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Breast cancer is a heterogeneous disease that comprises multiple histological and molecular subtypes. Breast cancer treatments are subtype-based but eventually cancer cells become resistant, with tumour relapse remaining a significant challenge for patient management. This thesis addresses tumour suppressor genes that drive breast tumorigenesis and potential mechanisms of therapy resistance. The first section of the thesis describes a framework for the identification and validation of novel tumour suppressor genes that were identified in an in vivo genome-wide CRISPR/Cas9-screen in Trp53+/– heterozygous mice. The genes that encode for the scaffold protein Axin1 and the regulatory subunit from the protein kinase A complex, Prkar1a were identified as tumour suppressor genes that collaborate with loss of Trp53 in mammary tumorigenesis. Axin1- and Prkar1a-deleted organoids showed a morphological change with an increased proliferative capacity. RNAseq analysis revealed potential deregulation of cellular energetics in Axin1 mutant organoids. In addition, direct in vivo genome editing via intraductal injection of lentiviral vectors engineered to express dual short-guide RNAs validated Axin1 and Prkar1a as potent tumour suppressor genes that cooperate with loss of Trp53 to accelerate tumorigenesis each combination gave rise to hormone receptor negative tumours. In the second part of this thesis, mass cytometry was applied to breast cancer patient-derived xenografts (PDXs) to understand changes in cell survival and signal transduction pathways at the single cell level. Mass cytometry can detect up to 40 different protein markers via antibodies conjugated to heavy metals. Two primary questions were investigated: (a) what is the proportion of cells within a tumour that respond to treatment and (b) what molecular changes contribute to therapy resistance and tumour relapse? Short- and long-term dual therapy consisting of ABT-199 plus fulvestrant for ER+ PDX models or S63845 plus

Breast cancer is a heterogenous disease that can be stratified into at least six subtypes based on gene expression profiling. Each of these subtypes likely arise from a different cell of origin, through a repertoire of genetic aberrations that influences treatment decisions and subsequent resistance to therapy. Multiple mechanisms of resistance exist, including evasion of apoptotic cell death, which is a hallmark of cancer. The development of BH3 mimetics, which antagonise pro-survival proteins of the BCL2 family, is a new field of targeted cancer therapy. Initial studies observed that BCL2 is overexpressed in the majority of primary and metastatic estrogen receptor positive (ER+) breast cancer, and targeting BCL2 with ABT-199 (venetoclax), a potent BCL2 inhibitor, synergises with endocrine therapy in preclinical models of ER+ breast cancer. This thesis builds on these insights into the molecular events that are responsible for resistance to cell death. ER+ breast cancers also frequently exhibit deregulation of the retinoblastoma (Rb) pathway that includes cyclin-dependent kinase 4 and 6 (CDK4/6)/cyclin D1 (CCND1)/retinoblastoma (Rb), resulting in uncontrolled cellular proliferation. CDK4/6 inhibitors significantly augment tumor response when combined with endocrine therapy in ER+ breast cancer. Despite their potent antiproliferative activity, CDK4/6 inhibitors fail to induce apoptotic cell death. This thesis investigates the addition of the BH3 mimetic ABT-199 to endocrine therapy (fulvestrant) and the CDK4/6 inhibitor palbociclib in breast tumor organoids and patient-derived xenograft models of ER+ breast cancer. Triple therapy, which was well tolerated in vivo, produced a superior and more durable tumor response compared to single or doublet therapy. Increased apoptosis was observed in vitro and in vivo, with improved survival in PDX models. Notably, MHC class I and PDL1 expression increased in a syngeneic mouse model of ER+ breast cancer, accompanied by a reduct

