediated inactivation of GSK-3, a major negative regulator of Mcl-1. The finding that BCR signaling inhibitors differ in their ability to target Mcl-1 is relevant for the design of clinical trials combining these agents with ABT-199.

Topics: Adenine; Antineoplastic Agents; Apoptosis; Bridged Bicyclo Compounds, Heterocyclic; Cell Line, Tumor; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Gene Expression Regulation, Leukemic; Gene Knockdown Techniques; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Myeloid Cell Leukemia Sequence 1 Protein; Oxazines; Piperidines; Purines; Pyrazoles; Pyridines; Pyrimidines; Quinazolinones; Receptors, Antigen, B-Cell; Sulfonamides

The survival and proliferation of CLL cells depends on microenvironmental contacts in lymphoid organs. CD38 is a cell surface receptor that plays an important role in survival and proliferation signaling in CLL. In this study we demonstrate SYK's direct involvement in the CD38 signaling pathway in primary CLL samples. CD38 stimulation of CLL cells revealed SYK activation. SYK downstream target AKT was subsequently induced and MCL-1 expression was increased. Concomitant inhibition of SYK by the SYK inhibitor R406 resulted in reduced activation of AKT and prevented upregulation of MCL-1. Moreover, short-term CD38 stimulation enhanced BCR-signaling, as indicated by increased ERK phosphorylation. CXCL12-dependent migration was increased after CD38 stimulation. Treating CLL cells with R406 inhibited CD38-mediated migration. In addition, we observed marked downregulation of CD38 expression for CLL cells treated with R406 compared to vehicle control. Finally, we observed a clear correlation between CD38 expression on CLL cells and SYK-inhibitor efficacy. In conclusion, our study provides deeper mechanistic insight into the effect of SYK inhibition in CLL.

Topics: ADP-ribosyl Cyclase 1; Adult; Aged; Aged, 80 and over; Apoptosis; Cell Movement; Cell Survival; Chemokine CXCL12; Extracellular Signal-Regulated MAP Kinases; Female; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Male; Membrane Glycoproteins; Middle Aged; Myeloid Cell Leukemia Sequence 1 Protein; Oxazines; Phosphorylation; Proto-Oncogene Proteins c-akt; Pyridines; Syk Kinase; Tumor Cells, Cultured

Chronic lymphocytic leukemia (CLL) is characterized by the progressive accumulation of clonal B lymphocytes. Proliferation occurs in lymphoid tissues upon interaction of leukemic cells with a supportive microenvironment. Therefore, the mobilization of tissue-resident CLL cells into the circulation is a useful therapeutic strategy to minimize the reservoir of tumor cells within survival niches. Because the exit of normal lymphocytes from lymphoid tissues depends on the presence of sphingosine-1 phosphate (S1P) and the regulated expression of S1P receptor-1 (S1PR1), we investigated whether the expression and function of S1PR1 can be modulated by key microenvironment signals. We found that activation of CLL cells with CXCL12, fibroblast CD40L(+), BCR cross-linking, or autologous nurse-like cells reduces their S1PR1 expression and the migratory response toward S1P. Moreover, we found that S1PR1 expression was reduced in the proliferative/activated subset of leukemic cells compared with the quiescent subset from the same patient. Similarly, bone marrow-resident CLL cells expressing high levels of the activation marker CD38 showed a lower expression of S1PR1 compared with CD38(low) counterparts. Finally, given that treatment with BCR-associated kinase inhibitors induces a transient redistribution of leukemic cells from lymphoid tissues to circulation, we studied the effect of the Syk inhibitors piceatannol and R406 on S1PR1 expression and function. We found that they enhance S1PR1 expression in CLL cells and their migratory response toward S1P. Based on our results, we suggest that the regulated expression of S1PR1 might modulate the egress of the leukemic clone from lymphoid tissues.

