Patients with prostate cancer frequently show resistance to androgen-deprivation therapy, a condition known as castration-resistant prostate cancer (CRPC). Acquiring a better understanding of the mechanisms that control the development of CRPC remains an unmet clinical need. The well-established dependency of cancer cells on the tumour microenvironment indicates that the microenvironment might control the emergence of CRPC. Here we identify IL-23 produced by myeloid-derived suppressor cells (MDSCs) as a driver of CRPC in mice and patients with CRPC. Mechanistically, IL-23 secreted by MDSCs can activate the androgen receptor pathway in prostate tumour cells, promoting cell survival and proliferation in androgen-deprived conditions. Intra-tumour MDSC infiltration and IL-23 concentration are increased in blood and tumour samples from patients with CRPC. Antibody-mediated inactivation of IL-23 restored sensitivity to androgen-deprivation therapy in mice. Taken together, these results reveal that MDSCs promote CRPC by acting in a non-cell autonomous manner. Treatments that block IL-23 can oppose MDSC-mediated resistance to castration in prostate cancer and synergize with standard therapies.
Summary Tumor-associated macrophages (TAMs) represent a major component of the tumor microenvironment supporting tumorigenesis. TAMs re-education has been proposed as a strategy to promote tumor inhibition. However, whether this approach may work in prostate cancer is unknown. Here we find that Pten -null prostate tumors are strongly infiltrated by TAMs expressing C-X-C chemokine receptor type 2 (CXCR2), and activation of this receptor through CXCL2 polarizes macrophages toward an anti-inflammatory phenotype. Notably, pharmacological blockade of CXCR2 receptor by a selective antagonist promoted the re-education of TAMs toward a pro-inflammatory phenotype. Strikingly, CXCR2 knockout monocytes infused in Pten pc−/− ; Trp53 pc−/− mice differentiated in tumor necrosis factor alpha (TNF-α)-releasing pro-inflammatory macrophages, leading to senescence and tumor inhibition. Mechanistically, PTEN -deficient tumor cells are vulnerable to TNF-α-induced senescence, because of an increase of TNFR1 . Our results identify TAMs as targets in prostate cancer and describe a therapeutic strategy based on CXCR2 blockade to harness anti-tumorigenic potential of macrophages against this disease.
Regulation of signal transduction networks depends on protein kinase and phosphatase activities. Protein tyrosine kinases of the JAK family have been shown to regulate integrin affinity modulation by chemokines and mediated homing to secondary lymphoid organs of human T lymphocytes. However, the role of protein tyrosine phosphatases in leukocyte recruitment is still elusive. In this study, we address this issue by focusing on protein tyrosine phosphatase receptor type γ (PTPRG), a tyrosine phosphatase highly expressed in human primary monocytes. We developed a novel methodology to study the signaling role of receptor type tyrosine phosphatases and found that activated PTPRG blocks chemoattractant-induced β2 integrin activation. Specifically, triggering of LFA-1 to high-affinity state is prevented by PTPRG activation. High-throughput phosphoproteomics and computational analyses show that PTPRG activation affects the phosphorylation state of at least 31 signaling proteins. Deeper examination shows that JAKs are critically involved in integrin-mediated monocyte adhesion and that PTPRG activation leads to JAK2 dephosphorylation on the critical 1007–1008 phosphotyrosine residues, implying JAK2 inhibition and thus explaining the antiadhesive role of PTPRG. Overall, the data validate a new approach to study receptor tyrosine phosphatases and show that, by targeting JAKs, PTPRG downmodulates the rapid activation of integrin affinity in human monocytes, thus emerging as a potential novel critical regulator of leukocyte trafficking.
