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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 WileySons, Ltd.

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.

In this issue of Blood , [Ellegast et al][1] demonstrate that MISTRG mice allow high-level, reproducible engraftment of favorable prognosis acute myeloid leukemia (AML), a group that has been recalcitrant to attempts to develop valuable preclinical patient-derived xenograft (PDX) models.[1][2] Over

Transgenic expression of key myelosupportive human cytokines in immune-deficient mice corrects for the lack of cross-species activities of stem cell factor (SCF), IL-3, and GM-CSF. When engrafted with human umbilical cord blood (UCB), these triple-transgenic mice produce BM and spleen grafts with much higher myeloid composition, relative to nontransgenic controls. Shortly after engraftment with UCB, these mice develop a severe, fatal macrophage activation syndrome (MAS) characterized by a progressive drop in rbc numbers, increased reticulocyte counts, decreased rbc half-life, progressive cytopenias, and evidence of chronic inflammation, including elevated human IL-6. The BM becomes strikingly hypocellular, and spleens are significantly enlarged with evidence of extramedullary hematopoiesis and activated macrophages engaged in hemophagocytosis. This manifestation of MAS does not respond to lymphocyte-suppressive therapies such as steroids, i.v. immunoglobulin, or antibody-mediated ablation of human B and T cells, demonstrating a lymphocyte-independent mechanism of action. In contrast, elimination of human myeloid cells using gemtuzumab ozogamicin (anti-CD33) completely reversed the disease. Additionally, the IL-6R antibody tocilizumab delayed progression and prolonged lifespan. This new model of MAS provides an opportunity for investigation of the mechanisms driving this disease and for the testing of directed therapies in a humanized mouse.

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.

Chromosomal translocation 8;21 is found in 40% of the FAB M2 subtype of acute myeloid leukemia (AML). The resultant in-frame fusion protein AML1-ETO (AE) acts as an initiating oncogene for leukemia development. AE immortalizes human CD34(+) cord blood cells in long-term culture. We assessed the transforming properties of the alternatively spliced AE isoform AE9a (or alternative splicing at exon 9), which is fully transforming in a murine retroviral model, in human cord blood cells. Full activity was realized only upon increased fusion protein expression. This effect was recapitulated in the AE9a murine AML model. Cotransduction of AE and AE9a resulted in a strong selective pressure for AE-expressing cells. In the context of AE, AE9a did not show selection for increased expression, affirming observations of human t(8;21) patient samples where full-length AE is the dominant protein detected. Mechanistically, AE9a showed defective transcriptional regulation of AE target genes that was partially corrected at high expression. Together, these results bring an additional perspective to our understanding of AE function and highlight the contribution of oncogene expression level in t(8;21) experimental models.

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.

Despite the improvement of chemotherapy and targeted therapy, drug resistance still remains a challenge for long term disease free survival in aggressive leukemia patients. Recently, enhanced glycolysis is observed in acute myeloid leukemia (AML), and in association with poor clinical outcomes and chemoresistance. The byproducts of glycolysis include lactate and protons (H+), which contribute to intracellular acidosis. The extrusion of protons further results in extracellular acidosis. A group of G protein-coupled receptors (GPCRs), including GPR4, GPR65 (TDAG8), GPR68 (OGR1) and GPR132 (G2A), have been demonstrated to respond to extracellular acidosis, resulting in activation of downstream signaling pathways that regulate pleotropic cellular processes. However, it remains unclear whether these proton-sensing GPCRs contribute to the etiology of AML. Here, we performed genomic examination of leukemia (via cBioPortal). Among 660 leukemia patients, only one patient exhibited deletion of GPR132. Other than this single case, we found no genetic mutations or cytogenetic abnormalities pertaining to proton-sensing GPCRs. Examination of transcripts of these proton-sensing GPCRs revealed that GPR68 was upregulated in both pediatric and adult AML. AML patients with higher levels of GPR68 were associated with shorter overall survival. To understand the function of GPR68 in AML, we knocked down GPR68 in AML cell lines with shRNA targeting GPR68 (shGPR68). GPR68 knockdown markedly induced apoptosis, and reduced colony formation and proliferation in AML cells. This result indicates that myeloid malignancies acquire a dependency on GPR68 function. In response to extracellular H+ or overexpression, GPR68 activates Ca2+ pathway. To determine the molecular mechanism by which GPR68 overexpression supports leukemia cell growth and survival, we examined the intracellular Ca2+ levels (i.e. [Ca2+]i) in primary AML samples. Compared with CD34+ normal hematopoietic cells, all primary AML specimens tested exhibited increased [Ca2+]i, consistent with GPR68 overexpression in AML cells. Meanwhile, shGPR68 reduced [Ca2+]i in all AML cell lines tested, indicating that overexpressed GPR68 activates the Ca2+ pathway in AML. Given that enhanced glycolysis leads to extracellular acidosis, we tested whether glycolysis-mediated local acidosis could also explain enhanced GPR68 activation in AML. Indeed, inhibition of glycolysis by 2-deoxyglucose (2-DG) reduced [Ca2+]i in most of the AML cell lines tested, indicating that glycolysis is likely responsible for enhanced GPR68 activation in AML as well. Next, we attempted to identify the Ca2+-dependent molecular mechanism that mediates the prosurvival effects due to GPR68 activation. We screened a series of pharmacological inhibitors for their efficacy in reducing cell growth and inducing apoptosis. Among the inhibitors tested, only a calcineurin (CaN) inhibitor, Cyclosporine, dramatically reduced cell growth and induced apoptosis in AML cells. This finding raises the possibility that GPR68 promotes AML cell survival through activating the Gq/11/Ca2+/CaN pathway. In summary, we find that the myeloid malignancies acquire a dependency on GPR68 signaling pathway, and inhibition of GPR68 might provide a novel therapeutic strategy for AML, especially in those developing chemoresistance. Disclosures No relevant conflicts of interest to declare.