Topics: ADP-ribosyl Cyclase 1; Adult; Aged; Aged, 80 and over; Animals; B-Lymphocytes; CD40 Ligand; Cell Movement; Chemokine CXCL12; Female; Gene Expression; Gene Expression Regulation, Neoplastic; Humans; Intracellular Signaling Peptides and Proteins; Leukemia, Lymphocytic, Chronic, B-Cell; Lysophospholipids; Male; Membrane Glycoproteins; Mice; Middle Aged; Oxazines; Protein-Tyrosine Kinases; Proto-Oncogene Proteins c-bcr; Pyridines; Receptors, CXCR4; Receptors, Lysosphingolipid; Sphingosine; Sphingosine-1-Phosphate Receptors; Stilbenes; Syk Kinase; Tumor Cells, Cultured; Tumor Microenvironment

The microenvironment provides essential growth and survival signals to chronic lymphocytic leukemia (CLL) cells and contributes to their resistance to cytotoxic agents. Pharmacologic inhibition of spleen tyrosine kinase (SYK), a key mediator of B-cell receptor (BCR) signaling, induces apoptosis in primary CLL cells and prevents stroma contact-mediated cell survival. This report demonstrates a role of SYK in molecularly defined pathways that mediate the CLL-microenvironmental crosstalk independent from the BCR. Chemokine and integrin stimulation induced SYK phosphorylation, SYK-dependent Akt phosphorylation, and F-actin formation in primary CLL cells. Inhibition of SYK by 2 pharmacologic inhibitors and siRNA-knockdown abrogated downstream SYK signaling and morphologic changes induced by these stimuli. CLL cell migration toward CXCL12, the major homing attractor, and CLL cell adhesion to VCAM-1, a major integrin ligand expressed on stromal cells, were markedly reduced by SYK inhibition. In combination with fludarabine, the SYK inhibitor R406 abrogated stroma-mediated drug resistance by preventing up-regulation of the antiapoptotic factor Mcl-1 in CLL cells. SYK blockade in CLL is a promising therapeutic principle not only for its inhibition of the BCR signaling pathway, but also by inhibiting protective stroma signals in a manner entirely independent of BCR signaling.

Topics: Actins; Aged; Animals; Apoptosis; Chemokine CXCL12; Chemokines; Chemotaxis; Coculture Techniques; Female; Fibronectins; Humans; Integrin alpha4; Integrins; Intracellular Signaling Peptides and Proteins; Leukemia, Lymphocytic, Chronic, B-Cell; Male; Mice; Middle Aged; Myeloid Cell Leukemia Sequence 1 Protein; Oxazines; Prognosis; Protein Kinase Inhibitors; Protein-Tyrosine Kinases; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Pyridines; Receptors, Antigen, B-Cell; RNA, Small Interfering; Signal Transduction; Stromal Cells; Syk Kinase; Tumor Cells, Cultured; Vascular Cell Adhesion Molecule-1

The protein kinase Syk is a key mediator of proximal B-cell receptor (BCR) signaling. Following antigen stimulation, Syk is recruited to the BCR and becomes activated by phosphorylation at Y352. Recently, Syk was found to be constitutively phosphorylated in several common B-cell lymphoma subtypes, indicating a role for antigen-independent Syk activation in the pathogenesis of these diseases. We now report that Syk is constitutively phosphorylated on the activating Y352 residue in chronic lymphocytic leukemia (CLL) B cells. To examine the effects of constitutive Syk activity on intracellular signaling and leukemic cell survival, we performed in vitro studies with the Syk inhibitor R406. Treatment with R406 induced leukemic cell apoptosis in the majority of investigated cases and affected the basal activity or expression of several pro-survival molecules regulated by Syk, including the Akt and extracellular signal-regulated (ERK) kinases, and the anti-apoptotic protein Mcl-1. In addition, R406 prevented the increase in leukemic cell viability induced by sustained BCR engagement and inhibited BCR-induced Akt activation and Mcl-1 upregulation. Collectively, these data identify Syk as a potential target for CLL treatment and suggest that inhibition of this kinase could provide a double therapeutic benefit by disrupting both antigen-dependent and antigen-independent signaling pathways that regulate leukemic cell survival.

