WORLD CLASS BASIC, TRANSLATIONAL AND CLINICAL RESEARCH

Overview

In keeping with the group’s goal of understanding and identifying new disease mechanisms in myeloid lineage blood cancers such as acute myeloid leukaemia (AML) and developing candidate therapeutic targets for development through to the clinic, we published two key studies in 2021. In the first (Simeoni et al., 2021, Cell Reports) we report our new insights into how the Forkhead transcription factor FOXC1 confers a differentiation block in human AML: the work reveals a number of potential therapeutic targets for future pre-clinical analysis. In a second publication (Williams et al., 2021, BMC Cancer) we report a comparison of paired presentation and primary resistant AML samples, which reveals that AML stem cells are cycling cells with a transcriptional signature indicative of Forkhead factor FOXM1 activity.

Acute myeloid leukaemia

Acute myeloid leukaemia (AML) is a blood cancer characterised by a block to normal myeloid lineage differentiation. This results in accumulation of myeloid blast cells in bone marrow (BM) and blood with consequent failure of normal haematopoiesis. While the range of balanced translocations, point mutations and indels associated with this malignancy is largely characterised, the mechanisms by which these genetic lesions confer a differentiation block is less well understood. This is emphasised by studies which show that many AML-associated mutations, including some chromosomal abnormalities, may be found in chemotherapy-treated patients in complete remission, in patients with myelodysplasia prior to evolution to AML or in aging individuals with normal blood counts – the latter being the so-called clonal hematopoiesis of indeterminate potential. This is consistent with an emergent theme in AML that many disease-associated mutations promote expansion of hematopoietic stem and progenitor cells (HSPCs), which otherwise retain relatively normal differentiation potential, rather than immediately conferring a differentiation block. Few AML-associated genetic lesions are exclusively found in AML and even those such as FLT3 internal tandem duplications and NPM1 mutations yield prominent myeloproliferative phenotypes when modelled in mice.

Forkhead transcription factor confers a differentiation block in human AML

The presence of certain combinations of genetic lesions within a long-lived progenitor cell is likely necessary for the generation of a differentiation block, but how mutations co-operate to arrest normal differentiation is often unclear. Improved understanding of the mechanisms involved is likely to facilitate development of therapeutic approaches to promote differentiation, an approach already exemplified by all-trans retinoic acid in the treatment of acute promyelocytic leukaemia. In addition to killing leukaemia cells with chemotherapy, induction of differentiation is a major goal of treatment.

We previously reported (Somerville et al, 2015, Cancer Cell) that the Forkhead family transcription factor gene FOXC1, which is a critical regulator of normal mesenchymal and mesodermal differentiation, is highly expressed in around 20% of cases of AML, but not expressed in normal hematopoietic lineages. High FOXC1 expression in AML is almost invariably found in association with high HOXA/B gene expression, and ~30% of human HOXA/B-expressing AML cases (e.g. those with NPM1 mutations) exhibit high FOXC1 expression. In vitro and in vivo experimental evidence confirms that FOXC1 confers a monocyte/macrophage lineage differentiation block and sustains clonogenic activity in both murine and primary human FOXC1high HOXAhigh AML cells. Co-expression of FOXC1 with Hoxa9 accelerates the onset of AML in murine modelling, with the resulting leukaemias exhibiting a higher level of differentiation block by comparison with those initiated by Hoxa9 alone. Further, patients with high FOXC1 expression exhibit inferior survival. More widely, high level FOXC1 expression is also observed in a multitude of solid malignancies, including breast, colorectal, cervical, gastric and liver (Gilding & Somervaille, 2019, Cancers (Basel)) where functional experiments confirm that it promotes increased migration and metastasis and, as in AML, typically confers an inferior survival.

