Grant Funds Research on Immunology in Chondrosarcoma

An ESUN Announcement

The Liddy Shriver Sarcoma Initiative has awarded a $108,640 grant to fund promising chondrosarcoma research at the Université Claude Bernard in Lyon, France.

Chondrosarcomas are rare cancers that are difficult to treat. They don't respond to radiation therapy and cannot be cured with available systemic treatments. It is clear that novel treatment approaches are needed for chondrosarcoma patients, but these tumors are rarely studied in the lab.

With this grant, Aurélie Dutor, PhD and Jean-Yves Blay, MD, PhD will perform the first known study of chondrosarcoma's interaction with its immune environment. They will attempt to identify the molecular mechanisms of tumor progression and to identify targets within chondrosarcoma for immunotherapy.

Why the Immune System?

The investigators were working on a different project when they began to ask questions that led to this study. Dr. Dutor explains: "A previous study, in which we demonstrated the efficacy of Everolimus in slowing down chondrosarcoma progression, generated questions regarding the involvement of the immune environment in chondrosarcoma progression and response to therapy. To our knowledge, such a question has never been addressed."

The Challenge and the Hope

According to Dr. Blay, the rarity of chondrosarcoma makes exploring and describing its interactions within human tissues very difficult. He says, "Describing chondrosarcoma's immune microenvironment, especially in human samples, is challenging given the rarity of this tumor."

Dr. Dutour explains that the investigators have come up with a way to overcome this challenge: "Our group has developed a chondrosarcoma model established in rats that mimics its human counterpart."

The investigators hope that their research will increase understanding of chondrosarcoma's biology and of its interaction with its environment, leading to further research and better treatments. Dr. Blay shares his encouraging perspective: "Studies performed by sarcoma research teams could bring new treatments to patients, as these research teams are dealing specifically with these tumors and gaining better insights into their biology."

Funding for this Study

The Liddy Shriver Sarcoma Initiative is indebted to the many friends and family who made generous donations in memory of Kenny Allen, Paul Hagan, Cheryl Mauldin, Frank Manganaro and Peter Cappuccio, all of whom lost their lives to chondrosarcoma, and to the friends and family of Gavin Kiener, a chondrosarcoma survivor who currently has no evidence of disease.

Investigation of Immune System Implication in Chondrosarcoma's Response to Targeted Therapy

By Aurélie Dutor, PhD and Jean-Yves Blay, MD, PhD

Background

Chondrosarcomas constitute a group of slow growing and heterogeneous tumors accounting for 20% of bone malignancies.¹ Despite distinct genetic, pathologic features, all these tumors are resistant to chemotherapy and radiations.¹ The treatment of choice in primary, recurrent or metastatic disease remains wide local excision when feasible. Thus the 10-year survival rate of chondrosarcoma has remained unchanged over the past 40 years and ranges from 29-83% depending on chondrosarcoma subtype and grade.^2-4 Improving this tumor's clinical management is challenging and novel therapeutic approaches are needed. Because different molecular pathways are altered in chondrosarcoma cell lines,² targeted therapy may represent an interesting option for chondrosarcoma treatment. Among them is the PI3K/Akt/mTOR signaling cascade as mutations or aberrant activation of its effectors are frequently detected in sarcoma.⁴

The PI3K/AKT/MTOR Pathway

The PI3K/AKT/MTOR pathway (Figure 1) is an intracellular signaling pathway is crucial and intensively explored in tumorigenesis. Indeed, mutations and aberrant activations of this pathway effectors are frequently encountered in malignancies including sarcoma and have been linked to cancer development, resistance mechanisms (chemoresistance as well as radiation resistance). In cancers, PI3K/mTOR promotes cell survival and proliferation, apoptotic resistance and increasing data also link this pathway to tumor invasion and metastasis. Effectors of this pathway are activated by phosphorylation: PI3K activation activates AKT which activates mTOR. mTOR is located downstream of this signaling cascade and integrates the input from upstream pathways, including insulin, growth factors; mTOR also senses cellular nutrient, oxygen, and energy levels.

The emergence of agents directed against mTOR represents a major breakthrough in the field of targeted therapies. First-generation mTOR inhibitors, i.e. rapalogs, have shown anticancer activities in preclinical trials and are now used in clinical trials.^5-7 Two recent sets of data suggest that mTOR pathway inhibition could be an effective treatment for chondrosarcoma. The first one was generated by the group of Merimsky who showed in a case report then in a clinical trial that the association of a rapalog with cyclophosphamide is efficient in controlling chondrosarcoma progression.^8,9 The second one generated by our group demonstrated in a rat chondrosarcoma model that the rapalog RAD001 significantly inhibited tumor progression and delayed tumor recurrence.¹⁰ Surprisingly, these in vivo antitumor effects could not be observed in vitro suggesting that tumor microenvironment plays a key role in chondrosarcoma response to rapalog, as it has been reported for other therapeutic agents.¹¹ Our hypothesis is that the immune microenvironment of the tumor may be involved in this response. Indeed, rapalogs are known to: i) induce the mobilization of CD8 effectors T cells; ii) increase the proliferation and differentiation of CD8 memory T cells; iii) increase the secretion of cytokines.^12,13 Therefore, such an immunostimulatory action could have contributed to the significant antitumor effects observed in our study.

