Introduction: Treating breast cancer is based on a combination of therapies: surgery, radiotherapy, chemotherapy, as well as hormonal and biological agents. The full impact of the disease and its treatment at a human level is often underestimated, and the benefits of holistic cancer care are increasingly recognised. Furthermore, patients often face a frightening and uncertain journey that presents a variety of needs. Moreover, recovery is not necessarily the end-point of the cancer experience. The many complexities and challenges in the identification of patient issues along this journey can lead to unmet needs. This can be particularly difficult in the confines of a busy clinic, where time constraints, together with an over-reliance on verbal communication, can pose significant barriers to effective consultations. A novel tool, known as the patient concerns inventory (PCI), has been successfully developed and introduced for use in patients with head and neck cancer. In this setting, it has helped to formulate an individualized record of patient concerns, needs, and priorities, thereby structuring outpatient consultations, and promoting and facilitating a multidisciplinary approach. This study aimed to develop and assess a PCI specific to breast cancer and to evaluate its impact on patient care; that is, to provide a “proof of concept” for a breast cancer PCI. Methods: This was a four-phase study, as follows. (1) Item generation through a literature review, input from clinicians (n = 10), four patient focus groups (n = 24), and national breast cancer charities (n = 3). (2) A survey of breast cancer patients (n = 200) for cross-sectional validation, to compare the PCI with an established quality of life tool and to look at the relative frequency of items and any associations. (3) A pilot, before and after study, assessing the PCI in a clinical setting with breast cancer patients (n = 53). (4) Semi-structured interviews with a breast surgeon (n = 1) and specialist nurses (n = 2) who used the PCI during clinics, to identify the perceived benefits of using the PCI. Results: In total 277 patients responded and participated in this work. The literature review identified 164 items; following input from clinicians, focus groups, and national charities, 56 items remained. The cross sectional study (phase 2; n=200, 80 % response rate) revealed that patients wanted to discuss the following: breast sensitivity or pain (46 %), fatigue (46 %), hot flushes (44 %), sleep (34 %); breast appearance (30 %), unable to control weight (28 %), mastectomy appearance (19 %), overall physical appearance (17 %); fear of recurrence (62 %), fear of cancer spreading (39 %), fear about the future (32 %), or one or more of these (72 %); ‘mood’ (15 %), ‘anxiety’ (21 %), ‘depression’ (17 %), or one or more of these (35 %); Phase 3 found that the PCI resulted in a focused consultation and no increase in consultation time. All the patients from phase 3 wanted to see a breast surgeon. Phase 4 revealed that clinicians involved with the PCI supported its use, and stated several advantages. In its final format, the breast cancer specific PCI had 57 items over several domains, with 16 referral options. Conclusions: The PCI could identify issues that patients would like to discuss in the breast oncology clinic. The routine use of the PCI in follow-up clinics could ultimately improve care for women with breast cancer; however, the clinical environment continues to make it difficult to screen for issues related to intimacy, relationship, and sex. Further research is essential to evaluate the breast cancer specific PCI. A larger patient cohort, a longitudinal approach, qualitative input, and a link to possible interventions, would each improve our understanding of the issues faced by breast cancer patients.

