Rip1 is required for IκB kinase activation in response to tumor necrosis factor α (TNF-α) and has been implicated in the Toll-like receptor 3 (TLR3) response to double-stranded RNA. Cytokine ...production is impaired when rip1–/– cells are treated with TNF-α, poly(I-C), or lipopolysaccharide, implicating Rip1 in the Trif-dependent TLR3 and TLR4 pathways. To examine the role of Rip1 in the Trif-dependent TLR4 pathway, we generated rip1–/– MyD88–/– cells. Lipopolysaccharide failed to stimulate NF-κB activation in rip1–/–MyD88–/– cells, revealing that Rip1 is also required for the Trif-dependent TLR4-induced NF-κB pathway. In addition to activating NF-κB, TLR3/4 pathways also stimulate interferon regulatory factor 3 activation. However, we find that Rip1 expression stimulates NF-κB but not interferon regulatory factor 3 activity. In the TNF-α pathway, Rip1 interacts with the E3 ubiquitin ligase Traf2 and is modified by polyubiquitin chains. Upon TLR3 activation, Rip1 is also modified by polyubiquitin chains and is recruited to TLR3 along with Traf6 and the ubiquitin-activated kinase Tak1. These studies suggest that Rip1 uses a similar, ubiquitin-dependent mechanism to activate IκB kinase-β in response to TNF-α and TLR3 ligands.
CD24 is frequently overexpressed in ovarian cancer and promotes immune evasion by interacting with its receptor Siglec10, present on tumor-associated macrophages, providing a “don’t eat me” signal ...that prevents targeting and phagocytosis by macrophages. Factors promoting CD24 expression could represent novel immunotherapeutic targets for ovarian cancer. Here, using a genome-wide CRISPR knockout screen, we identify GPAA1 (glycosylphosphatidylinositol anchor attachment 1), a factor that catalyzes the attachment of a glycosylphosphatidylinositol (GPI) lipid anchor to substrate proteins, as a positive regulator of CD24 cell surface expression. Genetic ablation of GPAA1 abolishes CD24 cell surface expression, enhances macrophage-mediated phagocytosis, and inhibits ovarian tumor growth in mice. GPAA1 shares structural similarities with aminopeptidases. Consequently, we show that bestatin, a clinically advanced aminopeptidase inhibitor, binds to GPAA1 and blocks GPI attachment, resulting in reduced CD24 cell surface expression, increased macrophage-mediated phagocytosis, and suppressed growth of ovarian tumors. Our study highlights the potential of targeting GPAA1 as an immunotherapeutic approach for CD24+ ovarian cancers.
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•GPAA1 promotes cell surface expression of the anti-phagocytic signal CD24•GPAA1 knockout enhances macrophage-mediated phagocytosis of ovarian cancer cells•GPAA1 knockout suppresses growth of ovarian tumors in mice xenografts•Bestatin, an aminopeptidase inhibitor, inhibits GPAA1 and suppresses tumor growth
Mishra et al. identify the GPI transamidase complex subunit GPAA1 as a factor required for cell surface expression of CD24 and show that genetic or pharmacological inhibition of GPAA1 enhances macrophage-mediated phagocytosis and suppresses ovarian tumor growth in mice. Their results highlight a potential immunotherapeutic approach for CD24+ ovarian cancers.
