Interferon-induced expression of programmed cell death ligands (PD-L1/PD-L2) may sustain tumour immune-evasion. Patients featuring MET amplification, a genetic lesion driving transformation, may ...benefit from anti-MET treatment. We explored if MET-targeted therapy interferes with Interferon-γ modulation of PD-L1/PD-L2 in MET-amplified tumours.
PD-L1/PD-L2 expression and signalling pathways downstream of MET or Interferon-γ were analysed in MET-amplified tumour cell lines and in patient-derived tumour organoids, in basal condition, upon Interferon-γ stimulation, and after anti-MET therapy.
PD-L1 and PD-L2 were upregulated in MET-amplified tumour cells upon Interferon-γ treatment. This induction was impaired by JNJ-605, a selective inhibitor of MET kinase activity, and MvDN30, an antibody inducing MET proteolytic cleavage. We found that activation of JAKs/ STAT1, signal transducers downstream of the Interferon-γ receptor, was neutralised by MET inhibitors. Moreover, JAK2 and MET associated in the same signalling complex depending on MET phosphorylation. Results were confirmed in MET-amplified organoids derived from human colorectal tumours, where JNJ-605 treatment revoked Interferon-γ induced PD-L1 expression.
These data show that in MET-amplified cancers, treatment with MET inhibitors counteracts the induction of PD-1 ligands by Interferon-γ. Thus, therapeutic use of anti-MET drugs may provide additional clinical benefit over and above the intended inhibition of the target oncogene.
Chimeric antigen receptor (CAR)-engineered T lymphocytes (CAR Ts) produced impressive clinical results against selected hematological malignancies, but the extension of CAR T cell therapy to the ...challenging field of solid tumors has not, so far, replicated similar clinical outcomes. Many efforts are currently dedicated to improve the efficacy and safety of CAR-based adoptive immunotherapies, including application against solid tumors. A promising approach is CAR engineering of immune effectors different from αβT lymphocytes. Herein we reviewed biological features, therapeutic potential, and safety of alternative effectors to conventional CAR T cells: γδT, natural killer (NK), NKT, or cytokine-induced killer (CIK) cells. The intrinsic CAR-independent antitumor activities, safety profile, and ex vivo expansibility of these alternative immune effectors may favorably contribute to the clinical development of CAR strategies. The proper biological features of innate immune response effectors may represent an added value in tumor settings with heterogeneous CAR target expression, limiting the risk of tumor clonal escape. All these properties bring out CAR engineering of alternative immune effectors as a promising integrative option to be explored in future clinical studies.
The MHC-unrestricted activity of cytokine-induced killer (CIK) cells against chemo-surviving melanoma cancer stem cells (mCSC) was explored, as CSCs are considered responsible for chemoresistance and ...relapses.
Putative mCSCs were visualized by engineering patient-derived melanoma cells (MC) with a lentiviral vector encoding eGFP under expression control by stemness gene promoter
Their stemness potential was confirmed
by limiting dilution assays. We explored the sensitivity of eGFP
mCSCs to chemotherapy (CHT), BRAF inhibitor (BRAFi) or CIK cells, as single agents or in sequence,
First, we treated MCs
with fotemustine or dabrafenib (BRAF-mutated cases); then, surviving MCs, enriched in mCSCs, were challenged with autologous CIK cells. CIK cell activity against chemoresistant mCSCs was confirmed
in two distinct immunodeficient murine models.
We visualized eGFP
mCSCs (14% ± 2.1%) in 11 MCs. The tumorigenic precursor rate
was higher within eGFP
MCs (1/42) compared with the eGFP
counterpart (1/4,870).
mCSCs were relatively resistant to CHT and BRAFi, but killed by CIK cells (
= 11, 8/11 autologous), with specific lysis ranging from 95% effector:tumor ratio (E:T), 40:1 to 20% (E:T 1:3).
infusion of autologous CIK cells into mice bearing xenografts from three distinct melanomas demonstrated significant tumor responses involving CHT-spared eGFP
mCSCs (
= 0.001). Sequential CHT-immunotherapy treatment retained antitumor activity (
= 12,
= 0.001) reducing mCSC rates (
= 0.01).
These findings are the first demonstration that immunotherapy with CIK cells is active against autologous mCSCs surviving CHT or BRAFi. An experimental platform for mCSC study and rationale for CIK cells in melanoma clinical study is provided.