The malignant cells of chronic lymphocytic leukemia (CLL) over-express PKCβII, a feature pathogenically important because the TCL1 mouse model of CLL fails to develop disease when PKCβ expression is disrupted. The purpose of this project was to generate transgenic mice in which PKCβII is over-expressed only in B cells. The central hypothesis of this thesis was that over-expression of PKCII in developing B cells will shift development to favour the generation of the B-1 and MZ B cell populations and eventually lead to the development of a CLL-like disease in the mouse. To construct the expression plasmid, an Eµ promoter was used to direct B cell-specific expression of PKCβII in transgenic mice (Eµ-PKCβII tg mice). PKCβII was tagged with haemagglutination antigen (HA) for identification in Western blots and immunohistochemistry (IHC). Also, mCherry was integrated within the expression plasmid construct in order to visualize transgenic B cells. Over-expressed PKCβII and mCherry fluorescence were detected when this plasmid was tested in A20 cells, a mouse B lymphoma cell line. The construct was then injected into pro-nuclei of fertilized ova isolated from pregnant mice. The injected ova were then transferred to recipient mice to generate potential founder mice. Sequential crossings of transgenic litters born from a single founder mouse led to the generation of homozygous Eµ-PKCβII tg mice. In the spleen of Eµ-PKCβII tg mice, the expression of the transgenic PKCIIHA was detected and quantification of PKCβII, using Western blotting, showed that PKCβII was over-expressed in Eµ-PKCβII tg mice compared with wild type mice. However, the mCherry could not be detected; this might be due to the fact that expression of the secondary gene(in this case mCherry) was not always efficient because of variable transcription from the IRES sequence. IHC analysis of the spleen of Eµ-PKCβII tg mice confirmed the expression of PKCIIHA in B cell-rich tissues where the staining for either HA or for PKCII showed the high expression of these proteins in the white pulp of the spleen specifically to the follicular region. There was no notable development of disease, CLL-like or otherwise in aged Eµ-PKCβII tg mice, but Flow cytometry analysis pointed out that the over-expression of PKCII resulted in a reduction in the proportion of follicular B cells and an increase in the proportion of MZ B cells in the spleen, and in B-1 cells in peritoneum and periphery of Eµ-PKCβII tg mice. This expansion of MZ B cells in the spleen was confirmed by H&E stains showing an enlarged marginal zone within the structure of the spleen, and IgM stains showed that this expanded MZ consists mainly of IgM+ cells. Thus, the generation of a mouse that over-expresses PKCII specifically within the B cell compartment led to an expansion in the populations of IgM+ B cells (MZ and B-1 B cells) and a reduction of follicular (mature) B cells. This mouse may be useful for the study of human CLL because of the potential to accelerate disease progression, and would therefore serve as a useful model for drug testing.

The unique situation where there is persistent clinical suspicion of prostate cancer (PCa) despite repeated benign histology from conventional prostate biopsy is a diagnostic dilemma for clinicians and patients. Transperineal template guided saturation biopsy (TTSB) has a high cancer yield in this group, but is associated with unacceptably high morbidity particularly high rates of acute urinary retention (AUR). As localised PCa rarely involves periurethral area at the base, we hypothesised that urethral trauma from extensive sampling of this area is a major trigger factor for AUR. This thesis presents data from modified periurethral sparing TTSB technique to determine whether the risk of AUR is reduced compared with rates reported in the literature, without compromising cancer yield and investigates biochemical predictors of pathological outcome and role of pre-biopsy MRI in this group. Three hundred and three men with persistent clinical suspicion of PCa despite a median of 2 (range 1-6) sets of negative biopsies were investigated. Patients prospectively completed a questionnaire evaluating bleeding, pain (visual analogue scale, range 0-10) and analgesic requirements which were assessed at 1 hour post biopsy and on days 1, 3 and 7. Serum PSA, PSAD and %fPSA were documented and evaluated for their ability to predict PCa diagnosis, Gleason score and cancer volume. Furthermore, a pre-biopsy magnetic resonance imaging (MRI) was performed and abnormalities in 4 anatomical quadrants were compared with TTSB to assess whether pre-biopsy MRI could defer need for TTSB or allow a more targeted biopsy regime. Median age was 64 years (range 43-85). PCa was diagnosed in 167 of 303 men (55.1%) from median of 29 cores [range 16-43, Gleason 6 (29.9%), 7 (45.5%) and 8-10 (24.6%)] and 140 (83.8%) were clinically significant with 77.2% of cancers involving the anterior region. AUR occurred in 23 men (7.6%). On multivariate analysis, only prostate volume predicted AUR (P=0.004). Pain was minimal (peak on day 1, mean 0.8 out of 10), requiring simple analgesia in 27% on day 1, mostly for mild perineal discomfort. Haematuria (75%) and rectal bleeding (20%) significantly decreased over one week, with 33% still experiencing haematospermia at this stage. PSAD (AUC 0.76) was more predictive of cancer diagnosis compared to 0.71 for %fPSA and pre-TTSB PSA respectively. The overall sensitivity and specificity of MRI for cancer diagnosis were 65% and 77% respectively with multiparametric MRI showing more accuracy with sensitivity and specificity of 83% and 95% respectively compared to other MRI sequences. Of the 24 false negative MRI cases, 16 (67%) were clinically significant. So complete prostate mapping should continue at present. In conclusion, this thesis demonstrates that clinical suspicion of PCa despite negative histology from conventional TRUS Biopsy should not be disregarded, as a significant proportion harbour aggressive disease. Modified TTSB is well tolerated with low risk of AUR. Presentations of aspects of this thesis at key regional, national and international urology meetings have generated interest and helped set up dedicated units in the region for PCa diagnosis in this group. Questions raised in this study provide backbone for future research in this field.

Following five decades of kidney transplantation, increasingly high risk immunological kidney transplantation (which previously was considered as sub-optimal) are carried out. The risk stratification with the current available assays have allowed safe transplantation in low risk non-sensitised patients and direct transplantation in high risk highly sensitised patients by removal of circulating donor specific antibodies (DSA) with reasonable outcomes. However, a large number of patients with chronic kidney disease and with low or intermediate antibody levels measured by current assay, the best way forward is uncertain resulting in denial of transplantation in some cases. Whilst in other cases, the solid phase Luminex assay may under or overestimate the risks of rejection and graft failure following direct kidney transplantation. Currently only IgG-class of DSA is considered immunologically important and routinely measured in clinical laboratories. Other bio-physiological characteristics such as class, sub-class and binding kinetics of DSA may be more specific for risk stratification of immunological risks. In this thesis, we studied effect of de novo IgM class of HLA-specific antibodies on outcome of kidney transplantation and characterised binding kinetics and strength of HLA-specific antibodies. De novo IgM or IgG HLA-specific responses alone were not associated with adverse outcomes following kidney transplantation. Presence of both IgM and IgG responses, however, was associated with poor graft function at 36 months. There was no temporal relationship of antibody response and episodes of rejections. De novo Donor specific responses were less frequent compared to non-specific responses. A shorter follow-up and use of modern triple immunosuppressant therapy (Tacrolimus, Mycophenolate and Steroid) may explain this. Binding kinetics measured by biosensor assay- surface plasmon resonance (SPR) on purified monoclonal HLA-specific antibodies showed binding kinetics and strength differed between HLA alleles despite same epitope and paratope interactions. There was a tendency towards higher affinity and faster association rate for HLA protein that was the initial immunizing antigen for the corresponding monoclonal HLA-specific antibodies. The dissociation constant (KD) of human monoclonal HLA-specific antibodies range between 10-8 to 10-10 M. Thermodynamic analysis showed higher Gibbs free energy released for interactions with higher binding strength. The binding strength of mixed monoclonal HLA-specific antibodies is generally average of the strength of individual monoclonal HLA-specific antibodies. Enriched polyclonal HLA-specific antibodies from clinical sample gave distinct binding response on bio-sensor based on SPR assay. Quantification of polyclonal HLA-specific antibodies using sandwich ELISA and SPR allowed quantitative measurement of binding kinetics and strengths. A range of binding strength was observed between patients and within same patient antibodies of different affinities was observed. Thus the antibodies could be grouped in four groups based on the strength of binding and this can serve as additional biomarker for risk stratifications.

