High caspase 3 and vulnerability to dual BCL2 family inhibition define ETO2::GLIS2 pediatric leukemia.

Pediatric acute myeloid leukemia expressing the ETO2::GLIS2 fusion oncogene is associated with dismal prognosis. Previous studies have shown that ETO2::GLIS2 can efficiently induce leukemia development associated with strong transcriptional changes but those amenable to pharmacological targeting remained to be identified. By studying an inducible ETO2::GLIS2 cellular model, we uncovered that de novo ETO2::GLIS2 expression in human cells led to increased CASP3 transcription, CASP3 activation, and cell death. Patient-derived ETO2::GLIS2+ leukemic cells expressed both high CASP3 and high BCL2. While BCL2 inhibition partly inhibited ETO2::GLIS2+ leukemic cell proliferation, BH3 profiling revealed that it also sensitized these cells to MCL1 inhibition indicating a functional redundancy between BCL2 and MCL1. We further show that combined inhibition of BCL2 and MCL1 is mandatory to abrogate disease progression using in vivo patient-derived xenograft models. These data reveal that a transcriptional consequence of ETO2::GLIS2 expression includes a positive regulation of the pro-apoptotic CASP3 and associates with a vulnerability to combined targeting of two BCL2 family members providing a novel therapeutic perspective for this aggressive pediatric AML subgroup.

Constitutive Activation of RAS/MAPK Pathway Cooperates with Trisomy 21 and Is Therapeutically Exploitable in Down Syndrome B-cell Leukemia.

PURPOSE: Children with Down syndrome (constitutive trisomy 21) that develop acute lymphoblastic leukemia (DS-ALL) have a 3-fold increased likelihood of treatment-related mortality coupled with a higher cumulative incidence of relapse, compared with other children with B-cell acute lymphoblastic leukemia (B-ALL). This highlights the lack of suitable treatment for Down syndrome children with B-ALL. EXPERIMENTAL DESIGN: To facilitate the translation of new therapeutic agents into clinical trials, we built the first preclinical cohort of patient-derived xenograft (PDX) models of DS-ALL, comprehensively characterized at the genetic and transcriptomic levels, and have proven its suitability for preclinical studies by assessing the efficacy of drug combination between the MEK inhibitor trametinib and conventional chemotherapy agents. RESULTS: Whole-exome and RNA-sequencing experiments revealed a high incidence of somatic alterations leading to RAS/MAPK pathway activation in our cohort of DS-ALL, as well as in other pediatric B-ALL presenting somatic gain of the chromosome 21 (B-ALL+21). In murine and human B-cell precursors, activated KRASG12D functionally cooperates with trisomy 21 to deregulate transcriptional networks that promote increased proliferation and self renewal, as well as B-cell differentiation blockade. Moreover, we revealed that inhibition of RAS/MAPK pathway activation using the MEK1/2 inhibitor trametinib decreased leukemia burden in several PDX models of B-ALL+21, and enhanced survival of DS-ALL PDX in combination with conventional chemotherapy agents such as vincristine. CONCLUSIONS: Altogether, using novel and suitable PDX models, this study indicates that RAS/MAPK pathway inhibition represents a promising strategy to improve the outcome of Down syndrome children with B-cell precursor leukemia.

Ontogenic Changes in Hematopoietic Hierarchy Determine Pediatric Specificity and Disease Phenotype in Fusion Oncogene-Driven Myeloid Leukemia.

Fusion oncogenes are prevalent in several pediatric cancers, yet little is known about the specific associations between age and phenotype. We observed that fusion oncogenes, such as ETO2-GLIS2, are associated with acute megakaryoblastic or other myeloid leukemia subtypes in an age-dependent manner. Analysis of a novel inducible transgenic mouse model showed that ETO2-GLIS2 expression in fetal hematopoietic stem cells induced rapid megakaryoblastic leukemia whereas expression in adult bone marrow hematopoietic stem cells resulted in a shift toward myeloid transformation with a strikingly delayed in vivo leukemogenic potential. Chromatin accessibility and single-cell transcriptome analyses indicate ontogeny-dependent intrinsic and ETO2-GLIS2-induced differences in the activities of key transcription factors, including ERG, SPI1, GATA1, and CEBPA. Importantly, switching off the fusion oncogene restored terminal differentiation of the leukemic blasts. Together, these data show that aggressiveness and phenotypes in pediatric acute myeloid leukemia result from an ontogeny-related differential susceptibility to transformation by fusion oncogenes. SIGNIFICANCE: This work demonstrates that the clinical phenotype of pediatric acute myeloid leukemia is determined by ontogeny-dependent susceptibility for transformation by oncogenic fusion genes. The phenotype is maintained by potentially reversible alteration of key transcription factors, indicating that targeting of the fusions may overcome the differentiation blockage and revert the leukemic state.See related commentary by Cruz Hernandez and Vyas, p. 1653.This article is highlighted in the In This Issue feature, p. 1631.

Pediatric Acute Megakaryoblastic Leukemia: Multitasking Fusion Proteins and Oncogenic Cooperations.

