Justin Loke

Azacitidine augments expansion of regulatory T cells after allogeneic stem cell transplantation in patients with acute myeloid leukemia (AML)

Strategies that augment a GVL effect without increasing the risk of GVHD are required to improve the outcome after allogeneic stem cell transplantation (SCT). Azacitidine (AZA) up-regulates the expression of tumor Ags on leukemic blasts in vitro and expands the numbers of immunomodulatory T regulatory cells (Tregs) in animal models. Reasoning that AZA might selectively augment a GVL effect, we studied the immunologic sequelae of AZA administration after allogeneic SCT. Twenty-seven patients who had undergone a reduced intensity allogeneic transplantation for acute myeloid leukemia were treated with monthly courses of AZA, and CD8(+) T-cell responses to candidate tumor Ags and circulating Tregs were measured. AZA after transplantation was well tolerated, and its administration was associated with a low incidence of GVHD. Administration of AZA increased the number of Tregs within the first 3 months after transplantation compared with a control population (P = .0127). AZA administration also induced a cytotoxic CD8(+) T-cell response to several tumor Ags, including melanoma-associated Ag 1, B melanoma antigen 1, and Wilm tumor Ag 1. These data support the further examination of AZA after transplantation as a mechanism of augmenting a GVL effect without a concomitant increase in GVHD.

Chronic FLT3-ITD Signaling in Acute Myeloid Leukemia Is Connected to a Specific Chromatin Signature

Summary Acute myeloid leukemia (AML) is characterized by recurrent mutations that affect the epigenetic regulatory machinery and signaling molecules, leading to a block in hematopoietic differentiation. Constitutive signaling from mutated growth factor receptors is a major driver of leukemic growth, but how aberrant signaling affects the epigenome in AML is less understood. Furthermore, AML cells undergo extensive clonal evolution, and the mutations in signaling genes are often secondary events. To elucidate how chronic growth factor signaling alters the transcriptional network in AML, we performed a system-wide multi-omics study of primary cells from patients suffering from AML with internal tandem duplications in the FLT3 transmembrane domain (FLT3-ITD). This strategy revealed cooperation between the MAP kinase (MAPK) inducible transcription factor AP-1 and RUNX1 as a major driver of a common, FLT3-ITD-specific gene expression and chromatin signature, demonstrating a major impact of MAPK signaling pathways in shaping the epigenome of FLT3-ITD AML.

Identification of Glycopeptides as Posttranslationally Modified Neoantigens in Leukemia

The identification of neoepitopes expressed by tumors will aid the effectiveness of antitumor therapies. Four classes of posttranslationally modified tumor neoantigens were identified on primary tumors. Healthy donors had detectable natural immunity to a subset of these. Leukemias are highly immunogenic, but they have a low mutational load, providing few mutated peptide targets. Thus, the identification of alternative neoantigens is a pressing need. Here, we identify 36 MHC class I–associated peptide antigens with O-linked β-N-acetylglucosamine (O-GlcNAc) modifications as candidate neoantigens, using three experimental approaches. Thirteen of these peptides were also detected with disaccharide units on the same residues and two contain either mono- and/or di-methylated arginine residues. A subset were linked with key cancer pathways, and these peptides were shared across all of the leukemia patient samples tested (5/5). Seven of the O-GlcNAc peptides were synthesized and five (71%) were shown to be associated with multifunctional memory T-cell responses in healthy donors. An O-GlcNAc-specific T-cell line specifically killed autologous cells pulsed with the modified peptide, but not the equivalent unmodified peptide. Therefore, these posttranslationally modified neoantigens provide logical targets for cancer immunotherapy. Cancer Immunol Res; 5(5); 376–84. ©2017 AACR.

