Screening of Biomarkers Associated with Osteoarthritis Aging Genes and Immune Correlation Studies.

Purpose: Osteoarthritis (OA) is a joint disease with a long and slow course, which is one of the major causes of disability in middle and old-aged people. This study was dedicated to excavating the cellular senescence-associated biomarkers of OA. Methods: The Gene Expression Omnibus (GEO) database was searched and five datasets pertaining to OA were obtained. After removing the batch effect, the GSE55235, GSE55457, GSE82107, and GSE12021 datasets were integrated together for screening of the candidate genes by differential analysis and weighted gene co-expression network analysis (WGCNA). Next, those genes were further filtered by machine learning algorithms to obtain cellular senescence-associated biomarkers of OA. Subsequently, enrichment analyses based on those biomarkers were conducted, and we profiled the infiltration levels of 22 types immune cells with the ERSORT algorithm. A lncRNA-miRNA-mRNA regulatory and drug-gene network were constructed. Finally, we validated the senescence-associated biomarkers at both in vivo and in vitro levels. Results: Five genes (BCL6, MCL1, SLC16A7, PIM1, and EPHA3) were authenticated as cellular senescence-associated biomarkers in OA. ROC curves demonstrated the reliable capacity of the five genes as a whole to discriminate OA samples from normal samples. The nomogram diagnostic model based on 5 genes proved to be a reliable predictor of OA. Single-gene GSEA results pointed to the involvement of the five biomarkers in immune-related pathways and oxidative phosphorylation in the development of OA. Immune infiltration analysis manifested that the five genes were significantly correlated with differential immune cells. Subsequently, a lncRNA-miRNA-mRNA network and gene-drug network containing were generated based on five cellular senescence-associated biomarkers in OA. Conclusion: A foundation for understanding the pathophysiology of OA and new insights into OA diagnosis and treatment were provided by the identification of five genes, namely BCL6, MCL1, SLC16A7, PIM1, and EPHA3, as biomarkers associated with cellular senescence in OA.

The pseudokinase Trib1 regulates the transition of exhausted T cells to a KLR+ CD8+ effector state, and its deletion improves checkpoint blockade.

CD8+ T cell exhaustion (TEX) impairs the ability of T cells to clear chronic infection or cancer. While TEX are hypofunctional, some TEX retain effector gene signatures, a feature associated with killer lectin-like receptor (KLR) expression. Although KLR+ TEX (TKLR) may improve control of chronic antigen, the signaling molecules regulating this population are poorly understood. Using single-cell RNA sequencing (scRNA-seq), flow cytometry, RNA velocity, and single-cell T cell receptor sequencing (scTCR-seq), we demonstrate that deleting the pseudokinase Trib1 shifts TEX toward CX3CR1+ intermediates with robust enrichment of TKLR via clonal T cell expansion. Adoptive transfer studies demonstrate this shift toward CD8+ TKLR in Trib1-deficient cells is CD8 intrinsic, while CD4-depletion studies demonstrate CD4+ T cells are required for improved viral control in Trib1 conditional knockout mice. Further, Trib1 loss augments anti-programmed death-ligand 1 (PD-L1) blockade to improve viral clearance. These data identify Trib1 as an important regulator of CD8+ TEX whose targeting enhances the TKLR effector state and improves checkpoint inhibitor therapy.

The pseudokinase Trib1 regulates the transition of exhausted T cells to a KLR + CD8 + effector state and its deletion improves checkpoint blockade.

T cell exhaustion (T EX ) impairs the ability of T cells to clear chronic infection or cancer. While exhausted T cells are hypofunctional, some exhausted T cells retain effector gene signatures, a feature that is associated with expression of KLRs (killer lectin-like receptors). Although KLR + T cells may improve control of chronic antigen, the signaling molecules regulating this population are poorly understood. Using scRNA-seq, flow cytometry, RNA velocity, and scTCR-seq, we demonstrate that deleting the pseudokinase Trib1 shifts T EX towards CX3CR1 + intermediates (T INT ) with robust enrichment of KLR + CD8 + T cells (T KLR ) via clonal T cell expansion. These changes are associated with globally increased KLR gene expression throughout the exhaustion program. Further, Trib1 loss augments anti-PD-L1 blockade to improve viral clearance by expanding the T KLR population. Together, these data identify Trib1 as an important regulator of T cell exhaustion whose targeting enhances the KLR + effector state and improves the response to checkpoint inhibitor therapy.

