Medulloblastoma (MB) is treated with surgery and chemotherapy, with or without irradiation, but unfortunately >20% of the patients are not cured, and treatment comes with serious long-term side effects, so novel treatments are urgently needed. Phosphoinositide 3-kinases (PI3K), fibroblast growth factor receptors (FGFR), and cyclin-D kinases (CDK) play critical roles in cancer, and especially PI3K is crucial in MB, so here targeted therapies against them were explored.MB cell lines DAOY and UW228-3 were exposed to PI3K (BYL719), FGFR (JNJ-42756493), and CDK4/6 (PD-0332991) inhibitors, as single or combined treatments, and their viability, cell confluence, apoptosis, and cytotoxicity were examined. Moreover, the inhibitors were combined with cisplatin, vincristine, or irradiation.Single treatments with FGFR, PI3K, or CDK4/6 inhibitors decreased viability and proliferation slightly; however, when combining two inhibitors, or the inhibitors with irradiation, sensitivity was enhanced and lower doses could be used. A more complex pattern was obtained when combining the inhibitors with cisplatin and vincristine.The data suggest that combination treatments with PI3K, FGFR, and CDK4/6 inhibitors for MB could be beneficial and their use should be pursued further. Likewise, their combination with irradiation gave positive effects, while the addition of cisplatin and vincristine resulted in more complex patterns, which need to be investigated further. Yearly, around 300,000 children aged 0 to 19 years old are diagnosed with cancer, and notably brain and central nervous system (CNS) tumors are among the most frequent childhood solid tumors . MedulloTreatment of MB consists of surgery, chemotherapy (CT), and usually radiation therapy (RT), depending also on the age of the individual; however, despite all efforts, around 30% of the patients succumb to disease, and those who survive often suffer from severe long-term side effects , 13. TheLately, great progress has been obtained in cancer treatment, and much of the advances have been due to the use of immunotherapy with PD1/PDL1 inhibitors, but also targeted therapy has been helpful with most experience in adult cancer , 15. ForThe PI3K pathway has been investigated to some extent in childhood cancer, while this is not as much the case for CDK4/6, and even less so for members of the FGFR family , 22. Mutin vitro and demonstrated that these inhibitors and especially in combination efficiently decreased viability as well as inhibition in the increase in cell confluence of the cell lines  and MB cell lines (with/without PI3K mutations) ll lines –30. The ll lines , 29. Howll lines , 19.Consequently, it is of great importance to accumulate more knowledge on the potential sensitivity of MB to FDA-approved PI3K, CDK4/6, and FGFR inhibitors, alone or combined, or in different combinations together with clinically used cytostatic drugs cisplatin or vincristine, or RT. Here, we therefore tested two frequently used MB cell lines, DAOY and UW228-3, the former with, and the latter without, a PI3K mutation, but both with TP53 mutations, and thereby representative for aggressive MB, for their sensitivity to inhibitors of PI3K (BYL719), FGFR (JNJ-42756493), or CDK4/6 (PD-0332991) alone, or in combination, and together with cisplatin, vincristine, or RT.https://depmap.org/portal/, neither of the cell lines had any FGFR3 mutations or MYC amplification, while DAOY had an in-frame deletion with a non-conserving PIK3R1 mutation, and they have both a mutation in TP53. DAOY, cultured in Minimum Essential Media (MEM), and UW228-3 in Dulbecco’s Modified Eagle Medium (DMEM/F-12), with both media supplemented with 10% fetal bovine serum (FBS), 1% L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin, were maintained at 37°C in a humidified incubator with 5% CO2. Media and FBS were purchased from Gibco, Waltham, MA, USA.MB group SHH cell lines, DAOY, purchased from ATCC, and UW228-3, kindly provided by Prof. M. Nistér (Karolinska Institutet), were used in this study. According to gene bank 3 and 5 × 103 cells, respectively, in 90–200 µl medium/well for the different assays, and the edges were filled with medium to avoid edge effects. Penicillin and streptomycin were excluded from the media in the different assays to avoid any interference with the drugs.DAOY and UW228-3 were seeded at 2.5 × 10Alpelisib (BYL719) used as PI3K inhibitor, JNJ-42756493 as FGFR inhibitor, and PD-032991 as CDK4/6 inhibitor were purchased from Selleckhem Chemicals, Munich, Germany, and introduced 24 h after cell seeding. The concentrations of the drugs were as follows: for BYL719, 1.0–10 µM; JNJ-42756493, 1.0–10.0 μM; and PD-032991, 5.0–20 μM.Stocks of cisplatin  and vincristine  were diluted in PBS, and the used concentrations were 1–10 μM for cisplatin and 5 nM–1 μM for vincristine.3 (DAOY) and 5×103 (UW228-3) were treated 24 h after seeding, and then they were exposed to 0, 2, or 10 Gy 3 h after treatment with the X-Rad 225XL machine .Cells at 2.5×10Cell viability was followed by the WST-1 assay . Here, WST-1  is cleaved to formazan by cellular enzymes in viable cells as presented before in detail .Cells seeded in 200 μl medium/well in a 96-well plate, were placed into the IncuCyte S3 Live−Cell Analysis System  for up to 72 h after seeding. The machine was set to scan the plates and take pictures every 2 h, with PBS as control and culture medium as background. Cell confluence was followed in these images.IncuCyte Red Cytotoxicity reagent and IncuCyte Caspase-3/7 Green Apoptosis assay  were utilized to follow cytotoxicity and apoptosis. After 24 h of seeding, the medium was replaced with fresh medium containing the cytotoxicity reagent  and the apoptosis reagent at a ratio of 1:1,000. The drugs were added thereafter, and for more details see , 30.“Highest Single Agent” and dose-effect-based approach “median-effect method” (based on Loewe Additivity) were used  as described before , JNJ-42756493 (1–10 μM), or PD-0332991 (5–20 μM), for 24, 48, and 72 h are compiled in Combinational effect analysis and dose-effect-based median-effect principle were also calculated as described below. Shortly, in the combinational effect analysis, having a combinatorial index (CI) CI>1 indicates a positive and a CI>1 a negative effect. For the dose-effect-based median-effect principle, a CI<0.7 indicates synergism, a 0.7<CI>1.45 indicates an additive effect, while a CI>1.45 indicates an antagonistic effect. The WST-1 assays were also complemented with assessment of cell confluence, cytotoxicity, and apoptosis using the IncuCyte S3 Live−Cell Analysis System, and the data extrapolated from these assays are presented below.BYL719 induced dose-dependent effects in both DAOY and UW228-3. The highest BYL719 dose induced a significant decrease in absorbance compared to PBS at all timepoints for DAOY , while this was the case for only the 5 μM dose at 48 h for UW228-3 (p<0.05) , , but not in UW228-3 (IC50 (inhibitory concentration 50%). To better evaluate the sensitivity of the two cell lines to the inhibitors, IC50 values for all cell lines were calculated and are presented in To summarize, the data indicated that DAOY was more sensitive than UW228-3 to all of the single inhibitor treatments.The highest BYL719 and JNJ-42756493 dose combination decreased viability at all timepoints in both DAOY and UW228-3, as did the intermediate dose combination at all timepoints for UW228-3 and at 48 h for DAOY, while with the lowest dose combination, this was the case only at 72 h for DAOY    , JNJ-42756493 (1–10 μM), or PD-0332991 (5–20 μM), respectively, and for combinations of BYL719, and JNJ-42756493 (both 1–10 µM) and PD-0331991 (5–10 µM) in comparison to PBS are depicted in The highest BYL719 dose inhibited an increase in cell confluence (when compared to PBS) in DAOY after 72 h, while the intermediate dose presented a marginal effect, and the lowest dose had no effect (The highest JNJ-42756493 inhibited an increase in the cell confluence (when compared to PBS) of both DAOY and UW228-3, while for both cell lines the intermediate dose had only a slight effect, while the lowest dose had no effect (The highest PD-0332991 significantly inhibited an increase in cell confluence the whole follow-up period (when compared to PBS) for both DAOY and UW228-3, and the intermediate dose had a marginal effect in DAOY, while no other effects were observed (Combinations of the two highest doses of BYL719 and JNJ-42756493 inhibited an increase in cell confluence completely (when compared to PBS) of DAOY, as was the case for UW228-3 with the highest dose combination  for DAOY, while for UW228-3, a similar but less prominent tendency was observed when compared to the use of the corresponding single drug doses (Combining JNJ-42756493 and PD-332991 (when compared to PBS) slightly improved the inhibition of an increase in cell confluence as compared to using each single drug alone in both cell lines .Combining the highest BYL719 and JNJ-42756493 doses resulted in pronounced cytotoxicity in DAOY and some cytotoxicity in UW228-3, while intermediate doses gave some cytotoxicity in DAOY, while all other effects were marginal .To summarize, inhibition of the increase in cell confluence paralleled the inhibition of viability for both cell lines. More limited effects were obtained with regard to cytotoxicity and apoptosis, with the exception of when high doses of PD-0332991 were used and to a lesser extent when BYL719 and JNJ-42756493 were combined.Data from at least three experiments with WST-1 viability assays 24, 48, and 72 h after single administrations of cisplatin (1–10 µM) and vincristine (5–100 nM) are depicted below, and with regard to IC50 values in Cisplatin induced dose-dependent responses, with DAOY being more sensitive than UW228-3 with both presenting significantly decreased viability compared to PBS with the highest dose the whole observation period, while only DAOY presented a significant decrease in viability at 72 h after treatment with the lower doses      and for UW228-3 with the highest JNJ-42756493 and vincristine combination   for UW228-3   (Combination indexes (CIs) of the inhibitors BYL719, JNJ-42756493, or PD-0332991 combined with cisplatin or vincristine were calculated 24 and 48 h after treatment of DAOY and UW228-3, and the data after 48 h treatment are shown in The overall combination effects show some variations with synergistic, additive, and antagonistic effects depending both on the specific drug and cell line. Treatment including cisplatin combinations showed a more complex picture than those including vincristine combinations.More specifically, combining cisplatin with BYL719 or JNJ-42756493 mainly gave additive or synergistic effects for DAOY, while for UW228-3, mainly neutral or antagonistic effects were observed, while upon combining cisplatin and PD-0332991, more neutral effects were obtained for both cell lines  and vincristine (5 nM–1 μM), BYL719 and JNJ-42756493 (both 1–10 µM), or PD-0332991 (5–20 μM) and their combinations in comparison to PBS are shown in In parallel, at least three experiments were also performed for cytotoxicity, but here only a representative figure for cytotoxicity is presented below. Apoptosis experiments were performed once, since no major effects were observed (data not shown).The two highest doses of cisplatin induced an almost complete inhibition of an increase in cell confluence (when compared to PBS) in DAOY, and this was also the case with the highest cisplatin dose for UW228-3, while the lower doses only gave slight inhibition (Dose-dependent inhibition of increase in cell confluence (when compared to PBS) was observed upon treatment of DAOY and UW228-3 with vincristine, and complete inhibition of proliferation was observed with the two highest doses for DAOY, while only intermediate effects were obtained for UW228-3 with the corresponding highest doses  for all combinations for DAOY, while for UW228-3, this was only the case for the combination including the highest cisplatin concentration  for the highest dose combinations in DAOY and UW228-3 and intermediate decreases with the other doses (Combining JNJ-42756493 and cisplatin gave dose-dependent responses and a complete inhibition of increased cell confluence (when compared to