Despite accumulated evidence in T-cell exhaustion in acute myeloid leukemia (AML), the immunotherapeutic targeting exhausted T cells such as programmed cell death protein 1 (PD-1) blockade in AML failed to achieve satisfying efficacy. Characteristics of exhausted T cells in AML remained to be explored.Phenotypic analysis of T cells in bone marrow (BM) using flow cytometry combining senescent and exhausted markers was performed in de novo AML patients and healthy donors as well as AML patients with complete remission (CR). Functional analysis of T-cell subsets was also performed in de novo AML patients using flow cytometry.+TIGIT+). Moreover, CD28- subsets rather than CD28+ subsets predominantly contributed to the significant accumulation of PD-1+TIGIT+ T cells in AML patients. Further comparison of de novo and CR AML patients showed that T-cell exhaustion status was improved after disease remission, especially in CD28+ subsets. Notably, higher frequency of CD28-TIGIT-CD4+ T cells correlated with the presence of minimal residual disease in AML-CR group. However, the correlation between CD28- exhausted T cells and cytogenetic risk or white blood cell count was not observed, except for that CD28- exhausted CD4+ T cells correlated with lymphocyte counts. Intriguingly, larger amount of CD28-TGITI+CD8+ T cells at diagnosis was associated with poor treatment response and shorter leukemia free survival.T cells experienced a phenotypic shift to terminal differentiation characterized by increased loss of CD28 expression and decrease of naïve T cells. Additionally, lack of CD28 expression could help define a severely exhausted subset from generally exhausted T cells (PD-1In summary, lack of CD28 expression defined a severely exhausted status from exhausted T cells. Accumulation of CD28- exhausted T cells was linked to occurrence of AML, and correlated to poor clinical outcome. Our data might facilitate the development of combinatory strategies to improve the efficacy of PD-1 blockade in AML. Immunotherapies revolute the therapeutic models of cancer by improving anti-tumor immunity, especially T cell function. However, the outcome of clinical trials using immunotherapies targeting T cells such as programmed cell death protein 1 (PD-1) blockade in acute myeloid leukemia (AML) remain frustrating  samples from AML patients. Since AML blasts as a key role in the compromising immune response are heterogenous and vary along with disease status, the alterations of severely exhausted T cells in different stages of the disease and the correlation to clinical features and prognosis were also evaluated.de novo AML patients and 32 AML patients in complete remission (CR) as well as from healthy donors  were collected after written informed consent was signed in accordance with the Declaration of Helsinki and approved by the ethics board of the First Affiliated Hospital of Xiamen University. All samples were taken from non-acute promyelocytic leukemia (APL) AML patients. Positivity of minimal residual disease (MRD) was defined as AML cells detected by real-time quantitative polymerase chain reaction (RQ-PCR) or multicolor flow cytometry in BM samples. CR, CR with in complete hematologic recovery (CRi) and partial remission (PR) and no response  were defined according to 2017 ELN recommendations (Bone marrow (BM) of 42 Surface staining was immediately performed after BM collection. A 100μl BM sample was incubated with directly conjugated monoclonal antibodies for 15 minutes at room temperature; after lysing red blood with 1ml lysing solution (BD Bioscience), the cells were washed and resuspended by phosphate buffered saline (PBS). The monoclonal antibodies used were anti-human CD3-Percp (UCHT1), CD28-FITC (CD28.2), CD4-APC-R700 (RPA-T4), CD8-APC-R700 (RPA-T8), CD45RA-APC-CY7 (HI100), CCR7-BV421 (G043H7), PD-1-PE-Cy7 (EH12.1), CD127-BV510 (A019D5), CD57-PE (HCD57), KLRG-1-APC (SA231A2), TIGIT-BV605 (A15153G) from Biolegend, CA, USA.BM was diluted 1:1 with PBS before separation of bone marrow mononuclear cells (BMMCs) with lymphocyte separation solution  density gradient centrifugation. The isolated BMMCs were cultured in RPMI-1640 medium (GIBCO) containing 10% fetal bovine serum and stimulated with anti-CD3/CD28 antibody (2 and 5μg/ml) or Phorbol-12-myristate-13-acetate  and ionmycin (1ug/ml) for 5 h. Anti-CD107a-APC (W18263B), CD28-FITC and Golgiplug  were added at the start of the incubation. Cells were harvested for surface staining with CD3-Percp, CD8-APCR700, CD45RA-APC-H7, CCR7-V450, PD-1-PE-Cy7, TIGIT-BV605 and intracellularly stained with IFN-γ-BV510(4S.B3).de novo AML and AML-CR group. P-values <0.05 were considered significant, where *, ** and *** indicated p-values between 0.01 to 0.05, 0.001 to 0.01 and 0.0001 