Medical Biology - Theses
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ItemNo Preview AvailableA Quantitative Analysis of Natural Killer Cell Homeostasis, Competition, and Collaboration( 2022-12)Contemporary Immunology views Natural Killer (NK) cells as critical facilitators of immune protection in various pathological settings. Still, this has not always been the case; a somewhat challenging history of NK cell research has delayed full scientific appreciation of their importance and modus operandi, which rendered NK cells a mysterious and misunderstood immune cell subset for several decades. In more recent years, NK cells are receiving a resurgence in clinical attention owing to characterisation of their potent anti-tumour and immunomodulatory properties; however, as modern Immunology remains in the aftermath of an uncertain era for NK cells, harnessing this revolutionary therapeutic potential has proven difficult. NK cells are key inducers of early inflammation and systemic immune activation, as well as expert decision makers in the destruction of harmful cells versus protection of healthy tissue. As may be expected, catastrophic consequences can occur to a host if these processes are not properly regulated. There is growing appreciation in the research community regarding the sheer complexity and redundancy in regulatory processes that maintain NK cell homeostasis and functions, as well as the plethora of cytokines and cell-cell interactions that govern this regulated behaviour. As a means of dissecting these complex processes, we have applied a reductionist approach to study how various individual signals are integrated into the internal machinery of an NK cell to produce different outcomes. To this end, we applied quantitative methods previously established in adaptive T and B lymphocytes to delineate and quantify parameters relating to survival and proliferation. In this work, we uncovered that stimulatory proliferative signals from the cytokines IL-15, IL-18, and IL-12 are offset by enhanced propensity for NK cell death, which limits the overall efficiency of their expansion during stimulation. These responses were largely dependent on direct interactions between NK cells via Fas and FasL, which induce fratricidal killing of each other. These competitive relationships between fellow NK cells were heavily dependent on the type and dose of cytokine present. Further, our investigation of NK cell interactions led us to identify that NK cells also facilitate advantageous interactions with other NK cells in more homeostatic contexts, which were dependent on IL-15. We discovered that these homotypic collaborative interactions are the result of complex interactions and bidirectional signalling events between SLAM family receptors 2B4 and CD48, which together facilitate IL-15 responsiveness and education events, thereby enhancing NK cell fitness and function, respectively. This work offers valuable insights to improve in vitro culture protocols in the clinical cultivation of NK cells for immunotherapies, such as Adoptive Cell Therapy, as well as indicating broader and nuanced roles of immune and target cell interactions in the stimulation and regulation of NK cell fitness, function, and homeostasis.
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ItemEngineering antibodies to trigger Bak-mediated apoptosis in cancer( 2022)Targeting the BCL-2 family with BH3-mimetics is proving an effective treatment for several cancers. These drugs initiate apoptosis by neutralising pro-survival BCL-2 members and allowing BH3-only proteins to activate BAK and BAX. However, to date there are no therapeutic agents to date that directly trigger BAK or BAX activation for subsequent apoptosis. Thus, agents that directly target BAK and BAX to trigger apoptosis may augment cancer therapy, and offer alternate means to induce apoptosis in cancer cells that are resistant to other forms of therapy. Although small molecule direct activators of BAX have been described (Lopez et al., 2022), there are none reported for BAK. An antibody, 7D10, that specifically binds and triggers activation of the intracellular pro-apoptotic protein BAK was reported by our group (Iyer et al., 2016), creating the opportunity to directly target the BAK protein to kill cancer cells. This thesis investigates various strategies adopted to obtain proof-of-concept that 7D10, when delivered into cancer cells, can bind, and activate BAK to induce apoptosis. Several approaches were employed to: (1) express 7D10 as "intrabodies" inside the target cells; (2) deliver recombinant 7D10 antibody inside cancer cells; (3) identify alternative "7D10-like" apoptosis inducing antibodies using new antibody discovery platforms; and (4) utilise newly identified antibodies