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Chromosomal rearrangements of the lysine methyltransferase 2A (KMT2A or MLL) gene are observed in ;10% of all acute leukemias, with particularly high frequency (;80%) in infant acute lymphoblastic leukemia (ALL),1 where, despite aggressive chemotherapy, patients still experience poor outcome and long-term side effects.2 Mixed lineage leukemia (MLL) rearrangements (MLL-r)also indicate particularly poor outcomes for patients with acute myeloid leukemia (AML).3 Mechanistically, MLL-r frequently generates fusion proteins involving partners that function in the super elongation complex,4 the result of which is aberrant recruitment to MLL target genes of the positive transcription elongation factor b (PTEFb), composed of cyclin-dependent kinase 9 (CDK9)as the catalytic subunit.5 CDK9 positively regulates transcription elongation through phosphorylation of serine 2 of RNA polymerase II (RNAPII).6 Given the central role of CDK9 in the leukemic MLL-r gene-expression program,7 and the well-described ability of CDK9 inhibitors to reduce levelsof the short-lived prosurvival protein MCL1,8 a number of CDK9 inhibitors have been selected forclinical trials focusing on acute leukemias, including those with MLL-r.8,9 In MLL-r leukemia, the bromodomain and extraterminal (BET) family member bromodomain-containing 4 (BRD4)10 acts to recruit PTEFb to super enhancers and together with CDK9 drives increased expression of many oncogenes including MYC.11,12 The roles of CDK9 and BRD4 in MLL-r leukemias present a strong case for testing inhibitors of these proteins in combination as a potential treatment of MLL-r acute leukemias. Refereed/Peer-reviewed

Acute myeloid leukemia (AML) is an aggressive malignancy where despite improvements in conventional chemotherapy and bone marrow transplantation, overall survival remains poor. Sphingosine kinase 1 (SPHK1) generates the bioactive lipid sphingosine 1- phosphate (S1P) and has established roles in tumor initiation, progression, and chemotherapy resistance in a wide range of cancers. The role and targeting of SPHK1 in primary AML, however, has not been previously investigated. HereweshowthatSPHK1is overexpressed and constitutively activated in primary AML patient blasts but not in normal mononuclear cells. Subsequent targeting of SPHK1 induced caspase-dependent cell death inAMLcell lines, primary AML patient blasts, and isolated AML patient leukemic progenitor/stem cells, with negligible effects on normal bone marrow CD34 + progenitors from healthy donors. Furthermore, administration of SPHK1 inhibitors to orthotopic AML patient-derived xenografts reduced tumor burden and prolonged overall survival without affecting murine hematopoiesis. SPHK1 inhibition was associated with reduced survival signaling from S1P receptor 2, resulting in selective down regulation of the prosurvival protein MCL1. Subsequent analysis showed that the combination of BH3 mimetics with either SPHK1 inhibition or S1P receptor 2 antagonism triggered synergistic AML cell death. These results support the notion that SPHK1 is a bona fide therapeutic target for the treatment of AML. Refereed/Peer-reviewed

Acute erythroid leukemia (AEL) is a high-risk leukemia of poorly understood genetic basis, with controversy regarding diagnosisin the spectrum of myelodysplasia and myeloid leukemia. We compared genomic features of 159 childhood and adult AEL cases with non-AEL myeloid disorders and defined five age-related subgroups with distinct transcriptional profiles: adult, TP53mutated; NPM1 mutated; KMT2A mutated/rearranged; adult, DDX41 mutated; and pediatric, NUP98 rearranged. Genomic features influenced outcome, with NPM1 mutations and HOXB9 overexpression being associated with a favorable prognosis andTP53, FLT3 or RB1 alterations associated with poor survival. Targetable signaling mutations were present in 45% of cases and included recurrent mutations of ALK and NTRK1, the latter of which drives erythroid leukemogenesis sensitive to TRK inhibition.This genomic landscape of AEL provides the framework for accurate diagnosis and risk stratification of this disease, and the rationale for testing targeted therapies in this high-risk leukemia. Refereed/Peer-reviewed

Sonya M. Diakiw, Michelle Perugini, Chung H. Kok, Grant A. Engler, Nik Cummings, Luen Bik To, Andrew H. Wei, Ian D. Lewis, Anna L. Brown, Richard J. D’Andrea

Self-renewal and differentiation block are two key features of leukemic stem cells (LSCs) in mixed-lineage leukemia (MLL)-rearranged acute myeloid leukemia (AML), which can originate from either hematopoietic stem cells (HSCs) or granulocyte macrophage progenitors (GMPs). We have previously demonstrated that β-catenin is essential for the establishment of MLL LSCs, where MLL-AF9 activates β-catenin, one role of which is to confer self-renewal capacity to GMPs, necessary for malignant transformation. Refereed/Peer-reviewed

