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Examines the unique properties of breast milk as a mediator of biochemical messages. Substances influencing the growth and development of the infant; Influence of milk nutrients on gastrointestinal development; Significance of breast feeding in the prevention of neonatal gastrointestinal diseases.

Novel chimeric antigen receptor (CAR) T-cell approaches are needed to improve therapeutic efficacy in solid tumors. High-risk neuroblastoma is an aggressive pediatric solid tumor that expresses cell-surface GPC2 and GD2 with a tumor microenvironment infiltrated by CD16a-expressing innate immune cells. Here we engineer T-cells to express a GPC2-directed CAR and simultaneously secrete a bispecific innate immune cell engager (BiCE) targeting both GD2 and CD16a. In vitro, GPC2.CAR-GD2.BiCE T-cells induce GPC2-dependent cytotoxicity and secrete GD2.BiCE that promotes GD2-dependent activation of antitumor innate immunity. In vivo, GPC2.CAR-GD2.BiCE T-cells locally deliver GD2.BiCE and increase intratumor retention of NK-cells. In mice bearing neuroblastoma patient-derived xenografts and reconstituted with human CD16a-expressing immune cells, GD2.BiCEs enhance GPC2.CAR antitumor efficacy. A CAR.BiCE strategy should be considered for tumor histologies where antigen escape limits CAR efficacy, especially for solid tumors like neuroblastoma that are infiltrated by innate immune cells., (© 2024. The Author(s).)

T-lineage acute lymphoblastic leukaemia (T-ALL) is a high-risk tumour 1 that has eluded comprehensive genomic characterization, which is partly due to the high frequency of noncoding genomic alterations that result in oncogene deregulation 2,3 . Here we report an integrated analysis of genome and transcriptome sequencing of tumour and remission samples from more than 1,300 uniformly treated children with T-ALL, coupled with epigenomic and single-cell analyses of malignant and normal T cell precursors. This approach identified 15 subtypes with distinct genomic drivers, gene expression patterns, developmental states and outcomes. Analyses of chromatin topology revealed multiple mechanisms of enhancer deregulation that involve enhancers and genes in a subtype-specific manner, thereby demonstrating widespread involvement of the noncoding genome. We show that the immunophenotypically described, high-risk entity of early T cell precursor ALL is superseded by a broader category of 'early T cell precursor-like' leukaemia. This category has a variable immunophenotype and diverse genomic alterations of a core set of genes that encode regulators of hematopoietic stem cell development. Using multivariable outcome models, we show that genetic subtypes, driver and concomitant genetic alterations independently predict treatment failure and survival. These findings provide a roadmap for the classification, risk stratification and mechanistic understanding of this disease., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Relapse rates in high-risk neuroblastoma remain exceedingly high. The malignant cells that are responsible for relapse have not been identified, and mechanisms of therapy resistance remain poorly understood. Here, we used single nucleus RNA sequencing and bulk whole genome sequencing to identify and characterize the residual malignant persister cells that survive chemotherapy from a cohort of 20 matched diagnosis and definitive surgery tumor samples from patients treated with high-risk neuroblastoma induction chemotherapy. We show that persister cells share common mechanisms of chemotherapy escape including suppression of MYCN activity and activation of NF-κB signaling, the latter is further enhanced by cell-cell communication between the malignant cells and the tumor microenvironment. Overall, our work dissects the transcriptional landscape of cellular persistence in high-risk neuroblastoma and paves the way to the development of new therapeutic strategies to prevent disease relapse.

Beckwith-Wiedemann Syndrome (BWS) is an epigenetic overgrowth syndrome caused by methylation changes in the human 11p15 chromosomal locus. Patients with BWS exhibit tissue overgrowth, as well as an increased risk of childhood neoplasms in the liver and kidney. To understand the impact of these 11p15 changes, specifically in the liver, we performed single-nucleus RNA sequencing (snRNA-seq) and single-nucleus assay for transposase-accessible chromatin with sequencing (snATAC-seq) to generate paired, cell-type-specific transcriptional and chromatin accessibility profiles of both BWS-liver and nonBWS-liver nontumorous tissue. Our integrated RNA+ATACseq multiomic approach uncovered hepatocyte-specific enrichment and activation of the peroxisome proliferator-activated receptor α (PPARA) - a liver metabolic regulator. To confirm our findings, we utilized a BWS-induced pluripotent stem cell (iPSC) model, where cells were differentiated into hepatocytes. Our data demonstrates the dysregulation of lipid metabolism in BWS-liver, which coincided with observed upregulation of PPARA during hepatocyte differentiation. BWS liver cells exhibited decreased neutral lipids and increased fatty acid β-oxidation, relative to controls. We also observed increased reactive oxygen species (ROS) byproducts in the form of peroxidated lipids in BWS hepatocytes, which coincided with increased oxidative DNA damage. This study proposes a putative mechanism for overgrowth and cancer predisposition in BWS liver due to perturbed metabolism.

Internal tandem duplication mutations in fms-like tyrosine kinase 3 (FLT3-ITD) are recurrent in acute myeloid leukemia (AML) and increase the risk of relapse. Clinical responses to FLT3 inhibitors (FLT3i) include myeloid differentiation of the FLT3-ITD clone in nearly half of patients through an unknown mechanism. We identified enhancer of zeste homolog 2 (EZH2), a component of polycomb repressive complex 2 (PRC2), as a mediator of this effect using a proteomic-based screen. FLT3i downregulated EZH2 protein expression and PRC2 activity on H3K27me3. FLT3-ITD and loss-of-function mutations in EZH2 are mutually exclusive in human AML. We demonstrated that FLT3i increase myeloid maturation with reduced stem/progenitor cell populations in murine Flt3-ITD AML. Combining EZH1/2 inhibitors with FLT3i increased terminal maturation of leukemic cells and reduced leukemic burden. Our data suggest that reduced EZH2 activity following FLT3 inhibition promotes myeloid differentiation of FLT3-ITD leukemic cells, providing a mechanistic explanation for the clinical observations. These results demonstrate that in addition to its known cell survival and proliferation signaling, FLT3-ITD has a second, previously undefined function to maintain a myeloid stem/progenitor cell state through modulation of PRC2 activity. Our findings support exploring EZH1/2 inhibitors as therapy for FLT3-ITD AML., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Histone modifications regulate chromatin remodeling and gene expression in development and diseases. DOT1L, the sole histone H3K79 methyltransferase, is essential for embryonic development. Here, we report that DOT1L regulates male fertility in mouse. DOT1L associates with MLLT10 in testis. DOT1L and MLLT10 localize to the sex chromatin in meiotic and post-meiotic germ cells in an inter-dependent manner. Loss of either DOT1L or MLLT10 leads to reduced testis weight, decreased sperm count and male subfertility. H3K79me2 is abundant in elongating spermatids, which undergo the dramatic histone-to-protamine transition. Both DOT1L and MLLT10 are essential for H3K79me2 modification in germ cells. Strikingly, histones are substantially retained in epididymal sperm from either DOT1L- or MLLT10-deficient mice. These results demonstrate that H3K79 methylation promotes histone replacement during spermiogenesis., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

