Emerging therapies for inv(16) AML.

The core binding factor composed of CBFß and RUNX subunits plays a critical role in most hematopoietic lineages and is deregulated in acute myeloid leukemia (AML). The fusion oncogene CBFß-SMMHC expressed in AML with the chromosome inversion inv(16)(p13q22) acts as a driver oncogene in hematopoietic stem cells and induces AML. This review focuses on novel insights regarding the molecular mechanisms involved in CBFß-SMMHC-driven leukemogenesis and recent advances in therapeutic approaches to target CBFß-SMMHC in inv(16) AML.

RUNX1 is required for oncogenic Myb and Myc enhancer activity in T-cell acute lymphoblastic leukemia.

The gene encoding the RUNX1 transcription factor is mutated in a subset of T-cell acute lymphoblastic leukemia (T-ALL) patients, and RUNX1 mutations are associated with a poor prognosis. These mutations cluster in the DNA-binding Runt domain and are thought to represent loss-of-function mutations, indicating that RUNX1 suppresses T-cell transformation. RUNX1 has been proposed to have tumor suppressor roles in T-cell leukemia homeobox 1/3-transformed human T-ALL cell lines and NOTCH1 T-ALL mouse models. Yet, retroviral insertional mutagenesis screens identify RUNX genes as collaborating oncogenes in MYC-driven leukemia mouse models. To elucidate RUNX1 function(s) in leukemogenesis, we generated Tal1/Lmo2/Rosa26-CreERT2Runx1f/f mice and examined leukemia progression in the presence of vehicle or tamoxifen. We found that Runx1 deletion inhibits mouse leukemic growth in vivo and that RUNX silencing in human T-ALL cells triggers apoptosis. We demonstrate that a small molecule inhibitor, designed to interfere with CBFß binding to RUNX proteins, impairs the growth of human T-ALL cell lines and primary patient samples. We demonstrate that a RUNX1 deficiency alters the expression of a crucial subset of TAL1- and NOTCH1-regulated genes, including the MYB and MYC oncogenes, respectively. These studies provide genetic and pharmacologic evidence that RUNX1 has oncogenic roles and reveal RUNX1 as a novel therapeutic target in T-ALL.

NrasG12D oncoprotein inhibits apoptosis of preleukemic cells expressing Cbfß-SMMHC via activation of MEK/ERK axis.

Acute myeloid leukemia (AML) results from the activity of driver mutations that deregulate proliferation and survival of hematopoietic stem cells (HSCs). The fusion protein CBFß-SMMHC impairs differentiation in hematopoietic stem and progenitor cells and induces AML in cooperation with other mutations. However, the combined function of CBFß-SMMHC and cooperating mutations in preleukemic expansion is not known. Here, we used Nras(LSL-G12D); Cbfb(56M) knock-in mice to show that allelic expression of oncogenic Nras(G12D) and Cbfß-SMMHC increases survival of preleukemic short-term HSCs and myeloid progenitor cells and maintains the differentiation block induced by the fusion protein. Nras(G12D) and Cbfß-SMMHC synergize to induce leukemia in mice in a cell-autonomous manner, with a shorter median latency and higher leukemia-initiating cell activity than that of mice expressing Cbfß-SMMHC. Furthermore, Nras(LSL-G12D); Cbfb(56M) leukemic cells were sensitive to pharmacologic inhibition of the MEK/ERK signaling pathway, increasing apoptosis and Bim protein levels. These studies demonstrate that Cbfß-SMMHC and Nras(G12D) promote the survival of preleukemic myeloid progenitors primed for leukemia by activation of the MEK/ERK/Bim axis, and define Nras(LSL-G12D); Cbfb(56M) mice as a valuable genetic model for the study of inversion(16) AML-targeted therapies.

N-MYC regulates cell survival via eIF4G1 in inv(16) acute myeloid leukemia.

