ABSTRACT
The treatment of patients with relapsed or refractory lymphoid neoplasms represents a significant clinical challenge. Here, we identify the pro-survival BCL-2 protein family member MCL-1 as a resistance factor for the BCL-2 inhibitor venetoclax in non-Hodgkin lymphoma (NHL) cell lines and primary NHL samples. Mechanistically, we show that the antibody-drug conjugate polatuzumab vedotin promotes MCL-1 degradation via the ubiquitin/proteasome system. This targeted MCL-1 antagonism, when combined with venetoclax and the anti-CD20 antibodies obinutuzumab or rituximab, results in tumor regressions in preclinical NHL models, which are sustained even off-treatment. In a Phase Ib clinical trial (NCT02611323) of heavily pre-treated patients with relapsed or refractory NHL, 25/33 (76%) patients with follicular lymphoma and 5/17 (29%) patients with diffuse large B-cell lymphoma achieved complete or partial responses with an acceptable safety profile when treated with the recommended Phase II dose of polatuzumab vedotin in combination with venetoclax and an anti-CD20 antibody.
Subject(s)
Immunoconjugates , Lymphoma, Non-Hodgkin , Humans , Myeloid Cell Leukemia Sequence 1 Protein/therapeutic use , Antineoplastic Combined Chemotherapy Protocols/adverse effects , Lymphoma, Non-Hodgkin/drug therapy , Lymphoma, Non-Hodgkin/pathology , Rituximab/therapeutic use , Immunoconjugates/therapeutic useABSTRACT
PURPOSE OF REVIEW: Rho kinases (ROCKs) are involved in regulating a variety of physiologic functions including cytoskeletal reorganization, migration, adhesion, survival and proliferation. They do so via activating several different downstream substrates such as myosin light chain phosphatase, LIM kinase and ezrin/radixin/moesin proteins. To date, most of the conclusions with regard to the function of ROCKs have involved the use of cell line models, pharmacologic inhibitors and dominant negative approaches. Importantly, the role of ROCK in hematopoiesis or leukemogenesis in the context of whole organism remains poorly understood. RECENT FINDINGS: Recent studies utilizing mice deficient in the expression of ROCK1 have begun to shed some light into the physiologic role(s) of ROCK in both normal and abnormal hematopoiesis. Findings, thus far, suggest that ROCK plays an essential role in regulating growth and survival in different hematopoietic lineages via distinct mechanisms, in part, by utilizing distinct downstream substrates including maintaining the activation of tumor-suppressor genes. SUMMARY: In blood cells, emerging data suggest that ROCK plays an essential role in negatively regulating inflammatory and erythropoietic stress and positively regulates the growth and survival of leukemic cells.
Subject(s)
Hematologic Diseases/genetics , Hematologic Diseases/metabolism , Hematopoiesis/physiology , rho-Associated Kinases/genetics , rho-Associated Kinases/metabolism , Animals , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/metabolism , Embryonic Stem Cells/cytology , Embryonic Stem Cells/metabolism , Erythropoiesis , Humans , Leukemia/genetics , Leukemia/metabolism , Myeloid Cells/physiology , Stress, PhysiologicalABSTRACT
Erythropoiesis is a dynamic, multistep process whereby hematopoietic stem cells differentiate toward a progressively committed erythroid lineage through intermediate progenitors. Although several downstream signaling molecules have been identified that regulate steady-state erythropoiesis, the major regulators under conditions of stress remain poorly defined. Rho kinases (ROCKs) belong to a family of serine/threonine kinases. Using gene-targeted ROCK1-deficient mice, we show that lack of ROCK1 in phenylhydrazine-induced oxidative stress model results in enhanced recovery from hemolytic anemia as well as enhanced splenic stress erythropoiesis compared with control mice. Deficiency of ROCK1 also results in enhanced survival, whereas wild-type mice die rapidly in response to stress. Enhanced survivability of ROCK1-deficient mice is associated with reduced level of reactive oxygen species. BM transplantation studies revealed that enhanced stress erythropoiesis in ROCK1-deficient mice is stem cell autonomous. We show that ROCK1 binds to p53 and regulates its stability and expression. In the absence of ROCK1, p53 phosphorylation and expression is significantly reduced. Our findings reveal that ROCK1 functions as a physiologic regulator of p53 under conditions of erythroid stress. These findings are expected to offer new perspectives on stress erythropoiesis and may provide a potential therapeutic target in human disease characterized by anemia.
