The RNA-binding protein IGF2BP3 is critical for MLL-AF4-mediated leukemogenesis.

Despite recent advances in therapeutic approaches, patients with MLL-rearranged leukemia still have poor outcomes. Here, we find that the RNA-binding protein IGF2BP3, which is overexpressed in MLL-translocated leukemia, strongly amplifies MLL-Af4-mediated leukemogenesis. Deletion of Igf2bp3 significantly increases the survival of mice with MLL-Af4-driven leukemia and greatly attenuates disease, with a minimal impact on baseline hematopoiesis. At the cellular level, MLL-Af4 leukemia-initiating cells require Igf2bp3 for their function in leukemogenesis. At the molecular level, IGF2BP3 regulates a complex posttranscriptional operon governing leukemia cell survival and proliferation. IGF2BP3-targeted mRNA transcripts include important MLL-Af4-induced genes, such as those in the Hoxa locus, and the Ras signaling pathway. Targeting of transcripts by IGF2BP3 regulates both steady-state mRNA levels and, unexpectedly, pre-mRNA splicing. Together, our findings show that IGF2BP3 represents an attractive therapeutic target in this disease, providing important insights into mechanisms of posttranscriptional regulation in leukemia.

Mouse Models of CMML.

Chronic myelomonocytic leukemia (CMML) is a rare and challenging type of myeloproliferative neoplasm. Poor prognosis and high mortality, associated predominantly with progression to secondary acute myeloid leukemia (sAML), is still an unsolved problem. Despite a growing body of knowledge about the molecular repertoire of this disease, at present, the prognostic significance of CMML-associated mutations is controversial. The absence of available CMML cell lines and the small number of patients with CMML make pre-clinical testing and clinical trials complicated. Currently, specific therapy for CMML has not been approved; most of the currently available therapeutic approaches are based on myelodysplastic syndrome (MDS) and other myeloproliferative neoplasm (MNP) studies. In this regard, the development of the robust CMML animal models is currently the focus of interest. This review describes important studies concerning animal models of CMML, examples of methodological approaches, and the obtained hematologic phenotypes.

RUNX1 and CBFß-SMMHC transactivate target genes together in abnormal myeloid progenitors for leukemia development.

Inversion of chromosome 16 is a consistent finding in patients with acute myeloid leukemia subtype M4 with eosinophilia, which generates a CBFB-MYH11 fusion gene. It is generally considered that CBFß-SMMHC, the fusion protein encoded by CBFB-MYH11, is a dominant negative repressor of RUNX1. However, recent findings challenge the RUNX1-repression model for CBFß-SMMHC-mediated leukemogenesis. To definitively address the role of Runx1 in CBFB-MYH11-induced leukemia, we crossed conditional Runx1 knockout mice (Runx1f/f) with conditional Cbfb-MYH11 knockin mice (Cbfb+/56M). On Mx1-Cre activation in hematopoietic cells induced by poly (I:C) injection, all Mx1-CreCbfb+/56M mice developed leukemia in 5 months, whereas no leukemia developed in Runx1f/fMx1-CreCbfb+/56M mice, and this effect was cell autonomous. Importantly, the abnormal myeloid progenitors (AMPs), a leukemia-initiating cell population induced by Cbfb-MYH11 in the bone marrow, decreased and disappeared in Runx1f/fMx1-CreCbfb+/56M mice. RNA-seq analysis of AMP cells showed that genes associated with proliferation, differentiation blockage, and leukemia initiation were differentially expressed between Mx1-CreCbfb+/56M and Runx1f/fMx1-CreCbfb+/56M mice. In addition, with the chromatin immunocleavage sequencing assay, we observed a significant enrichment of RUNX1/CBFß-SMMHC target genes in Runx1f/fMx1-CreCbfb+/56M cells, especially among downregulated genes, suggesting that RUNX1 and CBFß-SMMHC mainly function together as activators of gene expression through direct target gene binding. These data indicate that Runx1 is indispensable for Cbfb-MYH11-induced leukemogenesis by working together with CBFß-SMMHC to regulate critical genes associated with the generation of a functional AMP population.

