Therapy-related leukaemias are becoming an increasing healthcare problem as more patients survive their primary cancers. The nature of the causative agent has an important bearing upon the characteristics, biology, time to onset and prognosis of the resultant leukaemia. Agents targeting topoisomerase II induce acute leukaemias with balanced translocations that generally arise within 3 years, often involving the MLL, RUNX1 and RARA loci at 11q23, 21q22 and 17q21 respectively. Chromosomal breakpoints have been found to be preferential sites of topoisomerase II cleavage, which are believed to be repaired by the nonhomologous end-joining DNA repair pathway to generate chimaeric oncoproteins that underlie the resultant leukaemias. Therapy-related acute myeloid leukaemias occurring after exposure to antimetabolites and/or alkylating agents are biologically distinct with a longer latency period, being characterised by more complex karyotypes and loss of p53. Although treatment of therapy-related leukaemias represents a considerable challenge due to prior therapy and comorbidities, curative therapy is possible, particularly in those with favourable karyotypic features.
Key Points• CXXC5 inhibits Wnt signaling and is a candidate tumor suppressor in AML.• Low CXXC5 expression is an independent prognostic factor in AML.The gene CXXC5 on 5q31 is frequently deleted in acute myeloid leukemia (AML) with del(5q), suggesting that inactivation of CXXC5 might play a role in leukemogenesis. Here, we investigated the functional and prognostic implications of CXXC5 expression in AML. CXXC5 mRNA was downregulated in AML with MLL rearrangements, t(8;21) and GATA2 mutations. As a mechanism of CXXC5 inactivation, we found evidence for epigenetic silencing by promoter methylation. Patients with CXXC5 expression below the median level had a lower relapse rate (45% vs 59%; P 5 .007) and a better overall survival (OS, 46% vs 28%; P < .001) and event-free survival (EFS, 36% vs 21%; P < .001) at 5 years, independent of cytogenetic risk groups and known molecular risk factors. In gene-expression profiling, lower CXXC5 expression was associated with upregulation of cell-cycling genes and codownregulation of genes implicated in leukemogenesis (WT1, GATA2, MLL, DNMT3B, RUNX1). Functional analyses demonstrated CXXC5 to inhibit leukemic cell proliferation and Wnt signaling and to affect the p53-dependent DNA damage response. In conclusion, our data suggest a tumor suppressor function of CXXC5 in AML. Inactivation of CXXC5 is associated with different leukemic pathways and defines an AML subgroup with better outcome. (Blood.
Balanced chromosomal translocations that generate chimeric oncoproteins are considered to be initiating lesions in the pathogenesis of acute myeloid leukemia. The most frequent is the t(15;17)(q22;q21), which fuses the PML and RARA genes, giving rise to acute promyelocytic leukemia (APL). An increasing proportion of APL cases are therapy-related (t-APL), which develop following exposure to radiotherapy and/or chemotherapeutic agents that target DNA topoisomerase II (topoII), particularly mitoxantrone and epirubicin. To gain insights into molecular mechanisms underlying the formation of the t(15;17) we mapped the translocation breakpoints in a series of t-APLs, which revealed significant clustering according to the nature of the drug exposure. Remarkably, in approximately half of t-APL cases arising following mitoxantrone treatment for breast cancer or multiple sclerosis, the chromosome 15 breakpoint fell within an 8-bp “hotspot” region in PML intron 6, which was confirmed to be a preferential site of topoII-mediated DNA cleavage induced by mitoxantrone. Chromosome 15 breakpoints falling outside the “hotspot”, and the corresponding RARA breakpoints were also shown to be functional topoII cleavage sites. The observation that particular regions of the PML and RARA loci are susceptible to topoII-mediated DNA damage induced by epirubicin and mitoxantrone may underlie the propensity of these agents to cause APL.