to 0.001, respectively.All flow cytometric data were analyzed using Flowjo 10. software. All data were subjected to normal distribution analysis by the Kolmogorov-Smirnov test using SPSS 22.0 . Student’s t test was used to analyze data with a normal distribution, while the Mann-Whitney U test or Wilcoxon matched-pairs signed rank test (in paired setting) was used to analyze data without a normal distribution. Fisher’s exact test or chi-square test were used to analyze the constituent ratios between de novo AML patients and 32 AML patients in CR (AML-CR) were enrolled in the present study. The characteristics of de novo AML patients and AML-CR were summarized in de novo AML group, the percentages of patients at low risk and received idarubicin and cytarabine (IA) regimens tended to be higher in AML-CR group. Fewer patients in de novo AML group received low intensity chemotherapy in comparison to AML-CR group. Among 32 de novo AML patients, 18 patients achieved CR or CRi while 13 patients showed no response to induction therapy.Fifteen HDs , forty-two de novo AML patients (n=42) and HDs (n=15). A significant phenotypic shift to effector memory T cells (TEM) was observed in de novo AML patients (+ and CD4+ naïve T cells (Tnaïve) from de novo AML patients suffered a significant shrinkage compared with HDs (+ (TEMRA) in CD4+ and CD8+ T cells were comparable in two groups  or even terminal differentiated stage (TEMRA in CD4+ and CD8+T cells) of T cells rather than early stage such as Tnaive and TCM  was also evaluated in AML patients and HDs  production were evaluated in CD28- and CD28+ T cells  and lower WBC (<10×109/L) groups  in CD28-PD-1+TIGIT+ T cells, which might restrict the capacity of self-renewal capacity and the long-term living. Notably, we found higher expression of PD-1 and TIGIT mainly distributed in CD28- subsets especially in CD8+ compartment in de novo AML group. However, improvement of exhaustion status after CR mainly occurred in CD28+PD-1+TIGIT+ rather than CD28-PD-1+TIGIT+ subset of CD8+ T cells, neither was TIGIT+ T cells significantly changed after CR. Taken together, these data might provide a rational explanation for poor efficacy of PD-1 blockade in AML patients.CCR7 and CD45RA are generally used to define differentiated status of T cells. However, combination of CCR7 and CD45RA and inhibitory receptors like PD-1 and TIGIT could not well reflect the T-cell subsets correlated to AML occurrence. Kamphorst AO et al. demonstrated that rescue of exhausted T cells by anti-PD-1 therapy was CD28 dependent in mice model of life-long chronic lymphocytic choriomeningitis virus (LCMV) infection . However- exhausted subsets did not correlated to WBC or cytogenetic risk. Additionally, higher frequency of CD28- exhausted subsets in CD4+ T cells of BM was linked to lower lymphocyte count in PB. Previous studies showed that exhaustion linked to dysfunction and apoptosis of CD4+ T cells in BM (- exhausted subsets in CD4+ T cells could be more sensitive to reflect AML load than CD8+ T cells. Both CD28-PD-1+ and CD28-PD-1+TIGIT+CD4+ T cells were significantly decreased after CR while only CD28-PD-1+CD8+ T cells showed the same trend. Moreover, CD28-TIGIT+CD4+ T cells could even significantly elevate in AML with positive MRD. However, CD28-TIGIT+CD8+ T cells displayed the superior capacity to predict response to induction therapy and prognosis. Higher frequency of CD28-TIGIT+CD8+ T cells at diagnosis correlated to non-CR after induction and shorter LFS.WBC and cytogenetic alterations were considered as the main factors impact clinical outcome . Howeverls in BM , howeverde novo AML cohort than previous studies, a certain proportion of patients dropped out in our study, even if this did not affect the result at diagnosis. However, the small sample size and treatment variations might lead to the biased result in survival analysis.There are some limitations in our study. Despite a larger - exhausted T cells correlated to AML occurrence and poor prognosis. Our findings provide a novel insight into exhausted T cells in AML and the potential combinatory strategies to improve the efficacy of PD-1 blockade.In summary, lack of CD28 expression could define severely exhausted subsets of T cells with terminally differentiated and senescent features. Higher level of CD28The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Institutional Human Ethics Review Committee of the First Affiliated Hospital of Xiamen University. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.YTH, HZ, YZ, YH, and LL performed the experiments and data analysis. ZFL, ZJL, JZ, and ZF collected bone marrow samples and clinical data. XY, LL, and BX aided in interpreting the results and worked on the manuscript. XY, LL, and BX conceived and planned the experiments. All authors contributed to the article and approved the submitted version.