to better understand BAK regulation. Intracellular expression of 7D10 was achieved by grafting complementarity determining regions (CDRs) onto two intrinsically stable scaffolds and by increasing the net negative charge using negatively charged tags. Two out of nine 7D10 intrabodies were able to activate BAK when expressed in cells and release cytochrome c, thereby giving proof-of-concept that 7D10 can indeed activate BAK in cells. Delivery of recombinant BAK inside cancer cells was explored using receptor-mediated endocytosis through the EGF receptor. A 7D10 polyplex was generated wherein 7D10 fragments were conjugated or expressed as fusion proteins with an anti-EGFR affibody (targeting agent) and an endosomal escape agent. As only minimal uptake of 7D10 polyplexes was observed using this approach, an alternative delivery approach involving nano-assemblies was investigated. To identify alternative BAK-activating antibodies that might be more stable in the cytosolic environment, three antibody discovery platforms were pursued. Alpaca phage display identified nanobodies that bound BAK but did not activate or inhibit the protein. Mouse B cell cloning identified the first antibody that inhibits BAK activation, thereby facilitating better understanding of BAK inhibition as well as activation. A rat B cell cloning antibody discovery platform was developed and coupled with Oxford Nanopore long read sequencing to explore whether rare B cells that are specific to the BAK activation site might be identified. Though only naive B cells were sorted using FACS, a new sequencing workflow was established for the identification of full-length antibody genes. Thus, the results presented in this thesis, in addition to providing proof-of-concept that 7D10 can activate BAK in cells, gives insights into new strategies utilised to engineer antibodies for intracellular delivery, expression as well as improvements to antibody discovery platforms for streamlined identification of antibody genes.
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ItemInterfering with Host Cell Signalling and Cell Death Pathways to Promote Clearance of Viral and Parasitic Infections( 2022)Programmed cell death plays an essential role in homeostatic cell turnover and in eliminating diseased cells in complex organisms. Cell death processes can be divided into pathways that are non-lytic and favourable to adaptive immunity; intrinsic and extrinsic apoptosis, and pathways that are lytic and proinflammatory; pyroptosis and necroptosis. Intracellular pathogens rely on host cell resources for their replicative cycles and therefore must control cell death signalling to prevent premature termination of such processes. Induction of cell death as a therapeutic avenue for the treatment of malignancies is an established clinical paradigm but induction of cell death to manage intracellular infections has only recently attracted research interest. The following work explores the feasibility and efficacy of targeting cell death pathways to treat two different intracellular pathogens – hepatitis B virus (HBV) and Leishmania donovani. These pathogens result in great morbidity and mortality worldwide, and new therapies that would provide safer more effective treatments are desperately needed. Through the design of a novel, preclinical mouse model of HBV, which recapitulated an essential element in the viral lifecycle called covalently closed circular HBV DNA (cccDNA), I showed that promotion of extrinsic apoptosis could be targeted to eliminate cccDNA. Preferential death of infected hepatocytes was achieved by antagonising a set of host cell proteins called inhibitor of apoptosis (IAP) proteins. I showed that IAP inhibitors induce the extrinsic apoptosis of infected cells without causing collateral damage and this resulted in elimination of HBV infection in animal model. I also showed that this new therapeutic paradigm was applicable to parasitic infections of the liver. Beyond extrinsic apoptosis, I explored the relevance of other cell death pathways in the pathogenesis of visceral leishmaniasis. Pyroptosis and necroptosis were both shown to be redundant or rendered redundant in infection, indicating that targeting these pathways with inhibitors would be unlikely to be effective in treating L. donovani infection. In contrast, IAP inhibitors, particularly in combination with standard anti-parasitic drugs, promoted clearance of L. donovani in animals. My work provides a strong foundation underscoring the utility and applicability of harnessing cell death pathways to treat intracellular infections. This important discovery represents a novel therapeutic paradigm to treat infections that are intrinsically resistant or acquire resistance to antimicrobial agents.
