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Abstract

Abstract: EP487

Type: e-Poster

Background
The Poly (ADP-ribose) polymerase (PARP) protein family plays an important role in genome maintenance, with a major function in the correction of single strand breaks (SSB) via the base excision repair pathway. The significance of PARP inhibition and its potential for achieving synthetic lethality have been extensively characterized for cancers with germline BRCA mutations and/or HER2-negative breast cancer models. The knowledge incurred from these cancer models has already been applied to various other cancer models such as glioblastoma, prostrate and ovarian cancers. Although BRCA mutations are not amongst the major cluster of driver mutations, some reports have shown a hypermethylation of BRCA in acute myeloid leukemia (AML) patient samples suggesting dysfunctional DNA repair remains an untapped territory in targeting vulnerabilities of cancer cells.

Aims
AML is characterized by an increased cell proliferation, blocked differentiation, and aberrant self-renewal capabilities. In this project, we raised the hypothesis that further induction of replicative stress / DNA damage mediated by PARP inhibition in already highly proliferating cells potentiates the dependency on anti-apoptotic proteins and that a specific inhibition of anti-apoptotic pathways will cause cell death.

Methods
AML cell lines were treated with PARP 1/2 inhibitor (Olaparib) and characterized based on sensitivity to PARP inhibition using cell viability and cell cycle assays. Next, DNA damage biomarkers such a gH2AX, 53BP1, pRPA were measured using immunofluorescence and high-content screening approaches. Finally, combination treatments of Olaparib with antiapoptotic protein inhibitors of the B-cell lymphoma 2 (BCL2) family were performed and synergy was quantified using high-throughput screening.

Results

Preliminary data showed that mono-treatment with a PARP 1/2 inhibitor (Olaparib) induced a dose- and time-dependent accumulation of gH2AX foci. Olaparib neither affected the survival of the cells nor cell cycle progression. We next investigated, if the damage was cell cycle specific. To this end, we used high-content screening approach quantifying the DNA damage within each phase of the cell cycle. DNA damage accumulation was elevated in S/G2 phase, but cells managed to overcome this stress and continued cycling. Interestingly, the increase in DNA damage was accompanied by a dose-dependent increase in the anti-apoptotic protein B-cell lymphoma 2 (BCL2). We performed unsupervised hierarchal clustering of endogenous transcriptomic profiles of genes involved in DNA damage response pathways of various AML cell lines with distinct genetic background and correlated these profiles with the mutational status of major regulators of DNA damage. We observed that cell lines with wild type (wt) TP53 expression clustered together, and a subgroup of the cluster had FLT3 mutations. Consistently, when we combined PARP inhibition together with BCL2 inhibitor (Venetoclax) in a TP53wt FLT3mut background, cells underwent synergistic apoptotic death. Of note, although the combined treatment was most effective in cells with low or even undetectable BRCA1 or BRCA2 protein expression, cell lines with BRCA1 and BRCA2 expression also showed promising responses.

Conclusion
Our data indicate that inhibition of DNA damage repair in combination with inhibition of antiapoptotic pathways may provide a novel treatment strategy in highly proliferative cancers. Currently we are assessing this combinatory effect in primary AML patient samples and exploring the mechanisms by which AML cells manage to mitigate DNA damage.

Session topic: 03. Acute myeloid leukemia - Biology & Translational Research

Keyword(s): AML, Apoptosis, BCL2, DNA damage

Abstract: EP487

Type: e-Poster

Background
The Poly (ADP-ribose) polymerase (PARP) protein family plays an important role in genome maintenance, with a major function in the correction of single strand breaks (SSB) via the base excision repair pathway. The significance of PARP inhibition and its potential for achieving synthetic lethality have been extensively characterized for cancers with germline BRCA mutations and/or HER2-negative breast cancer models. The knowledge incurred from these cancer models has already been applied to various other cancer models such as glioblastoma, prostrate and ovarian cancers. Although BRCA mutations are not amongst the major cluster of driver mutations, some reports have shown a hypermethylation of BRCA in acute myeloid leukemia (AML) patient samples suggesting dysfunctional DNA repair remains an untapped territory in targeting vulnerabilities of cancer cells.

Aims
AML is characterized by an increased cell proliferation, blocked differentiation, and aberrant self-renewal capabilities. In this project, we raised the hypothesis that further induction of replicative stress / DNA damage mediated by PARP inhibition in already highly proliferating cells potentiates the dependency on anti-apoptotic proteins and that a specific inhibition of anti-apoptotic pathways will cause cell death.

Methods
AML cell lines were treated with PARP 1/2 inhibitor (Olaparib) and characterized based on sensitivity to PARP inhibition using cell viability and cell cycle assays. Next, DNA damage biomarkers such a gH2AX, 53BP1, pRPA were measured using immunofluorescence and high-content screening approaches. Finally, combination treatments of Olaparib with antiapoptotic protein inhibitors of the B-cell lymphoma 2 (BCL2) family were performed and synergy was quantified using high-throughput screening.

Results

Preliminary data showed that mono-treatment with a PARP 1/2 inhibitor (Olaparib) induced a dose- and time-dependent accumulation of gH2AX foci. Olaparib neither affected the survival of the cells nor cell cycle progression. We next investigated, if the damage was cell cycle specific. To this end, we used high-content screening approach quantifying the DNA damage within each phase of the cell cycle. DNA damage accumulation was elevated in S/G2 phase, but cells managed to overcome this stress and continued cycling. Interestingly, the increase in DNA damage was accompanied by a dose-dependent increase in the anti-apoptotic protein B-cell lymphoma 2 (BCL2). We performed unsupervised hierarchal clustering of endogenous transcriptomic profiles of genes involved in DNA damage response pathways of various AML cell lines with distinct genetic background and correlated these profiles with the mutational status of major regulators of DNA damage. We observed that cell lines with wild type (wt) TP53 expression clustered together, and a subgroup of the cluster had FLT3 mutations. Consistently, when we combined PARP inhibition together with BCL2 inhibitor (Venetoclax) in a TP53wt FLT3mut background, cells underwent synergistic apoptotic death. Of note, although the combined treatment was most effective in cells with low or even undetectable BRCA1 or BRCA2 protein expression, cell lines with BRCA1 and BRCA2 expression also showed promising responses.

Conclusion
Our data indicate that inhibition of DNA damage repair in combination with inhibition of antiapoptotic pathways may provide a novel treatment strategy in highly proliferative cancers. Currently we are assessing this combinatory effect in primary AML patient samples and exploring the mechanisms by which AML cells manage to mitigate DNA damage.

Session topic: 03. Acute myeloid leukemia - Biology & Translational Research

Keyword(s): AML, Apoptosis, BCL2, DNA damage

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