New CXCR1/CXCR2 inhibitors represent an effective treatment for kidney or head and neck cancers sensitive or refractory to reference treatments

Clear cell Renal Cell (RCC) and Head and Neck Squamous Cell Carcinomas (HNSCC) are characterized by a pro-angiogenic/pro-inflammatory context. Despite conventional or targeted therapies, metastatic RCC and HNSCC remain incurable. Alternative treatments to reference therapies (sunitinib, a multi tyrosine kinase inhibitor for RCC or cisplatin for HNSCC) are urgently needed on relapse. Here, we described the relevance of targeting the ELR+CXCL cytokines receptors, CXCR1/2, for the treatment of these two cancer types. Methods: The relevance to patient treatment was evaluated by correlating the ELR+CXCL/CXCR1/2 levels to survival using online available data. We report herein the synthesis of new pharmacological inhibitors of CXCR1/2 with anti-proliferation/survival activity. The latter was evaluated with the XTT assay with leukemic, breast, RCC and HNSCC cell lines. Their relevance as an alternative treatment was tested on sunitinib- and cisplatin- resistant cells. The most efficient compound was then tested in a mouse model of RCC and HNSCC. Results: RCC and HNSCC expressed the highest amounts of CXCR1/2 of all cancers. High levels of ELR+CXCL cytokines (CXCL1, 2, 3, 5, 6, 7, 8) correlated to shorter survival. Among the 33 synthesized and tested molecules, compound C29 reduced ELR+CXCL/CXCR1/2-dependent proliferation and migration of endothelial cells. C29 exerted an anti-proliferation/survival activity on a panel of cancer cells including naive and resistant RCC and HNSCC cells. C29 reduced the growth of experimental RCC and HNSCC tumors by decreasing tumor cell proliferation, angiogenesis and ELR+/CXCL-mediated inflammation. Conclusion: Our study highlights the relevance of new CXCR1/2 inhibitors for the treatment of RCC or HNSCC as first-line treatment or at relapse on reference therapies.


Introduction
Angiogenesis and inflammation are two closely interconnected hallmarks of cancer [1]. Inflammatory conditions induce the production of vascular endothelial growth factor (VEGF) by endothelial cells, tumor associated fibroblasts and tumor cells, resulting in angiogenesis. Hyper vascularization favors the transport of inflammatory cells and acute permeability of the neo-formed vessels facilitates the development of edema. Overall, inflammation induces angiogenesis, and angiogenesis enhances inflammation [2,3].
Therefore, we aimed at developing new molecules to tackle concomitantly these two phenomena. In this study, we focused our attention on the pro-angiogenic and pro-inflammatory ELR + CXCL cytokines that include CXCL1-3, 5-8. These cytokines bind to two seven-transmembrane heptahelical G protein-coupled receptors, namely CXCR1 and CXCR2. Receptor stimulation activates several signaling pathways including the protein kinase C, phospholipase C, PI3K/AKT/mTOR, RAS/RAF/MEK/ERK and NFĸB pathways, leading to tumor cell survival, proliferation, and dissemination [4].
The leading member of the ELR + CXCL cytokines is CXCL8 (also called interleukin 8 or IL-8), which promotes angiogenesis, tumorigenesis, and metastasis [5]. The levels also correlated with tumor burden in several cancer types including prostate, ovarian, brain, and skin cancers [6]. CXCL1 is involved in esophageal, gastric, colorectal and skin cancer cell proliferation [7][8][9][10]. CXCL7 is a key player in the development of renal cell carcinoma (RCC), and in its response to sunitinib [11,12]. Lastly, CXCL5 and CXCL8 correlated to head and neck squamous cell carcinoma (HNSCC) aggressiveness [13,14]. CXCR1/2 are expressed by endothelial and inflammatory cells and are key players in angiogenesis and inflammation, especially in a hypoxic environment [15]. Moreover, the intra-tumor expression of CXCR2 correlated with relapse and poor prognosis of RCC patients with non-metastatic disease (M0) [16].
Thus, we hypothesized that small-sized organic antagonists of the ELR + CXCL that bind to CXCR1/2 might exert a dual activity on both angiogenesis and inflammation. CXCR1/2 competitive and non-competitive inhibitors have been developed mainly for the treatment of pulmonary inflammatory disorders, and recently for advanced metastatic breast cancers [17].
We previously reported that the competitive CXCR1/2 inhibitor, SB225002, delays in vivo tumor growth and inflammation by antagonizing the signaling pathways induced by CXCL7. However, the anti-proliferative effect of SB225002 remains modest in vitro [12]. Therefore, the investigation of more potent analogues is of utmost interest. In line with these observations, two series of new and original N, N'-diaryl ureas and thioureas, featuring a nitro-benzothiazole moiety, were synthesized in an efficient way and evaluated as potential anti-cancer agents in two deadly cancers RCC and HNSCC. Among the 33 synthesized molecules, compound C29 emerged as a promising lead, with interesting dual anti-angiogenic and anti-proliferative activities. Moreover, C29 reduced significantly the growth of experimental tumors in mice. Therefore, this study paves the way for future clinical trials targeting a pathway leading to three major hallmarks of cancers, i.e., tumor cell proliferation, angiogenesis and inflammation.