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BRAF and MEK inhibitors (BRAF/MEKi) favor melanoma-infiltrating lymphocytes, providing the rationale for current combinatorial trials with anti-PD-1 antibody. A portion of melanoma cells may express ...PD-1, and anti-PD-1 antibody could have a direct antitumor effect. Here, we explore whether BRAF/MEKi modulate rates of PD-1
melanoma cells, supporting an additional-lymphocyte-independent-basis for their therapeutic combination with anti-PD-1 antibody.
With data mining and flow cytometry, we assessed PD-1, PD-L1/2 expression on melanoma cell lines (CCLE,
= 61; validation cell lines,
= 7) and melanoma tumors (TCGA,
= 214). We explored
how BRAF/MEKi affect rates of PD-1
, PD-L1/2
melanoma cells, and characterized the proliferative and putative stemness features of PD-1
melanoma cells. We tested the functional lymphocyte-independent effect of anti-PD-1 antibody alone and in combination with BRAF/MEKi
and in an
immunodeficient murine model.
PD-1 is consistently expressed on a small subset of melanoma cells, but PD-1
cells increase to relevant rates during BRAF/MEKi treatment 7.3% (5.6-14.2) vs. 1.5% (0.7-3.2),
= 0.0156;
= 7, together with PD-L2
melanoma cells 8.5% (0.0-63.0) vs. 1.5% (0.2-43.3),
= 0.0312;
= 7. PD-1
cells proliferate less than PD-1
cells (avg. 65% less;
= 7 days) and are preferentially endowed with stemness features.
, the direct anti-melanoma activity of PD-1 blockage as monotherapy was negligible, but its association with BRAF/MEKi significantly delayed the development of drug resistance and tumor relapse.
BRAF/MEKi increase the rates of PD-1
melanoma cells that may sustain tumor relapse, providing a lymphocyte-independent rationale to explore combinatory strategies with anti-PD-1 antibody.
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The multikinase inhibitor sorafenib displays antitumor activity in preclinical models of osteosarcoma. However, in sorafenib-treated patients with metastatic-relapsed osteosarcoma, disease ...stabilization and tumor shrinkage were short-lived and drug resistance occurred. We explored the sorafenib treatment escape mechanisms to overcome their drawbacks.
Immunoprecipitation, Western blotting, and immunohistochemistry were used to analyze the mTOR pathway mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). Cell viability, colony growth, and cell migration were evaluated in different osteosarcoma cell lines (MNNG-HOS, HOS, KHOS/NP, MG63, U-2OS, SJSA-1, and SAOS-2) after scalar dose treatment with sorafenib (10-0.625 μmol/L), rapamycin-analog everolimus (100-6.25 nmol/L), and combinations of the two. Cell cycle, reactive oxygen species (ROS) production, and apoptosis were assessed by flow cytometry. Nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice injected with MNNG-HOS cells were used to determine antitumor and antimetastatic effects. Angiogenesis and vascularization were evaluated in vitro by exploiting endothelial branching morphogenesis assays and in vivo in xenografted mice and chorioallantoic membranes.
After sorafenib treatment, mTORC1 signaling was reduced (downstream target P-S6), whereas mTORC2 was increased (phospho-mTOR Ser2481) in MNNG-HOS xenografts compared with vehicle-treated mice. Combining sorafenib with everolimus resulted in complete abrogation of both mTORC1 through ROS-mediated AMP-activated kinase (AMPK) activation and mTORC2 (through complex disassembly). The sorafenib/everolimus combination yielded: (i) enhanced antiproliferative and proapoptotic effects, (ii) impaired tumor growth, (iii) potentiated antiangiogenesis, and (iv) reduced migratory and metastatic potential.
mTORC2 activation is an escape mechanism from sorafenib treatment. When sorafenib is combined with everolimus, its antitumor activity is increased by complete inhibition of the mTOR pathway in the preclinical setting.
Enhancing the antitumor activity of the DNA-damaging drugs is an attractive strategy to improve current treatment options. Trabectedin is an isoquinoline alkylating agent with a peculiar mechanism of ...action. It binds to minor groove of DNA inducing single- and double-strand-breaks. These kinds of damage lead to the activation of PARP1, a first-line enzyme in DNA-damage response pathways. We hypothesized that PARP1 targeting could perpetuate trabectedin-induced DNA damage in tumor cells leading finally to cell death.
We investigated trabectedin and PARP1 inhibitor synergism in several tumor histotypes both in vitro and in vivo (subcutaneous and orthotopic tumor xenografts in mice). We searched for key determinants of drug synergism by comparative genomic hybridization (aCGH) and gene expression profiling (GEP) and validated their functional role.