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.

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., Ten-eleven translocation 1 (TET1) is a critical oncoprotein in AML. Here, the authors identify 2 compounds that target the binding of STAT3/5 specifically to the TET1 promoter, inhibiting its expression and AML cell viability.

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.

The t(16:16) and inv(16) are associated with FAB M4Eo myeloid leukemias and result in fusion of the CBFB gene to the MYH11 gene (encoding smooth muscle myosin heavy chain [SMMHC]). Knockout of CBFβ causes embryonic lethality due to lack of definitive hematopoiesis. Although knock-in of CBFB-MYH11 is not sufficient to cause disease, expression increases the incidence of leukemia when combined with cooperating events. Although mouse models are valuable tools in the study of leukemogenesis, little is known about the contribution of CBFβ-SMMHC to human hematopoietic stem and progenitor cell self-renewal. We introduced the CBFβ-MYH11 cDNA into human CD34+ cells via retroviral transduction. Transduced cells displayed an initial repression of progenitor activity but eventually dominated the culture, resulting in the proliferation of clonal populations for up to 7 months. Long-term cultures displayed a myelomonocytic morphology while retaining multilineage progenitor activity and engraftment in NOD/SCID-B2M-/- mice. Progenitor cells from long-term cultures showed altered expression of genes defining inv(16) identified in microarray studies of human patient samples. This system will be useful in examining the effects of CBFβ-SMMHC on gene expression in the human preleukemic cell, in characterizing the effect of this oncogene on human stem cell biology, and in defining its contribution to the development of leukemia.

Hematopoiesis is a complex process involving hematopoietic stem cell (HSC) self-renewal and lineage commitment decisions that must continue throughout life. Establishing a reproducible technique that allows for the long-term ex vivo expansion of human HSCs and maintains self-renewal and multipotential differentiation will allow us to better understand these processes, and we report the ability of the leukemia-associated AML1-ETO fusion protein to establish such a system. AML1-ETO-transduced human CD34+ hematopoietic cells routinely proliferate in liquid culture for more than 7 months, remain cytokine dependent for survival and proliferation, and demonstrate self-renewal of immature cells that retain both lymphoid and myeloid potential in vitro. These cells continue to express the CD34 cell surface marker and have ongoing telomerase activity with maintenance of telomere ends, however they do not cause leukemia in nonobese diabetic-severe combined immunodeficiency (NOD/SCID) mice. Identification of the signaling pathways that are modulated by AML1-ETO and lead to the self-renewal of immature human progenitor cells may assist in identifying compounds that can efficiently expand human stem and progenitor cells ex vivo. (Blood. 2003; 102:4369-4376)

The CCAAT/enhancer binding protein alpha (C/EBPalpha) belongs to a family of transcription factors that are involved in the differentiation process of numerous tissues, including the liver and hematopoietic cells. C/EBPalpha(-/-) mice show a block in hematopoietic differentiation, with an accumulation of myeloblasts and an absence of mature granulocytes, whereas expression of C/EBPalpha in leukemia cell lines leads to granulocytic differentiation. Recently, dominant-negative mutations in the C/EBPalpha gene and down-regulation of C/EBPalpha by AML1-ETO, an AML associated fusion protein, have been identified in acute myelogenous leukemia (AML). To better understand the role of C/EBPalpha in the lineage commitment and differentiation of hematopoietic progenitors, we transduced primary human CD34(+) cells with a retroviral construct that expresses the C/EBPalpha cDNA fused in-frame with the estrogen receptor ligand-binding domain. Induction of C/EBPalpha function in primary human CD34(+) cells, by the addition of beta-estradiol, leads to granulocytic differentiation and inhibits erythrocyte differentiation. Using Affymetrix (Santa Clara, CA) oligonucleotide arrays we have identified C/EBPalpha target genes in primary human hematopoietic cells, including granulocyte-specific genes that are involved in hematopoietic differentiation and inhibitor of differentiation 1 (Id1), a transcriptional repressor known to interfere with erythrocyte differentiation. Given the known differences in murine and human promoter regulatory sequences, this inducible system allows the identification of transcription factor target genes in a physiologic, human hematopoietic progenitor cell background.

The model systems available for studying human hematopoiesis, malignant hematopoiesis, and hematopoietic stem cell (HSC) function in vivo have improved dramatically over the last decade, primarily due to improvements in xenograft mouse strains. Several recent reviews have focused on the historic development of immunodeficient mice over the last 2 decades, as well as their use in understanding human HSC and leukemia stem cell (LSC) biology and function in the context of a humanized mouse. However, in the intervening time since these reviews, a number of new mouse models, technical approaches, and scientific advances have been made. In this review, we update the reader on the newest and best models and approaches available for studying human malignant and normal HSCs in immunodeficient mice, including newly developed mice for use in chemotherapy testing and improved techniques for humanizing mice without laborious purification of HSC. We also review some relevant scientific findings from xenograft studies and highlight the continued limitations that confront researchers working with human HSC and LSC in vivo.

