Tumor targeting by genetically modified mesenchymal stromal/stem cells (MSCs) carrying anti-cancer molecules represents a promising cell-based strategy. We previously showed that the pro-apoptotic agent tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can be successfully delivered by MSCs to cancer sites. While the interaction between TRAIL and its receptors is clear, more obscure is the way in which MSCs can selectively target tumors and their antigens. Several neuroectoderm-derived neoplasms, including glioblastoma (GBM), sarcomas, and neuroblastoma, express high levels of the tumor-associated antigen GD2. We have already challenged this cell surface disialoganglioside by a chimeric antigen receptor (CAR)-T cell approach against neuroblastoma. With the intent to maximize the therapeutic profile of MSCs delivering TRAIL, we here originally developed a bi-functional strategy where TRAIL is delivered by MSCs that are also gene modified with the truncated form of the anti-GD2 CAR (GD2 tCAR) to mediate an immunoselective recognition of GD2-positive tumors. These bi-functional MSCs expressed high levels of TRAIL and GD2 tCAR associated with a robust anti-tumor activity against GD2-positive GBM cells. Most importantly, the anti-cancer action was reinforced by the enhanced targeting potential of such bi-functional cells. Collectively, our results suggest that a truncated anti-GD2 CAR might be a powerful new tool to redirect MSCs carrying TRAIL against GD2-expressing tumors. This affinity-based dual targeting holds the promise to combine site-specific and prolonged retention of MSCs in GD2-expressing tumors, thereby providing a more effective delivery of TRAIL for still incurable cancers. Human mesenchymal stromal/stem cells (MSCs) are considered pivotal players in cellular therapy. Their isolation easiness from different sources and their capacity to be engineered by viral vectors make them cellular vehicles to deliver anti-tumor agents . As putaGlioblastoma (GBM) is the most common primary malignant brain tumor in human. Despite considerable advances in therapies, GBM remains one of the most challenging diseases . The invNew effective tools are needed to specifically target cancer cells within a context characterized by a high local metastatic potential . It has Therefore, parallel to homing, particular attention is needed on how to improve MSC affinity for the tumor site in order to achieve tumor-specific targeting and retention . Much ofThis targeting takes advantage of surface molecules uniquely or highly expressed by tumor cells. Regarding GBM, it has been demonstrated that a variety of GBM lines and primary biopsies commonly express high levels of disialoganglioside GD2 antigen, similarly to several pediatric and adult cancers , 17. GD2In recent years, our group developed T cells expressing anti-GD2 CAR . CAR affTaking inspiration from CAR T cells as successful examples for the specific recognition and binding to target cells, we originally further optimized tumor-localizing potential of TRAIL-expressing MSCs. Thus, we combined anti-GD2-targeted strategy where TRAIL is expressed on AD-MSCs together with a truncated form of a previously developed anti-GD2 CAR (GD2 tCAR) . This GDl-glutamine (200 mM) , and 1% penicillin–streptomycin . Human AD-MSCs were obtained as previously described [l-glutamine, 0.5% ciprofloxacin , and 0.2% heparin . Cells were incubated and maintained within a controlled atmosphere, 5% CO2, and temperature of 37 °C. Authentication of T98G, U87MG, and A172 cell lines has been recently performed by the Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany.Human GBM cell lines T98G, U87MG, and A172 are a kind gift from Dr. Franco Bambi . The primary C3c GBM cell line was obtained from a patient affected by GBM  and collected at the Department of Neurosurgery, Nuovo Ospedale Civile S. Agostino Estense, Modena, Italy. Tissue procurement was approved by the local Ethical Committee (#3600/27 September 2017). T98G and U87MG cell lines were cultivated in minimum essential medium (MEM) Eagle's with Earle's salts . A172 were maintained in Dulbecco’s modified Eagle's medium . The primary C3c GBM cell line was kept in DMEM/F-12 (Gibco). Media were supplemented with 10% fetal bovine serum , 1% BamHI and SalI restriction enzymes. Properly sequenced full-length human TRAIL gene has been cloned  into the lentiviral vector pCCL PGK WPRE to gene modify AD-MSCs for the surface expression of membrane-bound TRAIL (mTRAIL) molecule -conjugated anti-TRAIL antibody . Intracellular staining on transduced MSCs was performed with Becton Dickinson Cytofix/Cytoperm Kit . To detect GD2 tCAR expression on AD-MSCs, anti-idiotypic antibodies were raised as previously described , 24. AD-2. After 12 h, different concentrations  of recombinant human TRAIL (rhTRAIL)  were added in the culture media. Proliferation rate was assessed after 24 h by the colorimetric assay CellTiter 96 AQueous One Solution Cell Proliferation Assay . Alternatively, tumoricidal activity of rhTRAIL was evaluated by supravital propidium iodide  staining after 24 h by FACS.Tumor cell lines were seeded in 96-well cell culture plate at 6000 cells/cm2. After 12 h, all gene-modified MSCs were labeled with carboxyfluorescein succinimidyl ester (CFSE) cell tracker  and added at different target-to-effector (T:E) ratios . The tumoricidal activity of MSC-expressing TRAIL was evaluated by PI or Annexin V (BD)/PI staining after 12, 24, and 48 h by FACS gating on CFSE-negative cells and PI-positive or Annexin V/PI-double-positive events. Soluble rhTRAIL at 1 μg/ml was introduced as a positive control.Tumor cell lines were seeded in 12-well cell culture plate at 6000 cells/cmThe cell-to-cell interaction assay was established by the selective target cell recognition mediated by GD2 tCAR-functionalized MSCs against GBM cell lines that markedly differ for GD2 expression levels. A monocistronic murine stem cell virus-derived retroviral vector encoding for the red fluorescent protein DsRed was used to stably transduce GBM cell lines  and to distinguish them from MSCs into the cell-to-cell interaction assay. Retrovirus production was performed by FLYRD18 packaging cell line, as published . The priSimilarly, MSCs were labeled with the CFSE fluorescent dye according to the reagent’s manual. A cell-to-cell interaction assay was performed as described. CFSE-labeled MSCs were seeded into a 12-well cell culture plate at a high density  to create a confluent monolayer in 12 h. To test cell-to-cell interactions, a suspension of DsRed (or Deep Red for primary C3c GBM) tumor cells  were added to the MSC monolayer. After 1 h and half incubation time, media were removed and the coculture were washed twice in phosphate-buffered saine (PBS) and shaken for 3 min in order to lose weakly adherent tumor cells. An incubation time of 20 min has been identified as optimal for the primary C3c GBM line, as this line has a greater basal adherence to MSCs in comparison to that of GBM lines. The cells were then collected by trypsinization and suspended in a constant volume of PBS. The absolute number of MSC–tumor cell aggregates was quantified by FACS considering the CFSE/DsRed (or CFSE/Deep Red)-double-positive population in a constant time frame of 60 s. Data were expressed comparing the number of MSC–tumor cell aggregates acquired for all the conditions versus EV MSCs.2, and then used to set up a cell-to-cell interaction assay as described above. The percentage of Annexin V-positive MSC-GBM cell aggregates at a 7 h incubation was analyzed by FACS.A cell-to-cell interaction cytotoxicity assay was further challenged to detect the bi-functional MSC-specific cytotoxic effect against GD2-positive GBM cells at an early time point of 7 h incubation. The early cell death in MSC-GBM cell aggregates was evaluated by Annexin V staining and FACS analysis gating on CFSE/DsRed-double-positive conglomerates recording a fixed number of events, for all the conditions. Data were expressed as the percentage of Annexin V-positive MSC-GBM cell aggregates. Anti-idiotype mice sera against GD2 tCAR were further used to block the GD2 tCAR-mediated interaction between bi-functional MSCs and GD2-expressing GBM cells and the effect on apoptosis was investigated. Briefly, MSCs were incubated with a 1:20 dilution of mice sera for 40 min at 37 °C, 5% COp value of ≤ .05 from two-tailed Student’s t test was considered statistically significant. Normal distribution of data has been tested using Shapiro–Wilk normality test. For the assays, each experimental group was assayed at least twice in triplicate.Analyses were performed using Excel 2016 program . Data are expressed as mean ± SD. A Co-expression of GD2 tCAR together with mTRAIL in MSCs was obtained by lentiviral vector transduction. The presence of mTRAIL and GD2 tCAR molecules was verified by FACS on transduced MSCs Fig. . TRAIL aHaving generated the effectors, in order to challenge our cell therapy approach against three different GBM cell lines, we began testing both GD2 and TRAIL receptors, as predictive factor for affinity-based targeting and TRAIL sensitivity. FACS analyses revealed that the three selected GBM cell lines differ for GD2 expression Fig. . Specifip < .05) and U87MG the lowest one , after 24 h at the highest rhTRAIL dose (1 μg/ml).On TRAIL receptors expression Fig. , FACS anp < .001), provoking a mortality rate comparable to mTRAIL MSCs or rhTRAIL . The GBM cell apoptosis by bi-functional MSCs was further confirmed at 48 h . For all tested culture conditions and time points, cocultures of GBM cell lines with EV MSCs or GD2 tCAR MSCs did not impact tumor cell survival.Having proved the sensitivity of GBM lines to TRAIL pro-apoptotic effect, we performed cocultures to verify the effectiveness of our cell therapy strategy based on genetically modified bi-functional MSC-expressing mTRAIL and GD2 tCAR. The killing potential mediated by bi-functional MSCs was assessed testing different time points (24 and 48 h) and multiple target to effector ratios . Similarly to what observed for cell lines, EV MSC or GD2 tCAR MSC cocultures did not impact cell survival on primary C3c cells.The killing by bi-functional MSCs was further challenged against the primary C3c GBM line Fig. . Bi-funcThese in vitro data outline a significant, although variable, sensitivity of GBM towards MSCs delivering TRAIL. In particular, we demonstrated that the simultaneous expression of pro-apoptotic TRAIL ligand and GD2 tCAR does not affect the killing by engineered MSC, thus prompting our dual-targeting approach against GD2-postitive GBM.We hypothesized that MSCs surface functionalization with the GD2 tCAR is able to force interaction between MSCs and GBM cells expressing GD2. To investigate the new properties acquired by bi-functional MSC, we tested their ability to bind GBM in a cell-to-cell interaction assay. For this purpose, MSCs were labeled by CFSE, to acquire a green fluorescence; conversely, GBM cell lines were stably infected with viral vector coding for DsRed gene to exhibit a red fluorescence, while the primary C3c GBM line was labeled by the CellTracker Deep Red dye.By these different labeling strategies, MSCs and GBM cells were clearly distinguishable by FACS and their aggregates appeared as double positive for both CFSE and DsRed (or Deep Red) fluorescence. In a cell-to-cell interaction assay GBM cells were added to CFSE-labeled MSC monolayers and left in interaction for 1.5 h (or 20 min for primary C3c cells).p < .001; Fig. p < .01; Fig. p < .05; Fig. After sequential washing to lose weakly interactors, cells were collected by trypsinization and analyzed by FACS to determine the absolute number of MSC-GBM cell aggregates, visualized as CFSE/DsRed (or CFSE/Deep Red) double-positive events. The related gating strategy is described in Fig. p < .001) and non-expressing TRAIL ones, EV MSCs and GD2 tCAR MSCs  by Annexin V on CFSE/DsRed double-positive aggregates. A gating strategy Fig.  was applp > .05; Fig. Blocking the GD2-mediated interaction by anti-idiotype sera incubation , 24, bi-We have here explored how