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ItemDeveloping Preventative and Therapeutic Strategies Against HTLV-1 Using a Novel Humanised Mouse Model( 2022)Satisfactory preventative or therapeutic drugs are lacking for human T cell leukaemia virus 1 (HTLV-1), a disease which lags several decades behind its distantly related cousin HIV in this regard. Consequentially, 5-10 percent of the approximately 10 million people infected with HTLV-1 will progress to serious complications. These include fatalities resulting from a rapidly progressive blood cancer, adult T cell leukaemia/lymphoma, and inflammation of the spinal cord, HTLV-1-assocaited myelitis. Other clinically recognised disease associations include inflammatory diseases involving the eyes, lungs, and skin. In Central Australia, a region in which the adult prevalence of HTLV-1c approaches 40% in some remote Aboriginal communities, HTLV-1 subtype c (HTLV-1c) infection is strongly associated with chronic pulmonary disease and death due to bronchiectasis and is a significant health burden in these communities. Although HTLV-1 subtype a (HTLV-1a) is also associated with pulmonary disease, the extraordinarily high burden of pulmonary disease in Central Australia suggests that there may differences between HTLV-1a and HTLV-1c, which are the two most divergent subtypes. Novel preventative and therapeutic interventions are required to tackle HTLV-1. I investigated the efficacy of antiretroviral and pro-apoptotic BH3 mimetic compounds as preventative and therapeutic agents in a novel humanised mouse model of HTLV-1c infection, the first of its kind. I characterised infection in this model and compared disease to the globally prevalent HTLV-1a. Disease was indistinguishable in mice suggesting that HTLV-1 subtype does not drive distinct disease associations, including HTLV-1 associated pulmonary disease. Tenofovir, a reverse iv transcriptase inhibitor, significantly reduced HTLV-1 transmission in vivo at clinically relevant doses and attenuated de novo viral spread and disease progression during early infection in combination with dolutegravir, an integrase inhibitor. HTLV-1 infection was associated with dysregulation of the intrinsic apoptotic pathway at the transcriptional level, and pharmacological inhibition of MCL-1, but not BCL-2, BCL-xL or BCL-w, killed HTLV-1-infected cells ex vivo and mitigated disease progression in vivo in combination with tenofovir and dolutegravir. Collectively, these data provide evidence that combination antiretroviral and MCL1 antagonism may represent an effective, clinically relevant, curative strategy against HTLV-1. Routine clinical use of these compounds will facilitate their rapid translation to HTLV-1 treatment.
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ItemStructural and Functional Investigation of the BCL-2 Family Member, BCL-rambo( 2022)The BCL-2 family proteins are the faces of mitochondrial apoptosis, governing cell fate through interactions that mediate mitochondrial outer membrane permeabilisation. While the molecular intricacies of the pathway have been characterised in great detail, putative BCL-2 proteins of unknown function remain. BCL-rambo (or BCL-2-like protein 13) is one such protein. This enigmatic BCL-2 family member was first discovered in 2001 and 20 years later, a consensus concerning its role has yet to be reached. With reports suggesting a pro-apoptotic or an anti-apoptotic role for BCL-rambo, whilst others implicating a role in mitophagy, the deconvolution of its influence within these established fundamental signalling pathways is of great interest considering their potential for dysregulation in disease. This thesis aimed to characterise the BCL-rambo protein from the ground-up. Harnessing a combination of molecular, structural, and biochemical techniques, this work investigated the structure and function of BCL-rambo in the context of apoptosis and mitophagy. The effect of BCL-rambo expression on apoptosis is examined in Chapter 3, which details attempts to identify binding partners within the BCL-2 family of proteins. Chapter 4 describes the design and production of recombinant BCL-rambo protein used for X-ray crystallisation studies, and reveals the structure of the BCL-rambo BCL-2 homology region. Chapter 5 assesses the role of BCL-rambo in mitophagy and investigates its ability to bind ATG8-family proteins. This chapter also examines how phosphorylation of BCL-rambo affects its affinity for ATG8-family proteins and includes X-ray structures of a phosphorylated BCL-rambo peptide bound with ATG8-family proteins. Together, these studies address the discrepancy regarding the function of BCL-rambo, supporting a role for BCL-rambo as a mitophagy receptor, and elucidates the first experimental three-dimensional structure of BCL-rambo. This work also provides an intriguing example of divergent protein evolution and brings the field a step closer to completing the BCL-2 family portrait.
