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Abstract Background Several fusion oncogenes showing a higher incidence in pediatric acute myeloid leukemia (AML) are associated with heterogeneous megakaryoblastic and other myeloid features. Here we addressed how developmental mechanisms influence human leukemogenesis by ETO2::GLIS2, associated with dismal prognosis. Methods We created novel ETO2::GLIS2 models of leukemogenesis through lentiviral transduction and CRISPR-Cas9 gene editing of human fetal and post-natal hematopoietic stem/progenitor cells (HSPCs), performed in-depth characterization of ETO2::GLIS2 transformed cells through multiple omics and compared them to patient samples. This led to a preclinical assay using patient-derived-xenograft models to test a combination of two clinically-relevant molecules. Results We showed that ETO2::GLIS2 expression in primary human fetal CD34+ hematopoietic cells led to more efficient in vivo leukemia development than expression in post-natal cells. Moreover, cord blood-derived leukemogenesis has a major dependency on the presence of human cytokines, including IL3 and SCF. Single cell transcriptomes revealed that this cytokine environment controlled two ETO2::GLIS2-transformed states that were also observed in primary patient cells. Importantly, this cytokine sensitivity may be therapeutically-exploited as combined MEK and BCL2 inhibition showed higher efficiency than individual molecules to reduce leukemia progression in vivo. Conclusions Our study uncovers an interplay between the cytokine milieu and transcriptional programs that extends a developmental window of permissiveness to transformation by the ETO2::GLIS2 AML fusion oncogene, controls the intratumoral cellular heterogeneity, and offers a ground-breaking therapeutical opportunity by a targeted combination strategy.

Abstract Transcriptional cofactors of the ETO family are recurrent fusion partners in acute leukemia. We characterized the ETO2 regulome by integrating transcriptomic and chromatin binding analyses in human erythroleukemia xenografts and controlled ETO2 depletion models. We demonstrate that beyond its well‐established repressive activity, ETO2 directly activates transcription of MYB, among other genes. The ETO2‐activated signature is associated with a poorer prognosis in erythroleukemia but also in other acute myeloid and lymphoid leukemia subtypes. Mechanistically, ETO2 colocalizes with EP300 and MYB at enhancers supporting the existence of an ETO2/MYB feedforward transcription activation loop (e.g., on MYB itself). Both small‐molecule and PROTAC‐mediated inhibition of EP300 acetyltransferases strongly reduced ETO2 protein, chromatin binding, and ETO2‐activated transcripts. Taken together, our data show that ETO2 positively enforces a leukemia maintenance program that is mediated in part by the MYB transcription factor and that relies on acetyltransferase cofactors to stabilize ETO2 scaffolding activity.

Abstract Pediatric patients with recurrent and refractory cancers are in most need for new treatments. This study developed patient-derived-xenograft (PDX) models within the European MAPPYACTS cancer precision medicine trial (NCT02613962). To date, 131 PDX models were established following heterotopical and/or orthotopical implantation in immunocompromised mice: 76 sarcomas, 25 other solid tumors, 12 central nervous system tumors, 15 acute leukemias, and 3 lymphomas. PDX establishment rate was 43%. Histology, whole exome and RNA sequencing revealed a high concordance with the primary patient’s tumor profile, human leukocyte-antigen characteristics and specific metabolic pathway signatures. A detailed patient molecular characterization, including specific mutations prioritized in the clinical molecular tumor boards are provided. Ninety models were shared with the IMI2 ITCC Pediatric Preclinical Proof-of-concept Platform (IMI2 ITCC-P4) for further exploitation. This PDX biobank of unique recurrent childhood cancers provides an essential support for basic and translational research and treatments development in advanced pediatric malignancies.

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Abstract Background Anaplastic large cell lymphoma positive for ALK (ALK+ ALCL) is a rare type of non-Hodgkin lymphoma. This lymphoma is caused by chromosomal translocations involving the anaplastic lymphoma kinase gene (ALK). In this study, we aimed to identify mechanisms of transformation and therapeutic targets by generating a model of ALK+ ALCL lymphomagenesis ab initio with the specific NPM-ALK fusion. Methods We performed CRISPR/Cas9-mediated genome editing of the NPM-ALK chromosomal translocation in primary human activated T lymphocytes. Results Both CD4+ and CD8+ NPM-ALK-edited T lymphocytes showed rapid and reproducible competitive advantage in culture and led to in vivo disease development with nodal and extra-nodal features. Murine tumors displayed the phenotypic diversity observed in ALK+ ALCL patients, including CD4+ and CD8+ lymphomas. Assessment of transcriptome data from models and patients revealed global activation of the WNT signaling pathway, including both canonical and non-canonical pathways, during ALK+ ALCL lymphomagenesis. Specifically, we found that the WNT signaling cell surface receptor ROR2 represented a robust and genuine marker of all ALK+ ALCL patient tumor samples. Conclusions In this study, ab initio modeling of the ALK+ ALCL chromosomal translocation in mature T lymphocytes enabled the identification of new therapeutic targets. As ROR2 targeting approaches for other cancers are under development (including lung and ovarian tumors), our findings suggest that ALK+ ALCL cases with resistance to current therapies may also benefit from ROR2 targeting strategies.

