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Dual targeting of senescence and tumor immunity for cancer therapy

Periodic Reporting for period 2 - Immune-senescence (Dual targeting of senescence and tumor immunity for cancer therapy)

Reporting period: 2018-01-01 to 2019-06-30

Cancer immunotherapy is emerging as a relevant approach to treat several types of cancer. We have previously found that tumor-infiltrating myeloid cells can support the growth of prostate cancer and trigger treatment resistance. Therefore, a better characterization of the prostate tumor microenvironment and tumor immune response may contribute to the identification of novel immunotherapies to enhance the efficacy of standard of therapy in prostate cancer. An essential objective of this proposal through the task1 is to fully characterize the secretome and the immune landscape of prostate tumors driven by different genetic alterations. We will use gene expression analysis, proteomics and bioinformatics to identify secreted factors that shape the tumor microenvironment during different stages of prostate tumorigenesis and in mice undergoing treatment-induced senescence (TIS). This integrated analysis will allow us to develop novel “targeted” immunotherapies tailored on the genetic background of prostate tumors, thus setting the basis for future clinical trials in prostate cancer patients. In task2 of the proposal, we will assess the mechanisms that promote the recruitment and activation of tumor-associated macrophages (TAMs) and GR1+ myeloid cells, two immune subsets that are frequently found altered in Pten-deficient prostate cancers. In particular, we will focus on a novel class of secreted factors identified in Pten; Trp53 null tumors that are responsible for M1-M2 macrophages skewing and that can be targeted for cancer therapy. In task3 of the proposal we will assess whether the tumor secretome transmit senescence to immune subsets thereby affecting immune surveillance or potentiating the oncogenic capability of some immune subsets. Identification of secreted factors that promote premature immunosenescence will allow us to assess in vivo whether compounds that interfere with the secretome of Pten-null tumors can improve the efficacy of prostate cancer therapy. In task4 of the proposal, we will focus on tumor-infiltrating B cells (TILBs) in order to characterize their function in different Pten null mouse models. Final objective of this proposal is to identify and develop monoclonal antibodies directed towards senescence tumor cells. These antibodies will be used as biomarkers to detect senescence tumor cells in both mouse and human prostate cancers and will be tested in pre-clinical trials (task5) to assess whether they improve tumor clearance during TIS. In conclusion, many essential proves of the biological relevance of senescence in cancer have been currently discovered and strategies aimed to identify compounds which may enhance both senescence and immune surveillance in cancers would support the development of more efficient treatment modalities for cancer therapy. After the publication of our paper in Nature in 2018, there has been a lot of interest from both the scientific community and society in the field of therapies that block the function of MDSCs. This is documented by several articles that were published in scientific journals (commentaries), newspapers and media regaridng this descovery.
An essential objective of this proposal through the task1 and 2 is to characterize the secretome and the immune landscape of prostate tumors driven by different genetic alterations in order to identify secreted factors that can promote the recruitment and function of myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs). Our final aim is to develop novel “targeted” immunotherapies tailored on the genetic background of prostate tumors, that will set the basis for future clinical trials in prostate cancer patients (task 5). In task3 of the proposal, we aim to assess whether the secretome of Pten-deficient tumors can transmit senescence to different tumor-infiltrating immune subsets thereby compromising their function. Finally, in task4 of the proposal, we aim to develop monoclonal antibodies directed towards senescence tumor cells.

The main achievements of the project for the mid-term reporting period are:

a) Characterization of the secretome and immune response of prostate tumors of different mouse models.
We have completed the analysis of the immune landscape and the secretome of five different models of prostate cancer driven by different genetic alterations; specifically, we have so far analyzed Ptenpc-/-, Ptenpc-/-;Ergpc+/+, Ptenpc-/-;Timp1pc-/-, Ptenpc-/-;Cdcp1pc+/+ and Ptenpc-/-;P53pc-/-, characterized by different tumor cell invasiveness, metastatic potential and immune infiltration. This analysis has revealed that polymorphonuclears (PMN) MDSCs are clearly the most abundant immune cells population in all the mouse models of prostate cancer analyzed. However, we have found that the recruitment of these cells in cancer occurs through different modalities. To characterize the secretome of tumor cells driven by different genetic alterations, we have performed polysome profiles analysis, a technology that allows the identification of the tumors “translatome”, meaning the entire pool of mRNAs actively translating in the tumor cell. Through bioinformatics analysis performed by matching the upregulated secreted factors and receptors into the tumors with the receptors expressed by MDSCs, we found 12 ligand-receptor couples. We are currently studying 2 of these couples whose interaction can be blocked by available immunotherapies