The chromatin reader eleven-nineteen leukemia (ENL) has been identified as a critical dependency in acute myeloid leukemia (AML), but its therapeutic potential remains unclear. We describe a potent and orally bioavailable small-molecule inhibitor of ENL, TDI-11055, which displaces ENL from chromatin by blocking its YEATS domain interaction with acylated histones. Cell lines and primary patient samples carrying MLL rearrangements or NPM1 mutations are responsive to TDI-11055. A CRISPR-Cas9-mediated mutagenesis screen uncovers an ENL mutation that confers resistance to TDI-11055, validating the compound's on-target activity. TDI-11055 treatment rapidly decreases chromatin occupancy of ENL-associated complexes and impairs transcription elongation, leading to suppression of key oncogenic gene expression programs and induction of differentiation. In vivo treatment with TDI-11055 blocks disease progression in cell line- and patient-derived xenograft models of MLL-rearranged and NPM1-mutated AML. Our results establish ENL displacement from chromatin as a promising epigenetic therapy for molecularly defined AML subsets and support the clinical translation of this approach., Significance: AML is a poor-prognosis disease for which new therapeutic approaches are desperately needed. We developed an orally bioavailable inhibitor of ENL, demonstrated its potent efficacy in MLL-rearranged and NPM1-mutated AML, and determined its mechanisms of action. These biological and chemical insights will facilitate both basic research and clinical translation. This article is highlighted in the In This Issue feature, p. 2483., (©2022 The Authors; Published by the American Association for Cancer Research.)

Transcriptional dysregulation is a prominent feature in leukemia. Here, we systematically surveyed transcription factor (TF) vulnerabilities in leukemia and uncovered TF clusters that exhibit context-specific vulnerabilities within and between different subtypes of leukemia. Among these TF clusters, we demonstrated that acute myeloid leukemia (AML) with high IRF8 expression was addicted to MEF2D . MEF2D and IRF8 form an autoregulatory loop via direct binding to mutual enhancer elements. One important function of this circuit in AML is to sustain PU.1/MEIS1 co-regulated transcriptional outputs via stabilizing PU.1's chromatin occupancy. We illustrated that AML could acquire dependency on this circuit through various oncogenic mechanisms that results in the activation of their enhancers. In addition to forming a circuit, MEF2D and IRF8 can also separately regulate gene expression, and dual perturbation of these two TFs leads to a more robust inhibition of AML proliferation. Collectively, our results revealed a TF circuit essential for AML survival., Competing Interests: The authors declare no conflict of interest., (© 2022 The Author(s).)

Angiosarcoma is a rare, devastating malignancy with few curative options for disseminated disease. We analyzed a recently published genomic data set of 48 angiosarcomas and noticed recurrent amplifications of HOXA -cluster genes in 33% of patients. HOXA genes are master regulators of embryonic vascular development and adult neovascularization, which provides a molecular rationale to suspect that amplified HOXA genes act as oncogenes in angiosarcoma. HOXA amplifications typically affected multiple pro-angiogenic HOXA genes and co-occurred with amplifications of CD36 and KDR, whereas the overall mutation rate in these tumors was relatively low. HOXA amplifications were found most commonly in angiosarcomas located in the breast and were rare in angiosarcomas arising in sun-exposed areas on the head, neck, face and scalp. Our data suggest that HOXA -amplified angiosarcoma is a distinct molecular subgroup. Efforts to develop therapies targeting oncogenic HOX gene expression in AML and other sarcomas may have relevance for HOXA -amplified angiosarcoma.

Self-renewal of spermatogonial stem cells is vital to lifelong production of male gametes and thus fertility. However, the underlying mechanisms remain enigmatic. Here, we show that DOT1L, the sole H3K79 methyltransferase, is required for spermatogonial stem cell self-renewal. Mice lacking DOT1L fail to maintain spermatogonial stem cells, characterized by a sequential loss of germ cells from spermatogonia to spermatids and ultimately a Sertoli cell only syndrome. Inhibition of DOT1L reduces the stem cell activity after transplantation. DOT1L promotes expression of the fate-determining HoxC transcription factors in spermatogonial stem cells. Furthermore, H3K79me2 accumulates at HoxC9 and HoxC10 genes. Our findings identify an essential function for DOT1L in adult stem cells and provide an epigenetic paradigm for regulation of spermatogonial stem cells., (© 2022 Lin et al.; Published by Cold Spring Harbor Laboratory Press.)

KMT2A-rearranged (KMT2A-r) infant acute lymphoblastic leukemia (ALL) is a devastating malignancy with a dismal outcome, and younger age at diagnosis is associated with increased risk of relapse. To discover age-specific differences and critical drivers that mediate poor outcome in KMT2A-r ALL, we subjected KMT2A-r leukemias and normal hematopoietic cells from patients of different ages to single-cell multiomics analyses. We uncovered the following critical new insights: leukemia cells from patients <6 months have significantly increased lineage plasticity. Steroid response pathways are downregulated in the most immature blasts from younger patients. We identify a hematopoietic stem and progenitor-like (HSPC-like) population in the blood of younger patients that contains leukemic blasts and form an immunosuppressive signaling circuit with cytotoxic lymphocytes. These observations offer a compelling explanation for the ability of leukemias in young patients to evade chemotherapy and immune-mediated control. Our analysis also revealed preexisting lymphomyeloid primed progenitors and myeloid blasts at initial diagnosis of B-ALL. Tracking of leukemic clones in 2 patients whose leukemia underwent a lineage switch documented the evolution of such clones into frank acute myeloid leukemia (AML). These findings provide critical insights into KMT2A-r ALL and have clinical implications for molecularly targeted and immunotherapy approaches. Beyond infant ALL, our study demonstrates the power of single-cell multiomics to detect tumor intrinsic and extrinsic factors affecting rare but critical subpopulations within a malignant population that ultimately determines patient outcome., (© 2022 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