Some cancers, like acute myeloid leukemia (AML), use reactive oxygen species to endogenously activatecell proliferation and angiogenic signaling cascades. Thus many cancers display increases in reactiveoxygen like hydrogen peroxide concentrations. To translate this finding into a therapeutic strategy wedesigned new hydrogen peroxide-activated agents with two key molecular pharmacophores. The firstpharmacophore is a peroxide-acceptor and the second is a pendant amine. The acceptor is anN-(2,5-dihydroxyphenyl)acetamide susceptible to hydrogen peroxide oxidation. We hypothesized thatselectivity between AML and normal cells could be achieved by tuning the pendant amine. Synthesisand testing of fourteen compounds that differed at the pendent amine led to the identification of an agent(14) with 2 l M activity against AML cancer cells and an eleven fold-lower activity in healthy CD34+ bloodstem cells. Interestingly, analysis shows that upon oxidation the pendant amine cyclizes, ejecting water,with the acceptor to give a bicyclic compound capable of reacting with nucleophiles. Preliminarymechanistic investigations show that AML cells made from addition of two oncogenes (NrasG12D andMLL-AF9) increase the ROS-status, is initially an anti-oxidant as hydrogen peroxide is consumed toactivate the pro-drug, and cells respond by upregulating electrophilic defense as visualized by Westernblotting of KEAP1. Thus, using this chemical approach we have obtained a simple, potent, and selectiveROS-activated anti-AML agent.! 2014 Elsevier Ltd. All rights reserved.

Immunodeficient mice are now readily engrafted with human hematopoietic cells. However, these mice are susceptible to graft-versus-host disease (GVHD) induced by the engraftment and rapid expansion of coinjected human T cells. Therefore, highly purified sample populations must be used, adding significant time, expense, and effort. Here, we have explored in vivo and in vitro methods utilizing anti-T-cell antibodies to circumvent this problem. Intraperitoneal injection of the antibody within 48 hours prevented GVHD. Alternatively, short-term in vitro incubation of cells with antibody immediately before transplant was equally effective. Although in vitro antithymocyte globulin treatment resulted in a dramatic loss of SCID-repopulating cells (SRCs), treatment with OKT3 or UCHT1 abrogated GVHD risk and preserved engraftment potential. Leukemia samples that presented with substantial human T-cell contamination were effectively rescued from GVHD. In addition, OKT3 treatment of unfractionated cord blood resulted in robust engraftment of primary and secondary mice that was indistinguishable from grafts obtained using purified CD34(+) cells. Limiting dilution analysis of unfractionated blood demonstrated a SRC frequency of 1 in 300 to 500 CD34(+) cells, similar to that of purified hematopoietic stem and progenitor cells. This protocol streamlines xenograft studies while significantly reducing the cost and time of the procedure.

RUNX1/CBFβ (core binding factor [CBF]) is a heterodimeric transcription factor complex that is frequently involved in chromosomal translocations, point mutations, or deletions in acute leukemia. The mixed lineage leukemia (MLL) gene is also frequently involved in chromosomal translocations or partial tandem duplication in acute leukemia. The MLL protein interacts with RUNX1 and prevents RUNX1 from ubiquitin-mediated degradation. RUNX1/CBFβ recruits MLL to regulate downstream target genes. However, the functional consequence of MLL fusions on RUNX1/CBFβ activity has not been fully understood. In this report, we show that MLL fusion proteins and the N-terminal MLL portion of MLL fusions downregulate RUNX1 and CBFβ protein expression via the MLL CXXC domain and flanking regions. We confirmed this finding in Mll-Af9 knock-in mice and human M4/M5 acute myeloid leukemia (AML) cell lines, with or without MLL translocations, showing that MLL translocations cause a hypomorph phenotype of RUNX1/CBFβ. Overexpression of RUNX1 inhibits the development of AML in Mll-Af9 knock-in mice; conversely, further reducing Runx1/Cbfβ levels accelerates MLL-AF9–mediated AML in bone marrow transplantation assays. These data reveal a newly defined negative regulation of RUNX1/CBFβ by MLL fusion proteins and suggest that targeting RUNX1/CBFβ levels may be a potential therapy for MLLs.