N-MYC (encoded by MYCN) is a critical regulator of hematopoietic stem cell function. While the role of N-MYC deregulation is well established in neuroblastoma, the importance of N-MYC deregulation in leukemogenesis remains elusive. Here, we demonstrate that N-MYC is overexpressed in acute myeloid leukemia (AML) cells with chromosome inversion inv(16) and contributes to the survival and maintenance of inv(16) leukemia. We identified a previously unknown MYCN enhancer, active in multiple AML subtypes, essential for MYCN mRNA levels and survival in inv(16) AML cells. We also identified eukaryotic translation initiation factor 4 gamma 1 (eIF4G1) as a key N-MYC target that sustains leukemic survival in inv(16) AML cells. The oncogenic role of eIF4G1 in AML has not been reported before. Our results reveal a mechanism whereby N-MYC drives a leukemic transcriptional program and provides a rationale for the therapeutic targeting of the N-MYC/eIF4G1 axis in myeloid leukemia.

Cell-cycle regulator E2F1 and microRNA-223 comprise an autoregulatory negative feedback loop in acute myeloid leukemia.

Transcription factor CCAAT enhancer binding protein alpha (C/EBPalpha) is essential for granulopoiesis and its function is deregulated in leukemia. Inhibition of E2F1, the master regulator of cell-cycle progression, by C/EBPalpha is pivotal for granulopoiesis. Recent studies show microRNA-223 (miR-223), a transcriptional target of C/EBPalpha, as a critical player during granulopoiesis. In this report, we demonstrate that during granulopoiesis microRNA-223 targets E2F1. E2F1 protein was up-regulated in miR-223 null mice. We show that miR-223 blocks cell-cycle progression in myeloid cells. miR-223 is down-regulated in different subtypes of acute myeloid leukemia (AML). We further show that E2F1 binds to the miR-223 promoter in AML blast cells and inhibits miR-223 transcription, suggesting that E2F1 is a transcriptional repressor of the miR-223 gene in AML. Our study supports a molecular network involving miR-223, C/EBPalpha, and E2F1 as major components of the granulocyte differentiation program, which is deregulated in AML.

C/EBPα regulated microRNA-34a targets E2F3 during granulopoiesis and is down-regulated in AML with CEBPA mutations.

The transcription factor, CCAAT enhancer binding protein alpha (C/EBPα), is crucial for granulopoiesis and is deregulated by various mechanisms in acute myeloid leukemia (AML). Mutations in the CEBPA gene are reported in 10% of human patients with AML. Even though the C/EBPα mutants are known to display distinct biologic function during leukemogenesis, the molecular basis for this subtype of AML remains elusive. We have recently showed the significance of deregulation of C/EBPα-regulated microRNA (miR) in AML. In this study, we report that miR-34a is a novel target of C/EBPα in granulopoiesis. During granulopoiesis, miR-34a targets E2F3 and blocks myeloid cell proliferation. Analysis of AML samples with CEBPA mutations revealed a lower expression of miR-34a and elevated levels of E2F3 as well as E2F1, a transcriptional target of E2F3. Manipulation of miR-34a reprograms granulocytic differentiation of AML blast cells with CEBPA mutations. These results define miR-34a as a novel therapeutic target in AML with CEBPA mutations.

Small Molecule Inhibitor of CBFß-RUNX Binding for RUNX Transcription Factor Driven Cancers.

Transcription factors have traditionally been viewed with skepticism as viable drug targets, but they offer the potential for completely novel mechanisms of action that could more effectively address the stem cell like properties, such as self-renewal and chemo-resistance, that lead to the failure of traditional chemotherapy approaches. Core binding factor is a heterodimeric transcription factor comprised of one of 3 RUNX proteins (RUNX1-3) and a CBFß binding partner. CBFß enhances DNA binding of RUNX subunits by relieving auto-inhibition. Both RUNX1 and CBFß are frequently mutated in human leukemia. More recently, RUNX proteins have been shown to be key players in epithelial cancers, suggesting the targeting of this pathway could have broad utility. In order to test this, we developed small molecules which bind to CBFß and inhibit its binding to RUNX. Treatment with these inhibitors reduces binding of RUNX1 to target genes, alters the expression of RUNX1 target genes, and impacts cell survival and differentiation. These inhibitors show efficacy against leukemia cells as well as basal-like (triple-negative) breast cancer cells. These inhibitors provide effective tools to probe the utility of targeting RUNX transcription factor function in other cancers.