With the rapid developments in mass spectrometry (MS)-based proteomics methods, label-free semiquantitative proteomics has become an increasingly popular tool for profiling global protein abundances in an unbiased manner. However, the reproducibility of these data across time and LC-MS platforms is not well characterized. Here, we evaluate the performance of three LC-MS platforms (Orbitrap Elite, Q Exactive HF, and Orbitrap Fusion) in label-free semiquantitative analysis of cell surface proteins over a six-year period. Sucrose gradient ultracentrifugation was used for surfaceome enrichment, following gel separation for in-depth protein identification. With our established workflow, we consistently detected and reproducibly quantified >2300 putative cell surface proteins in a human acute myeloid leukemia (AML) cell line on all three platforms. To our knowledge this is the first study reporting highly reproducible semiquantitative proteomic data collection of biological replicates across multiple years and LC-MS platforms. These data provide experimental justification for semiquantitative proteomic study designs that are executed over multiyear time intervals and on different platforms. Multiyear and multiplatform experimental designs will likely enable larger scale proteomic studies and facilitate longitudinal proteomic studies by investigators lacking access to high throughput MS facilities. Data are available via ProteomeXchange with identifier PXD022721.

The transformed state in acute leukemia requires gene regulatory programs involving transcription factors and chromatin modulators. Here, we uncover an IRF8-MEF2D transcriptional circuit as an acute myeloid leukemia (AML)-biased dependency. We discover and characterize the mechanism by which the chromatin "reader" ZMYND8 directly activates IRF8 in parallel with the MYC proto-oncogene through their lineage-specific enhancers. ZMYND8 is essential for AML proliferation in vitro and in vivo and associates with MYC and IRF8 enhancer elements that we define in cell lines and in patient samples. ZMYND8 occupancy at IRF8 and MYC enhancers requires BRD4, a transcription coactivator also necessary for AML proliferation. We show that ZMYND8 binds to the ET domain of BRD4 via its chromatin reader cassette, which in turn is required for proper chromatin occupancy and maintenance of leukemic growth in vivo. Our results rationalize ZMYND8 as a potential therapeutic target for modulating essential transcriptional programs in AML., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Meningioma-1 (MN1) overexpression in AML is associated with poor prognosis, and forced expression of MN1 induces leukemia in mice. We sought to determine how MN1 causes AML. We found that overexpression of MN1 can be induced by translocations that result in hijacking of a downstream enhancer. Structure predictions revealed that the entire MN1 coding frame is disordered. We identified the myeloid progenitor-specific BAF complex as the key interaction partner of MN1. MN1 over-stabilizes BAF on enhancer chromatin, a function directly linked to the presence of a long polyQ-stretch within MN1. BAF over-stabilization at binding sites of transcription factors regulating a hematopoietic stem/progenitor program prevents the developmentally appropriate decommissioning of these enhancers and results in impaired myeloid differentiation and leukemia. Beyond AML, our data detail how the overexpression of a polyQ protein, in the absence of any coding sequence mutation, can be sufficient to cause malignant transformation., Competing Interests: Declaration of interests K.M.B. holds a patent on the use of DOT1L inhibitors for MN1 leukemia. K.M.B. has received research funding from Syndax and has previously consulted for Agios. I.H. received research funding from Bristol-Myers Squibb (BMS), Celgene, Merck Sharpe & Dohme (MSD), Astellas, Otsuka, Ono, Kyowa Kirin, Sanofi, Shionogi, Zenyaku, Daiichi Sankyo, Taiho, Takeda, Chugai, Eli Lilly, Nihon Shinyaku, Novartis, Pfizer, Fujimoto, Tanabe-Mitsubishi, Fukuyu Hospital, and Yamada Yohojo and received honoraria from or holds membership on an entity’s board of directors, speaker’s bureau, or its advisory committees for Celgene, Janssen, Takeda, Ono, BMS, Novartis, Daiichi Sankyo, Kyowa Kirin, Eisai, Nihon-Shinyaku, Pfizer, AbbVie, Otsuka, Shionogi, Mundi, CSL, and MSD. All other authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Translocations of Meningioma-1 (MN1) occur in a subset of acute myeloid leukemias (AML) and result in high expression of MN1, either as a full-length protein, or as a fusion protein that includes most of the N-terminus of MN1. High levels of MN1 correlate with poor prognosis. When overexpressed in murine hematopoietic progenitors, MN1 causes an aggressive AML characterized by an aberrant myeloid precursor-like gene expression program that shares features of KMT2A-rearranged (KMT2A-r) leukemia, including high levels of Hoxa and Meis1 gene expression. Compounds that target a critical KMT2A-Menin interaction have proven effective in KMT2A-r leukemia. Here, we demonstrate that Menin (Men1) is also critical for the self-renewal of MN1-driven AML through the maintenance of a distinct gene expression program. Genetic inactivation of Men1 led to a decrease in the number of functional leukemia-initiating cells. Pharmacologic inhibition of the KMT2A-Menin interaction decreased colony-forming activity, induced differentiation programs in MN1-driven murine leukemia and decreased leukemic burden in a human AML xenograft carrying an MN1-ETV6 translocation. Collectively, these results nominate Menin inhibition as a promising therapeutic strategy in MN1-driven leukemia.

Chromatin modifying enzymes play essential roles in skeletal development and bone maintenance, and deregulation of epigenetic mechanisms can lead to skeletal growth and malformation disorders. Here, we report a novel skeletal dysplasia phenotype in mice with conditional loss of Disruptor of telomeric silencing 1-like (Dot1L) histone methyltransferase in limb mesenchymal progenitors and downstream descendants. Phenotypic characterizations of mice with Dot1L inactivation by Prrx1-Cre (Dot1L-cKO Prrx1 ) revealed limb shortening, abnormal bone morphologies, and forelimb dislocations. Our in vivo and in vitro data support a crucial role for Dot1L in regulating growth plate chondrocyte proliferation and differentiation, extracellular matrix production, and secondary ossification center formation. Micro-computed tomography analysis of femurs revealed that partial loss of Dot1L expression is sufficient to impair trabecular bone formation and microarchitecture in young mice. Moreover, RNAseq analysis of Dot1L deficient chondrocytes implicated Dot1L in the regulation of key genes and pathways necessary to promote cell cycle regulation and skeletal growth. Collectively, our data show that early expression of Dot1L in limb mesenchyme provides essential regulatory control of endochondral bone morphology, growth, and stability., (Copyright © 2020 Elsevier Inc. All rights reserved.)