Acute promyelocytic leukemia (APL) is characterised by the t(15;17)(q22;q21) leading to fusion of PML to the gene encoding the myeloid transcription factor Retinoic Acid Receptor α (RARα). Chromosomal translocations such as the t(15;17) are considered to be initiating events in leukemogenesis; however, sequencing of APL genomes has provided further evidence that the PML-RARα fusion is insufficient to induce leukemia, which depends upon the acquisition of cooperating mutations. The PML-RARα oncoprotein exerts a profound effect on nuclear architecture, disrupting multiprotein structures known as PML nuclear bodies (NBs). The function of these structures remains an enigma; however, their disruption in PML-RARα+ APL and acute lymphoblastic leukemia with the t(9;15)(p13;q24)/PAX5-PML fusion is associated with delocalisation of a number of component proteins including PML, which have been implicated in growth control and neoplastic transformation. It is now established that the PML moiety contributes to APL pathogenesis by conferring via the translocation a novel dimerisation capacity to RARα, but it has been unclear whether deregulation of PML and other NB components cooperates in leukemic transformation or impacts the response to differentiating agents. To address these questions, we generated a knock-in mouse model with targeted NB disruption achieved through mutation of key zinc-binding cysteine residues in the amino-terminal RING domain of Pml. Homozygous Pml RING mutant mice are viable, with no overt developmental defect; however, analysis of the bone marrow revealed significant expansion of the Lin(-)Sca-1(+)c-Kit(+) (LSK) population compared to wild type (WT) controls (p<0.01), accompanied by increased LSK cell proliferation (p<0.0001) as evaluated by in vivo labelling through incorporation of 5-ethynyl-2'-deoxyuridine (EdU). In addition, hematopoietic cells derived from homozygous Pml RING mutant mice exhibited markedly elevated levels of DNA damage compared to WT cells from age-matched controls, as evidenced by increased numbers of γH2AX foci (p=0.009). This was associated with significantly delayed DNA damage repair responses in Pml RING mutant cells following γ-irradiation (p=0.005). Accordingly, expression of PML-RARα in human hematopoietic cells, which led to disruption of NBs, also induced a significant increase in γH2AX foci (p=0.0023). While no leukemias arose in homozygous Pml RING mutant mice, they developed an excess of T- and B-cell lymphomas (p=0.03), consistent with the proposed tumour suppressor function of PML and the NBs. Since a key property conferred by the PML moiety required for leukemogenicity of the PML-RARα oncoprotein is the capacity to dimerise, we evaluated whether Pml NB disruption could cooperate with forced RARα homodimerisation (mediated artificially by linking RARα to the p50 dimerisation motif of NFκB). While Pml NB disruption or p50-RARA expressed under the control of the MRP8 promoter in murine hematopoietic stem/progenitor cells conferred limited replating capacity, in combination they exhibited marked cooperativity, with a significant increase in third round colonies (p=0.03). Moreover, NB disruption was found to cooperate with forced RARα homodimerisation in vivo with a doubling in the rate of leukemia development in p50-RARα mice with mutated Pml (p<0.0001), leading to a penetrance comparable to that observed in previously published PML-RARα transgenic models. Moreover, the latency to onset of leukemia was significantly shorter in p50-RARα mice with the Pml RING mutation, occurring from 213 days of age vs 310 days with WT Pml (p=0.008). While Pml NB disruption did not affect engraftment of p50-RARα leukemias in serial transplantation, the in vitro differentiation response of p50-RARα leukemias to All transretinoic acid (ATRA) as determined by nitroblue tetrazolium assay was significantly impaired in the context of NB disruption (p<0.05). Moreover, prolongation of survival following ATRA treatment in mice transplanted with p50-RARα leukemic blasts was dependent upon Pml NB integrity (p=0.03). Overall, these data suggest that the NB disruption mediated by the PML-RARα oncoprotein plays a key role in APL pathogenesis contributing to expansion of the LSK population and defective DNA repair predisposing to the acquisition of cooperating mutations, but also implicate NBs in the response to differentiating agents. Disclosures: No relevant conflicts of interest to declare.
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