Krüppel-like factor 5 (KLF5) has been implicated as a tumor suppressor in various solid tumors such as breast and prostate, and recent studies have demonstrated a role for this protein in neutrophil differentiation of acute promyelocytic leukemia cells in response to ATRA. Here, we show that KLF5 expression increases during primary granulocyte differentiation and that expression of KLF5 is a requirement for granulocyte differentiation of 32D cells. In AML, we show that KLF5 mRNA expression levels are reduced in multiple French-American-British subtypes compared to normal controls, and also in leukemic stem cells relative to normal hematopoietic stem cells. We demonstrate that in selected AML cases, reduced expression is associated with hypermethylation of the KLF5 locus in the proximal promoter and/or intron 1, suggesting that this may represent a Class II genetic lesion in the development of AML. Refereed/Peer-reviewed

The prognosis of older patients with acute myelogenous leukemia is generally poor. The interleukin-3 receptor alpha-chain (CD123) is highly expressed on the surface of acute leukemia cells compared with normal hematopoietic stem cells. CSL362 is a fully humanized, CD123-neutralizing monoclonal antibody containing a modified Fc structure, which enhances human natural killer cell antibody-dependent cell-mediated cytotoxicity. Six continuous acute myelogenous leukemia xenografts established from patient explants and characterized by cell and molecular criteria, produced progressively lethal disease 42-202 days after transplantation. CSL362 alone reduced engraftment of one of four and three of four acute myelogenous leukemia xenografts in the bone marrow and peripheral organs, respectively. A cytarabine and daunorubicin regimen was optimized using this model to identify potentially synergistic interactions with CSL362. Cytarabine/daunorubicin improved the survival of mice engrafted with four of four acute myelogenous leukemia xenografts by 31-41 days. Moreover, CSL362 extended the survival of cytarabine/daunorubicin-treated mice for two of two acute myelogenous leukemia xenografts, while augmentation of natural killer cell-deficient NSG mice with adoptively transferred human natural killer cells improved survival against a single xenograft. Interestingly, this enhanced CSL362 efficacy was lost in the absence of chemotherapy. This study shows that acute myelogenous leukemia xenografts provide a platform for the evaluation of new therapeutics, simulating complex in vivo interactions, and that the in vivo efficacy of CSL362 supports continued clinical development of this drug. Refereed/Peer-reviewed

We describe methclone, a novel method to identify epigenetic loci that harbor large changes in the clonality of their epialleles (epigenetic alleles). Methclone efficiently analyzes genome-wide DNA methylation sequencing data. We quantify the changes using a composition entropy difference calculation and also introduce a new measure of global clonality shift, loci with epiallele shift per million loci covered, which enables comparisons between different samples to gauge overall epiallelic dynamics. Finally, we demonstrate the utility of methclone in capturing functional epiallele shifts in leukemia patients from diagnosis to relapse. Methclone is open-source and freely available at https://code.google.com/p/methclone. Electronic supplementary material The online version of this article (doi:10.1186/s13059-014-0472-5) contains supplementary material, which is available to authorized users.

Aberrant activation of β-catenin is a common event in AML and is an independent predictor of poor prognosis. Although increased β-catenin signaling in AML has been associated with oncogenic translocation products and activating mutations in the FLT3R, the mechanisms that activate β-catenin in AML more broadly are still unclear. Here, we describe a novel link between IL-3 signaling and the regulation of β-catenin in myeloid transformation and AML. In a murine model of HoxB8 and IL-3 cooperation, we show that β-catenin protein levels are modulated by IL-3 and that Cre-induced deletion of β-catenin abolishes IL-3-dependent growth and colony formation. In IL-3-dependent leukemic TF-1.8 cells, we observed increased β-catenin protein levels and nuclear localization in response to IL-3, and this correlated with transcriptional induction of β-catenin target genes. Furthermore, IL-3 promoted β-catenin accumulation in a subset of AML patient samples, and gene-expression profiling of these cells revealed induction of WNT/β-catenin and TCF4 gene signatures in an IL-3-dependent manner. This study is the first to link β-catenin activation to IL-3 and suggests that targeting IL-3 signaling may be an effective approach for the inhibition of β-catenin activity in some patients with AML.

