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BCR-ABL1 transforms hematopoietic stem cells into leukemia stem cells (LSCs) to induce chronic myeloid leukemia in chronic phase. Expression of BCR-ABL1 stimulates production of elevated levels of reactive oxygen species (ROS), which induce oxidative DNA damage. CML cells accumulate excessive amounts of ROS-induced DNA damage which can be converted to potentially lethal DNA double strand breaks (DSBs). BCR-ABL1 stimulates enhanced Rad51-mediated DSB repair by the homologous recombination repair (HRR) pathway. In these studies we show BCR-ABL1-transformed cells depend on Rad52-mediated HRR to promote repair of ROS-induced DSBs and that this activity is dependent on Rad52 binding to single-stranded DNA (ssDNA). Our results show in the absence of Rad52, BCR-ABL1-positive hematopoietic cells accumulated elevated numbers of DSBs as detected by enhanced γ--H2AX foci formation compared to cells with wild-type Rad52 which resulted in a decrease in proliferation and expansion of the Rad52-null LSC population. Expression of wild-type Rad52 in Rad52-null cells decreased the accumulation of DSBs and restored expansion of the LSC population. Inhibition of ROS with the antioxidants Vitamin E or N-acetyl cysteine exerted similar effects on the LSC population of Rad52-null cells as restoration of wild-type Rad52. Our studies also show Rad52's ssDNA-binding activity is required for the proliferation of CML cells as evidenced by the accumulation of DSBs and impairment of clonogenic potential in cells in which the Rad52-F79A ssDNA-binding deficient mutant was expressed. Inhibition of Rad52 DNA binding activity by a peptide aptamer targeting Rad52-F79 resulted in a synthetic lethal phenotype in BCR-ABL1-positive cells due to impairment of the Rad52-dependent HRR pathway, as demonstrated by immunofluorescence and HRR repair assays. Altogether we identify Rad52 as a novel target in the treatment of CML, and other BRCA1- and/or BRCA2-deficient cancers, by showing induction of synthetic lethality in proliferating BCR-ABL1-positive cells in which Rad52 ssDNA-binding activity is inhibited. / Biology

This thesis focuses on the development of molecules that target proteins in a previously undescribed manner for the treatment of BRCA deficient cancers. ZINC 13403027, a clerodan-based natural product, was shown to target a protein called Rad52. Cancers possessing loss of function mutations in BRCA1 and BRCA2 are dependent on Rad52 for DNA repair and replication while normal, healthy cells possess multiple DNA repair/replication pathways. Thus, inhibitors of Rad52 may serve as selective anti-cancer drugs for BRCA deficient tumors. ZNIC 13403027 was selected for its high activity in disrupting the ssDNA-Rad52 interaction in a gel-shift assay as well as exhibiting the required inactivity at disrupting the ssDNA-Rad51 interaction. Due to its lack of permeability, a synthetic route amenable to modification has been partially developed. It is thought that a prodrug or bioisostere of ZINC 13403027 could cross the membrane so that the cellular activity of this novel tool molecule may be established. Additionally, an allosteric PARP1 inhibitor, 5F02, was explored. Discussed here is the synthetic route to 5F02 and its analogs. Structure activity relationships were develop in an attempt to increase inhibitory activity and drug-like properties. This thesis reports the success to date on these two projects. / Pharmaceutical Sciences

Aberrant DNA synthesis and maintenance have been implicated in numerous human diseases. I describe here a novel strategy for systematically identifying budding yeast mutants with elevated levels of DNA damage foci, which represent hubs of DNA damage and repair. A previous study manually scored foci in single mutants but was limited in its ability to survey many conditions in large populations. I developed an automated and statistically robust method for identifying aberrant foci phenotypes by combining synthetic genetic array (SGA) and high-content screening (HCS) methodology. Using this approach, I scored thousands of essential and non-essential gene mutants subjected to environmental and genetic perturbations, including the DNA damaging agent, phleomycin, and deletions of DNA repair genes, SGS1 and YKU80. Collectively, I identified a functionally enriched set of 367 mutants that had increased frequencies of DNA damage foci and established SGA-HCS as a powerful tool for investigating the yeast DNA damage response.

Aberrant DNA synthesis and maintenance have been implicated in numerous human diseases. I describe here a novel strategy for systematically identifying budding yeast mutants with elevated levels of DNA damage foci, which represent hubs of DNA damage and repair. A previous study manually scored foci in single mutants but was limited in its ability to survey many conditions in large populations. I developed an automated and statistically robust method for identifying aberrant foci phenotypes by combining synthetic genetic array (SGA) and high-content screening (HCS) methodology. Using this approach, I scored thousands of essential and non-essential gene mutants subjected to environmental and genetic perturbations, including the DNA damaging agent, phleomycin, and deletions of DNA repair genes, SGS1 and YKU80. Collectively, I identified a functionally enriched set of 367 mutants that had increased frequencies of DNA damage foci and established SGA-HCS as a powerful tool for investigating the yeast DNA damage response.