Chemokines participate to B-cell chronic lymphocytic leukemia (B-CLL) pathogenesis by promoting cell adhesion and survival in bone marrow stromal niches and mediating cell dissemination to secondary lymphoid organs. In this study we investigated the role of JAK protein tyrosine kinases (PTK) in adhesion triggering by the CXC chemokine CXCL12 in normal versus CLL B-lymphocytes. We demonstrate that CXCL12 activates JAK2 in normal as well as CLL B-lymphocytes, with kinetics consistent with rapid adhesion triggering. By using complementary methodologies of signal transduction interference, we found that JAK2 mediates CXCL12-triggered activation of lymphocyte function-associated antigen-1 (LFA-1) and very late antigen-4 (VLA-4) integrins. We also show that JAK2 mediates the activation of the small GTP-binding protein RhoA, in turn controlling LFA-1 affinity triggering by CXCL12. Importantly, comparative analysis of 41 B-CLL patients did not evidence JAK2 functional variability between subjects, thus suggesting that JAK2, differently from other signaling events involved in adhesion regulation in B-CLL, is a signaling molecule downstream to CXCR4 characterized by a conserved regulatory role. Our results reveal JAK2 as critical component of chemokine signaling in CLL B-lymphocytes and indicate JAK inhibition as a potentially useful new pharmacological approach to B-CLL treatment.
JAK-dependent activation of the rho module of integrin affinity triggering mediates chemokine-induced leukocyte adhesion. However, the signaling events linking JAKs to rho small GTPase activation by chemokines is still incompletely described. In this study, we show that son of sevenless 1 (SOS1), rho guanine nucleotide exchange factor (GEF)1 (ARHGEF1), and dedicator of cytokinesis (DOCK)2 GEFs mediate CXCL12-induced LFA-1 activation in human primary T lymphocytes. Downregulated expression of SOS1, ARHGEF1, and DOCK2 impairs LFA-1-mediated rapid T lymphocyte adhesion as well as underflow arrest on ICAM-1 induced by CXCL12. Moreover, LFA-1 affinity triggering by CXCL12 is impaired by SOS1, ARHGEF1, and DOCK2 downregulation. Notably, the three GEFs are all critically involved in chemokine-induced RhoA and Rac1 activation, thus suggesting the occurrence of a SOS1 specificity shift in the context of chemokine signaling. Accordingly, SOS1, ARHGEF1, and DOCK2 are tyrosine phosphorylated upon chemokine signaling with timing coherent with rapid LFA-1 affinity activation. Importantly, chemokine-induced tyrosine phosphorylation of these GEFs is fully mediated by JAK protein tyrosine kinases. Unexpectedly, and differently from VAV1, tyrosine phosphorylation of SOS1, ARHGEF1, and DOCK2 is completely inhibited by pertussis toxin pretreatment, thus suggesting different routes of rho-GEF triggering upon CXCR4 engagement. Taken together, these findings reveal a deeper level of complexity in the rho-signaling module, with at least four different rho-GEFs cooperating in the regulation of chemokine-induced integrin activation, possibly suggesting the emergence of stochastic concurrency in signaling mechanisms controlling leukocyte trafficking.
We proved the ability of Fourier Transform Infrared microspectroscopy (microFTIR) complemented by Principal Component Analysis (PCA) to detect protein phosphorylation/de-phosphorylation in mammalian cells. We analyzed by microFTIR human polymorphonuclear neutrophil (PMNs) leukocytes, mouse-derived parental Ba/F3 cells (Ba/F3#PAR), Ba/F3 cells transfected with p210(BCR/ABL) (Ba/F3#WT) and expressing high levels of protein tyrosine kinase (PTK), and human-derived BCR/ABL positive K562 leukemic cell sub-clones engineered to differently express receptor-type tyrosine-protein phosphatase gamma (PTPRG). Synchrotron radiation (SR) and conventional (globar) IR sources were used to perform microFTIR respectively, on single cells and over several cells within the same sample. Ex vivo time-course experiments were run, inducing maximal protein phosphorylation in PMNs by 100 nM N-formylated tripeptide fMLP. Within the specific IR fingerprint 1800-850 cm(-1) frequency domain, PCA identified two regions with maximal signal variance. These were used to model and test the robustness of PCA in representing the dynamics of protein phosphorylation/de-phosphorylation processes. An IR signal ratio marker reflecting the homeostatic control by protein kinases and phosphatases was identified in normal leukocytes. The models identified by microFTIR and PCA in normal leukocytes also distinguished BCR/ABL positive Ba/F3#WT from BCR/ABL negative Ba/F3#PAR cells as well as K562 cells exposed to functionally active protein tyrosine phosphatase recombinant protein ICD-Tat transduced in cells by HIV-1 Tat technology or cells treated with the PTK inhibitor imatinib mesylate (IMA) from cells exposed to phosphatase inactive (D1028A)ICD-Tat recombinant protein and untreated control cells, respectively. The IR signal marker correctly reflected the degrees of protein phosphorylation associated with abnormal PTK activity in BCR/ABL positive leukemic cells and in general was inversely related to the expression/activity of PTPRG in leukemic sub-clones. In conclusion, we have described a new, reliable and simple spectroscopic method to study the ex vivo protein phosphorylation/de-phosphorylation balance in cell models: it is suitable for biomedical and pharmacological research labs but it also needs further optimization and its evaluation on large cohorts of patients to be proposed in the clinical setting of leukemia.
