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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.

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.

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.

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

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.

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.

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.

Therapies for pediatric acute myeloid leukemia (AML) remain unsatisfactory and generally do not incorporate molecularly-targeted agents aside from FLT3 inhibitors outside of the relapse setting. Patient-derived xenograft (PDX) models of AML are increasingly accessible for the preclinical evaluation of targeted therapies, though the degree to which these systems recapitulate the disease state as found in patients has not been well defined for AML. Gene expression profiling of patient blasts has been successfully used to discriminate distinct subtypes of AML, to uncover sub-type specific vulnerabilities, and to predict response to therapy and outcomes. We sought to systematically examine PDX models of pediatric AML for their ability to replicate global gene expression patterns and preserve mutational signatures found in patients. In addition, we conducted in-depth bioinformatic analyses of samples with cryptic CBA2T3-GLIS2 fusion generated by the inv(16)(p13.3q24.3) for identification of potential novel targeted therapies. We performed detailed analyses of RNA sequencing data from a diverse series of 24 pediatric AML PDX models established from samples obtained from patients with relapse and refractory disease. Initially we compared our PDX data against 49 selected relapse and refractory patient sample data files found in the NCI TARGET dataset of pediatric AML. When applying unsupervised hierarchical clustering to the PDX samples, we found that clustering was associated with MLL status. Clustering of the combined sets of samples by MLL status showed integration of samples according to mutation profile, regardless of data source (PDX or patient). The expression levels of all detectable transcripts were highly conserved between PDX and patient MLL-r samples. Separate analysis of each dataset yielded MLL specific gene lists that included a subset of overlapping genes which may point to a unique relapse and refractory pediatric MLL-r signature. This list contains several interesting new targets for further study. A subset of 12 PDX models were compared directly to the matched patient sample from which they were established. This analysis revealed strong similarity, with each PDX most closely related to its matched patient sample, suggesting retention of sample-specific gene expression in immune deficient mice. We set up our PDX models in NSG mice with transgenic expression of human myelo-supportive cytokines SCF, GM-CSF, and IL-3 in order to promote the most efficient and robust engraftment of precious patient material. In order to detect any skewing effects due to the host mouse strain, we compared NSGS PDX RNA sequencing data to 10 matched NSG PDX models. This comparison revealed consistent differences in only 9 transcripts, which were almost entirely related to increased JAK/STAT signaling and macrophage activation pathways in NSGS mice relative to NSG mice. Interestingly, during this analysis we observed a distinct PCA-driven clustering of a pair of PDX samples with previously clinically unidentified driver mutations. Reanalysis of the RNA sequencing data revealed evidence of a cryptic GLIS2 rearrangement (found in ~1% of pediatric AML cases) as the driver mutation, which was subsequently confirmed by RT-PCR in both samples. The unique CBFA2T3/GLIS2 RNA signature was mined to guide the composition of a focused 75-molecule in vitro drug screen against ex vivo PDX samples with an emphasis on the SHH, WNT, and BCL2 pathways. This screen identified the Wnt-C59 PORCN inhibitor as having specific activity against CBFA2T3/GLIS2+ AMLs. Further testing of C-59 in combinatorial studies revealed enhanced effects with the addition of the BCL2 inhibitor, venetoclax. In vivo experiments are currently underway to determine the pre-clinical efficacy of this novel combination. In summary, we found highly significant fidelity of gene expression in PDX models of relapse and refractory pediatric AML. Analysis of this dataset has led to several insights, including potential targeted therapies, highlighting how this system could be a valuable tool for discovery of novel targeted therapies, especially for very rare, distinct subtypes of disease. Disclosures Perentesis: Kurome Therapeutics: Consultancy.