BACKGROUNDInitial reports from the severe acute respiratory coronavirus 2 (SARS-CoV-2) pandemic described children as being less susceptible to coronavirus disease 2019 (COVID-19) than adults. Subsequently, a severe and novel pediatric disorder termed multisystem inflammatory syndrome in children (MIS-C) emerged. We report on unique hematologic and immunologic parameters that distinguish between COVID-19 and MIS-C and provide insight into pathophysiology.METHODSWe prospectively enrolled hospitalized patients with evidence of SARS-CoV-2 infection and classified them as having MIS-C or COVID-19. Patients with COVID-19 were classified as having either minimal or severe disease. Cytokine profiles, viral cycle thresholds (Cts), blood smears, and soluble C5b-9 values were analyzed with clinical data.RESULTSTwenty patients were enrolled (9 severe COVID-19, 5 minimal COVID-19, and 6 MIS-C). Five cytokines (IFN-γ, IL-10, IL-6, IL-8, and TNF-α) contributed to the analysis. TNF-α and IL-10 discriminated between patients with MIS-C and severe COVID-19. The presence of burr cells on blood smears, as well as Cts, differentiated between patients with severe COVID-19 and those with MIS-C.CONCLUSIONPediatric patients with SARS-CoV-2 are at risk for critical illness with severe COVID-19 and MIS-C. Cytokine profiling and examination of peripheral blood smears may distinguish between patients with MIS-C and those with severe COVID-19.FUNDINGFinancial support for this project was provided by CHOP Frontiers Program Immune Dysregulation Team; National Institute of Allergy and Infectious Diseases; National Cancer Institute; the Leukemia and Lymphoma Society; Cookies for Kids Cancer; Alex's Lemonade Stand Foundation for Childhood Cancer; Children's Oncology Group; Stand UP 2 Cancer; Team Connor; the Kate Amato Foundations; Burroughs Wellcome Fund CAMS; the Clinical Immunology Society; the American Academy of Allergy, Asthma, and Immunology; and the Institute for Translational Medicine and Therapeutics.

Understanding mechanisms of cooperation between oncogenes is critical for the development of novel therapies and rational combinations. Acute myeloid leukemia (AML) cells with KMT2A-fusions and KMT2A partial tandem duplications (KMT2APTD) are known to depend on the histone methyltransferase DOT1L, which methylates histone 3 lysine 79 (H3K79). About 30% of KMT2APTD AMLs carry mutations in IDH1/2 (mIDH1/2). Previous studies showed that 2-hydroxyglutarate produced by mIDH1/2 increases H3K79 methylation, and mIDH1/2 patient samples are sensitive to DOT1L inhibition. Together, these findings suggested that stabilization or increases in H3K79 methylation associated with IDH mutations support the proliferation of leukemias dependent on this mark. However, we found that mIDH1/2 and KMT2A alterations failed to cooperate in an experimental model. Instead, mIDH1/2 and 2-hydroxyglutarate exert toxic effects, specifically on KMT2A-rearranged AML cells (fusions/partial tandem duplications). Mechanistically, we uncover an epigenetic barrier to efficient cooperation; mIDH1/2 expression is associated with high global histone 3 lysine 79 dimethylation (H3K79me2) levels, whereas global H3K79me2 is obligate low in KMT2A-rearranged AML. Increasing H3K79me2 levels, specifically in KMT2A-rearrangement leukemias, resulted in transcriptional downregulation of KMT2A target genes and impaired leukemia cell growth. Our study details a complex genetic and epigenetic interaction of 2 classes of oncogenes, IDH1/2 mutations and KMT2A rearrangements, that is unexpected based on the high percentage of IDH mutations in KMT2APTD AML. KMT2A rearrangements are associated with a trend toward lower response rates to mIDH1/2 inhibitors. The substantial adaptation that has to occur for 2 initially counteracting mutations to be tolerated within the same leukemic cell may provide at least a partial explanation for this observation., (© 2020 by The American Society of Hematology.)

Inhibition of the H3K79 histone methyltransferase DOT1L has exhibited encouraging preclinical and early clinical activity in KMT2A (MLL)-rearranged leukemia, supporting the development of combinatorial therapies. Here, we investigated two novel combinations: dual inhibition of the histone methyltransferases DOT1L and EZH2, and the combination with a protein synthesis inhibitor. EZH2 is the catalytic subunit in the polycomb repressive complex 2 (PRC2), and inhibition of EZH2 has been reported to have preclinical activity in KMT2A-r leukemia. When combined with DOT1L inhibition, however, we observed both synergistic and antagonistic effects. Interestingly, antagonistic effects were not due to PRC2-mediated de-repression of HOXA9. HOXA cluster genes are key canonical targets of both KMT2A and the PRC2 complex. The independence of the HOXA cluster from PRC2 repression in KMT2A-r leukemia thus affords important insights into leukemia biology. Further studies revealed that EZH2 inhibition counteracted the effect of DOT1L inhibition on ribosomal gene expression. We thus identified a previously unrecognized role of DOT1L in regulating protein production. Decreased translation was one of the earliest effects measurable after DOT1L inhibition and specific to KMT2A-rearranged cell lines. H3K79me2 chromatin immunoprecipitation sequencing patterns over ribosomal genes were similar to those of the canonical KMT2A-fusion target genes in primary AML patient samples. The effects of DOT1L inhibition on ribosomal gene expression prompted us to evaluate the combination of EPZ5676 with a protein translation inhibitor. EPZ5676 was synergistic with the protein translation inhibitor homoharringtonine (omacetaxine), supporting further preclinical/clinical development of this combination. In summary, we discovered a novel epigenetic regulation of a metabolic process-protein synthesis-that plays a role in leukemogenesis and affords a combinatorial therapeutic opportunity., (Copyright © 2020 ISEH -- Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

More than 30% of genes in higher eukaryotes are regulated by promoter-proximal pausing of RNA polymerase II (Pol II). Phosphorylation of Pol II CTD by positive transcription elongation factor b (P-TEFb) is a necessary precursor event that enables productive transcription elongation. The exact mechanism on how the sequestered P-TEFb is released from the 7SK snRNP complex and recruited to Pol II CTD remains unknown. In this report, we utilize mouse and human models to reveal methylphosphate capping enzyme (MePCE), a core component of the 7SK snRNP complex, as the cognate substrate for Jumonji domain-containing 6 (JMJD6)'s novel proteolytic function. Our evidences consist of a crystal structure of JMJD6 bound to methyl-arginine, enzymatic assays of JMJD6 cleaving MePCE in vivo and in vitro, binding assays, and downstream effects of Jmjd6 knockout and overexpression on Pol II CTD phosphorylation. We propose that JMJD6 assists bromodomain containing 4 (BRD4) to recruit P-TEFb to Pol II CTD by disrupting the 7SK snRNP complex., Competing Interests: SL, HL, RH, CC, XH, FC, MK, QZ, XL, ZC, AL, KB, PM, JK, QZ, CL, YX, KH No competing interests declared, GZ has shares in NB Life Laboratory LLC, Colorado, (© 2020, Lee et al.)