engineered human MSCs by a truncated IgM-derived anti-GD2 CAR can be a powerful new tool to specifically redirect MSCs delivering TRAIL against a GD2-expressing tumor, such as GBM. The invasive nature of GBM is still the major reason for failure in surgical resection and after standard therapeutic approaches . The tumBy multiple in vitro assays, we explored whether the native tumor tropism of MSCs can be enhanced by engineered targeting via expression of a tumor-binding AR. First, we investigated the feasibility of expressing the GD2 tCAR on the cell surface of adipose MSCs by lentiviral transduction, in order to specifically target GD2-expressing tumor cells. The transduction of AD-MSCs with the vector encoding for GD2 tCAR was very efficient and did not interfere with the simultaneous expression of TRAIL.Previous works gave evidences of MSCs localization into glioma after systemic delivery or local intracranial injection followed by migration towards GBM , 29. OncConsidering this MSCs tropism for brain tumors, we hypothesized that strengthening adherence to tumor cells by GD2 tCAR could enhance bi-functional MSCs retention within GD2-positive GBM tumor bed while potentially sparing the normal brain tissue. This in turn may affect the efficacy of our cell-based approach, as pre-requisite for a local delivery of bi-functional MSCs into GBM site Fig. . Three dAll tested GBM cell lines were highly positive for the agonistic receptor DR5, implying a possible responsiveness to TRAIL apoptotic effect and did not express DcR1 and DcR2, exception made for U87MG where intermediate levels of DcR2 were detected, as previously described by Röhn et al. . For insLooking at first to cytotoxicity, bi-functional MSCs effectively killed all GBM lines in coculture assay; however, the effect was stronger against T98G and A172 than U87MG line. We assume that the lower sensitivity displayed by U87MG could be due to different molecular mechanisms impacting on the TRAIL pathway which need to be better investigated. For instance, a study by Qiu et al.  indicateIn parallel to the assessment of cytotoxicity, we investigated whether the GD2 tCAR was capable to improve MSCs affinity for GBM, hence extending TRAIL-mediated therapeutic effect and limiting off-target toxicities. Thus, we have challenged bi-functional MSCs ability to bind GD2-expressing GBM cells in a cell-to-cell interaction assay demonstrating that the number of MSC-GBM cell aggregates, collected after interaction, strictly correlated with both GD2-aboundance on GBM cell lines and the MSCs capability to interact via GD2 tCAR surface expression. The bi-functional MSCs selectively bound the highly T98G and the middle U87MG GD2-expressing lines at a higher extent than mTRAIL MSCs. Similarly, by challenging bi-functional MSCs interaction with primary C3c cells, we found a significantly higher number of aggregates compared to mTRAIL MSCs. For the A172 GD2-negative line, no differences in aggregates formation were detected, suggesting that MSCs functionalization by the anti-GD2 AR is associated with increased and specific binding restricted to GD2-expressing GBM cells.The cell-to-cell interaction cytotoxicity assay further demonstrated that the enhanced binding by bi-functional MSCs led to a more effective killing activity against GD2-positive T98G cells, compared to that mediated by mTRAIL MSCs. As a further confirmation, blocking the GD2-mediated interaction by bi-functional MSCs incubation with mice sera against GD2 tCAR restored a killing potential comparable to that observed with mTRAlL MSCs. These data demonstrated that GD2 tCAR is able to force the interaction between bi-functional MSCs and GBM-expressing GD2 antigen, potentiating the mTRAIL-mediated cytotoxic effect owing to a selective binding to GD2-positive GBM cells. Nevertheless, an optimization within a relevant animal model shall be necessary in order to better take advantage of the specific GD2 tCAR-binding capability to the GD2 antigen and to evaluate the therapeutic benefits of bi-functional MSCs carrying both GD2 tCAR and TRAIL.Targeted delivery of therapeutics has been so far restricted to lymphocytes based on CAR or bispecific adaptors . Recentl


RhoB) seems to be decisive for the ability of the conjugate to accumulate in mitochondria. So far, several triterpenoid rhodamine B conjugates have been prepared and screened for their cytotoxic activity. To obtain cytotoxic compounds with EC50 values in a low nano-molar range combined with good tumor/non-tumor selectivity, the Rho B unit has to be attached via an amine spacer to the terpenoid skeleton. To avoid spirolactamization, secondary amines have to be used. First results indicate that a homopiperazinyl spacer is superior to a piperazinyl spacer. Hybrids derived from maslinic acid or tormentic acid are superior to those from oleanolic, ursolic, glycyrrhetinic or euscaphic acid. Thus, a tormentic acid-derived RhoB conjugate 32, holding a homopiperazinyl spacer can be regarded, at present, as the most promising candidate for further biological studies.The combination of the “correct” triterpenoid, the “correct” spacer and rhodamine B ( Cancer remains one of the leading causes of death worldwide, and the incidence is increasing. Cancer is the second-leading cause of death globally, accounting for 9.6 million deaths in 2018 ,4,5,6. TBoth proteins and small molecules have proven their worth in therapy and are in use in the clinic. However, low-molecular-weight drugs often bear the stigma of reduced selectivity in which the cytotoxic drug not only targets cancerous cells, but it also damages healthy tissue. This increases serious side effects and stress symptoms in patients, including nausea, heart or brain damage, impairments to the central nervous system and damage to cells of the inner ear; losses of fertility, hearing and hair have also been noted .These serious side effects and impairments limit the use and acceptance of a drug, as they reduce patient compliance due to a significantly reduced quality of life. This not only endangers the chances of successful therapy, but it often also leads to discontinuation of the therapy ,10.Since the problems of reconversion of cancer cells into normal cells   have onlMitocans (as well as other cytotoxic agents) are often able to induce apoptosis; however, the death of a cell, irrespective of whether this cell is malignant, is not random at all. Triggering of controlled cell death is always preferable to an unselective rupture of membranes following the application of extreme but locally applied heat, freeze–thaw cycles or steep osmotic gradients. Controlled cell death can be triggered on a cellular level from nuclear, reticular, cytoskeletal, lysosomal, membrane or, most important, mitochondrial origins .Usually, the cells in a living organism closely cooperate, and cells are constantly in an equilibrium between life and death. Triggering programmed cell death routines removes damaged, infected and out-of-control cells from the organism. The problem arises from the latter cells, since most cancer cells do not respond to extrinsic apoptotic triggers. Thus, mitochondria present a target of emerging interest for cancer therapy as they can trigger apoptosis through an intrinsic pathway. Apoptosis usually starts with loss of mitochondrial membrane potential, followed by the release of cytochrome c and activation of caspase 3 . FurtherIn recent years, triterpenes have repeatedly and increasingly entered the focus of scientific interest. Extensive studies on their apoptotic and cytotoxic properties have been performed. A major concern in dealing with cancer is the MDR (multiple drug resistance) phenotype . These cN-oxides . In. In93]. 11, the S phase peak and the subG1/apoptosis peak increased significantly. However, for all compounds the selectivity between tumor cells and non-malignant fibroblasts NIH 3T3 was small and never exceeded 7.3  [A closer look at the cell cycle by FACS (with A2780 cells) showed a decrease of the G1 and G2/M peak with an increase of cells in the S phase. For cells treated with 7 cells) .13–16 (RhoB with yields between 47 and 50% [A similar behavior was observed for dehydroabietylamine (DHAA) derivatives 13–16 , Table 3 and 50% .16a/16b . From a photo-induced ring opening reaction, 16b was obtained from 16a very quickly within 10 s of irradiation either with visible light or with UV light (λ = 254 or 366 nm). This equilibrium is also strongly influenced by changes in temperature, and at room temperature 16a dominates the equilibrium [Although the cytotoxicity of these compounds was good, their pharmacological potential was restricted by low selectivity values. Interestingly enough, products 16a/16b , having c at all . As mentilibrium .RhoB conjugates are concerned, the RhoB moiety can be attached to the triterpenoid scaffold either directly  or with the aid of a suitable spacer. Pentacyclic triterpenoic acids , but it was not selective for human tumor cells. Extra staining experiments showed this compound to be accumulated in the mitochondria of A2780 cells and to act mainly by apoptosis [All of these compounds had EC 15.8 μM ; therebypoptosis .OA, UA or BA. Extensions in the design of these compounds led to the synthesis of triterpene conjugates with further modifications in the backbone (→ tormentic acid (TA) and euscaphic acid (EA)) as well as to changes in the ring size of the heterocyclic spacer between the backbone of the triterpene and the RhoB moiety  proved to be clearly superior to the piperazinyl moiety (as in 31). A similar trend was also noted for EA-derived compounds 29 and 30. On the other hand, TA-derived compounds were more cytotoxic than the corresponding EA derivatives. Interestingly, the absolute configuration at C–2 and C–3 in TA corresponds exactly to the configuration found in maslinic acid (MA). Several MA derivatives . Again, its acetylated piperazinyl-rhodamine B conjugate 33 was most cytotoxic to many human tumor cell lines, being accumulated in the mitochondria, and it also acted as a mitocan [The same configuration is found in asiatic acid  is given in 32  becomes clearly visible, making this compound an interesting drug for advanced testing and biological screening.From OA-derived RhoB conjugates appear to be superior to analog UA-derived compounds in the majority of cases with respect to their cytotoxicity. Although AKBA-derived derivatives have good cytotoxicity properties, they were found to be less cytotoxic compared to other triterpene carboxylic acid derivatives, but they often showed better tumor cell/non-tumor cell selectivity. So far, the best cytotoxicity properties have been found for MA-, EA- and TA-derived derivatives. These allowed the transition to compounds of nano-molar activity, while many other triterpene carboxylic acid derivatives were cytotoxic only on a micro-molar concentration range. MA- derived derivatives seem to be approximately equivalent to EA-derived compounds. They are currently only surpassed in many tumor cell lines only by the analogous derivatives from TA. From results available so far, it can be concluded that compounds holding a homopiperazinyl spacer are superior to those with a piperazinyl spacer. This underlines the importance of the spacer for obtaining good cytotoxicity properties. Replacement of the secondary amide derived spacer by a primary amine like ethylenediamine has invariably led to RhoB conjugates of insignificant cytotoxicity (EC50 > 30 μM) due to the formation of a spirolactam holding no positive charge in the RhoB part.RhoB part has been replaced by, for example, malachite green, a BODIPY residue or a safirinium group. In addition, the latter compounds do not act as mitocans, since their primary target is the endoplasmic reticulum.However, the presence of a distal cation is not sufficient to obtain compounds with excellent cytotoxicity, as has been shown for several quaternary ammonium compounds or compounds where the RhoB group with another rhodamine has a positive effect on biological activity cannot be made at present. The cytotoxic properties of these compounds, other spacers and other triterpene carboxylic acids are currently the subject of further investigation. The combination of the “correct” parent structure, the “correct” spacer and the “correct” RhoB seems to be decisive for the ability of the conjugate to accumulate in mitochondria. So far, a tormentic acid acid-derived RhoB conjugate 32 holding a homopiperazinyl spacer can be regarded as the most promising candidate for further biological studies. At present, no extended investigations have been carried out on the precise mode of action of these molecules.A statement on the extent to which the replacement of the


