Recently, 5-hydroxymethylcytosine (5-hmC), the 6th base of DNA, was discovered as the product of the hydroxylation of 5-methylcytosine (5-mC) by the ten-eleven translocation (TET) oncogene family ...members. One of them, TET oncogene family member 2 (TET2), is mutated in a variety of myeloid malignancies, including in 15% of myeloproliferative neoplasms (MPNs). Recent studies tried to go further into the biological and epigenetic function of TET2 protein and 5-hmC marks in the pathogenesis of myeloid malignancies. Although its precise function remains partially unknown, TET2 appears to be an important regulator of hematopoietic stem cell biology. In both mouse and human cells, its inactivation leads to a dramatic deregulation of hematopoiesis that ultimately triggers blood malignancies. Understanding this leukemogenic process will provide tools to develop new epigenetic therapies against blood cancers.
Epigenetic modifiers and signaling factors are frequently mutated and often co-occur in various myeloid malignancies. However, precisely how these mutations cooperate to cause myeloid leukemia is not ...fully understood. Here, we show that cells with concurrent Ten-eleven-translocation 2 (Tet2) loss and Nras mutation can cause lethal chronic myelomonocytic leukemia (CMML) like disease in vivo and synergistically activate Ras signaling through epigenetic silencing of Sprouty 2 (Spry2), thereby making cells with both disease alleles dependent on MAPK signaling and highly sensitive to MEK inhibition.
To assess if Tet2 loss and Nras mutation cooperate in myeloid transformation, we crossed Tet2 conditional knockout mice (Mx1-Cre+Tet2f/f) and Nras mutant mice (Mx1-Cre+Nras+/G12D) to generate Mx1-Cre+Tet2f/fNras+/G12D mice (Tet2Δ/ΔNras+/G12D). These mice, compared to single mutant mice with either allele alone, had more significant monocytosis, expansion of Lineage- Sca-1+ c-Kit+ (LSK) and myeloid progenitors in both bone marrow (BM) and spleen and development of lethal CMML-like disease (median survival 264 days). Moreover, serial transplantation of splenic cells derived from leukemic Tet2Δ/ΔNras+/G12D mice caused similar CMML-like disease in recipients, which emanates from LSK-positive stem/progenitor cells as the disease propagating population.
To delineate how Tet2 loss and Nras mutation synergize in leukemic transformation, we next performed western blot and phospho-flow analysis of MAPK and PI3K signaling in primary hematopoietic cells. Interestingly, pErk, pAkt and pS6 expression were significantly higher in Tet2Δ/ΔNras+/G12D cells compared to WT or single mutant cells, indicating that Tet2 loss and Nras mutation cooperates to further activate Ras signaling (Figure 1). Consistent with our murine model, TET2 silencing in NRAS mutant human leukemia cells increased MAPK output, consistent with augmentation of signaling by concurrent TET2/NRAS alterations in human leukemia cells.
To unravel the molecular mechanism of Ras signaling activation, we assessed mRNA / protein expression and performed bisulfite sequencing of known regulators of MAPK signaling, including Sprouty family members. We observed significant decrease of Spry2 expression, stepwise and specific hyper-methylation of CpG islands in the Spry2 promoter region in Tet2Δ/ΔNras+/G12D cells compared to WT or single mutant cells, consistent with progressive epigenetic remodeling in these leukemia cells in vivo (Figure 2). Genome wide methylation profiling of WT, single mutant and Tet2Δ/ΔNras+/G12D LSK cells using enhanced reduced representation bisulfite sequencing demonstrated clear separation of leukemic Tet2Δ/ΔNras+/G12D LSKs from WT or single mutant LSKs. Most importantly, restoration of Spry2 expression in Tet2Δ/ΔNras+/G12D cells led to decrease in pErk / pAkt level and significantly reduced colony formation, which functionally validates Spry2 as a key epigenetic target in Tet2/Nras mutant leukemia cells.
We next assessed whether the increased MAPK signaling seen in Tet2Δ/ΔNras+/G12D cells leads to differential sensitivity to MEK inhibition by performing studies with the clinical MEK inhibitor binimetinib (ARRY162). Tet2Δ/ΔNras+/G12D cells showed significantly higher sensitivity to binimetinib compared to Nras+/G12D cells in vitro (IC50, 6.948nM vs. 690.4nM). Moreover, in vivo treatment of Tet2Δ/ΔNras+/G12D leukemic recipients with binimetinib restored splenomegaly, significantly reduced disease burden in BM and spleen and improved overall survival compared to vehicle treatment (median survival 24.5 days vs. 44.5 days, p=0.0018, Figure 3). Of note, knockdown of human TET2 in NRAS mutant human leukemia cells sensitized to MEK inhibition in a similar manner demonstrating this approach may have value in leukemia patients with concurrent TET2 and NRAS mutations.
