Tasquinimod Treatment Research Articles

Tasquinimod enhances the sensitivity of ovarian cancer cells to cisplatin by regulating the Nur77-Bcl-2 apoptotic pathway.

Resistance to cisplatin (DDP) in ovarian cancer therapy has been a major clinical barrier. Drug-resistant cancers have been shown to downregulate the proapoptotic protein B-cell lymphoma-2 (Bcl-2) to inhibit apoptosis. Therefore, we explored whether tasquinimod could modulate resistance to DDP through apoptotic pathways. We aimed to explore the relationship between tasquinimod, Nur77-Bcl-2 apoptosis pathway and sensitivity of the ovarian carcinoma cell line SKOV3 and the DDP-resistant strain SKOV3/DDP cells to DDP. First, SKOV3 and SKOV3/DDP cells were treated with 2 μg/mL DDP or 40 μM tasquinimod. Western blot and quantitative real-time polymerase chain reaction (qPCR) were then used to analyze the expression of histone deacetylase 4 (HDAC4), Nur77, Bcl-2 (BH3 domain-specific), and caspase-3. Flow cytometry, scratch-wound assay and immunofluorescence were used to detect apoptosis, migration rate, and related expression of Nur77 and Bcl-2 (BH3 domain-specific). Subsequently, 5×107 SKOV3 or SKOV3/DDP cells cultured with 2 μg/mL DDP were injected into 4-week-old female BALB/c nude mice. Then, the mice were administered 4 mg/kg DDP and 50 mg/kg tasquinimod every 3 days. Finally, the changes in tumor diameter and weight were measured. After treatment of SKOV3 and SKOV3/DDP cells with tasquinimod, cell migration and HDAC4 expression levels were significantly reduced, while Nur77 expression was increased. Tasquinimod treatment enhanced the expression of Nur77 and caspase-3, and cells transfected with si-Nur77 showed the opposite result. Transfection of si-Nur77 reduced the expression of caspase-3 and Nur77 in the SKOV3/DDP cells that were treated with both DDP and tasquinimod. After injection of SKOV3/DDP cells into the mice, the tumor diameter, mass and in vivo HDAC4 level were significantly decreased by tasquinimod. Meanwhile, the levels of Nur77 and Bcl-2 (BH3 domain-specific) were increased. Tasquinimod upregulated the Nur77/Bcl-2 pathway to induce apoptosis in SKOV3/DDP cells and enhanced the anti-tumor effect of DDP in SKOV3/DDP xenografts. Therefore, tasquinimod can be expected to find clinical applications in enhancing DDP resistance.

Pharmacological and Genetic Inhibition of HDAC4 Alleviates Renal Injury and Fibrosis in Mice.

Histone deacetylase 4 (HDAC4) has been shown to be involved in cell proliferation, differentiation, and migration and is associated with a variety of cancers. However, the role of HDAC4 in renal fibrogenesis and its mechanisms are unclear. We assessed the role of HDAC4 and possible mechanisms of fibrosis in a murine model of kidney injury induced by unilateral ureteral obstruction (UUO) using tasquinimod, a highly selective HDAC4 inhibitor, and knockout mice with depletion of HDAC4 in renal tubular cells. UUO injury resulted in increased expression of HDAC4 and fibrotic proteins fibronectin and α-smooth muscle actin, while treatment with tasquinimod or knockout of HDAC4 significantly reduced their expression. Pharmacological and genetic inhibition of HDAC4 also decreased tubular epithelial cell arrest in the G2/M phase of the cell cycle, expression of transforming growth factor-β1 and phosphorylation of Smad3, signal transducer and activator of transcription 3, and extracellular signal-regulated kinase 1/2 in the injured kidney. Moreover, tasquinimod treatment or HDAC4 deletion inhibited UUO-induced renal tubular cell injury and apoptosis as indicated by reduced expression of neutrophil gelatinase–associated lipocalin, Bax, and inhibition of caspase-3. Finally, administration of tasquinimod or knockdown of HDAC4 prevented injury-related repression of Klotho, a renoprotective protein. Our results indicate that HDAC4 is critically involved in renal tubular injury and fibrosis and suggest that HDAC4 is a potential therapeutic target for treatment of chronic fibrotic kidney disease.

Tasquinimod efficacy and S100A9 expression in glucose-treated HREC cells.

Diabetic retinopathy (DR) is one of the leading causes of blindness in working-aged people. Few studies were on the relationship between S100 Calcium Binding Protein A9 (S100A9) protein and DR, and none on endothelial cells induced by tasquinimod in high glucose. Therefore, we assessed the relationship between tasquinimod and S100A9 in DR. DR pathogenesis was simulated using high-glucose-induced human retinal endothelial cells (HRECs) to study the mRNA expression of s100a9, thrombospondin-1 (tsp-1), hypoxia-inducible factor 1-alpha (hif1-α), intercellular adhesion molecule 1 (icam-1), and vascular endothelial growth factor (vegf) after tasquinimod treatment. The protein expression of S100A9, TSP-1, extracellular signal-regulated kinase (ERK), ICAM-1 and VEGF was also analyzed. A total of 28 eyes of 26 patients were included in this experiment. A significantly higher expression of S100A9 as well as enhanced proliferation and mobility was observed in the high-glucose-treated HRECs compared with that in low-glucose-treated cells. However, these were significantly inhibited when treated with high glucose with 50μM tasquinimod. The mRNA expression of tsp-1 was increased, whereas that of hif1-α, icam-1 and vegf was decreased after tasquinimod treatment. Western blot indicated the increased TSP-1 but decreased ERK, ICAM-1 and VEGF expression after treating with tasquinimod. High glucose promoted the expression of s100a9, S100A9 protein in DR patients and HRECs. Tasquinimod inhibited the proliferation, migration and lumen formation of HRECs under a high glucose environment. Tasquinimod might play a vital role in inhibiting angiogenesis through inducing TSP-1 and inhibiting VEGF, ICAM-1 and ERK.