Targeted inhibitors to oncogenic kinases demonstrate encouraging clinical responses early in the treatment course, however, most patients will relapse due to target-dependent mechanisms that mitigate enzyme-inhibitor binding or through target-independent mechanisms, such as alternate activation of survival and proliferation pathways, known as adaptive resistance. One example involves the FMS-like receptor tyrosine kinase (FLT3). Activating mutations of FLT3 result in its autophosphorylation and initiation of intracellular signaling pathways, which induce abnormal survival and proliferation of leukemic cells.One of the most common mutations in acute myeloid leukemia (AML) involves the internal tandem duplication (ITD) of FLT3, which occurs in ~25% of all cases of newly diagnosed AML and confers a particularly poor prognosis. FLT3 inhibitors (FLT3i) evaluated in clinical studies as monotherapy and combination therapies have shown good initial response rates; however, patients eventually relapse with FLT3i-resistant disease. The absence of durable remission in patients treated with potent and selective FLT3i highlights the need to identify resistance mechanisms and develop additional treatment strategies. Several mechanisms contribute to resistance to selective FLT3i, including mutations in the tyrosine kinase domain of FLT3 (20-50%) or activation of parallel signaling mechanisms that bypass FLT3 signaling, referred to as adaptive resistance (30-50%). Here we describe mechanisms of adaptive resistance in FLT3-mutant AML by examining in-cell kinase and gene regulatory network responses after oncogenic signaling blockade by FLT3 inhibitors (FLT3i). Through this integrative approach, we identified activation of innate immune stress response pathways after treatment of FLT3-mutant AML cells with FLT3i. Utilizing genetic approaches, we demonstrated that innate immune pathway activation via IRAK1 and IRAK4 contributes to adaptive resistance in FLT3-mutant AML cells. The immediate nature of IRAK1/4 activation in adaptively resistant FLT3-ITD AML cells requires concomitant inhibition of these targets to avoid compensatory signaling and cell survival. Achieving optimal multi-drug combination regimens that yield extended overlapping exposure while avoiding unwanted toxicities is challenging. Therefore, we desired a small molecule inhibitor that simultaneously targeted the FLT3 and IRAK1/4 kinases to eradicate adaptively resistant FLT3-ITD AML. To overcome this adaptive resistance mechanism, we developed and optimized a novel small molecule that simultaneously inhibits FLT3 and IRAK1/4 kinases. The FLT3-IRAK1/4 inhibitor exhibited potent binding affinity for IRAK1 (KD= 2.9 nM), IRAK4 (KD= 0.3 nM), and FLT3 (KD= 0.3 nM), as well as acceptable pharmacokinetic properties in mice. Moreover, a high-resolution crystal structure demonstrates that the FLT3-IRAK1/4 inhibitor binds as a type I inhibitor (ATP-competitive binding to the active state). The FLT3-IRAK1/4 inhibitor eliminated adaptively resistant FLT3-mutant AML cell lines and patient-derived samples in vitro and in vivo, and displayed superior efficacy as compared to current targeted FLT3 therapies. Our study demonstrates that therapies that simultaneously inhibit FLT3 signaling and compensatory IRAK1/4 activation have the potential to improve the therapeutic efficacy in patients with FLT3-mutant AML. In conclusion, these findings reveal that inflammatory stress response pathways contribute to adaptive resistance in FLT3-mutant AML and suggests that this mechanism may extend to other malignant cells undergoing a stress-induced response to therapy. Disclosures Hoyt: Kurome Therapeutics: Consultancy. Berman:Astellas: Membership on an entity's Board of Directors or advisory committees, Research Funding. Levine:Qiagen: Membership on an entity's Board of Directors or advisory committees; Prelude Therapeutics: Research Funding; Amgen: Honoraria; Lilly: Honoraria; Gilead: Consultancy; C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; Roche: Consultancy, Research Funding; Imago Biosciences: Membership on an entity's Board of Directors or advisory committees; Isoplexis: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Research Funding; Loxo: Membership on an entity's Board of Directors or advisory committees. Rosenbaum:Kurome Therapeutics: Consultancy, Employment. Perentesis:Kurome Therapeutics: Consultancy. Starczynowski:Kurome Therapeutics: Consultancy.

Acute myelogenous leukemia (AML) subtypes that result from oncogenic activation of homeobox (HOX) transcription factors are associated with poor prognosis. The HOXA9 transcription activator and growth factor independent 1 (GFI1) transcriptional repressor compete for occupancy at DNA-binding sites for the regulation of common target genes. We exploited this HOXA9 versus GFI1 antagonism to identify the genes encoding microRNA-21 and microRNA-196b as transcriptional targets of HOX-based leukemia oncoproteins. Therapeutic inhibition of microRNA-21 and microRNA-196b inhibited in vitro leukemic colony forming activity and depleted in vivo leukemia-initiating cell activity of HOX-based leukemias, which led to leukemia-free survival in a murine AML model and delayed disease onset in xenograft models. These data establish microRNA as functional effectors of endogenous HOXA9 and HOX-based leukemia oncoproteins, provide a concise in vivo platform to test RNA therapeutics, and suggest therapeutic value for microRNA antagonists in AML.

Genetic manipulation of NOD/SCID (NS) mice has yielded numerous sub-strains with specific traits useful for the study of human hematopoietic xenografts, each with unique characteristics. Here, we have compared the engraftment and output of umbilical cord blood (UCB) CD34+ cells in four immune-deficient strains: NS, NS with additional IL2RG knockout (NSG), NS with transgenic expression of human myeloid promoting cytokines SCF, GM-CSF, and IL-3 (NSS), and NS with both IL2RG knockout and transgenic cytokine expression (NSGS). Overall engraftment of human hematopoietic cells was highest in the IL2RG knockout strains (NSG and NSGS), while myeloid cell output was notably enhanced in the two strains with transgenic cytokine expression (NSS and NSGS). In further comparisons of NSG and NSGS mice, several additional differences were noted. NSGS mice were found to have a more rapid reconstitution of T cells, improved B cell differentiation, increased levels of NK cells, reduced platelets, and reduced maintenance of primitive CD34+ cells in the bone marrow. NSGS were superior hosts for secondary engraftment and both strains were equally suitable for experiments of graft versus host disease. Increased levels of human cytokines as well as human IgG and IgM were detected in the serum of humanized NSGS mice. Furthermore, immunization of humanized NSGS mice provided evidence of a functional response to repeated antigen exposure, implying a more complete hematopoietic graft was generated in these mice. These results highlight the important role that myeloid cells and myeloid-supportive cytokines play in the formation of a more functional xenograft immune system in humanized mice.