Uncontrolled cell proliferation is a hallmark of cancer and requires adequate nucleotide biosynthesis. Inosine monophosphate dehydrogenase (IMPDH) is a rate-limiting enzyme that catalyzes the conversion of IMP to XMP at the branch point of guanine nucleotide biosynthesis. Inhibition of IMPDH results in the depletion of guanine nucleotides, thereby suppressing the growth of cancers cells. Therefore, IMPDH inhibition constitutes a rational approach to treat cancers including acute myeloid leukemia (AML). We have developed an experimental system to model myeloid leukemogenesis using primary human cord blood (CB) cells. AML1-ETO is a leukemogenic fusion protein and promotes the self-renewal and long-term proliferation of CB cells in vitro. Another fusion protein MLL-AF9 immortalizes CB cells in vitro and produces human leukemia in immunodeficient mice. These engineered pre-leukemic and leukemic cells recapitulate many features of the clinical diseases and have been useful in testing potential anti-leukemic drugs. Using these human cell-based models, we assessed the effect of a specific IMPDH inhibitor, mycophenolic acid (MPA), on CB cells and those expressing AML1-ETO and MLL-AF9. MPA showed substantial growth-inhibitory effect against MLL-AF9-expressing CB cells, while it had only marginal effects on normal CB cells and AML1-ETO cells. Mechanistically, MPA treatment caused a cell cycle arrest at G0/G1-phase, triggered apoptosis, and induced upregulation of p53 and its downstream target genes including p21/CDKN1A in MLL-AF9 cells. The MPA-mediated changes in MLL-AF9 cells were almost fully reversed by the supplementation of guanosine, confirming that guanine nucleotide depletion underlies the effects of MPA. In contrast, knockdown of p53 or p21, or expression of a dominant-negative form of p53 (p53-DD), did not abrogate the growth-inhibitory effect of MPA on MLL-AF9 cells, indicating that MPA inhibits the leukemic growth largely through p53-independent mechanisms. To examine whether MPA has single-agent activity for MLL-fusion leukemia in vivo, we next established a mouse bone marrow transplant assay for MLL-AF9 leukemia. Bone marrow progenitors were transduced with MLL-AF9 and were transplanted into recipient mice. MLL-AF9-expressing bone marrow progenitors produced AML within 3 months. Leukemic cells were isolated from the spleens of moribund primary mice, and were transplanted into secondary recipient mice. These mice were treated either with injections of MPA (100 mg kg−1) every other day or with vehicle. MPA administration led to a delay in disease progression and significantly extended survival. We also tested the in vivo effect of another IMPDH inhibitor FF-10501-01 on MLL-AF9 leukemia. FF-10501-01 is a potent new competitive IMPDH inhibitor undergoing phase I clinical trials for patients with AML and high-risk myelodysplastic syndrome (MDS). As expected, FF-10501-01 also showed significant therapeutic value, providing survival benefit in a mouse MLL-AF9 leukemia model. Finally, we assessed the sensitivity of p53-deficient mouse MLL-AF9 leukemia cells to IMPDH inhibition. We generated p53-deficient leukemia cells by expressing MLL-AF9 into bone marrow progenitors derived from p53 knockout mice. Consistent with earlier results, the p53-deficient MLL-AF9 cells were still sensitive to MPA and FF-10501-01 both in vitro and in vivo, indicating that p53 activation is dispensable for the anti-leukemia effect of these IMPDH inhibitors. Taken together, these findings establish the inhibition of IMPDH as a promising therapy for MLL-fusion leukemia, including those with defective p53 signaling. Disclosures Goyama: Fuji Film: Research Funding. Iwamura:FUJIFILM Corporation: Employment. Saito:Fuji Film: Employment.

Macrophage Activation Syndrome (MAS) is a form of secondary hemophagocytic lymphohistiocytosis (HLH) that frequently complicates various rheumatoid disorders. Similar to HLH, these disorders present with chronic inflammation and immune dysfunction. Most typically, an identifiable infection triggers an immune response that is either ineffective due to genetic inactivating mutations that affect lymphocyte cytotoxicity, as in primary HLH, or uncontrolled and characterized by expansion of activated T and macrophage cells as in the case of MAS. While lymphocyte function is generally thought to be a crucial aspect of the disease state, abnormal myeloid cell activation by genetic activation of inflammasomes or repeated TLR stimulation was recently shown to result in MAS. These studies have implicated several cytokines, including SCF, IL-3, and GM-CSF in the disease process. A lymphocyte-independent model could be useful to isolate the myeloid contribution to MAS and would allow the testing of additional therapeutic strategies that target the effector cells directly. Such novel therapies are needed to address refractory cases that often prove fatal. Transgenic expression of key myelosupportive human cytokines in immune-deficient mice corrects for the lack of cross species activities of SCF, IL-3, and GM-CSF. When engrafted with human umbilical cord blood (UCB), these triple transgenic mice produce BM and spleen grafts with much higher myeloid composition, relative to non-transgenontrols. After engraftment with UCB, these mice develop severe, fatal MAS characterized by a progressive drop in rbc numbers, increased reticulocyte counts, decreased rbc half-life, progressive cytopenias and evidence of chronic inflammation. The BM becomes strikingly hypocellular and spleens are significantly enlarged with evidence of extramedullary hematopoiesis. Activated macrophages engaged in hemophagocytosis are readily detectable in the spleen and liver. Mice also show elevated body temperature and sCD25, indicating the presence of activated lymphocytes. Treatment of mice with the CD52 inhibitor alemtuzumab eliminated the human graft and resolved MAS, implicating a human cell and demonstrating the reversibility of the phenotype. However, there was no response to lymphocyte suppressive therapies such as steroids, intravenous immunoglobulin, or antibody-mediated ablation of human B and T cells, demonstrating a lymphocyte independent mechanism of action. In contrast, elimination of human myeloid cells using gemtuzumab ozogamicin (anti-CD33) completely reversed the disease, demonstrating the utility of targeting the effector macrophage cells directly in this model. Using a multiplex ELISA assay, TNF alpha and IFN gamma were low to absent in affected mice, however, IL-6, IL-10, MIP-1 alpha, and MIP-1 beta were consistently upregulated. IL-6, MIP-1 alpha, and MIP-1 beta were lost after gemtuzumab ozogamicin therapy, but not after combined B/T cell ablation with rituximab and OKT-3. Interestingly, IL-10 and sCD25 was suppressed by either treatment. Since the IL-6R antibody tocilizumab is currently being evaluated in a clinical trial for HLH, we examined the efficacy of tocilizumab injections in our model and found delayed progression of anemia and a prolonged lifespan for affected mice. This novel model of MAS provides a new tool for the investigation of the mechanisms driving MAS and for the testing of myeloid-directed therapies in a human cell model. Disclosures No relevant conflicts of interest to declare.