Subject(s)
Anemia, Hemolytic/mortality , Anemia, Hemolytic/prevention & control , Apoptosis , Erythropoiesis/physiology , Oxidative Stress/physiology , Tumor Suppressor Protein p53/metabolism , rho-Associated Kinases/physiology , Anemia, Hemolytic/chemically induced , Animals , Antimetabolites, Antineoplastic/toxicity , Blotting, Western , Bone Marrow/drug effects , Bone Marrow/metabolism , Bone Marrow/pathology , Erythroid Precursor Cells/drug effects , Erythroid Precursor Cells/metabolism , Erythroid Precursor Cells/pathology , Erythropoiesis/drug effects , Erythropoietin/blood , Female , Flow Cytometry , Fluorouracil/toxicity , Immunoprecipitation , Male , Mice , Mice, Knockout , Oxidants/toxicity , Oxidative Stress/drug effects , Phenylhydrazines/toxicity , Phosphorylation , RNA, Messenger/genetics , Reactive Oxygen Species/metabolism , Real-Time Polymerase Chain Reaction , Signal Transduction , Spleen/drug effects , Spleen/metabolism , Spleen/pathology , Survival Rate , Tumor Suppressor Protein p53/geneticsABSTRACT
We show that loss of p85α inhibits the growth and maturation of mast cells, whereas loss of p85ß enhances this process. Whereas restoring the expression of p85α in P85α(-/-) cells restores these functions, overexpression of p85ß has the opposite effect. Consistently, overexpression of p85ß in WT mast cells represses KIT-induced proliferation and IL-3-mediated maturation by inhibiting the expression of Microphthalmia transcription factor. Because p85α and p85ß differ in their N-terminal sequences, chimeric proteins consisting of amino or carboxy-terminal of p85α and/or p85ß do not rescue the growth defects of p85α(-/-) cells, suggesting cooperation between these domains for normal mast cell function. Loss of p85ß impaired ligand induced KIT receptor internalization and its overexpression enhanced this process, partly because of increased binding of c-Cbl to p85ß relative to p85α. In vivo, loss of p85ß resulted in increased mast cells, and bone marrow transplantation of cells overexpressing p85ß resulted in significant reduction in some tissue mast cells. Overexpression of p85ß suppressed the growth of oncogenic KIT-expressing cells in vitro and prolonged the survival of leukemic mice in vivo. Thus, p85α and p85ß differentially regulate SCF and oncogenic KIT-induced signals in myeloid lineage-derived mast cells.
Subject(s)
Class Ia Phosphatidylinositol 3-Kinase/physiology , Leukemia/etiology , Leukemia/pathology , Mast Cells/pathology , Animals , Blotting, Western , Cell Proliferation , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Leukemia/metabolism , Mast Cells/metabolism , Mice , Mice, Inbred C3H , Mice, Inbred C57BL , Mice, Knockout , Mutation/genetics , Proto-Oncogene Proteins c-kit/genetics , Proto-Oncogene Proteins c-kit/metabolism , RNA, Messenger/genetics , Real-Time Polymerase Chain ReactionABSTRACT
Intracellular mechanism(s) that contribute to promiscuous signaling via oncogenic KIT in systemic mastocytosis and acute myelogenous leukemia are poorly understood. We show that SHP2 phosphatase is essential for oncogenic KIT-induced growth and survival in vitro and myeloproliferative disease (MPD) in vivo. Genetic disruption of SHP2 or treatment of oncogene-bearing cells with a novel SHP2 inhibitor alone or in combination with the PI3K inhibitor corrects MPD by disrupting a protein complex involving p85α, SHP2, and Gab2. Importantly, a single tyrosine at position 719 in oncogenic KIT is sufficient to develop MPD by recruiting p85α, SHP2, and Gab2 complex to oncogenic KIT. Our results demonstrate that SHP2 phosphatase is a druggable target that cooperates with lipid kinases in inducing MPD.
