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Prostate cancer is the second most commonly diagnosed cancer in men with mortality rates, overtaking those for breast cancer in the last 2 years in the UK. Despite advances in prostate cancer treatments, over 25% of men do not survive over 5 years with advanced disease. Due to the success of immunotherapies in treating other cancers, this treatment modality has been investigated for Prostate cancer, however, the sole FDA approved immunotherapy so far (Provenge™) only extends life by a few months. Therefore, finding immunotherapeutic agents to treat prostate cancer is of major interest. Our group has previously shown that Interleukin-15 (IL-15), unlike other therapeutic cytokines such as IL-2 and IL-12, can stimulate expansion and activity of CD8 T cells and NK cells in vitro when they are exposed to prostate cancer cells, while studies in mice have shown a 50% reduction in tumor size with no apparent toxicity. In this study, we aim to examine potencies of IL-15 in combination with a cyclic dinucleotide (CDN) that activates the Stimulator of Interferon-Gene (STING) receptor. Selected CDNs (also known as STING agonists) have previously been shown to activate both T cells and dendritic cells through STING. We hypothesize that the combination of STING agonists and IL-15 can additively increase NK and T cell activity as they act to increase type I interferons (IFNs) through STING activation and IFN-γ through IL-15. In prostate cancer-lymphocyte co-cultures we now show that combination of IL-15 and the STING agonist ADU-S100 analog induces a marked killing of cancer cells above that seen with IL-15 or ADU-S100 alone. We show that this is related to a potent activation of NK cells resulting in increased perforin and CD69 expression, and up to a 13-fold increase in IFNγ secretion in the co-cultures. NK cells are responsible for killing of the cancer cells, as shown by a lack of cytotoxicity in NK depleted lymphocyte-tumor cell co-cultures, or in co-cultures of B and T cells with tumor cells. In summary, we propose that the combination of IL-15 and the sting agonist ADU-S100 analog may be potently effective in treatment of prostate cancer.

Background:WFDC1/Prostate stromal 20 (ps20) is a small secreted protein highly expressed within the prostate stroma. WFDC1/ps20 expression is frequently downregulated or lost in prostate cancer (PCa) and ps20 has demonstrated growth-suppressive functions in numerous tumour model systems, although the mechanisms of this phenomenon are not understood.Methods:Ps20 was cloned and overexpressed in DU145, PC3, LNCaP and WPMY-1 cells. Cellular growth, cell cycle and apoptosis were characterised. WPMY-1 stromal cells expressing ps20 were characterised by transcriptome microarray and the function of WPMY-1 conditioned media on growth of PCa cell lines was assessed.Results:Prostrate stromal 20 expression enhanced the proliferation of LNCaP cells, whereas stromal WPMY-1 cells were inhibited and underwent increased apoptosis. Prostrate stromal 20-expressing WPMY-1 cells secrete a potently proapoptotic conditioned media. Prostrate stromal 20 overexpression upregulates expression of cyclooxygenase-2 (COX-2) in LNCaP and WPMY-1 cells, and induces expression of a growth-suppressive phenotype, which inhibits proliferation of PCa cells by ps20-expressing WPMY-1 conditioned media. This growth suppression was subsequently shown to be dependent on COX-2 function.Conclusions:This work posits that expression of ps20 in the prostate stroma can regulate growth of epithelial and other tissues through the prostaglandin synthase pathway, and thereby restricts development and progression of neoplasms. This provides a rational for selective pressure against ps20 expression in tumour- associated stroma.

The KHP PCaBB was established in 2013 and recruits donors from the Urology or Oncology Departments at Guy’s Hospital in London (UK). Prostate cancer patients may be approached to give their consent for biobanking at any point in their treatment pathway, which allows residual material from their earlier diagnosis to be transferred and used by the Biobank. Currently, patients are specifically asked to donate samples of blood and surplus prostate tissue as well as permitting access to their clinical and pathological data that continues to be added throughout the course of their disease. Between 2013 and 2015, 549 prostate cancer patients gave their consent to the biobank and, the tissue repository collected 489 blood samples, 120 frozen prostate tissue samples and 1064 formalin fixed paraffin embedded diagnostic blocks. Prostate cancer has become a chronic disease in a large proportion of men, with many men receiving multiple subsequent treatments, and their treatment trajectory often spanning over decades. Therefore, this resource aims to provide an ideal research platform to explore potential variations in treatment response as well as disease markers in the different risk categories for prostate cancer. A recent audit of the KHP PCaBB revealed that between 2013 and 2015, 1796 patients were diagnosed with prostate cancer at King’s Health Partners (KHP), out of which 549 (30.6%) gave their consent to KHP PCaBB. Comparisons between demographic and clinical characteristics of patients who had consented compared to the total patient population revealed that the KHP PCaBB is demographically representative of the total prostate cancer patient population seen in Guy’s and St Thomas’ NHS Foundation Trust (GSTT). We observed no differences in distribution of ethnicity (p = 0.507) and socioeconomic status (p = 0.097). Some differences were observed in clinical characteristics, specifically with treatment type – which differed significantly between the patients who had given consent and total patient population. The KHP PCaBB has thereby amassed a rich data and tissue repository that is largely reflective of both the demographic and clinical diversity within the total prostate cancer patient population seen at KHP, making it an ideal platform for prostate cancer research.