These data clearly indicate that Tet2 loss and Nras mutation synergize in myeloid transformation and cooperatively remodel DNA methylation, which leads to epigenetic silencing of Spry2 and synergistic activation of MAPK signaling, which can be leveraged through therapeutic MEK inhibition. Our studies provide novel insights into how signaling and epigenetic mutations cooperate in leukemic transformation and provide a rationale for mechanism based therapy in CMML patients with these high risk genetic lesions.
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Melnick:Janssen: Research Funding. Levine:Qiagen: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy.
Abstract Hippo signaling emerged over the last decade as a major tumor-suppressing pathway. Its dysregulation is generally associated with abnormal expression levels of YAP1, WWTR1 (coding for the ...TAZ protein) and TEAD genes among others. This pathway has been shown to have a prognostic impact in several cancer types. In particular, the role of YAP1/TEAD activity across indications has been emphasized by several recent works, with potential implications on treatment options. Therefore, identifying patients with a deregulated pathway is key for a better clinical impact of the current potential therapies. Recent studies have been able to characterize RNA-seq based signatures of a deregulated Hippo pathway but a reproducible and cost-effective method to measure the activation of the pathway in clinical settings is needed. Here we first evaluate and confirm the robustness of a YAP1/TEAD activity signature recently published by Calvet et al. (RNA-based signature focused on TEAD downstream effectors), to predict the level of activity of this pathway across several cancer types available in TCGA. Our results confirmed that YAP1/TEAD activity is cancer type and subtype specific and that its high activity is correlated with poor prognosis in some of these cancers. We then trained deep learning models to predict YAP1/TEAD gene activation level, from H&E-stained histology slides in various cancer types from The Cancer Genome Atlas. We showed that histological markers associated with dysfunctional Hippo signaling are markers of disease aggressiveness and poor prognosis such as necrosis, poorly differentiated tumor, and inflammation. Altogether our results are opening the avenue of defining image-based biomarkers predictive of the YAP1/TEAD activity which could be used in clinical settings for better inclusion of subgroups of patients for targeted therapeutics against this hallmark of cancer development. Citation Format: Benoit Schmauch, Vincent Cabeli, Omar Darwiche-Domingues, Jean-Eudes Le Douget, Alexandra Hardy, Reda Belbahri, Charles Maussion, Alberto Romagnoni, Markus Eckstein, Florian Fuchs, Aurélie Swalduz, Sylvie Lantuejoul, Hugo Crochet, François Ghiringhelli, Valentin Derangere, Caroline Truntzer, Harvey Pass, Andre Moreira, Luis Chiriboga, Yuanning Zheng, Michael Ozawa, Brooke Howitt, Olivier Gevaert, Nicolas Girard, Elton Rexhepaj, Iris Valtingojer, Laurent Debussche, Eric Durand, Marion Classe, Katharina Von Loga, Elodie Pronier, Matteo Cesaroni. Deep learning uncovers morphological patterns of YAP1/TEAD activity related to disease aggressiveness in cancer patients abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7377.
Somatic mutation in TET2 gene is one of the most common clonal genetic events detected in age-related clonal hematopoiesis as well as in chronic myelomonocytic leukemia (CMML). In addition to being a ...pre-malignant state, TET2 mutated clones are associated with an increased risk of death from cardiovascular disease, which could involve cytokine/chemokine overproduction by monocytic cells. Here, we show in mice and in human cells that, in the absence of any inflammatory challenge, TET2 downregulation promotes the production of MIF (macrophage migration inhibitory factor), a pivotal mediator of atherosclerotic lesion formation. In healthy monocytes, TET2 is recruited to MIF promoter and interacts with the transcription factor EGR1 and histone deacetylases. Disruption of these interactions as a consequence of TET2-decreased expression favors EGR1-driven transcription of MIF gene and its secretion. MIF favors monocytic differentiation of myeloid progenitors. These results designate MIF as a chronically overproduced chemokine and a potential therapeutic target in patients with clonal TET2 downregulation in myeloid cells.
To improve our understanding of the pathological role of TET2 mutations, Pronier, Imanci et al. use mice and human cells to show that TET2 downregulation promotes the production of macrophage ...migration inhibitory factor (MIF). In addition they show that whilst TET2 is recruited to the MIF promoter in healthy monocytes, decreased TET2 expression results in chronic overproduction of MIF - suggesting that MIF signaling could therefore constitute a potential therapeutic target for conditions associated with TET2 mutations.