Runx1, a DNA binding subunit of core binding factors, is found frequently mutated in hematological malignancies. Runx1 mutation can be an early event in leukemogenesis endowing pre-leukemic stem cells with a selective advantage in the bone marrow, and is associated with an unfavorable outcome. In mouse models, loss of function (LOF) Runx1 mutations cause a broad decrease of ribosome biogenesis in hematopoietic stem and progenitor cells (HSPCs) by directly binding to ribosomal related genes essential for protein synthesis, and confers resistance to genotoxic stress (Cai et al. 2015 Cell Stem Cell 17(2):165-77). Paradoxially, leukemia cells generally require higher biosynthetic activity, and AML patients with LOF Runx1 mutations show upregulated ribosome signatures compared with those without Runx1 mutations (Silva et al., 2009 Blood 114:3001-3007). It remains unclear whether RUNX1 plays a role in regulating protein synthesis in leukemogenesis as in normal HSPCs, and if LOF Runx1 mutations are important for leukemia initiation, transformation and/or maintenance. To examine such mechanistic roles of RUNX1 in AML progression, we have used a previously reported MLL-PTD; Mx Cre; Runx1 Flox/Flox (Double mutant -DM) mouse model (Hayashi et al., 2015 Blood 126:303 ) that allows experimental tracking of the step-wise transition of HSPCs from pre-disease stage to a MDS-like stage, prior to full blown AML. Various subpopulations of the HSPCs, including genotypic HSCs, MPP, GMP, CMP, MEP, were isolated from the mice at pre-disease, MDS-like, and AML full-blown stages, and were assayed for protein synthesis rates by O-propargyl puromycin incorporation, DNA synthesis rates by BrdU labeling, and FACS analysis. At the pre-disease state, DM HSCs, as well as all the progenitor populations, had lower protein biosynthesis activity compared with similar populations of wild-type control or MLL-PTD mutant mice, consistent with LOF Runx1 mutations providing stress-resistance and survival advantage. As disease progressed, the DM mice developed MDS-like phenotypes including severe anemia and bone marrow fibrosis, with the HSCs (LSK CD34-Flt3- cells) showing increased protein synthesis rate compared with the pre-disease DM mice. Upon the onset of full-blown leukemia, the protein translation rates in all subpopulations of DM HSPCs were significantly faster than the control non-leukemic cells, regardless of the Runx1 mutant status. Importantly, preliminary analyses of two human AML samples found that CD34+ cells with LOF Runx1 mutations displayed a similarly enhanced protein synthesis rates than CD34+ leukemia bone marrow cells carrying wild type Runx1, as seen in the mouse model. Our results show that at early initiation, LOF Runx1 mutation supresses protein biosynthesis; during transition to MDS, the inhibitory regulation was bypassed in LT-HSCs (LSK CD34-Flt3-), suggesting that Runx1-controlled protein translation is involved in the early clonal selection of disease progression. In full-blown leukemia cells including the primitive subpopulations, however the protein synthesis rate appears to become uncoupled from Runx1 regulation possibly due to an activation of compensatory machineries. This study of the role of Runx1 mutation in pre-leukemia cell progression to full blown leukemia raises the question that while some tumor initiating mutations such as LOF Runx1 mutations contribute to the tumor initiation and transformation process, they may not be essential for maintaining certain crucial leukemia cell phenotypes such as protein biosynthesis. Disclosures No relevant conflicts of interest to declare.