A high-throughput screen indicates gemcitabine and JAK inhibitors may be useful for treating pediatric AML.

Improvement in survival has been achieved for children and adolescents with AML but is largely attributed to enhanced supportive care as opposed to the development of better treatment regimens. High risk subtypes continue to have poor outcomes with event free survival rates <40% despite the use of high intensity chemotherapy in combination with hematopoietic stem cell transplant. Here we combine high-throughput screening, intracellular accumulation assays, and in vivo efficacy studies to identify therapeutic strategies for pediatric AML. We report therapeutics not currently used to treat AML, gemcitabine and cabazitaxel, have broad anti-leukemic activity across subtypes and are more effective relative to the AML standard of care, cytarabine, both in vitro and in vivo. JAK inhibitors are selective for acute megakaryoblastic leukemia and significantly prolong survival in multiple preclinical models. Our approach provides advances in the development of treatment strategies for pediatric AML.

MLL is essential for NUP98-HOXA9-induced leukemia.

Rearrangements involving the NUP98 gene resulting in fusions to several partner genes occur in acute myeloid leukemia and myelodysplastic syndromes. This study demonstrates that the second FG repeat domain of the NUP98 moiety of the NUP98-HOXA9 fusion protein is important for its cell immortalization and leukemogenesis activities. We demonstrate that NUP98-HOXA9 interacts with mixed lineage leukemia (MLL) via this FG repeat domain and that, in the absence of MLL, NUP98-HOXA9-induced cell immortalization and leukemogenesis are severely inhibited. Molecular analyses indicate that MLL is important for the recruitment of NUP98-HOXA9 to the HOXA locus and for NUP98-HOXA9-induced HOXA gene expression. Our data indicate that MLL is crucial for NUP98-HOXA9 leukemia initiation.

Loss of p300 accelerates MDS-associated leukemogenesis.

The role that changes in DNA methylation and histone modifications have in human malignancies is poorly understood. p300 and CREB-binding protein (CBP), two distinct but highly homologous lysine acetyltransferases, are mutated in several cancers, suggesting their role as tumor suppressors. In the current study, we found that deletion of p300, but not CBP, markedly accelerated the leukemogenesis ofNup98-HoxD13 (NHD13) transgenic mice, an animal model that phenotypically copies human myelodysplastic syndrome (MDS). p300 deletion restored the ability of NHD13 expressing hematopoietic stem and progenitor cells (HSPCs) to self-renew in vitro, and to expand in vivo, with an increase in stem cell symmetric self-renewal divisions and a decrease in apoptosis. Furthermore, loss of p300, but not CBP, promoted cytokine signaling, including enhanced activation of the MAPK and JAK/STAT pathways in the HSPC compartment. Altogether, our data indicate that p300 has a pivotal role in blocking the transformation of MDS to acute myeloid leukemia, a role distinct from that of CBP.

Two types of BCR interactions are positively selected during leukemia development in the Eµ-TCL1 transgenic mouse model of CLL.

Chronic lymphocytic leukemia (CLL) is a common B-cell malignancy characterized by a highly variable course and outcome. The disease is believed to be driven by B-cell receptor (BCR) signals generated by external antigens and/or cell-autonomous BCR interactions, but direct in vivo evidence for this is still lacking. To further define the role of the BCR pathway in the development and progression of CLL, we evaluated the capacity of different types of antigen/BCR interactions to induce leukemia in the Eµ-TCL1 transgenic mouse model. We show that cell autonomous signaling capacity is a uniform characteristic of the leukemia-derived BCRs and represents a prerequisite for CLL development. Low-affinity BCR interactions with autoantigens generated during apoptosis are also positively selected, suggesting that they contribute to the pathogenesis of the disease. In contrast, high-affinity BCR interactions are not selected, regardless of antigen form or presentation. We also show that the capacity of the leukemic cells to respond to cognate antigen correlates inversely with time to leukemia development, suggesting that signals induced by external antigen increase the aggressiveness of the disease. Collectively, these findings provide in vivo evidence that the BCR pathway drives the development and can influence the clinical course of CLL.