Genetic lesions affecting polycomb repressive complex 2 (PRC2) have been found in more than 40% of pediatric cases of early T-cell precursor acute lymphoblastic leukemia. The functional role of these PRC2 alterations has been obscure. Our recent data suggest that compromise of PRC2 blocks differentiation and accentuates growth and survival signaling.

DOT1L is a protein methyltransferase involved in the development and maintenance of MLL -rearranged ( MLL -r) leukemia through its ectopic methylation of histones associated with well-characterized leukemic genes. Pinometostat (EPZ-5676), a selective inhibitor of DOT1L, is in clinical development in relapsed/refractory acute leukemia patients harboring rearrangements of the MLL gene. The observation of responses and subsequent relapses in the adult trial treating MLL -r patients motivated preclinical investigations into potential mechanisms of pinometostat treatment-emergent resistance (TER) in cell lines confirmed to have MLL -r. TER was achieved in five MLL -r cell lines, KOPN-8, MOLM-13, MV4-11, NOMO-1, and SEM. Two of the cell lines, KOPN-8 and NOMO-1, were thoroughly characterized to understand the mechanisms involved in pinometostat resistance. Unlike many other targeted therapies, resistance does not appear to be achieved through drug-induced selection of mutations of the target itself. Instead, we identified both drug efflux transporter dependent and independent mechanisms of resistance to pinometostat. In KOPN-8 TER cells, increased expression of the drug efflux transporter ABCB1 (P-glycoprotein, MDR1) was the primary mechanism of drug resistance. In contrast, resistance in NOMO-1 cells occurs through a mechanism other than upregulation of a specific efflux pump. RNA-seq analysis performed on both parental and resistant KOPN-8 and NOMO-1 cell lines supported two unique candidate pathway mechanisms that may explain the pinometostat resistance observed in these cell lines. These results are the first demonstration of TER models of the DOT1L inhibitor pinometostat and may provide useful tools for investigating clinical resistance. Mol Cancer Ther; 16(8); 1669-79. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Myeloid malignancies exist on a spectrum from asymptomatic clonal hematopoiesis to overt leukemia and exhibit substantial clonal heterogeneity. Both aspects are challenging to capture in experimental models. In two landmark studies in this issue of Cell Stem Cell, Kotini et al. (2017) and Chao et al. (2017) establish iPSC-based experimental platforms that recapitulate disease stages and clonal architecture., (Copyright © 2017. Published by Elsevier Inc.)

The mixed-lineage leukemia 1 (MLL1) gene (now renamed Lysine [K]-specific MethylTransferase 2A or KMT2A ) on chromosome 11q23 is disrupted in a unique group of acute leukemias. More than 80 different partner genes in these fusions have been described, although the majority of leukemias result from MLL1 fusions with one of about six common partner genes. Approximately 10% of all leukemias harbor MLL1 translocations. Of these, two patient populations comprise the majority of cases: patients younger than 1 year of age at diagnosis (primarily acute lymphoblastic leukemias) and young- to-middle-aged adults (primarily acute myeloid leukemias). A much rarer subgroup of patients with MLL1 rearrangements develop leukemia that is attributable to prior treatment with certain chemotherapeutic agents-so-called therapy-related leukemias. In general, outcomes for all of these patients remain poor when compared to patients with non- MLL1 rearranged leukemias. In this review, we will discuss the normal biological roles of MLL1 and its fusion partners, how these roles are hypothesized to be dysregulated in the context of MLL1 rearrangements, and the clinical manifestations of this group of leukemias. We will go on to discuss the progress in clinical management and promising new avenues of research, which may lead to more effective targeted therapies for affected patients.

Transient potential receptor melastatin-2 (TRPM2) is a nonselective cationic, Ca(2+)-permeable transmembrane pore that is preferentially expressed in cells of the myeloid lineage and modulates signaling pathways converging into NF-kB. This is of potential interest for acute myeloid leukemia (AML) therapy, as NF-κB signaling is emerging as a key pathway, mediating drug resistance and leukemia-initiating cell survival in AML. Inhibition of NF-κB signaling has been found to be synergistic with chemotherapy. TRPM2 is overexpressed in AML compared with normal bone marrow, with the highest levels in the FAB M3-6 subtypes. To determine the effect of TRPM2 depletions in a defined genetic model, we established MLL-AF9-driven AML on a Trpm2(-/-) genetic background. Trpm2(-/-) MLL-AF9 leukemias displayed reduced NF-κB phosphorylation as well as nuclear translocation. In vivo, primary and secondary recipients of Trpm2(-/-) MLL-AF9 leukemias exhibit increased latency compared with recipients of wild-type leukemia cells. However, the difference in latency was small and was lost in tertiary transplants. The effect of loss of Trpm2 in a BCR-ABL/NUP98-HOXA9 fusion model was even smaller. Given reports that loss or inhibition of TRPM2 enhanced killing by DNA-damaging agents in neuroblastoma, breast cancer, and prostate cancer cell lines, we exposed Trpm2(-/-) and Trpm2(wt) primary MLL-AF9 leukemias to doxorubicin, cytarabine, and etoposide, but found no difference in IC50 values. The in vitro response to decitabine was also unaffected. In summary, Trpm2 does not seem to play a major role in myeloid leukemogenesis. Additionally, loss of Trpm2 does not augment the cytotoxicity of standard AML chemotherapeutic agents., (Copyright © 2016 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.)

Early T-Cell precursor acute lymphoblastic leukemia (ETP-ALL) is a relatively newly identified subset of T-lineage ALL. There are conflicting results regarding prognosis, and the genetic basis of this condition is variable. Here, we summarize the current status of the field and discuss the role of mutations in the Polycomb Repressive Complex 2 frequently identified in ETP-ALL patients.