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ItemIdentifying Novel Strategies to Enhance the Anti-cancer Activity of Venetoclax by Manipulating NOXA Expression( 2021)Apoptosis is a form of programmed cell death. The intrinsic pathway of apoptosis is governed by the BCL2 family proteins. Targeting BCL2 proteins by small molecules that mimicking the BH3-only proteins to induce apoptosis has proven to be a successful strategy for cancer therapy. Venetoclax, a specific inhibitor of BCL2, has exhibited remarkable efficacy in treating cancers that rely on BCL2 for survival. However, the activity of venetoclax is often limited in other cancers whose survival relies on MCL1, another BCL2 family member. Selective MCL1 inhibitors have been developed and are currently being evaluated in clinical trials. However, the clinical development of these agents has been hampered by toxicity, especially cardiac toxicity. Potentially, another strategy to target MCL1 is by modulating NOXA, a BH3-only protein that selectively binds to MCL1 and mediates its degradation. I hypothesised that increased NOXA expression would prime cancer cells to venetoclax killing and that this would reduce their co-dependence on MCL1. In order to identify new targets to modulate NOXA expression, I generated and validated cell lines that report on NOXA transcription and then carried out CRISPR-Cas9 genetic screens in those NOXA reporter cell lines. In CRISPR-Cas9 loss-of-function screens focused on epigenetic regulators, I found several genes whose mutation or loss modulated NOXA expression, including CTBP1, CHTOP, ZMYM3, SPEN, HSPA1A, KEAP1, FOXA1, HDAC3 and SAP30. Some of these factors have been targeted for cancer therapies, for example KEAP1 and HDAC3, while the others have not yet been recognized for their therapeutic possibilities. Subject to their validation, my results have identified interesting novel mechanisms of NOXA regulation, thus providing the rationale basis for the development of new anti-cancer agents. In CRISPR-Cas9 tiling screens that focused on the NOXA promoter region, five cis-regulatory elements were identified that contributed to regulation of NOXA expression. Among them, a hypermethylated element on the NOXA promoter was found to be important for repressing NOXA expression across diverse cell lines derived from blood cancers. Disrupting this region led to NOXA induction. Potentially, the findings could provide a rational basis of combining hypomethylating agents with venetoclax in a range of haematological malignancies. In summary, several potential NOXA regulating proteins and DNA elements were discovered by the CRISPR-Cas9 screening approaches. Once validated, these findings should provide new insights into NOXA regulation.
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ItemCell Death Mechanisms in T cell Differentiation and Homeostasis( 2021)T cells are an essential component of the vertebrate adaptive immune system. In concert with the innate immune system, T cells protect the host from any number of pathogens that could be experienced over an organism’s lifetime. The hallmark of a T cell is its distinctive T cell receptor generated by somatic gene rearrangement. Variability in the T cell receptor repertoire arises during thymic T cell differentiation, which is then subjected to strict selection processes. Mature T cells in the periphery can undergo further differentiation upon the activation of naive cells to mount immune responses to pathogens. These differentiation events are accompanied by significant proliferative bursts, followed by the clearance of defective or superfluous cells. It follows then, that cell death is also an essential component of T cell differentiation and homeostasis. This PhD thesis explores the molecular mechanisms regulating the differentiation, proliferation and death of T cells, and how interplay among these mechanisms gives rise to immune homeostasis. This study examines how distinct cell death pathways, including the intrinsic and extrinsic apoptotic pathways and necroptosis, are differentially regulated through T cell differentiation and in the various subsets of mature T cells. We found that only inhibition of the intrinsic pathway of apoptosis overcomes failure of beta-selection in the absence of preTCR signalling or proliferation, enabling further differentiation. We also discovered that caspase 8 plays an important pro-survival role in inhibiting necroptosis in recent thymic emigrant T cells and regulatory T cells, and that this feature can be exploited in the case of regulatory T cells for therapeutic intervention in infection settings. In summary, this thesis defines context-specific roles of cell death modalities in controlling T cell differentiation and homeostasis, revealing the potential for immune interventions using targeted therapies.