Does post-operative radiotherapy improve the treatment outcomes of intracranial hemangiopericytoma? A retrospective study.

BACKGROUND: Intracranial hemangiopericytoma is a rare disease and surgery is the mainstay treatment. Although postoperative adjuvant radiotherapy is often used, there are no reports comparing different radiotherapy techniques. The purpose of this study is to analyze the impact of post-operative radiotherapy and different radiotherapy technique on the results in patients with intracranial hemangiopericytoma (HPC). METHODS: We retrospectively reviewed 66 intracranial HPC patients treated between 1999 and 2019 including 29 with surgery followed by radiotherapy (11 with intensity-modulated radiotherapy (IMRT) and 18 with stereotactic radiosurgery (SRS)) and 37 with surgery alone. Chi-square test was used to compare the clinical characteristic between the groups. The Kaplan-Meier method was used to analyze overall survival (OS) and recurrence-free survival (RFS). Multivariate Cox proportional hazards models were used to examine prognostic factors of survival. We also underwent a matched-pair analysis by using the propensity score method. RESULTS: The crude local control rates were 58.6% in the surgery plus post-operative radiotherapy group (PORT) and 67.6% in the surgery alone group (p = 0.453). In the subgroup analysis of the PORT patients, local controls were 72.7% in the IMRT group and 50% in the SRS group (p = 0.228). The median OS in the PORT and surgery groups were 122 months and 98 months, respectively (p = 0.169). The median RFS was 96 months in the PORT group and 72 months in the surgery alone group (p = 0.714). Regarding radiotherapy technique, the median OS and RFS of the SRS group were not significantly different from those in the IMRT group (p = 0.256, 0.960). The median RFS were 112 and 72 months for pathology grade II and III patients, respectively (p = 0.001). Propensity score matching did not change the observed results. CONCLUSION: In this retrospective analysis, PORT did not improve the local control rates nor the survivals. The local control rates after IMRT and SRS were similar even though the IMRT technique had a much higher biological dose compared with the SRS technique.

Novel frameshift mutations of ANKUB1, GLI3, and TAS2R3 associated with polysyndactyly in a Chinese family.

BACKGROUND: Polysyndactyly (PSD) is an autosomal dominant genetic limb malformation caused by mutations. METHODS: Whole exome sequencing and Sanger sequencing were used to determine the mutations in PSD patients. Luciferase reporter assay was performed to determine the effect of GLI3 mutation on its transcriptional activity. RESULTS: In this study, we investigated the gene mutations of three affected individuals across three generations. The frameshift mutations of GLI3 (NM_000168:c.4659del, NP_000159.3: p.Ser1553del), ANKUB1 (NM_001144960:c.1385del, NP_001138432.1: p.Pro462del), and TAS2R3 (NM_016943:c.128_131del, NP_058639.1: p.Leu43del) were identified in the three affected individuals, but not in three unaffected members by whole exome sequencing and sanger sequencing. Luciferase reporter assay demonstrated that GLI3 mutation reduced the transcriptional activity of GLI3. The results from SMART analysis showed that the frameshift mutation of TAS2R3 altered most protein sequence, which probably destroyed protein function. Although the frameshift mutation of ANKUB1 did not locate in ankyrin repeat domain and ubiquitin domain, it might influence the interaction between ANKUB1 and other proteins, and further affected the ubiquitinylation. CONCLUSION: These results indicated that the frameshift mutations of GLI3, ANKUB1, and TAS2R3 might alter the functions of these proteins, and accelerated PSD progression.

TooManyCells identifies and visualizes relationships of single-cell clades.

