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The fusion gene MLL-AF4 defines a high-risk subtype of pro-B acute lymphoblastic leukaemia. However, relapse can be associated with a switch from acute lymphoblastic to acute myeloid leukaemia. Here we show that these myeloid relapses share oncogene fusion breakpoints with their matched lymphoid presentations and can originate in either early, multipotent progenitors or committed B-cell precursors. Lineage switching is linked to substantial changes in chromatin accessibility and rewiring of transcriptional programmes indicating that the execution and maintenance of lymphoid lineage differentiation is impaired. We show that this subversion is recurrently associated with the dysregulation of repressive chromatin modifiers, notably the nucleosome remodelling and deacetylation complex, NuRD. In addition to mutations, we show differential expression or alternative splicing of NuRD members and other genes is able to reprogram the B lymphoid into a myeloid gene regulatory network. Lineage switching in MLL-AF4 leukaemia is therefore driven and maintained by defunct epigenetic regulation.Statement of SignificanceWe demonstrate diverse cellular origins of lineage switched relapse within MLL-AF4 pro-B acute leukaemia. Irrespective of the developmental origin of relapse, dysregulation of NuRD and/or other epigenetic machinery underpins fundamental lineage reprogramming with profound implications for the increasing use of epitope directed therapies in this high-risk leukaemia.

Human intestinal organoids (HIOs) derived from pluripotent stem cells provide a valuable model for investigating human intestinal organogenesis and physiology, but they lack the immune components required to fully recapitulate the complexity of human intestinal biology and diseases. To address this issue and to begin to decipher human intestinal–immune crosstalk during development, we generated HIOs containing immune cells by transplanting HIOs under the kidney capsule of mice with a humanized immune system. We found that human immune cells temporally migrate to the mucosa and form cellular aggregates that resemble human intestinal lymphoid follicles. Moreover, after microbial exposure, epithelial microfold cells are increased in number, leading to immune cell activation determined by the secretion of IgA antibodies in the HIO lumen. This in vivo HIO system with human immune cells provides a framework for future studies on infection- or allergen-driven intestinal diseases.

Inosine monophosphate dehydrogenase (IMPDH) is a rate-limiting enzyme in de novo guanine nucleotide synthesis pathway. Although IMPDH inhibitors are widely used as effective immunosuppressants, their antitumor effects have not been proven in the clinical setting. Here, we found that acute myeloid leukemias (AMLs) with MLL-fusions are susceptible to IMPDH inhibitors in vitro. We also showed that alternate-day administration of IMPDH inhibitors suppressed the development of MLL-AF9-driven AML in vivo without having a devastating effect on immune function. Mechanistically, IMPDH inhibition induced overactivation of Toll-like receptor (TLR)-TRAF6-NF-κB signaling and upregulation of an adhesion molecule VCAM1, which contribute to the antileukemia effect of IMPDH inhibitors. Consequently, combined treatment with IMPDH inhibitors and the TLR1/2 agonist effectively inhibited the development of MLL-fusion AML. These findings provide a rational basis for clinical testing of IMPDH inhibitors against MLL-fusion AMLs and potentially other aggressive tumors with active TLR signaling.

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Dysregulation of innate immune signaling pathways is implicated in various hematologic malignancies. However, these pathways have not been systematically examined in acute myeloid leukemia (AML). We report that AML hematopoietic stem and progenitor cells (HSPCs) exhibit a high frequency of dysregulated innate immune-related and inflammatory pathways, referred to as oncogenic immune signaling states. Through gene expression analyses and functional studies in human AML cell lines and patient-derived samples, we found that the ubiquitin-conjugating enzyme UBE2N is required for leukemic cell function in vitro and in vivo by maintaining oncogenic immune signaling states. It is known that the enzyme function of UBE2N can be inhibited by interfering with thioester formation between ubiquitin and the active site. We performed in silico structure-based and cellular-based screens and identified two related small-molecule inhibitors UC-764864/65 that targeted UBE2N at its active site. Using these small-molecule inhibitors as chemical probes, we further revealed the therapeutic efficacy of interfering with UBE2N function. This resulted in the blocking of ubiquitination of innate immune- and inflammatory-related substrates in human AML cell lines. Inhibition of UBE2N function disrupted oncogenic immune signaling by promoting cell death of leukemic HSPCs while sparing normal HSPCs in vitro. Moreover, baseline oncogenic immune signaling states in leukemic cells derived from discrete subsets of patients with AML exhibited a selective dependency on UBE2N function in vitro and in vivo. Our study reveals that interfering with UBE2N abrogates leukemic HSPC function and underscores the dependency of AML cells on UBE2N-dependent oncogenic immune signaling states.

