Leukaemia Biology - Tim Somervaille

Tim Somervaille is Senior Group Leader at the Cancer Research UK Manchester Institute where he leads the Leukaemia Biology Laboratory. He is also Honorary Consultant in Haematology at The Christie NHS Foundation Trust. His scientific and clinical research interest is in myeloid cancer, including acute myeloid leukaemia and the myeloproliferative disorders. Tim’s medical training was at Imperial College London and University College London. His scientific training was at University College London and Stanford University.

Introduction

Human acute myeloid leukaemias (AMLs) are heterogeneous with respect to both genetics and the function of the cells that make up the disease. A minority of the cells are leukaemia stem cells (LSCs) which have the ability to self-renew for an extended if not indefinite period, thus maintaining and expanding the disease. In order to cure a patient these cells must be eliminated completely, because if they are not they have the ability to regenerate the disease and induce relapse.

Understanding the cellular mechanisms that regulate the self-renewal of the LSC compartment represents a critical current problem in leukaemia biology. The Leukaemia Biology group is focused on understanding the biology of disordered LSCs in comparison with normal haematopoietic stem cells (HSCs) in a range of distinct categories of haematological malignancy, in order to identify new genes and cellular pathways that are critical for LSC function and which could be targeted by new treatments.

A particular area of interest is the burgeoning field of epigenetics. The group has recently discovered a new candidate therapeutic target in the mixed lineage leukaemia subtype of AML, called lysine specific demethylase 1 (LSD1). Working with the Drug Discovery Unit at the CRUK Manchester Institute and industrial partners (Oryzon Genomics and Roche) we synthesised novel compounds that inhibit the enzyme and showed they induce differentiation of LSCs in pre-clinical models. In early phase clinical trials in Manchester and Barcelona we also found that LSD1 inhibitors promote differentiation of blast cells in patients with acute leukaemia.

We have also recently uncovered a novel and frequent oncogenic mechanism in human acute myeloid leukaemia, active in 20% of patients. This is the tissue inappropriate mis-expression of a mesenchymal transcription factor called FOXC1. Expression of FOXC1 in bone marrow cells contributes to the differentiation block which is a cardinal feature of the disease. Developing therapies that target this transcription factor may benefit patients with high level FOXC1 expression.

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.

 

Figure 1. Model illustrating activity of mis-expressed FOXC1 in sequestering repressive activity of RUNX1 and TLE3 at key enhancers controlling differentiation genes in leukaemia.

 

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.

 

Figure 2. Transcriptional and cell cycle features of paired patient samples of primary AML from presentation and at the end of the first cycle of chemotherapy. Left panel: heat map illustrates differentially expressed genes. Right panel: exemplar cell cycle profiles.

 

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.

Selected Publications

 

Mead AJ, Butt NM, Nagi W, Whiteway A, Kirkpatrick S, Rinaldi C, Roughley C, Ackroyd S, Ewing J, Neelakantan P, Garg M, Tucker D, Murphy J, Patel H, Bains R, Chiu G, Hickey J, Harrison C, Somervaille TCP. (2022)
A retrospective real-world study of the current treatment pathways for myelofibrosis in the United Kingdom: the REALISM UK study.
Therapeutic Advances in Hematology 13:20406207221084487. PubMed abstract (PMID: 35371428)

Williams MS, Basma NJ, Amaral FMR, Wiseman DH, Somervaille TCP. (2021)
Blast cells surviving acute myeloid leukemia induction therapy are in cycle with a signature of FOXM1 activity.
BMC Cancer 21(1):1153. PubMed abstract (PMID: 34711181)

Simeoni F, Romero-Camarero I, Camera F, Amaral FMR, Sinclair OJ, Papachristou EK, Spencer GJ, Lie-A-Ling M, Lacaud G, Wiseman DH, Carroll JS, Somervaille TCP. (2021)
Enhancer recruitment of transcription repressors RUNX1 and TLE3 by mis-expressed FOXC1 blocks differentiation in acute myeloid leukemia.
Cell Reports 36(12):109725. PubMed abstract (PMID: 34551306)