Vacuolar ATPase (V-ATPase) is regarded as a possible target in cancer treatment. It is expressed in primary acute myeloid leukemia cells (AML), but the expression varies between patients and is highest for patients with a favorable prognosis after intensive chemotherapy. We therefore investigated the functional effects of two V-ATPase inhibitors  for primary AML cells derived from 80 consecutive patients. The V-ATPase inhibitors showed dose-dependent antiproliferative and proapoptotic effects that varied considerably between patients. A proteomic comparison of primary AML cells showing weak versus strong antiproliferative effects of V-ATPase inhibition showed a differential expression of proteins involved in intracellular transport/cytoskeleton functions, and an equivalent phosphoproteomic comparison showed a differential expression of proteins that regulate RNA processing/function together with increased activity of casein kinase 2. Patients with secondary AML, i.e., a heterogeneous subset with generally adverse prognosis and previous cytotoxic therapy, myeloproliferative neoplasia or myelodysplastic syndrome, were characterized by a strong antiproliferative effect of V-ATPase inhibition and also by a specific mRNA expression profile of V-ATPase interactome proteins. Furthermore, the V-ATPase inhibition altered the constitutive extracellular release of several soluble mediators , and increased mediator levels in the presence of AML-supporting bone marrow mesenchymal stem cells was then observed, especially for patients with secondary AML. Finally, animal studies suggested that the V-ATPase inhibitor bafilomycin had limited toxicity, even when combined with cytarabine. To conclude, V-ATPase inhibition has antileukemic effects in AML, but this effect varies between patients. Acute myeloid leukemia (AML) is a heterogeneous and aggressive malignancy characterized by proliferation of transformed immature hematopoietic cells in the bone marrow ,2. The oVacuolar ATPase (V-ATPase) is a proton pump that is regarded as a possible therapeutic target in human malignancies, and the available evidence suggests that this strategy has effects in various solid tumors and possibly hematological malignancies ,7,8,9,10The expression of V-ATPase in the plasma membrane is not only important for stabilization of pH gradients but also for the molecular organization of the membrane, e.g., formation of lipid rafts, interactions with caveola and regulation of receptor signaling and recycling ,12,13,14Bafilomycins  are macrolide antibiotics ,25,26,27A recent proteomic study suggested that low expression of V-ATPase is associated with clinical chemoresistance in AML  ,35, and The local Ethics Committee  approved the study (REK Vest 2017/305 14022017), and all patient samples were collected after written informed consent. AML blasts were derived from 80 consecutive patients . Our department is responsible for AML therapy in a defined geographical area, and our patient inclusion should therefore be regarded as population-based. The clinical and biological characteristics of the patients and their leukemia cells are summarized in The medium used for suspension culture of AML cells alone was serum-free Stem Span SFEM . Concanamycin A (CCA), Bafilomycin A1 (BAF) and chloroquine for the in vitro experiments were purchased from Sigma-Aldrich . The two drugs were initially dissolved in DMSO and further diluted in culture medium.3H-thymidine incorporation assay described below  . The culture medium was Stem Span SFEM™ supplemented with granulocyte–macrophage colony-stimulating factor (GM-CSF), Flt3 ligand (Flt3-L) and stem cell factor (SCF); all cytokines were purchased from PeproTech  and used at a final concentration of 20 ng/mL. After six days of incubation at 37 °C in an atmosphere of 5% CO2, 37 kBq/well of 3H-thymidine  was added in 20 μL, and the cells were thereafter incubated for 18 h before they were harvested and nuclear radioactivity measured by liquid scintillation counting . All experiments were performed in triplicates and the median cpm was used for all calculations.This assay has been described in detail previously . BrieflyRepeated negative controls showed a nuclear radioactivity corresponding to <180 cpm . Significant proliferation was therefore defined as a nuclear radioactivity corresponding to >1000 cpm. The results are presented as the relative response (RR), i.e., the median cpm for drug-containing cultures relative to the cpm for the corresponding drug-free control cultures.6 AML cells seeded in 2 mL/well (24-well culture plates) of Stem Span SFEM medium supplemented with GM-CSF+Flt3-L+SCF, with or