Meningioma-1 (MN1) overexpression is frequently observed in patients with acute myeloid leukemia (AML) and is predictive of poor prognosis. In murine models, forced expression of MN1 in hematopoietic progenitors induces an aggressive myeloid leukemia that is strictly dependent on a defined gene expression program in the cell of origin, which includes the homeobox genes Hoxa9 and Meis1 as key components. Here, we have shown that this program is controlled by two histone methyltransferases, MLL1 and DOT1L, as deletion of either Mll1 or Dot1l in MN1-expressing cells abrogated the cell of origin-derived gene expression program, including the expression of Hoxa cluster genes. In murine models, genetic inactivation of either Mll1 or Dot1l impaired MN1-mediated leukemogenesis. We determined that HOXA9 and MEIS1 are coexpressed with MN1 in a subset of clinical MN1hi leukemia, and human MN1hi/HOXA9hi leukemias were sensitive to pharmacologic inhibition of DOT1L. Together, these data point to DOT1L as a potential therapeutic target in MN1hi AML. In addition, our findings suggest that epigenetic modulation of the interplay between an oncogenic lesion and its cooperating developmental program has therapeutic potential in AML.

Early T cell precursor acute lymphoblastic leukemia (ETP-ALL) is an aggressive subtype of ALL distinguished by stem-cell-associated and myeloid transcriptional programs. Inactivating alterations of Polycomb repressive complex 2 components are frequent in human ETP-ALL, but their functional role is largely undefined. We have studied the involvement of Ezh2 in a murine model of NRASQ61K-driven leukemia that recapitulates phenotypic and transcriptional features of ETP-ALL. Homozygous inactivation of Ezh2 cooperated with oncogenic NRASQ61K to accelerate leukemia onset. Inactivation of Ezh2 accentuated expression of genes highly expressed in human ETP-ALL and in normal murine early thymic progenitors. Moreover, we found that Ezh2 contributes to the silencing of stem-cell- and early-progenitor-cell-associated genes. Loss of Ezh2 also resulted in increased activation of STAT3 by tyrosine 705 phosphorylation. Our data mechanistically link Ezh2 inactivation to stem-cell-associated transcriptional programs and increased growth/survival signaling, features that convey an adverse prognosis in patients., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Polycomb repressive complex 2 (PRC2) is a chromatin regulator with central roles in development and cancer. The canonical function of PRC2 is the trimethylation of histone 3 on lysine residue 27. This epigenetic modification is associated with gene silencing. Both tumor suppressor and oncogenic functions have been reported for PRC2, depending on cellular context. In leukemia mediated by the leukemogenic fusion MLL-AF9, complete ablation of canonical PRC2 function by genetic inactivation of the core component embryonic ectoderm development (Eed) or by combined pharmacologic inhibition of the PRC2 methyltransferases EZH2 and EZH1 has a strong anti-leukemic effect, and this effect has been linked to de-repression of the PRC2 target locus Cdkn2a. We asked whether inactivation of Cdkn2a is sufficient to restore leukemic activity of Eed-inactivated MLL-AF9 leukemia cells, using combined genetic inactivation of Cdkn2a and Eed. We found that Cdkn2a inactivation partially rescues in vitro and in vivo growth of Eed-inactivated MLL-AF9 cells. However, the growth of Eed-null Cdkn2a-null MLL-AF9 cells in the absence of Cdkn2a remained severely compromised in vitro and in vivo, compared with that of their Eed-floxed Cdkn2a-null counterparts. RNA sequencing analysis revealed that several genes previously implicated in inefficient growth of MLL-AF9-transformed cells, including Gata2, Egr1, and Cdkn2b were de-repressed as a consequence of Eed inactivation. Furthermore, we found that direct binding targets of MLL fusion proteins are negatively enriched in Eed-inactivated Cdkn2a-null MLL-AF9-transformed cells. Our data indicate that interference with PRC2 function affects MLL-AF9-mediated leukemogenesis by both Cdkn2a-dependent and Cdkn2a-independent mechanisms., (Copyright © 2015 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.)

While DNA abnormalities have long been recognized as the cause of cancer, the contribution of chromatin is a relatively recent discovery. Excitement in the field of cancer epigenetics is driven by 3 key elements: 1. Chromatin may play an active and often critical role in controlling gene expression, DNA stability and cell identity. 2. Chromatin modifiers are frequent targets of DNA aberrations, in some cancers reaching near 100%. Particularly in cancers with low rates of DNA mutations, the key "driver" of malignancy is often a chromatin modifier. 3. Cancer-associated aberrant chromatin is amenable to pharmacologic modulation. This has sparked the rapidly expanding development of small molecules targeting chromatin modifiers or reader domains, several of which have shown promise in clinical trials. In parallel, technical advances have greatly enhanced our ability to perform comprehensive chromatin/histone profiling. Despite the discovery that distinct histone profiles are associated with prognostic subgroups, and in some instances may point towards an underlying aberration that can be targeted, histone profiling has not entered clinical diagnostics. Even eligibility for clinical trials targeting chromatin hinges on traditional histologic or DNA-based molecular criteria rather than chromatin profiles. This review will give an overview of the philosophical debate around the role of histones in controlling or modulating gene expression and discuss the most common techniques for histone profiling. In addition, we will provide prominent examples of aberrantly expressed or mutated chromatin modifiers that result in either globally or locally aberrant histone profiles, and that may be promising therapeutic targets., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Rearrangements of MLL (encoding lysine-specific methyltransferase 2A and officially known as KMT2A; herein referred to as MLL to denote the gene associated with mixed-lineage leukemia) generate MLL fusion proteins that bind DNA and drive leukemogenic gene expression. This gene expression program is dependent on the disruptor of telomeric silencing 1-like histone 3 lysine 79 (H3K79) methyltransferase DOT1L, and small-molecule DOT1L inhibitors show promise as therapeutics for these leukemias. However, the mechanisms underlying this dependency are unclear. We conducted a genome-scale RNAi screen and found that the histone deacetylase SIRT1 is required for the establishment of a heterochromatin-like state around MLL fusion target genes after DOT1L inhibition. DOT1L inhibits chromatin localization of a repressive complex composed of SIRT1 and the H3K9 methyltransferase SUV39H1, thereby maintaining an open chromatin state with elevated H3K9 acetylation and minimal H3K9 methylation at MLL fusion target genes. Furthermore, the combination of SIRT1 activators and DOT1L inhibitors shows enhanced antiproliferative activity against MLL-rearranged leukemia cells. These results indicate that the dynamic interplay between chromatin regulators controlling the activation and repression of gene expression could provide novel opportunities for combination therapy.

Genome scale sequencing in patients with cancer has revealed a lower frequency of genetic aberrations in hematologic disorders compared with most other malignancies, suggesting a prominent role for epigenetic mechanisms. In parallel, epigenetic modifiers that are altered in cancer play critical roles in normal hematopoietic development, influencing both self-renewal of hematopoietic stem cells and differentiation into the different lineages. In this review, we aim to compare the role of several key DNA or histone modifying enzymes and complexes in normal development and hematopoietic malignancies, including DNMT3A, TET2, IDH1, IDH2, MLL1, MLL4, DOT1L, PRC1/2 and WSHC1/NSD2/MMSET. Insights into their biological mechanisms led to the development of therapies designed to target mutant IDH1 and IDH2, DOT1L in MLL-rearranged leukemias and EZH2 in several cancer types including lymphomas. Inhibitors for these enzymes are currently in clinical trials.

