Stefania Lamartina

Discovery of 2-{4-[(3S)-piperidin-3-yl]phenyl}-2H-indazole-7-carboxamide (MK-4827): a novel oral poly(ADP-ribose)polymerase (PARP) inhibitor efficacious in BRCA-1 and -2 mutant tumors.

We disclose the development of a novel series of 2-phenyl-2H-indazole-7-carboxamides as poly(ADP-ribose)polymerase (PARP) 1 and 2 inhibitors. This series was optimized to improve enzyme and cellular activity, and the resulting PARP inhibitors display antiproliferation activities against BRCA-1 and BRCA-2 deficient cancer cells, with high selectivity over BRCA proficient cells. Extrahepatic oxidation by CYP450 1A1 and 1A2 was identified as a metabolic concern, and strategies to improve pharmacokinetic properties are reported. These efforts culminated in the identification of 2-{4-[(3S)-piperidin-3-yl]phenyl}-2H-indazole-7-carboxamide 56 (MK-4827), which displays good pharmacokinetic properties and is currently in phase I clinical trials. This compound displays excellent PARP 1 and 2 inhibition with IC(50) = 3.8 and 2.1 nM, respectively, and in a whole cell assay, it inhibited PARP activity with EC(50) = 4 nM and inhibited proliferation of cancer cells with mutant BRCA-1 and BRCA-2 with CC(50) in the 10-100 nM range. Compound 56 was well tolerated in vivo and demonstrated efficacy as a single agent in a xenograft model of BRCA-1 deficient cancer.

Stringent control of gene expression in vivo by using novel doxycycline-dependent trans-activators.

The tetracycline (Tet)-dependent regulatory system has been widely used for controlling gene expression. The Tet-on version of the system, in which the reverse Tet-responsive transcriptional activator (rtTA) is positively regulated by Tet or its analogs, such as doxycycline (Dox), is of potential utility for gene therapy applications in humans. However, rtTA may display a high basal activity, especially when delivered in vivo by using episomal vectors such as plasmids. Two novel Dox-inducible activators, called rtTA2(S)-S2 and rtTA2(S)-M2, which have a significantly lower basal activity than rtTA in stably transfected cell lines, have been described. In this study we tested the capability of these trans-activators to control expression of mouse erythropoietin (mEpo) and to modulate hematocrit (Hct) increase in vivo on delivery of plasmids into quadriceps muscles of adult mice by DNA electroinjection. Both rtTA2(S)-M2 and rtTA2(S)-S2 displayed a considerably lower background activity and higher window of induction than rtTA in vivo. Moreover, a stringent control of mEpo gene expression and Hct levels in the absence of any background activity was maintained over a 10-month period by injecting as little as 1 microg of a single plasmid containing the rtTA2(S)-S2 expression cassette and the Tet-responsive mEpo cDNA. This constitutes the first report of a stringent ligand-dependent control of gene expression in vivo obtained by delivering a single plasmid encoding both the trans-activator and the regulated gene. Notably, the rtTA2(S)-S2-based system was induced by oral doses of doxycycline comparable to those normally used in clinical practice in humans.

Characteristics of the Adeno-Associated Virus Preintegration Site in Human Chromosome 19: Open Chromatin Conformation and Transcription-Competent Environment

ABSTRACT Adeno-associated virus (AAV) establishes latency in infected cells by integrating into the cellular genome, with a high preference for a unique region, called AAVS1, of the human chromosome 19. The AAV proteins Rep78 and -68 are postulated to initiate the site-specific integration process by binding to a Rep binding site (RBS) in AAVS1. We provide further evidence to corroborate this model by demonstrating that the AAVS1 RBS in human cell lines is located near a DNase I hypersensitive “open” chromatin region and therefore is potentially easily accessible to Rep proteins. This open conformation is maintained in transgenic rats which carry an AAVS1 3.5-kb DNA fragment and are proficient for Rep-mediated site-specific integration. Interestingly, the core of the DNAse I hypersensitive site in AAVS1 corresponds to a sequence displaying transcriptional enhancer-like properties, suggesting that AAVS1 constitutes a transcription-competent environment. The implications of our findings for AAV physiology and gene therapy are discussed.

Helper-dependent adenovirus for the gene therapy of proliferative retinopathies: stable gene transfer, regulated gene expression and therapeutic efficacy

Ocular neovascular disorders, such as diabetic retinopathy and age‐related macular degeneration, are the principal causes of blindness in developed countries. Current treatments are of limited efficacy, whereas a therapy based on intraocular gene transfer of angiostatic factors represents a promising alternative. For the first time we have explored the potential of helper‐dependent adenovirus (HD‐Ad), the last generation of Ad vectors, in the therapy of retinal neovascularization.

