DNA damage response defects in hematologic malignancies: mechanistic insights and therapeutic strategies.

ABSTRACT: The DNA damage response (DDR) encompasses the detection and repair of DNA lesions and is fundamental to the maintenance of genome integrity. Germ line DDR alterations underlie hereditary chromosome instability syndromes by promoting the acquisition of pathogenic structural variants in hematopoietic cells, resulting in increased predisposition to hematologic malignancies. Also frequent in hematologic malignancies are somatic mutations of DDR genes, typically arising from replication stress triggered by oncogene activation or deregulated tumor proliferation that provides a selective pressure for DDR loss. These defects impair homology-directed DNA repair or replication stress response, leading to an excessive reliance on error-prone DNA repair mechanisms that results in genomic instability and tumor progression. In hematologic malignancies, loss-of-function DDR alterations confer clonal growth advantage and adverse prognostic impact but may also provide therapeutic opportunities. Selective targeting of functional dependencies arising from these defects could achieve synthetic lethality, a therapeutic concept exemplified by inhibition of poly-(adenosine 5'-diphosphate ribose) polymerase or the ataxia telangiectasia and Rad 3 related-CHK1-WEE1 axis in malignancies harboring the BRCAness phenotype or genetic defects that increase replication stress. Furthermore, the role of DDR defects as a source of tumor immunogenicity, as well as their impact on the cross talk between DDR, inflammation, and tumor immunity are increasingly recognized, thus providing rationale for combining DDR modulation with immune modulation. The nature of the DDR-immune interface and the cellular vulnerabilities conferred by DDR defects may nonetheless be disease-specific and remain incompletely understood in many hematologic malignancies. Their comprehensive elucidation will be critical for optimizing therapeutic strategies to target DDR defects in these diseases.

Genome-scale clustered regularly interspaced short palindromic repeats screen identifies nucleotide metabolism as an actionable therapeutic vulnerability in diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is the most common malignancy that develops in patients with ataxia-telangiectasia, a cancer-predisposing inherited syndrome characterized by inactivating germline ATM mutations. ATM is also frequently mutated in sporadic DLBCL. To investigate lymphomagenic mechanisms and lymphoma-specific dependencies underlying defective ATM, we applied ribonucleic acid (RNA)-seq and genome-scale loss-offunction clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 screens to systematically interrogate B-cell lymphomas arising in a novel murine model (Atm-/-nu-/-) with constitutional Atm loss, thymic aplasia but residual T-cell populations. Atm-/-nu-/-lymphomas, which phenotypically resemble either activated B-cell-like or germinal center Bcell-like DLBCL, harbor a complex karyotype, and are characterized by MYC pathway activation. In Atm-/-nu-/-lymphomas, we discovered nucleotide biosynthesis as a MYCdependent cellular vulnerability that can be targeted through the synergistic nucleotidedepleting actions of mycophenolate mofetil (MMF) and the WEE1 inhibitor, adavosertib (AZD1775). The latter is mediated through a synthetically lethal interaction between RRM2 suppression and MYC dysregulation that results in replication stress overload in Atm-/-nu-/-lymphoma cells. Validation in cell line models of human DLBCL confirmed the broad applicability of nucleotide depletion as a therapeutic strategy for MYC-driven DLBCL independent of ATM mutation status. Our findings extend current understanding of lymphomagenic mechanisms underpinning ATM loss and highlight nucleotide metabolism as a targetable therapeutic vulnerability in MYC-driven DLBCL.

CRISPR screens identify genomic ribonucleotides as a source of PARP-trapping lesions.