HDAC4 Is Indispensable for Reduced Slow Myosin Expression at the Early Stage of Hindlimb Unloading in Rat Soleus Muscle.

It is well known that reduced contractile activity of the main postural soleus muscle during long-term bedrest, immobilization, hindlimb unloading, and space flight leads to increased expression of fast isoforms and decreased expression of the slow isoform of myosin heavy chain (MyHC). The signaling cascade such as HDAC4/MEF2-D pathway is well-known to take part in regulating MyHC I gene expression. Earlier, we found a significant increase of HDAC4 in myonuclei due to AMPK dephosphorylation during 24 h of hindlimb unloading via hindlimb suspension (HU) and it had a significant impact on the expression of MyHC isoforms in rat soleus causing a decrease in MyHC I(β) pre-mRNA and mRNA expression as well as MyHC IIa mRNA expression. We hypothesized that dephosphorylated HDAC4 translocates into the nuclei and can lead to a reduced expression of slow MyHC. To test this hypothesis, Wistar rats were treated with HDAC4 inhibitor (Tasquinimod) for 7 days before HU as well as during 24 h of HU. We discovered that Tasquinimod treatment prevented a decrease in pre-mRNA expression of MyHC I. Furthermore, 24 h of hindlimb suspension resulted in HDAC4 nuclear accumulation of rat soleus but Tasquinimod pretreatment prevented this accumulation. The results of the study indicate that HDAC4 after 24 h of HU had a significant impact on the precursor MyHC I mRNA expression in rat soleus.

Abstract 1100: Screening the chemical library against granulocytic MDSCs: Critical player in breast cancer metastasis

Abstract We recently demonstrated that infiltration of granulocytic MDSCs in distant organs is a critical step in metastatic process. The mouse transcriptome analysis of in vitro co-cultures and samples from syngeneic mouse model revealed that granulocytic subset of myeloid-derived suppressor cells (gMDSCs) from metastatic 4T1 tumor bearing mice are regulated a distinct set of genes. Two genes, S100A8 and S100A9 were highly upregulated in gMDSCs isolated from mice with metastatic 4T1 tumors compared to mice with non-invasive tumors. Using the recombinant single S100A8, S100A9 proteins or the S100A8/S100A9 heterotetramer (calprotectin), we demonstrated that calprotectin is a critical player in gMDSC induction and infiltration in distant organs particularly in lungs. Furthermore, injection of recombinant calprotectin but not single S100A8 or S100A9 proteins via tail vein enhanced the metastatic ability of nan-invasive EMT6 tumors in mice. We therefore performed computational screen of NCI compounds against the crystal structure of calprotectin and identified over 100 lead compounds. In vitro gMDSC differentiation assay identified 3 compounds with strong gMDSC inhibitory activity. We therefore designed a pilot experiment to test these compounds in preclinical mouse models. We also compared the activity of these compounds against tasquinimod, a small molecule targeting S100A9 only. However, tasquinimod treatment of 4T1 tumor-bearing mice had a moderate anti-tumor activity which may be due to a limited activity on granulocytic MDSC accumulation. In addition, we determined that there was a significant upregulation of S100A8 in MDSCs from tasquinimod treated mice. This data suggested that inhibiting only S100A9 leads to activation of S100A8 and thus may be ineffective targeting of MDSCs. In line with these data, one of the compounds (#10) showed a potent activity against gMDSCs in 4T1 tumor-bearing mice and significantly inhibited pulmonary metastasis. Moreover, this compound eliminated the cytotoxicity caused by cyclophosphamide and animals were free of cancer. In summary, we have identified a molecular target which regulates gMDSC induction and play critical role in metastatic process and our findings from in vitro screens and preclinical testing provide a strong rationale for targeting MDSCs. Citation Format: Hasan Korkaya, Eunmi Lee, Raziye Piranioglu, Thomas Albers, Maria Ouzounova, Charlie Weeks, Riley Rodier, Ahmet K. Korkaya, Khaled A. Hassan. Screening the chemical library against granulocytic MDSCs: Critical player in breast cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1100.

Abstract 2956: Chemical library screen identifies compounds that target S100A8/S100A9 complex and MDSC accumulation