As patient-derived xenograft (PDX) models of acute myeloid leukemia (AML) become increasingly common tools for preclinical evaluation of targeted therapies it becomes important to consider the fidelity with which this system recapitulates the disease state found in patients. Gene expression profiling of patient blasts has been successfully used to identify distinct subtypes of AML to uncover sub-type specific vulnerabilities and to predict response to therapy and outcomes. Currently, there is little information regarding how well PDX models of AML mimic global gene expression patterns found in patients. In order to address this point, we performed detailed RNA-Seq analysis of data obtained from a diverse series of pediatric AML PDXs, separately and compared to primary patient data. When unsupervised clustering was applied to the PDX sample dataset, we found grouping associated with KMT2A (MLL) gene status. Additionally, in combined analysis, PDX samples were found to align with primary patient samples harboring similar genetics. We found a strong correlation of expression levels of nearly all expressed transcripts in PDX and patient datasets thus demonstrating faithful recapitulation of gene expression signatures. Furthermore, paired patient/PDX samples showed strong concordance, suggesting retention of sample-specific gene expression in immune deficient mice. Comparisons of PDX models propagated in NOD/SCID/IL2rg-/-(NSG) mice compared to NSG mice with transgenic expression of human SCF, GM-CSF, and IL-3 (NSGS) revealed minimal differences related to increased JAK/STAT and macrophage activation pathways in NSGS. Additionally, a unique RAM immunophenotype associated expression signature pointed to discovery of cryptic CBFA2T3-GLIS2 rearrangement as the mechanistic driver mutation in two PDX models. Based on the relatively high BCL2 mRNA in these models, we tested the efficacy of venetoclax in combination with CPX-351 which resulted in reduced leukemia burden and prolonged survival. These results validate the PDX system as surrogate of the molecular signatures in high-risk pediatric AML and highlight this system’s utility for pre-clinical therapeutic discovery, especially in very rare subtypes of disease.

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Immune therapies such as blinatumomab, CD19-directed bispecific CD3 T-cell Engager (BiTE), have resulted in significant improvements in outcomes for relapsed B-cell acute lymphoblastic leukemia (B-ALL). However, up to half of blinatumomab treated patients do not respond completely or relapse after therapy. As a result, there is a need to identify potential strategies to improve the efficacy of BiTE therapy. The anti-PD-1 antibody pembrolizumab has been shown to successfully activate T cells against a wide range of cancer types. Here, we tested the ability of umbilical cord blood (UCB) reconstituted mice to respond to blinatumomab therapy with or without concurrent pembrolizumab treatment. Humanized mice were engrafted with patient-derived xenograft (PDX) cells derived from pediatric and adolescent/young adult (AYA) B-ALL patients who had either failed to achieve remission with negative minimum residual disease (MRD negative) or experienced a relapse. Mock-treated humanized mice engrafted with PDX cells efficiently developed overt disease within 30 days of engraftment of B-ALL. However, single agent therapy with either blinatumomab or pembrolizumab reduced disease burden in engrafted mice, with some mice observed to be MRD negative after the 28-day treatment course. Combination therapy significantly improved the percentage of MRD negative mice and improved long-term survival and cure rates as compared to mice that were given blinatumomab alone. Importantly, no benefits were observed in treated mice that lacked human immune cell reconstitution. These results indicate that UCB-humanized NRGS mice develop activatable immune function, and UCB-humanized PDX leukemia models can be used in preclinical studies to evaluate specificity, efficacy, and cooperativity of immune therapies in B-ALL.

Mixed-phenotype acute leukemia is a rare subtype of leukemia in which both myeloid and lymphoid markers are co-expressed on the same malignant cells. The pathogenesis is largely unknown, and the treatment is challenging. We previously reported the specific association of the recurrent t(8;12)(q13;p13) chromosomal translocation that creates the ETV6-NCOA2 fusion with T/myeloid leukemias. Here we report that ETV6-NCOA2 initiates T/myeloid leukemia in preclinical models; ectopic expression of ETV6-NCOA2 in mouse bone marrow hematopoietic progenitors induced T/myeloid lymphoma accompanied by spontaneous Notch1-activating mutations. Similarly, cotransduction of human cord blood CD34+ progenitors with ETV6-NCOA2 and a nontransforming NOTCH1 mutant induced T/myeloid leukemia in immunodeficient mice; the immunophenotype and gene expression pattern were similar to those of patient-derived ETV6-NCOA2 leukemias. Mechanistically, we show that ETV6-NCOA2 forms a transcriptional complex with ETV6 and the histone acetyltransferase p300, leading to derepression of ETV6 target genes. The expression of ETV6-NCOA2 in human and mouse nonthymic hematopoietic progenitor cells induces transcriptional dysregulation, which activates a lymphoid program while failing to repress the expression of myeloid genes such as CSF1 and MEF2C. The ETV6-NCOA2 induced arrest at an early immature T-cell developmental stage. The additional acquisition of activating NOTCH1 mutations transforms the early immature ETV6-NCOA2 cells into T/myeloid leukemias. Here, we describe the first preclinical model to depict the initiation of T/myeloid leukemia by a specific somatic genetic aberration.

Summary Fat mass and obesity-associated protein (FTO), an RNA N6-methyladenosine (m6A) demethylase, plays oncogenic roles in various cancers, presenting an opportunity for the development of effective targeted therapeutics. Here, we report two potent small-molecule FTO inhibitors that exhibit strong anti-tumor effects in multiple types of cancers. We show that genetic depletion and pharmacological inhibition of FTO dramatically attenuate leukemia stem/initiating cell self-renewal and reprogram immune response by suppressing expression of immune checkpoint genes, especially LILRB4. FTO inhibition sensitizes leukemia cells to T cell cytotoxicity and overcomes hypomethylating agent-induced immune evasion. Our study demonstrates that FTO plays critical roles in cancer stem cell self-renewal and immune evasion and highlights the broad potential of targeting FTO for cancer therapy.

