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Supplementary Figure from A Therapeutically Actionable Protumoral Axis of Cytokines Involving IL-8, TNFα, and IL-1β

Interleukin-8 (CXCL8) produced in the tumor microenvironment correlates with poor response to checkpoint inhibitors and is known to chemoattract and activate immunosuppressive myeloid leukocytes. In human cancer, IL8 mRNA levels correlate with IL1B and TNF transcripts. Both cytokines induced IL-8 functional expression from a broad variety of human cancer cell lines, primary colon carcinoma organoids, and fresh human tumor explants. Although IL8 is absent from the mouse genome, a similar murine axis in which TNFα and IL-1β upregulate CXCL1 and CXCL2 in tumor cells was revealed. Furthermore, intratumoral injection of TNFα and IL-1β induced IL-8 release from human malignant cells xenografted in immunodeficient mice. In all these cases, the clinically used TNFα blockers infliximab and etanercept or the IL-1β inhibitor anakinra was able to interfere with this pathogenic cytokine loop. Finally, in paired plasma samples of patients with cancer undergoing TNFα blockade with infliximab in a clinical trial, reductions of circulating IL-8 were substantiated.Significance:IL-8 attracts immunosuppressive protumor myeloid cells to the tumor microenvironment, and IL-8 levels correlate with poor response to checkpoint inhibitors. TNFα and IL-1β are identified as major inducers of IL-8 expression on malignant cells across cancer types and models in a manner that is druggable with clinically available neutralizing agents.This article is highlighted in the In This Issue feature, p. 2007

Interleukin-8 (CXCL8) produced in the tumor microenvironment correlates with poor response to checkpoint inhibitors and is known to chemoattract and activate immunosuppressive myeloid leukocytes. In human cancer, IL8 mRNA levels correlate with IL1B and TNF transcripts. Both cytokines induced IL-8 functional expression from a broad variety of human cancer cell lines, primary colon carcinoma organoids, and fresh human tumor explants. Although IL8 is absent from the mouse genome, a similar murine axis in which TNFα and IL-1β upregulate CXCL1 and CXCL2 in tumor cells was revealed. Furthermore, intratumoral injection of TNFα and IL-1β induced IL-8 release from human malignant cells xenografted in immunodeficient mice. In all these cases, the clinically used TNFα blockers infliximab and etanercept or the IL-1β inhibitor anakinra was able to interfere with this pathogenic cytokine loop. Finally, in paired plasma samples of patients with cancer undergoing TNFα blockade with infliximab in a clinical trial, reductions of circulating IL-8 were substantiated.Significance:IL-8 attracts immunosuppressive protumor myeloid cells to the tumor microenvironment, and IL-8 levels correlate with poor response to checkpoint inhibitors. TNFα and IL-1β are identified as major inducers of IL-8 expression on malignant cells across cancer types and models in a manner that is druggable with clinically available neutralizing agents.This article is highlighted in the In This Issue feature, p. 2007

Few therapies are currently available for patients with KRAS-driven cancers, highlighting the need to identify new molecular targets that modulate central downstream effector pathways. Here we found that the microRNA (miRNA) cluster including miR181ab1 is a key modulator of KRAS-driven oncogenesis. Ablation of Mir181ab1 in genetically engineered mouse models of Kras-driven lung and pancreatic cancer was deleterious to tumor initiation and progression. Expression of both resident miRNAs in the Mir181ab1 cluster, miR181a1 and miR181b1, was necessary to rescue the Mir181ab1-loss phenotype, underscoring their nonredundant role. In human cancer cells, depletion of miR181ab1 impaired proliferation and 3D growth, whereas overexpression provided a proliferative advantage. Lastly, we unveiled miR181ab1-regulated genes responsible for this phenotype. These studies identified what we believe to be a previously unknown role for miR181ab1 as a potential therapeutic target in 2 highly aggressive and difficult to treat KRAS-mutated cancers.