Chemistry
The N, N'-diarylureas and thioureas (C1-C12) were conveniently synthesized according to a one-step procedure (Figure 1 and Figure S1), that should be used on a large scale for in vivo tests and pilot extrapolation. Briefly, the reaction consists in the nucleophilic attack of substituted anilines on monoor di-substituted isocyanates and isothiocyanates, followed by spontaneous tautomerization. The expected N,N'-disubstituted ureas and thioureas have been obtained in good yields within a short reaction time by simple mixing of reactants at room temperature in the presence of an organic base (Figure 1, see also Supplementary Methods for details).

Molecular simulation studies
The RMN structure of CXCR1 (PDB ID : 2LNL) was retrieved from the Protein Data Bank (PDB) [18]. The RMN structure includes 10 conformations for CXCR1. Each conformation was considered separately, and the 10 resulting CXCR1 structures were prepared using MGL tools [19]. Three-dimensional structures of compound C29 were generated using iCon, the LigandScout v.4.3 conformer generator [20] (defaults settings of the BEST option were used, except for the maximum number of conformations generated that was set to 50 instead of 25). A total of 22 conformers of compound C29 was obtained. Protein -ligand docking of compound C29 into the CXCR1 structure was performed using AutoDock Vina v.1.1.2 [21]. As no information about the CXCR1 binding site of compound C29 was available, a blind docking approach was selected by docking compound C29 to the whole surface of CXCR1, without restriction of the search space. Each of the 22 conformers of compound C29 were docked into each of the 10 CXCR1 structures, resulting in 220 docking runs. For each run, 9 poses of compound C29 bound to CXCR1 were generated. The pose associated with the best score was considered for each run.

Reagents and antibodies
Sunitinib, SB225002 and danirixin were purchased from Selleckchem. Anti-HSP60 antibodies were purchased from Santa Cruz Biotechnology. Anti-AKT, anti-phospho-AKT, anti-ERK, anti-phospho-ERK antibodies were from Cell Signaling Technology. Methanol, ethyl acetate, diethyl ether and dichloromethane were purchased from Carlo Erba. Anhydrous DMF was purchased from Sigma Aldrich. All chemicals were purchased from Aldrich, Fisher or Alfa Aesar and used without further purification.  Tissue Culture  Collection. OCI-AML2, OCI-AML3, Molm13 and  Molm14 acute myeloid cell lines (AML), and K562  chronic  myeloid  cell  line  (CML), SKM1 myelodysplastic cell line (MDS) were a kind gift from Dr. P. Auberger. Two human HNSCC cell lines, CAL33 and CAL27, were provided through a Material Transfer Agreement with the Oncopharmacology Laboratory, Centre Antoine Lacassagne (CAL) where they were initially isolated [22]. Primary RCC cells (CC, TF and 15S) have already been described and cultured in a medium specific for renal cells (PromoCell) [23].