Abstract We previously demonstrated that MDSC subsets accumulated in primary tumor and distant organs regulate tumor plasticity. The mouse transcriptome analysis of in vitro co-cultures and samples from syngeneic mouse model revealed that granulocytic subset of myeloid-derived suppressor cells (gMDSCs) from metastatic 4T1 tumor bearing mice regulate several hundred genes in tumor cells upon co-culture. The top genes are S100A8, S100A9, MMP8, FPR1, CCL3, and TGFβ2 which also predicted poor survival in human solid tumors including breast cancer. Therefore, we called these 6 genes as “metastatic gene signature”. It has been reported that S100A8/S100A9 heterotetramer, called calprotectin, play a key role in inflammation-associated cancer progression. To investigate the role of calprotectin (S100A8/A9) in tumor-mediated immunosuppression and metastasis, we first utilized Tasquinimod, a small molecule targeting S100A9 only. However, Tasquinimod treatment of 4T1 tumor-bearing mice had a moderate anti-tumor activity which may be due to a limited activity on granulocytic MDSC accumulation. In addition, we determined that there was a significant upregulation of S100A8 in MDSCs from Tasquinimod treated mice. This data suggested that inhibiting only S100A9 leads to activation of S100A8 and thus may be ineffective targeting of MDSCs. We therefore run a computational screen of the NCI chemical library against the crystal structure of calprotectin (S100A8/S100A9) and identified 40 lead compounds. We then performed in vitro screening assay to identify compounds that inhibit MDSC induction. We identified 3 compounds that significantly suppressed gMDSC differentiation. We are currently performing in vivo studies with these 3 candidate drugs in our murine breast cancer model and will present our findings at the AACR meeting. We believe that this study will provide a novel drug targeting S100A8/S100A9 heterotetramer, a key molecule in MDSC induction and its regulatory functions during tumor progression. Citation Format: Eunmi Lee, Maria Ouzounova, Raziye Piranlioglu, Abdeljabar El Andaloussi, Sena Arbag, Gang Zhou, Hasan Korkaya. Chemical library screen identifies compounds that target S100A8/S100A9 complex and MDSC accumulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2956. doi:10.1158/1538-7445.AM2017-2956

Erratum to: The anti-tumor effect of the quinoline-3-carboxamide tasquinimod: blockade of recruitment of CD11b+ Ly6Chi cells to tumor tissue reduces tumor growth.

Previous work has demonstrated immunomodulatory, anti-tumor, anti-metastatic and anti-angiogenic effects of the small molecule quinoline-3-carboxamide tasquinimod in pre-clinical cancer models. To better understand the anti-tumor effects of tasquinimod in transplantable tumor models, we have evaluated the impact of the compound both on recruitment of myeloid cells to tumor tissue and on tumor-induced myeloid cell expansion as these cells are known to promote tumor development. Mice bearing subcutaneous 4 T1 mammary carcinoma tumors were treated with tasquinimod in the drinking water. A BrdU-based flow cytometry assay was utilized to assess the impact of short-term tasquinimod treatment on myeloid cell recruitment to tumors. Additionally, long-term treatment was performed to study the anti-tumor effect of tasquinimod as well as its effects on splenic myeloid cells and their progenitors. Myeloid cell populations were also immune-depleted by in vivo antibody treatment. Short-term tasquinimod treatment did not influence the proliferation of splenic Ly6Chi and Ly6Ghi cells, but instead reduced the influx of Ly6Chi cells to the tumor. Treatment with tasquinimod for various periods of time after tumor inoculation revealed that the anti-tumor effect of this compound mainly operated during the first few days of tumor growth. Similar to tasquinimod treatment, antibody-mediated depletion of Ly6Chi cells within that same time frame, caused reduced tumor growth, thereby confirming a significant role for these cells in tumor development. Additionally, long-term tasquinimod treatment reduced the splenomegaly and expansion of splenic myeloid cells during a later phase of tumor development. In this phase, tasquinimod normalized the tumor-induced alterations in myeloerythroid progenitor cells in the spleen but had only limited impact on the same populations in the bone marrow. Our results indicate that tasquinimod treatment reduces tumor growth by operating early after tumor inoculation and that this effect is at least partially caused by reduced recruitment of Ly6Chi cells to tumor tissue. Long-term treatment also reduces the number of splenic myeloid cells and myeloerythroid progenitors, but these effects did not influence established rapidly growing tumors.

The anti-tumor effect of the quinoline-3-carboxamide tasquinimod: blockade of recruitment of CD11b(+) Ly6C(hi) cells to tumor tissue reduces tumor growth.

BackgroundPrevious work has demonstrated immunomodulatory, anti-tumor, anti-metastatic and anti-angiogenic effects of the small molecule quinoline-3-carboxamide tasquinimod in pre-clinical cancer models. To better understand the anti-tumor effects of tasquinimod in transplantable tumor models, we have evaluated the impact of the compound both on recruitment of myeloid cells to tumor tissue and on tumor-induced myeloid cell expansion as these cells are known to promote tumor development.MethodsMice bearing subcutaneous 4 T1 mammary carcinoma tumors were treated with tasquinimod in the drinking water. A BrdU-based flow cytometry assay was utilized to assess the impact of short-term tasquinimod treatment on myeloid cell recruitment to tumors. Additionally, long-term treatment was performed to study the anti-tumor effect of tasquinimod as well as its effects on splenic myeloid cells and their progenitors. Myeloid cell populations were also immune-depleted by in vivo antibody treatment.ResultsShort-term tasquinimod treatment did not influence the proliferation of splenic Ly6Chi and Ly6Ghi cells, but instead reduced the influx of Ly6Chi cells to the tumor. Treatment with tasquinimod for various periods of time after tumor inoculation revealed that the anti-tumor effect of this compound mainly operated during the first few days of tumor growth. Similar to tasquinimod treatment, antibody-mediated depletion of Ly6Chi cells within that same time frame, caused reduced tumor growth, thereby confirming a significant role for these cells in tumor development. Additionally, long-term tasquinimod treatment reduced the splenomegaly and expansion of splenic myeloid cells during a later phase of tumor development. In this phase, tasquinimod normalized the tumor-induced alterations in myeloerythroid progenitor cells in the spleen but had only limited impact on the same populations in the bone marrow.ConclusionsOur results indicate that tasquinimod treatment reduces tumor growth by operating early after tumor inoculation and that this effect is at least partially caused by reduced recruitment of Ly6Chi cells to tumor tissue. Long-term treatment also reduces the number of splenic myeloid cells and myeloerythroid progenitors, but these effects did not influence established rapidly growing tumors.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2481-0) contains supplementary material, which is available to authorized users.

