Tracey Perry

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.

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.

Sequential Treatment with Cytarabine and Decitabine Has an Increased Anti-Leukemia Effect Compared to Cytarabine Alone in Xenograft Models of Childhood Acute Myeloid Leukemia

The current interest in epigenetic priming is underpinned by the belief that remodelling of the epigenetic landscape will sensitise tumours to subsequent therapy. In this pre-clinical study, paediatric AML cells expanded in culture and primary AML xenografts were treated with decitabine, a DNA demethylating agent, and cytarabine, a frontline cytotoxic agent used in the treatment of AML, either alone or in combination. Sequential treatment with decitabine and cytarabine was found to be more effective in reducing tumour burden than treatment with cytarabine alone suggesting that the sequential delivery of these agents may a have real clinical advantage in the treatment of paediatric AML. However we found no evidence to suggest that this outcome was dependent on priming with a hypomethylating agent, as the benefits observed were independent of the order in which these drugs were administered.

USP7 inhibition alters homologous recombination repair and targets CLL cells independently of ATM/p53 functional status

The role of deubiquitylase ubiquitin-specific protease 7 (USP7) in the regulation of the p53-dependent DNA damage response (DDR) pathway is well established. Whereas previous studies have mostly focused on the mechanisms underlying how USP7 directly controls p53 stability, we recently showed that USP7 modulates the stability of the DNA damage responsive E3 ubiquitin ligase RAD18. This suggests that targeting USP7 may have therapeutic potential even in tumors with defective p53 or ibrutinib resistance. To test this hypothesis, we studied the effect of USP7 inhibition in chronic lymphocytic leukemia (CLL) where the ataxia telangiectasia mutated (ATM)-p53 pathway is inactivated with relatively high frequency, leading to treatment resistance and poor clinical outcome. We demonstrate that USP7 is upregulated in CLL cells, and its loss or inhibition disrupts homologous recombination repair (HRR). Consequently, USP7 inhibition induces significant tumor-cell killing independently of ATM and p53 through the accumulation of genotoxic levels of DNA damage. Moreover, USP7 inhibition sensitized p53-defective, chemotherapy-resistant CLL cells to clinically achievable doses of HRR-inducing chemotherapeutic agents in vitro and in vivo in a murine xenograft model. Together, these results identify USP7 as a promising therapeutic target for the treatment of hematological malignancies with DDR defects, where ATM/p53-dependent apoptosis is compromised.

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.

S1PR1 drives a feedforward signalling loop to regulate BATF3 and the transcriptional programme of Hodgkin lymphoma cells

The Hodgkin/Reed–Sternberg cells of classical Hodgkin lymphoma (HL) are characterised by the aberrant activation of multiple signalling pathways. Here we show that a subset of HL displays altered expression of sphingosine-1-phosphate (S1P) receptors (S1PR)s. S1P activates phosphatidylinositide 3-kinase (PI3-K) in these cells that is mediated by the increased expression of S1PR1 and the decreased expression of S1PR2. We also showed that genes regulated by the PI3-K signalling pathway in HL cell lines significantly overlap with the transcriptional programme of primary HRS cells. Genes upregulated by the PI3-K pathway included the basic leucine zipper transcription factor, ATF-like 3 (BATF3), which is normally associated with the development of dendritic cells. Immunohistochemistry confirmed that BATF3 was expressed in HRS cells of most HL cases. In contrast, in normal lymphoid tissues, BATF3 expression was confined to a small fraction of CD30-positive immunoblasts. Knockdown of BATF3 in HL cell lines revealed that BATF3 contributed to the transcriptional programme of primary HRS cells, including the upregulation of S1PR1. Our data suggest that disruption of this potentially oncogenic feedforward S1P signalling loop could provide novel therapeutic opportunities for patients with HL.

The arginine metabolome in acute lymphoblastic leukemia can be targeted by the pegylated-recombinant arginase I BCT-100: Targeting arginine metabolism in ALL

Arginine is a semi‐essential amino acid that plays a key role in cell survival and proliferation in normal and malignant cells. BCT‐100, a pegylated (PEG) recombinant human arginase, can deplete arginine and starve malignant cells of the amino acid. Acute lymphoblastic leukemia (ALL) is the most common cancer of childhood, yet for patients with high risk or relapsed disease prognosis remains poor. We show that BCT‐100 is cytotoxic to ALL blasts from patients in vitro by necrosis, and is synergistic in combination with dexamethasone. Against ALL xenografts, BCT‐100 leads to a reduction in ALL engraftment and a prolongation of survival. ALL blasts express the arginine transporter CAT‐1, yet the majority of blasts are arginine auxotrophic due to deficiency in either argininosuccinate synthase (ASS) or ornithine transcarbamylase (OTC). Although endogenous upregulation or retroviral transduced increases in ASS or OTC may promote ALL survival under moderately low arginine conditions, expression of these enzymes cannot prevent BCT‐100 cytotoxicity at arginine depleting doses. RNA‐sequencing of ALL blasts and supporting stromal cells treated with BCT‐100 identifies a number of candidate pathways which are altered in the presence of arginine depletion. Therefore, BCT‐100 provides a new clinically relevant therapeutic approach to target arginine metabolism in ALL.

Co-Expression of the Epstein-Barr Virus-Encoded Latent Membrane Proteins and the Pathogenesis of Classic Hodgkin Lymphoma

The Epstein-Barr virus (EBV) is present in the tumour cells of a subset of patients with classic Hodgkin lymphoma (cHL), yet the contribution of the virus to the pathogenesis of these tumours remains only poorly understood. The EBV genome in virus-associated cHL expresses a limited subset of genes, restricted to the non-coding Epstein-Barr virus-encoded RNAs (EBERs) and viral miRNA, as well as only three virus proteins; the Epstein-Barr virus nuclear antigen-1 (EBNA1), and the two latent membrane proteins, known as LMP1 and LMP2, the latter of which has two isoforms, LMP2A and LMP2B. LMP1 and LMP2A are of particular interest because they are co-expressed in tumour cells and can activate cellular signalling pathways, driving aberrant cellular transcription in infected B cells to promote lymphomagenesis. This article seeks to bring together the results of recent studies of the latent membrane proteins in different B cell systems, including experiments in animal models as well as a re-analysis of our own transcriptional data. In doing so, we summarise the potentially co-operative and antagonistic effects of the LMPs that are relevant to B cell lymphomagenesis.

Accounting for systematic errors in bioluminescence imaging to improve quantitative accuracy

Bioluminescence imaging (BLI) is a widely used pre-clinical imaging technique, but there are a number of limitations to its quantitative accuracy. This work uses an animal model to demonstrate some significant limitations of BLI and presents processing methods and algorithms which overcome these limitations, increasing the quantitative accuracy of the technique. The position of the imaging subject and source depth are both shown to affect the measured luminescence intensity. Free Space Modelling is used to eliminate the systematic error due to the camera/subject geometry, removing the dependence of luminescence intensity on animal position. Bioluminescence tomography (BLT) is then used to provide additional information about the depth and intensity of the source. A substantial limitation in the number of sources identified using BLI is also presented. It is shown that when a given source is at a significant depth, it can appear as multiple sources when imaged using BLI, while the use of BLT recovers the true number of sources present.