PBS) for all dose combinations for both DAOY and UW228-3 (Combining JNJ-42756493 and vincristine gave dose-dependent responses and complete inhibition of increased cell confluence (when compared to PBS) within all dose combinations at all timepoints for both DAOY and UW228-3 (Combining PD-0332991 and cisplatin gave dose-dependent responses and a complete inhibition of increased cell confluence (when compared to PBS) for both dose combinations at all timepoints for DAOY, and with the highest dose combination for UW228-3 (Combining PD-0332991 and vincristine gave dose-dependent responses and a complete inhibition of increased cell confluence (when compared to PBS) for both dose combinations at all timepoints for DAOY, but only to some extent for UW228-3 .In summary, DAOY was more sensitive than UW228-3 when treated with inhibitors and cytostatic drugs. Inhibition of the increase in cell confluence when compared to PBS paralleled inhibition of viability for both cell lines, while limited effects, with few exceptions on DAOY, were obtained with regard to cytotoxicity and apoptosis upon combining the cytostatic drugs with the inhibitors.Data from at least three experiments with WST-1 viability assays 24, 48, and 72 h after treatment of DAOY and UW228-3, respectively, with BYL719, JNJ-42756493, and PD-0332991 followed by no irradiation, or 10 Gray (Gy); or no irradiation, and 2 Gy are depicted in Single treatments with 2 and 10 Gy did not affect viability of DAOY but did significantly affect viability of UW228-3 at 72 h         , the FGFR inhibitor erdafitinib (JNJ-42756493), and the CDK4/6 inhibitor palbociclib (PD-332991) were tested alone and combined or together with cisplatin, vincristine, or RT for their ability to inhibit the growth of SHH MB cell lines DAOY and UW228-3.Both DAOY and UW228-3, but the latter to a lower extent, showed dose-dependent decreases in viability and proliferation upon treatment with BYL719, JNJ-42756493, and PD-332991, cisplatin and vincristine, but only PD-332991 induced pronounced effects on cytotoxicity or apoptosis in the highest tested dose. Upon combining the inhibitors, additive/synergistic effects were observed on the decrease of viability and especially of the more resistant cell line UW228-3, as did the inhibition of an increase in cell confluence when compared to the PBS control. Likewise, upon combining the inhibitors with cisplatin or vincristine, some positive effects were observed, and the most prominent (additive/synergistic) effects on viability and cell confluence were observed when combining BYL719 and cisplatin or vincristine. Finally, both DAOY and UW228-3 were exposed to the inhibitors alone or combined, with and without irradiation with 2 or 10 Gy. Some positive effects were obtained with irradiation with the administration of inhibitors alone, but irradiation did not increase the efficacy when two inhibitors were combined.Others and we have previously reported that MB cell lines respond with decreased viability to PI3K inhibitors such as BEZ235, BKM120, and GDC-0941 and CDK4/6 inhibitors such SCH727965, and PD-332991, but only we have shown MB cell line sensitivity to FGFR inhibitors such as AZD4547 , 37. HowRemarkably, although marked inhibition of viability and an inhibition in increase in cell confluence when compared to the PBS control were obtained in both cell lines upon treatment with most of the single and combined inhibitor treatments, the effects on cytotoxicity and apoptosis were not as prominent. However, there were some exceptions, and when using PD-332991 or combining BYL719 and JNJ-42756493 in relatively high doses, cytotoxicity and apoptosis were observed. As mentioned above, there are limited or no reports on the use of the above drugs on MB cell lines; however, it has been reported previously that other PI3K inhibitors (BEZ235 and BYL719) do not have a major cytotoxic effect, while the cytotoxic effect of FGFR inhibitor AZD4547 is possibly somewhat increased . Nevertheless, the data described above underline that both MB cell lines, both exhibiting TP53 mutations, independently of having or not PI3K, CDK4/6, or FGFR mutations, can be sensitive to the tested inhibitors. This is in line with previous studies by others and us, where different tumors and tumor cell lines have been reported to be sensitive to PI3K and FGFR inhibitors, despite not having PI3K, CDK4/6, and or FGFR mutations . In the future, it would be valuable to pursue similar studies in a broader range of MB cell lines and under more strict conditions, e.g., to maintain SHH-signaling . In addiTo conclude, combination treatments of PI3K, FGFR, and/or CDK4/6 inhibitors, as well as especially the PI3K inhibitor BYL719 with cisplatin or vincristine on the MB cell lines DAOY and UW228-3, enhanced inhibition of cell viability and increase in cell confluence. In addition, irradiation of the corresponding cell lines tended to increase the effects of single but not combined inhibitor treatments. DAOY was generally more sensitive to the inhibitors compared to UW228-3, which could possibly be due to it having a PIK3R1 mutation, but further studies are needed to elucidate if indeed this is the case.Taken together our data suggest that PI3K and FGFR or CDK4/6 inhibitor combinations or single inhibitor CT/RT combinations may provide possible therapeutic opportunities for therapy of resistant MBs and should be investigated further.The original contributions presented in the study are included in the article/ML and OK did the majority of the experiments, interpreted the data, calculated the statistics, and contributed to the writing of the manuscript. HM and EP-S collaborated with OK and performed some experiments, contributed together with OK in the graphs of the manuscript. SH and ER initiated the experiments and the interpretation of the initial experiments and contributed to the writing of the Material and Methods section, all under the supervision of OK. ML, TD, and OK made substantial contributions to conception and design, acquisition of data, analysis and interpretation of data and have been involved in drafting the manuscript and revising it critically for important intellectual content. TD has also provided the sources of the performance of the experiments. TD and OK provided the financial support for conducting the research project. All authors contributed to the article and approved the submitted version.This research was funded by Swedish Childhood Cancer Fond , the Swedish Cancer Society , the Stockholm Cancer Society (181053), the Swedish Cancer and Allergy Foundation (190), Karolinska Institutet of Sweden (2018:0007), Stiftelsen AnnaBrita and Bo Casters Minne (LA2019-0080), Svenska Läkaresällskapet (SLS-934161), Åke wiberg foundation (M20-0061).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Corynebacterium glutamicum cell mass (CGCM) as a protein supplement and a source of cell wall fragments supporting the growth and intestinal health of nursery pigs. Thirty-two pigs (21 d of age) were allotted to four treatments (n = 8) based on the randomized block design with sex and initial body weight (BW) as blocks. The main effect was the dietary supplementation of lysed CGCM  replacing blood plasma and fed in two phases . Feed intake and BW were measured at the end of each phase. Pigs were euthanized on day 21 to collect jejunal tissue and mucosa to evaluate intestinal health. Ileal digesta were collected to measure the apparent ileal digestibility of nutrients in diets. Data were analyzed using Proc Mixed and Reg of SAS. Increasing daily intake of CGCM increased  ADG of pigs. Increasing CGCM supplementation affected  the relative abundance of Lactobacillaceae (minimum: 26.4% at 1.2% CGCM), Helicobacteraceae (maximum: 29.3% at 1.2% CGCM), and Campylobacteraceae (maximum: 9.0% at 1.0% CGCM). Increasing CGCM supplementation affected  the concentrations of immunoglobulin G (maximum: 4.94 µg/mg of protein at 1.0% CGCM) and protein carbonyl , whereas linearly decreased (P < 0.05) malondialdehyde (MDA) in the proximal jejunal mucosa. Increasing CGCM supplemention affected  intestinal enterocyte proliferation rate (maximum: 13.3% at 1.0% CGCM), whereas it did not affect intestinal morphology and the nutrient digestibility. In conclusion, supplementing 1.0% to 1.2%, reducing blood plasma supplementation by 0.7% to 0.9%, respectively, increased potential pathogenic microbiota associated in the jejunal mucosa resulting in increased immune response, enterocyte proliferation, and PC concentration. However, supplementing diets with 2.1% CGCM, replacing 1.5% blood plasma, improved growth performance, and reduced MDA without affecting nutrient digestibility, intestinal morphology, and microbiota in the jejunal mucosa. In this study, based on the polynomial contrast, supplementing 1.0% to 1.2% CGCM suppressed the benefits from blood plasma, whereas supplementing 2.1% CGCM showed functional benefits of CGCM with similar effects from blood plasma supplementation.The objective was to determine the nutritional and functional values of lysed  Soybean meal has been predominantly used as a protein supplement in pig production. However, the antinutritional factors such as allergenic proteins, trypsin inhibitors, and flatulence producing oligosaccharides limit its use in nursery pig diets . PreviouMethylococcus capsulatus , processed vegetable protein, and insect protein . The SCPpsulatus  and Coryutamicum  can overCorynebacterium glutamicum is a Gram-positive bacteria, commonly used to produce AA, and generally recognized as safe (Corynebacterium glutamicum cell mass (CGCM) is removed by filtration and then disposed of or used as fertilizers  at 900 bars for 4 s at 30 °C with a maximum allowance of 70 °C in the holding tube. After the holding time, the CGCM in deionized water flowed to a serpentine tube immersed into chilled water for rapid cooling. The lysis process was completed in four cycles in a continuous system . The lysed CGCM was dried using a freeze dryer . The lysing process was based on the study by Lysine broth including intact CGCM was obtained from CJ Bio . The lysine broth containing 15% to 20% dry matter (DM) was centrifuged at 3,100 × Images of CGCM before and after the lysis were taken using a variable pressure scanning electron microscope  at the 5 kV efficient voltage and 10 mm working distance to obtain the percentage of lysed cells. Each image of CGCM from before and after the lysis  was evenn = 8) based on the randomized complete block design with initial BW (light and heavy) and sex (gilts and barrows) as blocking criteria. All pigs were housed in individual pens and had ad libitum access to water and the assigned experimental diets for 21 d based on two phases: phase 1 (days 0 to 10) and phase 2 (days 11 to 21). Dietary treatments consisted of a basal diet with lysed CGCM at four levels  of CGCM replacing blood plasma, based on Lys content . Therefore, up to 1.5% blood plasma was replaced by 2.1% CGCM. All experimental diets were formulated to meet or exceed the nutrient requirements based on the study by Thirty-two nursery pigs (16 barrows and 16 gilts) at 21 d of age with initial body weight (BW) of 7.2 ± 0.6 kg were used for the experiment. Pigs were allotted to four dietary treatments  very hard and dry stool, (2) firm stool, (3) normal stool, (4) loose stool, and (5) watery stool with no shape following After 21 d of feeding, all pigs were euthanized by exsanguination after the penetration of a captive bolt to the head to collect samples. Sections of proximal  and distal  jejunum were collected and rinsed with 0.9% saline solution to collect mucosa and tissue. The jejunal mucosa was collected into 2 mL microcentrifuge tubes and immediately frozen in liquid nitrogen, and then stored at −80 °C for determination of immune status, oxidative stress markers, and microbiota. Proximal and distal jejunum tissues (5 cm) were collected into 50 mL polypropylene tubes with 40 mL of 10% buffered formaldehyde solution for histological evaluation. Ileal digesta was collected into 150 mL containers placed on ice and then stored at −20 °C for analysis of apparent ileal digestibility (AID).Distal jejunal mucosa samples were used to extract DNA using QIAamp Fast DNA Stool Mini Kit  following the description from g at 4 °C as described by Jejunal mucosa samples (1 g) from microcentrifuge tubes were taken and added with 2 mL of PBS solution into 5 mL polypropylene tubes. Mucosa samples were homogenized using a tissue homogenizer  for 30 s on ice and transferred to a new 2 mL microcentrifuge tube for centrifugation for 15 min at 14,000 × The concentrations of total protein, tumor necrosis factor alpha (TNFα), interleukin-8 (IL-8), malondialdehyde (MDA), protein carbonyl (PC), IgA, and IgG were analyzed by the colorimetric method and the absorbance was measured on a plate reader  and the Gen5 Data Analysis Software (BioTek Instruments).The concentration of total protein was analyzed by using Pierce BCA Protein Assay Kit  following procedures of The concentration of TNFα was analyzed in proximal and distal mucosa using the Porcine TNFα Immunoassay Kit  as described by Two sections of proximal and distal jejunum were cut and placed into cassettes and sent to North Carolina State University College of Veterinary Medicine Histopathology Lab  for immunohistochemistry staining with Ki-67 assay. One slide represented one pig. Pictures were taken using a microscope  and Infinity Analyze and Capture software .http://imagejs.org). The percentage of Ki-67-positive cells was used as an indicator of the enterocyte proliferation rate in the crypt (Pictures were taken at magnification 40× to measure villus height (VH) and width (VW), crypt depth (CD), and villus height to crypt depth ratio (VH:CD). Lengths of 15 well-shaped villi and corresponding crypts were measured in each slide. Pictures of 15 well-shaped crypts were taken from each slide at magnification 100× and cropped to calculate the percentage of the Ki-67-positive cells to the number of epithelial cells in a crypt by operating the ImageJS . Phase 2 diets and freeze dried ileal digesta were ground to fine powder form. The concentration of titanium dioxide in the diets and digesta was measured by following Apparent ileal digestibility of DM, GE, EE, CP, and AA was calculated as previously described by 2 in a diet/TiO2 in digesta) × (nutrient in digesta/nutrient in a diet)]AID% = 100 × {1 – [(TiOP < 0.05), the procedure RSREG was used to predict the critical value and the stationary point. The linear and quadratic effects of daily intake (g/d) of CGCM were tested by REG procedures. The analysis of fecal score data was performed by using Kruskal–Wallis test with Dwass, Steel, Critchlow-Fligner method option for pairwise two-sided multiple comparisons following P < 0.05 and 0.05 ≤ P < 0.10 were considered as a tendency.All data except for fecal score were analyzed based on a randomized complete block design using Mixed procedure by SAS 9.3 . Initial BW and sex were considered as blocks. Dietary treatments were defined as fixed effects and blocks were random effects. The LSMEANS statement was used to calculate mean values for all treatments. Linear and quadratic effects of increasing CGCM supplementation were tested by orthogonal polynomial contrasts using the CONTRAST statement. When a quadratic effect was significant (P < 0.05) the proportion of lysed Corynebacterium glutamicum cells from 4.3% in intact CGCM to 58%  fecal score than pigs fed 0% CGCM on day 19 of the experiment, whereas no differences were observed among other treatments (Pigs fed 0.7% CGCM had lower (eatments .P < 0.05) on the alpha diversity of jejunal mucosa-associated microbiota estimated with Shannon index at family (maximum 2.8 at 1.0% CGCM), genus (maximum 2.7 at 1.0% CGCM), and species (maximum 3.8 at 1.0% CGCM) levels (P < 0.05) on the alpha diversity of jejunal mucosa-associated microbiota estimated with Simpson index at family (maximum 0.8 at 1.1% CGCM), genus (maximum 0.7 at 1.1% CGCM), and species (maximum 0.9 at 1.1% CGCM) levels.Increasing CGCM supplementation had quadratic effects  levels . IncreasP = 0.094) at the family level with the maximum 0.75 at 5.4 g/d CGCM intake . Increasing the daily CGCM intake tended to have the quadratic effects on the alpha diversity of jejunal mucosa-associated microbiota estimated with Shannon (P = 0.091) and Simpson (P = 0.079) indexes at the genus level with the maximum at 2.71 and 0.74, when the daily CGCM intakes were 4.7  and 5.4 g/d , respectively. Increasing the daily CGCM intake had the quadratic effects (P < 0.05) on the alpha diversity of jejunal mucosa-associated microbiota estimated with Shannon and Simpson indexes at the species level with the maximum at 3.88 and 0.88, when the daily CGCM intake was 5.0 g/d , increasing the daily CGCM intake tended to have quadratic effects on the alpha diversity of jejunal mucosa-associated microbiota estimated with Simpson index  on the minimum relative abundances of Firmicutes (minimum 37.8% at 1.1% CGCM) and Proteobacteria (maximum 44.0% at 1.1% CGCM) and tended to have a quadratic effect (P = 0.058) on the relative abundance of Actinobacteria (P = 0.055) on the relative abundance of Firmicutes with minimum 37.5% at 5.4 g/d CGCM intake (P < 0.05) on the relative abundance of Proteobacteria with maximum abundance at 46.5%, when the daily CGCM intake was 5.6 g/d  on the relative abundance of Lactobacillaceae (minimum 26.4% at 1.2% CGCM), Helicobacteraceae (maximum 29.3% at 1.2% CGCM), and Campylobacteraceae (maximum 9.0% at 1.0% CGCM) (P = 0.060) on the relative abundance of Propionibacteriaceae, whereas linearly reduced (P < 0.05) the relative abundance of Bifidobacteriaceae. When the data were analyzed based on the daily CGCM intake (g/d), increasing the daily CGCM intake tended to have quadratic effects on the relative abundance of Helicobacteraceae (P = 0.053) and Campylobacteraceae (P = 0.098) with minimum abundance at 31.3% and 9.0% when the daily CGCM intake was 5.8 g/d  and 5.1 g/d , respectively. Increasing the daily CGCM intake tended to linearly reduce (P = 0.067) the relative abundance of Bifidobacteriaceae .At the family level, increasing CGCM supplementation had quadratic effects (0% CGCM) . IncreasLactobacillus (P = 0.056) and Propionibacterium (P = 0.073) and had quadratic effects (P < 0.05) on the relative abundance of Helicobacter (31.9%) and Campylobacter (9.4%) at 1.2% and 1.0% CGCM, respectively (P = 0.068) the relative abundance of Megasphaera and linearly reduced (P < 0.05) relative abundance of Bifidobacterium. When the data were analyzed based on the daily CGCM intake (g/d), increasing the daily CGCM intake had a quadratic effect (P < 0.05) on the relative abundance of Helicobacter with 33.9% at 5.7 g/d CGCM intake . Increasing the daily CGCM intake tended to linearly reduce (P = 0.064) the relative abundance of Bifidobacterium .At the genus level, increasing CGCM supplementation tended to have quadratic effects on the relative abundance of ectively . IncreasP = 0.023) the relative abundance of Prevotella sp. (P = 0.096) and tended to have a quadratic effect (P = 0.090) on the relative abundance of Lactobacillus kitasatonis and tended to linearly reduce (P = 0.053) the relative abundance of Corynebacterium glutamicum. Increasing CGCM supplementation had a quadratic effect (P < 0.05) on the relative abundance of Pelomonas puraquae (maximum 1.1% at 1.0% CGCM). When the data were analyzed based on the daily CGCM intake (g/d), increasing the daily CGCM intake tended to linearly reduce (P = 0.077) the relative abundance of Prevotella sp. (P < 0.05) on the relative abundances of Pelomonas puraquae with minimum abundance at 1.2%, when the daily CGCM intake was 4.8 g/d  on the concentration of IgA and had quadratic effects (P < 0.05) on the concentrations of IgG (maximum 4.94 µg/mg of protein at 1.0% CGCM) and PC (maximum 6.12 nmol/mg of protein at 1.1% CGCM). Whereas increasing CGCM supplementation linearly reduced (P < 0.05) the concentration of MDA. When the data were analyzed based on daily CGCM intake (g/d), increasing the daily intake of CGCM tended to have a quadratic effect (P = 0.080) on the concentration of PC with maximum 6.19 nmol/mg of protein at 4.9 g/d CGCM intake  and PC (P = 0.051).In the distal jejunal mucosa, increasing CGCM supplementation did not affect the concentration of TNFα, IgA, IgG, and MDA in nursery pigs . IncreasP = 0.090) VH:CD in the proximal jejunal mucosa and had a quadratic effect (P < 0.05) on the maximum intestinal enterocyte proliferation rate (13.3%) at 1.0% CGCM in the distal jejunal mucosa.Increasing CGCM supplementation did not affect VH, VW, and CD in proximal and distal jejunum . WhereasIncreasing CGCM supplementation did not affect the AID of DM, GE, EE, CP, and AA in nursery pig diets .This study demonstrated that CGCM can be supplemented up to 2.1%, replacing 1.5% blood plasma, in diets without affecting the growth performance and intestinal health of nursery pigs. Additionally, increasing daily CGCM intake increased the ADG of pigs. The results observed in this study are in accordance with previous studies showing that SCP can be used as alternative protein supplements to replace plant or animal protein supplements improving growth performance in broilers  and pigsHelicobacteraceae, which belongs to Proteobacteria, is commonly associated to unhealthy pigs (Lactobacillaceae in Firmicutes is predominant in healthy pigs (Previous studies have demonstrated that diet composition greatly affected the intestinal microbiota . Interesthy pigs , whereasthy pigs .Lactobacillaceae, which can increase the production of anti-inflammatory cytokines and, consequently, reduce proinflammatory cytokines (Corynebacterium glutamicum, contains TA and Slp that can affect the intestinal microbiota by immunomodulatory effects (Lactobacillus attaches to receptors on the intestinal epithelial cells and, consequently, reduces the availability of receptors for the adherence of potential pathogens (Helicobacteraceae. However, supplementing 2.1% CGCM, replacing 1.5% blood plasma, in diets may have provided sufficient TA and Slp for their functional roles reducing the relative abundance of Proteobacteria and increasing Firmicutes in the jejunal mucosa.Blood plasma not only is highly digestible but also provides around 20% of IgG that plays an essential role in the immune response by affecting the host immune system and intestinal microbiota , 2018. Iytokines . The cel effects  and by c effects . Previouathogens . In thisThe alpha diversity of intestinal microbiota can also be affected by the diet composition . The carHelicobacteraceae and Campylobacteraceae, can affect the immune system and oxidative stress status in the jejunum of nursery pigs (Lactobacillaceae can be associated with the synthesis of anti-inflammatory cytokines (The modulation of intestinal microbiota is generally associated with changes in the immune response (Duarte and ery pigs . In respery pigs . Conversytokines .Corynebacterium glutamicum, contains CWGs that have immunomodulatory functions, reducing excessive immune reactions (Previous study showed that pigs fed blood plasma in diets improved intestinal health by preventing from the production of proinflammatory cytokines, such as TNFα, IL-8, and interferon-γ, resulting in apoptosis of infected cells and the production of additional proinflammatory cytokines and chemokines . Therefoeactions . The CWGeactions . Accordieactions . In the The activation of the immune system can increase the production of reactive oxygen species leading to an increase in oxidative stress . MalondiThe oxidative stress can cause damage in the enterocytes by oxidizing lipids, proteins, and DNA resulting in cellular apoptosis, consequently, affecting the intestinal morphology . IncreasIn conclusion, based on the polynomial model, supplementing CGCM at 1.0% to 1.2%, reducing the blood plasma supplementation by 0.7% to 0.9%, respectively, increased potential pathogenic microbiota resulting in increased immune response, enterocyte proliferation, and PC concentration. However, supplementing CGCM at 2.1%, reducing blood plasma supplementation by 1.5%, improved growth performance, and reduced MDA concentration without affecting nutrient digestibility, intestinal morphology, and microbiota in the jejunal mucosa. Collectively, supplementing 1.0% to 1.2% CGCM suppressed the benefits from blood plasma, whereas supplementing 2.1% CGCM showed functional benefits of CGCM with similar effects from blood plasma supplementation.


