Victoria J Weston

BET inhibition as a single or combined therapeutic approach in primary paediatric B-precursor acute lymphoblastic leukaemia

Paediatric B-precursor ALL is a highly curable disease, however, treatment resistance in some patients and the long-term toxic effects of current therapies pose the need for more targeted therapeutic approaches. We addressed the cytotoxic effect of JQ1, a highly selective inhibitor against the transcriptional regulators, bromodomain and extra-terminal (BET) family of proteins, in paediatric ALL. We showed a potent in vitro cytotoxic response of a panel of primary ALL to JQ1, independent of their prognostic features but dependent on high MYC expression and coupled with transcriptional downregulation of multiple pro-survival pathways. In agreement with earlier studies, JQ1 induced cell cycle arrest. Here we show that BET inhibition also reduced c-Myc protein stability and suppressed progression of DNA replication forks in ALL cells. Consistent with c-Myc depletion and downregulation of pro-survival pathways JQ1 sensitised primary ALL samples to the classic ALL therapeutic agent dexamethasone. Finally, we demonstrated that JQ1 reduces ALL growth in ALL xenograft models, both as a single agent and in combination with dexamethasone. We conclude that targeting BET proteins should be considered as a new therapeutic strategy for the treatment of paediatric ALL and particularly those cases that exhibit suboptimal responses to standard treatment.

Frequent epigenetic inactivation of the SLIT2 gene in chronic and acute lymphocytic leukemia

Recently a mouse model of T/natural killer acute lymphoblastic leukemia was used to assess global promoter methylation across the mouse genome using the restriction landmark genomic scanning technique. One of the methylated mouse genes identified in this way was Slit2. There are three mammalian SLIT genes (SLIT1, SLIT2, SLIT3), that belong to a highly conserved family of axon guidance molecules. We have previously demonstrated that SLIT2 is frequently inactivated in lung, breast, colorectal and glioma tumors by hypermethylation of a CpG island in its promoter region, whilst inactivating somatic mutations are rare. Furthermore, we demonstrated that SLIT2 acts as a tumor suppressor gene in breast and colorectal cancer cells. In this report we determined the methylation status of the SLIT2 gene in leukemias (CLL and ALL). SLIT2 was methylated in all 10 leukemia cell lines analyzed (8 completely and 2 partially methylated). SLIT2 expression was restored after treating ALL lines with 5-aza-2dC. In primary ALL and CLL samples, SLIT2 was also frequently methylated, 58% (30/52) B-ALL; 83% (10/12) T-ALL and in 80% (24/30) CLL. Whilst DNA from peripheral blood and bone marrow from healthy control samples showed no SLIT2 methylation. Methylation results in leukemia cell lines and ALL and CLL primary samples were confirmed by direct sequencing of bisulfite modified DNA. Our results demonstrate that methylation of the SLIT2 5’ CpG island is conserved between mice and humans, and therefore is likely to be of functional importance.

Extreme telomere erosion in ATM-mutated and 11q-deleted CLL patients is independent of disease stage

Extreme telomere erosion in ATM-mutated and 11q-deleted CLL patients is independent of disease stage

The ability to cross the blood-cerebrospinal fluid barrier is a generic property of acute lymphoblastic leukemia blasts.

Prevention of central nervous system (CNS) relapse is critical for cure of childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Despite this, mechanisms of CNS infiltration are poorly understood, and the timing, frequency, and properties of BCP-ALL blasts entering the CNS compartment are unknown. We investigated the CNS-engrafting potential of BCP-ALL cells xenotransplanted into immunodeficient NOD.Cg- ITALIC! Prkdc (ITALIC! scid) ITALIC! Il2rg (ITALIC! tm1Wjl)/SzJ mice. CNS engraftment was seen in 23 of 29 diagnostic samples (79%): 2 of 2 from patients with overt CNS disease and 21 of 27 from patients thought to be CNS negative by diagnostic lumbar puncture. Histologic findings mimic human pathology and demonstrate that leukemic cells transit the blood-cerebrospinal fluid barrier situated close to the dural sinuses, the site of recently discovered CNS lymphatics. Retrieval of blasts from the CNS showed no evidence for chemokine receptor-mediated selective trafficking. The high frequency of infiltration and lack of selective trafficking led us to postulate that CNS tropism is a generic property of leukemic cells. To test this, we performed serial dilution experiments which showed CNS engraftment in 5 of 6 mice after transplant of as few as 10 leukemic cells. Clonal tracking techniques confirmed the polyclonal nature of CNS-infiltrating cells, with multiple clones engrafting in both the CNS and periphery. Overall, these findings suggest that subclinical seeding of the CNS is likely to be present in most BCP-ALL patients at original diagnosis, and efforts to prevent CNS relapse should concentrate on effective eradication of disease from this site rather than targeting entry mechanisms.

Stratification of pediatric ALL by in vitro cellular responses to DNA double-strand breaks provides insight into the molecular mechanisms underlying clinical response.

