Research
Leukaemia is the 12th most common cause of cancer death in the UK with ~4,600 deaths/year (3% of all cancer deaths). With the aim to benefit to future treatment strategies for patients with leukaemia, the Feldhahn lab investigates molecular mechanisms that promote leukaemia, especially those that are induced by an oncogene or that cause oncogene activation.
Recent Research by the lab:
Acute oncogene activation increases the vulnerability of B-lineage genes during the transformation of normal B-cells towards leukaemia. BCR-ABL1 is a powerful oncogene encoded by the Philadelphia (Ph) translocation that drives chronic myeloid leukaemia (CML) and the most common subtype of B-lineage acute lymphoblastic leukaemia (B-ALL) in adults, i.e. Ph+B-ALL. BCR-ABL1 is known to promote genome instability in leukaemia mouse models, and leukemic cells from Ph+B-ALL patients exhibit multiple secondary genome alterations, most prominently affecting B-lineage genes. Notably, oncogene expression and associated ‘oncogenic stress’ is a known cause of DNA damage, however the effect of BCR-ABL1 on genome stability in general and on B-lineage genes in particular had not been studied in detail. In our study published in Cell Reports (DOI: 10.1016/j.celrep.2017.01.057) we thus investigated which genomic regions become vulnerable upon acute oncogene expression in normal B-cell precursors during the process of malignant transformation, and whether these relate to genes that become altered in B-ALL. We used ChIP-Seq for the DNA damage response protein gH2AX to monitor DNA damage and break-apart probe DNA FISH probes to validate identified hotspots as actual sites of DNA double-strand breaks (DNA-DSBs). We observed that the expression of oncogenes such as BCR-ABL1 or MYC caused DNA damage at highly expressed genes, which in B-cell precursors often relate to genes with B-lineage specific function. Affected genes were further enriched for DNA sequences that promote DNA/RNA hybrids, called R-loops, which when colliding with replication forks can cause transcription/replication conflicts that cause DNA damage. Notably, the induction of a myeloid lineage phenotype in transformed B-cell precursors promoted de novo DNA damage at myeloid gene loci. Hence, lineage-specific transcription programs appeared to predispose lineage-specific genes to DNA damage in presence of oncogenic stress, which may promote the alteration of lineage-specific genes frequently observed in human leukaemia. Of note, for most lesions in B-ALL cells at least one breakpoint has been attributed to illegitimate activity of the RAG recombinase. However, oncogene stress could facilitate the generation of RAG induced lesions by providing additional DNA damage.
SSB proteins are critical safeguards for genome stability of bone marrow B-cells. Based on an initial observation that B-cell precursors transformed by leukaemia-inducing oncogenes exhibit increased expression of the DNA damage response factor SSB1, we investigated SSB1 function in B-cell precursors. Due to the compensatory function of its homolog SSB2, we required a conditional knockout model that allowed us to selectively delete both homologs in B-cells to study the effect of their combined loss; this model was established at The Rockefeller University by our lab. In our study published in The Journal of Immunology (DOI: 10.4049/jimmunol.1801618) we showed that the function of these factors appeared essential even in normal B-cells, leading to a defect in early B-cell differentiation upon combined SSB1/2 loss, most predominantly affecting the pre-B to immature B stage. This effect did not relate to their potential function in V(D)J recombination of immunoglobulin (Ig) genes but rather to their essential requirement during cell division as cultured, continuously dividing B-cell precursors that did not depend on V(D)J recombination similarly were affected by their loss. Further analysis showed that SSB1/2 loss caused increased disruption of genome fragile sites, inefficient cell cycle progression, and DNA damage if apoptosis was suppressed. As such, SSB1/2 appeared as safeguards of B-cell precursors and may similarly protect leukemic B-cell precursor cells.