Tasquinimod modulates tumor-infiltrating myeloid cells and improves the antitumor immune response to PD-L1 blockade in bladder cancer

ABSTRACTThe infiltration of myeloid cells helps tumors to overcome immune surveillance and imparts resistance to cancer immunotherapy. Thus, strategies to modulate the effects of these immune cells may offer a potential therapeutic benefit. We report here that tasquinimod, a novel immunotherapy which targets S100A9 signaling, reduces the immunosuppressive properties of myeloid cells in preclinical models of bladder cancer (BCa). As single anticancer agent, tasquinimod treatment was effective in preventing early stage tumor growth, but did not achieve a clear antitumor effect in advanced tumors. Investigations of this response revealed that tasquinimod induces an increase in the expression of a negative regulator of T cell activation, Programmed-death-ligand 1 (PD-L1). This markedly weakens its antitumor immunity, yet provokes an “inflamed” milieu rendering tumors more prone to T cell-mediated immune attack by PD-L1 blockade. Interestingly, the combination of tasquinimod with an Anti-PD-L1 antibody enhanced the antitumor immune response in bladder tumors. This combination synergistically modulated tumor-infiltrating myeloid cells, thereby strongly affecting proliferation and activation of effector T cells. Together, our data provide insight into the rational combination of therapies that activate both innate and adaptive immune system, such as the association of S100A9-targeting agents with immune checkpoints inhibitors, to improve the response to cancer immunotherapeutic agents in BCa.

Tasquinimod inhibits prostate cancer growth in bone through alterations in the bone microenvironment.

Tasquinimod (ABR-215050) is an orally active quinoline-3-carboxamide analog that inhibits occurrence of experimental metastasis and delays disease progression of castration resistant prostate cancer in humans. Its mechanism of action is not fully elucidated, but previous studies show immunomodulatory and anti-angiogenic effects. The aim of the present study was to investigate the tumor inhibiting effect of tasquinimod in bone of castrated mice as well as to elucidate its working mechanism related to bone microenvironment. Effects of tasquinimod on prostate cancer metastasis to bone was studied in an intratibial xenograft model. Animals were treated with tasquinimod and tumor establishment and growth, immunological status, as well as markers for bone remodeling were analyzed. Direct effects of tasquinimod on osteoblasts were studied in vitro. Establishment and growth of tumors in the bone after intratibial implantation in castrated mice was suppressed by tasquinimod treatment. The treatment effect was linked to decreased potential for immunosuppression in the pre-metastatic niche in bone (lower levels of CD206 and Arg1 expression in combination with increased iNOS expression) as well as in the tumor microenvironment (less Gr1 and CD206 staining). The shift to a pro-inflammatory, anti-tumorigenic milieu was also reflected in serum by increased levels of IFN-γ, CCL4, IL-5, LIX, IP-10, and MCP-1 as well as decreased TGF-β. Tasquinimod treatment also affected expression of factors involved in the pre-metastatic niche in the bone microenvironment (Lox, Cdh2, Cdh11, and Cxcl12). In addition, tasquinimod treatment caused a decreased osteogenic response indicated by decreased expression of Ocn, Runx2, and Col1a2 and increased expression of osteoclast stimulating CSF2. In vitro studies on mouse osteoblasts showed impaired osteoblast mineralization upon tasquinimod treatment. The present study shows that tasquinimod reduces establishment and progression of tumor growth in bone likely through a combination of effects on the pre-metastatic niche, homing, immunological status, and osteogenesis. It was concluded that tasquinimod interferes with the metastatic process, presumably by inhibition of tumor establishment. Hence, our data suggest that tasquinimod might be most effective in inhibiting the occurrence of new metastatic lesions.

Tasquinimod triggers an early change in the polarization of tumor associated macrophages in the tumor microenvironment.

BackgroundTasquinimod (a quinoline-3-carboxyamide) is a small molecule immunotherapy with demonstrated effects on the tumor microenvironment (TME) involving immunomodulation, anti-angiogenesis and inhibition of metastasis. A target molecule of tasquinimod is the inflammatory protein S100A9 which has been shown to affect the accumulation and function of suppressive myeloid cell subsets in tumors. Given the major impact of myeloid cells to the tumor microenvironment, manipulation of this cell compartment is a desirable goal in cancer therapeutics.MethodsTo understand the consequences of tasquinimod treatment on the TME, we evaluated early treatment effects in tumor infiltrating myeloid cells. Cellular phenotypes were studied by flow cytometry while gene expression both in tumor tissue and in isolated CD11b+ cells or tumor cells were measured by real time-PCR. Effects on angiogenesis were monitored by changes in CD31 levels and by gene expression in tumor tissue. Effects on cytokine levels in tumor tissue and serum were determined by multiplex analysis.ResultsThe MC38-C215 colon carcinoma tumors showed a substantial infiltration of primarily myeloid cells that were dominated by Ly6ClowF4/80+CD206+ M2-polarized tumor associated macrophages (TAMs), an immuno-suppressive and pro-angiogenic cell population. Here, we show that tasquinimod treatment induces an anti-tumor effect which is subsequent to a reduction in tumor infiltrating CD206+ M2 macrophages and a simultaneous increase in M1 macrophages expressing MHC class II and CD86. The tasquinimod-induced changes in TAM polarization were evident within 24 h of exposure, emphasizing the ability of tasquinimod to rapidly reprogram the tumor microenvironment. This change in the tumor associated myeloid compartment preceded an increased IL12-production within the tumor and a decrease in tumor neovascularization. The switch in TAM polarization by tasquinimod was confirmed in the 4T1 breast cancer model where tasquinimod also reduce lung metastasis development.ConclusionOur data show that tasquinimod affects tumor infiltrating myeloid cells early after exposure, leading to a change in phenotype from pro-angiogenic and immunosuppressive M2-like TAMs to pro-inflammatory M1-like macrophages. These changes are consistent with the effects of tasquinimod seen on tumor vascularization, immune suppression and metastasis giving further insights to the anti-tumor mechanism of action of tasquinimod.Electronic supplementary materialThe online version of this article (doi:10.1186/s40425-015-0098-5) contains supplementary material, which is available to authorized users.