Identifying and visualizing transcriptionally similar cells is instrumental for accurate exploration of the cellular diversity revealed by single-cell transcriptomics. However, widely used clustering and visualization algorithms produce a fixed number of cell clusters. A fixed clustering 'resolution' hampers our ability to identify and visualize echelons of cell states. We developed TooManyCells, a suite of graph-based algorithms for efficient and unbiased identification and visualization of cell clades. TooManyCells introduces a visualization model built on a concept intentionally orthogonal to dimensionality-reduction methods. TooManyCells is also equipped with an efficient matrix-free divisive hierarchical spectral clustering different from prevalent single-resolution clustering methods. TooManyCells enables multiresolution and multifaceted exploration of single-cell clades. An advantage of this paradigm is the immediate detection of rare and common populations that outperforms popular clustering and visualization algorithms, as demonstrated using existing single-cell transcriptomic data sets and new data modeling drug-resistance acquisition in leukemic T cells.

Trib1 regulates T cell differentiation during chronic infection by restraining the effector program.

In chronic infections, the immune response fails to control virus, leading to persistent antigen stimulation and the progressive development of T cell exhaustion. T cell effector differentiation is poorly understood in the context of exhaustion, but targeting effector programs may provide new strategies for reinvigorating T cell function. We identified Tribbles pseudokinase 1 (Trib1) as a central regulator of antiviral T cell immunity, where loss of Trib1 led to a sustained enrichment of effector-like KLRG1+ T cells, enhanced function, and improved viral control. Single-cell profiling revealed that Trib1 restrains a population of KLRG1+ effector CD8 T cells that is transcriptionally distinct from exhausted cells. Mechanistically, we identified an interaction between Trib1 and the T cell receptor (TCR) signaling activator, MALT1, which disrupted MALT1 signaling complexes. These data identify Trib1 as a negative regulator of TCR signaling and downstream function, and reveal a link between Trib1 and effector versus exhausted T cell differentiation that can be targeted to improve antiviral immunity.

Trib1 regulates eosinophil lineage commitment and identity by restraining the neutrophil program.

Eosinophils and neutrophils are critical for host defense, yet gaps in understanding how granulocytes differentiate from hematopoietic stem cells (HSCs) into mature effectors remain. The pseudokinase tribbles homolog 1 (Trib1) is an important regulator of granulocytes; knockout mice lack eosinophils and have increased neutrophils. However, how Trib1 regulates cellular identity and function during eosinophilopoiesis is not understood. Trib1 expression markedly increases with eosinophil-lineage commitment in eosinophil progenitors (EoPs), downstream of the granulocyte/macrophage progenitor (GMP). Using hematopoietic- and eosinophil-lineage-specific Trib1 deletion, we found that Trib1 regulates both granulocyte precursor lineage commitment and mature eosinophil identity. Conditional Trib1 deletion in HSCs reduced the size of the EoP pool and increased neutrophils, whereas deletion following eosinophil lineage commitment blunted the decrease in EoPs without increasing neutrophils. In both modes of deletion, Trib1-deficient mice expanded a stable population of Ly6G+ eosinophils with neutrophilic characteristics and functions, and had increased CCAAT/enhancer binding protein α (C/EBPα) p42. Using an ex vivo differentiation assay, we found that interleukin 5 (IL-5) supports the generation of Ly6G+ eosinophils from Trib1-deficient cells, but is not sufficient to restore normal eosinophil differentiation and development. Furthermore, we demonstrated that Trib1 loss blunted eosinophil migration and altered chemokine receptor expression, both in vivo and ex vivo. Finally, we showed that Trib1 controls eosinophil identity by modulating C/EBPα. Together, our findings provide new insights into early events in myelopoiesis, whereby Trib1 functions at 2 distinct stages to guide eosinophil lineage commitment from the GMP and suppress the neutrophil program, promoting eosinophil terminal identity and maintaining lineage fidelity.

MAFB enhances oncogenic Notch signaling in T cell acute lymphoblastic leukemia.