Acute leukemias with Mixed Lineage Leukemia gene (MLL, also called KMT2A) translocations are associated with poor outcomes. These leukemias are most frequently encountered in infants and as secondary malignancies, and can be either lymphoid or myeloid. In spite of aggressive treatments, including bone marrow transplantation, infants with MLL-leukemias face a grim prognosis, with predicted survival of only 20%. Novel, effective therapies are thus urgently needed. In search for molecular targets, we observed that MLL-leukemias uniquely display over-expression of Lysosome-associated Membrane Protein 5 (LAMP5, also known as C20orf103). This observation was consistent across several gene-expression-profiling studies and occurred in both ALL and AML. Moreover, data from the TCGA study on AML showed that patients with LAMP5 expression suffered worse prognosis compared to those lacking LAMP5. We first confirmed LAMP5 expression in human leukemia cell lines by immunoblot and RT-qPCR assays. We readily detected LAMP5 mRNA and protein in MLL-fusion leukemia cell lines (MV4;11, MOLM13, THP1, RS4;11, KOPN8), while no expression of LAMP5 was found in the non-MLL-cell lines (Kasumi, K562, REH). Published ChIP-seq studies on leukemia cell lines show the MLL-fusion protein directly bound at the promotor region of LAMP5. To validate that the MLL-fusion protein activates the expression of LAMP5, we transformed human CD34+ cord blood cells with an inducible (Tet-off) retrovirus carrying the MLL-AF9 fusion cDNA. In this system, addition of Doxycycline represses expression of the MLL-AF9 oncogene. We found that LAMP5 expression directly correlated with MLL-AF9 levels, with levels of both decreasing upon addition of Doxycycline. To investigate the role of LAMP5 in MLL-fusion leukemia, we studied the effect of shRNA-mediated knockdown. By screening several hairpin sequences, we identified one construct that efficiently inhibited LAMP5 expression in MLL-fusion leukemia cells but had no effect on LAMP5-negative cells, implying specificity. All MLL-fusion leukemia cell lines tested showed growth inhibition with LAMP5 knockdown. Specifically, growth of MV4;11, THP1 and MOLM13 cells was decreased by 69%, 73% and 80% respectively compared to controls (non-targeting shRNA). When cultured in semi-solid methylcellulose media for 10 days, LAMP-5-depleted MV4;11 cells formed significantly fewer colonies than control cells (64.3 ± 25.98 and 245.3 ± 27.42 colonies per 1000 cells respectively). To investigate the role of LAMP5 in leukemia-propagation in vivo, we transplanted control and LAMP5-depleted MV4;11 cells into Busulfan-conditioned immune-deficient (NRGS) mice (2x105cells/mouse). Preliminary results from bone marrow aspirates of transplanted mice at weeks 4 post-transplant showed abundant human leukemia cells in mice receiving control cells while the mice receiving LAMP5-knockdown cells showed near-absence of human leukemia cells. Collectively, these results demonstrate that LAMP5, a novel target of MLL-fusion proteins is required for the propagation of leukemia. In normal hematopoiesis, LAMP5 expression is restricted to non-activated plasmacytoid dendritic cells (pDC), where it localizes to the ER-Golgi intermediate compartment (ERGIC). In leukemia cells, using immunofluorescent confocal microscopy we detected LAMP5 in the perinuclear zones where it co-localized with ERGIC-53, a marker of the ERGIC compartment. While little is known about the functions of LAMP5 in normal pDC, studies suggest that it functions as a co-chaperone with UNC93B1, a known Toll-like Receptor (TLR) chaperone, to shuttle the TLRs to their respective locations in the plasma membrane or endosomes. In ongoing experiments, we are determining the functions of LAMP5 in leukemia, including its association with UNC93B1 and with the TLR-NFKB signaling pathway. Overall, based on our results and the limited expression in normal hematopoiesis, we postulate 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.

Leukemic stem cells (LSCs) are capable of limitless self-renewal and indefinitely propagating leukemia. Eradication of LSCs is the ultimate goal of treating acute myeloid leukemia (AML). Using a mouse model of AML induced by the MLL-fusion protein MLL-AF9, we recently showed that the combined loss of Runx1/Cbfb inhibited the development of leukemia in vivo (Goyama S…Mulloy JC. Transcription factor RUNX1 promotes survival of acute myeloid leukemia cells. Journal of Clinical Investigation 123(9): 3876-3888, 2013). However, LSC-enriched cells with immature surface phenotype (cKit+Gr1-) remained viable in Runx1/Cbfb-deleted MLL-AF9 cells, indicating that RUNX targeting may not eradicate the most immature LSCs. Gene expression analyses of Runx1/Cbfb-deleted MLL-AF9 cells revealed the upregulation of thrombin pathway genes including a thrombin-activatable receptor PAR-1. Interestingly, both overexpression and knockout of PAR-1 inhibit leukemogenesis but do so through distinct mechanisms. Similar to the effect of Runx1/Cbfb-depletion, PAR-1 overexpression induced 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 LSCs in vivo. 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 are a large number of LSCs, while a small numbers of LSCs required PAR-1 for their growth. Mechanistically, PAR-1 increased adhering properties of MLL-AF9 cells and promoted their engraftment to bone marrow. PAR-1-deficiency also reduced leukemogenicity of AML1-ETO-induced leukemia. Together, these data reveal a multifaceted role for PAR-1 in leukemogenesis, and highlight this receptor as a potential target to eradicate primitive LSCs in AML. Disclosures No relevant conflicts of interest to declare.