The observation that BRCA1- and BRCA2-deficient cells are sensitive to inhibitors of poly(ADP-ribose) polymerase (PARP) has spurred the development of cancer therapies that use these inhibitors to target deficiencies in homologous recombination1. The cytotoxicity of PARP inhibitors depends on PARP trapping, the formation of non-covalent protein-DNA adducts composed of inhibited PARP1 bound to DNA lesions of unclear origins1-4. To address the nature of such lesions and the cellular consequences of PARP trapping, we undertook three CRISPR (clustered regularly interspersed palindromic repeats) screens to identify genes and pathways that mediate cellular resistance to olaparib, a clinically approved PARP inhibitor1. Here we present a high-confidence set of 73 genes, which when mutated cause increased sensitivity to PARP inhibitors. In addition to an expected enrichment for genes related to homologous recombination, we discovered that mutations in all three genes encoding ribonuclease H2 sensitized cells to PARP inhibition. We establish that the underlying cause of the PARP-inhibitor hypersensitivity of cells deficient in ribonuclease H2 is impaired ribonucleotide excision repair5. Embedded ribonucleotides, which are abundant in the genome of cells deficient in ribonucleotide excision repair, are substrates for cleavage by topoisomerase 1, resulting in PARP-trapping lesions that impede DNA replication and endanger genome integrity. We conclude that genomic ribonucleotides are a hitherto unappreciated source of PARP-trapping DNA lesions, and that the frequent deletion of RNASEH2B in metastatic prostate cancer and chronic lymphocytic leukaemia could provide an opportunity to exploit these findings therapeutically.

TLR9 expression in chronic lymphocytic leukemia identifies a promigratory subpopulation and novel therapeutic target.

Chronic lymphocytic leukemia (CLL) remains incurable despite B-cell receptor-targeted inhibitors revolutionizing treatment. This suggests that other signaling molecules are involved in disease escape mechanisms and resistance. Toll-like receptor 9 (TLR9) is a promising candidate that is activated by unmethylated cytosine guanine dinucleotide-DNA. Here, we show that plasma from patients with CLL contains significantly more unmethylated DNA than plasma from healthy control subjects (P < .0001) and that cell-free DNA levels correlate with the prognostic markers CD38, ß2-microglobulin, and lymphocyte doubling time. Furthermore, elevated cell-free DNA was associated with shorter time to first treatment (hazard ratio, 4.0; P = .003). We also show that TLR9 expression was associated with in vitro CLL cell migration (P < .001), and intracellular endosomal TLR9 strongly correlated with aberrant surface expression (sTLR9; r = 0.9). In addition, lymph node-derived CLL cells exhibited increased sTLR9 (P = .016), and RNA-sequencing of paired sTLR9hi and sTLR9lo CLL cells revealed differential transcription of genes involved in TLR signaling, adhesion, motility, and inflammation in sTLR9hi cells. Mechanistically, a TLR9 agonist, ODN2006, promoted CLL cell migration (P < .001) that was mediated by p65 NF-κB and STAT3 transcription factor activation. Importantly, autologous plasma induced the same effects, which were reversed by a TLR9 antagonist. Furthermore, high TLR9 expression promoted engraftment and rapid disease progression in a NOD/Shi-scid/IL-2Rγnull mouse xenograft model. Finally, we showed that dual targeting of TLR9 and Bruton's tyrosine kinase (BTK) was strongly synergistic (median combination index, 0.2 at half maximal effective dose), which highlights the distinct role for TLR9 signaling in CLL and the potential for combined targeting of TLR9 and BTK as a more effective treatment strategy in this incurable disease.

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.

Aniline-containing derivatives of parthenolide: Synthesis and anti-chronic lymphocytic leukaemia activity.

Parthenolide exhibits anti-leukaemia activity, whilst its synthetic modification to impart improve drug-like properties, including 1,4-conjugate addition of primary and secondary amines, have previously been used, 1,4-addition of aniline derivatives to parthenolide has not been fully explored. A protocol for such additions to parthenolide is outlined herein. Reaction conditions were determined using tulipane as a model Michael acceptor. Subsequently, aniline-containing parthenolide derivatives were prepared under the optimised conditions and single crystal X-ray diffraction structures were resolved for three of the compounds synthesised. The synthesised derivatives, along with compounds resulting from a side reaction, were tested for their in vitro anti-leukaemia activity using the chronic lymphocytic leukaemia (CLL) MEC1 cell line. Computational studies with the 2RAM protein structure suggested that the activity of the derivatives was independent of their in silico ability to dock with the Cys38 residue of NF-κB.

Degradation of a Novel DNA Damage Response Protein, Tankyrase 1 Binding Protein 1, following Adenovirus Infection.