Cutaneous fatty acid-binding protein (C-FABP) or FABP5, is a FABP family member that can bind to long chain fatty acids with high affinity. C-FABP was identified by our research group as a gene involved in malignant progression of prostate cancer and able to promote the growth of primary tumours and induce metastasis when transfected into rat benign Rama 37 model cells. It was demonstrated that C-FABP was a prognostic marker for patient outcome and a target of tumour-suppression for prostate cancer. As an initial step to understanding the complex molecular mechanisms involved in the cancer promoting activity of C-FABP, this study investigated the possible relationship between tumorigenicity-promoting activity of C-FABP and its fatty acid-binding capability. After single and double mutations were generated in the fatty acid binding motif of the C-FABP cDNA, wild type and mutant C-FABP cDNAs were excised from the mammalian vector pIRES2-EGFP and inserted into pBluescript cloning vector to generate a complimentary restriction sites at both ends of the cDNAs. The C-FABP cDNAs were excised from the pBluescript vector with KpnI and PstI and inserted into pQEs expression vector to form three constructs that express the wild type and two mutant C-FABPs (C-FABP-WT, C-FABP-R109A and C-FABP-R109/129A), respectively. SDS-PAGE and sequence analysis confirmed the correct insertion of C-FABPs into expression vector pQE. The pure recombinant proteins were subsequently produced and purified. The importance of fatty acid-binding activity to the cancer promoting function was assessed by comparing the cancer promoting abilities exerted by C-FABPs with different fatty acid-binding capabilities. To test whether the recombinant proteins produced were biological active, the fatty acid binding ability of wild type and mutant C-FABPs were tested. When fatty acid binding ability of the wild type C-FABP is set at 1, the average fatty acid binding ability the C-FABP-R109A and C-FABP-R109/129A were significantly reduced to 0.32% and 0.09%, respectively (Student t-test, P<0.005). These results suggested that fatty acid binding ability of C-FABP depends on the structured integrity of the binding motif. Thus, changing one amino acid in the motif significantly reduced the fatty acid binding ability by 68%, and changing two amino acids almost completely abolished the fatty acid binding ability of C-FABP. To access the importance of the structural integrity of the fatty acid binding motif to the tumorigenicity-promoting activity of C-FABP in prostate cancer, the effect of wild type and mutant C-FABPs on cell proliferation, invasion and colony formation as indication of tumourigencity were analysed. The average growth rate of cells stimulated with C-FABP-WT was significantly increased by 17% (Student t-test, P<0.05) when compared to control cells. Whereas, the average growth rate of cells stimulated with C-FABP-R109A and C-FABP-R109/129A were significantly reduced by 33% and 47%, respectively (Student t-test, P<0.005) when compared to control cells. The invasiveness of cells stimulated with C-FABP-WT, C-FABP-R109, C-FABP-R109/129A and the control cells were 256±40, 163±32, 80±26 and 96.6±15.2, respectively. The number of invaded cells stimulated with C-FABP-WT was the highest in all cell lines and more than 2.6-fold higher than the number of invaded cells from control (Student t-test, P<0.05). The average number of colonies produced in the soft agar by selected cells stimulated with C-FABP-WT, C-FABP-R109A, C-FABP-R109/129A and control were 1050±132.29, 283±76.38, 157±38.1 and 155±68.74, respectively. In comparison with the control, the average number of colonies produced by C-FABP-R109A stimulated cells was increased by 45% (Student t-test, P<0.01) whereas the average number of colonies produced by C-FABP-R109/129A stimulated cells was at same level as the control cells (Student t-test, P>0.5). The most significant change occurred in C-FABP-WT stimulated cells that produced more than a 6.7-fold (85%) increase in the number of colonies formed in soft agar when compared to controls (Student t-test, P<0.001). These results showed that the increased wild type C-FABP stimulation in prostate cell line significantly increased cell proliferation, invasiveness, and tumorigenicity. Whereas, the increased expression of both mutant forms of C-FABP did not significantly affect these characteristics. Overall, this study has confirmed that the biological potential of C-FABP to promote tumourigencity of prostate cancer cells depends on its ability of binding to fatty acids. Thus, C-FABP may facilitate cancer development through a mechanism involving transportation of intracellular fatty acids into cells. These results were supported by our recent data obtained from in-vivo studies performed in a nude mouse model.