NrasG12D oncoprotein inhibits apoptosis of preleukemic cells expressing Cbfß-SMMHC via activation of MEK/ERK axis.

Acute myeloid leukemia (AML) results from the activity of driver mutations that deregulate proliferation and survival of hematopoietic stem cells (HSCs). The fusion protein CBFß-SMMHC impairs differentiation in hematopoietic stem and progenitor cells and induces AML in cooperation with other mutations. However, the combined function of CBFß-SMMHC and cooperating mutations in preleukemic expansion is not known. Here, we used Nras(LSL-G12D); Cbfb(56M) knock-in mice to show that allelic expression of oncogenic Nras(G12D) and Cbfß-SMMHC increases survival of preleukemic short-term HSCs and myeloid progenitor cells and maintains the differentiation block induced by the fusion protein. Nras(G12D) and Cbfß-SMMHC synergize to induce leukemia in mice in a cell-autonomous manner, with a shorter median latency and higher leukemia-initiating cell activity than that of mice expressing Cbfß-SMMHC. Furthermore, Nras(LSL-G12D); Cbfb(56M) leukemic cells were sensitive to pharmacologic inhibition of the MEK/ERK signaling pathway, increasing apoptosis and Bim protein levels. These studies demonstrate that Cbfß-SMMHC and Nras(G12D) promote the survival of preleukemic myeloid progenitors primed for leukemia by activation of the MEK/ERK/Bim axis, and define Nras(LSL-G12D); Cbfb(56M) mice as a valuable genetic model for the study of inversion(16) AML-targeted therapies.

Identification of renin progenitors in the mouse bone marrow that give rise to B-cell leukaemia.

The cell of origin and triggering events for leukaemia are mostly unknown. Here we show that the bone marrow contains a progenitor that expresses renin throughout development and possesses a B-lymphocyte pedigree. This cell requires RBP-J to differentiate. Deletion of RBP-J in these renin-expressing progenitors enriches the precursor B-cell gene programme and constrains lymphocyte differentiation, facilitated by H3K4me3 activating marks in genes that control the pre-B stage. Mutant cells undergo neoplastic transformation, and mice develop a highly penetrant B-cell leukaemia with multi-organ infiltration and early death. These renin-expressing cells appear uniquely vulnerable as other conditional models of RBP-J deletion do not result in leukaemia. The discovery of these unique renin progenitors in the bone marrow and the model of leukaemia described herein may enhance our understanding of normal and neoplastic haematopoiesis.

A CALM-derived nuclear export signal is essential for CALM-AF10-mediated leukemogenesis.

The t(10;11) chromosomal translocation gives rise to the CALM-AF10 fusion gene and is found in patients with aggressive and difficult-to-treat hematopoietic malignancies. CALM-AF10-driven leukemias are characterized by HOXA gene up-regulation and a global reduction in H3K79 methylation. DOT1L, the H3K79 methyltransferase, interacts with the octapeptide/leucine zipper domain of AF10, and this region has been shown to be necessary and sufficient for CALM-AF10-mediated transformation. However, the precise role of CALM in leukemogenesis remains unclear. Here, we show that CALM contains a nuclear export signal (NES) that mediates cytoplasmic localization of CALM-AF10 and is necessary for CALM-AF10-dependent transformation. Fusions of the CALM NES (NES(CALM)-AF10) or NES motifs from heterologous proteins (ABL1, Rev, PKIA, APC) in-frame with AF10 are sufficient to immortalize murine hematopoietic progenitors in vitro. The CALM NES is essential for CALM-AF10-dependent Hoxa gene up-regulation and aberrant H3K79 methylation, possibly by mislocalization of DOT1L. Finally, we observed that CALM-AF10 leukemia cells are selectively sensitive to inhibition of nuclear export by Leptomycin B. These findings uncover a novel mechanism of leukemogenesis mediated by the nuclear export pathway and support further investigation of the utility of nuclear export inhibitors as therapeutic agents for patients with CALM-AF10 leukemias.