Infection by most DNA viruses activates a cellular DNA damage response (DDR), which may be to the detriment or advantage of the virus. In the case of adenoviruses, they neutralize antiviral effects of DDR activation by targeting a number of proteins for rapid proteasome-mediated degradation. We have now identified a novel DDR protein, tankyrase 1 binding protein 1 (TNKS1BP1) (also known as Tab182), which is degraded during infection by adenovirus serotype 5 and adenovirus serotype 12. In both cases, degradation requires the action of the early region 1B55K (E1B55K) and early region 4 open reading frame 6 (E4orf6) viral proteins and is mediated through the proteasome by the action of cullin-based cellular E3 ligases. The degradation of Tab182 appears to be serotype specific, as the protein remains relatively stable following infection with adenovirus serotypes 4, 7, 9, and 11. We have gone on to confirm that Tab182 is an integral component of the CNOT complex, which has transcriptional regulatory, deadenylation, and E3 ligase activities. The levels of at least 2 other members of the complex (CNOT3 and CNOT7) are also reduced during adenovirus infection, whereas the levels of CNOT4 and CNOT1 remain stable. The depletion of Tab182 with small interfering RNA (siRNA) enhances the expression of early region 1A proteins (E1As) to a limited extent during adenovirus infection, but the depletion of CNOT1 is particularly advantageous to the virus and results in a marked increase in the expression of adenovirus early proteins. In addition, the depletion of Tab182 and CNOT1 results in a limited increase in the viral DNA level during infection. We conclude that the cellular CNOT complex is a previously unidentified major target for adenoviruses during infection.IMPORTANCE Adenoviruses target a number of cellular proteins involved in the DNA damage response for rapid degradation. We have now shown that Tab182, which we have confirmed to be an integral component of the mammalian CNOT complex, is degraded following infection by adenovirus serotypes 5 and 12. This requires the viral E1B55K and E4orf6 proteins and is mediated by cullin-based E3 ligases and the proteasome. In addition to Tab182, the levels of other CNOT proteins are also reduced during adenovirus infection. Thus, CNOT3 and CNOT7, for example, are degraded, whereas CNOT4 and CNOT1 are not. The siRNA-mediated depletion of components of the complex enhances the expression of adenovirus early proteins and increases the concentration of viral DNA produced during infection. This study highlights a novel protein complex, CNOT, which is targeted for adenovirus-mediated protein degradation. To our knowledge, this is the first time that the CNOT complex has been identified as an adenoviral target.

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.

ATR inhibition induces synthetic lethality and overcomes chemoresistance in TP53- or ATM-defective chronic lymphocytic leukemia cells.

TP53 and ataxia telangiectasia mutated (ATM) defects are associated with genomic instability, clonal evolution, and chemoresistance in chronic lymphocytic leukemia (CLL). Currently, therapies capable of providing durable remissions in relapsed/refractory TP53- or ATM-defective CLL are lacking. Ataxia telangiectasia and Rad3-related (ATR) mediates response to replication stress, the absence of which leads to collapse of stalled replication forks into chromatid fragments that require resolution through the ATM/p53 pathway. Here, using AZD6738, a novel ATR kinase inhibitor, we investigated ATR inhibition as a synthetically lethal strategy to target CLL cells with TP53 or ATM defects. Irrespective of TP53 or ATM status, induction of CLL cell proliferation upregulated ATR protein, which then became activated in response to replication stress. In TP53- or ATM-defective CLL cells, inhibition of ATR signaling by AZD6738 led to an accumulation of unrepaired DNA damage, which was carried through into mitosis because of defective cell cycle checkpoints, resulting in cell death by mitotic catastrophe. Consequently, AZD6738 was selectively cytotoxic to both TP53- and ATM-defective CLL cell lines and primary cells. This was confirmed in vivo using primary xenograft models of TP53- or ATM-defective CLL, where treatment with AZD6738 resulted in decreased tumor load and reduction in the proportion of CLL cells with such defects. Moreover, AZD6738 sensitized TP53- or ATM-defective primary CLL cells to chemotherapy and ibrutinib. Our findings suggest that ATR is a promising therapeutic target for TP53- or ATM-defective CLL that warrants clinical investigation.

Synthetic lethality in chronic lymphocytic leukaemia with DNA damage response defects by targeting the ATR pathway.