Les cassures double-brins d’ADN (CDBs) constituent une menace pour la viabilité cellulaire et l’intégrité du génome puisque l’absence de la réparation d’une CDB pourrait conduire à la mort cellulaire. En plus de la réparation par jonction d’extrémités nonhomologues (NHEJ) en phase G1 et de la recombinaison homologue (RH) en phase S et G2, les CDBs peuvent être réparées par l’ajout de télomères par l’action de la télomérase; un phénomène qui s’appelle l’ajout de télomères de novo. Ce phénomène pourrait mettre en danger la stabilité génomique parce qu’il engendre, dans la plupart des cas, une perte du bras chromosomique du fragment non-centromérique. En conséquence, ceci engendre soit une perte de l’hétérozygotie (LOH) dans les cellules diploïdes ou la mort cellulaire dans les cellules haploïdes. Dans le but d’empêcher la formation de télomères de novo, la cellule possède des mécanismes et des voies qui préviennent l’action inappropriée de la télomérase à des CDBs. Une des principales questions dans le domaine est de comprendre comment la cellule inhibe l’ajout de télomères de novo par la télomérase en favorisant la réparation des CDBs par les autres voies (NHEJ et la RH).Dans ce projet, nous utilisons la technique d’hybridation in situ en fluorescence (FISH) sur le facteur limitant de la télomérase, l’ARN TLC1 de la levure S. cerevisiae. Nous avons pu montrer que l’ARN TLC1 fait un trafic intranucléaire durant le cycle cellulaire des cellules sauvages. En phase G1/S, l’ARN TLC1 adopte une localisation nucléoplasmique avec les télomères, alors qu’il s’accumule au nucléole en phase G2/M. Nous avons fait l’hypothèse que l’accumulation de l’ARN TLC1 au nucléole en G2/M pourrait réduire la compétition entre la RH, qui est exclusivement nucléoplasmique, et la télomérase pour la réparation des CDBs. Pour tester cette hypothèse, nous avons employé la bléomycine (blm), un composé chimique générant des CDBs, pour traiter des cellules sauvages ou déficientes de la RH par la délétion du gène RAD52. Nous avons observé que l’ARN TLC1 conserve une localisation nucléolaire dans les cellules sauvages traitées par la blm en phase G2/M, alors que dans lescellules délétées de RAD52 exposées à la blm, l’ARN TLC1 se localise maintenant au nucléoplasme et s’associe partiellement aux sites de cassures. De plus, nous avons trouvé que l’accumulation nucléoplasmique de l’ARN TLC1 dans les cellules délétéées de RAD52 traitées à la blm, dépend de la voie de dommage à l’ADN (MRX, ATM/Tel1 et ATR/Mec1) et de la sumoylation par la SUMO E3ligase, Siz1. Plus particulièrement, l’association de la télomérase à des CDBs dépend de son interaction avec Cdc13, une protéine qui recrute la télomérase aux télomères. D’une manière surprenante, nous avons observé une accumulation rapide de Cdc13 à des CDBs en absence de Rad52, bien que nos résultats suggèrent que Rad52 empêche l’accumulation de l’ARN TLC1 au nucléoplasme par l’inhibition de l’accumulation de Cdc13 aux sites de cassures. L’ensemble de nos résultats ont mis en évidence que la télomérase est normalement exclue des sites de la réparation d’ADN. Cependant, en absence d’une voie fonctionnelle de la RH, la télomérase se localise du nucléole au nucléoplasme et s’accumule partiellement à des CDBs d’une manière dépendante de Cdc13 et Siz1. / DNA double-strand breaks (DSB) constitute a threat to genome integrity and cell survival if they are not repaired. In addition to canonical DNA repair systems such as nonhomologous end joining (NHEJ) in G1 and homologous recombination (HR) in S and G2 phases, DSBs can also be repaired by addition of new telomeres by telomerase. This phenomenon is referred to as telomere healing or de novo telomere addition. This process threatens genome stability since it results in chromosome arm loss, which could be lethal in haploid cells and lead to loss of heterozygosity (LOH) in diploid cells. Therefore, cells possess mechanisms that prevent the untimely action of telomerase on DSBs. One of the questions driving this field is to understand how telomere addition by telomerase is inhibited and DSBs repair can be efficiently performed by canonical DSB repair (NHEJ and HR). In this project, we used fluorescent in situ hybridization (FISH) to detect the endogenous TLC1 RNA, which is the limiting component of telomerase of the budding yeast. Using this technique, we found that TLC1 RNA traffics inside the nucleus during the cell cycle of wild-type cells. In G1 and S phases, TLC1 RNA adopts a nucleoplasmic localization, which is related to its function in telomere elongation, while it accumulates in the nucleolus in G2/M. We hypothesize that the nucleolar accumulation of TLC1 RNA in G2/M may reduce the possibility that telomerase interferes with HR to repair DNA DSB, since HR is excluded from the nucleolus and occurs only in the nucleoplasm. To test this hypothesis, we treated wild-type and rad52 (HR deficient cells) with bleomycin, a radiomimetic agent that generates preferentially DSBs. Our results show that after induction of DSB with bleomycin, TLC1 RNA remains nucleolar in wild-type cells in G2/M, but accumulates in the nucleoplasm and colocalizes partially with DSBs sites in rad52 cells, suggesting that RAD52 inhibits the nucleoplasmic accumulation of TLC1 RNA in the presence of DSBs. Nucleoplasmic accumulation of TLC1 RNA after DSB induction requires the DNA damage pathway (MRX, ATM/Tel1 and ATR/Mec1), and the SUMO ligase E3 Siz1. Interestingly, association of TLC1 RNA with DSBs depends on the single-strand telomeric binding protein Cdc13, which rapidly accumulates at sites of DNA damage, while Rad52 suppresses this process by inhibiting Cdc13 accumulation at DSBs. These results suggest that telomerase is normally excluded from sites of DNA repair. In the absence of functional homologous recombination, telomerase leaves the nucleolus and accumulates partially at DSB in the nucleoplasm in a Cdc13- and Siz1-dependent manner.