One of the major limits of chemotherapy is depending on the ability of the cancer cells to elude and adapt to different drugs. Recently, we demonstrated how the activation of the M2 muscarinic receptor could impair neuroblastoma cell proliferation. In the present paper, we investigate the possible effects mediated by the preferential M2 receptor agonist arecaidine propargyl ester (APE) on drug resistance in two neuroblastoma cell lines, SK-N-BE and SK-N-BE(2C), a sub-clone presenting drug resistance. In both cell lines, we compare the expression of the M2 receptor and the effects mediated by the M2 agonist APE on cell cycle, demonstrating a decreased percentage of cells in S phase and an accumulation of SK-N-BE cells in G1 phase, while the APE treatment of SK-N-BE(2C) cells induced a block in G2/M phase. The withdrawal of the M2 agonist from the medium shows that only the SK-N-BE(2C) cells are able to rescue cell proliferation. Further, we demonstrate that the co-treatment of low doses of APE with doxorubicin or cisplatin significantly counteracts cell proliferation when compared with the single treatment. Analysis of the expression of ATP-binding cassette (ABC) efflux pumps demonstrates the ability of the M2 agonist to downregulate their expression and that this negative modulation may be dependent on N-MYC decreased expression induced by the M2 agonist. Our data demonstrate that the combined effect of low doses of conventional drugs and the M2 agonist may represent a new promising therapeutic approach in neuroblastoma treatment, in light of its significant impact on drug resistance and the possible reduction in the side effects caused by high doses of chemotherapy drugs. Neuroblastoma (NB) is the most common type of solid extracranial tumor, with occurrence in childhood and showing a differentiated clinical behavior . The feaAlthough chemotherapy represents one of the most common treatments for neuroblastoma, chemoresistance tends to emerge as a major obstacle in its pharmacological treatments, as it does in many other forms of cancers . Thus, dNowadays, we know that chemoresistance is caused by the acquisition of a phenotype commonly known as MDR, determined by multiple factors, such as quantitative or qualitative alterations of the intracellular targets alteration, in response to cytotoxic damage or activation of detoxification systems in the cells ,6,7. NotStudies conducted in our laboratory have demonstrated that muscarinic receptors play relevant roles both in physiological and pathological conditions ,19,20,21More recently, we expanded this list including neuroblastoma, focusing our attention on the M2 muscarinic receptor subtype . What stThe results previously obtained have led us to investigate a possible involvement of M2 muscarinic receptors in chemoresistance processes. We used two neuroblastoma cell lines, the parental cell line SK-N-BE and one sub-clone resistant SK-N-BE(2C), characterized by a missense mutation of p53 in exon 5, with cysteine substitution in phenylalanine . In thesWe firstly evaluated the expression of muscarinic receptors in SK-N-BE and SK-N-BE(2C) cell lines by RT-PCR analysis. Both cell lines express all five muscarinic receptors at the transcriptional level, although the SK-N-BE(2C) cell line shows higher levels of the M1 and M3 subtypes A. ConsidPrevious data indicated that the selective activation of M2 receptors by the agonist APE caused a decreased cell proliferation in two different neuroblastoma cell lines and in neuroepithelioma cells (43). For this reason, we compared the response of SK-N-BE and SK-N-BE(2C) cell lines, characterized by a different resistance grade to pharmacological treatments, to the M2 agonist APE, in terms of cell proliferation. By MTT assay, we demonstrated that the APE treatment was able to reduce cell growth in a dose-dependent manner A,B. HoweIn order to demonstrate that the inhibition of cell growth was reversible, a recovery assay was performed. Cells were, thus, treated for 48 h with 50 or 100 µM APE, then the agonist was removed and a fresh medium was added to the cells. The cell number was assessed by cell count at 48 and 72 h after the M2 agonist withdrawal. Both cell lines were able to rescue the proliferation upon 50 µM APE treatment. Conversely, following treatment with 100 µM APE, cell proliferation was recovered only by SK-N-BE(2C), albeit at a lower proliferative rate than untreated cells C,D.To test our hypothesis regarding the M2 agonist’s ability to control cell proliferation in both neuroblastoma cell lines, we performed a FACS analysis in both cell lines that were kept either in the presence or absence of 100 μM APE at different experimental time points . As indicated in To explain the cause of these different responses, especially in terms of cell cycle perturbation, we analyzed the expression of the p53 protein. As reported in In order to test for potential chemoresistance, in particular for SK-N-BE(2C), we performed a chemosensitivity tests that allowed us to evaluate the pharmacological responses of both cell lines to conventional chemotherapy drugs, such as doxorubicin and cisplatin, and to further identify the minimum doses unable to produce significant changes in terms of cell proliferation. An MTT assay was performed in both cell lines in the presence of different concentrations of two drugs, demonstrating a dose-dependent effect on cell proliferation, in particular in SK-N-BE A,B. In fHowever, we found that within 48 h of treatment, the doses of 3 µM cisplatin A,C and 0From the MTT assay performed in the presence of different doses of APE A,B, we sConsidering the ability of M2 agonists to strengthen the inhibitory effects of chemotherapy drugs on neuroblastoma cell proliferation when low doses were used, we further evaluated the ability of the M2 agonist to modulate the drug efflux pumps in both cell lines. At first, using RT-PCR analysis, we demonstrated that both neuroblastoma cells expressed the ABC-B1, ABC-C1 and ABC-C4 pumps at transcriptional levels A,C.The treatment with 100 µM APE was able to significantly downregulate the expression of all three pumps in SK-N-BE B, while In order to explain how APE was able to control ABC pump expression, we analyzed the expression of one of the main oncogenes involved in neuroblastoma tumorigenesis, the proto-oncogene N-MYC, considered the main transcription factor involved in the regulation of ABC pumps ,13,14. BThe role of M2 muscarinic receptors has been largely investigated by our group in recent years, demonstrating its ability to inhibit cell proliferation and survival in primary glioblastoma cell lines ,40,41,42The different expression of muscarinic receptors in two different cell lines by RT-PCR analysis were also analysed, demonstrating that the two cell lines express all muscarinic receptor subtypes, albeit different levels of expression are observable. The M1 and M3 receptors, muscarinic receptor subtypes generally promoting tumor cell proliferation , are expConsidering the differences in terms of drug resistance and of the M2 receptor expression of the two NB cell lines, we analysed the ability of the preferential M2 agonist APE to counteract cell growth. The ability of APE to selectively bind the M2 receptors was previously demonstrated by pharmacological binding experiments and silencing of the receptors both in glioblastoma ,39; and In order to better understand the effects of APE on cell proliferation, we have analyzed, by FACS analysis, the cell cycle progression in the two cell lines. Interestingly, the M2 agonist treatment (100 µM) was able to arrest cell cycle progression in both cell lines causing a significant decrease in cells in S phase. However, we observed that while the SK-N-BE cells resulted preferentially accumulated in G1 phase, the SK-N-BE(2C) cells were accumulated in G2/M phase . The difRecent evidence indicates that metronomic poly-chemotherapy may represent a new generation of chemotherapy treatment showing more efficacy and reduced side effects. In our previous study, we demonstrated the ability of breast cancer to respond to low doses of muscarinic agonists in combination with low doses of paclitaxel . StartinThe metronomic combination of the drugs has clearly demonstrated that a low dose of APE with low doses of the chemotherapy drugs significantly decreased cell growth, while when low doses of the drugs were used alone, it did not show any effects on neuroblastoma cell growth . In ordeFinally, in order to identify possible mechanisms that could explain the ability of APE to reduce the expression of the efflux pumps, we have analyzed the effect of APE on the expression of N-MYC, the main transcriptional factor involved in the onset and progression of neuroblastoma  and able2 and 5% CO2.Human neuroblastoma cell lines SK-N-BE and SK-N-BE(2C) were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium  supplemented with 10% fetal bovine serum (FBS) , 50 mg/mL streptomycin, 50 IU/mL penicillin and 2 mM glutamine (Sigma) and maintained at 37 °C in atmosphere of 90% O3 cells/well. After 24 h, cells were treated with the cholinergic agonist arecaidine propargyl ester hydrobromide   at different times (ranging from 24 to 72 h). Cell proliferation was assessed by colorimetric assay based on 3-(4.5-dimethyl thiazol 2-y1)-2.5-diphenyl tetrazolium bromide  metabolization. The MTT assay was performed according the protocol optimized by Mosmann [Cells were seeded on 24-well plates at a density of 2 × 10 Mosmann . MTT wasTo assess whether inhibition of cell proliferation—APE induced was reversible, a recovery analysis was set up. The cells were treated for 48 h with APE. Then, the medium containing APE was withdrawn, the cells were washed with PBS and fresh complete medium without APE was added. Proliferation analysis was assessed by MTT assay . For each well, the OD at 570 nm was measured by the GloMax Multi Detection System  at different time points, after 48 and 72 h, after APE withdrawal.5 cells/dish. The day after plating, the cells, excluding control samples, were treated with 100 μM APE for 24, 48 and 72 h. At the end of the treatment, cells were incubated for 60 min with 45 µM bromodeoxyuridine  , collected by trypsinization, centrifuged for 10 min at 1000 rpm and then fixed in methanol/PBS 1:1 (v/v). To identify the cells in S phase, DNA content and BrdUrd incorporation were determined in simultaneous analyses by staining with propidium iodide (PI) and anti-BrdU, respectively. Partial DNA denaturation was performed by incubating the cells in 3N HCl for 45 min, followed by neutralization with 0.1 M sodium tetraborate. Samples were then incubated with monoclonal anti-BrdU  for a further 30 min at room temperature (RT), washed twice with 0.5% Tween-20 in PBS and incubated for 30 min with anti-mouse Alexa 488 fluor-conjugated (1:600) . After washing samples with PBS plus 0.5% Tween-20, they were stained with 10 µg/mL PI for 15 min at room temperature (RT). Flow cytometry analysis was performed with a flow cytometer Coulter Epics XL with 488 nm wavelength excitation and 104 events were collected for each sample. Biparametric (DNA content vs. BrdU content) analysis was performed using WinMDI 2.7 software.The cells were plated on 60-mm diameter dishes at a density of 2 × 10In order to evaluate the chemosensitivity of both cell lines to different concentrations of the chemotherapy drugs, we performed chemo-sensitivity tests, treating cells with decreasing doses of cisplatin