Subject(s)
Cell Transformation, Neoplastic/pathology , GRB2 Adaptor Protein/physiology , Mutation/genetics , Myeloproliferative Disorders/etiology , Myeloproliferative Disorders/prevention & control , Protein Tyrosine Phosphatase, Non-Receptor Type 11/physiology , Proto-Oncogene Proteins c-kit/genetics , Animals , Apoptosis , Blotting, Western , Bone Marrow Transplantation , Cell Proliferation , Cell Transformation, Neoplastic/genetics , Class Ia Phosphatidylinositol 3-Kinase/metabolism , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/drug effects , Hematopoietic Stem Cells/metabolism , Humans , Immunoprecipitation , Integrases/metabolism , Mice , Mice, Inbred C3H , Mice, Inbred C57BL , Myeloproliferative Disorders/mortality , Phosphorylation/drug effects , Protein Tyrosine Phosphatase, Non-Receptor Type 11/antagonists & inhibitors , Proto-Oncogene Proteins c-kit/metabolism , Signal Transduction/drug effects , Survival Rate , Tyrosine/metabolismABSTRACT
Although SHIP is a well-established suppressor of IgE plus Ag-induced degranulation and cytokine production in bone marrow-derived mast cells (BMMCs), little is known about its role in connective tissue (CTMCs) or mucosal (MMCs) mast cells. In this study, we compared SHIP's role in the development as well as the IgE plus Ag and TLR-induced activation of CTMCs, MMCs, and BMMCs and found that SHIP delays the maturation of all three mast cell subsets and, surprisingly, that it is a positive regulator of IgE-induced BMMC survival. We also found that SHIP represses IgE plus Ag-induced degranulation of all three mast cell subsets and that TLR agonists do not trigger their degranulation, whether SHIP is present or not, nor do they enhance IgE plus Ag-induced degranulation. In terms of cytokine production, we found that in MMCs and BMMCs, which are poor producers of TLR-induced cytokines, SHIP is a potent negative regulator of IgE plus Ag-induced IL-6 and TNF-α production. Surprisingly, however, in splenic or peritoneal derived CTMCs, which are poor producers of IgE plus Ag-induced cytokines, SHIP is a potent positive regulator of TLR-induced cytokine production. Lastly, cell signaling and cytokine production studies with and without LY294002, wortmannin, and PI3Kα inhibitor-2, as well as with PI3K p85α(-/-) BMMCs and CTMCs, are consistent with SHIP positively regulating TLR-induced cytokine production via an adaptor-mediated pathway while negatively regulating IgE plus Ag-induced cytokine production by repressing the PI3K pathway.
Subject(s)
Immunoglobulin E/immunology , Mast Cells/immunology , Mucous Membrane/immunology , Phosphoric Monoester Hydrolases/immunology , Animals , Antigens/immunology , Cell Degranulation/immunology , Cell Differentiation , Cell Lineage/immunology , Cell Survival/genetics , Cell Survival/immunology , Gene Expression Regulation , Inositol Polyphosphate 5-Phosphatases , Interleukin-6/biosynthesis , Interleukin-6/immunology , Mast Cells/cytology , Mice , Mice, Inbred C57BL , Mice, Knockout , Mucous Membrane/cytology , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol 3-Kinases/immunology , Phosphoinositide-3 Kinase Inhibitors , Phosphoric Monoester Hydrolases/antagonists & inhibitors , Phosphoric Monoester Hydrolases/genetics , Signal Transduction/immunology , Toll-Like Receptors/genetics , Toll-Like Receptors/immunology , Tumor Necrosis Factor-alpha/biosynthesis , Tumor Necrosis Factor-alpha/immunologyABSTRACT
The activated B cell (ABC) subset of diffuse large B-cell lymphoma (DLBCL) is characterized by chronic B-cell receptor signaling and associated with poor outcomes when treated with standard therapy. In ABC-DLBCL, MALT1 is a core enzyme that is constitutively activated by stimulation of the B-cell receptor or gain-of-function mutations in upstream components of the signaling pathway, making it an attractive therapeutic target. We discovered a novel small-molecule inhibitor, ABBV-MALT1, that potently shuts down B-cell signaling selectively in ABC-DLBCL preclinical models leading to potent cell growth and xenograft inhibition. We also identified a rational combination partner for ABBV-MALT1 in the BCL2 inhibitor, venetoclax, which when combined significantly synergizes to elicit deep and durable responses in preclinical models. This work highlights the potential of ABBV-MALT1 monotherapy and combination with venetoclax as effective treatment options for patients with ABC-DLBCL.
