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Homologous recombination repair (HRR) protects cells from the lethal effect of spontaneous and therapy-induced DNA double-stand breaks. HRR usually depends on BRCA1/2-RAD51, and RAD52-RAD51 serves as back-up. To target HRR in tumor cells, a phenomenon called "synthetic lethality" was applied, which relies on the addiction of cancer cells to a single DNA repair pathway, whereas normal cells operate 2 or more mechanisms. Using mutagenesis and a peptide aptamer approach, we pinpointed phenylalanine 79 in RAD52 DNA binding domain I (RAD52-phenylalanine 79 [F79]) as a valid target to induce synthetic lethality in BRCA1- and/or BRCA2-deficient leukemias and carcinomas without affecting normal cells and tissues. Targeting RAD52-F79 disrupts the RAD52-DNA interaction, resulting in the accumulation of toxic DNA double-stand breaks in malignant cells, but not in normal counterparts. In addition, abrogation of RAD52-DNA interaction enhanced the antileukemia effect of already-approved drugs. BRCA-deficient status predisposing to RAD52-dependent synthetic lethality could be predicted by genetic abnormalities such as oncogenes BCR-ABL1 and PML-RAR, mutations in BRCA1 and/or BRCA2 genes, and gene expression profiles identifying leukemias displaying low levels of BRCA1 and/or BRCA2. We believe this work may initiate a personalized therapeutic approach in numerous patients with tumors displaying encoded and functional BRCA deficiency.

Quiescent and proliferating leukemia cells accumulate highly lethal DNA double-strand breaks that are repaired by 2 major mechanisms: BRCA-dependent homologous recombination and DNA-dependent protein kinase-mediated (DNA-PK-mediated) nonhomologous end-joining, whereas DNA repair pathways mediated by poly(ADP)ribose polymerase 1 (PARP1) serve as backups. Here we have designed a personalized medicine approach called gene expression and mutation analysis (GEMA) to identify BRCA- and DNA-PK-deficient leukemias either directly, using reverse transcription-quantitative PCR, microarrays, and flow cytometry, or indirectly, by the presence of oncogenes such as BCR-ABL1. DNA-PK-deficient quiescent leukemia cells and BRCA/DNA-PK-deficient proliferating leukemia cells were sensitive to PARP1 inhibitors that were administered alone or in combination with current antileukemic drugs. In conclusion, GEMA-guided targeting of PARP1 resulted in dual cellular synthetic lethality in quiescent and proliferating immature leukemia cells, and is thus a potential approach to eradicate leukemia stem and progenitor cells that are responsible for initiation and manifestation of the disease. Further, an analysis of The Cancer Genome Atlas database indicated that this personalized medicine approach could also be applied to treat numerous solid tumors from individual patients.

Altered DNA repair mechanisms are responsible for survival of leukemia stem cells (LSCs) and/or leukemia progenitor cells (LPCs) accumulating numerous lethal DNA double-strand breaks (DSBs). DSBs resulting from stalled/broken replication forks in proliferating cells are primarily repaired by RAD51-mediated homologous recombination repair (HR), which depends on BRCA1-PALB2-BRCA2-RAD51 paralogs (BRCA pathway), while RAD52 pathway serves as redundant back-up mechanism. Enhanced self-renewal of LSCs and high proliferation rate of LPCs commit them to HR. It has been reported that inhibition of RAD52 either by the knockout, specific shRNA, or a small peptide aptamer induced synthetic lethality in BRCA pathway-deficient tumor cell lines and primary leukemia cells. Yet pharmacological inhibition of RAD52, which binds single-stranded DNA (ssDNA) and lacks enzymatic activity, has not been demonstrated. Here, we applied high-throughput screening and structure-based selection followed by biochemical assays and computer modeling to identify three leading compounds: (1) 20264, (2) RU-0084339, and (3) D-I03. Compound 20264 appeared to interact with the hotspot in RAD52 DNA binding domain 1 to interfere with ssDNA binding. RU-0084339 is a major allosteric inhibitor of RAD52 ssDNA binding domain which disassembles undecamer ring structure of RAD52. D-I03 abrogated RAD52-mediated ssDNA annealing and ssDNA pairing. RAD52 small molecule inhibitor (RAD52smi) reduced recruitment of RAD52 to DNA damage-induced nuclear foci and suppressed RAD52-mediated DNA double-strand break (DSB) repair activity in cells with negligible effects on other DSB repair pathways. Importantly, RAD52smi selectively eliminated cancer cell lines carrying BRCA1/2 inactivating mutations. Since inactivating mutations in BRCA pathway are rare in leukemias, individual BRCA pathway-deficient leukemias were identified by Gene Expression and Mutation Analysis (GEMA). Gene Expression approach applied microarrays, qRT-PCR, and/or flow cytometry to identify individual leukemias displaying downregulation of at least one gene in BRCA pathway. On the other hand, Gene Mutation strategy detected individual leukemias expressing an oncogene causing downregulation of BRCA pathway gene(s) (e.g., BCR-ABL1, MLL-AF9, AML1-ETO - mediated downregulation of BRCA1 and/or BRCA2) and harboring inactivating mutations in BRCA pathway (e.g., BRCA2 = FANCD1, and other FA genes). BRCA-deficient cells from individual patients indentified by GEMA were selectively sensitive to RAD52smi alone or in combination with already approved cytotoxic drugs. RAD52 is a promising new target because it is "druggable" by small molecule inhibitors. Moreover, inhibition of RAD52 by genetic knockout and small peptide aptamer did not exert any major negative effects in normal cells and tissues. Altogether, this work provided foundation for precision medicine guided synthetic lethality in BRCA-deficient leukemias exerted by small molecule inhibitors targeting novel mechanism - RAD52 dependent DSB repair. Disclosures No relevant conflicts of interest to declare.