Background: Myeloid-derived suppressor cells (MDSCs) are implicated in the complex interplay involving graft-versus-leukemia (GVL) effects and graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation  in hematologic malignancies. Methods: A review of literature through PubMed was undertaken to summarize the published evidence on the pathophysiology and clinical implications of MDSCs in allo-HCT. Literature sources published in English since 1978 were searched, using the terms Natural Suppressor (NS) cells, MDSCs, GVHD, and allo-HCT. Results: In vivo studies demonstrated that MDSCs derived from mobilization protocols could strongly suppress allo-responses mediated by T cells and enhance T-Reg activity, thus inhibiting GVHD toxicity. However, the influence of MDSCs on the GVL effect is not fully defined. Conclusions: The induction or maintenance of MDSC suppressive function would be advantageous in suppressing inflammation associated with GVHD. Pathways involved in MDSC metabolism and the inflammasome signaling are a promising field of study to elucidate the function of MDSCs in the pathogenesis of GVHD and translate these findings to a clinical setting. Allogeneic hematopoietic cell transplantation  is currently considered the only potentially curative option for various life-threatening malignant and benign hematologic conditions. However, allo-HCT poses two major limitations to its success; immune reactions of the (a) host versus graft (HVG), which may lead to engraftment failure, and (b) graft versus host (GVH), leading to the homonymous disease (GVHD). As the quest for the expansion of donor pool with alternative transplantations  continues, acute and/or chronic GVHD (aGVHD and cGVHD) are leading causes of morbidity and mortality ,2.The backbone of conventional treatment for aGVHD relies on the use of corticosteroids . CorticoMyeloid suppressor cells are implicated in the complex interplay involving graft-versus-leukemia (GVL) effects, GVHD, and hematological malignancies. In the microenvironment of hematological malignancies, several studies have highlighted the role of myeloid-derived suppressor cells (MDSCs) ,13. The In this review, we will try to shine a light on (a) key functions of MDSCs, (b) the role of MDSCs after allo-HCT on GVHD development, and (c) the implications of targeting MDSCs.MDSCs are a diverse population of naturally occurring immature myeloid cells characterized by their capacity to suppress responses mediated by both innate and adaptive immunity. MDSCs arise under chronic inflammatory conditions such as malignancy, infection, autoimmune diseases, trauma, GVHD, and so on. Although phenotypically similar to mature myeloid cells, they pose distinct genomic and biochemical profiles and function ,20. In tIn 1978, Bennett and colleagues were the first to demonstrate the presence of natural suppressor, myeloid cells in bone marrow and spleen of irradiated mice with the ability to suppress T cell activity . The potAs mentioned, MDSCs arise under chronic inflammatory stimuli. A two-signal model was described to explain the subsequent differentiation of myeloid suppressor cells ,32. A fiAvailable data nowadays support the notion that these gene and protein expression profiles allow MDSCs to utilize a number of mechanisms to suppress T cell responses, as demonstrated both in vitro and in vivo. The hyperproduction of inducible nitric oxidase (iNOS), arginase 1 (ARG1),  reactiveinitial tissue damage from the conditioning regimen, which activates host antigen-presenting cells (APC) by danger-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs), (2) afferent phase characterized by the stimulation and proliferation of donor T cells in response to alloantigen expressed either on host APCs, labeled as direct antigen presentation, or on donor APCs, labeled as indirect presentation, and the (3) effector phase represented by generated donor T cell-mediated cytotoxic damage against host cells through Fas–Fas ligand interaction, perforin–granzyme, and TNF-α   . On the = 0.002) .Considering their significant implications in GVHD, there is growing interest of developing agents targeting pathways where MDSCs are involved. The immunoregulatory properties of MDSCs have been demonstrated by several studies performed at different time points of a transplantation procedure: in the graft and in the donor along the post-transplantation period . Donor THowever, the hyper-responsiveness of MDSCs makes them difficult to track over time along the course of a transplanted patient. When exposed to inflammatory conditions, and in contrast to suppressed lymphocytic lineage, immature myeloid cells undergo emergency granulopoiesis  driven bp < 0.01, ANOVA) [p < 0.01, ANOVA and Tukey post-test) [The synergy between T Regs and MDSCs is another promising direction that warrants further investigation to diminish GVHD without a negative effect on GVL. In the presence of IFN-γ, MDSCs secrete IL-10 and TGFβ, activating thus T Regs  . In addist-test) . The usest-test) ,58. Suppst-test) . Howeverst-test) ,103. MorThe treatment of allo-HCT murine models with galectin-9 , a β-galactoside-binding soluble lectin family member, increased G-MDSC frequencies that showed enhanced expression of Arg-1 and iNOS and inhibited T cell proliferation, ameliorating thus the severity of aGVHD . SupportFinally, a better understanding of the role of microRNAs (miRs) in MDSC and subsequent T cell alloreactivity can be explored in terms of enhanced MDSC suppression and GVHD inhibition. miRs represent a family of small, non-coding RNAs that function as the significant endogenous triggers for RNA interference , whereasIn conclusion, MDSCs derived from mobilization protocols can strongly suppress allo-responses mediated primarily by T cells and to a lesser degree by B cells and NK cells. At the same time, MDSCs promote T Reg expansion. Transplants using mobilized peripheral blood stem cell products infuse approximately 10-fold more T cells than that found in bone marrow products. Nevertheless, the absence of GVHD in these patients has been associated with the infusion of MDSCs in these products . The indHowever, pathways involved in MDSC metabolism and the inflammasome signaling should be studied thoroughly in order to gain more knowledge regarding the applicability of these practices in the clinical setting. Furthermore, translational studies are highly needed in this field to confirm the role of MDSC in the pathogenesis of GVHD. While entering the era of CAR-T cell and other upcoming cellular therapies , appreci


TP53 (~ 60%) or PPM1D gain-of-function mutations (~ 30%) in DIPG cases. PPM1D gain-of-function mutations suppress p53 activity and result in DIPG tumorigenesis. While MDM2 is a major negative regulator of p53, the efficacy of MDM2 inhibitor has not been tested in DIPG preclinical models. In this study, we performed a comprehensive validation of MDM2 inhibitor RG7388 in patient-derived DIPG cell lines established from both TP53 wild-type/PPM1D-mutant and TP53 mutant/PPM1D wild-type tumors, as well in TP53 knockout isogenic DIPG cell line models. RG7388 selectively inhibited the proliferation of the TP53 wild-type/PPM1D mutant DIPG cell lines in a dose- and time-dependent manner. The anti-proliferative effects were p53-dependent. RNA-Seq data showed that differential gene expression induced by RG7388 treatment was enriched in the p53 pathways. RG7388 reactivated the p53 pathway and induced apoptosis as well as G1 arrest. In vivo, RG7388 was able to reach the brainstem and exerted therapeutic efficacy in an orthotopic DIPG xenograft model. Hence, this study demonstrates the pre-clinical efficacy potential of RG7388 in the TP53 wild-type/PPM1D mutant DIPG subgroup and may provide critical insight on the design of future clinical trials applying this drug in DIPG patients.Diffuse intrinsic pontine gliomas (DIPGs) are high-grade tumors of the brainstem that often occur in children, with a median overall survival of less than one year. Given the fact that DIPGs are resistant to chemotherapy and are not amenable to surgical resection, it is imperative to develop new therapeutic strategies for this deadly disease. The p53 pathway is dysregulated by The online version contains supplementary material available at 10.1186/s40478-021-01270-y. Diffuse intrinsic pontine gliomas (DIPGs) are malignant brainstem gliomas. In the United States, there are approximately 200–300 new cases of DIPG every year . DIPGs aTP53 and the genetic components of p53 signaling are also frequently mutated in DIPGs. TP53 mutations were identified in around 60% H3K27M mutant DIPGs [TP53 wild-type DIPGs, approximately 80% have mutations in exon 6 of Protein Phosphatase, Mg2 + /Mn2 + Dependent 1D (PPM1D) mutations, which result in truncated PPM1D mutant proteins. The mutations stabilize the truncated PPM1D protein and lead to increases in its native dephosphorylation activity toward p53 and other proteins in DNA damage response (DDR) and cellular checkpoint pathways [The mutational landscape of DIPG has been well characterized. Recurrent somatic Lys-27-Met mutations in histone H3 genes (either in H3.1 or H3.3) have been identified as an oncogenic driver in ~ 80% of DIPGs –9. In thnt DIPGs –15. Howepathways . Recent pathways , 17.Another approach to reactive p53 pathway is to reduce p53 protein ubiquitination and degradation by targeting Mouse double minute 2 homolog (MDM2), a key negative regulator of the p53 pathway. There are two major functions of MDM2: one as an E3 ubiquitin ligase of p53 and the other as an inhibitor of p53 transcriptional activation . In addiTP53 wild-type/PPM1D-mutant tumors, we integrate data from Mutations, Copy Number Alterations  and CRISPR screening data of 808 cancer cell lines  [TP53 and PPM1D status. We use Student’s t-test to compare the CERES score of each gene  in between molecular subgroups. These statistical analysis were performed in R, and 10% false-discovery rate  was applied using p.adjust function in R. Top dependency gene list were generated based on p value/adjusted p value and t value. CERES score of MDM2 across four subgroups were visualized with GraphPad Prism V9.0.To identify the top dependent gene of portal/) . These cH3F3A, TP53 and PPM1D genes are listed in Additional file TP53 KO isogenic lines were established and used in a previous study [Ten cell lines were obtained from biopsy or autopsy tissues of DIPG patient tumors. TT10714, TT10728 and TT10630 cell lines were used in a previous study . SF7761 us study .Cell pellets were lysed and resolved using (4–12%) NuPAGE Bis–Tris gradient gel. Gels were soaked in NuPAGE protein transfer buffer and transferred to PVDF membranes. Membranes were blocked in room temperature for 1 h and