Chromosome rearrangements involving the Mixed Lineage Leukemia (MLL) gene on chromosome 11q23 account for 15-20% of acute lymphoid leukemia (ALL) and confer poor prognosis. Such rearrangements generate the MLL-fusion proteins, in which the N-terminus of the MLL protein mediating chromatin interactions is fused with one of more than 70 different partner proteins. Proteins that are frequently involved in MLL translocations, including AF4, ENL, AF9 and AF10, were identified as components of the super elongation complex (SEC) or DOT1L complex (an H3K79 histone methyltransferase). Based on these observations, a consensus model of MLL-fusion leukemogenesis has been proposed, which suggests that all fusion proteins bind to the targets of wildtype MLL and lead to the aberrant transcriptional elongation and H3K79 methylation via the recruitment of SEC or DOT1L complex and thus the uncontrolled activation of the target genes. Therefore, regardless of the nature of fusion partners, all MLL-fusion proteins work in a similar fashion by dysregulating the same pathways. Our group has successfully established xenograft models of MLL-AF4 and MLL-AF9 B-ALL using human CD34+ hematopoietic stem and progenitor cells transduced with FLAG-tagged MLL-Af4 or MLL-AF9, which faithfully recapitulate the clinical features of the disease. We generated MLL-Af4 and -AF9 ALL using matched units of human CD34+ cells to directly test the consensus model. Interestingly, although having the same genetic background, the immunophenotype of the two ALL are unique, with CD34 expressed only in MLL-Af4 but not -AF9 cells. The transcriptomes of the two ALL were analyzed by RNAseq and dysregulated genes were defined by comparison with the transcriptome of normal pro-B cells (p≤0.05, fold-change≥2). Strikingly, only 40% of MLL-Af4-regulated genes overlap with those of MLL-AF9. This transcriptome heterogeneity is mirrored in clinical samples, where the gene signature generated from our model leukemia can be utilized to accurately classify patient samples in unsupervised hierarchical clustering analysis, with MLL-AF4 patient samples readily distinguishable from MLL-AF9 samples. To identify the mechanisms accounting for this heterogeneity, we performed ChIP-seq analysis using anti-FLAG antibody to compare the chromatin occupancy of MLL-Af4 and MLL-AF9 in our model ALL cells. The MLL-Af4 ChIP-seq signal displayed a clear correlation with those of published MLL-AF4 ChIP-seq datasets from patient-derived cell lines, in the range of 70-90%, highlighting the faithfulness of our model. Surprisingly, MLL-Af4 shows a distinct genome-wide distribution compared to MLL-AF9, with only 20% of MLL-Af4 peaks and 35% of MLL-AF9 peaks overlapping. In contrast to MLL-Af4 which predominantly binds to promoter regions, MLL-AF9 has a relatively greater enrichment at intra- and inter-genic regions. Intriguingly, MLL-AF9 tends to bind at repetitive sequences in introns, suggesting these repetitive sequences may serve as regulatory elements for gene expression. Integration with RNAseq data reveals a significant association between differentially-expressed MLL-Af4 and -AF9 targets and specific chromatin binding of different MLL-fusion proteins. This data demonstrates that chromatin binding is not solely controlled by the MLL portion of the fusion protein and that differential target recognition of different fusion proteins is one molecular mechanism driving gene expression heterogeneity. To test whether distinct co-factor recruitment by MLL-fusions adds another layer regulating gene expression heterogeneity beyond DNA binding, we purified the core complexes of MLL-Af4 and -AF9 from ALL cells by anti-FLAG immunoprecipitation and analyzed by mass spectrometry. These experiments identified both common and fusion-specific interacting proteins. MLL-Af4 showed a higher affinity with SEC component EAF2 but a lower affinity with DOT1L compared to MLL-AF9, suggesting that MLL-fusions have distinct associations with complex components which may achieve differential gene regulation. In summary, our data question the consensus model of MLL-fusion leukemia and emphasize that MLL-fusion ALL is a heterogeneous disease. These findings have important implications for therapy development as each MLL-fusion leukemia could have its own Achilles' heel and customized therapy may need to be introduced for each type of disease. Disclosures No relevant conflicts of interest to declare.

Approximately 25% of all acute myeloid leukemias (AMLs) contain an internal tandem duplication (ITD) in the juxtamembranal region of FLT3. Even with standard chemotherapies, FLT3-ITD AML is associated with poor prognosis, with a five-year survival rate of approximately 15% as compared to 40% for wild-type FLT3 AML. Therefore, FLT3 inhibitors have been developed as a targeted treatment approach. Although the inhibitors show considerable efficacy in vitro and in animal models, clinical trials using FLT3 inhibitors as single agents have been underwhelming, with few complete remissions and a high rate of relapse and resistance. Thus, there is a need for a FLT3 inhibitor that increases complete remission rates and reduces relapse in FLT3-ITD AML patients. Through chemical and structure-activity relationship studies, we have identified a novel class of FLT3 tyrosine kinase inhibitors. We have previously shown that our current lead compound (CTDS-004) has excellent selectivity against the kinase activity of FLT3-ITD and FLT-ITD D835Y at a subnanomolar concentration (IC50 Our lead compound has promising pharmacokinetic and pharmacodynamics properties in vivo, therefore, we tested CTDS-004 in a xenograft model of human AML. NRGS mice (NOD.Cg-Rag1tm1MomIL2rgtm1WjlTg[CMV-IL3,CSF2,KITLG]1EAv/J) were transplanted with human MLL-AF9 FLT3-ITD leukemic cells (2 x 105 cells/mouse) via tail vein injection. Starting on day 10 post-engraftment, the mice were treated with CTDS-004 (30 mg/kg; n=5), AC220 (15 mg/kg; n=5), or PBS (n=5) via intraperitoneal injection on a cycle of five days of daily injection followed by two days of rest for the duration of the experiment. Kaplan-Meier survival analysis revealed that CTDS-004 is extremely effective at prolonging survival in the human FLT3-ITD AML xenograft model. There was a significant increase in median survival for CTDS-004 and AC220 treatment groups compared to the PBS control group (Median survival post-xenograft: PBS: 28 days, AC220: 54 days, CTDS-004: 53 days; p=0.0002). Taken together, these findings suggest our novel FLT3 inhibitor shows promise for the treatment of FLT3-ITD positive AML, and particularly for patients that have intrinsic and/or acquired resistance to FLT3 tyrosine kinase inhibitors. Disclosures No relevant conflicts of interest to declare.