Williams MS, Basma NJ, Amaral FMR, Williams G, Weightman JP, Breitwieser W, Nelson L, Taylor SS, Wiseman DH, Somervaille TCP. (2020)
Targeted nanopore sequencing for the identification of ABCB1 promoter translocations in cancer. 
BMC Cancer 20(1):1075. PubMed abstract (PMID: 33167906)

Salamero O, Montesinos P, Willekens C, Pérez-Simón JA, Pigneux A, Récher C, Popat R, Carpio C, Molinero C, Mascaró C, Vila J, Arévalo MI, Maes T, Buesa C, Bosch F, Somervaille TCP. (2020)
First-in-Human Phase I Study of Iadademstat (ORY-1001): A First-in-Class Lysine-Specific Histone Demethylase 1A Inhibitor, in Relapsed or Refractory Acute Myeloid Leukemia. 
J Clin Oncol. 38:4260-4273. PubMed abstract (PMID: 33052756)

MS Williams, FMR Amaral, F Simeoni and TCP Somervaille. (2020)
Dynamic induction of drug resistance through a stress responsive enhancer in acute myeloid leukaemia. 
Journal of Clinical Investigation 130:1217-1232. PubMed abstract

Deb G, Wingelhofer B, Amaral FMR, Maiques-Diaz A, Chadwick JA, Spencer GJ, Williams EL, Leong HS, Maes T, Somervaille TCP. (2020)
Pre-clinical activity of combined LSD1 and mTORC1 inhibition in MLL-translocated acute myeloid leukaemia.
Leukemia 34(5):1266-1277. PubMed abstract

Maiques-Diaz A, Spencer GJ, Lynch JT, Ciceri F, Williams EL, Amaral FMR, Wiseman DH, Harris WJ, Li Y, Sahoo S, Hitchin JR, Mould DP, Fairweather EE, Waszkowycz B, Jordan AM, Smith DL, Somervaille TCP. (2018)
Enhancer Activation by Pharmacologic Displacement of LSD1 from GFI1 Induces Differentiation in Acute Myeloid Leukemia.
Cell Reports 22(13):3641-3659. PubMed abstract

Somerville TDD, Simeoni F, Chadwick JA, Williams EL, Spencer GJ, Boros K, Wirth C, Tholouli E, Byers RJ, Somervaille TCP. (2018)
Derepression of the iroquois homeodomain transcription factor gene IRX3 confers differentiation block in acute leukemia.
Cell Reports 22(3):638-652. PubMed abstract

Wiseman DH, Williams EL, Wilks DP, Sun Leong H, Somerville TD, Dennis MW, Struys EA, Bakkali A, Salomons GS, Somervaille TC. (2016)
Frequent reconstitution of IDH2 R140Q mutant clonal multilineage hematopoiesis following chemotherapy for acute myeloid leukemia.
Leukemia 30:1946-1950. Article

Somerville TD, Wiseman DH, Spencer GJ, Huang X, Lynch JT, Leong HS, Williams EL, Cheesman E, Somervaille TC. (2015)
Frequent derepression of the mesenchymal transcription factor gene FOXC1 in acute myeloid leukaemia.
Cancer Cell 14:329-342. PubMed abstract

Harris WJ, Huang X, Lynch JT, Spencer GJ, Hitchin JR, Li Y, Ciceri F, Blaser JG, Greystoke BF, Jordan AM, Miller CJ, Ogilvie DJ, Somervaille TC. (2012)
The histone demethylase KDM1A sustains the oncogenic potential of MLL-AF9 leukemia stem cells.
Cancer Cell 21:473-487. PubMed abstract

Wang Z, Smith KS, Murphy M, Piloto O, Somervaille TC, Cleary ML. (2008)
Glycogen synthase kinase 3 in MLL leukaemia maintenance and targeted therapy.
Nature 405:1205-1209. PubMed abstract

 

Postdoctoral Fellows
Isabel Romero-Camarero
Bettina Wingelhofer
Luciano Nicosia
Hannah Seberg
Teresita Flores-Tellez

Bioinformatician
Fabio Amaral

Senior Scientific Officer
Gary Spencer

Graduate Students
Francesco Camera
Bradley Revell
Oliver Sinclair
Naseer Basma
Michael Jones
Alexia Strickson

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