The success of tyrosine kinase inhibitors (TKIs) in treating chronic myeloid leukemia (CML) depends on the requirement for BCR-ABL1 kinase activity in CML progenitors. However, CML quiescent HSCs are TKI resistant and represent a BCR-ABL1 kinase–independent disease reservoir. Here we have shown that persistence of leukemic HSCs in BM requires inhibition of the tumor suppressor protein phosphatase 2A (PP2A) and expression — but not activity — of the BCR-ABL1 oncogene. Examination of HSCs from CML patients and healthy individuals revealed that PP2A activity was suppressed in CML compared with normal HSCs. TKI-resistant CML quiescent HSCs showed increased levels of BCR-ABL1, but very low kinase activity. BCR-ABL1 expression, but not kinase function, was required for recruitment of JAK2, activation of a JAK2/β-catenin survival/self-renewal pathway, and inhibition of PP2A. PP2A-activating drugs (PADs) markedly reduced survival and self-renewal of CML quiescent HSCs, but not normal quiescent HSCs, through BCR-ABL1 kinase–independent and PP2A-mediated inhibition of JAK2 and β-catenin. This led to suppression of human leukemic, but not normal, HSC/progenitor survival in BM xenografts and interference with long-term maintenance of BCR-ABL1–positive HSCs in serial transplantation assays. Targeting the JAK2/PP2A/β-catenin network in quiescent HSCs with PADs (e.g., FTY720) has the potential to treat TKI-refractory CML and relieve lifelong patient dependence on TKIs.

The ten-eleven translocation 1 ( TET1 ) gene is the founding member of the TET family of enzymes (TET1/2/3) that convert 5-methylcytosine to 5-hydroxymethylcytosine. Although TET1 was first identified as a fusion partner of the mixed lineage leukemia ( MLL ) gene in acute myeloid leukemia carrying t(10,11), its definitive role in leukemia is unclear. In contrast to the frequent down-regulation (or loss-of-function mutations) and critical tumor-suppressor roles of the three TET genes observed in various types of cancers, here we show that TET1 is a direct target of MLL-fusion proteins and is significantly up-regulated in MLL -rearranged leukemia, leading to a global increase of 5-hydroxymethylcytosine level. Furthermore, our both in vitro and in vivo functional studies demonstrate that Tet1 plays an indispensable oncogenic role in the development of MLL -rearranged leukemia, through coordination with MLL-fusion proteins in regulating their critical cotargets, including homeobox A9 ( Hoxa9 )/myeloid ecotropic viral integration 1 ( Meis1 )/pre-B-cell leukemia homeobox 3 ( Pbx3 ) genes. Collectively, our data delineate an MLL-fusion/Tet1/Hoxa9/Meis1/Pbx3 signaling axis in MLL -rearranged leukemia and highlight TET1 as a potential therapeutic target in treating this presently therapy-resistant disease.

Understanding and blocking the self-renewal pathway of preleukemia stem cells could prevent acute myeloid leukemia (AML) relapse. In this study, we show that increased FOXO1 represents a critical mechanism driving aberrant self-renewal in preleukemic cells expressing the t(8;21)-associated oncogene AML1-ETO (AE). Although generally considered as a tumor suppressor, FOXO1 is consistently upregulated in t(8;21) AML. Expression of FOXO1 in human CD34+ cells promotes a preleukemic state with enhanced self-renewal and dysregulated differentiation. The DNA binding domain of FOXO1 is essential for these functions. FOXO1 activates a stem cell molecular signature that is also present in AE preleukemia cells and preserved in t(8;21) patient samples. Genome-wide binding studies show that AE and FOXO1 share the majority of their binding sites, whereby FOXO1 binds to multiple crucial self-renewal genes and is required for their activation. In agreement with this observation, genetic and pharmacological ablation of FOXO1 inhibited the long-term proliferation and clonogenicity of AE cells and t(8;21) AML cell lines. Targeting of FOXO1 therefore provides a potential therapeutic strategy for elimination of stem cells at both preleukemic and leukemic stages.