The molecular basis of different outcomes in pediatric acute lymphoblastic leukemia (ALL) remains poorly understood. We addressed the clinical significance and mechanisms behind in vitro cellular responses to ionizing radiation (IR)-induced DNA double-strand breaks in 74 pediatric patients with ALL. We found an apoptosis-resistant response in 36% of patients characterized by failure to cleave caspase-3, -7, -9, and PARP1 by 24 hours after IR and an apoptosis-sensitive response with the cleavage of the same substrates in the remaining 64% of leukemias. Resistance to IR in vitro was associated with poor early blast clearance at day 7 or 15 and persistent minimal residual disease (MRD) at day 28 of induction treatment. Global gene expression profiling revealed abnormal up-regulation of multiple prosurvival pathways in response to IR in apoptosis-resistant leukemias and differential posttranscriptional activation of the PI3-Akt pathway was observed in representative resistant cases. Importantly, pharmacologic inhibition of selected prosurvival pathways sensitized apoptosis-resistant ALL cells to IR in vitro. We suggest that abnormal prosurvival responses to DNA damage provide one of the mechanisms of primary resistance in ALL, and that they should be considered as therapeutic targets in children with aggressive disease.

Targeting the Ataxia Telangiectasia Mutated-null phenotype in chronic lymphocytic leukemia with pro-oxidants

Inactivation of the Ataxia Telangiectasia Mutated gene in chronic lymphocytic leukemia results in resistance to p53-dependent apoptosis and inferior responses to treatment with DNA damaging agents. Hence, p53-independent strategies are required to target Ataxia Telangiectasia Mutated-deficient chronic lymphocytic leukemia. As Ataxia Telangiectasia Mutated has been implicated in redox homeostasis, we investigated the effect of the Ataxia Telangiectasia Mutated-null chronic lymphocytic leukemia genotype on cellular responses to oxidative stress with a view to therapeutic targeting. We found that in comparison to Ataxia Telangiectasia Mutated-wild type chronic lymphocytic leukemia, pro-oxidant treatment of Ataxia Telangiectasia Mutated-null cells led to reduced binding of NF-E2 p45-related factor-2 to antioxidant response elements and thus decreased expression of target genes. Furthermore, Ataxia Telangiectasia Mutated-null chronic lymphocytic leukemia cells contained lower levels of antioxidants and elevated mitochondrial reactive oxygen species. Consequently, Ataxia Telangiectasia Mutated-null chronic lymphocytic leukemia, but not tumors with 11q deletion or TP53 mutations, exhibited differentially increased sensitivity to pro-oxidants both in vitro and in vivo. We found that cell death was mediated by a p53- and caspase-independent mechanism associated with apoptosis inducing factor activity. Together, these data suggest that defective redox-homeostasis represents an attractive therapeutic target for Ataxia Telangiectasia Mutated-null chronic lymphocytic leukemia.

The subclonal complexity of STIL-TAL1+ T-cell acute lymphoblastic leukaemia

Single-cell genetics were used to interrogate clonal complexity and the sequence of mutational events in STIL-TAL1+ T-ALL. Single-cell multicolour FISH was used to demonstrate that the earliest detectable leukaemia subclone contained the STIL-TAL1 fusion and copy number loss of 9p21.3 (CDKN2A/CDKN2B locus), with other copy number alterations including loss of PTEN occurring as secondary subclonal events. In three cases, multiplex qPCR and phylogenetic analysis were used to produce branching evolutionary trees recapitulating the snapshot history of T-ALL evolution in this leukaemia subtype, which confirmed that mutations in key T-ALL drivers, including NOTCH1 and PTEN, were subclonal and reiterative in distinct subclones. Xenografting confirmed that self-renewing or propagating cells were genetically diverse. These data suggest that the STIL-TAL1 fusion is a likely founder or truncal event. Therapies targeting the TAL1 auto-regulatory complex are worthy of further investigation in T-ALL.

Synergistic Action of Dual IGF1/R and MEK Inhibition Sensitizes Childhood Acute Lymphoblastic Leukaemia (ALL) Cells to Cytotoxic Agents and Involves Down-Regulation of STAT6 and PDAP1

Highlights • Dual insulin-like growth factor 1 receptor (IGF1/R) + mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibition synergistically sensitize apoptosis-resistant acute lymphoblastic leukemia (ALL) cells.• Dual IGF1/R + MEK inhibition potentiates chemotherapy-induced killing of ALL cells.• Signal transducer and activator of transcription 6 (STAT6) and platelet-derived growth factor-associated protein 1 (PDAP1) are downregulated differentially by this synergistic action.• STAT6 and PDAP1 are predicted to act in a putative STAT6–ERK–nuclear factor kappa beta (NF-κB) survival network.• Targeting this network may be useful for sensitizing ALL to chemotherapy agents.

An unrelated cytogenetic karyotype and intra-lineage shift at relapse of an aggressive paediatric B-precursor ALL.

An unrelated cytogenetic karyotype and intra-lineage shift at relapse of an aggressive paediatric B-precursor ALL