Timed degradation of the cyclin-dependent kinase inhibitor p27Kip1 by the E3 ubiquitin ligase F-box protein SKP2 is critical for T-cell progression into cell cycle, coordinating proliferation and differentiation processes. SKP2 expression is regulated by mitogenic stimuli and by Notch signaling, a key pathway in T-cell development and in T-cell acute lymphoblastic leukemia (T-ALL); however, it is not known whether SKP2 plays a role in the development of T-ALL. Here, we determined that SKP2 function is relevant for T-ALL leukemogenesis, whereas is dispensable for T-cell development. Targeted inhibition of SKP2 by genetic deletion or pharmacological blockade markedly inhibited proliferation of human T-ALL cells in vitro and antagonized disease in vivo in murine and xenograft leukemia models, with little effect on normal tissues. We also demonstrate a novel feed forward feedback loop by which Notch and IL-7 signaling cooperatively converge on SKP2 induction and cell cycle activation. These studies show that the Notch/SKP2/p27Kip1 pathway plays a unique role in T-ALL development and provide a proof-of-concept for the use of SKP2 as a new therapeutic target in T-cell acute lymphoblastic leukemia (T-ALL).

Cell-line-derived xenografts (CDXs) or patient-derived xenografts (PDXs) in immune-deficient mice have revolutionized our understanding of normal and malignant human hematopoiesis. Transgenic approaches further improved in vivo hematological research, allowing the development of human-cytokine-producing mice, which show superior human cell engraftment. The most popular mouse strains used in research, the NOG (NOD.Cg-Prkdcscid Il2rγtm1Sug/Jic) and the NSG (NOD/SCID-IL2Rγ−/−, NOD.Cg-PrkdcscidIl2rγtm1Wjl/SzJ) mouse, and their human-cytokine-producing (interleukin-3, granulocyte-macrophage colony-stimulating factor, and stem cell factor) counterparts (huNOG and NSGS), rely partly on a mutation in the DNA repair protein PRKDC, causing a severe combined immune deficiency (SCID) phenotype and rendering the mice less tolerant to DNA-damaging therapeutics, thereby limiting their usefulness in the investigation of novel acute myeloid leukemia (AML) therapeutics. NRG (NOD/RAG1/2−/−IL2Rγ−/−) mice show equivalent immune ablation through a defective recombination activation gene (RAG), leaving DNA damage repair intact, and human-cytokine-producing NRGS (NRG-SGM3) mice were generated, improving myeloid engraftment. Our findings indicate that unconditioned NRG and NRGS mice can harbor established AML CDXs and can tolerate aggressive induction chemotherapy at higher doses than NSG mice without overt toxicity. However, unconditioned NRGS mice developed less clinically relevant disease, with CDXs forming solid tumors throughout the body, whereas unconditioned NRG mice were incapable of efficiently supporting PDX or human hematopoietic stem cell engraftment. These findings emphasize the contextually dependent utility of each of these powerful new strains in the study of normal and malignant human hematopoiesis. Therefore, the choice of mouse strain cannot be random, but must be based on the experimental outcomes and questions to be addressed.

Effective therapy of acute myeloid leukemia (AML) remains an unmet need. DNA methylcytosine dioxygenase Ten-eleven translocation 1 (TET1) is a critical oncoprotein in AML. Through a series of data analysis and drug screening, we identified two compounds (i.e., NSC-311068 and NSC-370284) that selectively suppress TET1 transcription and 5-hydroxymethylcytosine (5hmC) modification, and effectively inhibit cell viability in AML with high expression of TET1 (i.e., TET1-high AML), including AML carrying t(11q23)/MLL-rearrangements and t(8;21) AML. NSC-311068 and especially NSC-370284 significantly repressed TET1-high AML progression in vivo. UC-514321, a structural analog of NSC-370284, exhibited a more potent therapeutic effect and prolonged the median survival of TET1-high AML mice over three fold. NSC-370284 and UC-514321 both directly target STAT3/5, transcriptional activators of TET1, and thus repress TET1 expression. They also exhibit strong synergistic effects with standard chemotherapy. Our results highlight the therapeutic potential of targeting the STAT/TET1 axis by selective inhibitors in AML treatment.