Activating mutations in the gene encoding the cell-cell contact signaling protein Notch1 are common in human T cell acute lymphoblastic leukemias (T-ALLs). However, expressing Notch1 mutant alleles in mice fails to efficiently induce the development of leukemia. We performed a gain-of-function screen to identify proteins that enhanced signaling by leukemia-associated Notch1 mutants. The transcription factors MAFB and ETS2 emerged as candidates that individually enhanced Notch1 signaling, and when coexpressed, they synergistically increased signaling to an extent similar to that induced by core components of the Notch transcriptional complex. In mouse models of T-ALL, MAFB enhanced leukemogenesis by the naturally occurring Notch1 mutants, decreased disease latency, and increased disease penetrance. Decreasing MAFB abundance in mouse and human T-ALL cells reduced the expression of Notch1 target genes, including MYC and HES1, and sustained MAFB knockdown impaired T-ALL growth in a competitive setting. MAFB bound to ETS2 and interacted with the acetyltransferases PCAF and P300, highlighting its importance in recruiting coactivators that enhance Notch1 signaling. Together, these data identify a mechanism for enhancing the oncogenic potential of weak Notch1 mutants in leukemia models, and they reveal the MAFB-ETS2 transcriptional axis as a potential therapeutic target in T-ALL.

High selective pressure for Notch1 mutations that induce Myc in T-cell acute lymphoblastic leukemia.

Activating NOTCH1 mutations are frequent in human T-cell acute lymphoblastic leukemia (T-ALL) and Notch inhibitors (γ-secretase inhibitors [GSIs]) have produced responses in patients with relapsed, refractory disease. However, sustained responses, although reported, are uncommon, suggesting that other pathways can substitute for Notch in T-ALL. To address this possibility, we first generated KrasG12D transgenic mice with T-cell-specific expression of the pan-Notch inhibitor, dominant-negative Mastermind (DNMAML). These mice developed leukemia, but instead of accessing alternative oncogenic pathways, the tumor cells acquired Notch1 mutations and subsequently deleted DNMAML, reinforcing the notion that activated Notch1 is particularly transforming within the context of T-cell progenitors. We next took a candidate approach to identify oncogenic pathways downstream of Notch, focusing on Myc and Akt, which are Notch targets in T-cell progenitors. KrasG12D mice transduced with Myc developed T-ALLs that were GSI-insensitive and lacked Notch1 mutations. In contrast, KrasG12D mice transduced with myristoylated AKT developed GSI-sensitive T-ALLs that acquired Notch1 mutations. Thus, Myc can substitute for Notch1 in leukemogenesis, whereas Akt cannot. These findings in primary tumors extend recent work using human T-ALL cell lines and xenografts and suggest that the Notch/Myc signaling axis is of predominant importance in understanding both the selective pressure for Notch mutations in T-ALL and response and resistance of T-ALL to Notch pathway inhibitors.

Trib2 Suppresses Tumor Initiation in Notch-Driven T-ALL.

Trib2 is highly expressed in human T cell acute lymphoblastic leukemia (T-ALL) and is a direct transcriptional target of the oncogenic drivers Notch and TAL1. In human TAL1-driven T-ALL cell lines, Trib2 is proposed to function as an important survival factor, but there is limited information about the role of Trib2 in primary T-ALL. In this study, we investigated the role of Trib2 in the initiation and maintenance of Notch-dependent T-ALL. Trib2 had no effect on the growth and survival of murine T-ALL cell lines in vitro when expression was blocked by shRNAs. To test the function of Trib2 on leukemogenesis in vivo, we generated Trib2 knockout mice. Mice were born at the expected Mendelian frequencies without gross developmental anomalies. Adult mice did not develop pathology or shortened survival, and hematopoiesis, including T cell development, was unperturbed. Using a retroviral model of Notch-induced T-ALL, deletion of Trib2 unexpectedly decreased the latency and increased the penetrance of T-ALL development in vivo. Immunoblotting of primary murine T-ALL cells showed that the absence of Trib2 increased C/EBPα expression, a known regulator of cell proliferation, and did not alter AKT or ERK phosphorylation. Although Trib2 was suggested to be highly expressed in T-ALL, transcriptomic analysis of two independent T-ALL cohorts showed that low Trib2 expression correlated with the TLX1-expressing cortical mature T-ALL subtype, whereas high Trib2 expression correlated with the LYL1-expressing early immature T-ALL subtype. These data indicate that Trib2 has a complex role in the pathogenesis of Notch-driven T-ALL, which may vary between different T-ALL subtypes.