Topics: Aged; Aged, 80 and over; Apoptosis; Apoptosis Regulatory Proteins; B-Lymphocytes; Cell Survival; Down-Regulation; Female; Humans; Intracellular Signaling Peptides and Proteins; Leukemia, Lymphocytic, Chronic, B-Cell; Male; Middle Aged; Myeloid Cell Leukemia Sequence 1 Protein; Oxazines; Phosphorylation; Protein-Tyrosine Kinases; Proto-Oncogene Proteins c-bcl-2; Pyridines; Receptors, Antigen, B-Cell; Syk Kinase; Tumor Cells, Cultured

Antigenic stimulation through the B-cell antigen receptor (BCR) is considered to promote the expansion of chronic lymphocytic leukemia (CLL) B cells. The spleen tyrosine kinase (Syk), a key component of BCR signaling, can be blocked by R406, a small-molecule Syk inhibitor, that displayed activity in CLL patients in a first clinical trial. In this study, we investigated the effects of BCR stimulation and R406 on CLL cell survival and migration. The prosurvival effects promoted by anti-IgM stimulation and nurselike cells were abrogated by R406. BCR triggering up-regulated adhesion molecules, and increased CLL cell migration toward the chemokines CXCL12 and CXCL13. BCR activation also enhanced CLL cell migration beneath marrow stromal cells. These responses were blocked by R406, which furthermore abrogated BCR-dependent secretion of T-cell chemokines (CCL3 and CCL4) by CLL cells. Finally, R406 inhibited constitutive and BCR-induced activation of Syk, extracellular signal-regulated kinases, and AKT, and blocked BCR-induced calcium mobilization. These findings suggest that BCR activation favors CLL cell homing, retention, and survival in tissue microenvironments. R406 effectively blocks these BCR-dependent responses in CLL cells, providing an explanation for the activity of R406 in patients with CLL.

Topics: Adult; Aged; Aged, 80 and over; Animals; Antineoplastic Agents; Cell Adhesion Molecules; Cell Survival; Chemokine CCL3; Chemokine CCL4; Chemotaxis; Coculture Techniques; Drug Screening Assays, Antitumor; Female; Humans; Intracellular Signaling Peptides and Proteins; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphocyte Activation; Male; Mice; Middle Aged; Neoplasm Proteins; Oxazines; Protein Kinase Inhibitors; Protein Processing, Post-Translational; Protein-Tyrosine Kinases; Pyridines; Receptors, Antigen, B-Cell; Receptors, Chemokine; Signal Transduction; Stromal Cells; Syk Kinase

B-cell receptor signaling contributes to apoptosis resistance in chronic lymphocytic leukemia (CLL), limiting the efficacy of current therapeutic approaches. In this study, we investigated the expression of spleen tyrosine kinase (SYK), a key component of the B-cell receptor signaling pathway, in CLL and its role in apoptosis. Gene expression profiling identified enhanced expression of SYK and downstream pathways in CLL compared with healthy B cells. Immunoblotting showed increased expression and phosphorylation of SYK, PLCgamma(2), signal transducers and activators of transcription 3, and extracellular signal regulated kinase 1/2 in CLL compared with healthy B cells, suggesting enhanced activation of these mediators in CLL. SYK inhibitors reduced phosphorylation of SYK downstream targets and induced apoptosis in primary CLL cells. With respect to prognostic factors, SYK inhibitors exerted stronger cytotoxic effects in unmutated and ZAP70(+) cases. Cytotoxic effects of SYK inhibitors also associated with SYK protein expression, potentially predicting response to therapy. Combination of fludarabine with SYK Inhibitor II or R406 increased cytotoxicity compared with fludarabine therapy alone. We observed no stroma-contact-mediated drug resistance for SYK inhibitors as described for fludarabine treatment. CD40 ligation further enhanced efficacy of SYK inhibition. Our data provide mechanistic insight into the recently observed therapeutic effects of the SYK inhibitor R406 in CLL. Combination of SYK inhibitors with fludarabine might be a novel treatment option particularly for CLL patients with poor prognosis and should be further evaluated in clinical trials.

Topics: Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; B-Lymphocytes; Caspase 3; Cell Line, Tumor; Curcumin; Gene Expression Profiling; Humans; Immunoglobulin Variable Region; Intracellular Signaling Peptides and Proteins; Leukemia, Lymphocytic, Chronic, B-Cell; Mutation; Neoplasm Staging; Oxazines; Phosphorylation; Prognosis; Protein-Tyrosine Kinases; Proto-Oncogene Proteins c-akt; Pyridines; Syk Kinase; Vidarabine