Sustained, toxic elevations of pancreatic acinar cell cytosolic free calcium ion concentration ([Ca2+]C), such as those observed with supramaximal secretagogue stimulation (CCK) are implicated in acute pancreatitis. However, Cholecystokinin (CCK) has been thought to act only indirectly on human pancreatic acinar cells via vagal nerve stimulation, rather than by direct CCK receptor activation as observed in rodent pancreatic acinar cells. However, in the series of experiments presented here using human pancreatic acinar cells, CCK at physiological concentrations (1-20 pM) elicited rapid, robust, oscillatory rises of the cytosolic Ca2+ ion concentration ([Ca2+]C), showing apical to basal progression in acinar cells, in the presence of atropine and tetrodotoxin. The [Ca2+]C rises were followed by increases in mitochondrial ATP production and secretion, concluding that CCK acts directly on acinar cells in the human pancreas. The earliest pathological mechanisms, such as sustained, toxic elevations of the acinar cytosolic free calcium ion concentration ([Ca2+]C), incriminated in experimental pancreatitis have been previously demonstrated by non-oxidative metabolites of ethanol (FAEE’s), as well as bile salts, at supramaximal concentrations. However, in the clinical situation such hyperstimulation is unlikely to occur. To simulate a more clinically relevant stimulus, pancreatic acinar cells were stimulated with lower doses of FAEE’s and/or bile salts in combination with physiological doses of secretagogues - a process which may precipitate pancreatitis clinically. Illustrated here, the toxic transformation of secretagogue induced physiological Ca2+ signalling occurs with the perfusion of low doses of TLCS and POAEE resulting in cell injury. The intracellular second messengers implicated are IP3, cADPR and NAADP with the IP3 receptor channel pivotal with both toxins. However, as previously demonstrated with supramaximal concentrations of POAEE, if supplementary ATP is added to the intracellular milieu, cellular injury is avoided with continued extrusion of large quantities of Ca2+ from the cytosol indicating functional Ca2+ ATPase pumps. This is not observed in cells which do not receive supplementary ATP. The toxic sustained Ca2+ elevation is also be prevented by the removal of external Ca2+ or blockade of IP3 receptor using caffeine and cell injury is again avoided. Therefore, it may be concluded, that it is the large, sustained toxic [Ca2+]c load which impairs mitochondrial function and ATP production leading to Ca2+ATPase pump failure and ultimately cell death. Lowering sustained intracellular [Ca2+]c by blockade of IP3 receptor channels may reduce cell injury in clinical acute pancreatitis.

