Search

PARP inhibitors are clinically approved for cancer treatment, and the side effects are in general tolerable and transient. Tumor cells with defects in homologous recombination repair (HR), like BRCA1/2 mutations, are profoundly sensitive to the DNA damage induced by PARP inhibitors (PARPis). Repair by HR is linked to the cell cycle stage, yet when and how PARP inhibitors worked in the cell cycle was not well understood. The work presented in the thesis aimed to develop and refine methods to study the cell cycle by flow and mass cytometry. The improved methods were used to address the biological aims of the thesis; how PARPi affect induction of DNA-damage in relation to cell cycle phase and the possibility of repurposing PARPi towards lymphoma and leukemia with ATM loss-of-function. Ataxia telangiectasia mutated (ATM) transduce the DNA damage signal, and is thought to promote repair by HR. The effect of PARPi treatment was studied in a panel of malignant lymphocyte cell lines in the presence and absence of ATM inhibitor. PARPi toxicity was shown to require replication, and caused a prolonged G2 phase. Further, the study showed how the amount of replication-induced DNA damage of each cell determines its G2 phase duration, also in unperturbed culture. An improved cell cycle-panel and analysis pipeline for mass cytometry was developed in this work. The revised method made it possible to exclude all non-single cells while retaining the larger single cells. Thus, achieving accurate assessment of cell cycle distribution and enabling the use of up to 40 additional markers. The method strengthen the robustness of mass cytometry when used for both cycling and heterogeneous cell samples and may lead to new discoveries in the field. Overall, the thesis have shed light on the toxicity of PARPi and improved methods to study the cell cycle.

Master i biomedisin Cells that are deficient in proteins involved in homologous recombination repair (HRR) have been shown to be hypersensitive to PARP inhibitors. While a cell may tolerate PARP inhibition or HRR defects alone, the combination is lethal. This phenomenon is termed synthetic lethality. The function of HRR signaling protein ATM is lost in 50% of mantle cell lymphomas (MCL). MCL is an aggressive and currently incurable subtype of Non-Hodgkins lymphoma. PARP inhibition might prove to be a valuable treatment option for ATM deficient MCL cases. In this master thesis, we have investigated the alterations in cell growth, cell cycle distribution and DNA damage levels, as well as mode of cell death caused by PARP inhibitor treatment of ATM deficient lymphoid cells. Four lymphoid cancer cell lines (Reh, U698, JVM-2 and Granta-519) were continuously exposed to the clinically relevant PARP inhibitor (olaparib/AZD-2281), and/or ATM inhibitor (KU-55933). Cell growth was reduced or inhibited in all cell lines exposed to both ATM inhibitor and PARP inhibitor. The ATM inhibitor alone had little effect on the measured parameters in general, but increased the doubling time for all cell lines, and extended mitosis of U698 and Granta-519 cells. PARP inhibition caused a dose dependent-increase of DNA double strand breaks (DSB) during S phase. A G2 phase delay was induced by combined PARP and ATM inhibition. The cells repaired the DSBs associated with γH2AX foci during the prolonged G2 phase and entered mitosis without foci. Granta-519 and Reh cells became apoptotic from G2 or M in response to PARP and ATM inhibition, possibly because of high levels of DSBs. PARP and ATM inhibited U698 and JVM-2 cells suffered from mitotic catastrophe before necrosis. TP53 deficient U698 cells endoreduplicated extensively, while JVM-2 (wildtype TP53) cells arrested after failed cytokinesis. ATM deficient/inhibited lymphoid cells are sensitized to PARP inhibitors in a cell line specific manner, possibly because of other underlying genetic aberrations. We propose that the synthetic lethality of PARP and ATM inhibition was caused by repeated cycles of incorrect or failed repair of DNA DSBs that occurred during replication. Even though the HRR deficient cells have repaired the DSBs (possibly by error-prone non-homologous end joining), they may still accumulate translocations and/or other structural chromosome-defects that lead to apoptosis or mitotic catastrophe.