Integrative analysis of spontaneous CLL regression highlights genetic and microenvironmental interdependency in CLL.

Spontaneous regression is a recognized phenomenon in chronic lymphocytic leukemia (CLL) but its biological basis remains unknown. We undertook a detailed investigation of the biological and clinical features of 20 spontaneous CLL regression cases incorporating phenotypic, functional, transcriptomic, and genomic studies at sequential time points. All spontaneously regressed tumors were IGHV-mutated with no restricted IGHV usage or B-cell receptor (BCR) stereotypy. They exhibited shortened telomeres similar to nonregressing CLL, indicating prior proliferation. They also displayed low Ki-67, CD49d, cell-surface immunoglobulin M (IgM) expression and IgM-signaling response but high CXCR4 expression, indicating low proliferative activity associated with poor migration to proliferation centers, with these features becoming increasingly marked during regression. Spontaneously regressed CLL displayed a transcriptome profile characterized by downregulation of metabolic processes as well as MYC and its downstream targets compared with nonregressing CLL. Moreover, spontaneous regression was associated with reversal of T-cell exhaustion features including reduced programmed cell death 1 expression and increased T-cell proliferation. Interestingly, archetypal CLL genomic aberrations including HIST1H1B and TP53 mutations and del(13q14) were found in some spontaneously regressing tumors, but genetic composition remained stable during regression. Conversely, a single case of CLL relapse following spontaneous regression was associated with increased BCR signaling, CLL proliferation, and clonal evolution. These observations indicate that spontaneously regressing CLL appear to undergo a period of proliferation before entering a more quiescent state, and that a complex interaction between genomic alterations and the microenvironment determines disease course. Together, the findings provide novel insight into the biological processes underpinning spontaneous CLL regression, with implications for CLL treatment.

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.

Dynamic changes in clonal cytogenetic architecture during progression of chronic lymphocytic leukemia in patients and patient-derived murine xenografts.

Subclonal heterogeneity and clonal selection influences disease progression in chronic lymphocytic leukemia (CLL). It is therefore important that therapeutic decisions are made based on an understanding of the CLL clonal architecture and its dynamics in individual patients. Identification of cytogenetic abnormalities by FISH remains the cornerstone of contemporary clinical practice and provides a simple means for prognostic stratification. Here, we demonstrate that multiplexed-FISH can enhance recognition of CLL subclonal repertoire and its dynamics during disease progression, both in patients and CLL patient-derived xenografts (PDX). We applied a combination of patient-specific FISH probes to 24 CLL cases before treatment and at relapse, and determined putative ancestral relationships between subpopulations with different cytogenetic features. We subsequently established 7 CLL PDX models in NOD/Shi-SCID/IL-2Rγctm1sug/Jic (NOG) mice. Application of multiplexed-FISH to these models demonstrated that all of the identified cytogenetic subpopulations had leukemia propagating activity and that changes in their representation during disease progression could be spontaneous, accelerated by treatment or treatment-induced. We conclude that multiplexed-FISH in combination with PDX models have the potential to distinguish between spontaneous and treatment-induced clonal selection, and therefore provide a valuable tool for the pre-clinical evaluation of novel therapies.

Mitotic control of human papillomavirus genome-containing cells is regulated by the function of the PDZ-binding motif of the E6 oncoprotein.

The function of a conserved PDS95/DLG1/ZO1 (PDZ) binding motif (E6 PBM) at the C-termini of E6 oncoproteins of high-risk human papillomavirus (HPV) types contributes to the development of HPV-associated malignancies. Here, using a primary human keratinocyte-based model of the high-risk HPV18 life cycle, we identify a novel link between the E6 PBM and mitotic stability. In cultures containing a mutant genome in which the E6 PBM was deleted there was an increase in the frequency of abnormal mitoses, including multinucleation, compared to cells harboring the wild type HPV18 genome. The loss of the E6 PBM was associated with a significant increase in the frequency of mitotic spindle defects associated with anaphase and telophase. Furthermore, cells carrying this mutant genome had increased chromosome segregation defects and they also exhibited greater levels of genomic instability, as shown by an elevated level of centromere-positive micronuclei. In wild type HPV18 genome-containing organotypic cultures, the majority of mitotic cells reside in the suprabasal layers, in keeping with the hyperplastic morphology of the structures. However, in mutant genome-containing structures a greater proportion of mitotic cells were retained in the basal layer, which were often of undefined polarity, thus correlating with their reduced thickness. We conclude that the ability of E6 to target cellular PDZ proteins plays a critical role in maintaining mitotic stability of HPV infected cells, ensuring stable episome persistence and vegetative amplification.

T-cell number and subtype influence the disease course of primary chronic lymphocytic leukaemia xenografts in alymphoid mice.