Animal models that faithfully recapitulate clinical features of myelodysplastic syndrome (MDS) have been difficult to establish. MDS are hematologic disorders associated with ineffective hematopoiesis, blood cytopenias, myeloid dysplasia and an increased risk of acute myeloid leukemia (AML). Although transplantation of primary AML cells into immunocompromised mice has been met with much success, primary MDS patient samples exhibit poor engraftment with no evidence of disease in recipient animals.1–3 The engraftment of primary MDS samples is complicated further by frequent outgrowth of normal HSC clones.4 These challenges underscore the current limitations and inefficiencies of engrafting primary MDS cells. Recent improvements in engrafting primary MDS cells have been observed when transplanting immunophenotypically defined HSC from the marrow of MDS patients or when co-injecting stromal cells engineered to produce non-cross-reacting human cytokines.5,6 Unfortunately, even with the improved engraftment of primary MDS cells, mice do not succumb to features resembling human MDS, precluding the use of these models for pre-clinical testing. To circumvent the current limitations, we developed a model using immunocompromised recipient mice and a human MDS cell line (MDSL) derived from the non-leukemic phase of an MDS patient with refractory anemia-ringed sideroblasts.7–9 The MDSL line was derived as a subline of MDS92, and maintains factor dependency for cell growth, but has reduced differentiational capacity compared with MDS92.10 Herein, we report the successful engraftment of MDSL cells into NOD/SCID-IL2Rγ mice (NSG) and NSG-hSCF/hGM-CSF/hIL3 (NSGS) mice, and reproducible development of disease, including cytopenias, clonal expansion and host hematopoietic suppression. In addition, we show that the MDSL xenograft model is a useful tool for evaluating novel and existing therapeutics for MDS.

The human T-cell lymphotropic/leukemia virus type 1 (HTLV-1) genome spans approximately 9 kb and encodes the structural (gag and env) and enzymatic (reverse transcriptase, protease, and integrase) proteins (29) and, at the 3′ region, contains four different open reading frames (ORF; ORFsI to IV): ORFIII and ORFIV encode the viral transactivator p40 Tax, the p27Rex protein, a posttranscriptional regulator of RNA expression, and p21Rex, a protein with unknown function (8, 30). Singly and doubly spliced mRNA from the ORFI and the ORFII have been found in cultured T cells (1, 3, 10, 15) and macrophages infected by HTLV-1 (16), as well as in ex vivo samples of HTLV-1-infected individuals (1, 15). The singly spliced mRNA from the ORFI encodes a membrane-associated hydrophobic protein of 12 kDa (99 amino acids) (18) with two potential transmembrane regions, TM-1 and TM-2, and at least four putative SH3 binding motifs (PXXP) (8). To date, it has been difficult to assess ORFI protein(s) expression in infected cells; however, indirect evidence suggest its importance, since the ablation of the acceptor splice site for p12I-protein expression interferes with the ability of a biologically active HTLV-1 molecular clone to establish a persistent infection in the rabbit model (4). The p12I protein shares regions of genetic similarity with the E5 oncoprotein encoded by the bovine papillomavirus type 1 (27), enhances the E5 transforming ability (9) and, like E5, binds to the 16-kDa subunit of the vacuolar ATPase (9, 17). E5 forms dimers and binds to cellular receptors such as platelet-derived growth factor β, epithelial growth factors, and colony-stimulating factor receptors (6, 20, 22, 24). p12I specifically binds to the immature forms of both β and γc chains of the interleukin-2 receptor (IL-2R) and decreases their surface expression in transfected cells (21), presumably by retarding the translocation of the receptor chains from the endoplasmic reticulum compartment to the Golgi and subsequently to the plasma membrane. Since p12I interacts with both the β and γc chains of the IL-2R, we postulated that p12I may form dimers. Here, we demonstrate that p12I indeed forms dimers, providing a rational explanation for how the same region of p12I may interact with both IL-2R chains. In addition, we provide evidence that the p12I metabolic instability is mediated in part by ubiquitylation at a single Lys residue at position 88 and subsequent proteasomal degradation, as well as by destabilizing residues at its amino terminus. Finally, analysis of natural alleles of p12I in 53 cases of HTLV-1 infection suggests that the presence of a p12I allele carrying a lysine at position 88 could be important in tropical spastic paraparesis-HTLV-1-associated myelopathy (TSP-HAM).

Human T cell leukemia/lymphotropic virus type I (HTLV-I) induces adult T cell leukemia/lymphoma (ATLL). The mechanism of HTLV-I oncogenesis in T cells remains partly elusive. In vitro , HTLV-I induces ligand-independent transformation of human CD4 + T cells, an event that correlates with acquisition of constitutive phosphorylation of Janus kinases (JAK) and signal transducers and activators of transcription (STAT) proteins. However, it is unclear whether the in vitro model of HTLV-I transformation has relevance to viral leukemogenesis in vivo . Here we tested the status of JAK/STAT phosphorylation and DNA-binding activity of STAT proteins in cell extracts of uncultured leukemic cells from 12 patients with ATLL by either DNA-binding assays, using DNA oligonucleotides specific for STAT-1 and STAT-3, STAT-5 and STAT-6 or, more directly, by immunoprecipitation and immunoblotting with anti-phosphotyrosine antibody for JAK and STAT proteins. Leukemic cells from 8 of 12 patients studied displayed constitutive DNA-binding activity of one or more STAT proteins, and the constitutive activation of the JAK/STAT pathway was found to persist over time in the 2 patients followed longitudinally. Furthermore, an association between JAK3 and STAT-1, STAT-3, and STAT-5 activation and cell-cycle progression was demonstrated by both propidium iodide staining and bromodeoxyuridine incorporation in cells of four patients tested. These results imply that JAK/STAT activation is associated with replication of leukemic cells and that therapeutic approaches aimed at JAK/STAT inhibition may be considered to halt neoplastic growth.