Polycomb Cbx family members mediate the balance between haematopoietic stem cell self-renewal and differentiation.

The balance between self-renewal and differentiation of adult stem cells is essential for tissue homeostasis. Here we show that in the haematopoietic system this process is governed by polycomb chromobox (Cbx) proteins. Cbx7 is specifically expressed in haematopoietic stem cells (HSCs), and its overexpression enhances self-renewal and induces leukaemia. This effect is dependent on integration into polycomb repressive complex-1 (PRC1) and requires H3K27me3 binding. In contrast, overexpression of Cbx2, Cbx4 or Cbx8 results in differentiation and exhaustion of HSCs. ChIP-sequencing analysis shows that Cbx7 and Cbx8 share most of their targets; we identified approximately 200 differential targets. Whereas genes targeted by Cbx8 are highly expressed in HSCs and become repressed in progenitors, Cbx7 targets show the opposite expression pattern. Thus, Cbx7 preserves HSC self-renewal by repressing progenitor-specific genes. Taken together, the presence of distinct Cbx proteins confers target selectivity to PRC1 and provides a molecular balance between self-renewal and differentiation of HSCs.

Promoting regulation via the inhibition of DNAM-1 after transplantation.

Donor T cells play pivotal roles in graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL) effects following bone marrow transplantation (BMT). DNAX accessory molecule 1 (DNAM-1) is a costimulatory and adhesion molecule, expressed mainly by natural killer cells and CD8(+) T cells at steady state to promote adhesion to ligand-expressing targets and enhance cytolysis. We have analyzed the role of this pathway in GVHD and GVL. The absence of DNAM-1 on the donor graft attenuated GVHD in major histocompatibility complex (MHC)-mismatched and MHC-matched BMT following conditioning with lethal and sublethal irradiation. In contrast, DNAM-1 was not critical for GVL effects against ligand (CD155) expressing and nonexpressing leukemia. The effects on GVHD following myeloablative conditioning were independent of CD8(+) T cells and dependent on CD4(+) T cells, and specifically donor FoxP3(+) regulatory T cells (Treg). The absence of DNAM-1 promoted the expansion and suppressive function of Treg after BMT. These findings provide support for therapeutic DNAM-1 inhibition to promote tolerance in relevant inflammatory-based diseases characterized by T-cell activation.

Depletion of cytotoxic T-cells does not protect NUP98-HOXD13 mice from myelodysplastic syndrome but reveals a modest tumor immunosurveillance effect.

Myelodysplastic syndrome (MDS) and aplastic anemia (AA) patients both present with symptoms of bone marrow failure. In many AA patients, these features are thought to result from an oligoclonal expansion of cytotoxic T-cells that destroy haematopoietic stem or progenitor cells. This notion is supported by the observation that AA patients respond to immunosuppressive therapy. A fraction of MDS patients also respond well to immunosuppressive therapy suggesting a similar role for cytotoxic T-cells in the etiology of MDS, however the role of cytotoxic T-cells in MDS remains unclear. Mice that express a NUP98-HOXD13 (NHD13) transgene develop a MDS that closely mimics the human condition in terms of dysplasia, ineffective hematopoiesis, and transformation to acute myeloid leukemia (AML). We followed a cohort of NHD13 mice lacking the Rag1 protein (NHD13/Rag1KO) to determine if the absence of lymphocytes might 1) delay the onset and/or diminish the severity of the MDS, or 2) effect malignant transformation and survival of the NHD13 mice. No difference was seen in the onset or severity of MDS between the NHD13 and NHD13/Rag1KO mice. However, NHD13/Rag1KO mice had decreased survival and showed a trend toward increased incidence of transformation to AML compared to the NHD13 mice, suggesting protection from AML transformation by a modest immuno-surveillance effect. In the absence of functional Tcrb signaling in the NHD13/Rag1KO T-cell tumors, Pak7 was identified as a potential Tcrb surrogate survival signal.