Introduction & Objectives: Prostate cancer (PC) is the second most commonly diagnosed cancer in men and the eighth leading cause of cancer-related mortality in the world. The only successful immunotherapy for the disease (ProvengeTM) only extends life by a few months. Therefore the finding of immunotherapeutic agents for the treatment of PC is of major interest. It has been previously shown by our group that Interleukin-15 (IL-15), unlike other therapeutic cytokines such as IL-2 and IL-12, can stimulate expansion and activity of CD8 and NK cells in vitro when they are exposed to prostate cancer cells, while studies in mice have shown a reduction in tumor size by 50% with no apparent toxicity. In this study, we aim to examine potencies of IL-15 in combination with a cyclic dinucleotide (CDN) that activates the Stimulator of Interferon-Gene (STING) receptor. CDNs have previously been shown to activate both T cells and Dendritic cells through STING. We hypothesize that combination of CDNs and IL-15 can additively increase NK and T cell activity as they act to increase type 1 interferons through STING activation and IFN gamma through IL-15. Material & Methods: Peripheral blood mononuclear cells (PBMCs) were isolated and co-cultured with cancer cells (LNCaP, ATCC® CRL-1740™) in the presence of either IL-15, the CDN 2’3’-c-di-AM(PS)2, or a mixture of IL-15 with CDN. Control experiments were also prepared in which IL-15 was replaced by PBS and the CDN by a linear dinucleotide. Co-cultures were incubated for 48 hours at 37°C and 5% CO2. After this period, all cells from each well were collected and stained with fluorophore conjugated anti-CD3, -CD8 and -CD56 to identify CD8 T cells and NK cells, and anti-NKG2D and -perforin to analyze activation of NK and T lymphocytes. Tumor cell death was also evaluated and quantified using Live/Dead staining. All samples were acquired on FACS Canto. Results We observed that 1µg/ml of 2’3’-c-di-AM(PS)2 in combination with 2.5 ng/ml of IL-15 caused 81% of LNCaP killing in prostate cancer-lymphocyte co-cultures, while IL-15 by itself led to approximately 45% of cancer cell death. Little or no cancer cell killing was seen without IL-15 or CDN. Interestingly, the mixture of IL-15 with the CDN did not expand NK cells more than IL-15 alone. This suggests that the activity of NK cells is increased despite lack of their expansion. Preliminary data suggests that the level of perforin is increased by up to 35% in NK cells cultured with the mixture of both therapeutic drugs when compared to IL-15 alone in the cocultures. Of note, cell culture of either LNCaP or PMBCs with 1µg/ml of CDN did not affect cell growth. Conclusion: The combination of two immunotherapeutic drugs, IL-15 and 2’3’-c-di-AM(PS)2, lead to an increase in the activity of NK cells, with an apparent increase in the levels of perforin and doubling of cell death mediated by immune effector cells compared with IL-15 alone. Citation Format: Ana M. Esteves, Efthymia Papaevangelou, Prokar Dasgupta, Richard A.G. Smith, Christine Galustian. Combination of Interleukin-15 with a STING (Stimulator of Interferon Gene) agonist: A potential immunotherapy for prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4070.

BACKGROUND In this study, we investigated the effects of combining lenalidomide and docetaxel on in vitro and in vivo models of prostate cancer as a potential strategy for treatment of castrate resistant prostate cancer (CRPC). METHODS The effects of combining lenalidomide and docetaxel on proliferation, apoptosis, invasive potential, anchorage independent growth, and p53 activation in the PC3 and DU145 prostate cell lines were investigated. The effects of the lenalidomide and docetaxel combination on LNCaP prostate cancer cell growth and invasiveness in vitro was also studied. The combination of these two agents was finally tested on a xenograft model of PC3 tumor growth in nude mice. RESULTS Lenalidomide decreased the IC50 of docetaxel by up to 50% (P

Lenalidomide (REVLIMID®) CC-5013 (Celgene, NJ, USA) is approved, in both the USA and Europe, in combination with dexamethasone for the treatment of multiple myeloma patients who have received at least one prior therapy, and is rapidly being accepted worldwide for this condition. Lenalidomide is also approved in the USA and Canada for use in transfusion-dependent anemia in patients with low- and intermediate-1-risk myelodysplastic syndromes associated with del (5q) abnormality with or without additional abnormalities. Lenalidomide is an IMiD® immunomodulatory compound, incorporating structural modification of the drug thalidomide, which is active against a wide variety of autoimmune Th-2-dependent disorders, including erythema nodosum of leprosy, leishmaniasis, as well as severe ulcerative disorders such as Behcet’s syndrome. Unfortunately, long-term use of thalidomide is limited, particularly by neurotoxicity. To date, results suggest that lenalidomide is more active than thalidomide and does not cause the neurotoxicity seen with thalidomide. Lenalidomide has multiple properties, including anti-inflammatory, antiangiogenic and costimulatory effects, as well as being able to inhibit T-regulatory cells, all of which are properties deemed desirable for anticancer activity. This article covers the evidence that lenalidomide may have a major role in the treatment and control of many cancer types other than del (5q) myelodysplastic syndrome and multiple myeloma.

Recent years have seen improvements in strategies to treat pancreatic cancer. Pancreatic cancer is a leading cause of cancer-related death in the world, and is characterised by rapid disease progression, highly invasive tumour phenotype and resistance to chemotherapy. Patient prognosis is extremely grim, with a one-year survival rate of just 10% and only a 5% chance of surviving beyond five years. There has been little change in the treatment regimen, with 5-FU-based therapies being the usual route. Gemcitabine has also offered some relief over the past two decades, with modest improvements in median survival. This lack of choice has increased the call for new treatments, and indeed, novel drugs are now being investigated for their use in pancreatic cancer. These include those agents classically applied to other indications such as doxycycline and doxorubicin, as well as dietary components such as curcumin and genistein. Each of these drugs possesses different levels of activity both as single agents and in combinatorial approaches with gemcitabine. This review will discuss the difficulties in treating pancreatic cancer, and will summarise the progress and latest development in drugs used within this field of cancer.