Acute megakaryoblastic leukemia of Down syndrome (DS-AMKL) is a model of clonal evolution from a preleukemic transient myeloproliferative disorder requiring both a trisomy 21 (T21) and a GATA1s mutation to a leukemia driven by additional driver mutations. We modeled the megakaryocyte differentiation defect through stepwise gene editing of GATA1s, SMC3+/–, and MPLW515K, providing 20 different T21 or disomy 21 (D21) induced pluripotent stem cell (iPSC) clones. GATA1s profoundly reshaped iPSC-derived hematopoietic architecture with gradual myeloid-to-megakaryocyte shift and megakaryocyte differentiation alteration upon addition of SMC3 and MPL mutations. Transcriptional, chromatin accessibility, and GATA1-binding data showed alteration of essential megakaryocyte differentiation genes, including NFE2 downregulation that was associated with loss of GATA1s binding and functionally involved in megakaryocyte differentiation blockage. T21 enhanced the proliferative phenotype, reproducing the cellular and molecular abnormalities of DS-AMKL. Our study provides an array of human cell–based models revealing individual contributions of different mutations to DS-AMKL differentiation blockage, a major determinant of leukemic progression.

Loss of function mutations of NSD1 occur in blood cancers. Here, the authors report that NSD1 loss blocks erythroid differentiation which leads to an erythroleukemia-like disease in mice by impairing GATA1-induced target gene activation.

Malignancies of the erythroid lineage are rare but aggressive diseases. Notably, the first insights into their biology emerged over half a century ago from avian and murine tumor viruses-induced erythroleukemia models providing the rationale for several transgenic mouse models that unraveled the transforming potential of signaling effectors and transcription factors in the erythroid lineage. More recently, genetic roadmaps have fueled efforts to establish models that are based on the epigenomic lesions observed in patients with erythroid malignancies. These models, together with often unexpected erythroid phenotypes in genetically modified mice, provided further insights into the molecular mechanisms of disease initiation and maintenance. Here, we review how the increasing knowledge of human erythroleukemia genetics combined with those from various mouse models indicate that the pathogenesis of the disease is based on the interplay between signaling mutations, impaired TP53 function, and altered chromatin organization. These alterations lead to aberrant activity of erythroid transcriptional master regulators like GATA1, indicating that erythroleukemia will most likely require combinatorial targeting for efficient therapeutic interventions.

Summary: Circular RNAs constitute a unique class of RNAs whose precise functions remain to be elucidated. In particular, cancer-associated chromosomal translocations can give rise to fusion circular RNAs that play a role in leukemia progression. However, how and when fusion circular RNAs are formed and whether they are being selected in cancer cells remains unknown. Here, we used CRISPR/Cas9 to generate physiological translocation models of NPM1-ALK fusion gene. We showed that, in addition to generating fusion proteins and activating specific oncogenic pathways, chromosomal translocation induced by CRISPR/Cas9 led to the formation of de novo fusion circular RNAs. Specifically, we could recover different classes of circular RNAs composed of different circularization junctions, mainly back-spliced species. In addition, we identified fusion circular RNAs identical to those found in related patient tumor cells providing evidence that fusion circular RNAs arise early after chromosomal formation and are not just a consequence of the oncogenesis process. : Molecular Biology; Molecular Genetics; Cancer Subject Areas: Molecular Biology, Molecular Genetics, Cancer