Eradication of leukemia stem cells (LSCs) is the ultimate goal of treating acute myeloid leukemia (AML). We recently showed that the combined loss of Runx1/Cbfb inhibited the development of MLL-AF9-induced AML. However, c-Kit+/Gr-1− cells remained viable in Runx1/Cbfb-deleted cells, indicating that suppressing RUNX activity may not eradicate the most immature LSCs. In this study, we found upregulation of several hemostasis-related genes, including the thrombin-activatable receptor PAR-1 (protease-activated receptor-1), in Runx1/Cbfb-deleted MLL-AF9 cells. Similar to the effect of Runx1/Cbfb deletion, PAR-1 overexpression induced CDKN1A/p21 expression and attenuated proliferation in MLL-AF9 cells. To our surprise, PAR-1 deficiency also prevented leukemia development induced by a small number of MLL-AF9 leukemia stem cells (LSCs) in vivo. PAR-1 deficiency also reduced leukemogenicity of AML1-ETO-induced leukemia. Re-expression of PAR-1 in PAR-1-deficient cells combined with a limiting-dilution transplantation assay demonstrated the cell-dose-dependent role of PAR-1 in MLL-AF9 leukemia: PAR-1 inhibited rapid leukemic proliferation when there were a large number of LSCs, while a small number of LSCs required PAR-1 for their efficient growth. Mechanistically, PAR-1 increased the adherence properties of MLL-AF9 cells and promoted their engraftment to bone marrow. Taken together, these data revealed a multifaceted role for PAR-1 in leukemogenesis, and highlight this receptor as a potential target to eradicate primitive LSCs in AML.

Tyrosine-kinase inhibitor (TKI) therapy for human cancers is not curative, and relapse occurs owing to the continued presence of tumor cells, referred to as minimal residual disease (MRD). The survival of MRD stem or progenitor cells in the absence of oncogenic kinase signaling, a phenomenon referred to as intrinsic resistance, depends on diverse growth factors. Here we report that oncogenic kinase and growth-factor signaling converge to induce the expression of the signaling proteins FBJ osteosarcoma oncogene (c-FOS, encoded by Fos) and dual-specificity phosphatase 1 (DUSP1). Genetic deletion of Fos and Dusp1 suppressed tumor growth in a BCR-ABL fusion protein kinase-induced mouse model of chronic myeloid leukemia (CML). Pharmacological inhibition of c-FOS, DUSP1 and BCR-ABL eradicated MRD in multiple in vivo models, as well as in mice xenotransplanted with patient-derived primary CML cells. Growth-factor signaling also conferred TKI resistance and induced FOS and DUSP1 expression in tumor cells modeling other types of kinase-driven leukemias. Our data demonstrate that c-FOS and DUSP1 expression levels determine the threshold of TKI efficacy, such that growth-factor-induced expression of c-FOS and DUSP1 confers intrinsic resistance to TKI therapy in a wide-ranging set of leukemias, and might represent a unifying Achilles' heel of kinase-driven cancers.

One mechanism for disrupting the MLL gene in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) is through partial tandem duplication (MLL-PTD); however, the mechanism by which MLL-PTD contributes to MDS and AML development and maintenance is currently unknown. Herein, we investigated hematopoietic stem/progenitor cell (HSPC) phenotypes of Mll-PTD knock-in mice. Although HSPCs (Lin−Sca1+Kit+ (LSK)/SLAM+ and LSK) in MllPTD/WT mice are reduced in absolute number in steady state because of increased apoptosis, they have a proliferative advantage in colony replating assays, CFU-spleen assays, and competitive transplantation assays over wild-type HSPCs. The MllPTD/WT-derived phenotypic short-term (ST)–HSCs/multipotent progenitors and granulocyte/macrophage progenitors have self-renewal capability, rescuing hematopoiesis by giving rise to long-term repopulating cells in recipient mice with an unexpected myeloid differentiation blockade and lymphoid-lineage bias. However, MllPTD/WT HSPCs never develop leukemia in primary or recipient mice, suggesting that additional genetic and/or epigenetic defects are necessary for full leukemogenic transformation. Thus, the Mll-PTD aberrantly alters HSPCs, enhances self-renewal, causes lineage bias, and blocks myeloid differentiation. These findings provide a framework by which we can ascertain the underlying pathogenic role of MLL-PTD in the clonal evolution of human leukemia, which should facilitate improved therapies and patient outcomes.

AML1-ETO (AE) is a fusion product of translocation (8;21) that accounts for 40% of M2 type acute myeloid leukemia (AML). In addition to its role in promoting preleukemic hematopoietic cell self-renewal, AE represses DNA repair genes, which leads to DNA damage and increased mutation frequency. Although this latter function may promote leukemogenesis, concurrent p53 activation also leads to an increased baseline apoptotic rate. It is unclear how AE expression is able to counterbalance this intrinsic apoptotic conditioning by p53 to promote survival and self-renewal. In this report, we show that Bcl-xL is up-regulated in AE cells and plays an essential role in their survival and self-renewal. Further investigation revealed that Bcl-xL expression is regulated by thrombopoietin (THPO)/MPL-signaling induced by AE expression. THPO/MPL-signaling also controls cell cycle reentry and mediates AE-induced self-renewal. Analysis of primary AML patient samples revealed a correlation between MPL and Bcl-xL expression specifically in t(8;21) blasts. Taken together, we propose that survival signaling through Bcl-xL is a critical and intrinsic component of a broader self-renewal signaling pathway downstream of AML1-ETO–induced MPL.