Chronic lymphocytic leukaemia (CLL) cells are derived from mature B lymphocytes and are distinctive with respect to overexpression of the classical protein kinase C isoform protein kinase CβII (PKCβII), which is encoded by PRKCB. Expression of PKCβII in CLL plays a vital role in the pathogenesis of the malignant cells in this disease, and within the microenvironment cells where it provides signals for the production of factors which support the survival of CLL cells. In CLL cells PRKCB transcription is stimulated by vascular endothelial growth factor (VEGF) through a mechanism involving activated PKCβII. However, at the beginning of this thesis the molecular regulatory mechanism(s) governing expression of the PKCβ gene were poorly described. Thus, to characterise the factors regulating PRKCB transcription in CLL cells I used different approaches including mithramycin treatment, a drug which intercalates into GC-rich areas of DNA to inhibit binding of specificity protein 1 (Sp1), specific Sp1 siRNA, promoter function assays and site directed mutagenesis and chromatin immunoprecipitation (ChIP). Experiments using these techniques showed that Sp1 has a direct role in driving expression of the gene coding for PKCβII in CLL cells. My results also show that Sp1 is highly associated with the PRKCB promoter in CLL cells compared to that in normal B cells, and suggest that this is likely because of the presence of histone marks permissive of gene activation. Examination of other transcription factors such as Sp3, MITF, RUNX1 and E2F1 that potentially bind the PRKCB promoter showed that they have static or indirect effects in regulating transcription of this gene. The exception to this is STAT3 which my data suggests plays a role in suppressing PKCβ gene expression in CLL cells. Exploration of the mechanism through which VEGF induces PRKCB transcription revealed that this growth factor stimulates increased association of Sp1 and decreased association of STAT3 with the PRKCB promoter. Thus, VEGF-stimulated activation of PKCβII may play a role in this process. Taken together, Sp1 is the major driver for overexpression of PKCβII in CLL cells, and because this transcription factor is also overexpressed in these cells, the mechanisms I describe controlling PRKCB transcription potentially provide a foundation for further study of other genes contributing to the phenotype of CLL cells that are regulated by this pleiotropic transcription factor.

HA22 is an immunotoxin composed of an anti-CD22 variable fragment linked to a 38 kDa truncated protein derived from Pseudomonas exotoxin A. The mechanisms of cytotoxicity and resistance of HA22 against Acute Lymphoblastic Leukaemia (ALL) and Burkitt’s lymphoma were studied. Using a bone marrow mesenchymal cell culture assay to support ALL cell viability, I? investigated the in vitro cytotoxicity of HA22 against ALL blasts from newly diagnosed and relapsed patients. There was interpatient variability in sensitivity to HA22. There was no significant difference in HA22 sensitivity between diagnosis and relapse samples but peripheral blood ALL blasts were more sensitive to HA22 than those from bone marrow. The mechanisms of resistance to HA22 were studied, using cell lines as a model. The number of CD22 sites/ cell and the rates of immunotoxin internalisation did not affect HA22 cytotoxicity. HA22 mutants with resistance to lysosomal degradation and enhanced targeting to the endoplasmic reticulum had improved cytotoxicity. The role of apoptosis pathways proteins in HA22-mediated cell death was studied. Their role is complex but raised levels of the anti-apoptotic pathway protein Bcl-2 were found in the most resistant NALM6 cell line. Penetration of HA22 into Burkitt’s lymphoma masses was studied using a flow cytometric based method. HA22 rapidly penetrated into the lymphoma masses, however a barrier to further uptake is present which could not be overcome by the addition of adriamycin or taxol in the murine xenograft model. The ability of Acute Myeloid Leukaemia (AML) blasts to create an immunosuppressive niche was investigated using a cell line model and primary patient samples. AML blasts suppress T cell proliferation through altered arginine metabolism, dependent on the enzymes arginase II and iNOS. Small molecule inhibitors to arginase and iNOS restored T cell proliferation in vitro. AML further enhances its immunosuppressive niche by transforming surrounding monocytes into an M2-immunosuppressive phenotype, in an arginase dependent manner. The immunomodulatory protein Serum Amyloid A (SAA) was secreted by AML blasts, and leads to AML chemotaxis, IL-1production, and release of S100A9 protein. Finally, invariant Natural Killer T cells (iNKT) were shown to be cytotoxic to some AML blasts, in the presence of Galactosylceramide, and thus able to restore T cell proliferation. The results provide a strong rationale for the clinical testing of these novel immunotherapeutic and immunomodulatory strategies in patients with haematological malignancies.