Comparison of the expression of prognostic biomarkers between primary tumor and axillary lymph node metastases in breast cancer.

The prognosis and prediction of axillary lymph node (ALN) metastases in breast cancer is traditionally based upon the biomarkers status of the primary tumor. Some retrospective studies showed significant discordance in receptor expression between primary and metastatic tumors. We aim to prospectively assess the incidence of discordant biomarkers status in primary tumor and ALN metastases and to evaluate the role of ALN biopsies for the reassessment of receptor status. Tissue arrays were constructed from 54 breast cancer patients with ALN metastases diagnosed. Arrays were immuno-stained to compare protein expression of four biomarkers including estrogen receptor (ER), progesterone receptor (PR), HER2, and Ki67 by immunohistochemistry. The kappa value of consistency in the primary tumor and the metastatic lymph nodes were 0.465 for ER, 0.445 for PR, and 0.706 for HER2. Good consistency was shown for Ki67 expression in primary and metastases regions with T test. No significant difference is existed between primary tumor and ALN metastases. It is concluded that the good consistency is present for ER, PR, HER2 and Ki67 between the primary tumor and the metastatic lymph nodes, suggesting that ER, PR, HER2, or Ki67 status in primary tumors could reflect their status in ALN metastases.

The Notch1 transcriptional activation domain is required for development and reveals a novel role for Notch1 signaling in fetal hematopoietic stem cells.

Notch1 is required to generate the earliest embryonic hematopoietic stem cells (HSCs); however since Notch-deficient embryos die early in gestation, additional functions for Notch in embryonic HSC biology have not been described. We used two complementary genetic models to address this important biological question. Unlike Notch1-deficient mice, mice lacking the conserved Notch1 transcriptional activation domain (TAD) show attenuated Notch1 function in vivo and survive until late gestation, succumbing to multiple cardiac abnormalities. Notch1 TAD-deficient HSCs emerge and successfully migrate to the fetal liver but are decreased in frequency by embryonic day 14.5. In addition, TAD-deficient fetal liver HSCs fail to compete with wild-type HSCs in bone marrow transplant experiments. This phenotype is independently recapitulated by conditional knockout of Rbpj, a core Notch pathway component. In vitro analysis of Notch1 TAD-deficient cells shows that the Notch1 TAD is important to properly assemble the Notch1/Rbpj/Maml trimolecular transcription complex. Together, these studies reveal an essential role for the Notch1 TAD in fetal development and identify important cell-autonomous functions for Notch1 signaling in fetal HSC homeostasis.

Divergent effects of supraphysiologic Notch signals on leukemia stem cells and hematopoietic stem cells.

The leukemia stem cell (LSC) hypothesis proposes that a subset of cells in the bulk leukemia population propagates the leukemia.We tested the LSC hypothesis in a mouse model of Notch-induced T-cell acute lymphoblastic leukemia (T-ALL) in which the tumor cells were largely CD4+ CD8+ T cells. LSC activity was enriched but rare in the CD8+ CD4 HSA(hi) immature single-positive T-cell subset. Although our murine T-ALL model relies on transduction of HSCs, we were unable to isolate Notch-activated HSCs to test for LSC activity. Further analysis showed that Notch activation in HSCs caused an initial expansion of hematopoietic and T-cell progenitors and loss of stem cell quiescence, which was followed by progressive loss of long-term HSCs and T-cell production over several weeks. Similar results were obtained in a conditional transgenic model in which Notch activation is induced in HSCs by Cre recombinase. We conclude that although supraphysiologic Notch signaling in HSCs promotes LSC activity in T-cell progenitors, it extinguishes self-renewal of LT-HSCs. These results provide further evidence for therapeutically targeting T-cell progenitors in T-ALL while also underscoring the need to tightly regulate Notch signaling to expand normal HSC populations for clinical applications.