RUNX1 is essential for definitive hematopoiesis and T-cell differentiation. It has been shown that RUNX1 is phosphorylated at specific serine and threonine residues by several kinase families. However, it remains unclear whether RUNX1 phosphorylation is absolutely required for its biological functions. Here, we evaluated hematopoietic activities of RUNX1 mutants with serine (S)/threonine (T) to alanine (A), aspartic acid (D), or glutamic acid (E) mutations at phosphorylation sites using primary culture systems. Consistent with the results of knockin mice, RUNX1-2A, carrying two phospho-deficient mutations at S276 and S293, retained hematopoietic activity. RUNX1-4A, carrying four mutations at S276, S293, T300, and S303, showed impaired T-cell differentiation activity, but retained the ability to rescue the defective early hematopoiesis of Runx1-deficient cells. Notably, RUNX1-5A, carrying five mutations at S276, S293, T300, S303, and S462, completely lost its hematopoietic activity. In contrast, the phospho-mimic proteins RUNX1-4D/E and RUNX1-5D/E exhibited normal function. Our study identifies multiple phosphorylation sites that are indispensable for RUNX1 activity in hematopoiesis.

Extracellular matrix (ECM) actively participates in normal cell regulation and in the process of tumor progression. The Rho GTPase Cdc42 has been shown to regulate cell-ECM interaction in conventional two-dimensional culture conditions by using dominant mutants of Cdc42 in immortalized cell lines that may introduce nonspecific effects. Here, we employ three-dimensional culture systems for conditional gene targeted primary mouse embryonic fibroblasts that better simulate the reciprocal and adaptive interactions between cells and surrounding matrix to define the role of Cdc42 signaling pathways in ECM organization. Cdc42 deficiency leads to a defect in global cell-matrix interactions reflected by a decrease in collagen gel contraction. The defect is associated with an altered cell-matrix interaction that is evident by morphologic changes and reduced focal adhesion complex formation. The matrix defect is also associated with a reduction in synthesis and activation of matrix metalloproteinase 9 (MMP9) and altered fibronectin deposition patterning. A Cdc42 mutant rescue experiment found that downstream of Cdc42, p21-activated kinase (PAK), but not Par6 or WASP, may be involved in regulating collagen gel contraction and fibronectin organization. Thus, in addition to the previously implicated roles in intracellular regulation of actin organization, proliferation, and vesicle trafficking, Cdc42 is essential in ECM remodeling in three dimensions.

Epigenetic pathways can regulate gene expression by controlling and interpreting chromatin modifications. Cancer cells are characterized by altered epigenetic landscapes, and commonly exploit the chromatin regulatory machinery to enforce oncogenic gene expression programs1. Although chromatin alterations are, in principle, reversible and often amenable to drug intervention, the promise of targeting such pathways therapeutically has been limited by an incomplete understanding of cancer-specific dependencies on epigenetic regulators. Here we describe a non-biased approach to probe epigenetic vulnerabilities in acute myeloid leukaemia (AML), an aggressive haematopoietic malignancy that is often associated with aberrant chromatin states2. By screening a custom library of small hairpin RNAs (shRNAs) targeting known chromatin regulators in a genetically defined AML mouse model, we identify the protein bromodomain-containing 4 (Brd4) as being critically required for disease maintenance. Suppression of Brd4 using shRNAs or the small-molecule inhibitor JQ1 led to robust antileukaemic effects in vitro and in vivo, accompanied by terminal myeloid differentiation and elimination of leukaemia stem cells. Similar sensitivities were observed in a variety of human AML cell lines and primary patient samples, revealing that JQ1 has broad activity in diverse AML subtypes. The effects of Brd4 suppression are, at least in part, due to its role in sustaining Myc expression to promote aberrant self-renewal, which implicates JQ1 as a pharmacological means to suppress MYC in cancer. Our results establish small-molecule inhibition of Brd4 as a promising therapeutic strategy in AML and, potentially, other cancers, and highlight the utility of RNA interference (RNAi) screening for revealing epigenetic vulnerabilities that can be exploited for direct pharmacological intervention.

AML1-ETO (AE) is a fusion product of t(8;21) observed in 40% French-American-British M2 type of acute myeloid leukemia (AML). Clinical data suggest that Ras mutation is a frequent cooperating event in t(8;21) AML. Whether constitutively active Ras promotes leukemogenesis on the t(8;21) background has not been demonstrated experimentally. Here, we retrovirally expressed N-RasG12D in AE-expressing human hematopoieticcells to investigate cooperativity. The AE/N-RasG12D cultures were cytokine-independent, enriched for CD34 positivity, and possessed increased colony-forming and replating abilities. N-RasG12D expression led to Bcl-2 up-regulation and reduced apoptosis. Ectopic Bcl-2 expression also resulted in enhanced colony-forming and replating abilities but was insufficient to sustain cytokine independence. AE/N-RasG12D cells were more sensitive to Bcl-2 inhibition with ABT-737 than parent AE cells. Enhanced engraftment of AE/N-RasG12D cells was observed on intrafemoral injection into immunodeficient mice, presumably because of improved survival in the bone marrow microenvironment. N-RasG12D promotes progression toward transfor-mation in AE-expressing cells, partially through up-regulating Bcl-2.