Activation-induced cell death (AICD) is a complex immunoregulatory mechanism that causes the demise of a fraction of T-lymphocytes upon antigen-driven activation. In the present study we investigated the direct role of TNF in AICD of CD8 T lymphocytes. Methods: Human peripheral mononuclear cells were isolated from healthy donors and fresh tumor-infiltrating lymphocytes were obtained from cancer patients undergoing surgery. T cells were activated with anti-CD3/CD28 mAbs or with a pool of virus peptides, in combination with clinical-grade TNF blocking agents. Results: A portion of CD8 T cells undergoes apoptosis upon CD3/CD28 activation in a manner that is partially prevented by the clinically used anti-TNF agents infliximab and etanercept. TNF-mediated AICD was also observed upon activation of virus-specific CD8 T cells and tumor-infiltrating CD8 T lymphocytes. The mechanism of TNF-driven T cell death involves TNFR2 and production of mitochondrial oxygen free radicals which damage DNA. Conclusion: The use of TNF blocking agents reduces oxidative stress, hyperpolarization of mitochondria, and the generation of DNA damage in CD8 T celss undergoing activation. The fact that TNF mediates AICD in human tumor-reactive CD8 T cells suggests that the use of TNF-blocking agents can be exploited in immunotherapy strategies.

Background: Interleukin-8 produced in the tumor microenvironment correlates with poor response to checkpoint inhibitors and is known to chemoattract and activate immunosuppressive myeloid leukocytes. An important question is what are the mechanisms that control IL-8 transcription in tumors. Various factors have been described but the main common denominator is NF-кB activation. In this regard, considering inflammatory mediators present in the tumor and whose receptors act as NF-кB inducers, TNFα and IL-1β became obvious candidates. In this study, we provide multiple lines of evidence that TNFα and IL-1β are able to elicit functional IL-8 from malignant cells in a manner that can be inhibited by clinically grade blockade agents. Methods: Short-term cultured human tumor cell lines, fresh human tumor explants and primary organoids were stimulated with TNFα or IL-1β to test for IL-8 induction. Moreover, syngeneic and xenografted tumors were also used to study IL-8 induction in response to TNFα or to IL-1β. Correlation between these cytokines and IL-8 transcripts were studied in TCGA and other databases to search. Results: We have been able to correlate IL-8 mRNA levels with IL-1β and TNFα transcripts in human cancers. Both cytokines induced IL-8 functional expression from a broad variety of human cancer cell lines, primary colon carcinoma organoids and fresh human tumor explants. Although IL-8 is absent from the mouse genome, a similar murine axis in which TNFα and IL-1β upregulate CXCL1 and CXCL2 in several mouse tumor cells was revealed. Furthermore, intratumoral injection of TNFα and IL-1β induced IL-8 release from human malignant cells xenografted in immunodeficient mice. In all these cases, the clinically used TNFα-blockers infliximab and etanercept, or the IL-1β inhibitor anakinra were able to interfere with this pathogenic cytokine loop. Moreover, treatment with checkpoint inhibitors augments TNFα in syngeneic and xenografted tumors thereby turning on this immunosuppressive chemokine-driven axis. However, we have demonstrated that co-blockade of TNFα and IL-1β could equally enhance anti-tumor efficacy of αPD1+αCTLA-4 regimens as CXCR1/CXCR2 inhibitors, reparixin and AZD5069, did. Conclusions: In this study, we have identified TNFα and IL-1β as major inducers of IL-8 transcription on human cancers. In keeping with these findings, both TNFα and IL-1β induce functional IL-8 release from all tested human tumor cell lines representing a variety of tissue origins, as well as from primary organoids and fresh human cancer explants. In all these cases, including xenografted human tumors, IL-8 induction could be blunted by the pharmacological blockade of TNFα or IL-1β. Finally, inhibition of this pathogenic cytokine loop could increase the anti-tumoral effect of checkpoint inhibitors. Citation Format: Irene Olivera, Rebeca Sanz-Pamplona, Elixabet Bolaños, Inmaculada Rodriguez, Iñaki Etxeberria, Assunta Cirella, Saray Garasa, Itziar Migueliz, Iñaki Eguren, Miguel F. Sanmamed, Javier Glez-Vaz, Arantza Azpilikueta, Maite Alvarez, Maria C. Ochoa, Carlos E. De Andrea, Pedro Berraondo, Álvaro Teijeira, Ignacio Melero. A therapeutically actionable pro-tumoral axis of cytokines involving interleukin-8, TNFα and IL-1β [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3826.