without concanamycin A 1 nM or bafilomycin A1 10 nM. The cells were cultured in 24-well culture plates  for one week before harvesting, centrifugation and resuspension in 0.5 mL RPMI medium  before 2.5 mL methylcellulose medium was added  to reach a final concentration corresponding to 0.33 × 106 cells/mL of seeded cells from the initial suspension cultures. Cells were seeded in duplicates in 24-well plates . After 14 days of additional in vitro incubation, the number of colonies with ≥20 cells (corresponding to at least 4 cell divisions) was estimated via light microscopy. The results are presented as the number of colonies per well, i.e., the number of detectable clonogenic cells per 0.16 × 106 seeded cells at the initiation of suspension cultures.AML cells were first cultured in suspension cultures, i.e., 1 × 10®)  in Stem Span SFEM culture medium supplemented with GM-CSF, Flt3-L and SCF as described above. Cells were stained with propidium iodide (PI) and fluorescein isothiocyanate-conjugated Annexin V , and the percentages of viable Annexin V−PI−, early apoptotic Annexin V+PI− and late apoptotic/necrotic Annexin V+PI+ cells were determined by flow cytometry, as described in detail previously [Primary AML cells were cultured in flat-bottomed 24-well culture plates   in Stem Span SFEM medium supplemented with GM-CSF, Flt3-L and SCF. Supernatants were harvested after 48 h and stored at −80 °C until mediator levels were determined via Luminex analysis .As described in detail previously , primaryTM; Lonza) containing 10% inactivated fetal bovine serum (FBS) and 4 mM L-glutamine. Expanded MSCs were trypsinated and later used in our experiments in passage four.Normal human mesenchymal stem cells (MSC)  were expanded in complete mesenchymal stem cell growth medium (MSCGM4 cells/well) were added to the lower chamber and 1 × 106 AML cells to the upper chamber; the two cell populations were separated by a semipermeable membrane (0.4 μm pore size) and each transwell contained a total of 1.6 mL MSCGM™ medium [3H-thymidine  was added in 150 μL medium to each well after 6 days of culture, and the cells were incubated for an additional 18 h before nuclear 3H-thymidine incorporation was measured. The MSCs did not reach confluence in any experiments during the seven days of culture.Co-cultures were prepared in transwell plates . MSC  mix were performed as described in detail previously ,44,45.6 cells/100 μL/mouse) was performed on female NOD/SCID IL2Rγnull (NSG) mice . The AML cell lines were obtained from ATCC (USA) and cultured in accordance with the suppliers’ guidelines prior to engraftment. Both AML cell lines were routinely assessed for mycoplasma infection and were transduced using lentiviral particles to express red-shifted firefly luciferase and GFP as reporter genes . Animals harboring engrafted AML cells were randomized according to weight prior to being assigned to study groups.All experiments were approved by the Norwegian Animal Research Authority and performed in accordance with The European Convention for the Protection of Vertebrates Used for Scientific Purposes in an AAALAC accredited institution. The studies were approved by the Norwegian Food Safety Authority (FOTS), project number 28742, granted from 19082022–18082026). The animal models and the evaluation methodology have been described in detail previously . BrieflyFor in vivo studies, Bafilomycin A1  was dissolved in DMSO (10%), PEG 400 (40%) tween (5%) and saline (45%) and sterile filtered. The daily intraperitoneal doses used in the experiments were 0.1, 1.0 and 2.0 mg/kg. Mice were regularly evaluated by weight and bioluminescence for the ANL cell experiments as described in detail in a previous publication . The IVIAnimals inoculated with leukemic cells will suffer degrees of stress associated with disease progression and leukemia load. These animals were therefore scored daily and euthanized according to predefined and highly standardized criteria/endpoints including physical appearance/ruffled fur, behavior/activity, body movements  and nutrition/weight loss.Analysis of proteomic and phosphoproteomic data. The Perseus 2.0.7.0 platform was used to clean, normalize and run two-sample unequal variance t-test with the proteomic and phosphoproteomic data [2 tests (Pearson’s χ2 test and the likelihood ratio) were used to investigate correlations for continuous data. Fisher’s exact test was used for analysis of categorized data. Differences were regarded as statistically significant when p < 0.05.mic data . The idemic data ,44,45. Gmic data ,49. The Additional statistical and bioinformatical analyses. Unsupervised hierarchical clustering analysis was conducted in J-Express . Statistical differences in averages between animal treatment groups