Mutations in TET2 and IDH1/2, which result in reduced conversion of 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC), are observed in a significant proportion of patients with acute myeloid ...leukemia (AML). Hence these mutations define a novel class of AMLs with alterations in DNA methylation. However, it has been shown that not all AML cases with low levels of 5-hmC harbor somatic mutations in TET2 and IDH1/2. We hypothesized there are additional somatic mutations that can lead to alterations in TET enzyme function and resultant changes in the epigenetic state of hematopoietic cells.
Mutational studies of the ECOG 1900 trial allowed us to discover that somatic mutations in the WT1 gene were mutually exclusive of IDH1/2 mutations and negatively correlated with TET2 mutations (Figure 1A). 28/313 (9%) of TET2/IDH-wild-type patients had somatic WT1 mutations, whereas 2/85 (2%) TET2/IDH1/2-mutant patients had co-occurring WT1 mutations (p=0.026, Fisher’s Exact test). Analysis of combined data from the ECOG 1900 study and the AML TCGA dataset confirmed a significant anti-correlation between WT1 mutations and TET2/IDH1/2 mutations (p=0.016). We next compared the DNA methylation profiles of WT1, TET2, and IDH1/2 mutant AML patients to AML patients wild-type for these disease alleles. Comparative analysis of the three hypermethylation profiles revealed a near-complete overlap of TET2 and WT1 hypermethylated loci within the IDH1/2 hypermethylation signature, and a highly significant overlap between the TET2 and WT1 mutant signatures (p-value< 0.001 for all comparisons), consistent with convergent, site-specific effects on DNA methylation.
These genetic observations suggested a shared functional role for WT1, TET2, and IDH1/2 mutations in AML in regulating 5-hmC. WT1-mutant AML patients had significantly reduced 5-hmC (Liquid chromatography-electron spray ionization-tandem mass spectrometry) compared to AML patients wild-type for WT1, TET2 or IDH1/2 (p=0.016 T-test Figure 1B), similar to the reduction in 5-hmC observed in IDH1/2-mutant and TET2-mutant AML patient samples. In order to determine the impact of IDH1/2, WT1 and TET2 mutations on the distribution of epigenetic marks throughout the genome more precisely, we examined 5-hmC localization in primary AML specimens with next-generation sequencing. The average number of 5-hmC peaks present in samples from AML patients with TET2, WT1, IDH1 or IDH2 mutations was significantly reduced compared to AML patients wild-type for these disease alleles (t-test p-values between 0.0005 and 0.003). We observed highly significant overlap of differential 5-hmC peaks lost in WT1-mutant AML and TET2-mutant AML (Hypergeometric test p-value < 10-133). In a manner analogous to the findings for DNA methylation, the regions with reduced 5-hmC in WT1/TET2 mutant AML were a subset of those found in IDH1/2 mutant AML patients.
We next investigated whether perturbations in WT1 expression can directly alter 5-hmC levels. Knockdown of Wt1 significantly decreased 5-hmC levels in M15 cells (p<0.01) (Figure 1C). Conversely, overexpression of WT1 in 32D cells significantly increased 5-hmC levels compared to cells expressing a control vector or WT1-mutant (p<0.05) (Figure 1D). Thus, WT1 can directly regulate 5-hmC levels. Given the effects of WT1 on 5-hmC levels and the inverse correlation between WT1 and TET2 mutations in AML, we hypothesized that WT1 might modulate TET2 function through direct interaction.Co-immunoprecipitation experiments demonstrated WT1 interacts with TET2 and with TET3. Consistent with these effects, WT1 expression inhibited colony forming ability of TET2-deficient cells, but could not attenuate the colony growth of TET2/TET3-deficient cells in vitro.
Taken together, our genetic and functional studies demonstrate an inverse correlation between WT1 mutations and TET2/IDH1/2 mutations in AML and a functional role for WT1 in regulating DNA methylation. WT1 mutant AML is characterized by similar global and site-specific alterations in 5-hmC/5-mC as observed in TET2/IDH1/2 mutant AML. Furthermore, we demonstrate that alterations in WT1 levels directly regulate 5-hmC levels, which is due to an interaction between TET2/TET3 and WT1. These data provide a novel role for WT1 in regulating DNA hydroxymethylation and suggest that TET2 IDH1/2, and WT1 mutations define a novel AML subtype.
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Levine:Novartis: Consultancy, Grant support Other.