Introduction Chromosomal translocations involving the Mixed Lineage Leukemia (MLL, also called KMT2A) gene account for around 80% of the acute lymphoid leukemia (ALL) and 35%-50% of the acute myeloid leukemia (AML) diagnosed in infants, and are associated with poor outcomes. Despite improvements of conventional chemotherapies and treatments, patients with MLL-rearranged leukemia have weak response to treatment and poor prognosis. There is thus an urgent need to understand the molecular pathogenesis of these leukemias to develop more effective therapies. Gene expression studies show that MLL-leukemias uniquely display over-expression of Lysosome-associated Membrane Protein 5 (LAMP5) in both ALL and AML In normal hematopoiesis, LAMP5 expression is restricted to non-activated plasmacytoid dendritic cells (pDC), where it has been shown to be required for transport of TLR9 from VAMP3+/LAMP2+/LAMP1- endolysosomal vesicles to the LAMP1+ late lysosomal compartments, modulating the signaling and production of Type I interferon to TNF production. Here, we tested the hypothesis that LAMP5 is required for MLL leukemia wherein it modulates innate immune signaling. Methods and Results In previous studies, we found LAMP5 expression being restricted to MLL-fusion leukemia cell lines. Additionally, in an inducible model, LAMP5 expression directly correlated with that of the MLL-fusion protein MLL-AF9. . Knockdown on LAMP5 in MLL-fusion leukemia cells inhibited their propagation both in vitro and in vivo whereas the non-MLL leukemias were unaffected. To determine the mechanisms by which LAMP5 promotes growth in MLL leukemias, first we studied the localization of LAMP5 in these cells. Using immunofluorescent confocal microscopy we detected LAMP5 mainly in LAMP2+/LAMP1+ compartments. Additionally, LAMP5 co-localized with MYD88, a known scaffold protein required for IL1R/TLR signaling. Accordingly, LAMP5-knockdown abrogated TLR signaling as evidenced by reduced activation of JNK, MAPK p38, IRF7 and NFKB. Conversely, over-expression of LAMP5 in the non-MLL leukemia cell line Kasumi-1 led to increased activation of JNK, p38, IRF7 and NFKB, indicating that the innate-immune signaling pathway is regulated by LAMP5 expression in leukemias.r Conclusions Collectively, these results demonstrate that LAMP5 is required for the survival of MLL leukemia and that it plays an important role in the activation of the IL1/TLR signaling pathway. Overall, based on our results and the limited expression in normal hematopoiesis, we propose that LAMP5 could potentially serve as a therapeutic target with a wide therapeutic-window to treat MLL-leukemias. Disclosures No relevant conflicts of interest to declare.

Despite marked clinical successes in the treatment of childhood T-ALL, leukemia relapse, refractory disease and induction failure (around 30% of patients) remain significant clinical problems. New insights in the molecular alterations of ALL have revealed new molecular targets, such as activating mutations of Notch1 (N1), identified in more than 50% of T-ALL patients. However, disruption of N1 signaling by gamma-secretase inhibitors failed to fulfill its clinical promise. Furthermore, little is known about N1 downstream mediators that can be potential therapeutic targets in T-ALL.

Dominant RUNX1 inhibition has been proposed as a common pathway for CBF leukemia. CBF beta-SMMHC, a fusion protein in human acute myeloid leukemia (AML), dominantly inhibits RUNX1 largely through its RUNX1 high-affinity binding domain (HABD). However, the type I CBF beta-SMMHC fusion in AML patients lacks HABD. Here, we report that the type I CBF beta-SMMHC protein binds RUNX1 inefficiently. Knockin mice expressing CBF beta-SMMHC with a HABD deletion developed leukemia quickly, even though hematopoietic defects associated with Runx1-inhibition were partially rescued. A larger pool of leukemia-initiating cells, increased MN1 expression, and retention of RUNX1 phosphorylation are potential mechanisms for accelerated leukemia development in these mice. Our data suggest that RUNX1 dominant inhibition may not be a critical step for leukemogenesis by CBF beta-SMMHC.