Relapse in T-cell acute lymphoblastic leukemia (T-ALL) may signify the persistence of leukemia-initiating cells (L-ICs). Ectopic TAL1/LMO expression defines the largest subset of T-ALL, but its role ...in leukemic transformation and its impact on relapse-driving L-ICs remain poorly understood. In TAL1/LMO mouse models, double negative-3 (DN3; CD4
CD8
CD25
CD44
) thymic progenitors harbored L-ICs. However, only a subset of DN3 leukemic cells exhibited L-IC activity, and studies linking L-ICs and chemotolerance are needed. To investigate L-IC heterogeneity, we used mouse models and applied single-cell RNA-sequencing and nucleosome labeling techniques in vivo. We identified a DN3 subpopulation with a cell cycle-restricted profile and heightened TAL1/LMO2 activity, that expressed genes associated with stemness and quiescence. This dormant DN3 subset progressively expanded throughout leukemogenesis, displaying intrinsic chemotolerance and enrichment in genes linked to minimal residual disease. Examination of TAL/LMO patient samples revealed a similar pattern in CD7
CD1a
thymic progenitors, previously recognized for their L-IC activity, demonstrating cell cycle restriction and chemotolerance. Our findings substantiate the emergence of dormant, chemotolerant L-ICs during leukemogenesis, and demonstrate that Tal1 and Lmo2 cooperate to promote DN3 quiescence during the transformation process. This study provides a deeper understanding of TAL1/LMO-induced T-ALL and its clinical implications in therapy failure.
Glucocorticoid (GC) resistance remains a clinical challenge in pediatric acute lymphoblastic leukemia where response to GC is a reliable prognostic indicator. To identify GC resistance pathways, we ...conducted a genome-wide, survival-based, short hairpin RNA screen in murine T-cell acute lymphoblastic leukemia (T-ALL) cells. Genes identified in the screen interfere with cyclic adenosine monophosphate (cAMP) signaling and are underexpressed in GC-resistant or relapsed ALL patients. Silencing of the cAMP-activating Gnas gene interfered with GC-induced gene expression, resulting in dexamethasone resistance in vitro and in vivo. We demonstrate that cAMP signaling synergizes with dexamethasone to enhance cell death in GC-resistant human T-ALL cells. We find the E prostanoid receptor 4 expressed in T-ALL samples and demonstrate that prostaglandin E2 (PGE2) increases intracellular cAMP, potentiates GC-induced gene expression, and sensitizes human T-ALL samples to dexamethasone in vitro and in vivo. These findings identify PGE2 as a target for GC resensitization in relapsed pediatric T-ALL.
Ptpn6 is a cytoplasmic phosphatase that functions to prevent autoimmune and interleukin-1 (IL-1) receptor-dependent, caspase-1-independent inflammatory disease. Conditional deletion of Ptpn6 in ...neutrophils (Ptpn6
) is sufficient to initiate IL-1 receptor-dependent cutaneous inflammatory disease, but the source of IL-1 and the mechanisms behind IL-1 release remain unclear. Here, we investigate the mechanisms controlling IL-1α/β release from neutrophils by inhibiting caspase-8-dependent apoptosis and Ripk1-Ripk3-Mlkl-regulated necroptosis. Loss of Ripk1 accelerated disease onset, whereas combined deletion of caspase-8 and either Ripk3 or Mlkl strongly protected Ptpn6
mice. Ptpn6
neutrophils displayed increased p38 mitogen-activated protein kinase-dependent Ripk1-independent IL-1 and tumor necrosis factor production, and were prone to cell death. Together, these data emphasize dual functions for Ptpn6 in the negative regulation of p38 mitogen-activated protein kinase activation to control tumor necrosis factor and IL-1α/β expression, and in maintaining Ripk1 function to prevent caspase-8- and Ripk3-Mlkl-dependent cell death and concomitant IL-1α/β release.
The death domain kinase Rip1 is recruited to the tumor necrosis factor receptor type 1 and mediates the IκB kinase and p38 MAP kinase pathways. In response to tumor necrosis factor-α (TNF-α), we find ...Rip1 phosphorylated and ubiquitinated, suggesting that Rip1 phosphorylation may stimulate its ubiquitination. To address the contribution of the kinase activity of Rip1 to its ubiquitination and to TNF-α signaling, we introduced wild type Rip1 and a kinase-inactive form of Rip1, Rip1D138N, into rip1-/- murine embryonic fibroblast cells by retroviral infection. TNF-α-induced ubiquitination of Rip1 is observed in Rip1D138N cells, supporting the argument that Rip1 autophosphorylation is not required for Rip1 ubiquitination. TNF-α-induced Ikk and p38 MAP kinase activation is normal, and the Rip1D138N cells are resistant to TNF-α-induced cell death, indicating that the kinase activity of Rip1 is not required to mediate its antiapoptotic functions. In the absence of Traf2, TNF-α-induced ubiquitination of Rip1 is impaired, suggesting that Traf2 may be the E3 ubiquitin ligase responsible for the TNF-α-dependent, ubiquitination of Rip1. Finally, recruitment of the ubiquitinated Tak1 complex is dependent on the presence of Rip1, suggesting that Rip1 ubiquitination rather than its phosphorylation is critical in signaling.