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ItemIdentifying and Overcoming Therapeutic Resistance in High-Risk Acute Lymphoblastic Leukaemia( 2021)Background: Acute lymphoblastic leukaemia (ALL) is the most common childhood cancer and early disease eradication is critically important for long-term cure. ALL remains a leading cause of cancer death in children and young adults because of treatment toxicity and relapsed disease. Increased delineation of the key biological drivers in ALL as well as therapeutic response will present new opportunities to rationally combine existing and novel agents to improve outcomes whilst minimising toxicities. Blocks in apoptosis are now widely recognised as a hallmark of ALL but also a mechanism of resistance to standard chemotherapeutic agents. Co-targeting aberrant cell survival pathways, using novel combinations of BH3-mimetics, are an emerging therapeutic option. My thesis centres on translational and mechanistic studies of combining venetoclax (BCL-2 inhibitor) and S63845 (MCL-1 inhibitor) in high-risk (HR) ALL subtypes. Aim: To inform the clinical utility of BH3-mimetic combinations in HR-ALL by identifying synergistic combinations with each other as well as standard and targeted agents in vitro, evaluating the efficacy and tolerability of combination venetoclax (BCL-2 inhibitor) and S63845 (MCL-1 inhibitor) in vivo, and investigating mechanisms of therapeutic resistance. Methods: BH3-mimetics were combined with each other as well as dexamethasone and targeted tyrosine kinase inhibitors (TKI) to identify the most potent combinations across kinase-activated ALL cell lines and patient derived xenografts (PDX) in vitro and in vivo. The tolerability of combinations venetoclax and S63845 was detailed in NOD-SCID-IL7R (NSG) mice as well as healthy donor blood ex vivo. Loss-of-function mutations that confer resistance to TKIs and BH3-mimetics in a Ph+ALL cell line were identified using an unbiased genome-wide CRISPR-Cas9 loss-of-function screen. Differences in cell signalling and survival pathways following treatment with TKI and BH3-mimetics in vivo in PDX models of Ph-like ALL were identified using mass cytometry. Results: BCL-2 and MCL-1 protein expression remained high in kinase-activated B-ALL cell lines treated with targeted TKI despite up-regulation of BIM pro-apoptotic protein expression. Co-inhibition of BCL-2 and MCL-1, with combination venetoclax and S63845, induced synergistic killing in vitro and was comparable or superior to steroid or TKI combined with each BH3-mimetic, across a range of kinase-activated B-ALL cell lines and PDXs, including the Ph+ and Ph-like subtypes. The combination also had potent anti-leukaemia activity in vivo, which was demonstrated by rapid cytoreduction, but also acute tumour lysis syndrome (ATLS) in some PDX models of Ph-like B-ALL. Combining venetoclax and S63845 appeared tolerable, however, histologic evidence of haematopoietic toxicity was observed, at higher doses, in NSG mice, and synergistic cytotoxicity was observed in lymphocytes of healthy donor blood. Loss of function mutations in NOXA and BAX were identified from the CRISPR screen as important causes of resistance to dasatinib and venetoclax in Ph+ALL. Lastly, CyTOF demonstrated distinct single cell variability in response to TKI or BH3-mimetic treatment of Ph-like ALL, including differences in surface marker expression (CD38, CD179a, and CD34), and cell signalling pathways (pSTAT5). Conclusion: Co-inhibition of BCL-2 and MCL-1 induces synergistic killing in vitro and rapid cytoreduction in vivo in a range of HR B-ALL models, including the Ph+ and Ph-like subtypes. The combination of BCL-2 and MCL-1 inhibition was comparable or superior to steroid or TKI combined with each BH3-mimetic. Although this combination of BH3-mimetics was tolerable in vivo at lower doses, histologic evidence of haematopoietic toxicity and tumour lysis syndrome was observed in NSG mice and PDX models, respectively. The expression level of BCL-2 family anti-apoptotic genes (BCL-2 and MCL-1), BH3-only class of pro-apoptotic genes (NOXA and BAX), surface markers (CD38, CD179a, and CD34) and cell signalling pathways (pSTAT5), predicted treatment resistance. Co-targeting BCL-2 and MCL-1 warrants evaluation in clinical trials that incorporate supportive care including infection prophylaxis and tumour lysis precautions. The findings from this research may be exploited for future studies to test novel combinations of drugs, on the basis of their ability to act on non-overlapping mechanisms, which are likely to result in synergistic anti-leukaemia efficacy.