N6-methyladenosine (m6A), the most prevalent internal modification in eukaryotic messenger RNAs (mRNAs), plays critical roles in many bioprocesses. However, its functions in normal and malignant hematopoiesis remain elusive. Here, we report that METTL14, a key component of the m6A methyltransferase complex, is highly expressed in normal hematopoietic stem/progenitor cells (HSPCs) and acute myeloid leukemia (AML) cells carrying t(11q23), t(15;17), or t(8;21) and is downregulated during myeloid differentiation. Silencing of METTL14 promotes terminal myeloid differentiation of normal HSPCs and AML cells and inhibits AML cell survival/proliferation. METTL14 is required for development and maintenance of AML and self-renewal of leukemia stem/initiation cells (LSCs/LICs). Mechanistically, METTL14 exerts its oncogenic role by regulating its mRNA targets (e.g., MYB and MYC) through m6A modification, while the protein itself is negatively regulated by SPI1. Collectively, our results reveal the SPI1-METTL14-MYB/MYC signaling axis in myelopoiesis and leukemogenesis and highlight the critical roles of METTL14 and m6A modification in normal and malignant hematopoiesis.

MicroRNA (miRNA)- 17-92 cluster (miR-17-92), containing seven individual miRNAs, is frequently amplified and overexpressed in lymphomas and various solid tumors. We have found that it is also frequently amplified and the miRNAs are aberrantly overexpressed in mixed lineage leukemia ( MLL )-rearranged acute leukemias. Furthermore, we show that MLL fusions exhibit a much stronger direct binding to the locus of this miRNA cluster than does wild-type MLL; these changes are associated with elevated levels of histone H3 acetylation and H3K4 trimethylation and an up-regulation of these miRNAs. We further observe that forced expression of this miRNA cluster increases proliferation and inhibits apoptosis of human cells. More importantly, we show that this miRNA cluster can significantly increase colony-forming capacity of normal mouse bone marrow progenitor cells alone and, particularly, in cooperation with MLL fusions. Finally, through combinatorial analysis of miRNA and mRNA arrays of mouse bone marrow progenitor cells transfected with this miRNA cluster and/or MLL fusion gene, we identified 363 potential miR-17-92 target genes that exhibited a significant inverse correlation of expression with the miRNAs. Remarkably, these potential target genes are significantly enriched ( P < 0.01; >2-fold) in cell differentiation, hematopoiesis, cell cycle, and apoptosis. Taken together, our studies suggest that overexpression of miR-17-92 cluster in MLL -rearranged leukemias is likely attributed to both DNA copy number amplification and direct up-regulation by MLL fusions, and that the miRNAs in this cluster may play an essential role in the development of MLL -associated leukemias through inhibiting cell differentiation and apoptosis, while promoting cell proliferation, by regulating relevant target genes.

Rho family GTPases are intracellular signaling proteins regulating multiple pathways involved in cell actomyosin organization, adhesion, and proliferation. Our knowledge of their cellular functions comes mostly from previous biochemical studies that used mutant overexpression approaches in various clonal cell lines. Recent progress in understanding Rho GTPase functions in blood cell development and regulation by gene targeting of individual Rho GTPases in mice has allowed a genetic understanding of their physiologic roles in hematopoietic progenitors and mature lineages. In particular, mouse gene–targeting studies have provided convincing evidence that individual members of the Rho GTPase family are essential regulators of cell type–specific functions and stimuli-specific pathways in regulating hematopoietic stem cell interaction with bone marrow niche, erythropoiesis, and red blood cell actin dynamics, phagocyte migration and killing, and T- and B-cell maturation. In addition, deregulation of Rho GTPase family members has been associated with multiple human hematologic diseases such as neutrophil dysfunction, leukemia, and Fanconi anemia, raising the possibility that Rho GTPases and downstream signaling pathways are of therapeutic value. In this review we discuss recent genetic studies of Rho GTPases in hematopoiesis and several blood lineages and the implications of Rho GTPase signaling in hematologic malignancies, immune pathology. and anemia.

Summary 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 6 -methyladenosine (m 6 A) RNA modification in R-2HG-sensitive leukemia cells, which in turn decreases the stability of MYC/CEBPA transcripts, leading to the suppression of relevant pathways. Ectopically expressed mutant IDH1 and S-2HG recapitulate the effects of R-2HG. High levels of FTO sensitize leukemic cells to R-2HG, whereas hyperactivation of MYC signaling confers resistance that can be reversed by the inhibition of MYC signaling. R-2HG also displays anti-tumor activity in glioma. Collectively, while R-2HG accumulated in IDH1/2 mutant cancers contributes to cancer initiation, our work demonstrates anti-tumor effects of 2HG in inhibiting proliferation/survival of FTO -high cancer cells via targeting FTO/m 6 A/MYC/CEBPA signaling.