were determined using a two-tailed Student t test via Pearson correlation . A one-way analysis of variance (ANOVA) was performed to ensure no statistically significant difference in weights between the animals in the treatment groups.3H-thymidine incorporation assay was defined as nuclear radioactivity corresponding to at least 1000 cpm [p ≤ 0.001). However, the antiproliferative effect showed a wide variation between patients, and this variation was observed especially for bafilomycin, whereas concanamycin showed generally stronger effects for all three concentrations examined. Thus, even though the two inhibitors are regarded as structurally related [Detectable cytokine-dependent proliferation in the 1000 cpm . Seventy related ,21,26, tThere was a wide variation between patients with regard to the antiproliferative effect of V-ATPase inhibition when this effect was estimated as the relative responses . The patp = 0.0211). A significant difference was also seen when only the secondary AML patients but not the patients with AML relapse were compared in the statistical analysis (p = 0.0327).Secondary AML includes patients with antecedent myeloid malignancies or previous exposure to cytotoxic treatment, and clinical studies have shown that these patients have reduced AML-free survival after intensive therapy ,57,58,59The strong responders showed a wide variation with regard to genetic abnormalities; 15 of them showed a normal karyotype, whereas the other patients had both adverse  and favorable  genotypes ,60. The p = 0.0899).Targeted next-generation sequencing were available for 33 consecutive patients; a total of 57 mutations were analyzed, and 32 of them were detected for at least 1 of the patients . These aA previous study showed that AML patients could be subclassified based on the constitutive activation of the PI3K-Akt-mTor pathway in the AML cells , and theThe two groups (37 strong responders versus 33 intermediate/weak/no responders) did not differ with regard to age, sex or AML blast differentiation  . The cyt3H-thymidine incorporation assay. The relative antiproliferative effect of chloroquine was classified as weak or strong (>0.30 versus ≤0.30) based on the same criteria as used for bafilomycin A1 (see above); the majority of strong responders to chloroquine were also strong responders to bafilomycin 10 nM (15 out of 20), and the majority of weak responders to chloroquine were also classified as weak responders to bafilomycin A1 10 nm , thus indicating that there is an association between the antiproliferative effects of chloroquine and bafilomycin A1.Chloroquine is regarded as an inhibitor of autophagy ,64,65,66Based on the relative antiproliferative effect of bafilomycin A1 10 nM , we clasTo conclude, the antiproliferative effect of V-ATPase inhibition showed no association with the characterized cytogenetic or molecular genetic abnormalities, but there was an association between a strong antiproliferative effect and secondary AML.For the 48 patients with undetectable proliferation in concanamycin-containing cultures, the proliferation in cytarabine-containing cultures reached detectable levels for all patients and corresponded to a median relative response of 0.62 (range 0.06–1.26). A similar low/undetectable proliferation as for the concanamycin-containing cultures was also seen for all the corresponding cultures supplemented with concanamycin + cytarabine.The overall results for the 22 patients with detectable proliferation in the concanamycin-containing and cytarabine and concanamycin + cytarabine cultures are presented in We investigated the effect of combining concanamycin A 1 nM with cytarabine 1 μM on primary AML cell proliferation. All 70 patients showing detectable cytokine-dependent proliferation were included in these experiments. The antiproliferative effect of concanamycin A showed a wide variation between patients when comparing the relative responses ; for 48 Thus, even when using the stronger V-ATPase inhibitor, concanamycin A with low-dose cytarabine seems to have additive antiproliferative effects compared with the V-ATPase inhibition alone, at least for patients with relatively weak antiproliferative effects of concanamycin alone, and we found no evidence for counteracting effects between the two drugs.Proteomic and phosphoproteomic profiles were available for a consecutive subset of 15 younger AML patients (below 60 years of age), and we compared two contrasting patient groups with an antiproliferative effect corresponding to a relative response  <0.30 versus >0.60, respectively. These comparisons therefore included eight strong responders , as well as in previously identified potential prognostic AML markers.We thereafter compared the protein phosphorylation