Abstract 4120: Tasquinimod inhibits local invasion and metastases in two preclinical models of renal cell carcinoma

Abstract Renal clear cell carcinoma (RCC) is the most common type of kidney cancer in adults and represents 80% of cases. At the time of diagnosis, approximately 30% of patients have distant metastases. In addition, one third of patients who have surgical resection of the primary tumor eventually relapse and develop metastases. Effective agents that help to prevent the development of metastatic RCC are not available. Tasquinimod is a novel agent in clinical development for metastatic castration-resistant prostate cancer that has been shown to have immunomodulatory, anti-angiogenic, and anti-metastatic properties. In the current study, we tested whether tasquinimod effectively inhibits local invasion and spontaneous lung metastases in RCC. Two preclinical mouse models of RCC were used for this purpose: a xenograft nude model A498 and a recently established patient-derived xenograft model, RP-R-02 LM. In both models, tasquinimod had a modest effect on the growth of the primary tumors implanted subcutaneously. Interestingly, in the A498 RCC model, gene expression analysis in tumors revealed that tasquinimod increased E-cadherin and decreased vimentin expression, suggesting an effect on reversal of epithelial-mesenchymal transition. In addition, tasquinimod significantly decreased CXCR4 expression, suggesting a reduction in invasiveness and metastatic phenotype. To further test this hypothesis, we used the RP-R-02 LM model that is able to develop lung metastases. Tasquinimod induced a 66% reduction of lung metastases in comparison with the control group (p<0.01). Following tasquinimod treatment CD206+/Arginase 1+/high suppressive M2-polarized infiltrating macrophages were decreased in primary tumors, but even more so in the lung parenchyma and tumor nodules. In summary, our results demonstrate that tasquinimod has immunomodulatory and anti-metastatic properties in RCC models, providing a biological rationale for the clinical assessment of tasquinimod in RCC patients. Citation Format: Li Shen, Valerie Pierron, Anne-Laure Bauchet, Jessica Nakhle, Ashley Orillion, Remi Adelaiye, Eric Ciamporcero, Florence Meyer-Losic, Helena Eriksson, Nathalie Lassau, Ingrid Leguerney, Fabien Schmidlin, Anders Olsson, Roberto Pili. Tasquinimod inhibits local invasion and metastases in two preclinical models of renal cell carcinoma. [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 4120. doi:10.1158/1538-7445.AM2015-4120

Assessment of the bone scan index in a randomized placebo-controlled trial of tasquinimod in men with metastatic castration-resistant prostate cancer (mCRPC)

IntroductionDrug development and clinical decision making for patients with metastatic prostate cancer (PC) have been hindered by a lack of quantitative methods of assessing changes in bony disease burden that are associated with overall survival (OS). Bone scan index (BSI), a quantitative imaging biomarker of bone tumor burden, is prognostic in men with metastatic PC. We evaluated an automated method for BSI calculation for the association between BSI over time with clinical outcomes in a randomized double-blind trial of tasquinimod (TASQ) in men with metastatic castration-resistant PC (mCRPC). MethodsBone scans collected during central review from the TASQ trial were analyzed retrospectively using EXINIboneBSI, an automated software package for BSI calculation. Associations between BSI and other prognostic biomarkers, progression-free survival, OS, and treatment were evaluated over time. ResultsOf 201 men (57 TASQ and 28 placebo), 85 contributed scans at baseline and week 12 of sufficient quality. Baseline BSI correlated with prostate-specific antigen and alkaline phosphatase levels and was associated with OS in univariate (hazard ratio [HR] = 1.42, P = 0.013) and multivariate (HR = 1.64, P<0.001) analyses. BSI worsening at 12 weeks was prognostic for progression-free survival (HR = 2.14 per BSI doubling, P<0.001) and OS (HR = 1.58, P = 0.033) in multivariate analyses including baseline BSI and TASQ treatment. TASQ delayed BSI progression. ConclusionsBSI and BSI changes over time were independently associated with OS in men with mCRPC. A delay in objective radiographic bone scan progression with TASQ is suggested; prospective evaluation of BSI progression and response criteria in phase 3 trials of men with mCRPC is warranted.

Abstract 4746: Modulation of suppressive myeloid populations by tasquinimod.

