Simultaneous Targeting of PARP1 and RAD52 Triggers Dual Synthetic Lethality in BRCA-Deficient Tumor Cells.

PARP inhibitors (PARPis) have been used to induce synthetic lethality in BRCA-deficient tumors in clinical trials with limited success. We hypothesized that RAD52-mediated DNA repair remains active in PARPi-treated BRCA-deficient tumor cells and that targeting RAD52 should enhance the synthetic lethal effect of PARPi. We show that RAD52 inhibitors (RAD52is) attenuated single-strand annealing (SSA) and residual homologous recombination (HR) in BRCA-deficient cells. Simultaneous targeting of PARP1 and RAD52 with inhibitors or dominant-negative mutants caused synergistic accumulation of DSBs and eradication of BRCA-deficient but not BRCA-proficient tumor cells. Remarkably, Parp1-/-;Rad52-/- mice are normal and display prolonged latency of BRCA1-deficient leukemia compared with Parp1-/- and Rad52-/- counterparts. Finally, PARPi+RAD52i exerted synergistic activity against BRCA1-deficient tumors in immunodeficient mice with minimal toxicity to normal cells and tissues. In conclusion, our data indicate that addition of RAD52i will improve therapeutic outcome of BRCA-deficient malignancies treated with PARPi.

Gene expression and mutation-guided synthetic lethality eradicates proliferating and quiescent leukemia cells.

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.

Rb family proteins enforce the homeostasis of quiescent hematopoietic stem cells by repressing Socs3 expression.

Prolonged exit from quiescence by hematopoietic stem cells (HSCs) progressively impairs their homeostasis in the bone marrow through an unidentified mechanism. We show that Rb proteins, which are major enforcers of quiescence, maintain HSC homeostasis by positively regulating thrombopoietin (Tpo)-mediated Jak2 signaling. Rb family protein inactivation triggers the progressive E2f-mediated transactivation of Socs3, a potent inhibitor of Jak2 signaling, in cycling HSCs. Aberrant activation of Socs3 impairs Tpo signaling and leads to impaired HSC homeostasis. Therefore, Rb proteins act as a central hub of quiescence and homeostasis by coordinating the regulation of both cell cycle and Jak2 signaling in HSCs.

Normal ABL1 is a tumor suppressor and therapeutic target in human and mouse leukemias expressing oncogenic ABL1 kinases.

Leukemias expressing constitutively activated mutants of ABL1 tyrosine kinase (BCR-ABL1, TEL-ABL1, NUP214-ABL1) usually contain at least 1 normal ABL1 allele. Because oncogenic and normal ABL1 kinases may exert opposite effects on cell behavior, we examined the role of normal ABL1 in leukemias induced by oncogenic ABL1 kinases. BCR-ABL1-Abl1(-/-) cells generated highly aggressive chronic myeloid leukemia (CML)-blast phase-like disease in mice compared with less malignant CML-chronic phase-like disease from BCR-ABL1-Abl1(+/+) cells. Additionally, loss of ABL1 stimulated proliferation and expansion of BCR-ABL1 murine leukemia stem cells, arrested myeloid differentiation, inhibited genotoxic stress-induced apoptosis, and facilitated accumulation of chromosomal aberrations. Conversely, allosteric stimulation of ABL1 kinase activity enhanced the antileukemia effect of ABL1 tyrosine kinase inhibitors (imatinib and ponatinib) in human and murine leukemias expressing BCR-ABL1, TEL-ABL1, and NUP214-ABL1. Therefore, we postulate that normal ABL1 kinase behaves like a tumor suppressor and therapeutic target in leukemias expressing oncogenic forms of the kinase.

Genomic instability may originate from imatinib-refractory chronic myeloid leukemia stem cells.

Genomic instability is a hallmark of chronic myeloid leukemia in chronic phase (CML-CP) resulting in BCR-ABL1 mutations encoding resistance to tyrosine kinase inhibitors (TKIs) and/or additional chromosomal aberrations leading to disease relapse and/or malignant progression. TKI-naive and TKI-treated leukemia stem cells (LSCs) and leukemia progenitor cells (LPCs) accumulate high levels of reactive oxygen species (ROS) and oxidative DNA damage. To determine the role of TKI-refractory LSCs in genomic instability, we used a murine model of CML-CP where ROS-induced oxidative DNA damage was elevated in LSCs, including quiescent LSCs, but not in LPCs. ROS-induced oxidative DNA damage in LSCs caused clinically relevant genomic instability in CML-CP-like mice, such as TKI-resistant BCR-ABL1 mutations (E255K, T315I, H396P), deletions in Ikzf1 and Trp53, and additions in Zfp423 and Idh1. Despite inhibition of BCR-ABL1 kinase, imatinib did not downregulate ROS and oxidative DNA damage in TKI-refractory LSCs to the levels detected in normal cells, and CML-CP-like mice treated with imatinib continued to accumulate clinically relevant genetic aberrations. Inhibition of class I p21-activated protein kinases by IPA3 downregulated ROS in TKI-naive and TKI-treated LSCs. Altogether, we postulate that genomic instability may originate in the most primitive TKI-refractory LSCs in TKI-naive and TKI-treated patients.