Subject(s)
Drug Synergism , Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 Protein , Proto-Oncogene Proteins c-bcl-2 , Xenograft Model Antitumor Assays , Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 Protein/antagonists & inhibitors , Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 Protein/metabolism , Humans , Animals , Mice , Proto-Oncogene Proteins c-bcl-2/antagonists & inhibitors , Proto-Oncogene Proteins c-bcl-2/metabolism , Proto-Oncogene Proteins c-bcl-2/genetics , Cell Line, Tumor , Sulfonamides/pharmacology , Sulfonamides/therapeutic use , Cell Proliferation/drug effects , Lymphoma, Large B-Cell, Diffuse/drug therapy , Lymphoma, Large B-Cell, Diffuse/genetics , Lymphoma, Large B-Cell, Diffuse/pathology , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Bridged Bicyclo Compounds, Heterocyclic/therapeutic use , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Disease Models, AnimalABSTRACT
Mast cell maturation is poorly understood. We show that enhanced PI3K activation results in accelerated maturation of mast cells by inducing the expression of microphthalmia transcription factor (Mitf). Conversely, loss of PI3K activation reduces the maturation of mast cells by inhibiting the activation of AKT, leading to reduced Mitf but enhanced Gata-2 expression and accumulation of Gr1(+)Mac1(+) myeloid cells as opposed to mast cells. Consistently, overexpression of Mitf accelerates the maturation of mast cells, whereas Gata-2 overexpression mimics the loss of the PI3K phenotype. Expressing the full-length or the src homology 3- or BCR homology domain-deleted or shorter splice variant of the p85α regulatory subunit of PI3K or activated AKT or Mitf in p85α-deficient cells restores the maturation but not growth. Although deficiency of both SHIP and p85α rescues the maturation of SHIP(-/-) and p85α(-/-) mast cells and expression of Mitf; in vivo, mast cells are rescued in some, but not all tissues, due in part to defective KIT signaling, which is dependent on an intact src homology 3 and BCR homology domain of p85α. Thus, p85α-induced maturation, and growth and survival signals, in mast cells can be uncoupled.
Subject(s)
Cell Differentiation/genetics , Mast Cells/physiology , Microphthalmia-Associated Transcription Factor/physiology , Phosphatidylinositol 3-Kinases/physiology , Animals , Bone Marrow Cells/metabolism , Bone Marrow Cells/physiology , Cell Differentiation/physiology , Cell Proliferation , Cell Survival/genetics , Cells, Cultured , Mast Cells/metabolism , Mice , Mice, Inbred C57BL , Mice, Transgenic , Microphthalmia-Associated Transcription Factor/genetics , Microphthalmia-Associated Transcription Factor/metabolism , Models, Biological , Phosphatidylinositol 3-Kinases/metabolism , Signal Transduction/genetics , Signal Transduction/physiology , TransfectionABSTRACT
We evaluated immune cell subsets in patients with chronic lymphocytic leukemia (CLL) who received first-line therapy with 3 cycles of ibrutinib then 13 cycles of ibrutinib plus venetoclax in the minimal residual disease (MRD) cohort of the CAPTIVATE study (NCT02910583). Patients with Confirmed undetectable MRD (uMRD) were randomly assigned to placebo or ibrutinib groups; patients without Confirmed uMRD were randomly assigned to ibrutinib or ibrutinib plus venetoclax groups. We compared immune cell subsets in samples collected at 7 time points with age-matched healthy donors. CLL cells decreased within 3 cycles after venetoclax initiation; from cycle 16 onward, levels were similar to healthy donor levels (HDL; ≤0.8 cells per µL) in patients with Confirmed uMRD and slightly above HDL in patients without Confirmed uMRD. By 4 months after cycle 16, normal B cells had recovered to HDL in patients randomly assigned to placebo. Regardless of randomized treatment, abnormal counts of T cells, classical monocytes, and conventional dendritic cells recovered to HDL within 6 months (median change from baseline -49%, +101%, and +91%, respectively); plasmacytoid dendritic cells recovered by cycle 20 (+598%). Infections generally decreased over time regardless of randomized treatment and were numerically lowest in patients randomly assigned to placebo within 12 months after cycle 16. Sustained elimination of CLL cells and recovery of normal B cells were confirmed in samples from patients treated with fixed-duration ibrutinib plus venetoclax in the GLOW study (NCT03462719). These results demonstrate promising evidence of restoration of normal blood immune composition with ibrutinib plus venetoclax.
Subject(s)
Immune Reconstitution , Leukemia, Lymphocytic, Chronic, B-Cell , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Piperidines/therapeutic useABSTRACT
Heterozygous mutations in FLT3ITD, TET2, and DNMT3A are associated with hematologic malignancies in humans. In patients, cooccurrence of mutations in FLT3ITD combined with TET2 (TF) or FLT3ITD combined with DNMT3A (DF) are frequent. However, in some rare complex acute myeloid leukemia (AML), all 3 mutations cooccur - i.e., FLT3ITD, TET2, and DNMT3A (TFD). Whether the presence of these mutations in combination result in quantitative or qualitative differences in disease manifestation has not been investigated. We generated mice expressing heterozygous Flt3ITD and concomitant for either heterozygous loss of Tet2 (TF) or Dnmt3a (DF) or both (TFD). TF and DF mice did not induce disease early on, in spite of similar changes in gene expression; during the same time frame, an aggressive form of transplantable leukemia was observed in TFD mice, which was mostly associated with quantitative but not qualitative differences in gene expression relative to TF or DF mice. The gene expression signature of TFD mice showed remarkable similarity to the human TFD gene signature at the single-cell RNA level. Importantly, TFD-driven AML responded to a combination of drugs that target Flt3ITD, inflammation, and methylation in a mouse model, as well as in a PDX model of AML bearing 3 mutations.