incubated with primary antibodies at 4 degree overnight. Membranes were washed and then incubated with horseradish peroxidase (HRP) conjugated secondary antibody for one hour and HRP signals were detected by chemiluminescence using the BioRad ChemiDoc MP system. H3K27M , p53 , MDM2 , p21  and GAPDH .Cells were plated at a density of 3000 cells/well in 96-well white and clear bottom microplates (Greiner Bio-One) and were incubated for 24 h. For dose-dependent studies, cells were treated with RG7388  diluted in DMSO, ranging from 7.62 nM to 50 uM, or DMSO alone as a control, for 3 days. For time-dependent studies, cells were treated with DMSO, 50 nM and 100 nM RG7388 and luminescence signal was measured at Days 0, 2 and 4. Cell viability was measured by CellTiter-Glo Luminescent Cell Viability Assay (Promega #G7571) per manufacturer’s instruction. The luminescence signals were recorded using a Tecan Infinite M200 PRO microplate reader. Relative viabilities were calculated by normalizing luminescence values for each treatment condition to DMSO treated wells.TP53 KO were treated with 100 nM RG7388 or DMSO for 24 h. Three samples for each condition were harvested and RNA extractions were done using Maxwell RSC simplyRNA Cells Kit (Promega #AS1390). RNA samples were sent to for RNA-Seq and the resulting data was analyzed using the Galaxy/Europe platform [HSJD-DIPG-007-NTC and HSJD-DIPG-platform .Cells were plated at a density of 3000 cells/well in 96-well white and clear-bottom microplates (Greiner Bio-One). After 24-h incubation, cells were treated with 100 nM RG7388 or DMSO for 24 h. Cell viability was measured by Caspase-Glo® 3/7 Assay (Promega #G8091) per manufacturer’s instruction. The luminescence signals were recorded using a Tecan Infinite M200 PRO microplate reader. Relative viabilities were calculated by normalizing luminescence values for each treatment condition to DMSO treated wells.TP53 KO were treated with DMSO and 100 nM RG7388 for 24 h. 1 × 106 Cells were harvested and fixed in cold 70% ethanol for 30 min at 4℃. After centrifugation and washing with PBS, 0.5 ml FxCycle PI/RNAse Staining Solution (ThermoFisher #F10797) was added to the cell pellet and incubated for 15–30 min at room temperature in the dark. Flow cytometry analysis was performed using 532-nm excitation with a 585/42-nm bandpass filter. Data was analyzed using FlowJo software.HSJD-DIPG-007-NTC and HSJD-DIPG-5 of HSJD-DIPG-007 cells with CMV-Firefly luciferase lentivirus (Cellomics Technology cat#C839R47) were implanted stereotactically into the nude mouse brain stem region (n = 18). On the 21st-day post injection, mice were randomly assigned to two groups. For each group, nine mice were treated by oral gavage with 50 mg/kg of RG7388 (AstaTech cat#40916) or vehicle once per day, five days per week for three weeks. Mice were assessed for neurological symptoms and weight loss and sacrificed when either 20% weight loss was measured or when they exhibited neurologic symptoms. Kaplan–Meier curves were used for survival analysis.To access the pharmacokinetic of RG7388, 12 outbred athymic nude mice (J:NU Stock#007850) were treated with RG7388 delivered by oral gavage at 50 mg/kg. Plasma and tissues were collected and analyzed by Mass Spectrometry (AB Sciex 5500). To further access the in vivo efficacy of RG7388, 5 × 10Six mice were injected with DIPG cells and treated with RG7388 (n = 3) or vehicle (n = 3) beginning on day 21 as previously described. These mice were sacrificed on day 42 and tumor samples were collected for immunofluorescence staining. The mouse brain was fixed in 10% formalin, embedded, and cut into sections. Standard H&E staining procedures and immunofluorescent staining procedures were performed. Immunofluorescent images were imaged on the Zeiss 880 at Duke Light Microscopy Core Facility. Positively stained cells were both manually counted and counted with ImageJ. Those assigned to counting were blinded to animal genotype and were given designated quadrants of specific size and magnification to count.TP53 wild-type/PPM1D mutant cell lines, we combined data from Mutations, Copy Number Alterations  and CRISPR screening data of 808 cancer cell lines  [TP53 mutation while 28 cell lines contain activated PPM1D either through PPM1D amplification or exon 6 truncations. Notably, among these 28 cell lines, 11 cell lines are TP53 wild-type. To identify the top candidate genes for survival dependence in TP53 wild-type cell lines, Student’s t-test was performed to compare gene effect (CERES score) across 18,116 genes in-between different molecular subgroups. Compared to all TP53 mutant cell lines, MDM2 is among the top ten dependent genes in TP53 wild-type, PPM1D amplified or mutant cell lines  . We deteTP53, and PPM1D mutations  compared to the TP53 mutant cell lines   between RG7388 treatment and DMSO control were assessed, and differentially expressed genes  were selected. While the global gene expression was mostly unaffected in the HSJD-DIPG-007-TP53 KO cell line, 476 genes were significantly changed in the HSJD-DIPG-007-NTC line  included the down-regulation of the pathways involving DNA replication, cell cycle, mismatch repair and homologous recombination and most notably, the upregulation of the p53 signaling pathway [P-value < 0.05, fold change > 1.5)  revealed those related to cell cycle arrest and apoptosis were significantly changed, in that apoptosis related genes , TNFRSF10B (DR5) and ZMAT3 (PAG608) were up-regulated while cell cycle progression related genes  were down-regulated , 4 to 71  and 7 to 32   and collected blood samples, cerebrospinal fluid (CSF), cerebral hemispheres tissues and brainstem tissues from the mice at different time points 0.5/1/2/4/8/24 h) afterward and analyzed the presence and concentrations of RG7388 by mass spectrometry. The brains were perfused to remove blood before the submission for mass spectrometry. RG7388 was detectable in plasma with mean concentration of 263 ng/ml 30 min after the drug administration. Plasma concentration increased to the maximum (mean 570 ng/ml) after 1 h, remained relatively steady at least for the following 7 h, and decreased dramatically by 24 h (4.26 ng/ml)  .We then assessed the in vivo therapeutic efficacy of RG7388 in a DIPG orthotopic model derived from the HSJD-DIPG-007 cell line. Tumor cells expressing luciferase were injected into the brainstem of NSG mice, and the tumor development was monitored by the Luciferase imaging system. Host mice displaying similar bioluminescence signal on day 21 post-injection were randomly assigned to vehicle or RG7388 treatment groups. RG7388 or vehicle was orally administered once per day, five days per week for three weeks (Day 42 post-implantation) and the progression of tumors was determined. No overt toxicity to other organs was observed during the experiment. Notably, while the bioluminescence of orthotopic tumors was comparable between groups before the treatment (on Day 21), three weeks of RG7388 treatment resulted in measurably suppressed tumor progression, as demonstrated by reduced bioluminescence signals . A previous study has shown the effects of RG7388 treatment in glioblastoma orthotopic models . HoweverTP53 wild-type/PPM1D-mutant DIPG subgroup as a p53 pathway re-activation drug. One of the merits of RG7388 is that its safety and feasibility have already been confirmed by a variety of clinical trials, including a Phase III trial. RG7388 is currently undergoing clinical trials alone or in combination with other drugs for acute myeloid leukemia , myeloma (phase I/II: NCT02633059), lymphoma (phase I/II: NCT03135262), breast cancer (phase I/II: NCT03566485), a combination of acute leukemias and solid tumors (phase I/II: NCT04029688) and glioblastoma bearing an unmethylated MGMT promoter (phase I/II: NCT03158389). In general, RG7388 is well-tolerated by patients and the most common adverse events are limited to gastrointestinal system symptoms (diarrhea and vomiting), infections and myelosuppression [Based on our data, RG7388 could be a valuable therapeutic approach to develop clinically in the treatment of pression , 22.TP53 wild-type DIPGs, the mutant PPM1D can stabilize MDM2 and promotes MDM2-mediated p53 ubiquitination and degradation. Thus, inhibition of PPM1D may further potentiate the efficacy of RG7388. A small-molecule PPM1D inhibitor, GSK2830371, was recently shown to enhance DNA damage response and suppress cell growth in DIPGs [Several potential limitations of this MDM2-targeting therapeutic strategy are noted. First, in in DIPGs . Furtherin DIPGs . Additioin DIPGs , the CDKin DIPGs , the PARin DIPGs , may offAdditional file 1: Figure S1. (A) The characterization of ten patient-derived DIPG cell lines. Five cell lines (on the top) harbored H3K27M mutation and PPM1D truncating mutations, but were TP53 wild-type. The other five cell lines (on the bottom) harbored mutant H3K27M and TP53, but did not contain mutation in exon 6 of PPM1D. (B) Protein expression level of H3K27M and p53 by Western Blot in ten patient-derived DIPG cell lines. (C) Sanger sequencing result of H3K27M mutation. (D) Sanger sequencing result of TP53 mutation in SU-DIPG-XIII line. (E) Sanger sequencing result of PPM1D mutation in five TP53 wild-type DIPG cell lines.Additional file 2: Figure S2. (A) IC50 of RG7388 in five TP53 wild-type and five TP53 mutant DIPG lines. (B) The viabilities of the five TP53 mutant DIPG lines treated with DMSO, 50nM and 100nM RG7388 for two and four days. Relative viabilities were calculated by normalizing luminescence values for each time point to Day 0 at the same treatment conditions. Mean ± SEM, n=3 independent studies for each condition. P-values based on Two-way ANOVA. ns=non-significant. (C) IC50 of RG7388 in HSJD-DIPG-007 NTC and TP53 KO lines. (D) The viabilities of HSJD-DIPG-007 TP53 KO line treated with DMSO, 50nM and 100nM RG7388 for two and four days. Relative viabilities were calculated by normalizing luminescence values for each time point to Day 0 at the same treatment conditions. Mean ± SEM, n=3 independent studies for each condition. P-values based on Two-way ANOVA. ns=non-significant.Additional file 3: Figure S3. (A) Top five most differentially regulated KEGG pathways of HSJD-DIPG-007-NTC and HSJD-DIPG-007-TP53 KO lines response to RG7388. (B) Hierarchical clustering of significantly changed genes  within the DNA replication and cell cycle pathway of HSJD-DIPG-007-NTC response to RG7388. Columns in the heat map represent individual replicates (three for each condition). Rows represent genes, colored by log-transformed transcript intensity  in z-score. Blue shows replicates with low expression (z-score < 0); red shows replicates with high expression (z-score > 0). (C) Transcriptional levels of MDM2, TP53 and CDKN1A after RG7388 treatment in HSJD-DIPG-007-NTC line.Additional file 4: Figure S4. (A) Quantification of protein expression level of MDM2, p53 and p21 in HSJD-DIPG-007-NTC and HSJD-DIPG-007-TP53 KO treated with DMSO or 25nM RG7388 for 6 hours, 12 hours, 24 hours and 48 hours. Mean ± SEM, n=3 independent studies for each condition. P-values based on Two-way ANOVA. *P < 0.05. ***P < 0.001. ****P < 0.0001. (B) Quantification of protein expression level of MDM2, p53 and p21 in five TP53 wild-type and five TP53 mutant DIPG cell lines treated with DMSO and 100nM RG7388 for 24 hours. Mean ± SEM, n=3 independent studies for each condition. P-values based on Student’s t-test. ****P < 0.0001. (C) Representative H&E and immunofluorescence staining of brainstem xenograft. Scale bar: 100μm.Additional file 5: Supplementary MethodsAdditional file 6: Supplymentary Table S1. CLA analysis of tem DIPG cell linesAdditional file 7: Supplymentary Table S2. Primers for Sanger sequencing