Treatment of acute myeloid leukemia (AML) patients is still hindered by resistance and relapse, resulting in an overall poor survival rate. Recently, combining specific B-cell lymphoma (Bcl)-2 inhibitors with compounds downregulating myeloid cell leukemia (Mcl)-1 has been proposed as a new effective strategy to eradicate resistant AML cells. We show here that 1(R), 6(S), 1'(R), 6'(S), 11(R), 17(S)-fistularin-3, a bromotyrosine compound of the fistularin family, isolated from the marine sponge Suberea clavata, synergizes with Bcl-2 inhibitor ABT-199 to efficiently kill Mcl-1/Bcl-2-positive AML cell lines, associated with Mcl-1 downregulation and endoplasmic reticulum stress induction. The absolute configuration of carbons 11 and 17 of the fistularin-3 stereoisomer was fully resolved in this study for the first time, showing that the fistularin we isolated from the marine sponge Subarea clavata is in fact the (+)-11(R), 17(S)-fistularin-3 stereoisomer keeping the known configuration 1(R), 6(S), 1'(R), and 6'(S) for the verongidoic acid part. Docking studies and in vitro assays confirm the potential of this family of molecules to inhibit DNA methyltransferase 1 activity.

Treatment of acute myeloid leukemia (AML) patients is still hindered by resistance and relapse, resulting in an overall poor survival rate. Recently, combining specific B-cell lymphoma (Bcl)-2 inhibitors with compounds downregulating myeloid cell leukemia (Mcl)-1 has been proposed as a new effective strategy to eradicate resistant AML cells. We show here that 1(R), 6(S), 1'(R), 6'(S), 11(R), 17(S)-fistularin-3, a bromotyrosine compound of the fistularin family, isolated from the marine sponge Suberea clavata, synergizes with Bcl-2 inhibitor ABT-199 to efficiently kill Mcl-1/Bcl-2-positive AML cell lines, associated with Mcl-1 downregulation and endoplasmic reticulum stress induction. The absolute configuration of carbons 11 and 17 of the fistularin-3 stereoisomer was fully resolved in this study for the first time, showing that the fistularin we isolated from the marine sponge Subarea clavata is in fact the (+)-11(R), 17(S)-fistularin-3 stereoisomer keeping the known configuration 1(R), 6(S), 1'(R), and 6'(S) for the verongidoic acid part. Docking studies and in vitro assays confirm the potential of this family of molecules to inhibit DNA methyltransferase 1 activity.

Treatment of acute myeloid leukemia (AML) patients is still hindered by resistance and relapse, resulting in an overall poor survival rate. Recently, combining specific B-cell lymphoma (Bcl)-2 inhibitors with compounds downregulating myeloid cell leukemia (Mcl)-1 has been proposed as a new effective strategy to eradicate resistant AML cells. We show here that 1(R), 6(S), 1'(R), 6'(S), 11(R), 17(S)-fistularin-3, a bromotyrosine compound of the fistularin family, isolated from the marine sponge Suberea clavata, synergizes with Bcl-2 inhibitor ABT-199 to efficiently kill Mcl-1/Bcl-2-positive AML cell lines, associated with Mcl-1 downregulation and endoplasmic reticulum stress induction. The absolute configuration of carbons 11 and 17 of the fistularin-3 stereoisomer was fully resolved in this study for the first time, showing that the fistularin we isolated from the marine sponge Subarea clavata is in fact the (+)-11(R), 17(S)-fistularin-3 stereoisomer keeping the known configuration 1(R), 6(S), 1'(R), and 6'(S) for the verongidoic acid part. Docking studies and in vitro assays confirm the potential of this family of molecules to inhibit DNA methyltransferase 1 activity.

Treatment of acute myeloid leukemia (AML) patients is still hindered by resistance and relapse, resulting in an overall poor survival rate. Recently, combining specific B-cell lymphoma (Bcl)-2 inhibitors with compounds downregulating myeloid cell leukemia (Mcl)-1 has been proposed as a new effective strategy to eradicate resistant AML cells. We show here that 1(R), 6(S), 1'(R), 6'(S), 11(R), 17(S)-fistularin-3, a bromotyrosine compound of the fistularin family, isolated from the marine sponge Suberea clavata, synergizes with Bcl-2 inhibitor ABT-199 to efficiently kill Mcl-1/Bcl-2-positive AML cell lines, associated with Mcl-1 downregulation and endoplasmic reticulum stress induction. The absolute configuration of carbons 11 and 17 of the fistularin-3 stereoisomer was fully resolved in this study for the first time, showing that the fistularin we isolated from the marine sponge Subarea clavata is in fact the (+)-11(R), 17(S)-fistularin-3 stereoisomer keeping the known configuration 1(R), 6(S), 1'(R), and 6'(S) for the verongidoic acid part. Docking studies and in vitro assays confirm the potential of this family of molecules to inhibit DNA methyltransferase 1 activity.