Subject(s)
DNA Methyltransferase 3A , DNA-Binding Proteins , Dioxygenases , Leukemia, Myeloid, Acute , fms-Like Tyrosine Kinase 3 , Animals , DNA Methyltransferase 3A/genetics , DNA-Binding Proteins/genetics , Dioxygenases/genetics , Humans , Leukemia, Myeloid, Acute/genetics , Mice , Proto-Oncogene Proteins/genetics , fms-Like Tyrosine Kinase 3/geneticsABSTRACT
Studies of patients with acute myeloid leukemia (AML) have led to the identification of mutations that affect different cellular pathways. Some of these have been classified as preleukemic, and a stepwise evolution program whereby cells acquire additional mutations has been proposed in the development of AML. How the timing of acquisition of these mutations and their impact on transformation and the bone marrow (BM) microenvironment occurs has only recently begun to be investigated. We show that constitutive and early loss of the epigenetic regulator, TET2, when combined with constitutive activation of FLT3, results in transformation of chronic myelomonocytic leukemia-like or myeloproliferative neoplasm-like phenotype to AML, which is more pronounced in double-mutant mice relative to mice carrying mutations in single genes. Furthermore, we show that in preleukemic and leukemic mice there are alterations in the BM niche and secreted cytokines, which creates a permissive environment for the growth of mutation-bearing cells relative to normal cells.
Subject(s)
Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/pathology , Mutation/genetics , Animals , Bone Marrow Cells/metabolism , Cell Differentiation , Cell Proliferation , Cytokines/metabolism , DNA-Binding Proteins/metabolism , Dioxygenases , Heterozygote , Homozygote , Humans , Inflammation Mediators/metabolism , Mice, Inbred C57BL , Neoplastic Stem Cells/metabolism , Neoplastic Stem Cells/pathology , Proto-Oncogene Proteins/metabolism , Severity of Illness Index , Tumor Microenvironment , fms-Like Tyrosine Kinase 3/metabolismABSTRACT
Inflammation is a risk factor for cancer development. Individuals with preleukemic TET2 mutations manifest clonal hematopoiesis and are at a higher risk of developing leukemia. How inflammatory signals influence the survival of preleukemic hematopoietic stem and progenitor cells (HSPCs) is unclear. We show a rapid increase in the frequency and absolute number of Tet2-KO mature myeloid cells and HSPCs in response to inflammatory stress, which results in enhanced production of inflammatory cytokines, including interleukin-6 (IL-6), and resistance to apoptosis. IL-6 induces hyperactivation of the Shp2-Stat3 signaling axis, resulting in increased expression of a novel anti-apoptotic long non-coding RNA (lncRNAs), Morrbid, in Tet2-KO myeloid cells and HSPCs. Expression of activated Shp2 in HSPCs phenocopies Tet2 loss with regard to hyperactivation of Stat3 and Morrbid. In vivo, pharmacologic inhibition of Shp2 or Stat3 or genetic loss of Morrbid in Tet2 mutant mice rescues inflammatory-stress-induced abnormalities in HSPCs and mature myeloid cells, including clonal hematopoiesis.
Subject(s)
Benzoquinones/pharmacology , DNA-Binding Proteins/antagonists & inhibitors , Hematopoietic Stem Cells/drug effects , Inflammation/drug therapy , Myeloid Cells/drug effects , Piperidines/pharmacology , Propionates/pharmacology , Proto-Oncogene Proteins/antagonists & inhibitors , Pyrimidines/pharmacology , Animals , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Dioxygenases , Female , Hematopoiesis/drug effects , Hematopoietic Stem Cells/metabolism , Inflammation/genetics , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mutation , Myeloid Cells/metabolism , Oxidative Stress/drug effects , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , RNA, Long Noncoding/antagonists & inhibitors , RNA, Long Noncoding/genetics , Signal Transduction/drug effects , Signal Transduction/geneticsABSTRACT
Mutations in KIT and TET2 are associated with myeloid malignancies. We show that loss of TET2-induced PI3K activation and -increased proliferation is rescued by targeting the p110α/δ subunits of PI3K. RNA-Seq revealed a hyperactive c-Myc signature in Tet2-/- cells, which is normalized by inhibiting PI3K signaling. Loss of TET2 impairs the maturation of myeloid lineage-derived mast cells by dysregulating the expression of Mitf and Cebpa, which is restored by low-dose ascorbic acid and 5-azacytidine. Utilizing a mouse model in which the loss of TET2 precedes the expression of oncogenic Kit, similar to the human disease, results in the development of a non-mast cell lineage neoplasm (AHNMD), which is responsive to PI3K inhibition. Thus, therapeutic approaches involving hypomethylating agents, ascorbic acid, and isoform-specific PI3K inhibitors are likely to be useful for treating patients with TET2 and KIT mutations.