Leukemia stem cells (LSCs), and especially quiescent LSCs, have a dual role as tumor initiating and therapy-refractory cells. Therefore, even if anti-tumor treatment clears a disease burden consisting mostly of proliferating leukemia progenitor cells (LPCs), it usually fails to eradicate therapy-refractory LSCs and also therapy-resistant LPCs. LSCs, including quiescent LSCs and LPCs accumulate high numbers of spontaneous and drug-induced DNA lesions, including highly lethal DNA double-strand breaks (DSBs). Thus, LSCs and/or LPCs may be “addicted” to particular DNA repair mechanisms and targeting these pathways could sensitize LSCs to the lethal effect of unrepaired DSBs. DSBs are usually repaired by two major mechanisms, homologous recombination repair (HRR) and non-homologous end-joining (NHEJ). While NHEJ plays a major role in quiescent cells, HRR works predominantly on broken replication forks in proliferating cells. Normal quiescent cells employ DNA-PK (PRKDC, XRCC5, XRCC6, LIG4) –mediated NHEJ to repair DSBs whereas PARP1-dependent NHEJ serves as back-up. Normal proliferating cells use PARP1-mediated base excision repair (BER) to prevent DSBs at collapsed replication forks, whereas BRCA (BRCA1, BRCA2, PALB2, RAD51 paralogs, RAD51)-mediated HRR serves as back-up to repair stalled/broken forks. The existence of these pathways creates the opportunity to apply “synthetic lethality” triggered by PARP1 inhibitors (PARP1i) in DNA-PK –deficient therapy-refractory quiescent LSCs and BRCA-deficient therapy-resistant proliferating LSCs/LPCs. This hypothesis is supported by PARP1i-mediated synthetic lethality in BRCA1-/- proliferating and XRCC6-/-quiescent cells. Since inactivating mutations in BRCA and DNA-PK pathways are rather rare in leukemias we hypothesize that Gene Expression and Mutation Analysis (GEMA) can identify individual patients with leukemias displaying “spontaneous” and oncogene-induced downregulation of DSB repair genes which will be sensitive to “synthetic lethality” triggered by PARP1i +/- already approved drugs. GENE EXPRESSION ANALYSIS: Global mRNA microarrays and qRT-PCR followed by immunofluorescent protein staining identified primary AMLs and ALLs from individual patients displaying BRCA- and/or DNA-PK –deficient phenotype (downregulation of at least one member of BRCA and/or DNA-PK pathway). Quiescent DNA-PK –deficient LSCs and proliferating BRCA-deficient LSCs/LPCs, but not DNA-PK and BRCA –proficient counterparts were sensitive to olaparib and BMN673 +/- already approved drugs. GENE MUTATION ANALYSIS: BCR-ABL1 downregulates BRCA1 and DNA-PKcs proteins, FLT3(ITD) inhibits BRCA1 protein, and MLL-AF9 and AML1-ETO reduce BRCA2 and XRCC6 proteins, whereas HOXA9+MEIS1 do not affect the expression of DSB repair proteins. These effects were associated with reduced colony formation by BCR-ABL1, MLL-AF9 and AML1-ETO –transformed, but not HOXA9+MEIS1-treasformed Parp1-/-murine bone marrow cells. PARP1i olaparib and BMN673 +/- approved drugs induced DSBs resulting in abrogation of cell proliferation and/or apoptosis in DNA-PK –deficient quiescent LSCs and/or BRCA-deficient proliferating LSCs/LPCs from CML-CP/AP, Ph+ B-ALL, AML and ALL patients. Genetic (Parp1-/-) and pharmacological (olaparib, BMN673) inhibition of PARP1 prolonged survival of mice with CML-CP –like inducible disease and reduced engraftment/prolonged survival of NSG immunodeficient mice with DNA-PK and/or BRCA-deficient primary leukemia (CML-CP, AML and ALL) xenografts in combination with approved drugs. In conclusion, targeting PARP1 resulted in synthetic lethality in quiescent LSCs and/or proliferating LSCs/LPCs from individual patients identified by GEMA, which display specific defects in DSB repair mechanisms. Disclosures Minden: Celgene: Honoraria.