Conditional Site-Specific Integration into Human Chromosome 19 by Using a Ligand-Dependent Chimeric Adeno-Associated Virus/Rep Protein

ABSTRACT It is of great interest for gene therapy to develop vectors that drive the insertion of a therapeutic gene into a chosen specific site on the cellular genome. Adeno-associated virus (AAV) is unique among mammalian viruses in that it integrates into a distinct region of human chromosome 19 (integration site AAVS1). The inverted terminal repeats (ITRs) flanking the AAV genome and the AAV-encoded nonstructural proteins Rep78 and/or Rep68 are the only viral elements necessary and sufficient for site-specific integration. However, it is also known that unrestrained Rep activity may cause nonspecific genomic rearrangements at AAVS1 and/or have detrimental effects on cell physiology. In this paper we describe the generation of a ligand-dependent form of Rep, obtained by fusing a C-terminally deleted Rep68 with a truncated form of the hormone binding domain of the human progesterone receptor, which does not bind progesterone but binds only its synthetic antagonist RU486. The activity of this chimeric protein, named Rep1-491/P, is highly dependent on RU486 in various assays: in particular, it triggers site-specific integration at AAVS1 of an ITR-flanked cassette in a ligand-dependent manner, as efficiently as wild-type Rep68 but without generating unwanted genomic rearrangement at AAVS1.

Selective Cleavage of AAVS1 Substrates by the Adeno-Associated Virus Type 2 Rep68 Protein Is Dependent on Topological and Sequence Constraints

ABSTRACT The adeno-associated virus type 2 (AAV-2) Rep78 and Rep68 proteins are required for replication of the virus as well as its site-specific integration into a unique site, called AAVS1, of human chromosome 19. Rep78 and Rep68 initiate replication by binding to a Rep binding site (RBS) contained in the AAV-2 inverted terminal repeats (ITRs) and then specifically nicking at a nearby site called the terminal resolution site (trs). Similarly, Rep78 and Rep68 are postulated to trigger the integration process by binding and nicking RBS andtrs homologues present in AAVS1. However, Rep78 and Rep68 cleave in vitro AAVS1 duplex-linear substrates much less efficiently than hairpinned ITRs. In this study, we show that the AAV-2 Rep68 endonuclease activity is affected by the topology of the substrates in that it efficiently cleaves in vitro in a site- and strand-specific manner the AAVS1 trs only if this sequence is in a supercoiled (SC) conformation. DNA sequence mutagenesis in the context of SC templates allowed us to elucidate for the first time the AAVS1trs sequence and position requirements for Rep68-mediated cleavage. Interestingly, Rep68 did not cleave SC templates containing RBS from other sites of the human genome. These findings have intriguing implications for AAV-2 site-specific integration in vivo.

Abstract 685: PARP inhibitor MK-4827 is synthetic lethal for tumors with homologous recombination defects associated with ATM-deficiency, PTEN-deletion and microsatellite instability (MSI)

Poly(ADP-ribose) polymerase (PARP) −1 and −2 enzymes play a critical role in the base excision DNA repair (BER) pathway. PARP inhibition in oncology has the potential to target pre-defined sub-populations with higher sensitivity and to widen the therapeutic index of chemotherapy and radiotherapy. Pre-clinical and clinical evidence demonstrated that PARP inhibitors are synthetic lethal for tumors with defects in the homologous recombination (HR) pathway, such as tumors with mutations in the BRCA1 or BRCA2 genes. MK-4827 is a potent and selective orally available PARP-1/2 inhibitor currently in Phase 1 development. In pre-clinical models, MK-4827 displays excellent monotherapy activity in a large panel of BRCA mutant cell lines (10 nM ≤ CC50 ≤ 90nM) with at least 10-fold selectivity over BRCA wild-type celllines (CC50 ≥ 1.5 uM). We have explored the potential of MK-4827 for the therapy of tumors with defects in the HR pathway in addition to mutation in the BRCA genes. In this study we have evaluated the activity of MK-4827 on tumor cell lines with defects in the homologous recombination pathway as a consequence of 1) inactivation of ATM gene in melanoma and mantle cell lymphoma cell lines; 2) PTEN gene deletion in prostate cancer cells; 3) MRE11 expression level defects associated with the Microsatellite Instability (MSI) phenotype in colorectal cancer cells (CRCs). We demonstrated that cancer cells with an inactive ATM pathway as a consequence of homozygous ATM gene inactivation are very sensitive to MK-4827 inhibition (CC50 ≤ 50nM). MK-4827 also displays strong activity in ATM-deficient xenograft models. Heterozygous ATM inactivation is not sufficient to confer sensitivity to MK-4827. Our studies also showed that prostate cancer cells with homozygous-deletion of PTEN gene are sensitive to MK-4827 inhibition (100nM ≤ CC50 ≤ 600nM) as measured by long-term cell growth and proliferation assays. Homozygous, but not heterozygous, PTEN-deletion is required for MK-4827 sensitivity in agreement with data available for sensitivity of BRCA- and ATM-deficient cells to MK-4827. Finally, we have screened a panel of CRC cells with known Microsatellite Instability and have observed that MSI+ cells are sensitive to MK-4827 in long term proliferation assays, in contrast to Microsatellite stable (MSS) CRC cells which are resistant. In agreement with previous reports, we observed that MSI+ CRC cells show low expression levels of MRE11, a key protein for homologous recombination. Conclusion: Our pre-clinical data demonstrate that MK-4827 is synthetic lethal for tumors with ATM-deficiency, PTEN-deletion or MSI+ instability and suggest that patients with tumors carrying these genetic defects might benefit from treatment with PARP inhibitors Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 685.