Chronic lymphocytic leukaemia (CLL) cells require microenvironmental support for their proliferation. This can be recapitulated in highly immunocompromised hosts in the presence of T cells and other supporting cells. Current primary CLL xenograft models suffer from limited duration of tumour cell engraftment coupled with gradual T-cell outgrowth. Thus, a greater understanding of the interaction between CLL and T cells could improve their utility. In this study, using two distinct mouse xenograft models, we investigated whether xenografts recapitulate CLL biology, including natural environmental interactions with B-cell receptors and T cells, and whether manipulation of autologous T cells can expand the duration of CLL engraftment. We observed that primary CLL xenografts recapitulated both the tumour phenotype and T-cell repertoire observed in patients and that engraftment was significantly shorter for progressive tumours. A reduction in the number of patient T cells that were injected into the mice to 2-5% of the initial number or specific depletion of CD8(+) cells extended the limited xenograft duration of progressive cases to that characteristic of indolent disease. We conclude that manipulation of T cells can enhance current CLL xenograft models and thus expand their utility for investigation of tumour biology and pre-clinical drug assessment.

BOD1L Is Required to Suppress Deleterious Resection of Stressed Replication Forks.

Recognition and repair of damaged replication forks are essential to maintain genome stability and are coordinated by the combined action of the Fanconi anemia and homologous recombination pathways. These pathways are vital to protect stalled replication forks from uncontrolled nucleolytic activity, which otherwise causes irreparable genomic damage. Here, we identify BOD1L as a component of this fork protection pathway, which safeguards genome stability after replication stress. Loss of BOD1L confers exquisite cellular sensitivity to replication stress and uncontrolled resection of damaged replication forks, due to a failure to stabilize RAD51 at these forks. Blocking DNA2-dependent resection, or downregulation of the helicases BLM and FBH1, suppresses both catastrophic fork processing and the accumulation of chromosomal damage in BOD1L-deficient cells. Thus, our work implicates BOD1L as a critical regulator of genome integrity that restrains nucleolytic degradation of damaged replication forks.

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.

Lack of functional and expression homology between human and mouse aldo-keto reductase 1C enzymes: implications for modelling human cancers.

BACKGROUND: Over recent years, enzymes of the aldo-keto reductase (AKR) 1C subfamily have been implicated in the progression of prostate, breast, endometrial and leukemic cancers. This is due to the ability of AKR1C enzymes to modify androgens, estrogens, progesterone and prostaglandins (PGs) in a tissue-specific manner, regulating the activity of nuclear receptors and other downstream effects. Evidence supporting a role for AKR1C enzymes in cancer derives mostly from studies with isolated primary cells from patients or immortalized cell lines. Mice are ideal organisms for in vivo studies, using knock-out or over-expression strains. However, the functional conservation of AKR1C enzymes between human and mice has yet to be described. RESULTS: In this study, we have characterized and compared the four human (AKR1C1,-1C2, -1C3 and -1C4) and the eight murine (AKR1C6, -1C12, -1C13, -1C14, -1C18, -1C19, -1C20 and -1C21) isoforms in their phylogeny, substrate preference and tissue distribution. We have found divergent evolution between human and murine AKR1C enzymes that was reflected by differing substrate preference. Murine enzymes did not perform the 11beta-ketoreduction of prostaglandin (PG) D2, an activity specific to human AKR1C3 and important in promoting leukemic cell survival. Instead, murine AKR1C6 was able to perform the 9-ketoreduction of PGE2, an activity absent amongst human isoforms. Nevertheless, reduction of the key steroids androstenedione, 5alpha-dihydrotestosterone, progesterone and estrone was found in murine isoforms. However, unlike humans, no AKR1C isoforms were detected in murine prostate, testes, uterus and haemopoietic progenitors. CONCLUSIONS: This study exposes significant lack of phylogenetic and functional homology between human and murine AKR1C enzymes. Therefore, we conclude that mice are not suitable to model the role of AKR1C in human cancers and leukemia.

Combined bezafibrate and medroxyprogesterone acetate: potential novel therapy for acute myeloid leukaemia.

BACKGROUND: The majority of acute myeloid leukaemia (AML) patients are over sixty years of age. With current treatment regimens, survival rates amongst these, and also those younger patients who relapse, remain dismal and novel therapies are urgently required. In particular, therapies that have anti-leukaemic activity but that, unlike conventional chemotherapy, do not impair normal haemopoiesis. PRINCIPAL FINDINGS: Here we demonstrate the potent anti-leukaemic activity of the combination of the lipid-regulating drug bezafibrate (BEZ) and the sex hormone medroxyprogesterone acetate (MPA) against AML cell lines and primary AML cells. The combined activity of BEZ and MPA (B/M) converged upon the increased synthesis and reduced metabolism of prostaglandin D(2) (PGD(2)) resulting in elevated levels of the downstream highly bioactive, anti-neoplastic prostaglandin 15-deoxy Delta(12,14) PGJ(2) (15d-PGJ(2)). BEZ increased PGD(2) synthesis via the generation of reactive oxygen species (ROS) and activation of the lipid peroxidation pathway. MPA directed prostaglandin synthesis towards 15d-PGJ(2) by inhibiting the PGD(2) 11beta -ketoreductase activity of the aldo-keto reductase AKR1C3, which metabolises PGD(2) to 9alpha11beta-PGF(2alpha). B/M treatment resulted in growth arrest, apoptosis and cell differentiation in both AML cell lines and primary AML cells and these actions were recapitulated by treatment with 15d-PGJ(2). Importantly, the actions of B/M had little effect on the survival of normal adult myeloid progenitors. SIGNIFICANCE: Collectively our data demonstrate that B/M treatment of AML cells elevated ROS and delivered the anti-neoplastic actions of 15d-PGJ(2). These observations provide the mechanistic rationale for the redeployment of B/M in elderly and relapsed AML.