Immunoblotting
Cells were lysed in buffer containing 3% SDS, 10% glycerol and 0.825 mM Na 2 HPO 4 . 30 to 50 μg of proteins separated on 10% SDS-PAGE, transferred onto a PVDF membrane and then exposed to the appropriate antibodies. Proteins were visualized with the ECL system using horseradish peroxidase-conjugated anti-rabbit or anti-mouse antibodies.

Migration assay
CXCL7 or VEGFA-stimulated chemotaxis assays were monitored using modified Boyden chambers containing polycarbonate membranes (8-μm pores, Transwell; Corning, Sigma). Cells were seeded onto the upper side of the filters and chambers were placed on 24-well plates containing CXCL7 (50ng/ml) or VEGFA (50ng/ml). Cell migration was followed for 24 h at 37°C in 5% CO2. Migratory cells on the lower membrane surface were fixed in 3% paraformaldehyde, stained with 0.1% crystal violet.

Colony formation assay
Cells (5000 cells per condition) were treated or not with C29, sunitinib. Colonies were detected after 10 days of culture. Cells were then washed, fixed at room temperature for 20 min with 3% paraformaldehyde (PFA; Electron Microscopy Sciences) and colored with GIEMSA (Sigma).

Caspase assays
Caspase assays have already been described [24]. Briefly, the caspase 3 activity was assessed in quadruplicate using z-DEVD-AMC as substrate and the fluorescence were assessed.

Flow cytometry
CXCR2 measurement: After stimulation, cells were washed with PBS and stained with the CXCR2-PE antibody (Miltenyi) for 30 min at room temperature. Fluorescence was measured using the FL2 (PE) channels of a fluorescence-activated cell sorter apparatus (Calibur cytometer).
Apoptosis analysis: After stimulation, cells were washed with ice-cold PBS and were stained with the annexin-V-fluo staining kit (Roche, Meylan, France) according to the manufacturer's procedure. Fluorescence was measured using the FL2 (AV) and FL3 (propidium iodide, PI) channels of a fluorescence-activated cell sorter apparatus (Calibur cytometer).

XTT
Cells were incubated in a 96-well plate with different effectors for the times indicated in the figure legends.
Fifty microliters of sodium 3′-[1-phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-me thoxy-6-nitro) benzene sulfonic acid hydrate (XTT) reagent was added to each well. The assay is based on the cleavage of the yellow tetrazolium salt XTT to form an orange formazan dye by metabolically active cells. This bio-reduction occurs in viable cells only and is related to NAD(P)H production through glycolysis. Therefore, the amount of formazan dye measured at 490 nm directly correlated with the number of metabolically active cells reflecting cell proliferation and viability. Each assay was performed in quadruplicate.

Cell number -ADAM
For the experiments with C29, we confirmed all the results by evaluating the cell viability. Cell viability and cell death was assessed using the ADAM-MC apparatus (NanoEnTek, Guro-gu, Seoul, Korea) based on fluorescent propidium iodide staining according to the manufacturer's instructions.

Selectivity index
To determine the selectivity of the activity of the substances tested, the selectivity index (SI) was calculated according to the equation previously described [25] where SI = IC 50 of a compound in a normal cell line/IC 50 of the same compound in cancer cell line. The IC 50 is the concentration required to kill 50% of the cell population.

Quantitative Real-Time PCR (qPCR) experiments
One microgram of total RNA was used for the reverse transcription, using the QuantiTect Reverse Transcription kit (QIAGEN, Hilden, Germany), with blend of oligo (dT) and random primers to prime first-strand synthesis. SYBR master mix plus (Eurogentec) was used for qPCR. The mRNA level was normalized to 36B4 mRNA. The sequences of oligo nucleotides used in our experiments are describe in Table S1.

In vitro stability assay of C29
The in vitro stability was determined as followed: 786-O cells were treated with 2.5 µM of compound C29 for the defined time, then lysed with methanol. The lysates were filtered and analyzed by UPLCMS/MS.

Determination of the pharmacokinetic parameters
The in vivo pharmacokinetic parameters were determined in CD-1 mice at a dose of 50 mg/kg after oral administration. The plasma samples (400 µL) were mixed with acetonitrile (1 mL) to precipitate the proteins and extract the compound. After mixing and sonication, proteins were precipitated by centrifugation and the supernatants were analyzed by UPLCMS/MS.