BACKGROUND: DNA damage response (DDR) defects, particularly TP53 and biallelic ataxia telangiectasia mutated (ATM) aberrations, are associated with genomic instability, clonal evolution, and chemoresistance in chronic lymphocytic leukaemia (CLL). Therapies capable of providing long-term disease control in CLL patients with DDR defects are lacking. Using AZD6738, a novel ATR inhibitor, we investigated ATR pathway inhibition as a synthetically lethal strategy for targeting CLL cells with these defects. METHODS: The effect of AZD6738 was assessed by western blotting and immunofluorescence of key DDR proteins. Cytotoxicity was assessed by CellTiter-Gloluminescence assay (Promega, Madison, WI, USA) and by propidium iodide exclusion. Primary CLL cells with biallelic TP53 or ATM inactivation were xenotransplanted into NOD/Shi-scid/IL-2Rγ mice. After treatment with AZD6738 or vehicle, tumour load was measured by flow cytometric analysis of infiltrated spleens, and subclonal composition by fluorescence in-situ hybridisation for 17p(TP53) or 11q(ATM) deletion. FINDINGS: AZD6738 provided potent and specific inhibition of ATR signalling with compensatory activation of ATM/p53 pathway in cycling CLL cells in the presence of genotoxic stress. In p53 or ATM defective cells, AZD6738 treatment resulted in replication fork stalls and accumulation of unrepaired DNA damage, as evidenced by γH2AX and 53BP1 foci formation, which was carried through into mitosis, resulting in cell death by mitotic catastrophe. AZD6738 displayed selective cytotoxicity towards ATM or p53 deficient CLL cells, and was highly synergistic in combination with cytotoxic chemotherapy. This finding was confirmed in primary xenograft models of DDR-defective CLL, where treatment with AZD6738 resulted in decreased tumour load and selective reduction of CLL subclones with ATM or TP53 alterations. INTERPRETATION: We have provided mechanistic insight and demonstrated in-vitro and in-vivo efficacy of a novel therapeutic approach that specifically targets p53-null or ATM-null CLL cells. Such an approach can potentially help to avert clonal evolution, a major cause of therapeutic resistance and disease relapse. FUNDING: Leukaemia & Lymphoma Research.

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.

Induction of a CD8+ T-cell response to the MAGE cancer testis antigen by combined treatment with azacitidine and sodium valproate in patients with acute myeloid leukemia and myelodysplasia.

Epigenetic therapies, including DNA methyltransferase and histone deacetylase inhibitors, represent important new treatment modalities in hematologic malignancies, but their mechanism of action remains unknown. We reasoned that up-regulation of epigenetically silenced tumor antigens may induce an immunologically mediated antitumor response and contribute to their clinical activity. In this study, we demonstrate that azacitidine (AZA) and sodium valproate (VPA) up-regulate expression of melanoma-associated antigens (MAGE antigens) on acute myeloid leukemia (AML) and myeloma cell lines. In separate studies, we observed that prior exposure to AZA/VPA increased recognition of myeloma cell lines by a MAGE-specific CD8(+) cytotoxic T-lymphocyte (CTL) clone. We therefore measured CTL responses to MAGE antigens in 21 patients with AML or myelodysplasia treated with AZA/VPA. CTL responses to MAGE antigens were documented in only 1 patient before therapy; however, treatment with AZA/VPA induced a CTL response in 10 patients. Eight of the 11 patients with circulating MAGE CTLs achieved a major clinical response after AZA/VPA therapy. This is the first demonstration of a MAGE-specific CTL response in AML. Furthermore, it appears that epigenetic therapies have the capacity to induce a CTL response to MAGE antigens in vivo that may contribute to their clinical activity in AML.

Correction: Kwok et al. Targeting the p53 Pathway in CLL: State of the Art and Future Perspectives. Cancers 2021, 13, 4681.

The authors wish to make the following corrections to their paper [1] [...].

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 p53 Pathway in CLL: State of the Art and Future Perspectives.