Evasion of cell death is one of the hallmarks of cancer that has only recently become targetable using small molecule inhibitors. By antagonizing the pro-survival function of BCL-2 family proteins, these so-called BH3 mimetics skew cells towards undergoing apoptosis. Though single-agent approaches have yielded promising clinical efficacy in some contexts, it is often insufficient to induce substantial patient responses. This dissertation investigates the potential of using rational targeted therapies to enhance the efficacy of BH3 mimetics. In Chapter 2, we pursue the simple hypothesis that targeting a pathway whose aberrant activation is associated with cancer (the PI3K/AKT/mTOR pathway) can sensitize cells to BCL-2 inhibition. We demonstrate that inhibitors of this pathway synergize strongly with two BH3 mimetics, ABT-263 and ABT-199, and that the extent of this synergy can be predicted using dynamic BH3 profiling. By suppressing AKT activity, PI3K pathway inhibitors induced mitochondrial accumulation of pro-apoptotic proteins. This work provided insight into markers of pharmacodynamic response, tools for predicting efficacy, and evidence warranting further investigation. In Chapter 3 we pursue an alternative approach: that targeting a key metabolic pathway for which inhibitors are already FDA-approved, can also sensitize cells to BH3 mimetics. We show that targeting HMG-CoA reductase using statins selectively primes cancer cells for apoptosis and enhances the efficacy of ABT-199 in vitro and in vivo. We build on the work from Chapter 2 to demonstrate the predictive capabilities of dynamic BH3 profiling in cell lines and primary patient samples. Additionally, we identify downstream targets contributing to the sensitization effect, supporting further investigation. Lastly, in Chapter 4, I present incomplete or unpublished data that follows up on the work presented in Chapters 2 and 3. I also discuss potential future directions derived from those experiments as well

Evasion of cell death is one of the hallmarks of cancer that has only recently become targetable using small molecule inhibitors. By antagonizing the pro-survival function of BCL-2 family proteins, these so-called BH3 mimetics skew cells towards undergoing apoptosis. Though single-agent approaches have yielded promising clinical efficacy in some contexts, it is often insufficient to induce substantial patient responses. This dissertation investigates the potential of using rational targeted therapies to enhance the efficacy of BH3 mimetics. In Chapter 2, we pursue the simple hypothesis that targeting a pathway whose aberrant activation is associated with cancer (the PI3K/AKT/mTOR pathway) can sensitize cells to BCL-2 inhibition. We demonstrate that inhibitors of this pathway synergize strongly with two BH3 mimetics, ABT-263 and ABT-199, and that the extent of this synergy can be predicted using dynamic BH3 profiling. By suppressing AKT activity, PI3K pathway inhibitors induced mitochondrial accumulation of pro-apoptotic proteins. This work provided insight into markers of pharmacodynamic response, tools for predicting efficacy, and evidence warranting further investigation. In Chapter 3 we pursue an alternative approach: that targeting a key metabolic pathway for which inhibitors are already FDA-approved, can also sensitize cells to BH3 mimetics. We show that targeting HMG-CoA reductase using statins selectively primes cancer cells for apoptosis and enhances the efficacy of ABT-199 in vitro and in vivo. We build on the work from Chapter 2 to demonstrate the predictive capabilities of dynamic BH3 profiling in cell lines and primary patient samples. Additionally, we identify downstream targets contributing to the sensitization effect, supporting further investigation. Lastly, in Chapter 4, I present incomplete or unpublished data that follows up on the work presented in Chapters 2 and 3. I also discuss potential future directions derived from those experiments as well