Tumor xenograft experiments
These studies were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals. Our experiments were approved by the ''Comité National Institutionnel d'Ethique pour l'Animal de Laboratoire'' (reference: PEA-255 and PEA-277). Cells were injected subcutaneously into the flank of 5-week-old nude (nu/nu) female mice (Janvier). When the tumor reached 100 mm 3 , mice were treated. The tumor volume was determined with a caliper (v = L*l2*0.5).
Ectopic model of RCC 1-Seven million A498 cells were injected subcutaneously. Mice were treated five times a week for 4 weeks, by gavage with placebo (dextrose water vehicle) or C29 (50 mg/kg).

Ectopic model of HNSCC
One million CAL33 were injected subcutaneously. Mice were treated five times a week for two weeks, by gavage with placebo (dextrose water vehicle), with danirixin (100 mg/kg) or C29 (100 mg/kg) and once a week by intraperitoneal injection for cisplatin (4 mg/kg).

Immunohistochemistry (IHC)
Sections of formol-fixed and paraffin-embedded tumors were incubated with monoclonal anti-Ki67 (clone MIB1, DAKO) or anti-CD31 (clone MEC 13.3, BD Pharmingen) antibodies. A biotinylated secondary antibody (DAKO) was applied and binding was detected with the substrate diaminobenzidine against a hematoxylin counterstain.

Gene expression microarray analysis
Normalized RNA sequencing (RNA-Seq) data produced by The Cancer Genome Atlas (TCGA) were downloaded from cBioportal (www.cbioportal.org, TCGA Provisional; RNA-Seq V2) [26,27]. PFS was defined as the time between surgery and subsequent blood sampling and progression, or death from any cause, censoring live patients and progression free at last follow-up. OS was defined as the time from blood sample collection to the date of death from any cause, censoring those alive at last follow-up. The Kaplan Meier method was used to produce survival curves and significance was assessed using the log-rank test.

Statistical analysis
All data are expressed as the mean ± the standard error (SD). Statistical significance and p values were determined with the two-tailed Student's t-test. One-way ANOVA was used for statistical comparisons. Data were analyzed with Prism 5.0b (GraphPad Software) with a one-way ANOVA with Bonferroni post hoc.

The ELR + CXCL/CXCR pathway is linked to poor prognosis in RCC and HNSCC
The levels of CXCR1/2 were obtained from the database of patients "The Cancer Genome Atlas (TCGA)". Of the different tumors, HNSCC and RCC express the highest amounts of CXCR1 ( Figure S2A). HNSCC is the second and RCC the seventh tumor type expressing the highest amount of CXCR2 ( Figure  S2B).
The relevance of targeting the ELR + CXCL/CXCR1/2 axis was assessed by correlating the intra-tumor mRNA levels of ELR + CXCL cytokines to disease free/progression free (DFS/PFS) (  1A, B). RCC and HNSCC patients with multiple ELR + CXCL mRNA levels above the third quartile had the shortest OS ( Figure S3A-C). OS was even shorter for RCC and HNSCC patients with ELR + CXCL and CXCR1/2 mRNA levels above the third quartile ( Figure S3B-D). These results strongly suggest that CXCR1/2 may represent a relevant target for the treatment of RCC and HNSCC.