Notch ankyrin repeat domain variation influences leukemogenesis and Myc transactivation.

BACKGROUND: The functional interchangeability of mammalian Notch receptors (Notch1-4) in normal and pathophysiologic contexts such as cancer is unsettled. We used complementary in vivo, cell-based and structural analyses to compare the abilities of activated Notch1-4 to support T cell development, induce T cell acute lymphoblastic leukemia/lymphoma (T-ALL), and maintain T-ALL cell growth and survival. PRINCIPAL FINDINGS: We find that the activated intracellular domains of Notch1-4 (ICN1-4) all support T cell development in mice and thymic organ culture. However, unlike ICN1-3, ICN4 fails to induce T-cell acute lymphoblastic leukemia/lymphoma (T-ALL) and is unable to rescue the growth of Notch1-dependent T-ALL cell lines. The ICN4 phenotype is mimicked by weak gain-of-function forms of Notch1, suggesting that it stems from a failure to transactivate one or more critical target genes above a necessary threshold. Experiments with chimeric receptors demonstrate that the Notch ankyrin repeat domains differ in their leukemogenic potential, and that this difference correlates with activation of Myc, a direct Notch target that has an important role in Notch-associated T-ALL. CONCLUSIONS/SIGNIFICANCE: We conclude that the leukemogenic potentials of Notch receptors vary, and that this functional difference stems in part from divergence among the highly conserved ankyrin repeats, which influence the transactivation of specific target genes involved in leukemogenesis.

Identification of Flt3⁺CD150⁻ myeloid progenitors in adult mouse bone marrow that harbor T lymphoid developmental potential.

Common myeloid progenitors (CMPs) were first identified as progenitors that were restricted to myeloid and erythroid lineages. However, it was recently demonstrated that expression of both lymphoid- and myeloid-related genes could be detected in myeloid progenitors. Furthermore, these progenitors were able to give rise to T and B lymphocytes, in addition to myeloid cells. Yet, it was not known whether these progenitors were multipotent at the clonogenic level or there existed heterogeneity within these progenitors with different lineage potential. Here we report that previously defined CMPs possess T-lineage potential, and that this is exclusively found in the Flt3(+)CD150(-) subset of CMPs at the clonal level. In contrast, we did not detect B-lineage potential in CMP subsets. Therefore, these Flt3(+)CD150(-) myeloid progenitors were T/myeloid potent. Yet, Flt3(+)CD150(-) myeloid progenitors are not likely to efficiently traffic to the thymus and contribute to thymopoiesis under normal conditions because of the lack of CCR7 and CCR9 expression. Interestingly, both Flt3(+)CD150(-) and Flt3(-)CD150(-) myeloid progenitors are susceptible to Notch1-mediated T-cell acute lymphoblastic leukemia (T-ALL). Hence, gain-of-function Notch1 mutations occurring in developing myeloid progenitors, in addition to known T-lineage progenitors, could lead to T-ALL oncogenesis.

Notch dimerization is required for leukemogenesis and T-cell development.

Notch signaling regulates myriad cellular functions by activating transcription, yet how Notch selectively activates different transcriptional targets is poorly understood. The core Notch transcriptional activation complex can bind DNA as a monomer, but it can also dimerize on DNA-binding sites that are properly oriented and spaced. However, the significance of Notch dimerization is unknown. Here, we show that dimeric Notch transcriptional complexes are required for T-cell maturation and leukemic transformation but are dispensable for T-cell fate specification from a multipotential precursor. The varying requirements for Notch dimerization result from the differential sensitivity of specific Notch target genes. In particular, c-Myc and pre-T-cell antigen receptor α (Ptcra) are dimerization-dependent targets, whereas Hey1 and CD25 are not. These findings identify functionally important differences in the responsiveness among Notch target genes attributable to the formation of higher-order complexes. Consequently, it may be possible to develop a new class of Notch inhibitors that selectively block outcomes that depend on Notch dimerization (e.g., leukemogenesis).

Transformation by Tribbles homolog 2 (Trib2) requires both the Trib2 kinase domain and COP1 binding.