Retrovirus-transformed erythroleukemia cells induce central nervous system failure in a new syngeneic mouse model of meningeal leukemia.

Lack of suitable mouse models for central nervous system (CNS)-associated leukemias has hindered mechanism-guided development of therapeutics. By transplanting retrovirus-transformed mouse erythroleukemia cells into syngeneic mice, we developed a new animal model of meningeal leukemia associated with rapid paralysis. Necropsy revealed massive proliferation of the leukemic cells in the bone marrow (BM) followed by pathological angiogenesis and invasion of the leukemic cells into the meninges of the CNS. Further analysis demonstrated that the erythroleukemia cells secreted high levels of VEGF and preferentially adhered in vitro to fibronectin. This unique animal model for meningeal leukemia should facilitate studies of engraftment and proliferation of leukemic cells in the BM and their invasion of the CNS as well as pre-clinical evaluation of experimental therapeutics for CNS-associated leukemias.

Requirement for Dot1l in murine postnatal hematopoiesis and leukemogenesis by MLL translocation.

Disruptor of telomeric silencing 1-like (Dot1l) is a histone 3 lysine 79 methyltransferase. Studies of constitutive Dot1l knockout mice show that Dot1l is essential for embryonic development and prenatal hematopoiesis. DOT1L also interacts with translocation partners of Mixed Lineage Leukemia (MLL) gene, which is commonly translocated in human leukemia. However, the requirement of Dot1l in postnatal hematopoiesis and leukemogenesis of MLL translocation proteins has not been conclusively shown. With a conditional Dot1l knockout mouse model, we examined the consequences of Dot1l loss in postnatal hematopoiesis and MLL translocation leukemia. Deletion of Dot1l led to pancytopenia and failure of hematopoietic homeostasis, and Dot1l-deficient cells minimally reconstituted recipient bone marrow in competitive transplantation experiments. In addition, MLL-AF9 cells required Dot1l for oncogenic transformation, whereas cells with other leukemic oncogenes, such as Hoxa9/Meis1 and E2A-HLF, did not. These findings illustrate a crucial role of Dot1l in normal hematopoiesis and leukemogenesis of specific oncogenes.

Mutant nucleophosmin and cooperating pathways drive leukemia initiation and progression in mice.

Acute myeloid leukemia (AML) is a molecularly diverse malignancy with a poor prognosis whose largest subgroup is characterized by somatic mutations in NPM1, which encodes nucleophosmin. These mutations, termed NPM1c, result in cytoplasmic dislocation of nucleophosmin and are associated with distinctive transcriptional signatures, yet their role in leukemogenesis remains obscure. Here we report that activation of a humanized Npm1c knock-in allele in mouse hemopoietic stem cells causes Hox gene overexpression, enhanced self renewal and expanded myelopoiesis. One third of mice developed delayed-onset AML, suggesting a requirement for cooperating mutations. We identified such mutations using a Sleeping Beauty transposon, which caused rapid-onset AML in 80% of mice with Npm1c, associated with mutually exclusive integrations in Csf2, Flt3 or Rasgrp1 in 55 of 70 leukemias. We also identified recurrent integrations in known and newly discovered leukemia genes including Nf1, Bach2, Dleu2 and Nup98. Our results provide new pathogenetic insights and identify possible therapeutic targets in NPM1c+ AML.