Selection of C29 as a lead compound
A small focused chemical library of thirty-three new molecules was synthesized and evaluated for anti-proliferative activity against a panel of human tumor cell lines including breast, head and neck, hematologic and kidney tumor cells. The IC 50 values for each compound were determined and compared to those of SB225002, a CXCR1/2 competitive inhibitor already tested for its effect on the growth of RCC tumor [12], and used as a reference compound; the results are listed in Table 2.
N, N'-diaryureas and thioureas (C1-12) showed limited effect on malignant cells. However, two compounds sharing a common chlorobenzene ring (C9, C12) showed an IC50 in the 10 -20 µmol/L range for solid tumors (breast and kidney), which was higher than the reference molecule SB225002. Other structural changes in the N, N'-disubstituted ureas, i.e., switching from a phenyl derivative to a benzimidazole or benzothiazole motif, resulted in a higher IC 50 (compounds C1-12 vs C13-33). However, the introduction of a benzoxazole ring (C18, C19) into the compounds did not give anti-proliferation/survival activity. Among the most potent derivatives, compounds C16, C25, C28, C29 and C30, exhibited IC50 values below 20 µmol/L for several cancer cell lines. All these hits had a chlorophenyl ring, except compound C30. Among these five hits, only C16 contained an unsubstituted benzimidazole ring, the four other molecules were benzothiazoles substituted at position 6 with a methyl (C25), a nitro (C28, C29) or an ethoxy (C30). Lastly, in the specific case of kidney and head and neck tumor cells, C28 and C29 exerted a dramatic effect on cell proliferation/survival (A498: IC50 5 and 2.5 µmol/L respectively; 786-O: IC 50 5 and 2 µmol/L respectively; CAL27: IC 50 7 and 2.5 µmol/L respectively; CAL33: IC 50 5 and 4 µmol/L respectively), underlying the potency of this class of molecules for treatment of RCC and HNSCC.
Importantly, at a dose of 2.5 µmol/L, C29 did not affect the proliferation/survival of uveal melanoma cells (Mel202), which is consistent with a low level of expression of CXCR1/2 in this tumor type (Figure S2  and S4). Based on this screening, we selected C29 as a lead compound for the following studies. It is worth nothing that C29 features a nitro-benzothiazole moiety that has not been reported before, attesting to the originality of this new class of bioactive compounds.

C29 docking to CXCR1
To address the specificity of C29 for CXCR1/2, we performed a blind docking approach. Several areas of CXCR1 were identified as possible C29 binding sites. However, we noted that 14 out of the 15 top poses ranked according to their AutoDock Vina scores, i.e. the 5% poses with the best scores, were all located at the same binding site. This binding site is a central buried pocket, the volume of which varies among the ten available conformations of CXCR1. The cytokine binding site contains Arg203, a residue previously identified by Alanine scanning experiments to be involved in the CXCR1/CXCL8 interaction [28]. We thus propose a binding model, in which C29 is located in a buried central pocket of CXCR1 close to Arg203 (Figure 2). This finding supports the hypothesis that C29-CXCR1 binding will impede CXCL8 interaction with CXCR1.

C29 inhibited ELR+CXCL-mediated proliferation and migration of endothelial cells
CXCR1/2 are expressed on endothelial cells and participate in ELR + CXCL-mediated angiogenesis. We assessed the ability of C29 to inhibit CXCR1/2 in normal endothelial cells (HuVECs). CXCR1/2 are internalized upon stimulation, a process mainly involved in signal desensitization associated with G protein coupled receptors and resulting in the transfer of receptors from the plasma membrane to the endosomal compartment [29]. Hence, modification of plasma membrane levels reflects, in part, stimulation. Following CXCL7 or CXCL8 stimulation in the presence of C29 (2.5 µmol/L), the CXCR1 or CXCR2 levels at the plasma membrane were increased suggesting that C29 prevented CXCL7/8-dependent internalization of their receptors (Figure 3A-B).
C29 decreased CXCL7-dependent but not VEGFA-dependent migration of HuVECs at the same concentration (2.5 µmol/L). Danirixin, a competitive inhibitor of CXCR2 and to a lesser extent of CXCR1 [30], tested in phase II clinical trials for the treatment of Respiratory Syncytial Virus (RSV) infections, had no effect at this concentration ( Figure 3C, Figure S5). This result suggests stronger efficacy of C29 compared to danirixin. C29 also inhibited basal and CXCL5/CXCL7-dependent HuVECs viability ( Figure  3 D-E), which was consistent with inhibition of the activity of basal and CXCL5-dependent ERK/MAP Kinase signaling pathway ( Figure 3F). Hence, C29 has a strong inhibitory effect on an alternative angiogenic pathway that compensates for the inhibition of the VEGF/VEGFR axis [31].