Abstract Purpose: Suppressive myeloid cells such as myeloid derived suppressor cells (MDSCs) and tumor associated macrophages (TAMs) are a major barrier of cancer immunotherapy. These populations suppress immune responses, promote angiogenesis, and facilitate cancer progression and metastasis. Tasquinimod is an experimental anti-angiogenic agent tested in prostate cancer. In the current study, we investigated the novel immunomodulatory effects of tasquinimod. We focused on the modulation of myeloid populations by tasquinimod since tasquinimod binds to the inflammatory protein S100A9, which potentially activates MDSCs. Thus, we studied whether tasquinimod affects the induction/function of MDSCs and TAMs, and whether this agent can facilitate vaccine therapy in prostate and kidney cancer models. Experimental Design: The effects of tasquinimod on myeloid cells were tested in Myc-CaP and TRAMP transplantable prostate cancer models, and the RENCA orthotopic model of renal cell carcinoma. Splenocytes, blood cells, and cells from tumor samples were isolated from tumor bearing mice. The samples were subjected to staining with MDSC markers (Gr1 and CD11b) and macrophage markers (F4/80 and CD206), and to flow cytometry analysis. An immunotherapy combining tasquinimod with a modified survivin peptide vaccine, SurVaxM, was administered in Myc-CaP and TRAMP models. Tumor growth, MDSCs, and TAMs accumulation and their function after different treatments were monitored. Tasquinimod's effect on metastases is currently being tested in a LuCaP 23.1 orthotopic prostate cancer model. Results: Tasquinimod treatment reduced peripheral Gr1−CD11b+ monocytes, but did not affect the number of peripheral Gr1+CD11b+ MDSCs. However, tumor infiltrating Gr1+CD11b+ MDSCs significantly decreased following tasquinimod treatment (60% reduction, p = 0.03). In addition, Gr1−CD11b+ monocytes gained the macrophage marker, F4/80, in tumors, and tasquinimod reduced the M2 macrophages (immunosuppressive macrophages) in both peripheral sites and tumors (56% reduction, p = 0.04). Tasquinimod had a modest effect on the suppressive function of MDSCs. In the vaccine therapy, tasquinimod, combined with SurVaxM, improved antitumor immune responses and reduced tumor growth (48% inhibition, p = 0.03), as compared to vehicle and single treatments (no inhibition by SurVaxM, 15% inhibition by tasquinimod). Conclusions: Our study suggests that tasquinimod does not affect the expansion of Gr1+CD11b+ MDSCs induced by tumor, but interferes with the migration of MDSCs to tumor sites. Moreover, tasquinimod targets M2 macrophages, possibly by depleting the monocytic precursor populations such as Gr1−CD11b+ monocytes. By inhibiting MDSCs and TAMs, tasquinimod has an immunomodulatory potential to target cancer progression and metastasis, and to improve immunotherapies. Citation Format: Li Shen, Michael Ciesielski, Kiersten M. Miles, Robert Fenstermaker, Roberto Pili. Modulation of suppressive myeloid populations by tasquinimod. [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 4746. doi:10.1158/1538-7445.AM2013-4746

Abstract B17: Immunomodulation by tasquinimod: Combination with immunotherapy results in enhanced CD8 T cell responses and improved antitumor effects.

Abstract Purpose: The novel anti-tumor agent tasquinimod (ABR-215050) is being developed for treatment of metastatic castration-resistant prostate cancer. Proof of concept has been demonstrated in a Phase II trial by delaying disease progression and increasing overall survival, and enrollment of patients to a global pivotal Phase III trial (10TASQ10) is ongoing. Tasquinimod targets the tumor microenvironment resulting in anti-angiogenesis, inhibition of metastasis and immunomodulation. A target molecule of tasquinimod is the S100A9 protein which has been demonstrated to affect the accumulation and function of CD11b+Gr-1+ regulatory myeloid cell subsets. Given the major contribution of these cells to the immunosuppressive tumor environment, modulation of this cell population could contribute to the anti-tumor effects by tasquinimod and enhance the effects of cancer immunotherapy. In this study, we evaluated the immunomodulatory effects of tasquinimod when combined with tumor-targeted superantigens (TTS); an immunotherapeutic strategy which is highly dependent on activation of effector T cells directed against the tumor. Experimental design: The effect of tasquinimod treatment on CD11b+ myeloid cell populations was analyzed by flow cytometry in cell suspensions from spleens and from B16 tumors growing subcutaneously. In addition, B16-5T4 expressing tumors were treated with tasquinimod (30 mg/kg), the TTS fusion protein 5T4Fab-SEA (25 μg/kg), or the combination. Tumor growth and tumor-directed T cell responses were monitored. Results: The number of CD11b+ myeloid cells significantly increased in tumor bearing mice. Tasquinimod treatment inhibited tumor growth and reduced the CD11b+Ly6G-Ly6Chigh monocytic subpopulation of CD11b+Gr-1+ cells. In addition, less CD206-expressing tumor-associated macrophages were detected. Combining tasquinimod and TTS immunotherapy significantly enhanced the anti-tumor effects compared to the mono therapies. This was accompanied with increased numbers of TTS-activated CD8+ T cells in the tumors over a prolonged period of time. Conclusions: This study demonstrates that tasquinimod modulates the tumor infiltrating myeloid cell subsets and suppresses tumor growth. In line with an effect on the immunosuppressive tumor microenvironment, combining tasquinimod with the immunotherapy TTS gave rise to significantly improved anti-tumor effects. These data suggest that tasquinimod reduces tumor-induced immune suppression and that tasquinimod combined with immunotherapy could represent a novel and promising anti-cancer strategy. Citation Format: Anette Sundstedt, Anders Olsson, Martin Stenström, Mona Celander, Marie Törngren, Helena Eriksson, Gunnar Hedlund, David Liberg, Tomas Leanderson. Immunomodulation by tasquinimod: Combination with immunotherapy results in enhanced CD8 T cell responses and improved antitumor effects. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B17.