and doxorubicin. In this regard, our experiments were based on the treatment of SK-N-BE cells and SK-N-BE(2C) cells with decreasing concentrations of doxorubicin (range 25–0.01 μM) and cisplatin (range 25–3 μM) for 24, 48 72 h. The data obtained allowed to identify the lower concentration that showed reduced effects on cell proliferation in both cell lines. Therefore, for the following experiments, we decided to select the concentration of 0.01 μM for doxorubicin and 3 µM for cisplatin within the 24 h of treatment.Cells were lysed with lysis buffer  containing protease inhibitors . The total amount of protein loaded for Western blot was 30 or 40 μg . After protein extraction, the quantification of the total amount of protein was determined by the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific) according to the manufacturer’s protocol. Then, the sample buffer supplemented with 5% β-mercaptoethanol was added to protein lysates and heated for 5 min at 95 °C. Protein extracts were loaded on 10% SDS-polyacrylamide gel (PAGE) and transferred to PVDF membrane . Membranes were blocked for 1 h in 5% non-fat milk powder  in PBS containing 0.1% Tween-20  and then incubated with monoclonal anti-M2 antibody , polyclonal anti-N-MYC antibody  and monoclonal anti-p53 antibody  overnight at 4 °C. Blots were washed with PBS + Tween and then incubated for 1 h with anti-mouse secondary antibody horseradish-peroxidase-conjugated . Signal was revealed by Chemiluminescence (ECL) reagents . Immunoreactive bands were visualized in a Chemidoc system  and the optical density of the bands was quantified by ImageJ software . Beta-actin was used as protein reference for loading control .CHRM1: (Acc. N. AW49504)Total RNA was extracted using the Total RNA extraction Mini Kit , following the manufacturer’s instructions, and then digested with DNAse I . For each sample, 1 μg of total RNA was reverse transcribed for 60 min at 37 °C with Random Primers  and M-MLV reverse transcriptase . Primers and GoTaq Green Master Mix  were added to 100 ng of cDNA. The expression of all the five muscarinic receptors transcripts was evaluated by semi-quantitative RT-PCR analysis using the following primers:F 5′-AGAGAGACCCTGCCAACTTT-3′CHRM2: (Acc. N. NM_000739.3)R 5′-CTCCTGACTTTCCTGCCTAAA-3′F 5′-CTCCAGCCATTCTCTTCTGG-3′CHRM3: (Acc. N. NM_000740.4)R 5′-CTCCAGCCATTCTCTTCTGG-3′F 5′-CGCTCCAACAGGAGGAAGTA-3′CHRM4: (Acc. N. NM_000741.5)R 5′-GGAGTTGAGGATGGTGCTGT-3′F 5′-AATGAAGCAAGAGCGTCAAGAA-3′CHRM5 (Acc. N. NM_001320917.2)R 5′-TCATTGGAAGTGTCCTTATCA-3′F 5′-CCTGGCTGATCTCCTTCATC-3′TP53 (Acc. N. NM_000546.6)R 5′-GTCCTTGGTTCGCTTCTCTG-3′F 5′-CCCCTGTCATCTTCTGTCCCTT-3′18S (Acc. N. NR_1461152)R 5′-CAGACCATCGTCATCTGAGCAG-3′F 5′-CCAGTAAGTGCGGGTCATAAGC-3′R 5′-AACGATCCAATCGGTAGTAGCG-3′RT-PCR starts with denaturation step at 95 °C for 4 min, followed by 25 cycles of amplification at 60 °C for 30 s (18 s gene), 32 cycles at 60 °C for 30 s (M1-M5 muscarinic receptors) or 34 cycles at 64 °C for 30 s (ABC pumps) and final extension at 72 °C for 30 s.The expression of the efflux pumps was also analyzed by qRT-PCR. For qRT-PCR, a quantity of 50 ng of each cDNA was used as a template in each tube for real time-PCR assay. SYBER green PCR Master Mix   and primers  were also added at the respective reaction tubes and analyzed by Thermofisher Quantstudio3. All samples were run in triplicate. The real-time PCR conditions included a denaturing step at 95 °C for 3 min followed by 40 cycles at 95 °C for 30 s, 60 °C for 30 s and 75 °C for 45 s. Two cycles were included as final steps: one at 95 °C (1 min) and the other at the annealing temperature specific for each couple of primers used (1 min).−ΔΔCT where ΔΔCT = ΔCTsample − ΔCTcalibrator.Quantification was expressed as 2ABCB1: (Acc. N. NM_000927.5)The primers used for qRT-PCR are the following:F 5′-CGACAGGAGATAGGCTGGTT-3′ABCC1: (Acc. N. XM_017023237.1)R 5′-AGAACAGGACTGATGGCCAA-3′F 5′-TGCTCACTTTCTGGCTGGTA-3′ABCC4: (Acc. N. NM_001105515.3)R 5′-ACAGGACAAGACGAGCTGAA -3′F 5′- AGACCCCAACTCTACAAGGC-3′R 5′-ATTCTTCCATGCACGCTGAC-3′t-test and one-way ANOVA test followed by Bonferroni’s post-test were used to evaluate statistical significance within the different samples. Results were considered statistically significant at p < 0.05 (*), p < 0.01 (**) and p < 0.001 (***).The Student’s The data reported in the present work, together with the results obtained in other tumor types ,40,41,42


Background: Impaired B-cell receptor (BCR) function has been associated with the progress of several B-cell malignancies. The spleen tyrosine kinase (SYK) represents a potential therapeutic target in a subset of B-cell neoplasias. In precursor B-acute lymphoblastic leukemia (B-ALL), the pathogenic role and therapeutic potential of SYK is still controversially discussed. We evaluate the application of the SYK inhibitor entospletinib (Ento) in pre- and pro-B-ALL cell lines, characterizing the biologic and molecular effects. Methods: SYK expression was characterized in pre-B-ALL (NALM-6) and pro-B-ALL cell lines . The cell lines were exposed to different Ento concentrations and the cell biological response analyzed by proliferation, metabolic activity, apoptosis induction, cell-cycle distribution and morphology. BCR pathway gene expression and protein modulations were further characterized. Results: Ento significantly induced anti-proliferative and pro-apoptotic effects in NALM-6 and SEM, while barely affecting RS4;11. Targeted RNAseq revealed pronounced gene expression modulation only in NALM-6, while Western Blot analyses demonstrated that vital downstream effector proteins, such as pAKT, pERK, pGSK3β, p53 and BCL-6, were affected by Ento exposure in the inhibitor-sensitive cell lines. Conclusion: Different acting modes of Ento, independent of pre-BCR dependency, were characterized, unexpected in SEM. Accordingly, SYK classifies as a potential target structure in a subset of pro-B-ALLs. In B-lyansition ,4,5,6. Dansition . As a reansition . Now, SYansition ,10,11,12ansition , diffuseansition , mantle ansition  and follansition , SYK actansition .Indeed, Goodman et al. reported a SYK deficiency in pediatric precursor pro-B-ALL , whereasBased on pre-clinical evaluation, several SYK inhibitors are developed and under clinical investigation, e.g., fostamatinib disodium (R788) , entospl+), whereas pro-B-ALL cell lines were not affected (BCR−) [The observed effects based on SYK inhibition in precursor B-ALL in vitro are mostly defined by BCR or pre-BCR dependency. Köhrer et al. likewise showed an anti-tumorigenic potential of SYK inhibition by PRT318 in pre-B-ALL cell lines (BCRd (BCR−) .Due to the controversially discussed SYK function, especially in precursor B-ALL, the characterization of SYK as a potential target structure is an essential need. Here, we provide an in vitro-based study, which aimed to illustrate the application of entospletinib as a specific SYK inhibitor on pre- and pro-B-acute lymphoblastic leukemia cell lines, in order to further demonstrate a possible application in a subset of precursor B-ALL and, moreover, to elucidate the underlying diverging acting modes.+) revealed the highest amount of total SYK during Western blot analyses (−) showed less total SYK expression. Total SYK expression was also visualized by immunofluorescence staining in all cell lines and the strongest signal was confirmed in the pre-B-ALL cell line NALM-6 within all tested B-ALL cell lines  revealed significant differences between the B-ALL cell lines showing highest MFI values for NALM-6.In contrast to the total form of SYK, the amount of the activated and phosphorylated SYK form  varied within the analyzed cell lines using Western blot detection a. Two diTo evaluate the basal RNA expression level of SYK as well as key regulators in the B-cell-receptor pathway, we performed targeted RNAseq with 179 genes involved in the BCR, PI3K/AKT pathway signaling and more. The highest SYK expression was detected in pre-B-ALL NALM-6 with two-fold higher expression compared to both the pro-B-ALL cell lines  showed a concentration and slightly time-dependent decrease in cell proliferation, most affected at 72 h (−) (−) showed only a moderate reduction in proliferation at the highest concentration 10 µM and 20 µM applied for 48 h and 72 h (Ento exposure on pre-B-ALL NALM-6 (pre-BCR at 72 h a. The me72 h (−) b. Signifand 72 h c. The ICand 72 h , where tHigh Ento concentrations induced significant early and late apoptosis/necrosis in the pre-B-ALL NALM-6 cell line during all tested time points a. HoweveThe representative images of the Ento-exposed B-ALL cells d indicatCell-cycle distribution was investigated in the pre-B-ALL cell line NALM-6 and pro-B-ALL cell lines SEM and RS4;11 after Entospletinib exposure. Both concentrations of Ento (low concentration 1 µM or high concentration 10 µM) were not able to induce changes in cell-cycle distribution after 72 h incubation in the tested B-ALL cell lines a–c. ExemPTPN6 is the most downregulated gene in Ento-exposed NALM-6 cells compared to the DMSO control (fold change: −10.99), followed by BCL-6 (fold change: −9.62). Furthermore, we detected the downregulation of LAT2, IL7R, DAPP1, CD22 and more. Ento administration only upregulated a minor set of some BCR genes like AKT3 and RPS6KB1 in NALM-6. However, the pro-B-ALL SEM showed only 1.13% up- and 1.13% downregulated genes (CD79B (fold change: 2.41) and FOXO1 (fold change 2.04) were upregulated, whereas VEGFA (fold change: −2.01) and SPRY2 (fold change: −2.01) were downregulated after Ento exposure. In contrast, in the pro-B-ALL RS4;11, no relevant gene expression changes could be identified after Ento mono exposure during RNA panel sequencing. All read counts and normalized read counts are shown in Principle component analysis (PCA) indicates clear clustering of the appropriate cell line samples a. NALM-6ed genes a,b,d,e. Tyr525/526 was detected, while SYKY352 slightly increased during the tested time points  and pro-B-ALL (SEM) cell lines, while a second pro-B-ALL  remained resistant, independently of the applied Ento concentration. Currently, BCR dependency is considered as a response marker to SYK inhibition. Here, pre-BCRhibition . Howeverhibition ,28. CLL hibition . Disrupthibition , which mhibition . Presumahibition . Moreovehibition . In B-ALTyr525/526 and p53 and the increase of pERK1/2. It is already known, that SYK is able to modulate p53 activity [TP53 gene) normally bears a tumor-suppressor function, where a high p53 induces apoptosis, cell-cycle arrest and senescence [TP53 is frequently mutated [TP53 hot-spot mutants, as R157H, R282W, R248Q and R249, can destabilize the DNA-binding domain, which can lead to partially unfolded p53 proteins and therefore loss of the p53 wild type (wt) function [We further investigated the Ento-induced effects on BCR and PI3K/AKT downstream signaling by Western blot analyses. Here, we focused on the pivotal kinases. All tested cell lines exhibit different pathway modulation after Ento exposure. Whereas RS4;11 protein expression was only moderately modified by Ento treatment, SEM and NALM-6 were markedly affected. The most conspicuous alterations in the pro-B-ALL cell line SEM was the reduction of pSYKactivity ,34. p53 nescence . In canc mutated , resultifunction . The subfunction ,39. Furtfunction ,39. Aggrfunction .TP53 p.R248Q mutation [The pro-B-ALL cell line SEM carries the mutation   and consequently regulates BIM expression. The deletion of BCL-6 induced an increased BIM level [MLL-rearranged B-ALL. Low BIM expression or BIM loss correlates with a pro-survival behavior and was already observed in many cancers as a survival strategy [MLL-rearranged sensitive SEM cells, high BCL-6 expression in MLL-rearranged resistant RS4;11 and the reduction of BCL-6 expression in NALM-6 could be explained by the BCL-6–BIM axis and with that the sensitivity to Ento exposure. Concerning this, a new study from Toscan et al. revealed a strategy to overcome glucocorticoid resistance in ALL due to recovering the BIM function [However, Western blot analyses revealed a downregulation of BCL-6 and pSHP-1 in