T-regulatory cells suppress anti-tumour immunity in cancer patients and in murine tumour models. Furthermore, their activity is likely to have an effect on the effectiveness of immunotherapeutic treatments for cancer. Here we describe the current status of developing clinical strategies for modulating Treg activity in cancer patients.

Lenalidomide (Revlimid; CC-5013) and pomalidomide (CC-4047) are IMiDs proprietary drugs having immunomodulatory properties that have both shown activity in cancer clinical trials; lenalidomide is approved in the United States for a subset of MDS patients and for treatment of patients with multiple myeloma when used in combination with dexamethasone. These drugs exhibit a range of interesting clinical properties, including anti-angiogenic, anti-proliferative, and pro-erythropoietic activities although exact cellular target(s) remain unclear. Also, anti-inflammatory effects on LPS-stimulated monocytes (TNF-alpha is decreased) and costimulatory effects on anti-CD3 stimulated T cells, (enhanced T cell proliferation and proinflammatory cytokine production) are observed. These drugs also cause augmentation of NK-cell cytotoxic activity against tumour-cell targets. Having shown that pomalidomide confers T cell-dependent adjuvant-like protection in a preclinical whole tumour-cell vaccine-model, we now show that lenalidomide and pomalidomide strongly inhibit T-regulatory cell proliferation and suppressor-function. Both drugs inhibit IL-2-mediated generation of FOXP3 positive CTLA-4 positive CD25high CD4+ T regulatory cells from PBMCs by upto 50%. Furthermore, suppressor function of pre-treated T regulatory cells against autologous responder-cells is abolished or markedly inhibited without drug related cytotoxicity. Also, Balb/C mice exhibit 25% reduction of lymph-node T regulatory cells after pomalidomide treatment. Inhibition of T regulatory cell function was not due to changes in TGF-beta or IL-10 production but was associated with decreased T regulatory cell FOXP3 expression. In conclusion, our data provide one explanation for adjuvant properties of lenalidomide and pomalidomide and suggest that they may help overcome an important barrier to tumour-specific immunity in cancer patients.

Cells used in adoptive cell-transfer immunotherapies against cancer include dendritic cells (DCs), natural-killer cells, and CD8+ T-cells. These cells may have limited efficacy due to their lifespan, activity, and immunosuppressive effects of tumor cells. Therefore, increasing longevity and activity of these cells may boost their efficacy. Four cytokines that can extend immune effector-cell longevity are IL-2, IL-7, IL-21, and IL-15. This review will discuss current knowledge on effector-cell lifespans and the mechanisms by which IL-2, IL-7, IL-15, and IL-21 can extend effector-cell longevity. We will also discuss how lifespan and efficacy of these cells can be regulated to allow optimal clinical benefits.

The concept that cancer can be eliminated by the immune system has been put forward over 100 years ago [1]. At this time, it was already thought that immune effector cells can recognize cancer cells as non-self and can eliminate them in the same way as viral or microbial pathogens. Both the innate immune system and the adaptive immune system have a major role in the control of tumor cell growth. The innate immune system consists of nonantigen-specific cells including macrophages, dendritic cells, neutrophils, natural killer cells, gamma delta T cells, and complement. The adaptive immune system consists of cells such as antigen-specific cytotoxic and helper T cells and antibody-producing B cells which can obtain a memory phenotype against specific antigenic challenge. The result is the ability of the different immune cell types to recognize cancer cells as foreign [2]. Antigens produced by tumor cells are known to be recognized by T cells and B cells, and both tumor antigen-specific T cells and antibodies against tumor antigens can be detected in patients with cancers such as melanoma, ovarian cancer, colorectal carcinoma, and hepatocellular cell carcinoma [3, 4]. Tumor-related antigens fall into a number of types including unique patient or shared tumor-specific antigens, antigens which are in both tumors and normal tissues, and antigens derived from tumor-associated viruses. In prostate cancer, a number of antigens are expressed which can be used for prostate cancer diagnosis or monitoring (Table 5.1).