Abstract B91: Tasquinimod targets immuno-suppressive myeloid cell populations and enhances immunotherapy in murine prostate cancer models.

Abstract Purpose: A major component of the immunosuppressive environment responsible for immune tolerance in cancer is represented by myeloid cell populations such as myeloid derived suppressor cells (MDSCs) and tumor associated macrophages (TAMs). These cells facilitate carcinogenesis and tumor progression by impairing T and NK cells activation and contributing to modulate angiogenesis. Tasquinimod is an anti-angiogenic and immunomodulatory agent that has shown clinical benefit in patients with prostate cancer. This agent has been found to bind to the inflammatory protein S100A9, to block the interaction of S100A9 with myeloid cells surface receptors, and therefore has the potential to affect accumulation and function of these cells. Experiments and Objectives: We tested tasquinimod in combination with a vaccine therapy in two castration resistant, syngenic transplantable murine prostate cancer models: CR Myc-Cap and TRAMP-C2. This study had the following objectives: 1) to explore tasquinimod's novel immunomodulatory activities and underlining mechanism; 2)To investigate whether tasquinimod inhibits the induction and function of immunosuppressive myeloid cell populations; 3) To assess whether tasquinimod can facilitate vaccine therapy in the castrate resistant prostate cancer models. Results: We observed that the peripheral MDSCs (in blood and spleen) dramatically increased in tumor bearing mice as compared to naïve mice. The MDSCs number increase correlated with disease progression in the transgenic model. Tasquinimod treatment depleted Gr1-CD11b+, Ly6G-CD11b+ (monocytic MDSCs) myeloid cells and M2 macrophages in peripheral sites, and reduced Gr1+CD11b+ MDSCs infiltrates in the tumors. In addition, tasquinimod (10mg/kg) dramatically improved tumor-specific immune responses, including the granzyme B expression in CD8 cells and cytotoxicity of splenocytes, and enhanced anti-tumor activity of a survivin-mimetic peptide vaccine. Conclusions: 1. MDSCs accumulate in prostate cancer models suggesting that MDSCs may contribute to immune tolerance in prostate cancer. 2. Tasquinimod reduces immunosuppressive MDSCs and M2 macrophage and may facilitate vaccine therapy by enhancing cell-mediated cytotoxic tumor cell killing and the anti-tumor immune response. Citation Format: Li Shen, Kiersten Marie Miles, Michael Ciesielski, Leigh Ellis, Robert Fenstermaker, Roberto Pili. Tasquinimod targets immuno-suppressive myeloid cell populations and enhances immunotherapy in murine prostate cancer models. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B91.

Tasquinimod and survival in men with metastatic castration-resistant prostate cancer: Results of long-term follow-up of a randomized phase II placebo-controlled trial.

4550 Background: Tasquinimod (T) is an oral quinoline-3-carboxamide derivative that binds S100A9 protein and has preclinical anti-angiogenic and anti-tumor activity. Between 12/07-6/09, 201 (134 T, 67 Placebo (P)) men with metastatic CRPC were randomized and received treatment once-daily at an initial dose of 0.25 mg/day escalated to 1.0 mg/day over 4 weeks. Placebo patients could cross over to T after 6 months or at disease progression. The primary endpoint of improved PCWG2 criteria-defined progression at 6 months was met (69 vs. 37% of patients (T/P) were progression free) with PFS of 7.6 vs. 3.3 months for pts on T vs. P1 with acceptable toxicity. This abstract provides the first analysis on symptomatic progression, overall survival (OS) as well as a multivariate analysis for PFS and OS. Methods: Survival data were collected between June 2011 and January 2012 with a median time to censoring of 32 months. Survival data was also evaluated in an exploratory multivariate model of known prognostic factors in CRPC. Results: An imbalance of several baseline prognostic criteria favored placebo (e.g. baseline PSA of 29 vs. 19 (T/P)) (JCO 2011;20:4022). Time to symptomatic progression was longer in T treated patients (p=0.039, HR=0.42). Record of death (97 events) or survival &gt;13 months was documented in 182 patients. Median time to death was 34.2 vs. 30.2 months (T/P). Median time to death in the PCWG2 bone-metastatic subgroup (N=92/44) was 34.2 vs. 25.6 months. A multivariate analysis of known prognostic factors including PSA, LDH, PSA kinetics, and hemoglobin demonstrated an adjusted HR for PFS of 0.54 (95% CI 0.37,0.81) and OS of 0.72 (95% CI 0.46,1.12) in the total population and 0.63 (95% CI 0.37,1.07, n=136) in the bone-metastatic group. Conclusions: OS observed after tasquinimod treatment is longer than previously reported in this patient population. The current exploratory data indicates that the prolongation in PFS observed with tasquinimod treatment may lead to a survival advantage in men with metastatic CRPC. A phase III placebo-controlled study (NCT01234311) is ongoing in men with bone-metastatic CRPC powered to detect an OS improvement.