Mutations in NOTCH1 are frequently detected in patients with T-cell acute lymphoblastic leukemia (T-ALL) and in mouse T-ALL models. Treatment of mouse or human T-ALL cell lines in vitro with ...γ-secretase inhibitors (GSIs) results in growth arrest and/or apoptosis. These studies suggest GSIs as potential therapeutic agents in the treatment of T-ALL. To determine whether GSIs have antileukemic activity in vivo, we treated near-end-stage Tal1/Ink4a/Arf+/− leukemic mice with vehicle or with a GSI developed by Merck (MRK-003). We found that GSI treatment significantly extended the survival of leukemic mice compared with vehicle-treated mice. Notch1 target gene expression was repressed and increased numbers of apoptotic cells were observed in the GSI-treated mice, demonstrating that Notch1 inhibition in vivo induces apoptosis. T-ALL cell lines also exhibit PI3K/mTOR pathway activation, indicating that rapamycin may also have therapeutic benefit. When GSIs are administered in combination with rapamycin, mTOR kinase activity is ablated and apoptosis induced. Moreover, GSI and rapamycin treatment inhibits human T-ALL growth and extends survival in a mouse xenograft model. This work supports the idea of targeting NOTCH1 in T-ALL and suggests that inhibition of the mTOR and NOTCH1 pathways may have added efficacy.
Leukemia Propagating Cells Akt Up Gutierrez, Alejandro; Roderick, Justine E.; Kelliher, Michelle A.
Cancer cell,
03/2014, Letnik:
25, Številka:
3
Journal Article
Recenzirano
Odprti dostop
Individual cancer cells can exhibit striking differences in tumorigenic potential following experimental transplantation, but the molecular pathways that regulate this activity remain poorly ...understood. In this issue of Cancer Cell, Blackburn and colleagues report that Akt signaling regulates both leukemia-propagating potential and proliferation rate via distinct pathways in T-ALL.
Targeted molecular therapy has yielded remarkable outcomes in certain cancers, but specific therapeutic targets remain elusive for many others. As a result of two independent RNA interference (RNAi) ...screens, we identified pathway dependence on a member of the Janus-activated kinase (JAK) tyrosine kinase family, TYK2, and its downstream effector STAT1, in T-cell acute lymphoblastic leukemia (T-ALL). Gene knockdown experiments consistently showed TYK2 dependence in both T-ALL primary specimens and cell lines, and a small-molecule inhibitor of JAK activity induced T-ALL cell death. Activation of this TYK2-STAT1 pathway in T-ALL cell lines occurs by gain-of-function TYK2 mutations or activation of interleukin (IL)-10 receptor signaling, and this pathway mediates T-ALL cell survival through upregulation of the antiapoptotic protein BCL2. These findings indicate that in many T-ALL cases, the leukemic cells are dependent upon the TYK2-STAT1-BCL2 pathway for continued survival, supporting the development of molecular therapies targeting TYK2 and other components of this pathway.
In recent years, "pathway dependence" has been revealed in specific types of human cancer, which can be important because they pinpoint proteins that are particularly vulnerable to antitumor-targeted inhibition (so-called Achilles’ heel proteins). Here, we use RNAi technology to identify a novel oncogenic pathway that involves aberrant activation of the TYK2 tyrosine kinase and its downstream substrate, STAT1, which ultimately promotes T-ALL cell survival through the upregulation of BCL2 expression
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