Acute myeloid leukemia (AML) is associated with poor survival, and there is a strong need to identify disease vulnerabilities that might reveal new treatment opportunities. Here, we found that Toll-like receptor 1 (TLR1) and TLR2 are upregulated on primary AML CD34 + CD38 − cells relative to corresponding normal bone marrow cells. Activating the TLR1/TLR2 complex by the agonist Pam3CSK4 in MLL-AF9 -driven human AML resulted in induction of apoptosis by p38 MAPK-dependent activation of Caspase 3 and myeloid differentiation in a NFκB-dependent manner. By using murine Trp53 −/− MLL-AF9 AML cells, we demonstrate that p53 is dispensable for Pam3CSK4-induced apoptosis and differentiation. Moreover, murine AML1-ETO9a -driven AML cells also were forced into apoptosis and differentiation on TLR1/TLR2 activation, demonstrating that the antileukemic effects observed were not confined to MLL -rearranged AML. We further evaluated whether Pam3CSK4 would exhibit selective antileukemic effects. Ex vivo Pam3CSK4 treatment inhibited murine and human leukemia-initiating cells, whereas murine normal hematopoietic stem and progenitor cells (HSPCs) were relatively less affected. Consistent with these findings, primary human AML cells across several genetic subtypes of AML were more vulnerable for TLR1/TLR2 activation relative to normal human HSPCs. In the MLL-AF9 AML mouse model, treatment with Pam3CSK4 provided proof of concept for in vivo therapeutic efficacy. Our results demonstrate that TLR1 and TLR2 are upregulated on primitive AML cells and that agonistic targeting of TLR1/TLR2 forces AML cells into apoptosis by p38 MAPK-dependent activation of Caspase 3, and differentiation by activating NFκB, thus revealing a new putative strategy for therapeutically targeting AML cells.

Chromosome rearrangements involving the mixed-lineage leukemia gene (MLL) create MLL-fusion proteins, which could drive both acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). The lineage decision of MLL-fusion leukemia is influenced by the fusion partner and microenvironment. To investigate the interplay of fusion proteins and microenvironment in lineage choice, we transplanted human hematopoietic stem and progenitor cells (HSPCs) expressing MLL-AF9 or MLL-Af4 into immunodeficient NSGS mice, which strongly promote myeloid development. Cells expressing MLL-AF9 efficiently developed AML in NSGS mice. In contrast, MLL-Af4 cells, which were fully oncogenic under lymphoid conditions present in NSG mice, displayed compromised transformation capacity in a myeloid microenvironment. MLL-Af4 activated a self-renewal program in a lineage-dependent manner, showing the leukemogenic activity of MLL-Af4 was interlinked with lymphoid lineage commitment. The C-terminal homology domain (CHD) of Af4 was sufficient to confer this linkage. Although the MLL-CHD fusion protein failed to immortalize HSPCs in myeloid conditions in vitro, it could successfully induce ALL in NSG mice. Our data suggest that defective self-renewal ability and leukemogenesis of MLL-Af4 myeloid cells could contribute to the strong B-cell ALL association of MLL-AF4 leukemia observed in the clinic.

The CD44 gene encodes several protein isoforms due to alternative splicing and post translational modifications. Given that CD44 variant isoform 9 (CD44v9) is expressed within Spasmolytic Polypeptide/TFF2-Expressing Metaplasia (SPEM) glands during repair, CD44v9 may be play a funcitonal role during the process of regeneration of the gastric epithelium. Here we hypothesize that CD44v9 marks a regenerative cell lineage responsive to infiltrating macrophages during regeneration of the gastric epithelium. Ulcers were induced in CD44-deficient (CD44KO) and C57BL/6 (BL6) mice by a localized application of acetic acid to the serosal surface of the stomach. Gastric organoids expressing CD44v9 were derived from mouse stomachs and transplanted at the ulcer site of CD44KO mice. Ulcers, CD44v9 expression, proliferation and histology were measured 1, 3, 5 and 7-days post-injury. Human-derived gastric organoids were generated from stomach tissue collected from elderly (>55 years) or young (14-20 years) patients. Organoids were transplanted into the stomachs of NOD scid gamma (NSG) mice at the site of injury. Gastric injury was induced in NRG-SGM3 (NRGS) mice harboring human-derived immune cells (hnNRGS) and the immune profile anlayzed by CyTOF. CD44v9 expression emerged within regenerating glands the ulcer margin in response to injury. While ulcers in BL6 mice healed within 7-days post-injury, CD44KO mice exhibited loss of repair and epithelial regeneration. Ulcer healing was promoted in CD44KO mice by transplanted CD55v9-expressing gastric organoids. NSG mice exhibited loss of CD44v9 expression and gastric repair. Transplantation of human-derived gastric organoids from young, but not aged stomachs promoted repair in NSG mouse stomachs in response to injury. Finally, compared to NRGS mice, huNRGS animals exhibited reduced ulcer sizes, an infiltration of human CD162+ macrophages and an emergence of CD44v9 expression in SPEM. Thus, during repair of the gastic epithelium CD44v9 emerges within a regenerative cell lineage that coincides with macrophage inflitration within the injured mucosa. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Recently, macroautophagy/autophagy has emerged as a promising target in various types of solid tumor treatment. However, the impact of autophagy on acute myeloid leukemia (AML) maintenance and the validity of autophagy as a viable target in AML therapy remain unclear. Here we show that Kmt2a/Mll-Mllt3/Af9 AML (MA9-AML) cells have high autophagy flux compared with normal bone marrow cells, but autophagy-specific targeting, either through Rb1cc1-disruption to abolish autophagy initiation, or via Atg5-disruption to prevent phagophore (the autophagosome precursor) membrane elongation, does not affect the growth or survival of MA9-AML cells, either in vitro or in vivo. Mechanistically, neither Atg5 nor Rb1cc1 disruption impairs endolysosome formation or survival signaling pathways. The autophagy inhibitor chloroquine shows autophagy-independent anti-leukemic effects in vitro but has no efficacy in vivo likely due to limited achievable drug efficacy in blood. Further, vesicular exocytosis appears to mediate chloroquine resistance in AML cells, and exocytotic inhibition significantly enhances the anti-leukemic effect of chloroquine. Thus, chloroquine can induce leukemia cell death in vitro in an autophagy-independent manner but with inadequate efficacy in vivo, and vesicular exocytosis is a possible mechanism of chloroquine resistance in MA9-AML. This study also reveals that autophagy-specific targeting is unlikely to benefit MA9-AML therapy.