INTRODUCTION AND OBJECTIVES: Infiltration of cytotoxic T effector and NK cells in the tumour microenvironment has been a major goal for effective immunotherapy. The prostate cancer microenviroment is a challenge considering its poor immunogenicity. Although T and NK cells infiltrate these tumours, they are frequently anergic or suppressive. Secretion of TGF beta and IL-10 by prostate cancer cells is thought to influence the suppressive microenvironment. Cytokines that enhance effector T and NK cell infiltration without regulatory T cell expansion are therefore desirable. The aim of this study was to identify cytokines that can expand effector cells in prostate cancer-effector cell co-cultures. METHODS: Non adherent PBMCs were isolated and cultured for 7 days with irradiated PC3 cancer cells in a 8:1 ratio with cytokines IL-2, IFN gamma, IL-12, IL-15 and IL-21 used at ED50 doses. Expansion of effector cells was measured using anti-CD3, CD56, CD4, CD8, CD25 and FOXP3. Results were analyzed on a FACSCalibur flow cytometer. RESULTS: In the absence of tumour cells, IL-2, IL-15, IL-21 and IFN gamma had similar effects on expanding effector T and NK cells resulting in a population of 10 –11% NK cells, 20 –21% CD8 effector T cells, and 35– 40% CD4 CD25T cells ( % of gated lymphocytes). The cytokine IL-12 expanded NK cells (13%), CD8 T cells (24%) and CD4 CD25effector cells (43%) to a greater extent in the absence of tumour cells compared to other cytokines (p 0.05 by 1 way anova and Newman Keuls). Similar numbers of CD4 CD25 FOXP3 Tregs arose with all cytokines (25–30% of the CD4 CD25 cells). However, with tumour cells present, there were less NK cells (2–7%), CD8 T cells (6–9%) and CD4 CD25T cells (13–19%) after 7 days: IL21 or IFN gamma did not affect NK cell numbers compared to PBS control, (3% of the gated lymphocytes); IL-2 and IL-12 gave a significant increase (3–4%) but the greatest effect was with IL-15 (7%).CD8 cells were also increased significantly by IL-15 (9% compared to 6% with the other cytokines) and CD4 25cells were increased by IL-12 and IL-15 (19% and 18% respectively compared to 13% with the other cytokines). Treg expansion occurred with IL-2 (44% compared to 30% of CD4 CD25 FOXP3 T cells in the PBS control) but Tregs were decreased by IL-12(14%)and IFN gamma (17%). CONCLUSIONS: IL-15 and IL-12 are most effective in restoring effector cell numbers in the immunosuppressive prostate tumour environment, with reduced Tregs in the presence of IL-12 and increased CD8 T cells in the presence of IL-15. Combinations of these cytokines may therefore be the basis for new immunotherapeutic modalities in prostate cancer.

Background: Thalidomide and lenalidomide are FDA approved for the treatment of multiple myeloma and, along with pomalidomide, are being investigated in various other cancers. Although these agents display immunomodulatory, anti-angiogenic and anti-apoptotic effects, little is known about their primary mode of therapeutic action in patients with cancer. Methods: As part of a continuing research effort, we have investigated the effects of these agents on the metastatic capacity of murine colorectal cancer cell lines both in vivo and in vitro. Allied to these, we have studied their effects on the molecular pathways associated with metastasis. Results: Results indicate that thalidomide, lenalidomide and pomalidomide significantly inhibit the metastatic capability of colorectal carcinoma cells. Anchorage-independent growth, used as a coarse indicator of transformation, was significantly reduced, as were migratory capacity and invasive competence. In addition, an in vivo experimental metastasis model also showed that treatment with the drugs resulted in a significantly lower number of metastatic pulmonary nodules relative to control mice. Allied to these cellular and phenotypic changes were alterations in molecular markers of metastasis and in intracellular signalling competency. Conclusions: These results provide evidence that in addition to their immunomodulatory effects, thalidomide, lenalidomide and pomalidomide can impair the metastatic capacity of tumours, and that this mechanism may involve alterations to cell signalling functionality.

Introduction & Objectives: Prostate cancer progression can arise by tolerance to, and evasion of tumour cells from immune effector cells such as CD8 T cells and NK cells due to the immunosuppressive tumour microenvironment. Immunotherapeutic drugs can eliminate tumours by breaking this tolerance, and such drugs that can be localized to the prostate will have greater efficacy and less systemic toxicity than drugs administered systemically. It has also been shown that a localized stimulation of the immune system can boost immunity all over the body to clear distant metastases. However, in the highly immunosuppressive prostate cancer microenvironment, most activated effector immune cells are rendered anergic or even regulatory. We have shown that IL-15 in a localizable form retains its ability to activate NK and CD8 T cells in the presence and absence of prostate cancer cells. We have now used a similar approach to modify two antibodies (patent pending) which have been shown previously to be potent in inhibiting checkpoint blockade of the immune response in clinical trials. Material & Methods: The antibodies were modified to enable their conjugation to a cytotopic “tail” molecule. The “tail” consists of three regions - a thiol reactive region which interacts with free cysteines on the protein of interest, a cationic region, which binds to the negatively charged cell membrane surface, and a hydrophobic region that enters the cell membrane. The cytotopic antibodies can then adhere to cell membranes and can thus localize to any lesion where it is potentially injected. The tailed agents were then compared to non-tailed ones in in-vitro assays of T cell inhibition to measure their neutralizing ability and an in-vivo prostate tumour challenge model in C57/BL6 mice. Results: We have shown that the modified antibodies can attach to cell membranes through their cytotopic tails. The two cytotopically modified antibodies were both active in reconstituting T cell activation as shown by IL-2 secretion assays in Jurkats and EL4 by upto 50% (p Furthermore we have also demonstrated in vivo in a TRAMP-C2 tumour challenge model using C57/BL6 wild type mice model that a cocktail of tailed antibodies and cytotopically modified IL-15 can completely clear animals of tumour by day 73 whereas tumour size is only reduced by 50% with the non-tailed cocktail. Conclusion: We have identified that a cocktail of a modified cytokine, IL-15, together with two antibodies directed to immune checkpoint proteins can break tolerance to immunity in the prostate cancer microenvironment and cause disease regression. A tailed form of these molecules, has equivalent or greater activity in in vivo model and makes it therefore a promising new agent for immunotherapy of prostate cancer. Citation Format: Dorota Smolarek, Christina A. Sakellariou, Prokar Dasgupta, Richard A. G. Smith, Christine Galustian. Cytotopically modified antibodies to checkpoint proteins can actively reconstitute immune checkpoint blockade and inhibit tumor growth in a prostate cancer mouse model. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 268. doi:10.1158/1538-7445.AM2015-268