C29 is equally efficient on naive and resistant RCC and HNSCC cells
The first-line treatments for RCC and HNSCC are respectively, sunitinib and cisplatin but relapse is ineluctably observed. Hence, our objectives were to; i) compare the efficacy of reference treatments to that of C29; ii) evaluate if C29 may be relevant at relapse as current standard of care. Dose responses and time courses were performed on naive cells and on sunitinib [32] and cisplatin-resistant cells to compare the C29 efficiency. C29 inhibited the viability of sensitive 786-O RCC cells as efficiently as sunitinib (786, Figure 4A-E, Figure S6A). The effect of C29 on cell viability was conserved in sunitinib-resistant 786-O cells (786R, Figure 4B-F, Figure S6A). The effect of C29 on CAL27 HNSCC cell viability was greater than that of cisplatin (Figure 4C-G, Figure S6B), and the effect was equivalent for cisplatin-resistant CAL27 cells (CAL27R, Figure 4D-H, Figure S6B). Compared to C29, the reference CXCR1/2 inhibitor SB225002 had a modest effect on RCC and HNSCC cells ( Figure  S7). C29 inhibited the ERK/MAP Kinase and PI3K/AKT signaling pathways, two major pathways involved in cell proliferation, of naive and resistant RCC and HNSCC cells (Figure 5A-B). This result suggests that ELR + CXCL cytokines produced by RCC [31] and HNSCC [33] cells stimulate a CXCR1/2-dependent autocrine proliferation loop. C29 induced cell death in sensitive and resistant cells ( Figure 5C-D). The apoptotic cell death induced by C29 was confirmed by an increase in the caspase 3 activity in both cell types (Figure 5E-F).  To further explore the effect of C29 we compared its activity on tumor kidney cells to that on primary normal cells [34]. C29 significantly decreased the proliferation of primary kidney tumor cells (CC, TF, Figure S8A-B) but had no effect on primary normal kidney cells (15S), even when C29 was used at a higher concentration (5 µmol/L). FACS analysis detected apoptotic markers in TF and CC cells in the presence of C29 at 1 µmol/L, which was not the case for normal cells (15S, Figure S8C). The results with primary cells were similar to those described above for kidney cell lines.
We then calculated the selectivity index (SI) to evaluate the selectivity to the targets. The normal primary kidney cells (15S) served as the reference value. The SI was superior to that of Mel202 for all tumor cells (Table 3), which is in favor of a specific effect on CXCR1/2 receptors.

C29 inhibited the growth of experimental RCC and HNSCC
To be confident about using C29 for in vivo experiments in mice, we first tested its stability by performing UPLC/HRMS analyzes on the 786-O cells. No degradation of C29 was observed after a 24 h-treatment at room temperature (Figure 6A), attesting to its high stability in cellulo. C29 was then formulated at 7.6 mg/mL and administrated by oral gavage at 50 mg/kg. C29 exhibited a half-life of 190 min, combined with a C MAX of 0.9 µg/ml at 30 min ( Figure 6B). Global exposure remained high and the AUC was close to 85000 min.ng/m. These results prompted us to evaluate C29 on the growth of experimental tumors in mice.
C29 slowed-down the growth of experimental RCC or HNSCC with a decrease of more than 65% of the tumor volume at the end of the experiment (Figure 7A-8A). This result correlated with the decrease in the tumor weight of 40 or 65% for RCC and HNSCC, respectively (Figure 7B-8B). Moreover, we did not observe any weight loss of the animals in the treated group, which suggests that this molecule did not exert acute toxicity (Figure 7C-8C). C29 decreased the proliferation marker Ki-67 ( Figure  7D-8D) and inhibited PI3K/AKT but not the ERK pathway in experimental RCC (Figure 7E-F) and both pathways in experimental HNSCC (Fig. 8E-F). The mRNA level of murine CD31, a relevant marker of blood vessels, was decreased for both tumor types (Figure 7G-8G). The mRNA levels of ERL + CXCL cytokines (CXCL5/7/8), but not of VEGFA, were significantly decreased by C29 in experimental RCC ( Figure 7H-K). Only CXCL5 was decreased in experimental HNSCC (Figure 8H-K). These results were consistent with the decrease in blood vessels visualized by the down-regulation of CD31 levels on IHC (Figure 7G-8G).
The concentration in blood (CMAX) of C29 once administered by oral gavage was 2.6 µmol/L (comparable to the in vitro IC 50 ), which means that a small amount of the drug crossed the gastric barrier. Therefore, we tested the efficacy of C29 administered by intra-peritoneal injection. Three injections per week of 100 µg C29 was as efficient as a dose of 50 mg/kg administered by oral gavage (Figure S9A-B). An equivalent efficacy was obtained with danirixin at 200 µg three times a week or sunitinib 50 mg/kg by oral gavage five times a week (Figure S9C-D). C29 administered by oral gavage at a dose of 100 mg/kg five times a week was more efficient than danirixin, at the same dose, or than the reference treatment with cisplatin on experimental HNSCC (Figure S9E-F). C29 treatment (100mg/kg by oral gavage five times a week for three weeks) did not induce hematological, renal or hepatic toxicity (Figure S10A-D). These results strongly suggest that C29 may represent a relevant therapeutic tool for RCC and HNSCC.