Abstract: JAK3-activating mutations are commonly seen in chronic or acute hematologic malignancies affecting the myeloid, megakaryocytic, lymphoid, and natural killer (NK) cell compartment. Overexpression models of mutant JAK3 or pharmacologic inhibition of its kinase activity have highlighted the role that these constitutively activated mutants play in the T-cell, NK cell, and megakaryocytic lineages, but to date, the functional impact of JAK3 mutations at an endogenous level remains unknown. Here, we report a JAK3A572V knockin mouse model and demonstrate that activated JAK3 leads to a progressive and dose-dependent expansion of CD8+ T cells in the periphery before colonization of the bone marrow. This phenotype is dependent on the γc chain of cytokine receptors and presents several features of the human leukemic form of cutaneous T-cell lymphoma (L-CTCL), including skin involvements. We also showed that the JAK3A572V-positive malignant cells are transplantable and phenotypically heterogeneous in bone marrow transplantation assays. Interestingly, we revealed that activated JAK3 functionally cooperates with partial trisomy 21 in vivo to enhance the L-CTCL phenotype, ultimately leading to a lethal and fully penetrant disorder. Finally, we assessed the efficacy of JAK3 inhibition and showed that CTCL JAK3A572V-positive T cells are sensitive to tofacitinib, which provides additional preclinical insights into the use of JAK3 inhibitors in these disorders. Altogether, this JAK3A572V knockin model is a relevant new tool for testing the efficacy of JAK inhibitors in JAK3-related hematopoietic malignancies.

Abstract: The TET2 gene encodes an α-ketoglutarate–dependent dioxygenase able to oxidize 5-methylcytosine into 5-hydroxymethylcytosine, which is a step toward active DNA demethylation. TET2 is frequently mutated in myeloid malignancies but also in B- and T-cell malignancies. TET2 somatic mutations are also identified in healthy elderly individuals with clonal hematopoiesis. Tet2-deficient mouse models showed widespread hematological differentiation abnormalities, including myeloid, T-cell, and B-cell malignancies. We show here that, similar to what is observed with constitutive Tet2-deficient mice, B-cell–specific Tet2 knockout leads to abnormalities in the B1-cell subset and a development of B-cell malignancies after long latency. Aging Tet2-deficient mice accumulate clonal CD19+ B220low immunoglobulin M+ B-cell populations with transplantable ability showing similarities to human chronic lymphocytic leukemia, including CD5 expression and sensitivity to ibrutinib-mediated B-cell receptor (BCR) signaling inhibition. Exome sequencing of Tet2−/− malignant B cells reveals C-to-T and G-to-A mutations that lie within single-stranded DNA–specific activation-induced deaminase (AID)/APOBEC (apolipoprotein B messenger RNA editing enzyme, catalytic polypeptide-like) cytidine deaminases targeted motif, as confirmed by the lack of a B-cell tumor in compound Tet2-Aicda–deficient mice. Finally, we show that Tet2 deficiency accelerates and exacerbates T-cell leukemia/lymphoma 1A–induced leukemogenesis. Together, our data establish that Tet2 deficiency predisposes to mature B-cell malignancies, which development might be attributed in part to AID-mediated accumulating mutations and BCR-mediated signaling.

This review aims to provide an overview of the current knowledge of the genetic lesions driving pediatric acute myeloid leukemia (AML), emerging biological concepts, and strategies for therapeutic intervention. Hereby, we focus on lesions that preferentially or exclusively occur in pediatric patients and molecular markers of aggressive disease with often poor outcome including fusion oncogenes that involve epigenetic regulators like KMT2A, NUP98, or CBFA2T3, respectively. Functional studies were able to demonstrate cooperation with signaling mutations leading to constitutive activation of FLT3 or the RAS signal transduction pathways. We discuss the issues faced to faithfully model pediatric acute leukemia in mice. Emerging experimental evidence suggests that the disease phenotype is dependent on the appropriate expression and activity of the driver fusion oncogenes during a particular window of opportunity during fetal development. We also highlight biochemical studies that deciphered some molecular mechanisms of malignant transformation by KMT2A, NUP98, and CBFA2T3 fusions, which, in some instances, allowed the development of small molecules with potent anti-leukemic activities in preclinical models (e.g., inhibitors of the KMT2A–MENIN interaction). Finally, we discuss other potential therapeutic strategies that not only target driver fusion-controlled signals but also interfere with the transformed cell state either by exploiting the primed apoptosis or vulnerable metabolic states or by increasing tumor cell recognition and elimination by the immune system.

The ETO2-GLIS2 fusion oncoprotein is associated with poor prognosis pediatric acute megakaryoblastic leukemia. Recently, we observed that ETO2-GLIS2 controls enhancers activity at genes regulating haematopoietic progenitor self-renewal and differentiation toward the megakaryocytic lineage. We also showed that targeting ETO2-GLIS2 complex stability inhibits these properties and may represent a novel therapeutic strategy.