Subject(s)
Cell Transformation, Neoplastic/genetics , DNA-Binding Proteins/metabolism , Epigenesis, Genetic , Gene Expression Regulation, Neoplastic/genetics , Mast Cells/pathology , Mastocytosis/genetics , Proto-Oncogene Proteins c-kit/metabolism , Proto-Oncogene Proteins/metabolism , Animals , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Cell Differentiation/drug effects , Cell Differentiation/genetics , Cell Proliferation/drug effects , Cell Proliferation/genetics , Cell Transformation, Neoplastic/drug effects , Cell Transformation, Neoplastic/pathology , DNA Methylation/drug effects , DNA-Binding Proteins/genetics , Dioxygenases , Disease Models, Animal , Gain of Function Mutation , Gene Expression Profiling , Gene Expression Regulation, Neoplastic/drug effects , Gene Knock-In Techniques , Humans , Mastocytosis/drug therapy , Mastocytosis/pathology , Mice , Mice, Knockout , Myeloid Cells/drug effects , Myeloid Cells/physiology , Phosphatidylinositol 3-Kinases/metabolism , Phosphoinositide-3 Kinase Inhibitors , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins c-kit/geneticsABSTRACT
Understanding mast cell development is essential due to their critical role in regulating immunity and autoimmune diseases. Here, we show how Rho kinases (ROCK) regulate mast cell development and can function as therapeutic targets for treating allergic diseases. Rock1 deficiency results in delayed maturation of bone marrow derived mast cells (BMMCs) in response to IL-3 stimulation and reduced growth in response to stem cell factor (SCF) stimulation. Further, integrin-mediated adhesion and migration, and IgE-mediated degranulation are all impaired in Rock1-deficient BMMCs. To understand the mechanism behind altered mast cell development in Rock1-/- BMMCs, we analyzed the activation of ROCK and its downstream targets including LIM kinase (LIMK). We observed reduced activation of ROCK, LIMK, AKT and ERK1/2 in Rock1-deficient BMMCs in response to SCF stimulation. Further, loss of either Limk1 or Limk2 also demonstrated altered BMMC maturation and growth; combined deletion of both Limk1 and Limk2 resulted in further reduction in BMMC maturation and growth. In passive cutaneous anaphylaxis model, deficiency of Rock1 or treatment with ROCK inhibitor Fasudil protected mice against IgE-mediated challenge. Our results identify ROCK/LIMK pathway as a novel therapeutic target for treating allergic diseases involving mast cells.
Subject(s)
Cell Differentiation/immunology , Lim Kinases/metabolism , Mast Cells/cytology , Mast Cells/metabolism , rho-Associated Kinases/metabolism , Actins/metabolism , Animals , Cell Adhesion/immunology , Cell Degranulation/immunology , Cell Movement/immunology , Hypersensitivity/immunology , Mice , Mice, KnockoutABSTRACT
Hyperactivation of the mTOR pathway impairs hematopoietic stem cell (HSC) functions and promotes leukemogenesis. mTORC1 and mTORC2 differentially control normal and leukemic stem cell functions. mTORC1 regulates p70 ribosomal protein S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E-binding (eIF4E-binding) protein 1 (4E-BP1), and mTORC2 modulates AKT activation. Given the extensive crosstalk that occurs between mTORC1 and mTORC2 signaling pathways, we assessed the role of the mTORC1 substrate S6K1 in the regulation of both normal HSC functions and in leukemogenesis driven by the mixed lineage leukemia (MLL) fusion oncogene MLL-AF9. We demonstrated that S6K1 deficiency impairs self-renewal of murine HSCs by reducing p21 expression. Loss of S6K1 also improved survival in mice transplanted with MLL-AF9-positive leukemic stem cells by modulating AKT and 4E-BP1 phosphorylation. Taken together, these results suggest that S6K1 acts through multiple targets of the mTOR pathway to promote self-renewal and leukemia progression. Given the recent interest in S6K1 as a potential therapeutic target in cancer, our results further support targeting this molecule as a potential strategy for treatment of myeloid malignancies.