The above described results and data prompted us to further explore the role played by the immune system in chondrosarcoma progression and response to PI3K/Akt/mTOR pathway targeting agents. We will therefore assess whether a targeted therapy could generate an immune response that reinforces its direct therapeutic efficacy. We will also investigate the role of the immune checkpoint PD1/PDL1, involved in immunosurveillance escape mechanisms, in chondrosarcoma and study whether: i) its blockade could inhibit tumor progression and affect chondrosarcoma response to a therapy; ii) combining PDL1 blockade with a targeted therapy inhibiting the PI3K/Akt/mTOR pathway would have an antitumor activity. These questions will be addressed at 3 levels:

Aim 1: Describe chondrosarcoma immunological niche and analyze the effect of PI3K/Akt/mTOR therapy on chondrosarcoma immunological niche.

Aim 2: Evaluate the impact of mTOR pathway inhibition on the immune response both on immune effectors cells (Aim 2.a) and on systemic and tumor cytokines secretion profile (Aim 2.b).

Aim 3: Analyze the involvement of PD1/PDL1 in chondrosarcoma progression and response to targeted therapy and define whether this immune checkpoint could be a target for immunotherapy approaches.

Immune Checkpoints

Regulation and activation of T lymphocytes depend on signaling by the T cell receptor (TCR), and also by cosignaling receptors that deliver negative or positive signals. The amplitude and quality of the immune response of T cells is controlled by an equilibrium between these positive and negative signals, called immune checkpoints. Under normal physiological conditions, immune checkpoints are crucial to protect tissues from damage during pathogenic infection.

One of the mechanisms by which cancer cells escape immunosurveillance is by hijacking immune checkpoint pathways that regulate T-cell responses. Because many of the immune checkpoints are initiated by ligand–receptor interactions, they can be r blocked by antibodies. Thus, significant research efforts have focused on the development of antibodies targeting these proteins. The checkpoint protein that has garnered the most attention is cytotoxic T-lymphocyte antigen-4 (CTLA-4). But a number of other checkpoint proteins are also being examined. One of these is The programmed-death 1 (PD-1) receptor and its ligands PD-L1 and PD-L2 which are attracting a lot of research in the past few years

Model and Treatment

In this project, the evaluation of the immune system's and immunocheckpoints implication in chondrosarcoma response to targeted therapys will be assessed in the Swarm rat chondrosarcoma model (Figure 2). Animal care and procedures will be performed according to institutional and national guidelines by habilitated investigators in structures approved for housing and small animal experimentations. Animals will be euthanized if tumors become too bulky (> 2cm in diameter) of if any signs of distress are observed.

Treatments: mTOR inhibitor (RAD001) ; ii) PI3K inhibitor (GDC-941); iii) Akt inhibitor (G-0584) will be used. The different treatments will be applied at doses that have demonstrated their antitumor efficiency in previous studies. For in vivo assays, treatments will be administered twice a week by IP injection for a duration of three weeks. At the end of the treatment period, tumors, blood, splenocytes will be collected, and stored for analyses.

Aim 1: Analysis of chondrosarcoma immunological niche.

Chondrosarcoma immunological microenvironment remaining largely unknown, we will first characterize the immunological niche of this tumor and its potential involvement in chondrosarcoma progression. The description of chondrosarcoma immunological environment will be done by immunohistochemistry on rat chondrosarcoma samples (Figure 3). The following immune populations will be analysed: macrophages, CD8 effector and memory T cells, NK cells, CD4 Regulatory T cells. The expression of immune checkpoint molecules PD1 and PDL1 will be analyzed respectively in lymphocytes and tumor cells.

The role of immunological environment in chondrosarcoma progression will be assessed by performing depletions of CD3 and CD163 cells in rat chondrosarcoma model. In parallel, chondrosarcoma's immune environment will be analyzed on human samples provided by Centre Leon BERARD and TMA from the Conticabase network. Chondrosarcoma immunological environment will be compared to the one observed in benign cartilaginous tumors (enchondroma). For the first time, we will define the immune infiltrates within or surrounding the chondrosarcoma both in animal and human samples. We expect to demonstrate the presence of PD1 and PDL1 in this tumor environment. We expect that the CD3 and CD163 cell depletion will downregulate chondrosarcoma progression. The descriptions of the potential involvement of immunological microenvironment in chondrosarcoma progression will enable us to gain insight on the importance of this tumor's immune niche particularly in its mobilization in response to treatments (aim 2).