profiles for the same two contrasting patient groups, and 100 differentially regulated phosphorylation sites were identified. Fifty-seven phosphosites were increased for patients with a strong antiproliferative effect of bafilomycin A1 10 nM, whereas fourty-three phosphosites were increased for the low responders . First, GO term analyses of differentially expressed phosphorylation sites showed that strong responders to bafilomycin A1 were characterized by increased phosphorylation, especially for proteins involved in transcriptional regulation/RNA functions, whereas the weak responders showed increased levels of phosphorylation of proteins involved in the regulation of proliferation/cytoskeleton/signal transduction/metabolism . Furtherp = 0.012). The number of colonies was reduced by concanamycin A for all except one patient. Finally, the effect of bafilomycin A 10 nM was similar to the concanamycin A effect for the three patients examined. Thus, V-ATPase inhibition has an antiproliferative effect also when analyzing the clonogenic AML cell subset.We investigated the effect of V-ATPase inhibitors on the clonogenic proliferation of primary human AML cells derived from 10 unselected patients that show an antiproliferative effect of concanamycin A I nM corresponding to a relative response < 0.30 in the suspension culture assay. The overall results are presented in Analyses of the global gene expression profiles were available for 32 consecutive patients, and we did an unsupervised hierarchical clustering analysis based on identified members of the V-ATPase interactome  3,67,68,6867,68.p = 0.0016). Furthermore, the frequency of patients with Flt3-ITD did not differ between the two main clusters, but the majority of seven out of the 10 Flt3-ITD positive patients clustered close to each other in the upper main cluster .Our clustering analysis identified two main patient subsets including 18 (upper) and 14 patients (lower), respectively. The upper main cluster was characterized by relatively high levels of CCT expression, whereas the lower main cluster showed relatively low CCT expression but generally high expression of ATP molecules/genes. The clinical and biological characteristics of the patients in each of the two main patient clusters are presented in −PI−) together with minor subsets of early apoptotic (Annexin+PI−) and late apoptotic/necrotic cells (Annexin V+PI+) immediately after thawing [We used cryopreserved primary AML cells in all our present experiments. Previous studies have demonstrated that cryopreserved AML cells usually include at least 70% viable cells , the effect was generally weaker for bafilomycin A1  than for concanamycin A, which showed a wider variation with a strong viability-decreasing effect for several patients. The relative viability in concanamycin A-containing cultures showed no significant association with the degree of spontaneous apoptosis in drug-free control cultures.Primary human AML cells from all 80 patients were also incubated with bafilomycin A1 10 nM and concanamycin A 10 nM for 48 h before viability was examined by flow cytometry. The overall results when comparing the relative viability  are presented in To investigate the variation in spontaneous in vitro apoptosis between our 80 patients, we analyzed the results for concanamycin A 10 nM in more detail. Sixty of the eighty patients showed more than 30% viable cells (variation range 97.9–31.5%), whereas only twenty of the patients showed a viability below 30% (range 28.6–2.6%) after 48 h of culture in medium alone. This variation of viability in control cultures did not show any significant associations with secondary versus de novo AML, differentiation , genetic abnormalities  or cytokine-dependent proliferation.+, propidium-iodide−) cells for drug-containing and control cultures. The percentage of early apoptotic cells was significantly increased especially for concanamycin A-containing cultures compared with the corresponding drug-free controls (p < 0.005), suggesting that the decreased viability associated with V-ATPase inhibition was caused by increased apoptosis. Finally, as expected, the percentage of late apoptotic/necrotic cells was significantly increased for the drug-containing cultures compared with the drug-free controls (p < 0.0005 for both drugs). The effects on apoptotic/necrotic cells did not show any significant associations with differentiation , genetic abnormalities  or antiproliferative effects. Finally, the percentage of viable cells in bafilomycin A1 and concanamycin A cultures showed significant correlation with the viability in drug-free control cultures  was maintained also in the presence of cytarabine, but when comparing the overall results, cytarabine significantly increased