EVI1 oncogene activation in poor prognosis myeloid leukemia. While oncogenes can initiate leukaemia, such as BCR-ABL1 in CML and Ph+B-ALL, secondary activation of additional oncogenes can further worsen the disease. An example of such secondary oncogene activation is the transcriptional activation of EVI1, which expression is associated with poor prognosis in acute myeloid leukaemia (AML) and CML. Activation of EVI1, encoded by the MECOM locus on chromosome 3, is caused in a small fraction of AML cases (~2%) by chromosomal rearrangements involving 3q. However, in a larger fraction of AML (~10%) and myeloid blast crisis CML (CML-MBC, >50%), its activation occurs in the absence of 3q rearrangements. While multiple factors have been identified that can promote EVI1 expression, it is still largely unknown what additional circumstances are required to promote the unfortunate expression of this oncogene in leukemic cells. In a current study funded by The Kay Kendall Leukaemia Fund, we functionally investigate chromatin states of EVI1+3q- compared to EVI1-3q- leukaemia cells to define chromatin environment and regulatory elements that further support its transcriptional activation.
The oncogenic herpesvirus KSHV triggers hallmarks of an alternative pathway that maintains the length of telomeres.To achieve replicative immortality, cancer cells must activate telomere maintenance mechanisms to prevent progressive telomere shortening that occurs with each cell division. ~85% of cancers circumvent telomeric attrition by re-expressing telomerase, while the remaining ~15% of cancers induce alternative lengthening of telomeres (ALT), which relies on break-induced replication (BIR) and telomere recombination. Although ALT tumours were first reported over 20 years ago, the mechanism of ALT induction remains unclear and no study before had described a cell-based model that permits the induction and sustained switch to ALT. In a collaborative study with the Boulton lab at The Francis Crick Institute and published in Nature Communications (DOI: 10.1038/s41467-020-20819-4), we were able demonstrate that infection with Kaposi’s sarcoma herpesvirus (KSHV) induces sustained acquisition of ALT in previously non-ALT/telomerase positive cell lines. KSHV is the causative agent that promotes Kaposi’s sarcoma, primary effusion lymphoma (PEL) and a proportion multicentric Castleman’s disease (MCD). KSHV-infected cells acquire an ALT-associated telomeric proteome and molecular hallmarks of ALT activity that are also observed in KSHV-associated patient tumour biopsies. We further showed that down-regulating BIR impairs KSHV latency, suggesting that KSHV induces and then co-opts ALT as part of its own life cycle. As such, our study uncovered KSHV infection as a means to induce telomere maintenance by ALT and reveals features of ALT in KSHV-associated cancer.
Transcriptional deregulation in non-transcription factor driven B-lineage precursor leukaemias. Leukaemia sub-types can be differentiated by their initiating genetic lesions. Likewise, each subtype exhibits a unique transcriptional program that defines and drives the malignancy and that can be used to differentiate the subtypes. Together, currently 23 different B-lineage leukaemia (B-ALL) types can be distinguished by the combined analysis of genetic lesions and transcriptional programs. While this comes to no surprise as most B-ALLs are driven by genetic alterations affecting transcription factors (TFs), for B-ALLs that are not driven by TF alterations it is less clear how the transcriptional program is established. In a study funded by Blood Cancer UK, the Feldhahn lab investigates this for a historically poor prognosis B-ALL called Ph+B-ALL that is driven by the BCR-ABL1 oncogene, a constitutive tyrosine kinase. The project investigates different aspects of the transcriptional program, how the respective genes are further regulated by so called enhancers, and which TFs are utilised by BCR-ABL1 to orchestrate the transcriptional program and the underlying changes in the chromatin.
Transcriptional regulation of leukaemia-inducing oncogenes. While BCR-ABL1 is the driving lesion of Ph+B-ALL, a recent study suggested that the promoter that drives BCR-ABL1 expression alone is not sufficient to cause BCR-ABL1-induced malignancy. Instead, for leukaemia development, BCR-ABL1 needs to be transcribed at very high levels and, in line with this, in the study where BCR-ABL1 does not cause neoplasia, it is transcribed at very low levels. However, little is known about how the BCR promoter that drives BCR-ABL1 in leukaemia becomes enhanced to yield oncogenic BCR-ABL1 levels. Promoter activity of most genes is elevated by gene-regulatory elements, so-called enhancers, which physically interact with promoters via chromatin interactions. In a current study, the Feldhahn lab identified such an enhancer that likely regulates BCR-ABL1 expression and as such appears to be essential for BCR-ABL1-driven leukaemia cells such as Ph+B-ALL and CML. The lab further investigates enhancers associated with other leukaemia-inducing oncogenes to understand if regulation by enhancers is a general feature of leukaemia-inducing lesions.