The p53 pathway is a desirable therapeutic target, owing to its critical role in the maintenance of genome integrity. This is exemplified in chronic lymphocytic leukemia (CLL), one of the most common adult hematologic malignancies, in which functional loss of p53 arising from genomic aberrations are frequently associated with clonal evolution, disease progression, and therapeutic resistance, even in the contemporary era of CLL targeted therapy and immunotherapy. Targeting the 'undruggable' p53 pathway therefore arguably represents the holy grail of cancer research. In recent years, several strategies have been proposed to exploit p53 pathway defects for cancer treatment. Such strategies include upregulating wild-type p53, restoring tumor suppressive function in mutant p53, inducing synthetic lethality by targeting collateral genome maintenance pathways, and harnessing the immunogenicity of p53 pathway aberrations. In this review, we will examine the biological and clinical implications of p53 pathway defects, as well as our progress towards development of therapeutic approaches targeting the p53 pathway, specifically within the context of CLL. We will appraise the opportunities and pitfalls associated with these therapeutic strategies, and evaluate their place amongst the array of new biological therapies for CLL.

Depletion of the Ras association domain family 1, isoform A-associated novel microtubule-associated protein, C19ORF5/MAP1S, causes mitotic abnormalities.

Ras association domain family 1, isoform A (RASSF1A) is a novel tumor suppressor gene that is found to be inactivated in more than 40 types of sporadic cancers. In addition, mouse Rassf1a knockout models have an increased frequency of spontaneous and induced tumors. The mechanisms by which RASSF1A exerts its tumor suppression activities or the pathways it can regulate are not yet fully understood. Using yeast two-hybrid system, we have previously identified C19ORF5/MAP1S as the major RASSF1A-interacting protein. C19ORF5 has two conserved microtubule-associated regions and may function to anchor RASSF1A to the centrosomes. In this study, we have analyzed the cellular functions of C19ORF5. By using small interfering RNA-mediated depletion and time-lapse video microscopy, we show that C19ORF5 knockdown causes mitotic abnormalities that consist of failure to form a stable metaphase plate, premature sister chromatid separation, lagging chromosomes, and multipolar spindles. We also show that a fraction of C19ORF5 localizes to the spindle microtubules. Additionally, we show here that C19ORF5 localizes to the microtubule-organizing centers during microtubule regrowth after nocodazole washout. Knockdown of C19ORF5 disrupts the microtubule-organizing center and results in microtubule nucleation from several sites. Whereas the localization of pericentrin is not affected, alpha- and gamma-tubulin localization and sites of nucleation are greatly altered by C19ORF5 depletion. This may indicate that C19ORF5 plays a role in anchoring the microtubule-organizing center to the centrosomes. In addition, we show that the NH2 terminus of C19ORF5 is essential for this process. This novel role for C19ORF5 could explain the resulting mitotic abnormalities that occur on its depletion and can potentially provide an underlying mechanism for the frequent centrosome and microtubule abnormalities detected in several cancers.

Derivatisation of parthenolide to address chemoresistant chronic lymphocytic leukaemia.

Parthenolide is a natural product that exhibits anti-leukaemic activity, however, its clinical use is limited by its poor bioavailability. It may be extracted from feverfew and protocols for growing, extracting and derivatising it are reported. A novel parthenolide derivative with good bioavailability and pharmacological properties was identified through a screening cascade based on in vitro anti-leukaemic activity and calculated "drug-likeness" properties, in vitro and in vivo pharmacokinetics studies and hERG liability testing. In vitro studies showed the most promising derivative to have comparable anti-leukaemic activity to DMAPT, a previously described parthenolide derivative. The newly identified compound was shown to have pro-oxidant activity and in silico molecular docking studies indicate a prodrug mode of action. A synthesis scheme is presented for the production of amine 7 used in the generation of 5f.

Role of the Ras-association domain family 1 tumor suppressor gene in human cancers.