Chemical biology. A small-molecule inhibitor of the aberrant transcription factor CBFß-SMMHC delays leukemia in mice.

Acute myeloid leukemia (AML) is the most common form of adult leukemia. The transcription factor fusion CBFß-SMMHC (core binding factor ß and the smooth-muscle myosin heavy chain), expressed in AML with the chromosome inversion inv(16)(p13q22), outcompetes wild-type CBFß for binding to the transcription factor RUNX1, deregulates RUNX1 activity in hematopoiesis, and induces AML. Current inv(16) AML treatment with nonselective cytotoxic chemotherapy results in a good initial response but limited long-term survival. Here, we report the development of a protein-protein interaction inhibitor, AI-10-49, that selectively binds to CBFß-SMMHC and disrupts its binding to RUNX1. AI-10-49 restores RUNX1 transcriptional activity, displays favorable pharmacokinetics, and delays leukemia progression in mice. Treatment of primary inv(16) AML patient blasts with AI-10-49 triggers selective cell death. These data suggest that direct inhibition of the oncogenic CBFß-SMMHC fusion protein may be an effective therapeutic approach for inv(16) AML, and they provide support for transcription factor targeted therapy in other cancers.

Target proteins of C/EBPalphap30 in AML: C/EBPalphap30 enhances sumoylation of C/EBPalphap42 via up-regulation of Ubc9.

CCAAT/enhancer-binding protein alpha (C/EBPalpha) is a critical regulator for early myeloid differentiation. Mutations in C/EBPalpha occur in 10% of patients with acute myeloid leukemia (AML), leading to the expression of a 30-kDa dominant-negative isoform (C/EBPalphap30). In the present study, using a global proteomics approach to identify the target proteins of C/EBPalphap30, we show that Ubc9, an E2-conjugating enzyme essential for sumoylation, is increased in its expression when C/EBPalphap30 is induced. We confirmed the increased expression of Ubc9 in patients with AML with C/EBPalphap30 mutations compared with other subtypes. We further confirmed that the increase of Ubc9 expression was mediated through increased transcription. Furthermore, we show that Ubc9-mediated enhanced sumoylation of C/EBPalphap42 decreases the transactivation capacity on a minimal C/EBPalpha promoter. Importantly, overexpression of C/EBPalphap30 in granulocyte colony-stimulating factor (G-CSF)-stimulated human CD34(+) cells leads to a differentiation block, which was overcome by the siRNA-mediated silencing of Ubc9. In summary, our data indicate that Ubc9 is an important C/EBPalphap30 target through which C/EBPalphap30 enhances the sumoylation of C/EBPalphap42 to inhibit granulocytic differentiation.

Proteomic analysis of acute promyelocytic leukemia: PML-RARalpha leads to decreased phosphorylation of OP18 at serine 63.

In the present study, we employed 2-DE to characterize the effect of the acute promyelocytic leukemia (APL)-specific PML-RARalpha fusion protein on the proteome. Differentially expressed proteins, a number of which are related to the cell cycle function, including oncoprotein18 (OP18), heat shock protein70, glucose-regulated protein75, and peptidyl-prolyl isomerase, were identified by MS. Subsequent bioinformatic pathway discovery revealed an integrated network constituting SMARCB1, MYC, and TP53-regulated pathways. The data from the DNA microarray and proteomic experiments demonstrated the correlation between the translocation and higher expression of OP18 at mRNA and protein levels. Transient cotransfection assay revealed that PML-RARalpha is a potent activator of OP18 promoter and this transcriptional activation is retinoic acid sensitive. PML-RARalpha induction also leads to decreased phosphorylation on Ser63 residue of OP18, which is okadaic acid sensitive suggesting the involvement of a phosphatase pathway. Overexpression of a constitutively phosphorylated Ser63 mutant of OP18 in PML-RARalpha expressing APL patient, PR9, and NB4 cells led to a G2/M-phase arrest in contrast to a phosphorylation-deficient Ser63 mutant and untransfected control. Taken together, our results demonstrate the significance of decreased Ser63 phosphorylation of OP18 in PML-RARalpha-mediated effects on cell cycle.