Trabectedin activated PARP1 enzyme and the combination with PARP1 inhibitors potentiated DNA damage, cell cycle arrest at G2/M checkpoint and apoptosis, if compared to single agents. Olaparib was the most active PARP1 inhibitor to combine with trabectedin and we confirmed the antitumor and antimetastatic activity of trabectedin/olaparib combination in mice models. However, we observed different degree of trabectedin/olaparib synergism among different cell lines. Namely, in DMR leiomyosarcoma models the combination was significantly more active than single agents, while in SJSA-1 osteosarcoma models no further advantage was obtained if compared to trabectedin alone. aCGH and GEP revealed that key components of DNA-repair pathways were involved in trabectedin/olaparib synergism. In particular, PARP1 expression dictated the degree of the synergism. Indeed, trabectedin/olaparib synergism was increased after PARP1 overexpression and reduced after PARP1 silencing.
PARP1 inhibition potentiated trabectedin activity in a PARP1-dependent manner and PARP1 expression in tumor cells might be a useful predictive biomarker that deserves clinical evaluation.
Unresectable metastatic bone sarcoma and soft-tissue sarcomas (STS) are incurable due to the inability to eradicate chemoresistant cancer stem-like cells (sCSC) that are likely responsible for ...relapses and drug resistance. In this study, we investigated the preclinical activity of patient-derived cytokine-induced killer (CIK) cells against autologous bone sarcoma and STS, including against putative sCSCs. Tumor killing was evaluated both in vitro and within an immunodeficient mouse model of autologous sarcoma. To identify putative sCSCs, autologous bone sarcoma and STS cells were engineered with a CSC detector vector encoding eGFP under the control of the human promoter for OCT4, a stem cell gene activated in putative sCSCs. Using CIK cells expanded from 21 patients, we found that CIK cells efficiently killed allogeneic and autologous sarcoma cells in vitro. Intravenous infusion of CIK cells delayed autologous tumor growth in immunodeficient mice. Further in vivo analyses established that CIK cells could infiltrate tumors and that tumor growth inhibition occurred without an enrichment of sCSCs relative to control-treated animals. These results provide preclinical proof-of-concept for an effective strategy to attack autologous sarcomas, including putative sCSCs, supporting the clinical development of CIK cells as a novel class of immunotherapy for use in settings of untreatable metastatic disease.
Background: The immunotherapy of head and neck cancer induces a limited rate of long-term survivors at the cost of treating many patients exposed to toxicity without benefit, regardless of PD-L1 ...expression. The identification of better biomarkers is warranted. We analyzed a panel of cytokines, chemokines and growth factors, hereinafter all referred to as ‘cytokines’, as potential biomarkers in patients with head and neck cancer treated with nivolumab. Materials and methods: A total of 18 circulating cytokines were analyzed. Samples were gathered at baseline (T0) and after 3 courses of nivolumab (T1) in patients with relapsed/metastatic disease. The data extracted at T0 were linked to survival; the comparison of T0–T1 explored the effect of immunotherapy. Results: A total of 22 patients were accrued: 64% current heavy smokers, 36% female and 14% had PS = 2. At T0, ROC analysis showed that IL-6, IL-8, IL-10 and TGF-β were higher in patients with poor survival. Cox analysis demonstrated that only patients with the IL-6 and TGF-β discriminate had good or poor survival, respectively. Longitudinal increments of CCL-4, IL-15, IL-2 and CXCL-10 were observed in all patients during nivolumab treatment. Conclusion: In this small population with poor clinical characteristics, this study highlights the prognostic role of IL-6 and TGF-β. Nivolumab treatment is associated with a positive modulation of some Th1 cytokines, but it does not correlate with the outcome.
Mechanisms governing stress-induced hematopoietic progenitor cell mobilization are not fully deciphered. We report that during granulocyte colony-stimulating factor–induced mobilization c-Met ...expression and signaling are up-regulated on immature bone marrow progenitors. Interestingly, stromal cell–derived factor 1/CXC chemokine receptor-4 signaling induced hepatocyte growth factor production and c-Met activation. We found that c-Met inhibition reduced mobilization of both immature progenitors and the more primitive Sca-1+/c-Kit+/Lin− cells and interfered with their enhanced chemotactic migration to stromal cell–derived factor 1. c-Met activation resulted in cellular accumulation of reactive oxygen species by mammalian target of rapamycin inhibition of Forkhead Box, subclass O3a. Blockage of mammalian target of rapamycin inhibition or reactive oxygen species signaling impaired c-Met–mediated mobilization. Our data show dynamic c-Met expression and function in the bone marrow and show that enhanced c-Met signaling is crucial to facilitate stress-induced mobilization of progenitor cells as part of host defense and repair mechanisms.
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