In recent years, the list of tumor suppressor genes (or candidate TSG) that are inactivated frequently by epigenetic events rather than classic mutation/deletion events has been growing. Unlike mutational inactivation, methylation is reversible and demethylating agents and inhibitors of histone deacetylases are being used in clinical trails. Highly sensitive and quantitative assays have been developed to assess methylation in tumor samples, early lesions, and bodily fluids. Hence, gene silencing by promoter hypermethylation has potential clinical benefits in early cancer diagnosis, prognosis, treatment, and prevention. The hunt for a TSG located at 3p21.3 resulted in the identification of the RAS-association domain family 1, isoform A gene (RASSF1A). RASSF1A falls into the category of genes frequently inactivated by methylation rather than mutational events. This gene is silenced and frequently inactivated by promoter region hypermethylation in many adult and childhood cancers, including lung, breast, kidney, gastric, bladder, neuroblastoma, medulloblastoma, gliomas and it has homology to a mammalian Ras effector (i.e., Nore1). RASSF1A inhibits tumor growth in both in vitro and in vivo systems, further supporting its role as a TSG. We and others identified the gene in 2000, but already there are over a 150 publications demonstrating RASSF1A methylation in a large number of human cancers. Many laboratories including ours are actively investigating the biology of this novel protein family. Thus far, it has been shown to play important roles in cell cycle regulation, apoptosis, and microtubule stability. This review summarizes our current knowledge on genetic, epigenetic, and functional analysis of RASSF1A tumor suppressor gene and its homologues.

Transcriptional regulation of cyclin A2 by RASSF1A through the enhanced binding of p120E4F to the cyclin A2 promoter.

Recent advances in the study of RASSF1A, the candidate tumor suppressor gene, indicate a possible role of RASSF1A in cell cycle regulation; however, very little is known regarding molecular mechanisms underlying this control. Using small interfering RNA to knockdown endogenous RASSF1A in the breast tumor cell line HB2 and in the cervical cancer cell line HeLa, we identify that a key player in cell cycle progression, cyclin A2, is concomitantly increased at both protein and mRNA levels. In A549 clones stably expressing RASSF1A, cyclin A2 levels were diminished compared with vector control. A known transcriptional regulator of cyclin A2, p120(E4F) (a repressor of cyclin A2), has been shown previously by our group to interact with RASSF1A. We show that levels of p120(E4F) are not affected by RASSF1A small interfering RNA in HB2 and HeLa cells. However, electrophoretic mobility shift assays indicate that knockdown of endogenous RASSF1A in HB2 and HeLa cells leads to a reduction in the binding capacity of p120(E4F) to the cyclin A2 promoter, whereas in the A549 clone stably expressing RASSF1A the binding capacity is increased. These data are further corroborated in vitro by the luciferase assay and in vivo by chromatin immunoprecipitation experiments. Together, these data identify the cyclin A2 gene as a cellular target for RASSF1A through p120(E4F) and for the first time suggest a transcriptional mechanism for RASSF1A-dependent cell cycle regulation.

Involvement of the RASSF1A tumor suppressor gene in controlling cell migration.

We have previously shown that RASSF1A associates with the microtubules. This association alters the microtubule dynamics and seems essential for RASSF1A tumor suppressive function. Mutant variants of RASSF1A that do not associate fully with the microtubules have reduced ability to stabilize them and cause cell cycle arrest. Here we show that overexpression of RASSF1A diminished the ability of A549 non-small cell lung cancer cells to migrate either through a transwell filter or to close a wound. In addition, we employed gene knockdown as well as mouse embryonic fibroblasts (MEFs) from Rassf1a knockout mice to analyze RASSF1A function in controlling cell motility. A549 cells stably transfected with RASSF1A exhibited increased cell-cell adhesion and less refractive morphology compared with controls. Conversely, RASSF1A knockdown in HeLa caused loss of cell-cell adhesion and a more refractive morphology. RASSF1A-depleted HeLa cells as well as Rassf1a-/- MEFs displayed increased cell migration that could be partly phosphatidylinositol 3-kinase dependent. Time-lapse microscopy showed the RASSF1A-depleted cells are highly motile with fibroblast-like morphology and diminished cell-cell adhesion. Staining of the cytoskeleton in RASSF1A-depleted HeLa cells and MEFs show marked differences in terms of microtubules outgrowth and actin stress fibers formation. This observation was associated with increased activation of Rac1 in RASSF1A-knockdown cells and the Rassf1a-/- MEFs. In addition, expression of a dominant-negative variant of Rac1 in the RASSF1A-depleted HeLa cells reduced their ability to form lamellipodia and other protrusions. These findings represent a novel function for RASSF1A, which may help explain its tumor suppression ability independently of its effects on cell cycle and apoptosis.