NALM-6 after Ento exposure. This observation was also confirmed by RNA panel sequencing, where LL cells . FurtherBCR+ ALL . It was BCR+ ALL . HoweverIM level . Altogetstrategy ,51. For function .TP53 variant in pro-B-ALL SEM and the BCL-6–BIM axis can probably explain the entospletinib sensitivity and therefor provides a rationale for Ento application also in a subset of pro-B-ALL.Altogether, our present study demonstrates anti-proliferative, metabolism modulating and pro-apoptotic effects in pre- and pro-B-ALL cell lines in vitro, independent of pre-BCR presence. We further unveiled that the +), pro-B-ALL cell lines SEM and RS4;11 (pre-BCR−), B lymphoma cell line SU-DHL-4 and T lymphoma cell line SUP-T1 were purchased from the German Collection of Microorganisms and Cell Cultures . The cells were cultured as recommended by the manufacturer at 37 °C and 5% CO2 in media supplemented with 10 % heat-inactivated FCS  and 100 µg/mL penicillin and streptomycin . Mycoplasma contamination was regularly tested.Human B-ALL pre-B-ALL cell line NALM-6 (pre-BCREntospletinib (GS-9973) was purchased from Selleck Chemicals . A 10 mM Stock solution was prepared according to the manufacturer’s instructions and stored at −80 °C.5 cells/mL) were treated with serial end-concentrations (0.001–10 μM) of entospletinib. Cells were cultured in the appropriate medium containing 0.1% (v/v) DMSO as the solvent of the drug or dose ranges of entospletinib as single substance for 24 h, 48 h and 72 h. Following, the effect on cell proliferation (trypan blue staining), metabolism (WST-1 assay), apoptosis/necrosis (annexin V/PI staining), cell morphology and protein expression were determined. All experiments were performed at least in three biological replicates.B-ALL cell lines . The B-ALL cell lines were exposed to the entospletinib or 0.1% (v/v) DMSO as control. The cells were harvested 24 h, 48 h and 72 h after drug incubation and washed with PBS , . The number of viable cells was determined by cell count with a hemocytometer after trypan blue staining .Pro-B-ALL cells SEM and RS4;11 as well as pre-B-ALL cell line NALM-6 were seeded at a density of 5 × 104 cells/well in 96-well plates with 150 µL cell culture medium containing DMSO as the control or entospletinib, in triplicate. Evaluation of the metabolic activity was analyzed via tetrazolium compound WST-1  according to the manufacturer’s protocol. Briefly, after an appropriate incubation time, the cells were incubated with 15 µL WST-1 premix WST-1 for up to 3 h. The absorbance at 450 nm and a reference wavelength at 750 nm were determined using the GloMax- Multi Microplate Multimode Reader . The absorbance of only culture medium containing the WST-1 reagent was used as the background control.Cells were seeded at a density of 5 × 10TM  and BD FACSuite Software .Early and late apoptosis was analyzed by annexin V FITC  and propidium iodide (PI)  staining according to the manufacturer’s protocol. Drug-exposed and control cells were harvest from 24-well plates and washed twice with cold PBS . The cell pellets were resuspended in a 100 µL annexin binding buffer  and stained with 5 µL annexin V FITC for 15 min at room temperature in the dark. Cell suspensions were adjusted to a final volume of 500 μL with the annexin binding buffer and stained with PI (20 μg/mL) immediately before measurement. Unstained and single stained control cells were included in each experiment. Measurements were performed using FACS Verseg for 10 min at 4 °C the cells were washed twice with PBS, followed by fixing the cells with 70 % ice cold ethanol and a freezing cycle at −20 °C (≥24 h). Subsequently, the fixation solution was removed by centrifugation at 180× g for 10 min at 4 °C. The fixed cells were then washed twice with PBS. After centrifugation the cell pellet was then resuspended in 500 µL ribonuclease A (1 mg/mL) . RNase treatment ensued 45 min at 37 °C. After a centrifugation step at 180× g for 10 min at 4 °C, the cells were washed again with PBS for two times. PBS was removed by a centrifugation, the pellet was resuspended in 400 µL PI Solution (50 µg/mL)  and analyzed by flow cytometry  and BD FACSuite Software .The DNA intercalating dye propidium iodide (PI) was used for cell-cycle analysis to quantify the DNA content. Cells were treated with 1 µM and 10 µM entospletinib or DMSO as the vehicle. After 48 h and 72 h at 37 °C entospletinib exposure, cells were harvested. After centrifugation at 180× 4 cells per slide  in a Cytospin 3 centrifuge . Slides were fixed in ice-cold methanol for 10 min. Afterward, the cells were permeabilized, blocked and stained with SYK antibody (1:400 diluted in 1% BSA) . Slides were washed and stained with secondary goat Anti-mouse IgG Alexa Fluor Plus 488 (1:1000 diluted in 1% BSA) . The antibodies used are listed in Coated cytoslides  were prepared with 5 × 104 cells per slide  in a Cytospin 3 centrifuge . The slides were stained for 6 min with May-Grünwald working solution , rinsed with microscopy buffer according to WEISE (pH7.2) , stained for 20 min in a Giemsa working solution  and rinsed thoroughly with the microscopy buffer again. The slides were air dried at room temperature before analysis. To evaluate the morphological changes of the cells, the slides were analyzed by EVOS® XL Core Imaging System .Coated cytoslides  were prepared with 5 × 10® TurboTM Transfer System . After blockage in 1:3 diluted Odyssey Blocking Buffer  for one hour, the membranes were incubated with the primary antibody overnight at 4 °C. The antibodies used are listed in Treated cells were lysed using RIPA buffer ) and ultrasound. Protein concentrations were determined by Bradford Protein Assay . For each sample, 30 µg protein were separated on Criterion TGX Precast Gels . They were blotted onto a PVDF membrane  using Trans-BlotTM, BD Biosciences, Heidelberg, Germany). The SUP-T1 T-cell lymphoma cell line acted as the negative control due to lacking SYK expression. The IgG-stimulated B-lymphoma cell line SU-DHL-4 acted as the positive control. AffiniPure F(ab’)2 Fragment Goat Anti-Human IgG, F(ab’)2 fragment specific was used for stimulation and purchased from Dianova . Here, SU-DHL-4 was seeded in 24-well plates (106 cells/mL) and stimulated for 15 min with 5 µg/mL IgG at 37 °C and 5 % CO2 [TM, BD Biosciences, Heidelberg, Germany). For all experiments, the unstimulated, autofluorescence and isotype controls were analyzed simultaneously. All experiments were performed at least in three biological replicates.The detection of the intracellular targets pSYK and SYK was carried out by intracellular flow cytometry  and RNA Isolation was performed with miRNeasy Mini Kit  according to the manufacturer’s instructions. In brief, after lysis, the aqueous phase that contains the total RNA of the lysed cells were extracted and purified by a silica membrane of RNeasy Mini spin columns. The remaining DNA was digested by RNase-free DNase  and the contaminants were washed away. Total RNA was eluted by nuclease-free water and RNA concentration measured on a Nanodrop Spectrophotometer ND1000 .Treated cells or control cells were grown in 6-well plates for up to 72 h, harvested and washed three times with cold PBS by centrifugation steps at 180× Expression analyses were performed with AmpliSeq RNA targeted sequencing on the Ion Personal Genome Machine™ (PGM™) System (Ion Torrent™) . For targeted RNA sequencing, an in-house custom panel was designed with the Ion AmpliSeq Designer (Thermo Fisher Scientific), containing 179 genes mainly involved in B-cell-receptor and PI3K/AKT pathway signaling and more. Ion AmpliSeq RNA Libraries were prepared according to the manufacturer’s protocol (MAN0006735). In brief, RNA was quantified with the Qubit RNA HS Assay Kit  on the Qubit 2.0 Fluorometer  and transcribed into cDNA using SuperScript VILO cDNA Synthesis Kit . cDNA targets were amplified, amplicons partially digested, ligated to the adapters and purified with the Ion AmpliSeq™ Library Kit 2.0 . All steps were performed in the ProFlex PCR System . The final library was quantified by the Ion Library TaqMan Quantification Kit  with the ViiA 7 Real-Time PCR System . Thereafter, template preparation was carried out with the Ion PGM Hi-Q View OT2 Kit—200 in the Ion One Touch 2 Instrument  and enrichment of template-positive Ion Sphere Particles (ISPs) with the Ion OneTouch ES . Finally, the sequencing run was performed with the PGM System. The evaluations of the data sets were performed using Transcriptome Analysis Console (TAC) Software . The following filter criteria were set to identify relevantly regulated genes: Avg (log2) > 5, fold change > 2 or < −2.p < 0.05, ** p < 0.001, *** p < 0.0001.Statistical data are presented as the mean ± standard deviation (SD) from at least three biologically independent samples using GraphPad Prism Software . Statistical differences between multiple comparisons were analyzed using one-way ANOVA followed by Dunnett’s multiple comparison test as a post-hoc analysis. Differences were considered statistically significant for * TP53 variant in pro-B-ALL SEM and the BCL-6–BIM axis are probably Ento key targets and can explain the Ento sensitivity, therefor providing a rationale for Ento application also in a subset of pro-B-ALL.In this study, we provided evidence for a potential anti-leukemic effect of entospletinib in pre- and pro-B-ALL cell lines in vitro, independent of their pre-BCR status. Opposed to the expectation, the pro-B-ALL cell line SEM showed a good Ento response, such as anti-proliferative, metabolism and pro-apoptotic effects. We identified a diverse acting mode of Ento, independent of pre-BCR dependency, based on gene and protein modulation after Ento exposure. Here, the


RB1 few gene-copy number (CN) alterations are associated with irreversible-resistance to endocrine therapy and subsequent secondary resistance to palbociclib. Resistance to palbociclib occurred as a result of tumour cell re-wiring leading to increased expression of EGFR, MAPK, CDK4, CDK2, CDK7, CCNE1 and CCNE2. Resistance altered the ER genome wide-binding pattern, leading to decreased expression of ‘classical’ oestrogen-regulated genes and was accompanied by reduced sensitivity to fulvestrant and tamoxifen. Persistent CDK4 blockade decreased phosphorylation of tuberous sclerosis complex 2 (TSC2) enhancing EGFR signalling, leading to the re-wiring of ER. Kinome-knockdown confirmed dependency on ERBB-signalling and G2/M–checkpoint proteins such as WEE1, together with the cell cycle master regulator, CDK7. Noteworthy, sensitivity to CDK7 inhibition was associated with loss of ER and RB1 CN. Overall, we show that resistance to CDK4/6 inhibitors is dependent on kinase re-wiring and the redeployment of signalling cascades previously associated with endocrine resistance and highlights new therapeutic networks that can be exploited upon relapse after CDK4/6 inhibition.Combination of CDK4/6 inhibitors and endocrine therapy improves clinical outcome in advanced oestrogen receptor (ER)-positive breast cancer, however relapse is inevitable. Here, we show in model systems that other than loss of  The majority of breast tumours at primary diagnosis are oestrogen receptor alpha positive (ER). This knowledge has been exploited clinically by the development of endocrine therapies, which seek to deprive the hormone dependent tumour cells of oestrogen (E) using aromatase inhibitors (AI), or the use of antiestrogens such as tamoxifen or fulvestrant (Fulv) both of which compete with E for the ER. Antagonising ER leads to inhibition of cyclin dependent kinase (CDK)/cyclin activity and the maintenance of retinoblastoma protein (RB) in a hypophosphorylated state suppressing transcription of E2F-regulated genes and inhibiting progression through S-phase.CCND1 [kip1 can reduce the efficacy of ER inhibition [CCND1 occurs in ~15% of breast cancer (BC) [Unfortunately, the beneficial actions of existing endocrine treatments are attenuated by the ability of tumours to circumvent the need for steroid hormones, whilst in most cases, retaining the nuclear steroid receptors (reviewed by ). In thiCCND1 . In addihibition . Amplificer (BC)  and overcer (BC) –8. This As proliferation is a hallmark of endocrine resistant tumours, targeting cell cycle regulation has provided an attractive proposition. Indeed, recent studies suggest many cancer cells might be addicted to high CDK4/6 activity . A numbeRB1, few genetic changes are associated with resistance to CDK4/6 inhibition. Upregulation of CCNE1 and CCNE2 was evident in all resistant models. We provide evidence that prolonged CDK4 blockade enhances EGFR/ERBB signalling, as a result of reduced TSC2-phosphorylation, which impacts downstream on ER signalling leading to an altered ER-cistrome and reduced sensitivity to subsequent endocrine blockade. Overall, we show that resistance to CDK4/6 inhibitors is dependent on kinase re-wiring and the redeployment of signalling cascades previously associated with endocrine resistance. Our study highlights the potential clinical utility of targeting the ERBB-signalling axis or cell cycle via perturbation of CDK7 (cell cycle master regulator) or WEE1 (G2/M Checkpoint), according to the mode of resistance acquired to long-term CDK4/6 treatment.The combination of CDK4/6 inhibitors and endocrine therapy have been shown to improve clinical outcome in patients with ER+ metastatic BC –13, and Detailed materials and methodology is provided in the Supplementary STAR file.Human BC cell lines MCF7, T47D, HCC1428, ZR75.1 and SUM44 were purchased from ATCC and Asterand. Cells were cultured in phenol red free RPMI containing 10% foetal bovine serum (FBS) and 1 nM estradiol (E2). LTED derivatives were cultured in phenol red free RPMI supplemented with 10% dextran charcoal stripped (DCC) FBS, as previously described [Cell viability were carried as detailed previously . In briegave for 10 min. Spheres were formed over 72 h and subsequently treated with the drugs as indicated for 7 days. Proliferation was assessed using Celigo S (Nexcelom Bioscience).Spheroid cultures were generated by seeding 2500 cells per well of a 96 well ultra-low-attachment plate (Costar). Plates were spun at 900 × Immunoblotting was carried out, as previously described . In brieTFF1 (Hs00907239_m1), CCND1 (Hs00765553_m1), PDZK1 (Hs00275727_m1), RB1 (Hs01078066_m1), EGFR (HS01076090_m1), MAPK1 (Hs01046830_m1), MAP3K1 (Hs00394890_m1), CDK2 (Hs01548894_m1), CDK4 (Hs00364847_m1), CDK7 (Hs00361486_m1), CCNE1 (Hs01026536_m1), CCNE2 (Hs00180319_m1) together with FKBP15 (Hs00391480_m1) as housekeeping gene to normalise the data. The relative quantity was determined using ΔΔCt, according to the manufacturer’s instructions (Applied Biosystems).Taqman gene expression assays (Applied Biosystems) were used to quantify http://software.broadinstitute.org/GSEA/msigdb/annotate.jsp and G-profiler).Global gene expression analysis was carried out for MCF7, MCF7 LTED and T47D human BC cell lines along with their palbociclib-resistant derivatives (GSE98987). Data was normalised and handled, as previously described . Gene seDNA was extracted from the target cell lines using QIAamp DNA mini kit, according to the manufacturer’s instructions (Qiagen). Exome libraries were generated, as previously described . Data waChIP-seq was performed, as previously described , 16 usinDimethyl labelling was carried out, as reported previously . Samplest-tests were used.In vivo studies were carried out in ovariectomized 8- to 12-week-old female BALB/c- nude mice in accordance with Home Office guidelines and approved by the Institute of Cancer Research Ethics Committee. Xenografts modelling patients resistant to AI (MCF7 LTED) were treated with 100 mg/kg palbociclib administered daily by oral gavage or vehicle control. The study operator was blinded to the treatments. Overall statistical differences were calculated using the Wilcoxon signed-rank test if the variance was not equal and failed the normality test otherwise paired t-test.Palbociclib resistant PDX (see star file) were generated and transplanted into female 8-week-old Swiss nude mice in accordance with institutional guidelines and the rules of the French Ethics Committee (project authorisation no. 02163.02). Mice were randomly assigned to the control or treated groups (4–7 mice per group). Palbociclib was administered orally twice per week at 50 mg/kg. Everolimus was administered orally at a dose of 2.5 mg/kg, 3 days per week. Fulv was administered by intramuscular injection at a dose of 50 mg/kg once a week. AZD1775 and neratinib were administered orally at 90 mg/kg and 40 mg/kg, 5 days per week, respectively. Treatments were administered for 60 days. The statistical significance was determined by unpaired PalboR, MCF7 LTEDPalboR, T47DPalboR and T47D LTEDPalboR cell lines were transfected using the ON-TARGETplus siRNA Library-Human Tyrosine Kinases (Dharmacon) targeting 709 protein kinases. After 6 days, cell viability was assessed using CellTiterGlo assay (Promega). A library screen for each cell line was performed in duplicate and repeated 2–3 times. The dynamics of each library screen was assessed by calculating Z prime values. The threshold of acceptance was set as Z′ > 0.3 [MCF7Z′ > 0.3 .A panel of endocrine-sensitive and long-term oestrogen deprived (LTED) BC cell lines modelling relapse on an AI, were evaluated for their sensitivity to the CDK4/6 inhibitor palbociclib: MCF7, SUM44, T47D, ZR75.1 and HCC1428 cell lines along with their LTED derivatives were assessed both in the presence and absence of 17β−estradiol (E2).50 values ranging between 100 and 300 nM. In the absence of E2, modelling the effect of an AI, proliferation dropped significantly in all cell lines and the addition of palbociclib at concentrations between 500 and 1000 nM showed a further reduction of ~50%. This suggested that in the absence of E2, palbociclib targeted those cells which had partial resistance to E2 deprivation.The cell lines showed varying degrees of sensitivity to palbociclib . Resistance was authenticated by culturing the resistant cell lines with escalating concentrations of palbociclib in comparison with their wild-type progenitor  long-term in the presence of palbociclib (1μM). Of the 8 cell lines, 5 palbociclib-resistant models were successfully generated, three of which, were also resistant to E-deprivation  alterations using exome sequencing Fig. . Initialderation  and a codel Fig. . No othePalboR, as a comparator based on their overall RB1 CN loss. As expected, pathway analysis , which showed almost a 10-fold shift in sensitivity. Inhibition of CDK9 had no significant impact in the palbociclib-resistant lines tested  targeting 709 kinases in the palbociclib-resistant cell lines Fig. . T47DPalnes Fig. . PerturbTaken together, these data confirm our finding that growth factor receptor signalling associates with resistance to CDK4/6 inhibition and highlights the potential to target this, CDK7 or G2/M checkpoint proteins such as WEE1 upon progression on palbociclib.p < 0.0001). Treatment by AZD1775 inhibited tumour growth with a TGI of 70% (p = 0.0025) and 54% when combined to Fulv (p = 0.046). Everolimus alone or in combination with Fulv inhibited tumour growth with TGI of 75% (p = 0.0006) and 78% (p = 0.0004), respectively.To validate the results obtained in our in vitro experiments, we tested the anti-tumour activity of AZD1775, neratinib or everolimus in the presence or absence of Fulv in an in vivo PDX model of metastatic ER + CCND1-driven BC with acquired resistance to palbociclib  Fig. . PalbociTaken together, these data highlight the potential of targeting upstream growth factor signalling receptors, downstream signalling pathways such as PI3K/mTORC and G2/M associated checkpoint proteins such as WEE1 in palbociclib resistant tumours.CDK4 and CCND1 amplification is associated with endocrine resistance . Recentiewed by  that tariewed by , 12, or iewed by . While tE2F signature, prior to surgery may provide utility in identifying those patients most likely to benefit from such combinations [In this study, we sought to answer some of these issues by addressing the mechanisms involved in the development of resistance to CDK4/6 inhibition. We initially showed that CDK4/6 inhibition in combination with endocrine therapy targets de novo ligand-independent proliferation providing further justification for the use of these agents to target intrinsic endocrine resistance earlier. Indeed, this notion is supported by the recent NeoPalAna study, which showed patients with Ki67 > 10% gained benefit from the combination of an AI and palbociclib, resulting in complete cell cycle arrest (Ki67 < 2.7%) . In addiinations , 37 and RB1 CN were associated with resistance to palbociclib in ER + BC models and this appeared restricted to one isogenic cell line. Furthermore, the genetic make-up of the parental cell lines did not inform on the mechanism of resistance to CDK4/6 inhibition. This observation is in keeping with our previous studies [RB-null population. The parental tumour, which was isolated from a patient previously treated with endocrine therapy harbouring an ESR1Y537S, TP53 mutation and loss of genes encoding p16INK4A, p15INK4B and p14ARF, was shown to be initially sensitive to palbociclib treatment suggesting that these genetic alterations do not impact on response to CDK4/6 perturbation and that long-term exposure led to the expansion of the pre-existing RB-null sub-population. In our current study, despite the primary tumour from which our palbociclib resistant PDX was derived harbouring a TP53 mutation, the resultant CDK4/6 resistant tumour maintained RB1 expression. This suggests, loss of TP53 does not influences RB1 expression and does not determine sensitivity to CDK4/6 inhibition.We showed that few genetic changes other than loss of  studies , in whicTP53 mutations were shown to be maintained from day one. In conclusion, the genetic make-up of the primary tumour appears insufficient to determine sensitivity or resistance to CDK4/6 inhibition [Clinical studies from PALOMA-3 indicate that loss of RB1 is a rare event, occurring in only 4.7% of patients, whilst hibition .PalboR cells. ChIP-seq analysis showed a loss of ER-binding to ESR1 and FOXA1 motifs and enrichment at SP1 and AP2 motifs, which were concomitant with a decrease in the expression of classical E-regulated genes PDZK1 and TFF1 but not CCND1. Further interrogation of the transcriptome showed a reduction in dependence on ER for cell cycle progression and a shift towards dependence on SP1 transcription factors, which have been associated with modulation of EGFR expression [Further interrogation using transcriptomic and phosphoproteomic profiling showed cell lines retaining RB used kinase re-wiring to circumvent perturbation of CDK4/6. Noteworthy, an increase in growth factor signalling and downstream activation of MAPK was evident, pathways previously associated with resistance to endocrine therapy. As preceding studies have highlighted the ability of altered MAPK  and AKT pression . Taken tCDK6 CN was evident in our cell line models. We therefore hypothesised that signalling via EGFR/ERBB may provide the dominant mitogenic drive accounting for the enhanced sensitivity to the pan-ERBB inhibitors neratinib and AZD8931. Mechanistically, we showed that blockade of cyclin D1/CDK4 reduced the phosphorylation of tuberous sclerosis 2 (TSC2), relieving the negative feedback loop and increasing expression of EGFR family members [In keeping with an earlier report, those CDK4/6 resistant cell lines that maintained ER expression, showed a reduction in sensitivity to Fulv and tamoxifen compared with their respective parental cell lines . However members  which in members . Previou members .CDK4, CCNE1 and CCNE2. The Cyclin E1/CDK2 complex inactivates RB by quantum hyper-phosphorylation [CCNA2. Moreover, increases in cyclin D1 can aid in cyclin E1/CDK2 complex formation by sequestering CDK2 negative regulators p21CIP1 and p27KIP1 promoting aberrant RB-phosphorylation and cell cycle progression [Taken together these data suggest altered growth factor signalling pathways, which have previously been associated with endocrine resistance