Applying CaDRReS-Sc to rapidly expanding scRNA-seq compendiums can serve as in silico screen to accelerate drug-repurposing studies. Availability: https://github.com/CSB5/CaDRReS-Sc.While understanding molecular heterogeneity across patients underpins precision oncology, there is increasing appreciation for taking intra-tumor heterogeneity into account. Based on large-scale analysis of cancer omics datasets, we highlight the importance of intra-tumor transcriptomic heterogeneity (ITTH) for predicting clinical outcomes. Leveraging single-cell RNA-seq (scRNA-seq) with a recommender system (CaDRReS-Sc), we show that heterogeneous gene-expression signatures can predict drug response with high accuracy (80%). Using patient-proximal cell lines, we established the validity of CaDRReS-Sc’s monotherapy (Pearson The online version contains supplementary material available at 10.1186/s13073-021-01000-y. Tumors comprise heterogeneous populations of malignant cells that display cellular plasticity and phenotypic heterogeneity, as determined by genetic and environmental cues –3. PhenoAdvances in high-throughput sequencing have enabled large-scale studies into inter-patient tumor heterogeneity at the molecular level –12, servIn this work, we begin by highlighting the impact of intra-tumor transcriptomic heterogeneity on clinical outcomes based on large-scale re-analysis of TCGA data , 25. We https://www.cancer.gov/tcga) [Response Evaluation Criteria in Solid Tumors (RECIST) values were obtained from prior work to curate drug response information [n=8 with ≥15 patients in “Complete Response” and “Clinical Progressive Disease” classes).Gene expression  and patient survival data for 10,956 tumors from 33 cancer types were obtained from The Cancer Genome Atlas (TCGA) Research Network (ov/tcga) . Clinicaormation , and sta2 expression fold-change was calculated with respect to its average expression across cell lines, and cell line kernel features were calculated using Pearson correlation based on 1856 essential genes [Drug response data and RMA-normalized gene expression data for 1074 cancer cell lines and 226 drugs were obtained from the Genomics of Drug Sensitivity in Cancer (GDSC) database  to be usal genes .n=14,558) were removed  were identified using the same process as the first round clustering. In total, 23 clusters were identified for the Sharma et al. dataset (1171 cells) and 62 clusters for the Puram et al. dataset (5902 cells)  to obtain a signature matrix with 1529 marker genes.Percentages of pre-defined cancer cell types in each tumor were identified by using CIBERSORT , a tool Pi is the fraction of cells with cell type 푖 identified in the tumor  and an average ITTH score was used. Patients were classified at the Pan-cancer level into three categories  based on the first and third quartiles of ITTH scores. Gene expression profiles for tumors from TCGA were clustered using nonnegative matrix factorization  [To measure the degree of heterogeneity for each sample based on the deconvolution result, we defined an intra-tumor transcriptomic heterogeneity score (ITTH) as information entropy of the corresponding cell type profile, i.e., lusters) .Pj is the fraction of cells that belong to cluster j.For scRNA-seq data, a proxy for bulk gene expression values was obtained by calculating the average gene expression across all cells and used to compute ITTH scores as described above. Gold-standard ITTH scores (single-cell ITTH) were then computed based on single-cell clustering (as described above) and then computing information entropy as before, i.e., https://github.com/CSB5/CaDRReS-Sc) [A pharmacogenomic space is a latent space that captures the relationship between drugs and samples , where a dot product between drug and sample vectors captures drug sensitivity. The pharmacogenomic space is learned in CaDRReS-Sc (RReS-Sc)  based onRReS-Sc)  was defisiu, the observed sensitivity score of sample u to drug i, is defined by siu =  − log2(IC50), Κ is the total number of drug-sample pairs, μ is the overall mean drug response, i and sample u, vectors qi,pu ∈ ℝf represent drug i and sample u in the f-dimensional latent space, and WP ∈ ℝd × fis a transformation matrix that projects transcriptomic kernel features xu ∈ ℝd for each sample onto the pharmacogenomic space. Based on this objective function, the cell line u is sensitive to drug i when qi and pu are near each other in the pharmacogenomic space.pu. Furthermore, to reduce noise from extrapolation errors for IC50 values , learning rate of 0.01, and maximum number of epochs set to 100,000. All training samples were used for updating the trainable parameters in each epoch  does not take into account the sigmoid shape of the curve, resulting in inaccurate aggregate IC50 values  were used to train a head and neck cancer-specific model  and cell cluster (weighted average) predictions.Drugs that elicit a response in at least 30% (13 out of 42) of head and neck cancer cell lines in the GDSC dataset .Cell lines were isolated from patients with oral squamous cell carcinoma (OSCC) as mentioned in previous work . BrieflyDrug/ Luminescence DMSO) and expressed as percentage cell viability  were seeded per well of a 96-well plate, 24 h prior to drug treatment. Drugs that were used for treatment were obtained from SelleckChem, MedChemExpress, and Cayman Chemical. Docetaxel (cat. no. S1148), Doxorubucin hydrochloride (cat. no. S1208), Epothilone B (cat. no. S1364), Obatoclax Meylate (cat. no. S1057), PHA-793887 (cat. no. S1487), PI-103 (cat. no. S1038), and Vorinostat (cat. no. S1047) were obtained from Selleckchem, while Gefitinib was purchased from Cayman Chemical (cat. no. 13166) and Staurosporin (cat. no. HY-15141) from MedChemExpress. Cells were treated at a drug concentration that corresponds to the median IC50 value for head and neck cancer cell lines seen in the GDSC database [r=0.55 and 0.59; Fig. To investigate the relationship between intra-tumor transcriptomic heterogeneity (ITTH) and clinical outcomes, we leveraged transcriptomic data from The Cancer Genome Atlas (TCGA) for 10,956 tumors across 33 cancer types, and an in silico deconvolution approach , to defip value<2.39×10-5) and sarcoma , in agreement with prior work on the impact of cell-type diversity in low-grade glioma (LGG) [Survival data for various cancer types was then analyzed to detect differences in patients with low, medium, and high transcriptomic heterogeneity (24/33 cancer types with ≥15 samples in each class). Significant associations between ITTH and survival were observed in 5 cancer types, with high heterogeneity associated with poorer outcomes in some cancer types and low heterogeneity in others   on treatp value<0.05), and these transcriptomic clusters were typically observed to be orthogonal to ITTH clusters . For example, in low-grade glioma, the low ITTH cluster is characterized by a better survival rate compared to transcriptomic clusters 1 and 2 , while in rectum adenocarcinoma, the high ITTH cluster is characterized by a lower survival rate compared to all three transcriptomic groups  and cancer types (n=24), we systematically assessed associations between ITTH scores and clinical drug response . This response pattern for Doxorubicin in patients with high ITTH scores could indicate pre-existing resistant populations [n=138; CR vs CPD Wilcoxon p value<2.06×10-2), and Leucovorin , the opposite trend was observed where responders showed significantly higher transcriptomic heterogeneity, consistent with prior work on other platinum compounds such as Cisplatin [Drawing on the availability of patient response data in TCGA for a few drugs , especially for drugs with a smaller proportion of sensitive cell lines. By focusing on predicting response values for sensitive cell lines, we observed that the overall median absolute error (MAE) was significantly reduced in a majority of the drugs , with an average prediction accuracy of around 80% compared to <60% for other methods  and yet provide discriminatory drug response predictions for the drug dosages used in future experiments (accuracy). Benchmarking against other machine learning approaches for drug response prediction trained on the same cancer line dataset, such as ElasticNet , cwKBMF Besides prediction accuracy , it is essential to predict precise dosages. Precision in IC50 predictions also allows us to infer responses such as cell death percentage at a given dosage. We observed that by aggregating information across drugs, CaDRReS-Sc showed the lowest prediction error, reducing MAE by >30% compared to cwKBMF, ElasticNet, and SRMF Fig. f. By com5 reads/cell, >5×103 detected genes, >1200 cells in total; Additional file As commonly used cancer cell lines typically lack significant transcriptomic heterogeneity, we leveraged patient-derived cell lines (PDCs) to serve as model systems where sensitivity measurements can be systematically and conveniently made across multiple drugs, while capturing in vivo transcriptomic heterogeneity . In totar=0.68, 0.66, 0.62, p value<1.11×10-6, 3.59×10-6, and 1.93×10-5, respectively; Additional file r=0.68/0.66 vs 0.62; consistently across drug dosages; Additional file r≤0.59 with ElasticNet and RWEN; Additional file We explored several strategies to utilize scRNA-seq data—ranging from using transcriptomic profiles of individual cells, aggregating profiles within a cluster of cells, to combining profiles at the patient-level—for predicting drug response , in comparison to weaker correlations with other methods  for five candidate drug combinations, including Docetaxel:Epothilone B, Docetaxel:Gefitinib, Gefitinib:Epothilone B, Epothilone B:PI-103, and Doxorubicin:Vorinostat, based on predicted cluster-specific drug responses and the