INTRODUCTION The immunosuppressive Prostate Cancer (PCa) microenvironment has a major influence on the progression of the disease and is a key challenge for immunotherapy considering that immune effector cells activated by such modalities, will be rendered anergic or regulatory once they enter this microenvironment. We have previously shown that IL-15 can activate NK and NKT cells to mediate cytotoxicity against PCa cells in lymphocyte-PCa cocultures and also that IL-15 can modulate NK receptors to shift the balance between activation and inhibition of NK cells. From initial data in PCa-lymphocyte cocultures, we also observed that IL-15 downregulated the inhibitory NK ligand HLA-BW4 on PCa cells, whereas the ligands MICA/MICB were not significantly affected. Due to the critical role of MICA and other activatory NK receptor ligands (aNKRL) such as Nectin-2 and ULBP1 on NK cell function we have now studied effects of IL-15 on the expression of aNKRLs MICA, Nectin-2 and ULBP1 both on the cell surface of PCa cells and in the supernatants of PCa-lymphocyte co-cultures. METHODS PC-3 and LNCaP PCa cell-lines were incubated with non-adherent PBMCs at effector:target ratios of 8:1 and cytokines IL-2 or IL-15. After 1 week, tumour cells were stained with antibodies to Nectin-2, MICA, and ULBP1 ligands. Flow cytometric analysis followed staining. MICA, Nectin-2 and ULBP1 ELISAs were performed with supernatants from these co-cultures of PCa cells and lymphocytes (and from PCa cells alone, treated with or without cytokines) to determine whether there was any effect of IL-15 on shedding of these ligands from the PCa cells. RESULTS We now show that MICA, Nectin-2 and ULBP1 ligands expressed on PC-3 cells were not significantly changed with IL-15 treatment in PCa-lymphocyte cocultures, however, on LNCaP cells, IL-15, (but not IL-2) downregulated MICA, Nectin-2 and ULBP1 ligands by upto 90%* (* = p In ELISAs measuring concentrations of MICA and Nectin-2 in the PCa-lymphocyte co-culture sups, IL-15, but not IL-2, upregulated concentrations of MICA by upto 145%* (* = p CONCLUSIONS In contrast to previous findings with IL-15 where aNKRLs (e.g. MICA, ULBP1) on certain cell types are upregulated, we see a downregulation of MICA, ULBP1 and Nectin-2 ligand expression on LNCaP cells with a concomitant increase in shed MICA. Although the current paradigm has been that shed MICA can inhibit NK cell function, other groups are now showing that the shed ligand may actually activate NKG2D on NK cells and our findings that IL-15 dramatically increases NK cell functions against PCa cells may confirm this alternative paradigm. Citation Format: Christina Alexandra Sakellariou, Oussama Elhage, Richard A. Smith, Prokar Dasgupta, Christine Galustian. IL-15 increases NK functions in the PCa-lymphocyte microenvironment by a profound increase in shedding of MICA from PCa cells - a novel paradigm. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2340. doi:10.1158/1538-7445.AM2015-2340

Introduction & Objectives: Prostate cancer progression can arise by tolerance to, and evasion of tumor cells from immune effector cells such as CD8 T cells and NK cells due to the immunosuppressive tumor microenvironment. Immunotherapeutic drugs can eliminate tumors by breaking this tolerance, and such drugs that can be localized to the prostate will have greater efficacy and less systemic toxicity than drugs administered systemically. It has also been shown that a localized stimulation of the immune system can boost immunity all over the body to clear distant metastases. However, in the highly immunosuppressive prostate cancer microenvironment, most activated effector immune cells are rendered anergic or even regulatory. We have previously shown that IL-15, unlike other therapeutic cytokines such as IL-2 and IL-12, can stimulate expansion and activity of CD8 and NK cells when they are exposed to prostate cancer cells. Therefore, we decided to create a localizable form of this cytokine to study its activity on NK and CD8 T cells in the presence and absence of prostate cancer cells. Material & Methods: The cytokine IL-15 was produced in a modified form to enable its conjugation to a cytotopic “tail” molecule. The “tail” consists of three regions - a thiol reactive region which interacts with free cysteines on the protein of interest, a cationic region, which binds to the negatively charged cell membrane surface, and a hydrophobic region that enters the cell membrane. The cytotopic IL-15 can therefore adhere to cell membranes and can thus localize to any lesion where it is potentially injected. The tailed IL-15 was then compared to non-tailed IL-15 and other cytokines in assays to measure T cell proliferation, and NK and CD8 T cell expansion in the presence and absence of prostate cancer cells. Results: Compared to non-tailed forms of IL-15, the tailed IL-15 induced greater proliferation of a murine T cell line, CTLL-2 and PBMCs . Proliferation levels were up to 20 times greater with the tailed IL-15. In initial experiments, activity of the tailed IL-15 was also greater or equivalent to non tailed IL-15 in its ability to expand NK cells in the presence of a prostate cancer cell line (TRAMP-C1). Conclusion: We have identified a cytokine, IL-15, that can break tolerance to immunity in the prostate cancer microenvironment. A tailed form of this molecule, has equivalent or greater activity in expanding immune effector cells in the presence or absence of prostate cancer cells and its potential for regioselective action makes it therefore a promising new agent for immunotherapy of prostate cancer. Citation Format: Dorota A. Smolarek, Christina A. Sakellariou, Oussama Elhage, Osamu Ukimura, Inderbir Gill, Angus G. Dalgleish, Prokar Dasgupta, Richard A. G. Smith, Christine Galustian. Cytotopic IL-15 as a novel therapeutic for prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2582. doi:10.1158/1538-7445.AM2014-2582