appear further enhanced in CDK4/6 resistant cell lines that retain functional RB. This altered signalling network leads to increased expression of rylation  leading gression . In turnCCND3, CCNE1 and CDKN2D) was evident, indicating that continued activation of E2F transcription in resistant tumours, equates to loss of the RB regulon [Clinical support for this hypothesis comes from the NeoPalAna study, where association between palbociclib resistance and persistent on-treatment expression of E2F targets ( regulon .PIK3CA loss caused a marked drop in proliferation of all cell lines irrespective of RB status. The mTORC1 inhibitor everolimus and PI3K inhibitor pictisilib suppressed proliferation of all palbociclib-resistant models, suggesting that sequencing of these agents after resistance to CDK4/6 inhibition may provide clinical utility.In order to take a global view of kinases associated with the palbociclib-resistant phenotype, and to identify new therapeutic targets, we carried out siRNA kinome knockdown studies. Not surprisingly, PalboR cells, which lose expression of both ER and RB. Interestingly, THZ1 has been proposed to be effective in triple negative BC [Multiple CDKs were highlighted as potential targets including CDK9. However, the CDK9 inhibitor LDC000067 had little impact on cell proliferation. One explanation is that kinases can have both catalytic and structural modalities . CDK9 caative BC . Nonetheative BC , 51.In summary, the majority of common kinases associated with resistance were connected with growth factor signalling, PI3K/mTOR and G2/M checkpoint including WEE1, which is required for phosphorylation and inactivation of CDK1 and determines cell size prior to mitosis . In confTaken together, these data suggest that multiple mechanisms of resistance to CDK4/6 inhibitors can occur leading to deregulation of RB1 regulon either by CN loss, methylation or increased growth factor signalling. We show pathways associated with endocrine resistance can be further upregulated in RB competent yet CDK4/6 resistant cell lines suggesting that some tumours may remain hardwired to the RB/E2F transcriptional axis and use flexibility in kinase signalling to circumvent the G1/S checkpoint by increasing expression of CCNE1 leading to hyperphosphorylation of RB and reducing dependence on ER signalling as a mitogenic driver. This data highlights the high degree of adaptability to CDK4/6 inhibition and the need to screen patients prior to and after relapse on these inhibitors in order to inform on effective drug sequencing.Supplementary Figure S1Supplementary Figure S2Supplementary Figure S3Supplementary Figure S4Supplementary Figure S5Supplementary Figure S6Supplementary Figure S7Supplementary File S1Supplementary File S2Supplementary File S3Supplementary File S4


The treatment of Ewing sarcoma, an aggressive bone and soft tissue sarcoma, is associated with suboptimal outcomes and significant side-effects. Consequently, there is an urgent need to identify novel therapies that will improve outcomes for children and adults with Ewing sarcoma tumors while also decreasing treatment-related toxicities.We analyzed data from the PRISM drug repurposing screen, which tested the activity of 4518 drugs across 578 cancer cell lines, to identify drugs that selectively inhibit the growth of Ewing sarcoma cell lines. We then tested the effects of a top hit from the screen on cell proliferation, cell cycle progression, and activation of the DNA damage pathway using Ewing sarcoma cell lines. We also used a CRISPR/Cas9 gene knockout approach to investigate the role of Schlafen 11 (SLFN11), a restriction factor for DNA replication stress that is overexpressed in Ewing sarcoma tumors, in mediating the sensitivity of Ewing sarcoma cells to the drug.We found that eltrombopag, an FDA-approved thrombopoietin-receptor agonist (TPO-RA) that is currently being evaluated as a treatment for chemotherapy-induced thrombocytopenia, inhibits the growth of Ewing sarcoma cell lines in vitro in proliferation and colony formation assays. However, from a mechanistic standpoint, the thrombopoietin receptor is not expressed in Ewing sarcoma cells and we show that eltrombopag impairs DNA replication and causes DNA damage in Ewing sarcoma cells by chelating iron, a known “off-target” effect of the drug. We also found that the sensitivity of Ewing sarcoma cells to eltrombopag is mediated, in part, by SLFN11, which regulates the cellular response to DNA replication stress.Ewing sarcoma cell lines are sensitive to eltrombopag and this drug could improve outcomes for patients with Ewing sarcoma tumors by both targeting the tumor, via chelation of iron and inhibition of DNA replication, and reducing chemotherapy-induced thrombocytopenia, via stimulation of the thrombopoietin receptor.Supplementary information accompanies this paper at 10.1186/s12885-020-07668-6. Ewing sarcoma is an aggressive bone and soft tissue cancer that occurs primarily in children and young adults , 2. The The VDC/IE chemotherapy regimen also causes significant on- and off-treatment toxicities, including myelosuppression, heart failure, infertility, and renal damage , 6. NotaIn the current study, we analyzed the Broad Institute’s PRISM drug repurposing screen, which includes 12 Ewing sarcoma cell lines, to identify drugs that selectively impair the growth of the Ewing sarcoma cell lines compared to cell lines derived from other types of cancer . Eltrombo C in a 5% CO2 atmosphere. The A673, TC32, TC71, and EW8 cell lines were provided by Dr. Kimberly Stegmaier . The ES6 cell line was kindly provided by the St. Jude Childhood Solid Tumor Network. The CB-AGPN cell line was obtained from the Childhood Cancer Repository (Children’s Oncology Group). The HT1080 and U2OS cell lines were obtained from ATCC. Cells were grown in Dulbecco’s Modified Eagle’s Media (DMEM) supplemented with 10% FBS, 100 IU ml− 1 penicillin and 100 μg ml− 1 streptomycin. Cell lines were used within 8–10 thawing passages and DNA fingerprinting confirmation was performed using the short tandem repeat method.Cell lines were maintained at 37p-value, and T-score.Data obtained from the PRISM repurposing drug screen , which tested the activity of 4518 drugs across 578 cancer cell lines, were used to identify drugs that selectively reduce the growth of Ewing sarcoma cell lines compared to other cell lines . CompounMPL mRNA expression data for cell lines was obtained from the Cancer Dependency Map (Broad Institute) [MPL mRNA expression data for primary tumors was obtained from The Cancer Genome Atlas (TCGA) via cBioPortal for Cancer Genomics [stitute) . MPL mRNGenomics .Eltrombopag was obtained from MedChemExpress.4 cells were plated per well of a 96-well plate, after which the cells were exposed to a range of drug concentrations for 72 h. Fluorescence readings were then obtained after adding AlamarBlue (Sigma) using a FLUOstar Omega microplate reader (BMG Labtech). IC50 values were calculated using log-transformed and normalized data (GraphPad Prism 8.3).Cell proliferation was measured using the AlamarBlue (resazurin) fluorescence assay, as previously described . ApproxiA673, EW8, TC71, CB-AGPN, and U2OS cells growing in 6-well plates in triplicate were exposed to DMSO or 5 μM eltrombopag for 14 days. Crystal Violet was used to stain the colonies and the number of colonies per well were counted manually.o C. The BCA reagent (Pierce) was used to determine the protein concentrations in the samples. SDS-PAGE was used to separate proteins, which were then transferred to polyvinylidene difluoride membranes (Millipore). Antibodies to the following proteins were used in the immunoblots: phospho-Histone H2A.X , phospho-CHK1 , CHK1 , SLFN11 , and Actin .Protein extracts for immunoblotting were prepared by incubating cells in RIPA buffer (Boston BioProducts), supplemented with protease and phosphatase inhibitors , for 20 min. Supernatants were collected after centrifugation, 17,000 r.c.f. for 15 min, at 45 cells/well) were plated in a 6-well plate and allowed to adhere overnight. The cells were then treated with eltrombopag, or vehicle, for 48 h and then labeled with 5-ethynyl-2′-deoxyuridine (EdU) for 2 h. Flow cytometry for γH2AX and EdU was then performed on a Becton Dickinson LSR II instrument as described [Cells  that co-expresses Cas9 and a gRNA (GAGTCCTGAGAGCAGCGCAG) targeting sduction , 19, 20.Cell-cycle analysis was performed in duplicate using the Click-iT EdU-488 Kit for flow cytometry (Thermo Fisher Scientific). Cells were labeled with EdU for 2 h and analysis was performed according to the manufacturer’s instructions. Flow cytometry was performed on a Becton Dickinson LSR II instrument.A lentiviral (pLV) plasmid expressing the PIP-FUCCI (PCNA-interacting protein-Fluorescent Ubiquitination-based Cell Cycle Indicator) construct was obtained from VectorBuilder . Lentivit-test was used to calculate p-values. Statistical analyses were conducted using GraphPad Prism 8.3.Student’s MPL mRNA [MPL mRNA is expressed in some acute myeloid leukemias (AML), but not in sarcomas . Figure OS. FigurEltrombopag, in addition to its actions as a TPO-RA, also binds and chelates iron –14. NotaIn previous work, we identified that the iron-dependent RRM2 subunit of ribonucleotide reductase (RNR), which is the rate limiting and iron-dependent enzyme in the synthesis of deoxyribonucleotides and required for DNA replication, is one target of iron chelators in Ewing sarcoma cells , 17, 20.DNA replication stress, due to inhibition of RRM2 or other causes, generates DNA damage and activates the CHK1 DNA damage response pathway , 37–39. s, in part, the enhanced sensitivity to eltrombopag.SLFN11 is overexpressed in Ewing sarcoma tumors and is known to sensitize cells to a range of drugs that cause DNA replication stress, including iron chelators , 43–48. The standard therapy for Ewing sarcoma, which consists of alternating cycles of vincristine-doxorubicin-cyclophosphamide and ifosfamide-etoposide (VDC/IE) administered on an interval-compressed schedule, is associated with overall survival rates of ~ 20% and ~ 70% for patients with metastatic and localized tumors, respectively , 2, 5. TEltrombopag and other thrombopoietin-receptor agonists are currently under investigation as potential supportive care agents to help reduce the duration and severity of thrombocytopenia in patients receiving myelosuppressive chemotherapy, in a similar fashion as to how granulocyte-colony stimulating factor (G-CSF) is used to help reduce the time to neutrophil recovery , 11, 51.In this work, we used data available through the PRISM drug repurposing screen to identify that eltrombopag selectively reduces the growth of Ewing sarcoma cell lines compared to cell lines from other tumor types , 15. We Identification of this off-target mechanism of eltrombopag in inhibiting the growth of Ewing sarcoma cells could aid in optimizing the anti-cancer efficacy of the drug. For example, we showed that eltrombopag activates the CHK1 DNA damage response pathway. Combining eltrombopag with CHK1 inhibitors, which are being tested in early phase clinical trials, could generate synergistic toxicity , 38, 39.In conclusion, our data suggest that eltrombopag, a well-tolerated drug used to treat pediatric chronic ITP and aplastic anemia, could provide therapeutic benefit for patients with Ewing sarcoma by both mitigating treatment-related thrombocytopenia and contributing additional anti-tumor toxicity via iron chelation and RNR inhibition.Additional file 1: Supplementary Figure 1. Ewing sarcoma cell lines were treated with vehicle, eltrombopag 5 μM, Z-VAD-FMK 10 μM, or the combination of eltrombopag and Z-VAD-FMK for 18 h and then cellular lysates were collected for immunoblotting. The blots have been cropped and full-length blots are presented in Supplementary Figure Additional file 2: Supplementary Figure 2. Full-length blots for Figs. Additional file 3: Supplementary Table 1. Ewing sarcoma cell lines included in the PRISM drug screen.Additional file 4: Supplementary Table 2. Drug dependencies enriched in the Ewing sarcoma cell lines.