We have previously characterized an acute myeloid leukemia (AML) chemotherapy model for SCID-based immune deficient mice (NSG and NSGS), consisting of 5 days of cytarabine (AraC) and 3 days of anthracycline (doxorubicin), to simulate the standard 7+3 chemotherapy regimen many AML patients receive. While this model remains tractable, there are several limitations, presumably due to the constitutional Pkrdcscid (SCID, severe combined immune deficiency) mutation which affects DNA repair in all tissues of the mouse. These include the inability to combine preconditioning with subsequent chemotherapy, the inability to repeat chemotherapy cycles, and the increased sensitivity of the host hematopoietic cells to genotoxic stress. Here we attempt to address these drawbacks through the use of alternative strains with RAG-based immune deficiency (NRG and NRGS). We find that RAG-based mice tolerate a busulfan preconditioning regimen in combination with either AML or 4-drug acute lymphoid leukemia (ALL) chemotherapy, expanding the number of samples that can be studied. RAG-based mice also tolerate multiple cycles of therapy, thereby allowing for more aggressive, realistic modeling. Furthermore, standard AML therapy in RAG mice was 3.8-fold more specific for AML cells, relative to SCID mice, demonstrating an improved therapeutic window for genotoxic agents. We conclude that RAG-based mice should be the new standard for preclinical evaluation of therapeutic strategies involving genotoxic agents.

SummaryThe translocation MLL-AF4 defines a high-risk subtype of acute leukaemia which, uniquely amongst MLL translocations, is almost exclusively associated with a pro-B lymphoid phenotype. However, the ability to switch lineage at relapse allowed interrogation of the cellular origin and lineage determinants of this leukaemia. The origin of MLL-AF4 lymphoid leukaemia was commonly within a multipotent precursor population. In contrast, relapse as MLL-AF4 AML occurs from either a multipotent progenitor or a committed pro-B leukaemic clone. Lineage switching results from the dysregulation of chromatin modifiers, including the nucleosome remodelling and deacetylation complex, NuRD, completely extinguishing the B lymphoid gene regulatory network, instead establishing a myeloid epigenotype. We propose that, in contrast to mixed phenotype acute leukaemias, lineage switch in MLL-AF4 leukemia is driven and maintained by a reorganised epigenome.

The t(4;11)(q21;q23) fuses mixed-lineage leukemia (MLL) to AF4, the most common MLL-fusion partner. Here we show that MLL fused to murine Af4, highly conserved with human AF4, produces high-titer retrovirus permitting efficient transduction of human CD34+ cells, thereby generating a model of t(4;11) pro-B acute lymphoblastic leukemia (ALL) that fully recapitulates the immunophenotypic and molecular aspects of the disease. MLL-Af4 induces a B ALL distinct from MLL-AF9 through differential genomic target binding of the fusion proteins leading to specific gene expression patterns. MLL-Af4 cells can assume a myeloid state under environmental pressure but retain lymphoid-lineage potential. Such incongruity was also observed in t(4;11) patients in whom leukemia evaded CD19-directed therapy by undergoing myeloid-lineage switch. Our model provides a valuable tool to unravel the pathogenesis of MLL-AF4 leukemogenesis.

// Shan Lin 1,* , Junping Wei 1,* , Mark Wunderlich 1 , Fu-Sheng Chou 1 and James C. Mulloy 1 1 Cancer and Blood Disease Institute, Cincinnati Children’s Hospital Research Center, Cincinnati, OH, USA * These authors have contributed equally to this work Correspondence to: James C. Mulloy, email: // Keywords : immortalization, hTERT, t(8;21) fusion genes, human HSPC Received : April 14, 2016 Accepted : June 13, 2016 Published : August 05, 2016 Abstract Human CD34+ hematopoietic stem and progenitor cells (HSPC) expressing fusion protein AML1-ETO (AE), generated by the t(8;21)(q22;q22) rearrangement, manifest enhanced self-renewal and dysregulated differentiation without leukemic transformation, representing a pre-leukemia stage. Enabling replicative immortalization via telomerase reactivation is a crucial step in cancer development. However, AE expression alone is not sufficient to maintain high telomerase activity to immortalize human HSPC cells, which may hamper transformation. Here, we investigated the cooperativity of telomerase reverse transcriptase (hTERT), the catalytic subunit of telomerase, and AE in disease progression. Enforced expression of hTERT immortalized human AE pre-leukemia cells in a telomere-lengthening independent manner, and improved the pre-leukemia stem cell function by enhancing cell proliferation and survival. AE-hTERT cells retained cytokine dependency and multi-lineage differentiation potential similar to parental AE clones. Over the short-term, AE-hTERT cells did not show features of stepwise transformation, with no leukemogenecity evident upon initial injection into immunodeficient mice. Strikingly, after extended culture, we observed full transformation of one AE-hTERT clone, which recapitulated the disease evolution process in patients and emphasizes the importance of acquiring cooperating mutations in t(8;21) AML leukemogenesis. In summary, achieving unlimited proliferative potential via hTERT activation, and thereby allowing for acquisition of additional mutations, is a critical link for transition from pre-leukemia to overt disease in human cells. AE-hTERT cells represent a tractable model to study cooperating genetic lesions important for t(8;21) AML disease progression.