Abstract 1584
TET2, a gene frequently mutated in myeloid malignancies, encodes an oxygenase that may convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in hematopoietic cells. Using ...high performance liquid chromatography coupled to tandem mass sprectrometry (HPLC-MS-MS), we found lower levels of 5hmC in erythroblasts from patients with myeloproliferative neoplasms and TET2 mutations than in erythroblasts from non mutated patients. To study the function of TET2 during myelopoiesis, we used lentiviruses expressing short hairpin RNA (shRNA) designed to inhibit TET2 expression in CD34+ cells from normal bone marrow or umbilical cord blood. TET2 mRNA was knocked down by 56 to 74% in several human leukemia cell lines expressing shRNA-TET2, compared to cell lines expressing scramble shRNA. TET2 expression was then assessed in these cell lines by western blot, which showed a 49% reduction of TET2 protein in TF1 cell line, and 80 to 90% reduction in Kasumi, Uke1, and Mo7E cell lines. In addition, knock down of TET2 led to a 73% decrease in 5hmC in Mo7E cell line DNA whereas 5mC and cytosine contents remained unchanged as measured by HPLC-MS-MS. These results indicate that TET2 has a role in the hydroxylation of 5mC in human cells of hematopoietic origin. We then studied the consequences of TET2 knock-down in umbilical cord blood CD34+ cells in vitro. We observed a skewing of CD34+CD38- progenitor differentiation toward the myeloid lineage (52 +/− 4% of the TET2 knock down cells versus 35 +/− 3% of the control cells, n=3, p=0.001) at the expense of lympho-myeloid development and B cell and natural killer (NK) lymphoid lineages in a B/myeloid/NK culture condition. Methylcellulose in the presence of EPO, IL3, SCF, G-CSF showed greater numbers of CFU-G/GM (66 +/− 4 versus 58 +/− 3 colonies per 1000 input CD34+ cells) and lower numbers of BFU-E (60 +/−19 versus 78 +/− 19) in TET2 knock-down samples than in control samples (n= 4, p<0.05) indicating that there was also a skewing toward the granulo-monocytic differentiation at the expense of the erythroid lineage. In addition, in presence of G-CSF, IL3, and FLT3-ligand granulocytic differentiation was delayed in TET2 knock-down cells with a relative excess of monocytic cells at day 10 of culture as assessed by the analysis of cell morphology (47 +/− 4% monocytic cells versus 37 +/− 3%, n=5, p=0.006) and immunophenotype (52 +/− 3% of CD14+ cells versus 39+/−8%, n=5, p=0.002). We then analyzed the expression of a set of transcription factors at the mRNA level that confirmed that some transcription factors specific to granulocytic differentiation were under-expressed in TET2 knocked down cells between day 5 and day 10 of culture. Together, our results show that TET2 participates to the conversion of 5mC to 5hmC in hematopoietic cells, and suggest that TET2 inactivation may have a role in the pathogenesis of myeloid malignancies through the disturbance of myeloid differentiation.
No relevant conflicts of interest to declare.
Bone Morphogenetic Proteins (BMPs) are morphogens that play a major role in regulating development and homeostasis. Although BMPs are used for the treatment of bone and kidney disorders, their ...clinical use is limited due to the supra-physiological doses required for therapeutic efficacy causing severe side effects. Because recombinant BMPs are expensive to produce, small molecule activators of BMP signaling would be a cost-effective alternative with the added benefit of being potentially more easily deliverable. Here, we report our efforts to identify small molecule activators of BMP signaling. We have developed a cell-based assay to monitor BMP signaling by stably transfecting a BMP-responsive human cervical carcinoma cell line (C33A) with a reporter construct in which the expression of luciferase is driven by a multimerized BMP-responsive element from the Id1 promoter. A BMP-responsive clone C33A-2D2 was used to screen a bioactive library containing ~5,600 small molecules. We identified four small molecules of the family of flavonoids all of which induced luciferase activity in a dose-dependent manner and ventralized zebrafish embryos. Two of the identified compounds induced Smad1, 5 phosphorylation (P-Smad), Id1 and Id2 expression in a dose-dependent manner demonstrating that our assays identified small molecule activators of BMP signaling.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
During the synthesis of geopolymer materials, all of the phenomena and reactions that occur should be included in the analysis of the synthesis. This work aims to study the role that siliceous ...species play in an activation solution in the presence of pure metakaolin. The formation of a gel phase during the synthesis of a K-geopolymer was shown. Different mixtures were analyzed by infrared spectroscopy. During material consolidation, there is always competition between the geopolymer network and the gel that is governed by a change in the siliceous species. The heat treatment of various gels and solids provided evidence of various networks in geopolymer materials. Finally, the change in the crystallinity of the silica in the activation solution led to a change in the kinetics of the polycondensation reactions, in agreement with previous work.
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