Pediatric leukemia presents specific clinical and genetic features from adult leukemia but the underpinning mechanisms of transformation are still unclear. Acute megakaryoblastic leukemia (AMKL) is the malignant accumulation of progenitors of the megakaryocyte lineage that normally produce blood platelets. AMKL is diagnosed de novo, in patients showing a poor prognosis, or in Down syndrome (DS) patients with a better prognosis. Recent data show that de novo AMKL is primarily associated with chromosomal alterations leading to the expression of fusions between transcriptional regulators. This review highlights the most recurrent genetic events found in de novo pediatric AMKL patients and, based on recent functional analyses, proposes a mechanism of leukemogenesis common to de novo and DS-AMKL.

Chronic Type I IFN Is Sufficient To Promote Immunosuppression through Accumulation of Myeloid-Derived Suppressor Cells.

Failure of the immune system to eradicate viruses results in chronic viral infections, which are associated with increased susceptibility to secondary infections. Pathogenic HIV or lymphocytic choriomeningitis virus chronic infections display a persistent type I IFN signature. In chronic lymphocytic choriomeningitis virus infection, blockade of type I IFN signaling partially restores antiviral responses. In a mouse model, we tested whether chronic administration of type I IFN, at doses mimicking chronic viral infection, induced immunosuppression. Chronic exposure of mice to IFN-α alone was sufficient to strongly suppress specific CD8+ T cells responses to subsequent vaccinia virus infection. It resulted in the accumulation of Ly6Chi monocytes. These monocytes were similar, phenotypically and functionally, to the myeloid-derived suppressor cells found in cancer because they exerted a potent suppression on CD8+ T cell responses in vitro. They acted at least partly through the l-arginine pathway. In vivo, their elimination restored antiviral CD8+ T cell responses. Our work provides a specific mechanism accounting for the role of IFN-α in immunosuppression and predicts that type I IFN modulation will be pivotal to cure human chronic infections, cancer, or autoimmune diseases.

The AMPKγ1 subunit plays an essential role in erythrocyte membrane elasticity, and its genetic inactivation induces splenomegaly and anemia.

AMP-activated protein kinase (AMPK) is an αßγ heterotrimer conserved throughout evolution and important for energy sensing in all eukaryote cells. AMPK controls metabolism and various cellular events in response to both hormones and changes in cellular energy status. The γ subunit senses intracellular energy status through the competitive binding of AMP and ATP. We show here that targeted disruption of the mouse AMPKγ1 gene (Prkag1) causes regenerative hemolytic anemia by increasing the sequestration of abnormal erythrocytes. Prkag1(-/-) mice displayed splenomegaly and iron accumulation due to compensatory splenic erythropoiesis and erythrophagocytosis. Moreover, AMPKγ1-deficient erythrocytes were highly resistant to osmotic hemolysis and poorly deformable in response to increasing shear stress, consistent with greater membrane rigidity. No change in cytoskeletal protein composition was observed; however, the phosphorylation level of adducin, a protein promoting the binding of spectrin to actin, was higher in AMPKγ1-deficient erythrocytes. Together, these results demonstrate that AMPKγ1 subunit is required for the maintenance of erythrocyte membrane elasticity.

Transferrin receptor 2 is a component of the erythropoietin receptor complex and is required for efficient erythropoiesis.

Erythropoietin (Epo) is required for erythroid progenitor differentiation. Although Epo crosslinking experiments have revealed the presence of Epo receptor (EpoR)-associated proteins that could never be identified, EpoR is considered to be a paradigm for homodimeric cytokine receptors. We purified EpoR-binding partners and identified the type 2 transferrin receptor (TfR2) as a component of the EpoR complex corresponding to proteins previously detected in cross-linking experiments. TfR2 is involved in iron metabolism by regulating hepcidin production in liver cells. We show that TfR2 and EpoR are synchronously coexpressed during the differentiation of erythroid progenitors. TfR2 associates with EpoR in the endoplasmic reticulum and is required for the efficient transport of this receptor to the cell surface. Erythroid progenitors from TfR2(-/-)mice show a decreased sensitivity to Epo and increased circulating Epo levels. In human erythroid progenitors, TfR2 knockdown delays the terminal differentiation. Erythroid cells produce growth differentiation factor-15, a cytokine that suppresses hepatic hepcidin production in certain erythroid diseases such as thalassemia. We show that the production of growth differentiation factor-15 by erythroid cells is dependent on both Epo and TfR2. Taken together, our results show that TfR2 exhibits a non hepatic function as a component of the EpoR complex and is required for efficient erythropoiesis.

Differentiation of PLB-985 myeloid cells into mature neutrophils, shown by degranulation of terminally differentiated compartments in response to N-formyl peptide and priming of superoxide anion production by granulocyte-macrophage colony-stimulating factor.

Mature blood neutrophils have a short lifespan in vitro and are not easily transfectable. We obtained terminally mature neutrophils after differentiation of immature transfectable PLB-985 myeloid cells by treatment with dimethylformamide (0.5%), Nutridoma SP (1%) and fetal calf serum (0.5%). Maturation was shown by functional degranulation, in response to bacterial N-formyl peptide (fMLP), of specific granules and secretory vesicle contents; the latter emerge during the last step of normal neutrophil differentiation into bone marrow. These differentiated cells also produced quantities of superoxide anion similar to those produced by blood neutrophils, in response to physiological stimuli (fMLP); in addition, the fMLP-induced respiratory burst was primed by the proinflammatory cytokine granulocyte-macrophage colony-stimulating factor. Thus, in our experimental conditions, PLB-985 cells transformed into fully differentiated neutrophils capable of fine regulation by inflammatory agents. This cell model will help in the understanding of the molecular mechanisms underlying neutrophil functions.