distribution of cell clusters. These combinations were then functionally validated on five pooled PDCs via cell-based viability assays using low drug dosages to circumvent off-target effects resulting from extreme inhibition . Despitep value<3.39×10-2), but no significant associations for other methods . These results indicate that CaDRReS-Sc can sufficiently capture therapeutic response for mono- and combinatorial therapy, enabling prioritization of drugs and combinations for in vitro and in vivo studies.Testing drugs in each combination at low dosages also helped to mimic what might be needed to support the mitigation of side effects from combinatorial treatment . We thenWhile the role of intra-patient heterogeneity in genetic mutations has been extensively explored with respect to tumor biology , 55, fewPredicting treatment response in silico in the presence of intra-tumor heterogeneity requires models that provide calibrated values for a single drug across many cell types, while prior work has focused on calibrated predictions for a cell type across many drugs . To addrPatient-derived cell lines (PDCs) serve as ideal systems for drug sensitivity measurements in vitro while capturing intra-tumor transcriptomic heterogeneity , 9, and r=0.68, Fig. Several sources of noise in drug response measurement experiments have been reported in previous studies, including lack of experimental replication , 15, andIn predicting response to monotherapies, we observed consistently higher correlations with in vitro measurements when using transcriptomic profiles with higher granularity . This prompted us to consider prioritizing combinatorial therapy options based on CaDRReS-Sc predictions for different subclones, assuming that the combinatorial effect can be approximated in many cases through independent drug action on distinct subclonal cancer cell populations . IndepenDeveloping in silico tools for predicting in vivo treatment response remains a challenge as multiple factors  can impact patient trajectories. The ability to predict the synergistic effect of multiple drugs on a given sub-population would also be useful information that complements the additive effects across sub-populations that is captured by our framework. In this study, we aimed to bridge the in silico to in vitro gap for predicting response to mono- and combinatorial therapy in the presence of transcriptomic heterogeneity. Together with improved technologies for patient-derived cancer cell models, this combined in silico/in vitro approach could form the basis of a first-cut precision oncology platform that prioritizes mono- and combinatorial therapy options in a clinically relevant timeframe.Additional file 1: Fig S1. Single-cell RNA-seq statistics for 12 patient-derived cell lines. Fig S2. Impact of dose-response curves from in vitro cell viability assays on IC50 estimates. Fig S3. Training and validation loss. Fig S4. CaDRReS-Sc accurately estimates aggregate IC50 values in the presence of transcriptomic heterogeneity. Fig S5. Survival analysis for clusters based on bulk transcriptomic profiles. Fig S6. Boxplots comparing ITTH scores across clinical response categories for various cancer drugs. Fig S7. Additional performance evaluation per drug. Fig S8. Pairwise comparison of CaDRReS-SC’s performance on unseen cell types. Fig S9. Transcriptomic patterns of cells from HN120 and HN137. Fig S10. Detailed comparison between predicted and observed cell death percentages. Fig S11. Pharmacogenomic space of GDSC cell lines and HNSC patient-derived cell clusters. Fig S12. Comparison of observed and predicted drug response across 5 pooled PDCs and 8 drugs. Fig S13. Predictive performance of ElasticNet and RWEN based on cell clusters. Fig S14. Comparison of drug response between tumor types and pathway activity groups.Additional file 2: Table S1: TPM values of scRNA-Seq for patient-derived cell lines.Additional file 3: Table S2: Cell clustering results of Sharma et al dataset. Table S3: Cell clustering results of Purum et al dataset.Additional file 4: Table S4: ITTH score and drug response. Table S5: CIBERSORT's TCGA deconvolution. Table S6: CIBERSORT's signature matrix.Additional file 5: Table S7: GDSC drug statistics. Table S8: GDSC cell lines.Additional file 6: Table S9: Drug combinatorial prediction. Table S10: Monotherapy prediction.Additional file 7: Table S11: Drug dosages in experimental validation. Table S12: Experimental results of monotherapy. Table S13: Experimental results of drug combination


PTEN protein deletion and/or activation of the PI3K/Akt signaling pathway leading to altered cell growth and metabolism are emerging as a common feature in T-ALL. We herein investigated the relationship amongst PTEN deletion, ASNase sensitivity and glucose metabolism in T-ALL cells. First, we found significant differences in the sensitivity to ASNase amongst T-ALL cell lines. While cell lines more sensitive to ASNase were PTEN wild type (WT) and had no detectable level of phosphorylated Akt (P-Akt), cell lines less sensitive to ASNase were PTEN-null with high P-Akt levels. Pharmacological inhibition of Akt in the PTEN-null cells rendered them more sensitive to ASNase and lowered their glycolytic function which then resembled PTEN WT cells. In primary T-ALL cells, although P-Akt level was not dependent exclusively on PTEN expression, their sensitivity to ASNase could also be increased by pharmacological inhibition of Akt. In summary, we highlight a promising therapeutic option for T-ALL patients with aberrant PTEN/PI3K/Akt signaling.Childhood T-cell acute lymphoblastic leukemia (T-ALL) still remains a therapeutic challenge due to relapses which are resistant to further treatment.  Despite the fact that pediatric T-ALL can be cured in approximately 80% of patients3, relapse still occurs in 15–20% of cases5 which then achieve an event-free and overall survival in less than 25%6. For standardized treatment, there has been a stable repertoire of cytostatic drugs used for the past several decades. l-asparaginase (ASNase) has been part of a first-line therapy since 19677 and still remains crucial. Extensive clinical data in pediatric ALL support the benefit of intensive ASNase treatment compared with less intensive regimens10. Unfortunately, there are inter-individual differences in ASNase sensitivity and some patients also develop hypersensitivity, which could influence treatment efficacy since the resistance to ASNase is considered an unfavorable prognostic factor13.Acute lymphoblastic leukemia (ALL) is the most prevalent pediatric malignancy, of which T-cell ALL (T-ALL) comprises about 15% of cases14. PTEN is critical in preventing the malignant transformation of T-cells and its expression and functions are altered in human T-ALL15. PTEN is an inositol lipid phosphatase that acts as a major negative regulator of the phosphatidylinositol-3 kinase (PI3K)/Akt/mechanistic target of rapamycin (mTOR) signaling pathway16. This cascade plays a key role in controlling a wide range of essential cellular processes including cell proliferation, growth, survival and metabolism18.Major advances in the understanding of genes, signaling networks and mechanisms that play crucial roles in the development and progression of T-ALL have led to the identification of key drivers of this disease. The phosphatase and tensin homolog (PTEN) is one of the most frequently-mutated/functionally-inactivated oncosuppressors in cancer19. Moreover, we observed a correlation of specific metabolic parameters with the sensitivity to ASNase20. Here, we focused on the PTEN deletion in T-ALL and its effect on cell metabolism and sensitivity to ASNase. PTEN loss-of-function mutations/gene deletions are detected in approximately 20% of pediatric T-ALL cases22 and PTEN abnormalities are associated with an unfavorable long‐term outcome in some pediatric and adult T‐ALL patient cohorts25. Moreover, the PI3K/Akt pathway in T-ALL could also be affected by many other mechanisms, e.g. by PI3K and Akt mutations or by the decreased activity of PP2A26. Since PTEN functions as a negative regulator of Akt, whose activity promotes glucose metabolism27 and renders cancer cells dependent on aerobic glycolysis28, we pursued the connection between PTEN deletion, ASNase sensitivity (i.e. asparagine and glutamine depletion) and glucose metabolism. In the current study, we have observed that T-ALL cells with PTEN wild type (WT) are more sensitive to ASNase (compared to the PTEN-null cells) and that resistance to ASNase in the PTEN-null cells could be reversed by inhibiting Akt signaling.We have previously reported that leukemia cells treated with ASNase reprogrammed their metabolism6. Furthermore, the search for new approaches for the treatment of recurrent disease is highly warranted. In order to find new possible therapeutic windows, we measured the sensitivity to ASNase, VCR and DNR, drugs used in T-ALL therapy, in five human T-ALL cell lines. We discovered that although their sensitivities to VCR and DNR are quite similar, the cell lines differ in their sensitivity to ASNase  between the PTEN-positive and the PTEN-null T-ALL cell lines  levels in T-ALL cell lines , predominantly produced truncated NICD compared to cell lines less sensitive to ASNase (PTEN-null) which produced WT NICD. We did not see any difference in NOTCH1 levels between the PTEN-positive and the PTEN-null cells. Regarding c-Myc, a glycolysis activator, we did not see any significant differences between cell lines more sensitive and resistant to ASNase  similarly. Therefore, the sole effect of the Akt inhibitor is not selective and we could test its impact on the sensitivity of T-ALL cells to ASNase. Akt inhibition significantly increased the sensitivity of the PTEN-null, but not the PTEN-positive T-ALL cell lines to ASNase  is driven by a hyperactivated Akt and by higher glycolytic function. We also tested structurally different Akt inhibitor Ipatasertib which affected the sensitivity of T-ALL cells to ASNase similarly as GSK690693 , effectively inhibited NOTCH1 cleavage in both the PTEN-positive and the PTEN-null cells , were comparable with endogenous PTEN levels in ALL-SIL and DND-41 cells . We saw lowered P-S6 levels in cells transduced with pHIV-PTEN compared to empty vector (EV)  Fig. E. This cEV) Fig. C. MoreovEV) Fig. D. AltogeEV) Fig. E. FurtheThe results from the T-ALL cell lines indicated a possible therapy target for T-ALL patients with a PTEN deletion/mutation. Therefore, we decided to examine the relative activity of the tested signaling pathways in cell lines Fig. , Fig. S1PTEN mRNA of patient P5. Therefore, the kinase activity of P-Akt could be disturbed.The patients were initially diagnosed between 1 and 20 years of age and then treated