Subject(s)
Carrier Proteins/metabolism , Hematopoietic Stem Cells/cytology , Leukemia/blood , Multiprotein Complexes/metabolism , Phosphoproteins/metabolism , Ribosomal Protein S6 Kinases, 90-kDa/metabolism , TOR Serine-Threonine Kinases/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Animals , Cell Cycle Proteins , Disease Progression , Eukaryotic Initiation Factors , Humans , Leukemia/metabolism , Mechanistic Target of Rapamycin Complex 1 , Mechanistic Target of Rapamycin Complex 2 , Mice , Mice, Inbred C57BL , Phosphorylation , Proto-Oncogene Proteins c-akt/metabolism , Ribosomal Protein S6 Kinases, 70-kDa/metabolism , Signal Transduction , Stem Cells/metabolismABSTRACT
Oncogenic mutations of FLT3 and KIT receptors are associated with poor survival in patients with acute myeloid leukemia (AML) and myeloproliferative neoplasms (MPNs), and currently available drugs are largely ineffective. Although Stat5 has been implicated in regulating several myeloid and lymphoid malignancies, how precisely Stat5 regulates leukemogenesis, including its nuclear translocation to induce gene transcription, is poorly understood. In leukemic cells, we show constitutive activation of focal adhesion kinase (FAK) whose inhibition represses leukemogenesis. Downstream of FAK, activation of Rac1 is regulated by RacGEF Tiam1, whose inhibition prolongs the survival of leukemic mice. Inhibition of the Rac1 effector PAK1 prolongs the survival of leukemic mice in part by inhibiting the nuclear translocation of Stat5. These results reveal a leukemic pathway involving FAK/Tiam1/Rac1/PAK1 and demonstrate an essential role for these signaling molecules in regulating the nuclear translocation of Stat5 in leukemogenesis.
Subject(s)
Carcinogenesis/metabolism , Focal Adhesion Protein-Tyrosine Kinases/metabolism , Leukemia, Myeloid, Acute/metabolism , Proto-Oncogene Proteins c-kit/metabolism , STAT5 Transcription Factor/metabolism , fms-Like Tyrosine Kinase 3/metabolism , p21-Activated Kinases/metabolism , Animals , Benzothiazoles/pharmacology , Carcinogenesis/drug effects , Carcinogenesis/pathology , Cell Nucleus/drug effects , Cell Nucleus/metabolism , Cell Proliferation/drug effects , Focal Adhesion Protein-Tyrosine Kinases/antagonists & inhibitors , Guanine Nucleotide Exchange Factors/metabolism , Humans , Leukemia, Myeloid, Acute/pathology , Mastocytosis, Systemic/pathology , Mice, Inbred C57BL , Mutant Proteins/metabolism , Mutation/genetics , Phenylurea Compounds/pharmacology , Phosphorylation/drug effects , Protein Kinase Inhibitors/pharmacology , Protein Transport/drug effects , Signal Transduction/drug effects , Survival Analysis , T-Lymphoma Invasion and Metastasis-inducing Protein 1 , p21-Activated Kinases/antagonists & inhibitors , rac1 GTP-Binding Protein/metabolismABSTRACT
An acquired somatic mutation at codon 816 in the KIT receptor tyrosine kinase is associated with poor prognosis in patients with systemic mastocytosis and acute myeloid leukemia (AML). Treatment of leukemic cells bearing this mutation with an allosteric inhibitor of p21-activated kinase (Pak) or its genetic inactivation results in growth repression due to enhanced apoptosis. Inhibition of the upstream effector Rac abrogates the oncogene-induced growth and activity of Pak. Although both Rac1 and Rac2 are constitutively activated via the guanine nucleotide exchange factor (GEF) Vav1, loss of Rac1 or Rac2 alone moderately corrected the growth of KIT-bearing leukemic cells, whereas the combined loss resulted in 75% growth repression. In vivo, the inhibition of Vav or Rac or Pak delayed the onset of myeloproliferative neoplasms (MPNs) and corrected the associated pathology in mice. To assess the role of Rac GEFs in oncogene-induced transformation, we used an inhibitor of Rac, EHop-016, which specifically targets Vav1 and found that EHop-016 was a potent inhibitor of human and murine leukemic cell growth. These studies identify Pak and Rac GTPases, including Vav1, as potential therapeutic targets in MPN and AML involving an oncogenic form of KIT.