b) Defining the mechanism leading to M1-M2 macrophages polarization in Pten-null tumors and asses how M1-like TAMs drive senescence in prostate cancer
By using the method described above we have found that CXCL1, 2 3 and CXCL5are strongly upregulated in Ptenpc-/-; Trp53pc-/- mice and that TAMs express the CXCR2 receptor both in murine and human prostate tumors. We have found that these chemokines, in particular, CXCL2, are able to promote macrophages M2-like polarization. Inhibition of the CXCR2 receptor in these TAMs blocked their polarization towards M2-like macrophages enhancing the tumor suppressive capability of these macrophages. By performing a cytokine profiling on sorted pro-inflammatory macrophages, we identified TNF as the factor capable of inducing growth inhibition and senescence in tumors treated with CXCR2 antagonists (Di Mitri D. Cell Reports, Under Revision).

c) To assess whether the secretome of prostate tumors induces “senescence” in tumor-infiltrating immune cells
By using the senescence markers FDG in prostate tumors we have found that the majority of C12FDG+ cells in the CD45+ population are PMN-MDSCs. These cells were also characterized by an up-regulation of several senescence markers such as p16 and PAI-1. In addition, we have found that chemotherapies such as docetaxel increase the number of tumor-infiltrating senescent-like myeloid cells in line with our hypothesis that the senescence secretome can transmit senescence to immune cells. Based on these promising results and the important role played by MDSCs in the tumor microenvironment of prostate cancer, we are now focusing our investigation on the function and role played by senescent-like myeloid cells by performing RNA-seq and functional assays. This will allow us to identify novel entry points to improve the efficacy of currently available immunotherapies

d) Identify novel senescence biomarkers by developing novel antibodies.
In order to identify transmembrane proteins specifically present on the membrane of senescent tumor cells we have performed proteomic analysis of FDG positive and FDG negative Epcam+ epithelial prostate cancer cells sorted from Ptenpc-/- tumors. The proteomic analysis from FDG positive, senescent cells and FDG negative not senescent epithelial prostate cancer cells, allowed to identify 2165 proteins, of which 247 upregulated proteins and 277 downregulated proteins. Between the upregulated proteins, we identified 33 plasma membrane proteins selectively upregulated in FDG positive cells compared to FDG negative cells. We are in the process to develop antibodies against these targets. This tool will allow to label and track in vivo senescent cells and the coupling of the specific antibody to senolytic compounds.

e) Evaluating whether immunotherapy improve the efficacy of standard of therapy in prostate cancer
The aim of this task was to develop the most effective immunotherapy targeting MDSCs to combine with the standard of therapy in prostate cancer. By analyzing both human and murine tumor biopsies from castration-sensitive (HS) and resistant prostate cancer patients (CRPC) we found that tumors from CRPC patients were massively infiltrated by MDSC and that MDSCs promotes resistance to androgen-deprivation therapies by releasing in the tumor microenvironment IL23. IL23 was the most abundant factor present in the blood of CRPC patients when compared to HS patients. Treatment of different mouse models of CRPC with IL23 antibody synergies with enzalutamide a standard of therapy in CRPC patients (Calcinotto A. et al, Nature, 2018). This paper has opened at novel exciting prospective for the therapy of CRCP patients
When I started this project it was unclear how prostate cancer recruits and controls the function of tumor-infiltrating myeloid cells. Moreover, the majority of the clinically available immunotherapies used in cancer therapy were directed to enhance the function of tumor-infiltrating T lymphocytes. Thanks to this project we have systemically analyzed the secretome of both prostate cancer and tumor-infiltrating myeloid cells and found a number of druggable factors that are responsible for the recruitment and function of MDSCs and TAMs. This knowledge has allowed us to develop novel immunotherapies directed to block MDSCs. In particular, we have found that tumor-infiltrating MDSCs promote resistance to different androgen-deprivation therapies (ADTs) in prostate cancer by secreting IL23 in the tumor microenvironment (Calcinotto, Nature 2018). IL23 secreted by MDSCs binds the IL23 receptor presents on prostate tumor cells and activate the RORg/STAT3 pathway leading to ADT insensitivity. We have found that the efficacy of enzalutamide treatment, the standard of therapy for patients affected by metastatic prostate cancer patients is enhanced by an antibody that blocks the IL23 receptor. Given that these antibodies are already available in the clinic for the treatment of autoimmune disorders, in the near future we plan to bring this therapy in the clinic. In the meantime, we have started a trial in collaboration with Prof. De Bono team that combines a drug that blocks the intratumoral recruitment of MDSCs with enzalutamide. This innovative trial is the first trial assessing the tolerability and efficacy of this class of compounds in prostate cancer. Thus in the near future, I expect to identify at least 2 additional druggable targets involved in inhibition of MDSCs function that we can use to enhance the efficacy of standard of therapy in CRPC.