according to the INTERIM ALL 2007  or the AIEOP BFM 2017 P3, 4, 5) protocols. We did not detect PTEN using western blot in T-ALL cells from four of the patients  using Cisplatin staining on mass cytometry. Our results showed that Akt inhibition combined with ASNase treatment increased the number of Cisplatin-positive cells compared with cells treated with ASNase only Fig. D. Moreovnull cells with constitutively activated Akt and with a higher glycolytic function, and cell lines more sensitive to ASNase, which produced PTEN, did not have phosphorylated Akt and had a lower glycolytic function. In addition, we also explored the sole influence of PTEN expression on ASNase sensitivity, glycolysis and the activation of Akt. Our results confirmed that introduction of PTEN into PTEN-null cells increased their sensitivity to ASNase and lowered their glycolytic function, both probably via the inhibition of Akt. The connection between PTEN and P-Akt levels in the T-ALL cell lines fully corresponds with the phosphatase activity of PTEN26. Moreover, it has been shown that Akt signaling regulates nutrient uptake and metabolism. More specifically, Akt stimulates glucose uptake36. Higher glycolytic function reported by us thus corresponds to higher P-Akt levels. Metabolic reprogramming in general is now considered as one of the hallmarks of cancer37. Therefore, targeting cancer metabolism has become an attractive therapeutic approach, one which, nevertheless requires a therapeutic window in which tumor cells have a great dependence on specific enzymes38. Constitutive activation of Akt in T-ALL cell lines less sensitive to ASNase inspired us to pursue Akt as a possible therapeutic target. We discovered that resistance to ASNase in T-ALL cell lines with constitutively active Akt could be decreased by the inhibition of Akt by two structurally different inhibitors. Although several Akt inhibitors are now undergoing clinical trials, only one has been evaluated in hematological malignancies to this day (GSK2110183 in multiple myeloma and chronic lymphocytic leukemia)39.PTEN, whose activity antagonizes the PI3K/Akt/mTOR pathway and therefore represses tumor cell growth and survival, is one of the most frequently disrupted tumor suppressors in cancer. The significance and complexity of the PI3K signaling makes PTEN an important but challenging therapeutic target. In childhood T-ALL, PTEN deletions/mutations are quite frequent, but so far, the therapy does not reflect the possibility of the PI3K/Akt/mTOR signaling deregulation. Our results from the T-ALL cell lines gave us a fairly complex information about the part which PTEN/Akt plays in the signaling pathway. Moreover, we focused on the sensitivity of the cell lines to some of the currently used cytostatic drugs. Although the sensitivity to VCR and DNR did not vary between the five tested T-ALL cell lines, the sensitivity of these cell lines to ASNase differed dramatically. Interestingly, we could distinguish cell lines more resistant to ASNase, which were PTEN-41. The loss of PTEN in 20% of pediatric T-ALL cases is thus definitely worthy of attention. In this study, we discovered that in primary T-ALL the relationship between PTEN and P-Akt is not as straightforward as in cell lines. Similar ambiguous findings were reported in endometrioid cancers. In these malignancies PI3K pathway aberrations occur in more than 80% of cases and it appears that the PTEN protein loss serves as a dominant activation event of this particular pathway. However, in tumors with retained PTEN protein, PI3K pathway mutations phenocopy PTEN loss, resulting in pathway activation42. When we focused on Akt, we were able to increase the sensitivity to ASNase by inhibiting Akt in primary cells from T-ALL patient with high P-Akt level. Noteworthy, T-ALL cells from this patient also produced PTEN. On the other hand, Akt inhibition did not change the sensitivity to ASNase in cells with low P-Akt level. Using mass cytometry, we confirmed the increase of the sensitivity to ASNase in Akt-inhibited cells from three other T-ALL patients. Importantly, we discovered that mass cytometry presents a suitable tool for evaluating drug efficacy in frozen T-ALL samples. In summary, regardless of the presence of PTEN, the sensitivity to ASNase could be increased in some primary T-ALL cells which have constitutively active Akt. Altogether, the results of our study suggest possible improvements of T-ALL therapy for patients with higher levels of P-Akt.PTEN deletion occurs in a relevant fraction of a variety of cancers (e.g. breast or prostate), where it results in a worse prognosis and outcome2). The cultured cells were split every two to three days and maintained in exponential growth phase. JURKAT stable clones that express the WT or mutant PTEN (G129R) under the control of the tetracycline-inducible system (Tet-on)34 were kindly provided by Dr. Arthur Weiss from the Howard Hughes Medical Institute . PTEN G129R is a tumor-derived mutation that is defective in both the protein and the lipid phosphatase activity34. Clones were maintained in RPMI-1640 medium with GlutaMAX™ supplemented with 10% Tet system-proved fetal calf serum and 2 mg/mL G418 and 300 µg/mL hygromycin B and expression of PTEN was induced by doxycycline (DOX), a tetracycline analog.Human T-cell leukemia cell lines  were purchased from German Collection of Microorganisms and Cell Cultures . The cell lines were negative for mycoplasma contamination and were cultivated in RPMI-1640 medium with GlutaMAX™ supplemented with 10% fetal calf serum, penicillin (100 U/mL) and streptomycin (100 μg/mL) under controlled conditions . PTEN cDNA was amplified with 5′-TATTCTAGAGTACCCGGGATGACAGCCATCATCAAAGAG-3′ and 5′-GGAGAGGGGCGGATCCCATGGTGTTTTATCCCTCTTGAT-3′ primers and cloned under the control of the EF-1α promoter into the pHIV-EGFP vector. Lentiviral particles were produced in HEK-293T cells using 30 µg of the lentiviral vector (pHIV-EGFP or pHIV-PTEN), 24 µg of the packaging vector, 2.8 µg of the envelope-plasmid and polyethylenimine. Lentiviral particles were collected from the supernatant after 48 and 72 h and concentrated using the Lenti-X Concentrator . JURKAT cells were transduced using spinoculation by centrifuging the cells for 2 h at 800×g at RT. Forty-eight hours after transduction, GFP+ cells were sorted. Transduced JURKAT cells were maintained as GFP + cells (> 90%). pHIV-EGFP was a gift from Bryan Welm & Zena Werb 43.pHIV-EGFP lentiviral vector containing l-glutamine and gentamicin supplemented with 10% fetal calf serum, penicillin (100 U/mL), streptomycin (100 μg/mL), IL-7 (50 ng/mL) and insulin-transferrin-sodium selenite supplement .Bone marrow or peripheral blood samples from untreated children initially diagnosed with T-ALL were collected from the Czech Pediatric Hematology Centers. To be able to perform the here described experiments, only patients with the blast percentage higher than 80% and with a high cellularity were included. Within 24 h after aspiration, the mononuclear cells were isolated by density gradient centrifugation using Ficoll-Paque PLUS . All the samples were obtained with the informed consent of the children’s parents or guardians as well as the approval of the Ethical Committee of the University Hospital Motol, Prague, Czech Republic, study no. 201528848A. All experiments were performed in accordance with relevant guidelines and regulations. The isolated blasts were frozen in 90% fetal calf serum and 10% DMSO. After thawing, the blasts were maintained in X-VIVO™ 15 Medium with 4 cells) and primary cells (1–3 × 105 cells) were seeded. The MTS assay was performed at least in three independent experiments.To evaluate the cytotoxicity of ASNase, vincristine (VCR), and daunorubicin (DNR), MTS (dimethylthiazol carboxymethoxyphenyl sulfophenyl tetrazolium) assays were performed using a CellTiter 96 AQueous One Solution Cell Proliferation Assay  according to the manufacturer’s instructions. Leukemia cell lines  using a Glycolysis stress test. Cells were plated in XF RPMI medium pH 7.4, at a density of 100,000 cells/well in XFp tissue culture plates coated with CellTak  according to the Agilent Seahorse protocol for the seeding of suspension cells. Final concentrations of the injected drugs were 10 mM glucose, 2 μM Oligomycin A and 100 mM 2-deoxy glucose (2-DG). All of the Seahorse measurements were done at least in biological triplicates and three technical replicates.Copy number aberrations (CNA) in the PTEN gene were screened by single-nucleotide polymorphism array . DNA labeling and hybridization were performed according to the manufacturer’s instructions. The GenomeStudio software v2011.1 (Illumina) was used for genotype calling and quality control. Copy number variations (CNV) were called using the CNV Partition 2.4.4 algorithm plug-in within the GenomeStudio software. The resulting data  were visually inspected in the Illumina Chromosome Browser and the CNV calls were manually curated.44. Proteins (30 μg per well) were resolved by NuPAGE Novex 4–12% Bis–Tris Gels  and transferred onto a nitrocellulose membrane . The membrane was probed overnight with the primary antibodies listed in Table Protein lysates were prepared as previously describedg, 5 min, RT), and barcoding monoclonal antibodies (moAbs) were added . The cells were washed, pooled together and washed in CSB . To minimize technical variability between stimulated and unstimulated samples, differently treated samples from particular donors were barcoded, mixed and further processed in one tube. The samples were stained according to the MaxPar Phosphoprotein Staining with Fresh Fix (Fluidigm) protocol as recommended by the manufacturer. The instrument  was prepared for acquisition according to the manufacturer’s recommendation. Signal spillover between channels was corrected using the CATALYST R package as described previously45. Compensated fcs files were further processed using FlowJo . T-ALL blasts were gated as CD7+CD45dimCD3+CD19- and homogenous population on CD4 vs CD8 scatterplot. Heatmaps were created in MeV software46 by displaying arcsinh transformed absolute values of median intensities.For evaluation of cell viability, 1 µM Cell-ID Cisplatin-198Pt (Fluidigm) was added into the cell culture. After 5 min incubation cells were washed twice in MaxPar Cell Staining Buffer . Samples were fixed with 1.6% paraformaldehyde and stored . The samples were thawed, washed with MaxPar Cell Staining Buffer , EC50 was calculated using non-linear regression (variable slope method). Calculations were performed using GraphPad Prism 6 software. Group data are expressed as the mean ± standard deviation (SD).The statistical analysis was performed using a paired Student’s Supplementary Information.