Targeting 6-phosphogluconate dehydrogenase (6PGD) can inhibit cancer cell proliferation and tumor growth. However, the relationship between 6PGD and cisplatin resistance still needs further study. Cisplatin-sensitive and cisplatin-resistant ovarian cancer OV2008 and C13* lines and lung cancer A549 and A549DDP lines were treated with different concentrations of cisplatin and cell viability was evaluated. We also compared the growth rates and the cell cycle distributions between cisplatin-sensitive and cisplatin-resistant cells. The expression level of 6PGD was detected by immunoblotting. The Chou-Talalay method was used to evaluate the effect of a combination treatment using cisplatin and the small molecule inhibitor 1-Hydroxy-8-methoxy-anthraquinon (S3) that targets 6PGD. The cisplatin-resistant ovarian and lung cancer cell lines grew faster than the cisplatin- sensitive cell lines, with more cells in S and G2 phases in cisplatin-resistant cell lines. The expression level of 6PGD in cisplatin-resistant cell lines was significantly increased compared with cisplatin-sensitive cell lines. Furthermore, treatment of cells with the S3 small molecule inhibitor of 6PGD together with cisplatin could overcome cisplatin resistance. The expression level of 6PGD in cisplatin-resistant cells lines was significantly upregulated, and the resistance to cisplatin of drug-resistant cells lines could be overcome when treated with the small molecule inhibitor S3 that specifically targets 6PGD. Cisplatin can then bind to negatively charged molecules in the cell, including proteins, RNA and DNA. Cisplatin mainly forms cytotoxic adducts with DNA dinucleotides, causing intrastrand or interstrand DNA crosslinks; this blocks the replication and transcription processes in the cell, which halts normal cell division and leads to cell apoptosis [Cisplatin (DDP) is a platinum-based first-line anti-cancer drug with wide applications. Presently, cisplatin is mainly used for chemotherapy of solid tumors in the clinic , includipoptosis .Despite the extensive use of cisplatin in treating cancers, many patients with tumors develop cisplatin resistance at an earlier stage or after several courses of therapy. In addition, cisplatin has side effects and shows toxicity, thus seriously limiting its clinical applications. Therefore, elucidating the mechanism of cisplatin resistance in tumors has great clinical benefit ,6.Increasing evidence has indicated that abnormalities of cellular energy are associated with the occurrence of drug resistance in tumor cells during the course of chemotherapy ,8. Howev+ to NADPH and participates in the synthesis of lipids and nucleic acids. Studies show that 6PGD expression is markedly upregulated in various tumors, including colon cancer, cervical intraepithelial neoplasia, thyroid tumor and lung cancer and ovarian cancer [The pentose phosphate pathway (PPP) is the main source of synthesis for NADPH. The three key enzymes of PPP include G6PD, 6-phosphogluconolactonase (PGLS) and 6-phosphogluconate dehydrogenase (6PGD). The third enzyme in the PPP is 6PGD, which converts 6-phosphogluconate into ribulose-5-phosphate. Additionally, 6PGD reduces NADPn cancer , 14, 15.n cancer . The smaHerein, we investigated whether specific inhibition of 6PGD by the small molecule inhibitor S3 can reverse cisplatin resistance in cancer cells.22.1Human ovarian cancer cell lines OV2008 and C13* were kindly provided by Dr. Benjamin K. Tsang from University of Ottawa, USA. Human lung cancer cell lines A549 and A549DDP were kindly provided by Professor Zhi Shi from Jinan University, China. All cell lines were subjected to cell identification.β-actin and 6PGD antibodies were purchased from Santa Cruz Biotechnology . Goat anti-rabbit and goat anti-mouse secondary antibodies were purchased from Sanjian Biotech Co., Ltd. . Fetal bovine serum was purchased from Gibco Life Technologies . RPMI 1640, trypsin and the small molecule compound S3 were purchased from Sigma-Aldrich LLC .2.2The following instruments were used in this study: a SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) system ; membrane transfer system ; gel imaging system ; and multi-function microplate reader .2.32. Cells were passaged or cryopreserved based on their proliferation rate.Cisplatin resistance in the cisplatin-resistant cell line A549DDP was maintained by culturing cells with a low concentration of cisplatin; the ovarian cancer cell line C13* is naturally resistant to cisplatin. All cell lines were cultured in an incubator at 37℃ with 5% CO2.44 cells in 6-well plates. Relative cell proliferation was determined bycell counting at 3 days after being seeded and the percentage cell proliferation of the control. Cell growth was determined by cell numbers recorded at 0, 1, 2, 3, and 4 days after being seeded.Cell proliferation assays were performed by seeding 5×102.5OV2008, C13*, A549 and A549DDP cells were seeded in six-well plates respectively and cultured for 24 h. For cell cycle analysis, treated cells were fixed using 70% ethanol for 24 h followed by propidium iodide staining. Cells were analyzed using FACScantoII cytometers . All analyses were repeated in triplicate.2.6+ to NADPH, 6PGD enzyme activity is determined through the accumulation of NADPH produced. An aliquot of cell lysate (10 μg protein) was added into the reaction buffer , followed by the addition of NADP+ with a final concentration of 0.1 mmol·L- 1. Finally, an enzyme kinetics program was set in the multifunctional microplate reader (one measurement every 20 s for 10 min). The absorbance was measured at a wavelength of 340 nm.Total protein was extracted from the cells using RIPA Lysis and Extraction Buffer . The protein in cell lysates was quantified and samples were adjusted to the same protein concentration. Because 6PGD catalyzes the reduction of NADP2.7When cells reached 80%–90% confluence, cells were harvested and lysed using RIPA Lysis buffer, and total protein was quantified. Samples were separated by SDS-PAGE and then transferred to a polyvinylidene fluoride membrane. The membrane was blocked with 5% skimmed milk powder for 2 h and then incubated with primary antibodies  on a shaker at 4℃ overnight. Membranes were then incubated with the corresponding secondary antibody (anti-mouse 1:1000 or anti-rabbit 1:1000) for 2 h. The membranes were then incubated with a chemiluminescent solution for 1 min and exposed with a Bio-Rad gel imaging system.2.8Cells were plated in 96-well plates at a density of 4000 cells per well in a volume of 200 μL per well. After incubation overnight, cells were treated with drugs for 24 h  and 2 d (for cell proliferation assay), respectively. Subsequently, 20 μL of MTT (thiazolyl blue tetrazolium bromide) solution  was added to each well and cells were incubated for another 4 h. The supernatant was carefully aspirated from the wells and 150 μL of DMSO (dimethyl sulfoxide) was added into each well, followed by oscillation for 10 min. The absorbance in each well was measured and recorded at a wavelength of 570 nm. Cell growth curves were drawn with drug concentration as the abscissa and the absorbance as the ordinate.2.9P value of less than 0.05 was considered to indicate statistical significance.All experiments were performed with three replicates and data were subjected to an analysis of variance by t-test using GraphPad Prism 5.0 . A 33.1To explore the mechanism of cisplatin resistance in lung and ovarian cancers, we used cisplatin-sensitive and cisplatin-resistant ovarian cancer cell lines  and lung cancer cell lines . We confirmed that OV2008 and A549 cells were more sensitive to cisplatin compared with their counterparts, C13* and A549DDP cells  can quantitatively describe the additive (CI =1), synergetic (CI <1) or antagonistic (CI >1) effects of drug combination . The com4Chemotherapy is one of the most important strategies for the treatment of malignant tumors. However, tumor cells can show resistance to chemotherapeutic drugs, which often leads to failure of chemotherapy . For exaActivation of 6PGD has great implications for the PPP and tumor growth in various tumors. 6PGD can inhibit LKB1-AMPK signaling and link the PPP with the lipid synthesis pathway, accelerating tumor growth. Thus, 6PGD can be used as a potential anti-cancer target. S3 is aIn this study, we found a higher cell proliferation rate in cisplatin-resistant cell lines compared with cisplatin-sensitive cell lines. We also demonstrated the upregulation of 6PGD, an enzyme in the PPP, in cisplatin-resistant cell lines compared with cisplatin-sensitive lines. We found that S3 specifically targeted 6PGD to inhibit its enzyme activity and attenuate cell proliferation in ovarian and lung cancer cells. A combination treatment of the small molecule inhibitor S3 with cisplatin significantly improved cisplatin sensitivity and increased cell mortality in cisplatin-resistant cell lines, with a synergistic effect. Taken together, these results suggest that cisplatin resistance in cisplatin-resistant cell lines can be overcome by specific inhibition of 6PGD enzyme activity using the small molecule inhibitor S3.The process of developing new anti-cancer drugs is highly expensive and time consuming. Therefore, current cancer treatment strategies often involve combining one or several other drugs, which has reduced the overall cost of combined combination therapy studies and simultaneously increased the cost-effectiveness of therapy. Additionally, the combination of drugs with other therapeutic agents has shown promising results with respect to reducing the tumor burden. Therefo