Background Gemcitabine resistance is a major challenge in pancreatic cancer where the drug is considered a “gold standard” treatment. Although many studies have attempted to address mechanisms of gemcitabine resistance, these are not yet clear. The complete elimination of these resistant cells will be a major breakthrough in treatment. In many cancers, curcumin is of interest due to its inherently low toxicity to patients even at high doses: The drug can inhibit pancreatic cancer cell growth, and also acts in synergy with gemcitabine reducing viability of several established cancer cell lines. Identifying curcumins efficacy after transition from gemcitabine sensitive to resistant disease is therefore a key question for progress toward treatment. We have created a novel cell line from a parental MIA-PaCa-2 pancreatic adenocarcinoma which is 286 times more resistant to gemcitabine exposure than the parent line. Here we investigated the cell cycling properties of this cell line and its ability to be killed by curcumin. Methods The gemcitabine resistant MIA-PaCa-2 cell line was created by continuous gemcitabine dosing which increased its EC50 for the drug to 286-fold greater than that of the parent line. The extent of resistance was confirmed by MTT for viability and BrdU ELISA for proliferation. The sensitive and resistant Mia-PaCa-2 cells were then incubated in the absence, or presence of varying doses of curcumin (0.01-150μM). Cells were analysed with respect to their cell cycling profiles by staining initially with EdU over a time course, followed by 7-AAD DNA staining of permeabilised cells with subsequent flow cytometric analysis. Early apoptotic, late apoptotic and necrotic fractions of these cells were determined by staining with annexin-V and propidium iodide. Results: Curcumin inhibited growth of both gemcitabine sensitive and resistant cell lines to a similar extent (IC50s =13.7 μM and 16.6 μM for sensitive and resistant lines respectively). Whereas gemcitabine was incapable of causing apoptosis in the resistant strain, curcumin induced similar levels of apoptosis in both lines at doses between 1 and 56μM. With respect to the cell cycle, gemcitabine caused complete S-phase arrest in the sensitive strain and had no effect on the resistant strain, whereas curcumin caused G2/M phase arrest (no increase in G1 and 100% loss of second cycle S-phase) in both cell lines. Conclusions Curcumin is effective as a chemotherapeutic drug against gemcitabine resistant cells due to its ability to inhibit the cell cycle at the G2/M boundary after gemcitabine fails to activate the restriction point. The ability of curcumin to avoid cross resistance after gemcitabine is rendered ineffective should be studied as this could provide mechanisms for the creation of new drugs or highlight the possibilities of curcumin analogues as a second-line therapeutic for the placation of pancreatic adenocarcinoma. Citation Format: Alan Levett, Rachel Allen, Christine Galustian, Angus George Dalgleish. Curcumin is effective in killing gemcitabine resistant pancreatic cancer cells: Studies of the cell cycle, and apoptosis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3426. doi:10.1158/1538-7445.AM2013-3426

Introduction The protein ps20 encoded by the WFDC1 gene, is a member of a protein family containing conserved whey-4-disulphide-core (WFDC) domains. Ps20 is upregulated in prostate cancer epithelium and expression is thought to correlate with disease progression. The aim of this study was to determine whether ps20 expressed by prostate cancer cells also increases docetaxel resistance. Methods The cell-lines LNCaP and PC3 express low endogenous ps20, but were transfected with empty vector (EV) or WFDC1 gene vectors that increase WFDC1 RNA and ps20 protein expression by over 100% as measured by qPCR and ELISA. Docetaxel was added from 0.001nM –1µM with or without ps20 antibody (1G7) and cell-killing was assessed by MTT after 48 hours. Results EV-transfected PC3 and LNCaP cells have docetaxel IC50s of 0.223+/-0.05 and 0.16+/- 0.09nM respectively. In WFDC1-transfected PC3 and LNCAP, IC50s are increased to 0.677+/-0.11 and 0.326+/-0.05nM respectively (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4233. doi:1538-7445.AM2012-4233

Introduction The protein ps20 encoded by the WFDC1 gene contains a highly conserved whey 4-disulphide core (WFDC) domain and is a member of a protein family that regulates inflammation. Ps20/WFDC1 is expressed by prostate cancer stromal cells but its expression by cancerous prostate epithelium is associated with tumour progression in patients. The aim of this study was to determine how WFDC1expression by prostate cancer cells alters their tumourigenic properties. Methods The cell-lines LNCAP and PC3 were transfected with WFDC1 vector or empty vector (EV) and their growth, adhesive and migratory characteristics were investigated by MTT proliferation, transwell migration and ICAM-1 adhesion assays. Results PC3 and LNCAP cells transfected with WFDC1 increased in numbers by 3148 +/−151% and 523+/−311% after 48 hours respectively compared to EV transfected cell lines (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2833. doi:1538-7445.AM2012-2833