RUNX1-ETO and RUNX1-EVI1 Differentially Reprogram the Chromatin Landscape in t(8;21) and t(3;21) AML

Summary Acute myeloid leukemia (AML) is a heterogeneous disease caused by mutations in transcriptional regulator genes, but how different mutant regulators shape the chromatin landscape is unclear. Here, we compared the transcriptional networks of two types of AML with chromosomal translocations of the RUNX1 locus that fuse the RUNX1 DNA-binding domain to different regulators, the t(8;21) expressing RUNX1-ETO and the t(3;21) expressing RUNX1-EVI1. Despite containing the same DNA-binding domain, the two fusion proteins display distinct binding patterns, show differences in gene expression and chromatin landscape, and are dependent on different transcription factors. RUNX1-EVI1 directs a stem cell-like transcriptional network reliant on GATA2, whereas that of RUNX1-ETO-expressing cells is more mature and depends on RUNX1. However, both types of AML are dependent on the continuous expression of the fusion proteins. Our data provide a molecular explanation for the differences in clinical prognosis for these types of AML.

C/EBPα overrides epigenetic reprogramming by oncogenic transcription factors in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a heterogeneous disease caused by recurrent mutations in the transcription regulatory machinery, resulting in abnormal growth and a block in differentiation. One type of recurrent mutations affects RUNX1, which is subject to mutations and translocations, the latter giving rise to fusion proteins with aberrant transcriptional activities. We recently compared the mechanism by which the products of the t(8;21) and the t(3;21) translocation RUNX1-ETO and RUNX1-EVI1 reprogram the epigenome. We demonstrated that a main component of the block in differentiation in both types of AML is direct repression of the gene encoding the myeloid regulator C/EBPα by both fusion proteins. Here, we examined at the global level whether C/EBPα is able to reverse aberrant chromatin programming in t(8;21) and t(3;21) AML. C/EBPα overexpression does not change oncoprotein expression or globally displace these proteins from their binding sites. Instead, it upregulates a core set of common target genes important for myeloid differentiation and represses genes regulating leukemia maintenance. This study, therefore, identifies common CEBPA-regulated pathways as targets for therapeutic intervention.

Identification of common and distinct epigenetic reprogramming properties of RUNX1 fusion proteins in acute myeloid leukaemia

Abstract Background Regulation of gene expression by transcription factors such as RUNX1 is crucial for haemopoiesis. The most common RUNX1 translocation resulting in acute myeloid leukaemia is t(8;21), which forms RUNX1–ETO; a second translocation—t(3;21)—results in RUNX1–EVI-1. Although, these two fusion proteins have the same DNA binding domain, the prognoses of patients with these translocations differ greatly. Whether different RUNX1 fusion proteins deregulate the same genes is unknown. We sought to understand the differences in the epigenome that underlie these prognostic differences. Methods DNase-seq maps regions of open chromatin that represent active gene regulatory elements. By using this technique with RNA-seq, we were able to describe the epigenetic landscape in CD34+ purified, primary material from patients with RUNX1–EVI-1 and RUNX1–ETO leukaemias, and in healthy controls. We used ChIP-seq to map the binding sites of both normal RUNX1 and the fusion proteins. We integrated these analyses to determine transcription factor networks that characterise each type of leukaemia. Findings RUNX1–EVI-1, but not RUNX1–ETO, directly regulated a unique subset of genes required for stem-cell function. We found that these differences in binding sites of the two fusion proteins were associated with differences in the transcription factor complexes that collaborate with them, and were directly related to the differences in the epigenetic landscape of each leukaemia. RUNX1–EVI-1 knockdown restored differentiation of t(3;21) cells and this finding was associated with upregulation of genes crucial for myeloid differentiation, including C/EBPα. We showed that C/EBPα was necessary and sufficient for the response of t(3;21) cells to RUNX1–EVI-1 knockdown and that C/EBPα was commonly deregulated in both leukaemias. Interpretation The differences in the clinical outcomes of each RUNX1 mutant leukaemia was reflected by differences in their epigenetic landscape. This finding was driven by differences in the transcription factor networks in each type of leukaemia. Despite these differences, both leukaemias were dependent on downregulating C/EBP, thereby providing a common therapeutic route for both RUNX1–ETO and RUNX1–EVI-1 leukaemia. Funding Kay Kendall Leukaemia Fund, Bloodwise.

Acquired isodisomy on chromosome 13 at diagnosis results in impaired overall survival in patients with FLT3-ITD mutant acute myeloid leukaemia.

Acquired isodisomy on chromosome 13 at diagnosis results in impaired overall survival in patients with FLT3- ITD mutant acute myeloid leukaemia