Homeotic (HOX) genes are dysregulated in multiple malignancies, including several AML subtypes. We demonstrate that H3K79 dimethylation (H3K79me2) is converted to monomethylation (H3K79me1) at HOX loci as hematopoietic cells mature, thus coinciding with a decrease in HOX gene expression. We show that H3K79 methyltransferase activity as well as H3K79me1-to-H3K79me2 conversion is regulated by the DOT1L cofactor AF10. AF10 inactivation reverses leukemia-associated epigenetic profiles, precludes abnormal HOXA gene expression, and impairs the transforming ability of MLL-AF9, MLL-AF6, and NUP98-NSD1 fusions-mechanistically distinct HOX-activating oncogenes. Furthermore, NUP98-NSD1-transformed cells are sensitive to small-molecule inhibition of DOT1L. Our findings demonstrate that pharmacological inhibition of the DOT1L/AF10 complex may provide therapeutic benefits in an array of malignancies with abnormal HOXA gene expression., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The t(9;22)(q34;q11) or Philadelphia chromosome creates a BCR-ABL1 fusion gene encoding for a chimeric BCR-ABL1 protein. It is present in 3-4% of pediatric acute lymphoblastic leukemia (Ph(+) ALL), and about 25% of adult ALL cases. Prior to the advent of tyrosine kinase inhibitors (TKI), Ph(+) ALL was associated with a very poor prognosis despite the use of intensive chemotherapy and frequently hematopoietic stem-cell transplantation (HSCT) in first remission. The development of TKIs revolutionized the therapy of Ph(+) ALL. Addition of the first generation ABL1 class TKI imatinib to intensive chemotherapy dramatically increased the survival for children with Ph(+) ALL and established that many patients can be cured without HSCT. In parallel, the mechanistic understanding of Ph(+) ALL expanded exponentially through careful mapping of pathways downstream of BCR-ABL1, the discovery of mutations in master regulators of B-cell development such as IKZF1 (Ikaros), PAX5, and early B-cell factor (EBF), the recognition of the complex clonal architecture of Ph(+) ALL, and the delineation of genomic, epigenetic, and signaling abnormalities contributing to relapse and resistance. Still, many important basic and clinical questions remain unanswered. Current clinical trials are testing second generation TKIs in patients with newly diagnosed Ph(+) ALL. Neither the optimal duration of therapy nor the optimal chemotherapy backbone are currently defined. The role of HSCT in first remission and post-transplant TKI therapy also require further study. In addition, it will be crucial to continue to dig deeper into understanding Ph(+) ALL at a mechanistic level, and translate findings into complementary targeted approaches. Expanding targeted therapies hold great promise to decrease toxicity and improve survival in this high-risk disease, which provides a paradigm for how targeted therapies can be incorporated into treatment of other high-risk leukemias.

Methylation of H3K79 is associated with chromatin at expressed genes, though it is unclear if this histone modification is required for transcription of all genes. Recent studies suggest that Wnt-responsive genes depend particularly on H3K79 methylation, which is catalyzed by the methyltransferase DOT1L. Human leukemias carrying MLL gene rearrangements show DOT1L-mediated H3K79 methylation and aberrant expression of leukemogenic genes. DOT1L inhibitors reverse these effects, but their clinical use is potentially limited by toxicity in Wnt-dependent tissues such as intestinal epithelium. Genome-wide positioning of the H3K79me2 mark in Lgr5(+) mouse intestinal stem cells and mature intestinal villus epithelium correlated with expression levels of all transcripts and not with Wnt-responsive genes per se. Selective Dot1l disruption in Lgr5(+) stem cells or in whole intestinal epithelium eliminated H3K79me2 from the respective compartments, allowing genetic evaluation of DOT1L requirements. The absence of methylated H3K79 did not impair health, intestinal homeostasis, or expression of Wnt target genes in crypt epithelium for up to 4 months, despite increased crypt cell apoptosis. Global transcript profiles in Dot1l-null cells were barely altered. Thus, H3K79 methylation is not essential for transcription of Wnt-responsive or other intestinal genes, and intestinal toxicity is not imperative when DOT1L is rendered inactive in vivo.

The t(10;11)(p12;q23) translocation and the t(10;11)(p12;q14) translocation, which encode the MLL (mixed lineage leukemia)-AF10 and CALM (clathrin assembly lymphoid myeloid leukemia)-AF10 fusion oncoproteins, respectively, are two recurrent chromosomal rearrangements observed in patients with acute myeloid leukemia and acute lymphoblastic leukemia. Here, we demonstrate that MLL-AF10 and CALM-AF10-mediated transformation is dependent on the H3K79 methyltransferase Dot1l using genetic and pharmacological approaches in mouse models. Targeted disruption of Dot1l using a conditional knockout mouse model abolished in vitro transformation of murine bone marrow cells and in vivo initiation and maintenance of MLL-AF10 or CALM-AF10 leukemia. The treatment of MLL-AF10 and CALM-AF10 transformed cells with EPZ004777, a specific small-molecule inhibitor of Dot1l, suppressed expression of leukemogenic genes such as Hoxa cluster genes and Meis1, and selectively impaired proliferation of MLL-AF10 and CALM-AF10 transformed cells. Pretreatment with EPZ004777 profoundly decreased the in vivo spleen-colony-forming ability of MLL-AF10 or CALM-AF10 transformed bone marrow cells. These results show that patients with leukemia-bearing chromosomal translocations that involve the AF10 gene may benefit from small-molecule therapeutics that inhibit H3K79 methylation.