Discussion
The therapeutic options for RCC and HNSCC at a metastatic stage are anti-angiogenic drugs or immunotherapy, alone or in combination [35,36], and a combination of radio/chemotherapy, respectively [37]. Despite an increase in time to progression, the patients ineluctably relapse within a few months [38]. We have already demonstrated that the CXCL/CXCR axis is involved in relapse of RCC patients [12,33]. We report herein the identification and biological characterization of a new series of CXCR1/2 inhibitors with significant anti-cancer effects. The lead compound of this series, C29, inhibited tumor cell viability and angiogenesis, two major hallmarks of aggressive cancers [39]. This study highlighted the possibility of targeting CXCL/CXCR crosstalk using a potent CXCR1/2 inhibitor, as a relevant therapeutic option to treat incurable RCC and HNSCC. C29 showed higher efficacy in two independent tumor types as compared to the older generation of competitive inhibitors SB225002 or danirixin.
Targeting the CXCL/CXCR signaling pathway induced; i) inhibition of proliferation of tumor and endothelial cells, ii) tumor cell death, and iii) inhibition of tumor vascularization. A high expression level of ELR + CXCL cytokines and their receptors in RCC and HNSCC suggested that CXCR inhibitors may be considered as relevant first-line treatment. Since C29 may represent an option on relapse when on reference therapies early phase clinical trials can be rapidly performed. The rationale in the case of therapeutic failure is sound for both tumors.
We observed that ELR + CXCL cytokines were induced in response to the reference anti-angiogenic treatment sunitinib in RCC [24] and in response to radiotherapy in HNSCC [26]. These results represent a relevant rationale to administer CXCR inhibitors on relapse.
RCC and HNSCC are inflamed tumors and therefore eligible for immunotherapy. Indeed, immunotherapy improved the outcome of RCC [35,40] and HNSCC [41]. However, only 30% of patients benefit from these treatments [35,42]. The presence of myeloid-derived suppressor cell (MDSC) partly explains the limited effect of immunotherapies in some patients [31,33]. A correlation between the intra-tumor expression of CXCL5, CXCL8 and IL1β, and the presence of MDSC, creating an immuno-suppressive environment, has been recently highlighted in RCC [43]. Moreover, we demonstrated the IL1β controls the expression of CXCL7, one of the main CXCL cytokines involved in RCC aggressiveness [12]. Thus, by decreasing CXCR-expressing MDSC, C29 might reactivate the anti-tumor immune response. In addition to its anti-viability and anti-angiogenic effects, C29 may serve indirectly as an immune check point inhibitor and can also sensitize tumors to immunotherapy by decreasing the level of cytokines favoring anergia. Therefore, C29 has the ability to target four hallmarks of cancers; proliferation of tumor cells, tumor angiogenesis, chronic inflammation and immune-tolerance. Our efforts are currently directed towards the validation of this hypothesis, and the results will be reported in due course. upon work form COST Action CA15135, supported by COST. This work was performed using the animal and cytometry (CYTOMED) facilities of IRCAN. The materials of CytoMed were supported by the Conseil Général 06, the FEDER, the Ministère de l'Enseignement Supérieur, the Région Provence Alpes-Côte d'Azur and the INSERM.