The tissue factor-thrombin pathway is known to mediate proliferation, adhesion, invasion and metastasis of many types of cancer cells. These effects are additional to the thromboembolic processes commonly occurring in cancer, particularly in patients on hormonal therapies. In prostate cancer, there is an overexpression of the thrombin receptor PAR1 correlating with tumor progression. Anticoagulants such as heparin and hirudin are known to inhibit metastasis of tumors and improve survival rates in prostate cancer patients with localized disease. However, anticoagulants given systemically have an associated risk of bleeding which is higher in cancer patients and can be compounded by an increased risk of thrombocytopenia. Anticoagulants which can be localized to a tumor lesion are therefore highly favorable. The aim of the following study was to determine the efficacy of membrane-tethered anticoagulants on thrombin mediated migration and proliferation of prostate cancer cells in vitro. The hirudin cytotopic analogs PTL004 and PTL006, previously designed for use with anti-complement therapeutics in transplantation, were assessed as inhibitors of proliferation and migration of prostate cancer cells, in comparison to non-tailed hirudin-derived peptide, a PAR-1 antagonist and hirudin itself. Methods: For proliferation assays, 2000 PC3 prostate cancer cells were seeded in 96 well plates in serum free culture medium. Cells were preincubated for 30 mins with anticoagulant inhibitors at concentrations from 1 µM to 0.001 µM, and then 0.5 nM thrombin was added. Proliferation was measured after 48 hrs using MTT. To measure effects on cell migration, 10,000 PC3 cells were seeded onto porous membrane inserts in 24 well plates in serum free medium and then incubated with the inhibitors at 1 µM for 30 mins followed by 0.5 nM thrombin. Migration of cells through the membrane was measured after a 24 hr period. Results: Thrombin increased proliferation of PC3 cells in serum-free medium with a 1.5 fold increase in cell number after 48h. The tailed derivative PTL004 inhibited proliferation with an IC50 of 1 µM, equivalent to hirudin and the PAR1 antagonist. Liposomes prepared with PTL004 on the surface were more active than the tailed preparation in free solution with an IC50 10-50 fold lower. However, the non-tailed hirudin-derived peptide, and the derivative PTL006, which is known to have a more rapid dissociation rate when used with other cell types, were not active under these assay conditions. In the migration assays, PTL004 was also most active among the derivatives we tested. Conclusions: The membrane localizing anti-coagulant PTL004 has comparable anti-migratory and anti-proliferative properties to hirudin, and liposomes bearing this compound are even more effective at inhibition of proliferation of PC3 cells. The results demonstrate the potential of a cytotopic approach using tailed anticoagulants in localized prostate cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 673. doi:10.1158/1538-7445.AM2011-673

e13630 Background: Gemcitabine is the leading therapeutic for the treatment of pancreatic cancer, despite emergence of resistance. Understanding the mechanisms of gemcitabine resistance and discove...

e13155 Background: Docetaxel in combination with prednisone is approved for castrate-resistant metastatic prostate cancer (CRPC). However, docetaxel-based drug combinations may exhibit significant ...

1α,25-Dihydroxyvitamin D3, (1,25 D3) the biologically active form of vitamin D, is well established as an inhibitor of cancer cell growth in addition to its primary role in maintaining bone mineralization. In breast cancer cell lines, inhibitory effects on cell cycle arrest, angiogenesis, invasion and metastasis have been observed in addition to pro-apoptotic effects. 1,25 D3 also inhibits and prevents breast cancer growth in several mouse models, and a correlation between vitamin D receptor expression on breast cancer cells and disease free survival of breast cancer patients has also been observed. However, resistance to vitamin D and hypercalcaemia at higher doses are key limiting factors in clinical use. The drug Revlimid ® (lenalidomide) which has shown great promise in multiple myeloma, can also modulate signalling in apoptotic and cell growth pathways leading to inhibition of cell growth, inhibition of metastasis and invasion. Our study aimed to determine whether lenalidomide treatment of breast cancer cells resistant to vitamin D would result in an acquisition of sensitivity to vitamin D and a resultant inhibition of cell growth. The cell lines MCF-12A, MCF-7 and MDA-MB-231,representing non-tumorigenic, tumorigenic and metastatic breast lines respectively were used. The latter line was also vitamin D resistant. Cells were treated with lenalidomide and/or 1,25 D3 using a dose of 100 nM 1,25 D3 (a clinically tolerable dose giving IC50 inhibition of MCF-7 and MCF-12A cells). Results showed that whereas lenalidomide had no effect on the growth of the vitamin D sensitive lines, and gave only 20% inhibition of growth of MDA-MB-231 at 10 µM, a 50% inhibition of cell growth by 1,25 D3 (equivalent to that seen with the sensitive lines) was achieved in the presence of lenalidomide at a concentration of 1 µM. Further investigation revealed that the mechanism of this effect was an increase in apoptosis of the cell line, shown by an increase in parp cleavage and annexin V expression. An array measuring proteins associated with several signalling cascades showed that the combination of 1,25 D3 and lenalidomide resulted in an increase in proapoptotic proteins (phosphorylated P53, P21 and claspin, in addition to a decrease in BCL-2. Although both drugs had individual effects on pro-apototic proteins, and anti-apoptotic proteins, the combination resulted in an overall increase of pro-apoptotic protein expression leading to the observed inhibition of cell viability and growth. These results demonstrate the potential for the use of lenalidomide and 1,25 D3 in combination for tumours that are refractory to vitamin D. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5418.