Antisense RNAs regulate the transcription and translation of the corresponding sense genes. Here, we report that an antisense RNA, AS‐RBM15, is transcribed in the opposite direction within exon 1 of RBM15 . RBM15 is a regulator of megakaryocyte (MK) differentiation and is also involved in a chromosome translocation t(1;22) in acute megakaryocytic leukemia. MK terminal differentiation is enhanced by up‐regulation of AS‐RBM15 expression and attenuated by AS‐RBM15 knockdown. At the molecular level, AS‐RBM15 enhances RBM15 protein translation in a CAP‐dependent manner. The region of the antisense AS‐RBM15 RNA, which overlaps with the 5′UTR of RBM15, is sufficient for the up‐regulation of RBM15 protein translation. In addition, we find that transcription of both RBM15 and AS‐RBM15 is activated by the transcription factor RUNX1 and repressed by RUNX1‐ETO, a leukemic fusion protein. Therefore, AS‐RBM15 is a regulator of megakaryocyte differentiation and may play a regulatory role in leukemogenesis. ![][1] The lncRNA AS‐RBM15 is transcriptionally up‐regulated by RUNX1 and enhances RBM15 protein translation via its overlapping region with the RBM15 5′UTR. [1]: /embed/graphic-1.gif

Transcription factor RUNX1 plays a crucial role in hematopoiesis and its activity is tightly regulated at both the transcriptional and posttranslational levels. However, translational control of RUNX1 expression has not been fully understood. In this study, we demonstrated that RUNX1b mRNA is translated from two alternative initiation sites, Met-1 and Met-25, giving full-length RUNX1b and a shorter protein lacking the first 24 amino acids (RUNX1ΔN24). Presence/absence of strong Kozak consensus sequences around Met-1 determines which initiation site is mainly used in RUNX1b cDNA. Selective disruption of either Met-1 or Met-25 abrogates expression of the corresponding protein while facilitating the production of another protein. The RUNX1b cDNA containing 65bp natural promoter sequences mainly produces full-length RUNX1b in human cord blood cells, but disruption of Met-1 in this cDNA also induced translation from Met-25. Consistent with these data, disruption of endogenous RUNX1b around Met-1 using CRISPR/Cas9 induced selective expression of RUNX1ΔΝ24 in several leukemia cell lines. RUNX1ΔN24 protein is more stable than full-length RUNX1b and retains hematopoietic activity. We also found that FLAG-tagged full-length RUNX1 showed altered activity, indicating the influence of N-terminal FLAG-tag on RUNX1 function. The alternative translation initiation of RUNX1b may participate in fine tuning RUNX1 activity.

Nature Communications 3: Article number: 688 (2012); Published: 21 February 2012; Updated: 10 April 2018 The original HTML version of this Article had an incorrect volume number of 2; it should have been 3. This has now been corrected in the HTML; the PDF version of the Article was correct from the time of publication.

Summary FTO, an mRNA N6-methyladenosine (m6A) demethylase, was reported to promote leukemogenesis. Using structure-based rational design, we have developed two promising FTO inhibitors, namely FB23 and FB23-2, which directly bind to FTO and selectively inhibit FTO's m6A demethylase activity. Mimicking FTO depletion, FB23-2 dramatically suppresses proliferation and promotes the differentiation/apoptosis of human acute myeloid leukemia (AML) cell line cells and primary blast AML cells in vitro. Moreover, FB23-2 significantly inhibits the progression of human AML cell lines and primary cells in xeno-transplanted mice. Collectively, our data suggest that FTO is a druggable target and that targeting FTO by small-molecule inhibitors holds potential to treat AML.

Cdc42 was originally discovered as a key regulator of bud site assembly and polarity in S. cerevisiae. Recent genetic studies have shown that the function of Cdc42 in regulating cell polarity appears highly conserved from budding yeast to humans. The role of Cdc42 in hematopoietic cell transformation and leukemia progression has been studied in an acute myeloid leukemia model using the MLL-AF9 oncogene-induced transformation and a Cdc42 conditional gene-targeted mouse model. Here we describe the leukemia cell polarity and division symmetry assays in the context of leukemia cell fate determination.