Author: Jürgen Reidla, BSc, Masterarbeit University of Innsbruck 2020

Author: Jürgen Reidla, BSc, Masterarbeit University of Innsbruck 2020

Direct co-operation between sensitiser molecules BAD and NOXA in mediating apoptosis suggests that therapeutic agents which sensitise to BAD may complement agents which sensitise to NOXA. Dynamic BH3 profiling is a novel methodology that we have applied to the measurement of complementarity between sensitiser BH3 peptide mimetics and therapeutic agents. Using dynamic BH3 profiling, we show that the agent TG02, which downregulates MCL-1, sensitises to the BCL-2-inhibitory BAD-BH3 peptide, whereas the BCL-2 antagonist ABT-199 sensitises to MCL-1 inhibitory NOXA-BH3 peptide in acute myeloid leukaemia (AML) cells. At the concentrations used, the peptides did not trigger mitochondrial outer membrane permeabilisation in their own right, but primed cells to release Cytochrome C in the presence of an appropriate trigger of a complementary pathway. In KG-1a cells TG02 and ABT-199 synergised to induce apoptosis. In heterogeneous AML patient samples we noted a range of sensitivities to the two agents. Although some individual samples markedly favoured one agent or the other, in the group as a whole the combination of TG02 + ABT-199 was significantly more cytotoxic than either agent individually. We conclude that dynamic NOXA and BAD BH3 profiling is a sensitive methodology for investigating molecular pathways of drug action and complementary mechanisms of chemoresponsiveness.

Direct co-operation between sensitiser molecules BAD and NOXA in mediating apoptosis suggests that therapeutic agents which sensitise to BAD may complement agents which sensitise to NOXA. Dynamic BH3 profiling is a novel methodology that we have applied to the measurement of complementarity between sensitiser BH3 peptide mimetics and therapeutic agents. Using dynamic BH3 profiling, we show that the agent TG02, which downregulates MCL-1, sensitises to the BCL-2-inhibitory BAD-BH3 peptide, whereas the BCL-2 antagonist ABT-199 sensitises to MCL-1 inhibitory NOXA-BH3 peptide in acute myeloid leukaemia (AML) cells. At the concentrations used, the peptides did not trigger mitochondrial outer membrane permeabilisation in their own right, but primed cells to release Cytochrome C in the presence of an appropriate trigger of a complementary pathway. In KG-1a cells TG02 and ABT-199 synergised to induce apoptosis. In heterogeneous AML patient samples we noted a range of sensitivities to the two agents. Although some individual samples markedly favoured one agent or the other, in the group as a whole the combination of TG02 + ABT-199 was significantly more cytotoxic than either agent individually. We conclude that dynamic NOXA and BAD BH3 profiling is a sensitive methodology for investigating molecular pathways of drug action and complementary mechanisms of chemoresponsiveness.

Direct co-operation between sensitiser molecules BAD and NOXA in mediating apoptosis suggests that therapeutic agents which sensitise to BAD may complement agents which sensitise to NOXA. Dynamic BH3 profiling is a novel methodology that we have applied to the measurement of complementarity between sensitiser BH3 peptide mimetics and therapeutic agents. Using dynamic BH3 profiling, we show that the agent TG02, which downregulates MCL-1, sensitises to the BCL-2-inhibitory BAD-BH3 peptide, whereas the BCL-2 antagonist ABT-199 sensitises to MCL-1 inhibitory NOXA-BH3 peptide in acute myeloid leukaemia (AML) cells. At the concentrations used, the peptides did not trigger mitochondrial outer membrane permeabilisation in their own right, but primed cells to release Cytochrome C in the presence of an appropriate trigger of a complementary pathway. In KG-1a cells TG02 and ABT-199 synergised to induce apoptosis. In heterogeneous AML patient samples we noted a range of sensitivities to the two agents. Although some individual samples markedly favoured one agent or the other, in the group as a whole the combination of TG02 + ABT-199 was significantly more cytotoxic than either agent individually. We conclude that dynamic NOXA and BAD BH3 profiling is a sensitive methodology for investigating molecular pathways of drug action and complementary mechanisms of chemoresponsiveness.