In humans, IL-8 (CXCL8) is a key chemokine for chemotaxis of polymorphonuclear leukocytes and monocytes/macrophages when acting on CXCR1 and CXCR2. CXCL8 activity on neutrophils includes chemotaxis and eliciting the extrusion of neutrophil extracellular traps (NETs). In this study, we show that concentrations of IL-8 that induce NETosis surpass in at least one order of magnitude those required to elicit chemoattraction in human neutrophils. IL-8-induced NETosis was less dependent on G-proteins than migration, while extracellular Ca+2 chelation similarly inhibited both processes. Reactive oxygen species (ROS) were more important for NETosis than for chemotaxis as evidenced by neutralization with N-acetyl -cysteine. Interestingly, selective blockade with anti-CXCR1 mAb inhibited NETosis much more readily than chemotaxis, while pharmacological inhibition of both CXCR1 and CXCR2, or selective inhibition for CXCR2 alone, similarly inhibited both functions. Together, these results propose a model according to which low concentrations of IL-8 in a gradient attract neutrophils to the inflammatory foci, while high receptor-saturating concentrations of IL-8 give rise to NETosis once leukocytes reach the core of the inflammatory insult.

Phenolic compounds might modulate adiposity. Here, we report our observation that polyphenols and phenolic acids inhibit adipogenesis in 3T3-L1 with different intensity depending on the family and the stage of differentiation. While quercetin and resveratrol inhibited lipid accumulation along the whole process of differentiation, apigenin and myricetin were active during the early and latest stages, but not intermediate, contrary to hesperidin. The activity of phenolic acids was limited to the early stages of the differentiation process, except p-coumaric and ellagic acids. This anti-adipogenic effect was accompanied by down-regulation of Scd1 and Lpl. Molecular docking analysis revealed that the inhibitory activity of these phenolic compounds over the early stages of adipogenesis exhibits a significant correlation (r = 0.7034, p = 0.005) with their binding affinity to the ligand-binding domain of PPAR&gamma, Results show that polyphenols and phenolic acids would interact with specific residues of the receptor, which could determine their potential anti-adipogenic activity during the early stages of the differentiation. Residues Phe264, His266, Ile281, Cys285 and Met348 are the most frequently involved in these interactions, which might suggest a crucial role for these amino acids modulating the activity of the receptor. These data contribute to elucidate the possible mechanisms of phenolic compounds in the control of adipogenesis.

Recurrent loss of heterozygosity (LOH) in 4q24 in patients with myeloid malignancies has led to the identifi cation of mutations in TET2 in diff erent myeloid neoplasms including myeloproliferative neoplasm (MPN), myelodysplastic syndrome (MDS), mixed myelodysplastic/myeloproliferative neoplasm (MDS/MPN) and acute myeloid leukemia (AML) [1 – 13]. Th eir impact on prognosis remains unresolved, and the mechanisms by which TET2 leads to transformation remain unclear. Based on its homology to TET1, it is possible that TET2 may play a role in epigenetic regulation [14] via the presence of catalytic domains in the two highly conserved regions of the gene. Mutations in these two regions could, by this mechanism, lead to inactivation of a specifi c tumor suppressor gene and/or activation of pro-proliferative pathways, although mutations in other areas of the gene could also result in a modifi cation of the protein. Studies using murine models of Tet2 inactivation show that inactivation of this gene establishes important roles for Tet2 in hematopoiesis and the development of myeloproliferative disease. Beacuse TET2 alterations in patients are commonly heterozygous, TET2 haploinsuffi ciency could be enough to promote defects in a hematopoietic progenitor and myeloproliferation [15]. We have collected data on all TET2 mutations published to date [1 – 13] (a total of 725 mutations in 3274 patients) (Table I) in order to analyze whether: (a) they are evenly distributed throughout the gene, (b) diff erent mutation types (missense, nonsense or frameshift) are more prevalent in diff erent regions of the gene and (c) distinct mutational patterns are found in diff erent types of myeloid neoplasms. To this end, we divided the sequence of the TET2 gene into four regions. Region B1 ( Box 1 ) and region B2 ( Box 2 ) are highly conserved in several species and in all three proteins of the TET family. Th e other two regions, which we denominated F1 and F2, are non-conserved regions (Figure 1). We calculated the frequency of mutations per amino acid in each of these regions (number of mutations divided by the number of amino acids