Acute myeloid leukemia (AML) is one of the most common and fatal forms of hematopoietic malignancies. Over 70% of patients with AML cannot survive over 5 years. Many AML subtypes, such as the MLL -rearranged AMLs, are often associated with unfavorable outcome. Current treatment frequently involves intensive chemotherapy, which impairs the quality of life of the patients. While the incidence of AML is continually rising due to aging, most elder patients cannot bear intensive chemotherapy and are associated with very poor survival. Thus, improved therapeutic strategies with less intensive treatment but a higher cure rate are urgently needed. The Ten-eleven translocation (TET) proteins (including TET1/2/3) are known to be able to convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), leading to DNA demethylation. In contrast to the repression and tumor-suppressor role of TET2 observed in hematopoietic malignancies, we recently showed that TET1, the founding member of the TET family, was significantly up-regulated in MLL -rearranged AML and played an essential oncogenic role. An independent study by Zhao et al. confirmed the essential oncogenic role of Tet1 in the development of myeloid malignancies. Thus, TET1 is an attractive therapeutic target for AML. In order to identify chemical compounds that may target TET1 signaling, we searched the drug-sensitivity/gene expression database of a total of 20,602 chemical compounds in the NCI-60 collection of cancer cell samples. The expression levels of endogenous TET1 showed a significant positive correlation with the responsiveness of cancer cells across the NCI-60 panel to 953 compounds (r > 0.2; P Further, secondary bone marrow transplantation followed by drug treatment was carried out to test the in vivo therapeutic effects of NSC-X1 and NSC-X2. Both candidate compounds significantly inhibited MLL-AF9 -induced AML, by prolonging the median survival from 49 days (control) to 94 (NSC-X1) or >200 (NSC-X2) days. Notably, 57% of the NSC-X2 treated mice were cured. In another AML model induced by AML-ETO9a (AE9a), NSC-X1 and NSC-X2 also exhibited remarkable therapeutic effects, with an elongated median survival from 46 days (control) to 95 (NSC-X1) and 122 (NSC-X2) days, respectively. To decipher the molecular mechanism by which NSC-X2 represses TET1 expression, we treated THP-1 AML cells with moderate to high concentration of NSC-X2 for over 100 days and then isolated a set of individual drug-resistant THP-1 single clones. Through RNA-seq of 6 of the NSC-X2-resistant clones, recurrent mutations were found in 14 genes, including JAK1 . Ingenuity pathway analysis (IPA) was used to analyze biological relationships amongst the 14 mutated genes. The top one network identified by IPA involving all of the 14 genes is closely associated with the JAK/STAT5 pathway. Results of chromatin immunoprecipitation (ChIP) assays showed TET1 is one of the direct downstream gene targets of STAT3 and STAT5. Through NMR chemical shift perturbation (CSP) and electrophoretic mobility shift assays (EMSAs), we showed a strong direct association between STAT3/5 DNA binding domain and the TET1 promoter, which could be severely interrupted by NSC-X2. Compared to currently available JAK/STAT inhibitors (e.g., Pacritinib, KW-2449, Stattic, and sc-355979), our compounds (NSC-370284 and UC-514321) exhibit a much higher selectivity and also a higher efficacy in targeting TET1 -high AML. Taken together, we identified chemical compounds NSC-X1 and especially, NSC-X2, as potent inhibitors that significantly and selectively suppress the viability of AML cells with high level of TET1 expression, and dramatically repress the progression of TET1 -high AML in mice. NSC-X2 directly binds STAT3/5 as STAT inhibitors and thereby suppress TET1 transcription and TET1 signaling, leading to potent anti-leukemic effects. Our results highlight the therapeutic potential of targeting the STAT/TET1 axis by selective inhibitors in AML treatment. Disclosures No relevant conflicts of interest to declare.

Rac1, a member of the Rho family of GTPases, is an intracellular transducer known to regulate multiple signaling pathways that control cytoskeleton organization, transcription, and cell proliferation. Deregulated expression or activation patterns of Rac1 can result in aberrant cell signaling and numerous pathological conditions. Here, we highlight the physiological functions and signaling mechanisms of Rac1 and their relevance to disease.

The t(8;21) rearrangement, which creates the AML1-ETO fusion protein, represents the most common chromosomal translocation in acute myeloid leukemia (AML). Clinical data suggest that CBL mutations are a frequent event in t(8;21) AML, but the role of CBL in AML1-ETO-induced leukemia has not been investigated. In this study, we demonstrate that CBL mutations collaborate with AML1-ETO to expand human CD34+ cells both in vitro and in a xenograft model. CBL depletion by shRNA also promotes the growth of AML1-ETO cells, demonstrating the inhibitory function of endogenous CBL in t(8;21) AML. Mechanistically, loss of CBL function confers hyper-responsiveness to thrombopoietin and enhances STAT5/AKT/ERK/Src signaling in AML1-ETO cells. Interestingly, we found the protein tyrosine phosphatase UBASH3B/Sts-1, which is known to inhibit CBL function, is upregulated by AML1-ETO through transcriptional and miR-9-mediated regulation. UBASH3B/Sts-1 depletion induces an aberrant pattern of CBL phosphorylation and impairs proliferation in AML1-ETO cells. The growth inhibition caused by UBASH3B/Sts-1 depletion can be rescued by ectopic expression of CBL mutants, suggesting that UBASH3B/Sts-1 supports the growth of AML1-ETO cells partly through modulation of CBL function. Our study reveals a role of CBL in restricting myeloid proliferation of human AML1-ETO-induced leukemia, and identifies UBASH3B/Sts-1 as a potential target for pharmaceutical intervention.