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ItemIdentifying Novel Regulators of Intrinsic Apoptosis( 2021)Abstract: Apoptosis is a conserved cellular process of programmed cell death. The intrinsic pathway of apoptosis is principally regulated by three functional and structural subgroups of the BCL-2 protein family: pro-survival proteins, pro-apoptotic effector proteins and apoptotic initiator BH3-only proteins. The interactions between these proteins on the mitochondrial outer membrane (MOM) determine whether a cell survives or dies. In healthy cells, pro-survival proteins inhibit the pro-apoptotic effector proteins BAK and BAX. In response to death stimuli, BH3-only proteins are activated to promote apoptosis either by activating BAK and BAX or neutralising pro-survival proteins. As well as BCL-2 family proteins, non-BCL-2 proteins such as VDAC2 are emerging as important regulators of apoptosis. However, the different impacts are observed in different contexts – with VDAC2 promoting apoptosis in some cases and restricting apoptosis in others. For BAK-dependent apoptosis, VDAC2 inhibits BAK function by forming complexes with BAK on the MOM. Therefore, loss of VDAC2 sensitises the cells to apoptotic stimuli in some contexts. This suggests that there may be proteins that accelerate BAK-dependent apoptosis in VDAC2-deficient cells. A previous genome-wide CRISPR-Cas9 library screen was performed in Bax-/-Vdac2-/-Mcl-1-/- MEFs to identify genes that when deleted inhibited the response to BH3-mimetics. In this screen, the absence of MCL1 enabled BH3-mimetic drug ABT-737 that inhibits BCL-2, BCL-XL and BCL-W to drive apoptosis in these cells, while the absence of BAX and VDAC2 ensured that apoptosis was mediated by BAK and controlled independently of VDAC2. From this screen, three proteins involved in ubiquitin signalling were identified as potential candidates: MARCHF5, UBQLN1 and USP24. In Chapter 3, I focused on validating each of these candidates in BAK-dependent apoptosis and identified that deletion of Marchf5 in Bax-/-Vdac2-/-Mcl-1-/- MEFs had the greatest impact on BAK-driven apoptosis. The effect of Marchf5/MARCHF5 on BAK function was further explored across different contexts using both Mcl1+/+ and Mcl1-/- MEFs and two human cell lines (HeLa and KMS-12-PE). Consistent with the initial validation results, deleting Marchf5/MARCHF5 in both murine and human cells provided long-term protection from BAK-driven apoptosis induced by BH3 mimetic drugs. Having successfully identified MARCHF5 as an important regulator of BAK-dependent apoptosis in Chapter 3, I further investigated how MARCHF5 modulated BAK apoptotic function in Chapter 4. My research indicated that BAK adopted an activated conformation and formed stable Mode 2 inhibitory complexes with MCL-1 and BCL-XL in MARCHF5-deficient cells, thereby providing protection from BH3 mimetic drugs. Given that MARCHF5 is an E3 ubiquitin ligase, I further identified that the drug resistance observed upon MARCHF5 deletion could be phenocopied by abrogating MARCHF5 enzymatic activity. Finally, in Chapter 5 I performed proteomics and functional CRISPR/Cas9 genetic screens to identify substrates of MARCHF5 that could account for the mechanism driving BAK into Mode 2 complexes in MARCHF5-deficient cells. Through these complementary approaches, several potential candidates were revealed, that may represent novel mediators of BAK apoptotic activity.
