Targeting of Quiescent and Proliferating CML Stem Cells By DNA Repair Inhibitors
Authors: Sullivan Katherine , Bolton-Gillespie Elisabeth , Nieborowska-Skorska Margaret , Cerny-Reiterer Sabine , Valent Peter , Muschen Markus , Pomerantz Richard , Mazin Alexander , Skorski Tomasz 
Published: October 2019

Tyrosine kinase inhibitors (TKIs) such as imatinib, dasatinib and nilotinib revolutionized the treatment of chronic myeloid leukemia in chronic phase (CML-CP). However, it is unlikely that TKIs will "cure" a majority of CML patients due to the presence of TKI-refractory quiescent leukemia stem cells (LSCs) and/or TKI-resistant proliferating LSCs. Moreover, a sub-population of patients does not respond favorably to TKI therapy and/or accumulates TKI-resistant BCR-ABL1 mutants and additional chromosomal aberrations (high risk/poor responders = HR/PR). This process may lead to the disease relapse and/or malignant progression to fatal chronic myeloid leukemia in accelerated phase or blast phase (CML-AP/BP). Therefore new treatment modalities are necessary to improve therapeutic outcome of CML HR/PRs.

We found that CML LSCs and LPCs, including quiescent LSCs, from HR/PRs accumulate the highest levels of reactive oxygen species (ROS)-induced DNA double strand breaks (DSBs) in comparison to "good responders" and normal counterparts (Bolton-Gillespie et al., Blood, 2013). DSBs are the most lethal DNA lesions, but CML cells can tolerate them because two major repair mechanisms, homologous recombination (HR) and non-homologous end-joining (NHEJ), are hyper-activated (Slupianek et al., Mol. Cell, 2001; Oncogene, 2005; DNA Repair, 2006; Cancer Res., 2011; Blood, 2011; Nowicki et al., Blood, 2005). It appears that CML cells, and especially these from HR/PRs are "addicted" to DSB repair pathways to survive pro-apoptotic challenge from lethal DSBs.

There are critical differences between DSB repair in normal and CML cells due to downregulation of BRCA1 in HR pathway and DNA-Pkcs in NHEJ pathway (Slupianek et al., Cancer Res., 2011; Podszywalow-Bartnicka et al., Cell Cycle, 2014). Therefore, proliferating LSCs/LPCs employ RAD52-dependent alternative HR, in contrast to BRCA1-mediated HRR in normal counterparts. Quiescent LSCs use PARP1-mediated alternative NHEJ instead of DNA-PKcs -dependent NHEJ, which is predominant in normal quiescent HSCs. We hypothesized that simultaneous targeting of PARP1 and RAD52 will trigger "dual synthetic lethality" eradicating quiescent LSCs and proliferating LSCs/LPCs.

To test this hypothesis we generated Parp1-/-Rad52-/- double knockout mice, which did not display any detectable problems in hematopoietic system and other organs. Leukemogenesis in SCLtTA/p210BCR-ABL1/Parp1-/-Rad52-/- mice was much prolonged in comparison to SCLtTA/p210BCR-ABL1/Parp1-/-, SCLtTA/p210BCR-ABL1/Rad52-/- and SCLtTA/p210BCR-ABL1 animals; 33% of SCLtTA/p210BCR-ABL1/Parp1-/-Rad52-/- mice did not develop leukemia. Moreover, BCR-ABL1 transfected Parp1-/-Rad52-/- bone marrow cells formed the lowest number of colonies when compared to BCR-ABL1 transfected Parp1-/-, Rad52-/-, and wild-type cells.

Next, we tested anti-leukemia effect of a combination of PARP1 inhibitor (FDA approved Lynparza) and RAD52 small molecule inhibitor (RAD52smi) identified by high-throughput screen. Simultaneous administration of RAD52 and PARP1 inhibitors completely eradicated clonogenic activity of TKI-treated CML-CP HR/PRs and CML-AP LSCs/LPCs and almost exhausted quiescent LSCs without affecting normal cells. These inhibitors were also very effective against Ph+ ALL cells.

In conclusion "dual synthetic lethality" triggered by simultaneous targeting of PARP1 and RAD52 represents a novel strategy which may eradicate quiescent LSCs and proliferating LSCs/LPCs not only from CML but also from BCR-ABL1 -positive ALL.

Disclosures

Valent: Novartis: Consultancy, Honoraria, Research Funding; Celgene: Honoraria; Bristol-Myers Squibb: Honoraria; Ariad: Honoraria, Research Funding; Pfizer: Honoraria.