Aim 2: Effect of PI3K/Akt/mTOR pathway inhibition on chondrosarcoma immune response.

Here we will investigate whether drugs targeting the PI3K/Akt/mTOR pathway could exert an immunostimulatory effect and initiate an immune response in chondrosarcoma. For this purpose, we will study changes in immune cell populations and cytokines secretion arising in response to the evaluated treatments. We will evaluate the effect of the different treatments on immune effectors cells (NK and T cells) and immune cells of the bone niche determined above (aim 1). If the effects of mTOR inhibition on NK cells are still unknown, mTOR inhibition promotes the proliferation and the differentiation of naïve CD8+ T cells and the expansion of CD4+ regulatory T cells. To elucidate whether the immunostimulatory effects of mTOR inhibition are superior to the immunosuppressive ones in rat chondrosarcoma, we will evaluate the effect of the different treatments on the establishment of an immune response mediated by NK cells and CD8+ effector T cells. For this purpose, specific cytotoxic activity of splenocytes (collected from animals of the different treatment groups) directed against chondrosarcoma will be evaluated (by cytotoxicity assays, measure of IFNγ release) using rat primary chondrosarcoma cell line and normal chondrocytes as target cells. The effect of the different treatments on CD8+ T cells (naïve and memory), NK cells and CD4+ regulatory T cells will be analyzed by flow cytometry performed on splenocytes from animals of the different treatment groups.

It was shown that cytokines and growth factors secreted by tumor cells are involved in chondrosarcoma progression and that mTOR inhibitors increase the secretion of several immunostimulatory cytokines like IL-12, IL-23, IL-17,IFN-γ. We propose to determine if this occurs in response to targeted therapies in our chondrosarcoma model by evaluating the levels of cytokines involved in immune response (IL-12, IL-17, IL-23 and INF-γ) and in chondrosarcoma progression (IL-1, SDF-1, RANTES, TNF-α, osteopontin and TGF-β). The levels of cytokines measured both in the plasma and in tumors extracts from all treatment groups will be correlated with the immune population present in the tumor microenvironment and in response to therapy observed. PI3K/mTOR pathway inhibition will certainly affect the cytokines secretion profile and its analysis could enable us to identify cytokines involved in the inhibition of chondrosarcoma progression after inhibition of the PI3K/mTOR pathway.

Aim 3: Involvement of immune checkpoint in chondrosarcoma response to PI3K/Akt/mTOR targeted therapy.

PD1/PDL1 is one the 2 main immunocheckpoints reported today.^14-16 PD1 is expressed by immune cells in peripheral tissue whereas its ligand PDL1 is expressed by tumor cells. After analyzing the expression of PD1/PDL1 in chondrosarcoma microenvironment (aim 1), we will evaluate its role in tumor progression by blocking its activity and overexpressing PDL1 in vitro and in vivo. To confirm the role of PD1/PDL1 in chondrosarcoma response to a targeted agent, we will: i) analyze the changes in PD1/PDL1 expression after inhibition of the PI3K/mTOR pathway; ii) combine PDL1 blockade with an inhibitor of PI3K/mTOR pathway and evaluate the therapeutic efficiency of this combination. The inhibition of PD1/PDL1 will be done if feasible by using antibodies directed against these molecules. If antibodies specific for the rat can not be found or developed we will design anti-PDL1 SiRNA and evaluate their efficacy to silence PDLI in vitro. In vitro, chondrosarcoma cells will be cultured in hypoxia conditions and as 3D pellet model. After analyzing the effects of PDL1 silencing on cell proliferation, cell cultures will be submitted to PI3K/mTOR pathway inhibitors. The effects of mTOR pathway inhibition on PDL1 expression will be assessed by immunohistochemistry. Using the most efficient approach (antibody or SiRNA) to inhibit PDL1 expression in vivo, we will then evaluate the effects of its inhibition on chondrosarcoma progression and tumor immune microenvironment. Then, in the rat model, we will combine PDL1 blockade with an inhibitor of PI3K/mtOR pathway and evaluate the antitumor activity of this combination.

Conclusion

The data obtained in this study will allow the characterization of the chondrosarcoma immunological niche and determine its potential role in tumor progression and response to targeted therapy. Understanding this relation between the chondrosarcoma and its immune environment will help us to better characterize and treat this complex tumor. This project would open novels approaches of combined therapies associating immunotherapy and targeted therapy for the treatment of resistant or refractory bone sarcoma.