We designed ROS-activated cytotoxic agents (RACs) that are active against AML cancer cells. In this study, the mechanism of action and synergistic effects against cells coexpressing the AML oncogenes MLL-AF9 fusion and FLT3-ITD were investigated. One RAC (RAC1) had an IC50 value of 1.8 ± 0.3 µm, with ninefold greater selectivity for transformed cells compared to untransformed cells. Treatment induced DNA strand breaks, apoptosis, and cell cycle arrest. Proteomics and transcriptomics revealed enhanced expression of the pentose phosphate pathway, DNA repair, and pathways common to cell stress. Western blotting confirmed repair by homologous recombination. Importantly, RAC1 treatment was synergistic in combination with multiple pathway-targeting therapies in AML cells but less so in untransformed cells. Together, these results demonstrate that RAC1 can selectively target poor prognosis AML and that it does so by creating DNA double-strand breaks that require homologous recombination.

Hematopoietic stem and progenitor cells (HSPCs) reside in a specialized niche that regulates their proliferative capacity and their fate. There is increasing evidence for similar roles of marrow niches on controlling the behavior of leukemic cells; however, whether normal hematopoietic stem cell (HSC) and leukemic cells reside in or functionally compete for the same marrow niche is unclear. We used the mixed lineage leukemia-AF9 (MLL-AF9) murine acute myeloid leukemia (AML) in a competitive repopulation model to investigate whether normal HSPC and leukemic cells functionally compete for the same marrow niches. Irradiated recipient mice were transplanted with fixed numbers of MLL-AF9 cells mixed with increasing doses of normal syngeneic whole bone marrow (WBM) or with purified HSPC (LSK). Survival was significantly increased and leukemic progression was delayed proportional to increasing doses of normal WBM or normal LSK cells in multiple independent experiments, with all doses of WBM or LSK cells studied above the threshold for rapid and complete hematopoietic reconstitution in the absence of leukemia. Confocal microscopy demonstrated nests of either leukemic cells or normal hematopoietic cells but not both in the marrow adjacent to endosteum. Early following transplantation, leukemic cells from animals receiving lower LSK doses were cycling more actively than in those receiving higher doses. These results suggest that normal HSPC and AML cells compete for the same functional niche. Manipulation of the niche could impact on response to antileukemic therapies, and the numbers of normal HSPC could impact on leukemia outcome, informing approaches to cell dose in the context of stem cell transplantation. Stem Cells 2015;33:3635–3642

Acute myeloid leukemia (AML) is associated with a poor survival rate, and there is an urgent need for novel and more efficient therapies, ideally targeting AML stem cells that are essential for maintaining the disease. The interleukin 1 receptor accessory protein (IL1RAP; IL1R3) is expressed on candidate leukemic stem cells in the majority of AML patients, but not on normal hematopoietic stem cells. We show here that monoclonal antibodies targeting IL1RAP have strong antileukemic effects in xenograft models of human AML. We demonstrate that effector-cell-mediated killing is essential for the observed therapeutic effects and that natural killer cells constitute a critical human effector cell type. Because IL-1 signaling is important for the growth of AML cells, we generated an IL1RAP-targeting antibody capable of blocking IL-1 signaling and show that this antibody suppresses the proliferation of primary human AML cells. Hence, IL1RAP can be efficiently targeted with an anti-IL1RAP antibody capable of both achieving antibody-dependent cellular cytotoxicity and blocking of IL-1 signaling as modes of action. Collectively, these results provide important evidence in support of IL1RAP as a target for antibody-based treatment of AML.

Leukemias with MLL translocations are often found in infants and are associated with poor outcomes. The pathogenesis of MLL-fusion leukemias has been linked to upregulation of HOX/MEIS1 genes. The functions of the Hox/Meis1 complex in leukemia, however, remain elusive. Here, we used inducible Meis1-knockout mice coupled with MLL-AF9 knockin mice to decipher the mechanistic role of Meis1 in established MLL leukemia. We demonstrate that Meis1 is essential for maintenance of established leukemia. In addition, in both the murine model and human leukemia cells, we found that Meis1 loss led to increased oxidative stress, oxygen flux, and apoptosis. Gene expression and chromatin immunoprecipitation studies revealed hepatic leukemia factor (HLF) as a target gene of Meis1. Hypoxia or HLF expression reversed the oxidative stress, rescuing leukemia development in Meis1-deficient cells. Thus, the leukemia-promoting properties of Meis1 are at least partly mediated by a low-oxidative state, aided by HLF. These results suggest that stimulants of oxidative metabolism could have therapeutic potential in leukemia treatment.

R-2-hydroxyglutarate (R-2HG), produced at high levels by mutant isocitrate dehydrogenase 1/2 (IDH1/2) enzymes, was reported as an oncometabolite. We show here that R-2HG also exerts a broad anti-leukemic activity in vitro and in vivo by inhibiting leukemia cell proliferation/viability and by promoting cell-cycle arrest and apoptosis. Mechanistically, R-2HG inhibits fat mass and obesity-associated protein (FTO) activity, thereby increasing global N

AML1-ETO leukemia is the most common cytogenetic subtype of acute myeloid leukemia, defined by the presence of t(8;21). Remarkable progress has been achieved in understanding the molecular pathogenesis of AML1-ETO leukemia. Proteomic surveies have shown that AML-ETO forms a stable complex with several transcription factors, including E proteins. Genome-wide transcriptome and ChIP-seq analyses have revealed the genes directly regulated by AML1-ETO, such as CEBPA. Several lines of evidence suggest that AML1-ETO suppresses endogenous DNA repair in cells to promote mutagenesis, which facilitates acquisition of cooperating secondary events. Furthermore, it has become increasingly apparent that a delicate balance of AML1-ETO and native AML1 is important to sustain the malignant cell phenotype. Translation of these findings into the clinical setting is just beginning.