Inhibition of metastasis in a castration resistant prostate cancer model by the quinoline‐3‐carboxamide tasquinimod (ABR‐215050)

Tasquinimod (ABR-215050) is an orally active quinoline-3-carboxamide analog that has completed phase II clinical trial in patients with castration resistant prostate cancer, showing promising inhibiting effects on the occurrence of metastasis and delayed disease progression. Its mechanism of action is not fully elucidated, but previous studies show anti-angiogenic effects and strong interaction with the S100A9 protein. This study was performed to evaluate if tasquinimod inhibits prostate cancer metastasis, by using both orthotopic and intratibial xenograft models. Animals were treated with tasquinimod, and tumor growth characteristics as well as molecular markers for metastasis and angiogenesis were analyzed. The results show that formation of lung and lymph node metastases from orthotopic castration resistant prostate tumors was inhibited by tasquinimod treatment. Importantly, establishment of tumors in the bone after intratibial implantation was suppressed by tasquinimod. In addition, establishment and growth of subcutaneous tumors were affected. Both in primary tumors and serum from treated mice an upregulation of thrombospondin 1 was observed. Further, downregulation of the hypoxia driven genes VEGF, CXCR4, and LOX was detected in the primary tasquinimod-treated tumors and decreased expression of chemotactic ligand SDF-1 was demonstrated in the lungs. Thus, these molecular changes could contribute to the anti-angiogenic and anti-metastatic effects of tasquinimod. In conclusion, this study and clinical data show that tasquinimod interferes with the metastatic process, presumably by inhibition of tumor establishment. Therefore, tasquinimod is an interesting treatment option for patients with prostate cancer prone to metastasis.

Phase II study of tasquinimod in chemotherapy-naive patients with metastatic castrate-resistant prostate cancer (CRPC): Safety and efficacy analysis including subgroups.

126 Background: Tasquinimod (TASQ) is an oral quinoline-3-carboxamide derivative that binds to S100A9 and displays anti-angiogenic and anti-tumor activity in prostate cancer (PC) models. In a randomized blinded phase II study, 206 (136 TASQ, 70 placebo [P]) men with metastatic castrate resistant (CRPC) were assigned to TASQ/P once-daily at an initial dose level of 0.25 mg/day escalating to 1.0 mg/day over 4 weeks. The primary endpoint to demonstrate an improvement in PCWG2 criteria-defined progression at 6 months was met and presented at ASCO 2010. This abstract provides an update on safety and efficacy including CRPC subgroups. Methods: Subgroups of patients based on baseline criteria were investigated for safety using NCI CTC v 3.0 criteria, PK and efficacy. Results: 201 (134 TASQ, 67 P) pts with a median age of 72.6 years received treatment and were evaluable for efficacy and safety. The updated analysis based on 5 additional PFS events confirmed an improved PFS of 7.6 vs. 3.3 months for pts on TASQ vs. P. Most progression events in both arms were radiological, but more pts progressed on bone scan in the P group. Radiographic PFS was 8.8 vs 4.4 months. Significant PFS improvements were observed in the PCWG2 risk groups with bone metastatic and visceral disease. TASQ treatment led to a transient increase in inflammatory lab markers such as CRP and fibrinogen, as well as asymptomatic increases in amylase/lipase. CRP increase was associated with adverse events (AEs) such as muscle and joint pain, while increased amylase was associated with a lower risk for gastrointestinal AEs. TASQ treatment was associated with anaemia, but did not affect CV risk factors such as hypertension or QTc prolongation, and the rate of composite cardiac events was acceptably low. Clearance of TASQ is decreased with age (1.4 % per year) and patients over 80 often required dose reduction due to increased exposure and toxicities. Conclusions: TASQ improved PFS in men with metastatic CRPC. Side effects are manageable and seem to correlate with laboratory markers. Individualized dosing based on tolerability is recommended and a phase III placebo-controlled study is being initiated. [Table: see text]

The quinoline‐3‐carboxamide anti‐angiogenic agent, tasquinimod, enhances the anti‐prostate cancer efficacy of androgen ablation and taxotere without effecting serum PSA directly in human xenografts

Tasquinimod is a second-generation orally active quinoline-3-carboxamide analog with enhanced potency against prostate cancer via its anti-angiogenic activity. It is presently undergoing clinical trials. Androgen ablation and taxanes are standard therapies for metastatic prostate cancer. This raises the issue of whether combining tasquinimod with either of these approaches enhances therapeutic efficacy. The tumor growth of a series of human prostate cancer xenografts (CWR-22Rv1, CWR-22R-H, LAPC-4, LNCaP, PC-3 and DU-145) in male nude mice given nothing versus tasquinimod alone or in combination with androgen ablation or with androgen ablation plus taxotere were evaluated as model systems to resolve these issues. These studies documented that daily oral treatment with tasquinimod consistently, statistically (P < 0.05) inhibited the tumor growth of each of the xenografts in a dose-dependent manner via an anti-angiogenic response as monitored by a significant (P < 0.05) decrease in the tumor blood vessel density. Tasquinimod's anti-prostate cancer efficacy is enhanced when combined with androgen ablation and this enhancement was observed even when androgen ablation was either subsequent to or proceeded by tasquinimod treatment. In addition, tasquinimod also enhanced the tumor growth inhibition and survival when combined with androgen ablation plus taxotere. Companion studies documented that tasquinimod has no direct effect on serum PSA in these xenografts. These results documented that differences in serum PSA in tasquinimod-treated hosts are related to inhibition in tumor growth. This suggests that in clinical trials, changes in PSA slope or doubling time are indicative of therapeutic response to tasquinimod.