Despite the importance of FOXC1 in human AML, and more broadly in solid malignancies, the mechanisms by which FOXC1 confers adverse outcomes in human cancers remain largely unexplored. To address this in AML, we performed an integrated analysis of the protein:protein interactions and genome-wide binding sites of FOXC1 in human cell line and primary AML cells. In work led by Fabrizio Simeoni (Simeoni et al., 2021, Cell Reports) we discovered that FOXC1 interacts with the key myeloid lineage transcription factor RUNX1 through its Forkhead DNA binding domain and that the two factors co-occupy a discrete set of primed and active enhancers distributed close to monocyte/macrophage differentiation genes. FOXC1 stabilises association of RUNX1, HDAC1 and the Groucho family repressor protein TLE3 at these sites to limit enhancer activity: FOXC1 knockdown induced loss of repressor proteins, gain of CEBPA binding, enhancer acetylation and upregulation of nearby genes involved in monocyte differentiation, including KLF2 (Figure 1). Furthermore, it triggered genome-wide redistribution of RUNX1, TLE3 and HDAC1 from enhancers to promoters, leading to repression of self-renewal genes including MYC and MYB. Our studies highlight RUNX1 and CEBPA transcription factor swapping at enhancers and promoters as a feature of leukaemia cell differentiation, and reveal that FOXC1 prevents this by stabilising enhancer binding of a RUNX1/HDAC1/TLE3 transcription repressor complex, to oncogenic effect. A number of the protein: protein interactions we identified in this study as required for retarding leukaemia cell differentiation are candidate therapeutic targets and will be evaluated in future pre-clinical studies. This work further emphasises the importance and multi-faceted roles of RUNX1 as a critical protein in human myeloid lineage blood cancers, over and above its well described point mutations and chromosomal translocations.

AML treatment and chemotherapy resistance

While development of drugs which relieve the differentiation block in AML is an important therapeutic approach, it remains the case that standard-of-care AML treatments are predominantly DNA damaging chemotherapies, which induce leukaemia cell death. Disease relapse remains sadly all too common following treatment of AML and is generally due to chemoresistance of leukaemia cells with disease repopulating potential. To date, attempts to define the characteristics of in vivo resistant blasts have focused on comparisons between leukemic cells at presentation and relapse. However, further treatment responses are often seen following relapse, suggesting that most blasts remain chemosensitive. In work led by Mark Williams (Williams et al., 2021, BMC Cancer), we sought to characterise in vivo chemoresistant AML blast cells by studying their transcriptional and genetic features before and shortly after induction chemotherapy using paired samples from patients with primary refractory AML.

Leukemic blasts were isolated by flow sorting and subjected to fluorescence in situ hybridisation, targeted genetic sequencing, detailed immunophenotyping and RNA sequencing. Molecular genetic analysis revealed early clonal selection occurring after induction chemotherapy. Immunophenotypic characterisation found leukaemia-associated immunophenotypes in all cases that persisted following treatment. Despite the genetic heterogeneity of the leukaemias studied, transcriptional analysis found concerted changes in gene expression in resistant blasts. Remarkably, the gene expression signature suggested that post-chemotherapy blasts were more proliferative than those at presentation (Figure 2).

Resistant blasts also appeared less differentiated and expressed leukaemia stem cell maintenance genes. However, the proportion of immunophenotypically defined LSCs appeared to decrease following treatment, with implications for the targeting of these cells on the basis of cell surface antigen expression. The refractory gene signature was highly enriched with targets of the transcription factor FOXM1. Lentiviral vectors expressing short hairpin RNAs were used to assess the effect of FOXM1 knockdown on colony forming capacity, proliferative capacity and apoptosis in cell lines, primary AML cells and CD34+ cells from healthy donors. These knockdown experiments demonstrated that the viability of primary AML cells, but not normal CD34+ cells, depended on FOXM1 expression.

Thus in summary, we found that chemorefractory blasts from leukaemias with varied genetic backgrounds expressed a common transcriptional program. In contrast to the notion that LSC quiescence confers resistance to chemotherapy we find that refractory blasts are both actively proliferating and enriched with LSC maintenance genes. Using primary patient material from a relevant clinical context we also provided support for the role of FOXM1 in chemotherapy resistance, proliferation and stem cell function in AML.