Granulocyte–macrophage colony stimulating factor (GM-CSF) promotes the growth, survival, differentiation and activation of normal myeloid cells and is essential for fully functional macrophage differentiation in vivo. To better understand the mechanisms by which growth factors control the balance between proliferation and self-renewal versus growth-suppression and differentiation we have used the bi-potent FDB1 myeloid cell line, which proliferates in IL-3 and differentiates to granulocytes and macrophages in response to GM-CSF. This provides a manipulable model in which to dissect the switch between growth and differentiation. We show that, in the context of signaling from an activating mutant of the GM-CSF receptor β subunit, a single intracellular tyrosine residue (Y577) mediates the granulocyte fate decision. Loss of granulocyte differentiation in a Y577F second-site mutant is accompanied by enhanced macrophage differentiation and accumulation of β-catenin together with activation of Tcf4 and other Wnt target genes. These include the known macrophage lineage inducer, Egr1. We show that forced expression of Tcf4 or a stabilised β-catenin mutant is sufficient to promote macrophage differentiation in response to GM-CSF and that GM-CSF can regulate β-catenin stability, most likely via GSK3β. Consistent with this pathway being active in primary cells we show that inhibition of GSK3β activity promotes the formation of macrophage colonies at the expense of granulocyte colonies in response to GM-CSF. This study therefore identifies a novel pathway through which growth factor receptor signaling can interact with transcriptional regulators to influence lineage choice during myeloid differentiation. Refereed/Peer-reviewed

The Acute Myeloid Leukemia (AML) subtype characterised by translocations of the Mixed-Lineage Leukemia gene, MLL (t11q23; MLL-AML), is a particularly devastating disease with a median overall survival of only 9 months with current standard therapy. Cyclin dependent kinase (CDK) 9 inhibitors (CDK9i) directly target the CDK9/cyclin T complex (pTEFb) that is essential for activity of the MLL-fusion proteins and for transcriptional elongation, and therefore leads to reduction of transcript levels for multiple key leukemic oncogenes e.g. HOXA9, MYC and MCL1. Several observations suggest that utilising CDK9i to simultaneously target these oncogenes will be an effective strategy for AML, and MLL-AML in particular: (i) Leukemic stem cell (LSC) fractions of AML cells express a high level of MCL1, (ii) Targeting MCL1 has been demonstrated to reduce leukaemia cell survival in a murine model of MLL-ENL, (iii) MCL1 is consistently elevated in AML patients at relapse, (iv) HOXA9 is critical for leukemogenesis in many AMLs, in particular MLL-AML, (v) MYC has been shown to be a critical oncogene in MLL-AML, and (vi) CDK9 function has been shown to be important for MYC-driven tumorigenesis. Our in vitro and in vivo data support the clinical potential of a novel orally bioavailable inhibitor of CDK9, CDKI-73, as an effective therapy for MLL-AML patients. CDKI-73 is a potent inhibitor of CDK9 (Ki 3.5nM)1 and has been shown to induce down-regulation of MCL1, and cell death of Chronic Lymphocytic Leukemia (CLL) B-cells2 and Ovarian Cancer (OvCa) cells3 with nanomolar potency. At doses that are highly toxic for tumour cells, CDKI-73 shows limited toxicity for normal T- and B- Lymphocytes, Bone Marrow Mononuclear cells (BMMNC) and normal colony forming cells (CFC) from the BMMNC fraction. CDKI-73 has many favorable properties also making it an excellent clinical candidate for AML when compared to other CDK9i; in particular, CDKI-73 is (i) unique in its spectrum of inhibition, including targeting CDK6 (IC50 = 0.038 µM; a critical kinase for MLL-AML4), and is (ii) orally bioavailable (F = 56%)2, facilitating sustained in vivo target inhibition. Here we present data showing that in MLL-AML cell lines, CDKI-73 induces growth suppression and apoptosis associated with rapid loss of Myc and MCL1, and activation of PARP. In primary AML patient samples treated with 200nM CDKI-73, we have observed a similar decrease in MCL1 protein levels, with increased 7AAD uptake and Annexin-V staining, consistent with apoptotic cell death. Using a subcutaneous MV4;11 nude mouse xenograft model, we have shown that oral dosing of CDKI-73 (100 mg/kg once every 3 days for 18 days) resulted in a high level of anti-tumour efficacy (p Given this data, our priority now is to establish the effectiveness of CDKI-73 across a larger panel of primary MLL-AML samples, in further patient derived AML xenografts, and as a combination treatment with AML chemotherapy. REFERENCES: 1. Shao H, Shi S, et al. (2013). J Med Chem. 56(3):640-59. 2. Walsby E, Pratt G, et al. (2014). Oncotarget. 5(2):375-85. 3. Lam F, Abbas AY, et al. (2014). Oncotarget. 5(17):7691-704. 4. Placke T, Faber K, et al. (2014). Blood. 124(1):13-23. Disclosures No relevant conflicts of interest to declare.