Personalized synthetic lethality induced by targeting RAD52 in leukemias identified by gene mutation and expression profile

Abstract

• Targeting RAD52 DNA binding domain I by peptide aptamer induces synthetic lethality in BRCA-deficient leukemias.• Individual patients with BRCA-deficient leukemias could be identified by genetic and epigenetic profiling.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. (Blood. 2013;122(7):1293-1304

shRNA library screening identifies nucleocytoplasmic transport as a mediator of BCR-ABL1 kinase-independent resistance

“…44 In addition, 3 of the top 50 genes (TXN, RPA3, and MUS81) are associated with DNA damage or repair pathways, adding to the observation of increased homologous recombination repair in samples from imatinib nonresponders, 45 and a report implicating RAD52 as a target to enhance the effects of TKIs on CML cells. 46 Drug resistance remains a significant clinical problem in targeted cancer therapy. In the case of CML, our function-first shRNA library approach led to the identification of RAN and XPO1 as critical mediators of BCR-ABL1 kinase-independent TKI resistance.…”

MLL-AF9 leukemias are sensitive to PARP1 inhibitors combined with cytotoxic drugs

“…Primary human hematopoietic cells were incubated in Iscove modified Dulbecco medium supplemented with 10% FBS and growth factors (100 ng/mL SCF, 20 ng/mL IL-3, 100 ng/mL Flt-3 ligand, 20 ng/mL granulocyte colony-stimulating factor, and 20 ng/mL IL-6), as previously described. 13 Cells were plated in Methocult (Stemcell Technologies) and colonies were scored after 5 to 7 days. For quiescent/proliferating cells, Lin -cells were stained with 5(6)-carboxyfluorescein diacetate succinimidyl ester (CFSE), cell trace violet, or CellProliferation Dye eFluor670 (eBioscience) and incubated for 5 days in StemSpan serum-free expansion medium (Stem Cell Technologies, Vancouver, Canada) supplemented with the cocktail of growth factors (see above) and inhibitors when indicated.…”

“…Cell death and g-H2AX were measured by flow cytometry using Fixable Viability Dye eFluor 660 (eBioscience) and Alexa Fluor 488 Mouse anti-H2AX (phosphoserine 139) (BD Pharmingen) as described before. 13 …”

CML cells actively evade host immune surveillance through cytokine-mediated downregulation of MHC-II expression

“…15 Although some genes are not downregulated individually, there is a trend for downregulation of the MHC-II genes in the primitive (Lin Figure 1B). Overall, this in silico validation process considered 19 independent CML samples and 10 independent normal samples (supplemental Table 1).…”