By Aurélie Dutor, PhD
and Jean-Yves Blay, MD, PhD
Université Claude Bernard in Lyon, France

References

1. Gelderblom H, Hogendoorn PC, Dijkstra SD, van Rijswijk CS, Krol AD, et al. (2008) The clinical approach towards chondrosarcoma. Oncologist 13: 320-329.

2. Dickey ID, Rose PS, Fuchs B, Wold LE, Okuno SH, et al. (2004) Dedifferentiated chondrosarcoma: the role of chemotherapy with updated outcomes. J Bone Joint Surg Am 86-A: 2412-2418.

3. Kalinski T, Sel S, Kouznetsova I, Ropke M, Roessner A (2009) Heterogeneity of angiogenesis and blood vessel maturation in cartilage tumors. Pathol Res Pract 205: 339-345.

4. Bovee JV, Cleton-Jansen AM, Taminiau AH, Hogendoorn PC (2005) Emerging pathways in the development of chondrosarcoma of bone and implications for targeted treatment. Lancet Oncol 6: 599-607.

5. Ito D, Fujimoto K, Mori T, Kami K, Koizumi M, et al. (2006) In vivo antitumor effect of the mTOR inhibitor CCI-779 and gemcitabine in xenograft models of human pancreatic cancer. Int J Cancer 118: 2337-2343.

6. Mabuchi S, Altomare DA, Connolly DC, Klein-Szanto A, Litwin S, et al. (2007) RAD001 (Everolimus) delays tumor onset and progression in a transgenic mouse model of ovarian cancer. Cancer Res 67: 2408-2413.

7. Nathan CO, Amirghahari N, Rong X, Giordano T, Sibley D, et al. (2007) Mammalian target of rapamycin inhibitors as possible adjuvant therapy for microscopic residual disease in head and neck squamous cell cancer. Cancer Res 67: 2160-2168.

8. Merimsky O., R Bernstein-Molho R., Sagi-Eisenberg et al. Targeting the mammalian target of rapamycin in myxoid chondrosarcoma: Anticancer Drugs 2008; 19: 1019-1021.

9. Bernstein-Molho R, Kollender Y, Issakov J et al. Clinical activity of mTOR inhibition in combination with cyclophosphamide in the treatment of recurrent unresectable chondrosarcomas. Cancer Chemother Pharmacol. 2012; 70: 855-860.

10. Perez J, Decouvelaere AV, Pointecouteau T, Pissaloux D, Michot JP, et al. (2012) Inhibition of Chondrosarcoma Growth by mTOR Inhibitor in an In Vivo Syngeneic Rat Model. PLoS One 7: e32458.

11. Zitvogel L, Apetoh L, Ghiringhelli F, Kroemer G (2008) Immunological aspects of cancer chemotherapy. Nat Rev Immunol 8: 59-73.

12. Araki K, Turner AP, Shaffer VO, Gangappa S, Keller SA, et al. (2009) mTOR regulates memory CD8 T-cell differentiation. Nature 460: 108-112.

13. Weichhart T, Costantino G, Poglitsch M, Rosner M, Zeyda M, et al. (2008) The TSC-mTOR signaling pathway regulates the innate inflammatory response. Immunity 29: 565-577.

14. Dong H, Strome SE, Salomao DR, Tamura H, Hirano F, et al. (2002) Tumor-associated B7-H1 promotes T-cell apoptosis: a potential mechanism of immune evasion. Nat Med 8: 793-800.

15. Hathcock KS, Laszlo G, Dickler HB, Bradshaw J, Linsley P, et al. (1993) Identification of an alternative CTLA-4 ligand costimulatory for T cell activation. Science 262: 905-907.

16. Freeman GJ, Long AJ, Iwai Y, Bourque K, Chernova T, et al. (2000) Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation. J Exp Med 192: 1027-1034.

Schematic representation of PI3K/mTOR signaling pathway, its activation and interaction with MEK pathway.
This orthotopic rat chondrosarcoma model is obtained by paratibial implantation of tumor fragment onto the right posterior paw of 4 weeks old Sprague Dawley rats. Ten days after implantation a tumor can be found at the transplantation site. This model mimics a grade II human chondrosarcoma with a macroscopic and microscopic lobular pattern visible on MRI imaging (A) and histologic analyses (B). A: MRI showing a progressive chondrosarcoma implanted in the right posterior paw of Sprague Dawley rat. B: Histopathologic analysis of rat chondrosarcoma model shows the presence of mitotic cells, a high density of cartilaginous matrix and a tumor with lobular pattern.
In the periphery of the tumor, CD3, CD8 T cells and macrophages (CD163+ cells) could be found. A proportion of CD3 T cells expressed the immune checkpoint PD1 whereas the expression of its ligand (PD-L1) was found in chondrosarcoma cells.