AML1-ETO leukemia is the most common cytogenetic subtype of acute myeloid leukemia, defined by the presence of t(8;21). Remarkable progress has been achieved in understanding the molecular pathogenesis of AML1-ETO leukemia. Proteomic surveies have shown that AML-ETO forms a stable complex with several transcription factors, including E proteins. Genome-wide transcriptome and ChIP-seq analyses have revealed the genes directly regulated by AML1-ETO, such as CEBPA. Several lines of evidence suggest that AML1-ETO suppresses endogenous DNA repair in cells to promote mutagenesis, which facilitates acquisition of cooperating secondary events. Furthermore, it has become increasingly apparent that a delicate balance of AML1-ETO and native AML1 is important to sustain the malignant cell phenotype. Translation of these findings into the clinical setting is just beginning.

The transcription factor RUNX1 is a master regulator of hematopoiesis. Disruption of RUNX1 activity has been implicated in the development of hematopoietic neoplasms. Recent studies also highlight the importance of RUNX1 in solid tumors both as a tumor promoter and a suppressor. Given its central role in cancer development, RUNX1 is an excellent candidate for targeted therapy. A potential strategy to target RUNX1 is through modulation of its posttranslational modifications (PTMs). Numerous studies have shown that RUNX1 activity is regulated by PTMs, including phosphorylation, acetylation, methylation and ubiquitination. These PTMs regulate RUNX1 activity either positively or negatively by altering RUNX1-mediated transcription, promoting protein degradation and affecting protein interactions. In this review, we first summarize the available data on the context- and dosage-dependent roles of RUNX1 in various types of neoplasms. We then provide a comprehensive overview of RUNX1 PTMs from biochemical and biologic perspectives. Finally, we discuss how aberrant PTMs of RUNX1 might contribute to tumorigenesis and also strategies to develop anticancer therapies targeting RUNX1 PTMs.

Overactive RAS signaling is prevalent in juvenile myelomonocytic leukemia (JMML) and the myeloproliferative variant of chronic myelomonocytic leukemia (MP-CMML) in humans, and both are refractory to conventional chemotherapy. Conditional activation of a constitutively active oncogenic Nras (NrasG12D/G12D) in murine hematopoietic cells promotes an acute myeloproliferative neoplasm (MPN) that recapitulates many features of JMML and MP-CMML. We found that NrasG12D/G12D-expressing HSCs, which serve as JMML/MP-CMML-initiating cells, show strong hyperactivation of ERK1/2, promoting hyperproliferation and depletion of HSCs and expansion of downstream progenitors. Inhibition of the MEK pathway alone prolonged the presence of NrasG12D/G12D-expressing HSCs but failed to restore their proper function. Consequently, approximately 60% of NrasG12D/G12D mice treated with MEK inhibitor alone died within 20 weeks, and the remaining animals continued to display JMML/MP-CMML-like phenotypes. In contrast, combined inhibition of MEK and JAK/STAT signaling, which is commonly hyperactivated in human and mouse CMML, potently inhibited human and mouse CMML cell growth in vitro, rescued mutant NrasG12D/G12D-expressing HSC function in vivo, and promoted long-term survival without evident disease manifestation in NrasG12D/G12D animals. These results provide a strong rationale for further exploration of combined targeting of MEK/ERK and JAK/STAT in treating patients with JMML and MP-CMML.

As a central regulator of cell polarity, the activity of CDC42 GTPase is tightly controlled in maintaining normal hematopoietic stem and progenitor cell (HSC/P) functions. We found that transformation of HSC/P to acute myeloid leukemia (AML) is associated with increased CDC42 expression and activity in leukemia cells. In a mouse model of AML, the loss of Cdc42 abrogates MLL-AF9-induced AML development. Furthermore, genetic ablation of CDC42 in both murine and human MLL-AF9 (MA9) cells decreased survival and induced differentiation of the clonogenic leukemia-initiating cells. We show that MLL-AF9 leukemia cells maintain cell polarity in the context of elevated Cdc42-guanosine triphosphate activity, similar to nonmalignant, young HSC/Ps. The loss of Cdc42 resulted in a shift to depolarized AML cells that is associated with a decrease in the frequency of symmetric and asymmetric cell divisions producing daughter cells capable of self-renewal. Importantly, we demonstrate that inducible CDC42 suppression in primary human AML cells blocks leukemia progression in a xenograft model. Thus, CDC42 loss suppresses AML cell polarity and division asymmetry, and CDC42 constitutes a useful target to alter leukemia-initiating cell fate for differentiation therapy.