AKR1C isoforms represent a novel cellular target for jasmonates alongside their mitochondrial-mediated effects.

Members of the aldo-keto reductase (AKR) superfamily, particularly the AKR1C subfamily, are emerging as important mediators of the pathology of cancer. Agents that inhibit these enzymes may provide novel agents for either the chemoprevention or treatment of diverse malignancies. Recently, jasmonates, a family of plant stress hormones that bear a structural resemblance to prostaglandins, have been shown to elicit anticancer activities both in vitro and in vivo. In this study, we show that jasmonic acid (JA) and methyl jasmonate (MeJ) are capable of inhibiting all four human AKR1C isoforms. Although JA is the more potent inhibitor of recombinant AKR1C proteins, including the in vitro prostaglandin F synthase activity of AKR1C3, MeJ displayed greater potency in cellular systems that was, at least in part, due to increased cellular uptake of MeJ. Moreover, using the acute myelogenous leukemia cell lines HL-60 and KG1a, we found that although both jasmonates were able to induce high levels of reactive oxygen species in a dose-dependent fashion, only MeJ was able to induce high levels of mitochondrial superoxide (MSO), possibly as an epiphenomenon of mitochondrial damage. There was a strong correlation observed between MSO formation at 24 hours and reduced cellularity at day 5. In conclusion, we have identified AKR1C isoforms as a novel target of jasmonates in cancer cells and provide further evidence of the promise of these compounds, or derivatives thereof, as adjunctive therapies in the treatment of cancer.

The aldo-keto reductase AKR1C3 contributes to 7,12-dimethylbenz(a)anthracene-3,4-dihydrodiol mediated oxidative DNA damage in myeloid cells: implications for leukemogenesis.

The aldo-keto reductase AKR1C3, has been shown to regulate myelopoiesis via its ability to metabolise prostaglandin D2 (PGD2). Other studies have demonstrated the oxidative activation of polycyclic aromatic hydrocarbon (PAH) procarcinogens by AKR1C3 in cell-free systems. This is the first study that addresses whether AKR1C3 mediates carcinogen activation within intact living cells following manipulation of AKR1C3 by molecular intervention. Quantitative RT-PCR identified AKR1C3 as the predominant AKR1C isoform expressed in acute myeloid leukemia (AML). Exposure of K562 and KG1a myeloid cell lines to the known AKR1C3 substrate 7,12-dimethylbenz(a)anthracene-3,4-dihydrodiol (7,12-DMBA-3,4-diol) resulted in both single strand DNA breaks and oxidative DNA damage as measured using conventional and FPG-modified comet assays respectively. PGD2-keto reductase activity was shown to be correlated with relative AKR1C3 expression and together with quantitative real time PCR was used to validate the RNAi-knockdown of AKR1C3 in K562 cells. Knockdown of AKR1C3 did not alter single strand DNA breaks following 7,12-DMBA-3,4-diol exposure but significantly decreased oxidative DNA damage. A similar interrelationship between AKR1C3 activity and 7,12-DMBA-3,4-diol mediated oxidative DNA damage but not single strand breaks was observed in KG1a cells. Finally, AKR1C3 knockdown also resulted in spontaneous erythroid differentiation of K562 cells. Since K562 cells are a model of AML blast crisis of chronic myeloid leukemia (CML) the data presented here identify AKR1C3 as a novel mediator of carcinogen-induced initiation of leukemia, as a novel regulator of erythroid differentiation and paradoxically as a potential new target in the treatment of CML.

Making sense of molecular signatures in the immune system.

The development of Functional Genomics technologies has opened new avenues to investigate the complexity of the immune system. Microarray technology has been particularly successful because of its relatively low cost and high genome coverage. Consequently to our ability to monitor the expression of a significant proportion of an organism genome, our understanding of the molecular dynamics behind cell differentiation and cell response has greatly improved. Molecular signatures associated to immune cells have provided important tools to investigate the molecular basis of diseases and have been often associated to diagnostic and prognostic markers. The availability of such large collection of data has stimulated the application of complex machine learning techniques in the attempt to link molecular signatures and cell physiology. Here we review the most recent developments in the analysis of molecular signatures in the immune system.