The t(6;11)(q27;q23) is a recurrent chromosomal rearrangement that encodes the MLLAF6 fusion oncoprotein and is observed in patients with diverse hematologic malignancies. The presence of the t(6;11)(q27;q23) has been linked to poor overall survival in patients with AML. In this study, we demonstrate that MLL-AF6 requires continued activity of the histone-methyltransferase DOT1L to maintain expression of the MLL-AF6-driven oncogenic gene-expression program. Using gene-expression analysis and genome-wide chromatin immunoprecipitation studies followed by next generation sequencing, we found that MLL-fusion target genes display markedly high levels of histone 3 at lysine 79 (H3K79) dimethylation in murine MLL-AF6 leukemias as well as in ML2, a human myelomonocytic leukemia cell line bearing the t(6;11)(q27;q23) translocation. Targeted disruption of Dot1l using a conditional knockout mouse model inhibited leukemogenesis mediated by the MLL-AF6 fusion oncogene. Moreover, both murine MLL-AF6-transformed cells as well as the human MLL-AF6-positive ML2 leukemia cell line displayed specific sensitivity to EPZ0004777, a recently described, selective, small-molecule inhibitor of Dot1l. Dot1l inhibition resulted in significantly decreased proliferation, decreased expression of MLL-AF6 target genes, and cell cycle arrest of MLL-AF6-transformed cells. These results indicate that patients bearing the t(6;11)(q27;q23) translocation may benefit from therapeutic agents targeting aberrant H3K79 methylation.

Generation of induced pluripotent stem cells (iPSCs) by somatic cell reprogramming involves global epigenetic remodelling. Whereas several proteins are known to regulate chromatin marks associated with the distinct epigenetic states of cells before and after reprogramming, the role of specific chromatin-modifying enzymes in reprogramming remains to be determined. To address how chromatin-modifying proteins influence reprogramming, we used short hairpin RNAs (shRNAs) to target genes in DNA and histone methylation pathways, and identified positive and negative modulators of iPSC generation. Whereas inhibition of the core components of the polycomb repressive complex 1 and 2, including the histone 3 lysine 27 methyltransferase EZH2, reduced reprogramming efficiency, suppression of SUV39H1, YY1 and DOT1L enhanced reprogramming. Specifically, inhibition of the H3K79 histone methyltransferase DOT1L by shRNA or a small molecule accelerated reprogramming, significantly increased the yield of iPSC colonies, and substituted for KLF4 and c-Myc (also known as MYC). Inhibition of DOT1L early in the reprogramming process is associated with a marked increase in two alternative factors, NANOG and LIN28, which play essential functional roles in the enhancement of reprogramming. Genome-wide analysis of H3K79me2 distribution revealed that fibroblast-specific genes associated with the epithelial to mesenchymal transition lose H3K79me2 in the initial phases of reprogramming. DOT1L inhibition facilitates the loss of this mark from genes that are fated to be repressed in the pluripotent state. These findings implicate specific chromatin-modifying enzymes as barriers to or facilitators of reprogramming, and demonstrate how modulation of chromatin-modifying enzymes can be exploited to more efficiently generate iPSCs with fewer exogenous transcription factors.

The histone 3 lysine 79 (H3K79) methyltransferase Dot1l has been implicated in the development of leukemias bearing translocations of the Mixed Lineage Leukemia (MLL) gene. We identified the MLL-fusion targets in an MLL-AF9 leukemia model, and conducted epigenetic profiling for H3K79me2, H3K4me3, H3K27me3, and H3K36me3 in hematopoietic progenitor and leukemia stem cells (LSCs). We found abnormal profiles only for H3K79me2 on MLL-AF9 fusion target loci in LSCs. Inactivation of Dot1l led to downregulation of direct MLL-AF9 targets and an MLL translocation-associated gene expression signature, whereas global gene expression remained largely unaffected. Suppression of MLL translocation-associated gene expression corresponded with dependence of MLL-AF9 leukemia on Dot1l in vivo. These data point to DOT1L as a potential therapeutic target in MLL-rearranged leukemia., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Mislocated enzymatic activity of DOT1L has been proposed as a driver of leukemogenesis in mixed lineage leukemia (MLL). The characterization of EPZ004777, a potent, selective inhibitor of DOT1L is reported. Treatment of MLL cells with the compound selectively inhibits H3K79 methylation and blocks expression of leukemogenic genes. Exposure of leukemic cells to EPZ004777 results in selective killing of those cells bearing the MLL gene translocation, with little effect on non-MLL-translocated cells. Finally, in vivo administration of EPZ004777 leads to extension of survival in a mouse MLL xenograft model. These results provide compelling support for DOT1L inhibition as a basis for targeted therapeutics against MLL., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Rearrangements of the Mixed-Lineage Leukemia (MLL) gene are found in > 70% of infant leukemia, ~ 10% of adult acute myelogenous leukemia (AML), and many cases of secondary acute leukemias. The presence of an MLL rearrangement generally confers a poor prognosis. There are more than 60 known fusion partners of MLL having some correlation with disease phenotype and prognosis. The most common fusion proteins induce the inappropriate expression of homeotic (Hox) genes, which, during normal hematopoiesis, are maintained by wild-type MLL. MLL-rearranged leukemias display remarkable genomic stability, with very few gains or losses of chromosomal regions. This may be explained by recent studies suggesting that MLL-rearranged leukemias are largely driven by epigenetic dysregulation. Several epigenetic regulators that modify DNA or histones have been implicated in MLL-fusion driven leukemogenesis, including DNA methylation, histone acetylation, and histone methylation. The histone methyltransferase DOT1L has emerged as an important mediator of MLL-fusion-mediated leukemic transformation. The clinical development of targeted inhibitors of these epigenetic regulators may therefore hold promise for the treatment of MLL-rearranged leukemia.

Children with relapsed and refractory acute lymphoblastic leukemia (ALL) still face a critical prognosis. We tested the hypothesis that targeted calicheamicin theta (θ) using an anti-CD19-immunoconjugate may provide an effective treatment strategy for CD19(+) ALL. Calicheamicin θ is a rationally designed prodrug of the natural enediyene calicheamicin γ, obtained by total synthesis. It offers the advantage of increased in vivo stability and 1000-fold higher antitumor potency over calicheamicin γ. First, we demonstrate efficacy of calicheamicin θ against primary pre-B leukemic cells and multidrug-resistant leukemia cell lines (IC(50) = 10(-9) to 10(-12) M). Second, conjugation of calicheamicin θ to an internalizing murine anti-CD19 monoclonal antibody was demonstrated to affect neither calicheamicin θ mediated cytotoxicity nor binding of the antibody to the target molecule. Third, anti-CD19-calicheamicin θ immunoconjugate revealed a maximum tolerated dose of 10 μg/kg and CD19-specific and long-lasting eradication of established leukemia was demonstrated in a xenograft model. Finally, we show that the antileukemic effect of anti-CD19-calicheamicin θ is mediated by induction of apoptosis proceeding through the caspase-mediated mitochondrial pathway. On the basis of these results, we conclude that anti-CD19-calicheamicin θ immunoconjugates may offer a novel and effective approach for the treatment of relapsed CD19(+) ALL.