Introduction: Prostate cancer accounts for approximately a third of all cancers in men. The FDA has approved the use of docetaxel in combination with prednisone for the treatment of Castrate Resistant Prostate Cancer (CRPC). However, it commonly elicits toxic reactions at effective doses. Thus combination of docetaxel with agents exhibiting non-overlapping toxicities is required. Combined, docetaxel and thalidomide are effective in treating patients with metastatic CRPC. Lenalidomide (Revlimid®), a thalidomide analogue, is FDA approved for multiple myeloma (MM) in combination with dexamethasone and in del 5q myelodysplastic Syndrome (MDS). Phase I study results suggest that docetaxel and lenalidomide actively combine in HRPC and a phase III trial is underway. Lenalidomide is known to exert direct tumoricidal effects only against subsets of hematological tumors. However, there are multiple effects on signalling pathways related to cell survival and proliferation which may also affect solid tumor biology. Aims: We have evaluated direct single agent lenalidomide activity as well as potential synergistic effects of combining docetaxel and lenalidomide using in vitro and in vivo models of prostate cancer. Methods: The effect of lenalidomide alone (up to 1 microM) or in combination with docetaxel (0.32-200nM) was assessed on the proliferation of EGF-stimulated prostate cancer cells. In vitro transwell invasion assays were also performed to assess the ability of lenalidomide to inhibit EGF-induced PC3 and DU145 cell invasion. The effect of combining docetaxel and lenalidomide in an in vivo model of tumor growth was studied by injecting 5×106 PC3 cells into the flanks of nude mice. Mice were treated with docetaxel (8mg/kg, bi-weekly) and lenalidomide (50mg/kg, daily) until tumors reached 1400mm3. Results: Single agent lenalidomide did not significantly inhibit EGF-induced PC3 proliferation; however at 1 microM, lenalidomide reduced the IC50 of docetaxel by an average of 42%. Lenalidomide displayed potent single agent activity by completely inhibiting EGF-induced PC3 and DU145 cell invasion (p Conclusions: These results demonstrate that lenalidomide alone displays direct effects against prostate cancer cells by inhibiting growth factor induced invasion. Lenalidomide enhances the anti-proliferative effect of docetaxel in vitro. Combinatorial activity was further confimed in a xenograft model showing that combination of lenalidomide and docetaxel produced a hyperadditive effect that slows tumor progression leading to enhanced median overall survival. These results support the use of lenalidomide in combination with docetaxel as an effective treatment for patients with CRPC. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5386.

Small cell lung cancer (SCLC) represents approximately 15% of all lung cancers, and is the most aggressive form of lung cancer. Left untreated, the time from diagnosis to death is 2–3 months. With current treatment, expected survival is 7–20 months, depending on the stage of disease. A new drug, amrubicin, is approved in Japan for lung cancer and has demonstrated efficacy in U.S. and European phase II trials of SCLC patients with either untreated disease or relapsed refractory illness. In a phase II study of amrubicin in previously untreated patients, response rates reached 75% with a median survival time of almost 1 year. Amrubicin is a fully synthetic 9-aminoanthracycline, and an analog of doxorubicin and epirubicin. The major mechanism of action of amrubicin is inhibition of topoisomerase II. Unlike doxorubicin, however, it exhibits little or no cardiotoxicity in clinical studies and preclinical models. In preclinical rodent tumor models, it is selectively distributed to tumour tissue and is not detected in the heart when compared with doxorubicin, which is distributed equivalently to these sites. The primary metabolite of amrubicin, amrubicinol, is up to 100 times more cytotoxic in vitro than the parent compound. This review describes the mechanisms of action of amrubicin as well as clinical studies which demonstrate the potential of this drug in future SCLC treatment. The review also puts forward hypothetical considerations for the use of other drugs such as lenalidomide, an immunomodulatory drug acting on multiple signalling pathways, or histone deacetylase inhibitors, in combination with amrubicin in SCLC.

The discovery of effective cancer treatments is a key goal for pharmaceutical companies. However, the current costs of bringing a cancer drug to the market in the USA is now estimated at $1 billion per FDA approved drug, with many months of research at the bench and costly clinical trials. A growing number of papers highlight the use of data mining tools to determine associations between drugs, genes or protein targets, and possible mechanism of actions or therapeutic efficacy which could be harnessed to provide information that can refine or direct new clinical cancer studies and lower costs. This report reviews the paper by R.J. Epstein, which illustrates the potential of text mining using Boolean parameters in cancer drug discovery, and other studies which use alternative data mining approaches to aid cancer research.

e14635 Background: Lenalidomide is an immunomodulatory and anti-angiogenic agent that has demonstrated activity against a range of hematological malignancies. Despite evidence of direct anti-proliferative activity against hematological cells in vitro, there is no evidence of single agent direct activity against solid tumor cells in vitro. To take advantage of its known immune-enhancing properties alongside direct anti-tumor agents, lenalidomide is being advanced in solid tumor indications in combination with other agents. There are few data regarding the combination of lenalidomide and standard of care chemotherapeutic agents, such as gemcitabine. Methods: Here, we assess the effects of lenalidomide alone, and in combination with gemcitabine, on pancreatic cancer cell growth and survival, and the ability of lenalidomide to enhance the ability of human PBMC to kill allogeneic pancreatic tumor cells (BxPC3, PANC-1 and MiaPaCa) in a PBMC:tumor cell co-culture model. Results: Lenalidomide alone had no effect on the proliferation of pancreatic cancer cells (BxPC-3 and Panc-1) whereas gemcitabine had moderate anti-proliferative activity. With combination therapy there was clear synergistic enhancement of anti-proliferative activity in both cell lines and additive effects were observed in a BxPC-3 xenograft mouse model of pancreatic cancer. About 20% of tumor cells were sensitive to immune-mediated cell death and, for BxPC3, this was increased significantly in the presence of lenalidomide. Lenalidomide significantly and dose-dependently enhanced immune-mediated killing (both T and NK cells are required for tumor cell killing in this model). For PANC-1 and MiaPaCa, immune-mediated killing was also increased by lenalidomide, albeit non-significantly. Conclusions: These results suggest that, in addition to anti-angiogenic and other effects within the tumor microenvironment, lenalidomide may act as an immune adjuvant to enhance the recognition and apoptosis of tumor cells by host T and NK cells. These studies support the potential utility of lenalidomide in combination with chemotherapeutic agents, gemcitabine in particular, in the treatment of patients with solid tumors including pancreatic cancer. [Table: see text]