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.
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.