STAT3 protein phosphorylation is a frequent event in various hematologic malignancies and solid tumors. Acquired STAT3 mutations have been recently identified in 40% of patients with T-cell large granular lymphocytic leukemia, a rare T-cell disorder. In this study, we investigated the mutational status of STAT3 in a large series of patients with lymphoid and myeloid diseases. STAT3 mutations were identified in 1.6% (4 of 258) of patients with T-cell neoplasms, in 2.5% (2 of 79) of patients with diffuse large B-cell lymphoma but in no other B-cell lymphoma patients (0 of 104) or patients with myeloid malignancies (0 of 96). Functional in vitro assays indicated that the STAT3Y640F mutation leads to a constitutive phosphorylation of the protein. STA21, a STAT3 small molecule inhibitor, inhibited the proliferation of two distinct STAT3 mutated cell lines. Using a mouse bone marrow transplantation assay, we observed that STAT3Y640F expression leads to the development of myeloproliferative neoplasms with expansion of either myeloid cells or megakaryocytes. Together, these data indicate that the STAT3Y640F mutation leads to constitutive activation of STAT3, induces malignant hematopoiesis in vivo, and may represent a novel therapeutic target in some lymphoid malignancies.

Background Cdx4 is a homeobox gene essential for normal blood formation during embryonic development in the zebrafish, through activation of posterior Hox genes. However, its role in adult mammalian hematopoiesis has not been extensively studied and its requirement in leukemia associated with Hox gene expression alteration is unclear.Design and Methods We inactivated Cdx4 in mice through either a germline or conditional knockout approach and analyzed requirement for Cdx4 in both normal adult hematopoiesis and leukemogenesis initiated by the MLL-AF9 fusion oncogene.Results Here, we report that loss of Cdx4 had a minimal effect on adult hematopoiesis. Indeed, although an increase in white blood cell counts was observed, no significant differences in the distribution of mature blood cells, progenitors or stem cells were observed in Cdx4-deficient animals. In addition, long-term repopulating activity in competitive transplantation assays was not significantly altered. In vitro, B-cell progenitor clonogenic potential was reduced in Cdx4-deficient animals but no significant alteration of mature B cells was detected in vivo. Finally, induction of acute myeloid leukemia in mice by MLL-AF9 was significantly delayed in the absence of Cdx4 in a retroviral transduction/bone marrow transplant model.Conclusions These observations indicate that Cdx4 is dispensable for the establishment and maintenance of normal hematopoiesis in adult mammals. These results, therefore, outline substantial differences in the Cdx-Hox axis between mammals and zebrafish and support the hypothesis that Cdx factors are functionally redundant during mammalian hematopoietic development under homeostatic conditions. In addition, our results suggest that Cdx4 participates in MLL-AF9-mediated leukemogenesis supporting a role for Cdx factors in the pathogenesis of myeloid leukemia.

The JAK2V617F allele has recently been identified in patients with polycythemia vera (PV), essential thrombocytosis (ET), and myelofibrosis with myeloid metaplasia (MF). Subsequent analysis has shown that constitutive activation of the JAK-STAT signal transduction pathway is an important pathogenetic event in these patients, and that enzymatic inhibition of JAK2V617F may be of therapeutic benefit in this context. However, a significant proportion of patients with ET or MF are JAK2V617F-negative. We hypothesized that activation of the JAK-STAT pathway might also occur as a consequence of activating mutations in certain hematopoietic-specific cytokine receptors, including the erythropoietin receptor (EPOR), the thrombopoietin receptor (MPL), or the granulocyte-colony stimulating factor receptor (GCSFR).DNA sequence analysis of the exons encoding the transmembrane and juxtamembrane domains of EPOR, MPL, and GCSFR, and comparison with germline DNA derived from buccal swabs, identified a somatic activating mutation in the transmembrane domain of MPL (W515L) in 9% (4/45) of JAKV617F-negative MF. Expression of MPLW515L in 32D, UT7, or Ba/F3 cells conferred cytokine-independent growth and thrombopoietin hypersensitivity, and resulted in constitutive phosphorylation of JAK2, STAT3, STAT5, AKT, and ERK. Furthermore, a small molecule JAK kinase inhibitor inhibited MPLW515L-mediated proliferation and JAK-STAT signaling in vitro. In a murine bone marrow transplant assay, expression of MPLW515L, but not wild-type MPL, resulted in a fully penetrant myeloproliferative disorder characterized by marked thrombocytosis (Plt count 1.9-4.0 x 10(12)/L), marked splenomegaly due to extramedullary hematopoiesis, and increased reticulin fibrosis.Activation of JAK-STAT signaling via MPLW515L is an important pathogenetic event in patients with JAK2V617F-negative MF. The bone marrow transplant model of MPLW515L-mediated myeloproliferative disorders (MPD) exhibits certain features of human MF, including extramedullary hematopoiesis, splenomegaly, and megakaryocytic proliferation. Further analysis of positive and negative regulators of the JAK-STAT pathway is warranted in JAK2V617F-negative MPD.