Current autologous cell therapies, with blockbuster products on the market, have been leading for a decade to unprecedented clinical successes in patients with hematological malignancies. However, these patient-derived T-cell therapies are facing many challenges. The use of GMP iPSC lines to produce immune effector cells will reduce the complexity of the manufacturing process and will provide an unlimited source of starting material. The goal of the EVOcells Oncology platform is to offer a truly allogeneic cell therapy platform to treat a broad number of cancer patients with consistent quality and scalability of the final product. Besides, the versatility of our platform to produce different immune cell types combined to customized genetic engineering strategies will bring cell therapy to the level of personalized medicine. Our “off-the-shelf” cell therapy platform has already validated two pillars: iPSC-derived NK cells (iNK) and iPSC-derived Macrophages (iMACs). Through multiple genetic engineering strategies specific to each immune cell type, we are developing a comprehensive portfolio of cell therapy products to address specific tumor escape mechanism in liquid and solid tumors. Our initial effort aimed to develop these two innate immune cell types to propose efficacious cell therapies with an increased safety profile as they have low risk of graft-versus-host disease (GvHD) or CRS (Cytokine Release Syndrome). Thanks to the expression of a broad pattern of activatory receptors, iNK cells form Immunological Synapses with tumor cells leading in turn to efficient killing with and without addition of a CAR construct. Besides, we demonstrated the possibility to combine “naked” iNK cells with marketed therapeutic monoclonal antibodies (mAb) to further improve their efficacy. At the end of the differentiation process, iMACs are showing a M0 like phenotype with high plasticity allowing the in vitro differentiation of the cells towards either a M1 or a M2 polarization in response to the appropriate stimulations. iMACs produce key macrophages cytokines and are able to kill tumor cells via ADCP (Antibody-Dependent-Cell-Phagocytosis) mechanism when combined to a therapeutic mAb. Thanks to our collaboration with clinicians at the IUCT-Oncopole (Toulouse Cancer Hospital), we were able to identify appropriate cancer indications and further demonstrate in a translational fashion that both iNK and iMACs are able to kill primary resistant tumor cells which were isolated from patient’ samples. Taken together, these results are showing the versatility and the breadth of our EVOcells Oncology platform to produce a true arsenal of cell therapies and its potential for future clinical development. Citation Format: Michael Esquerré, Audrey Holtzinger, Nadja Wagner, Monika Braun, Mélanie Pichery, Stefanie Pfaender, Stephanie Sontag, Kathrin Haake, Michela Mirenda, Michael Paillasse, Davide Grandolfo, Chloé Beuraud, Mandy Richter, Philip Hublitz, Julien Bousquet, Marion Fabre, Mylène Gador, Daniel Sommermeyer, Tanja Schneider, Oriane Bombarde, Camille Esquerré, Loic Ysebaert, Fabien Despas, Matthias Austen, Andreas Scheel, Markus Dangl. EVOcells Oncology: Tailored genetic engineering of iPSC-derived immune effector cells and combination with the right biologic therapeutics result in optimal killing of primary tumor cells from patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3203.
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