Subject(s)
Aminoquinolines/pharmacology , Antineoplastic Agents/pharmacology , Carbazoles/pharmacology , Leukemia, Myeloid, Acute/drug therapy , Proto-Oncogene Proteins c-kit/genetics , Pyrimidines/pharmacology , p21-Activated Kinases/physiology , rac GTP-Binding Proteins/physiology , Animals , Carcinogenesis/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival , Enzyme Activation , Humans , Mastocytosis/drug therapy , Mice , Mice, Inbred C3H , Mice, Inbred C57BL , Mutation, Missense , Proto-Oncogene Proteins c-kit/metabolism , Proto-Oncogene Proteins c-vav/metabolism , Xenograft Model Antitumor Assays , p21-Activated Kinases/antagonists & inhibitors , rac GTP-Binding Proteins/antagonists & inhibitorsABSTRACT
If ROCKs are going to be targeted as anti-leukemic agents either as a monotherapy or in combination with existing drugs, a better characterization of the relative contribution of ROCK1 and ROCK2 in disease progression is essential.
Subject(s)
Leukemia/drug therapy , Leukemia/enzymology , Myeloproliferative Disorders/drug therapy , Myeloproliferative Disorders/enzymology , rho-Associated Kinases/metabolism , Humans , Isoenzymes , Leukemia/genetics , Molecular Targeted Therapy/methods , Myeloproliferative Disorders/genetics , rho-Associated Kinases/antagonists & inhibitors , rho-Associated Kinases/geneticsABSTRACT
Gain-of-function mutations in KIT receptor in humans are associated with gastrointestinal stromal tumors, systemic mastocytosis and acute myelogenous leukemia. The intracellular signals that contribute to oncogenic KIT-induced myeloproliferative disease (MPD) are poorly understood. Here, we show that oncogenic KITD814V-induced MPD occurs in the absence of ligand stimulation. The intracellular tyrosine residues are important for KITD814V-induced MPD, albeit to varying degrees. Among the seven intracellular tyrosines examined, tyrosine 719 alone has a unique role in regulating KITD814V-induced proliferation and survival in vitro, and MPD in vivo. Importantly, the extent to which AKT, extracellular signal-regulated kinase and Stat5 signaling pathways are activated via the seven intracellular tyrosines in KITD814V impacts the latency of MPD and severity of the disease. Our results identify critical signaling molecules involved in regulating KITD814V-induced MPD, which might be useful for developing novel therapeutic targets for hematologic malignancies involving this mutation.
Subject(s)
Mutation/genetics , Myeloproliferative Disorders/metabolism , Myeloproliferative Disorders/pathology , Proto-Oncogene Proteins c-kit/genetics , Proto-Oncogene Proteins c-kit/metabolism , Signal Transduction , Tyrosine/metabolism , Animals , Blotting, Western , Bone Marrow Transplantation , Cell Proliferation , Cell Transformation, Neoplastic/genetics , Extracellular Signal-Regulated MAP Kinases/metabolism , Mice , Mice, Inbred C57BL , Myeloproliferative Disorders/genetics , Phosphatidylinositol 3-Kinases/metabolism , Phosphorylation , Proto-Oncogene Proteins c-akt/metabolism , STAT5 Transcription Factor/metabolism , TOR Serine-Threonine Kinases/metabolism , Tumor Cells, CulturedABSTRACT
Stem cell factor (SCF) mediated KIT receptor activation plays a pivotal role in mast cell growth, maturation and survival. However, the signaling events downstream from KIT are poorly understood. Mast cells express multiple regulatory subunits of class 1(A) PI3Kinase (PI3K) including p85α, p85ß, p50α, and p55α. While it is known that PI3K plays an essential role in mast cells; the precise mechanism by which these regulatory subunits impact specific mast cell functions including growth, survival and cycling are not known. We show that loss of p85α impairs the growth, survival and cycling of mast cell progenitors (MCp). To delineate the molecular mechanism (s) by which p85α regulates mast cell growth, survival and cycling, we performed microarray analyses to compare the gene expression profile of MCps derived from WT and p85α-deficient mice in response to SCF stimulation. We identified 151 unique genes exhibiting altered expression in p85α-deficient cells in response to SCF stimulation compared to WT cells. Functional categorization based on DAVID bioinformatics tool and Ingenuity Pathway Analysis (IPA) software relates the altered genes due to lack of p85α to transcription, cell cycle, cell survival, cell adhesion, cell differentiation, and signal transduction. Our results suggest that p85α is involved in mast cell development through regulation of expression of growth, survival and cell cycle related genes.