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ItemAdvancing a functional cure for HIV by identifying therapeutics that promote the death of latently infected cells( 2020)The persistence of a replication-competent HIV reservoir necessitates life-long antiretroviral adherence and precludes the possibility of a HIV cure via conventional therapy alone. Furthermore, recent clinical studies have made it increasingly clear that the predominant strategy for reservoir elimination, enforced transcriptional reactivation, does not diminish the size of the latent reservoir or reduce the time to viral rebound following treatment interruption. A novel approach seeks to purge the HIV reservoir by activating apoptotic pathways in latently infected cells and shifting the balance away from survival and towards cell death. Several lines of evidence implicate Bcl-2 family proteins in the long-term survival of memory CD4+ T cells – the major reservoir for HIV. Bcl-2 antagonism thus represents a viable strategy for sensitizing latent cells to death and delaying viral rebound. The development and clinical progression of BH3-mimetics, which induce apoptosis by binding pro-survival Bcl-2 homologs, has resulted in a well- characterised class of inhibitors with relatively few unknowns regarding toxicity, side effects and dosage. In this thesis, I hypothesise that there are apoptotic blocks in place, specifically a greater dependence on pro-survival Bcl-2 proteins, which prevent a minority of infected CD4+ T cells from dying during active infection. I hypothesise that latently infected cells are distinct from other infected or healthy cells, and that this pro-survival phenotype allows them to persist in such a way that renders them susceptible to pro- apoptotic therapeutics which target the intrinsic pathway, such as BH3-mimetics. In Chapter 3, I infect primary human CD4+ T cells with HIV in vitro to assess the ability of BH3-mimetics to kill actively infected cells. I demonstrate that ABT-737 and Venetoclax, but not the Mcl-1 inhibitor S63845, preferentially kill activated, HIV infected CD4+ T cells in the setting of productive viral replication. These results shed light on the pro-survival role of Bcl-2 proteins during active HIV infection, and inform our progression into a preclinical model of HIV latency. Chapter 4 uses a humanized mouse model of HIV latency to further interrogate the importance of Bcl-2 pro-survival proteins in reservoir survival. I investigate the ability of Venetoclax, a clinically-approved Bcl-2 antagonist, as well as S63845, a preclinical Mcl-1 inhibitor, to delay viral rebound following analytical treatment interruption. This work provides the first compelling evidence that BH3-mimetics, either as monotherapy or in combination, can eliminate latently infected cells in vivo. In Chapter 5 I perform a tat/rev Induced Limiting Dilution Assay (TILDA) on CD4+ T cells from latently infected mice in order to quantify the impact of Venetoclax on the magnitude of the latent HIV reservoir. I confirm the existence of an inducible reservoir in our mouse latency model, although I do not observe a significant effect of Venetoclax treatment as measured by TILDA. I also use single-cell RNA sequencing to characterize peripheral CD4+ T cells from ART-suppressed human donors following Venetoclax treatment ex vivo, arriving at the suggestion that Venetoclax may target CD4+ T cells that are enriched for a gene signature associated with activation and cell metabolism. This work lays the foundation for furthering our understanding of which cells may contribute to HIV persistence and which may be susceptible to death- inducing compounds. Overall, this thesis represents a comprehensive assessment of the ability of BH3-mimetics to kill HIV active and latently infected cells, offering a strong justification for the translation of pro-apoptotic therapeutics such as Venetoclax into a clinical setting where reservoir eradication is the goal.