Tribbles homolog 2 (Trib2) is a pseudokinase that induces acute myelogenous leukemia (AML) in mice and is highly expressed in a subset of human AML. Trib2 has 3 distinct regions, a proline-rich N-terminus, a serine/threonine kinase homology domain, and a C-terminal constitutive photomorphogenesis 1 (COP1)-binding domain. We performed a structure-function analysis of Trib2 using in vitro and in vivo assays. The N-terminus was not required for Trib2-induced AML. Deletion or mutation of the COP1-binding site abrogated the ability of Trib2 to degrade CCAAT/enhancer-binding protein-α (C/EBP-α), block granulocytic differentiation, and to induce AML in vivo. Furthermore, COP1 knockdown inhibited the ability of Trib2 to degrade C/EBP-α, showing that it is important for mediating Trib2 activity. We also show that the Trib2 kinase domain is essential for its function. Trib2 contains variant catalytic loop sequences, compared with conventional kinases, that we show are necessary for Trib2 activity. The kinase domain mutants bind, but cannot efficiently degrade, C/EBP-α. Together, our data demonstrate that Trib2 can bind both COP1 and C/EBP-α, leading to degradation of C/EBP-α. Identification of the functional regions of Trib2 that are essential to its oncogenic role provides the basis for developing inhibitors that will block Trib functions in cancer.

Deletion-based mechanisms of Notch1 activation in T-ALL: key roles for RAG recombinase and a conserved internal translational start site in Notch1.

Point mutations that trigger ligand-independent proteolysis of the Notch1 ectodomain occur frequently in human T-cell acute lymphoblastic leukemia (T-ALL) but are rare in murine T-ALL, suggesting that other mechanisms account for Notch1 activation in murine tumors. Here we show that most murine T-ALLs harbor Notch1 deletions that fall into 2 types, both leading to ligand-independent Notch1 activation. Type 1 deletions remove exon 1 and the proximal promoter, appear to be RAG-mediated, and are associated with mRNA transcripts that initiate from 3' regions of Notch1. In line with the RAG dependency of these rearrangements, RAG2 binds to the 5' end of Notch1 in normal thymocytes near the deletion breakpoints. Type 2 deletions remove sequences between exon 1 and exons 26 to 28 of Notch1, appear to be RAG-independent, and are associated with transcripts in which exon 1 is spliced out of frame to 3' Notch1 exons. Translation of both types of transcripts initiates at a conserved methionine residue, M1727, which lies within the Notch1 transmembrane domain. Polypeptides initiating at M1727 insert into membranes and are subject to constitutive cleavage by γ-secretase. Thus, like human T-ALL, murine T-ALL is often associated with acquired mutations that cause ligand-independent Notch1 activation.

Differential ability of Tribbles family members to promote degradation of C/EBPalpha and induce acute myelogenous leukemia.

Trib1, Trib2, and Trib3 are mammalian homologs of Tribbles, an evolutionarily conserved Drosophila protein family that mediates protein degradation. Tribbles proteins function as adapters to recruit E3 ubiquitin ligases and enhance ubiquitylation of the target protein to promote its degradation. Increased Trib1 and Trib2 mRNA expression occurs in human myeloid leukemia and induces acute myeloid leukemia in mice, whereas Trib3 has not been associated with leukemia. Given the high degree of structural conservation among Tribbles family members, we directly compared the 3 mammalian Tribbles in hematopoietic cells by reconstituting mice with hematopoietic stem cells retrovirally expressing these proteins. All mice receiving Trib1 or Trib2 transduced hematopoietic stem cells developed acute myeloid leukemia, whereas Trib3 mice did not. Our previous data indicated that Trib2-mediated degradation of the transcription factor, CCAAT/enhancer-binding protein-alpha (C/EBPalpha), is important for leukemogenesis. Similar to Trib2, Trib1 induced C/EBPalpha degradation and inhibited its function. In contrast, Trib3 failed to inactivate or promote efficient degradation of C/EBPalpha. These data reveal that the 3 Tribbles homologs differ in their ability to promote degradation of C/EBPalpha, which account for their differential ability to induce leukemia.