Core-binding factor acute myeloid leukemia (CBF-AML) is an acute myeloid neoplasm characterized by the presence of t(8;21)(q22;q22) [abbreviated as t(8;21)] or inv(16)(p13q22)/t(16;16)(p13;q22) [in...

Background Despite the discovery of the p.V617F in JAK2 , the molecular pathogenesis of some chronic myeloproliferative neoplasms remains unclear. Although very rare, different studies have identified CBL (Cas-Br-Murine ecotropic retroviral transforming sequence) mutations in V617F JAK2 -negative patients, mainly located in the RING finger domain. In order to determine the frequency of CBL mutations in these diseases, we studied different regions of all CBL family genes ( CBL , CBLB and CBLC ) in a selected group of patients with myeloproliferative neoplasms. We also included V617F JAK2- positive patients to check whether mutations in CBL and JAK2 are mutually exclusive events. Design and Methods Using denaturing high performance liquid chromatography, we screened for mutations in CBL , CBLB and CBLC in a group of 172 V617F JAK2 -negative and 232 V617F JAK2 -positive patients with myeloproliferative neoplasms not selected for loss of heterozygosity. The effect on cell proliferation of the mutations detected was analyzed on a 32D(FLT3) cell model. Results An initial screening of all coding exons of CBL , CBLB and CBLC in 44 V617F JAK2 -negative samples revealed two new CBL mutations (p.C416W in the RING finger domain and p.A678V in the proline-rich domain). Analyses performed on 128 additional V617F JAK2 -negative and 232 V617F JAK2 -positive samples detected three CBL changes (p.T402HfsX29, p.P417R and p.S675C in two cases) in four V617F JAK2 -positive patients. None of these mutations was found in 200 control samples. Cell proliferation assays showed that all of the mutations promoted hypersensitivity to interleukin-3 in 32D(FLT3) cells. Conclusions Although mutations described to date have been found in the RING finger domain and in the linker region of CBL, we found a similar frequency of mutations in the proline-rich domain. In addition, we found CBL mutations in both V617F JAK2 -positive (4/232; 1.7%) and negative (2/172; 1.2%) patients and all of them promoted hypersensitivity to interleukin-3.

We developed a new targeted cationic nanoparticulate system composed of poly(D,L-lactic-co-glycolic acid) (PLGA), 1,2-dioleoyl-3-(trimethylammonium) propane (DOTAP) and asialofetuin (AF), and found it to be a highly effective formulation for gene delivery to liver tumor cells. The nanoparticles (NP) were prepared by a modified solvent evaporation process that used two protocols in order to encapsulate (NP1 particles) or adsorb (NP2 particles) plasmid DNA. The final particles are in the nanoscale range. pDNA loaded in PLGA/DOTAP/AF particles with high loading efficiency showed a positive surface charge. Targeted asialofetuin-nanoparticles (AF-NP) carrying genes encoding for luciferase and interleukin-12 (IL-12) resulted in increased transfection efficiencies compared to free DNA and to plain (non-targeted) systems, even in the presence of 60% fetal bovine serum (FBS). The results of transfections performed on HeLa cells, defective in asialoglycoprotein receptors (ASGPr-), confirmed the receptor-mediated endocytosis mechanism. In summary, this is the first time that asialoglycoprotein receptor targeting by PLGA/DOTAP/DNA nanoparticles carrying the therapeutic gene IL-12 has been shown to be efficient in gene delivery to liver cancer cells in the presence of a very high concentration of serum, and this could be a potential system for in vivo application.