Breast cancer is a complex heterogenous disease that consists of several different subtypes displaying distinct behaviors and responses to different treatments. It is the second leading cause of cancer death among women, and is the most commonly diagnosed cancer in women. Although recent developments have helped shed light into this disease, there is still much to investigate. One particular subtype of breast cancer, known as triple-negative breast cancer, remains the most aggressive, as this tumor type is of high histological grade and preferentially affects women with BRCA1 mutations and women who are younger than 40 years of age. Unlike other subtypes with better prognoses, triple-negative breast cancer still has no targeted therapy, and chemotherapy remains the primary systemic treatment. Recently, there has been an increase of interest in the SOXC family of high mobility group transcription factors and their roles in tumor development. Studies have revealed some of the effects that SOXC genes may have on various tumor types. However, further studies are still needed to elucidate the roles, functions, regulations, and mechanisms of these transcription factors. This study aims to focus on one particular gene in the SOXC family known as sex determining region Y-box 11. Recent studies have shown that sex determining region Y-box 11, also known as SOX11, is one of the factors required for maintaining the basal-like breast cancer phenotype and is also critical in regulating growth, migration, invasion, and expression of signature basal-like breast cancer genes. Emerging evidence also reveals that this transcription factor may have an impact on homologous recombination repair when DNA damage occurs, in triple-negative breast cancer. Using SOX11 overexpression and knockout cell models combined with basic science laboratory techniques and omics, the next generation of laboratory tools, this study seeks to explore the role and function of SOX11 in DNA damage in triple-negative breast cancer. The results of this study have confirmed the recent findings of the role of SOX11 in cell proliferation and growth in triple-negative breast cancer. It has also revealed that overexpression of SOX11 in triple-negative breast cancer cell lines leads to an increase in DNA damage, loss of BRCA1 function, and dysregulation in the cell cycle. High expression of SOX11 is also associated with worse prognostic outcomes in triple-negative breast cancer patients. Because overexpression of SOX11 resulted in a loss of BRCA1 function, there may be a potential role for SOX11 in inducing the BRCAness phenotype commonly seen in basal-like breast cancers. The results of this study strongly suggest that SOX11 is involved in defective DNA damage repair pathways. Further studies need to be conducted in order to evaluate SOX11 as an inducer of the BRCAness phenotype, which occurs when there is a homologous recombination repair defect and no germline BRCA1 mutation present. Because of this, SOX11 may also have the potential to act as a functional biomarker for therapies targeting DNA damage, as recent developments in identifying therapies that could potentially target homologous recombination repair defects have been promising.

Tuberous sclerosis complex (TSC) is a multisystem genetic disease that is caused by a germ line mutation in the genes TSC1 and TSC2. Patients with the disease tend to suffer from benign tumors of the brain, heart, kidneys, skin, and other organs that contain giant cells. Although mTORC1 inhibitors (rapamycin and rapalogs) are often used to treat TSC because of their efficacy in promoting tumor shrinkage, clinical studies in the past have shown that when treatment is taken away, the tumor size returns to its original state. The objective of this study was to identify compounds that selectively inhibit proliferation of TSC2-deficient cells. A high-throughput screen of about 4000 compounds was performed using a lysosomal inhibitor (chloroquine [CQ], 5 μM) and a “repurposing” library of compounds. Through some yet to be determined mechanism, the combination of ritanserin (a selective serotonin reuptake inhibitor [SSRI]) and chloroquine was found to synergize to selectively inhibit the cell viability of DJK MEFs and TSC2-/-KO cells (TFFs) starting at 48 hours after treatment. The effects of this combination treatment were confirmed in a second cell line (TFFs) exhibiting similar reduction in proliferation. Interestingly, treatment with CQ (5 μM) and ritanserin (20 μM) showed synergistic action (combination index [CI] = 0.6) against TSC2-deficient cells. This combination treatment induced apoptosis (41%) in TSC2-deficient cells but not in TSC2-expressing cells. These results suggest a novel treatment approach in tuberous sclerosis complex and provide an incentive for further investigation of the mechanisms contributing to the vulnerability of TSC2-deficient cells. Moreover, the use of an already Food and Drug Administration (FDA)-approved compound can lead to a more rapid pharmacologic approach for TSC patients.