the levels of TNFα, whereas the levels of SerpinE1, MMP2, MMP1, IL1RA, CCL3 and CCL4 were significantly decreased . HoweverWe then investigated the effect of concanamycin A 1 nM on the constitutive AML cell release of soluble mediators in the presence of cytarabine 1 nM. All 80 patients were included in these studies. A wide variation in the constitutive release of 10 soluble mediators were seen both when cytarabine was tested alone and in combination with concanamycin. Concanamycin A altered the soluble mediator profile of primary AML cells also in the presence of cytarabine ; when cop = 0.0126) and concanamycin A (p = 0.000068) had significant antiproliferative effects on primary human AML cells, even in the presence of the leukemia-supporting MSCs.The local soluble mediator network enhances the cell proliferation of primary human AML cells during co-culture of the leukemic cells with normal MSCs . For thip < 0.005) significantly decreased the relative proliferative responses of the MSCs (p = 0.000062 for both drugs).We also investigated whether bafilomycin A1 10 nM and concanamycin A 1 nM had any effect on bone marrow MSC proliferation when these cells were co-cultured with the same 18 primary human AML cells . The medTaken together, these observations show that (i) V-ATPase inhibition has an antiproliferative effect on primary AML cells even in the presence of leukemia-supporting MSCs; and (ii) the antiproliferative effect of V-ATPase inhibitors is not specific for AML cells but is also observed for non-leukemic bone marrow MSCs.We investigated the effect of the two inhibitors on the release of 17 soluble mediators during co-culture of AML cells and MSCs; the leukemia cells derived from 18 unselected/consecutive patients were then examined . The ovep = 0.294).We also investigated the effects of concanamycin A 1 nM on the co-culture mediator levels for individual patients. These analyses were based on the relative responses, i.e., the level of a certain mediator in drug-containing co-cultures relative to the levels of the same mediator in the corresponding drug-free control culture. The results for concanamycin A are presented in To summarize, V-ATPase inhibitors can modulate the extracellular network of soluble mediators both for primary AML cells cultured alone and when AML cells are co-cultured with MSCs. However, there is a considerable variation between AML patients with regard to the capacity of their leukemic cells to constitutively release soluble mediator  on xenografted MV4-11 cells was tested; during the experiment, we evaluated body weight, AML progression by bioluminescence, with quantification of leukemia cell burden, and survival. Drug administration was started 20 days post-transplant to allow engraftment of the leukemic cells, and the drug was administered for 3 days every week (Q.Dx3) for the next three weeks, i.e., the overall observation period was five weeks. This pilot study included three mice in each group, and after five weeks, neither the body weight, the leukemia cell burden evaluated by bioluminescence imaging nor the survival  differedp = 0.0078). However, this effect was not reflected in bioluminescence imaging, where no significant difference was observed between groups  on xenografted HL-60 cells was also tested by using the same drug regimen as for the MV4-11 xenograft model. In this experiment, bafilomycin treatment was initiated on day 13 post-transplant, and animals received four cycles of therapy before the first control group animals reached endpoint criteria. Six mice were included per group, though one animal in the bafilomycin group was lost prematurely (day 28), likely due to the toxicity associated with the drug administration procedure because leukemic burden, as determined by bioluminescence imaging, was comparable with other animals within the group. Interestingly, the survival curve  indicaten groups . Thus, tTo conclude, our studies of two different animal models indicate that there is also an in vivo diversity in the antileukemic effect of bafilomycin, but we would emphasize that the antileukemic effect of bafilomycin alone in the HL-60 model is weak and its clinical relevance should be regarded as uncertain. Even though only three mice were examined in each group of MV4-11 xenografted mice, the clinical course of the leukemic mice was as would be expected from previous studies , and neiV-ATPase is regarded as a possible therapeutic target in cancer treatment ,70,71,72We investigated enriched primary human AML cells derived from consecutive and thereby unselected patients. Our hospital is responsible for diagnosis and treatment of AML in a defined geographical area, and the study should therefore be regarded as population-based. However, we only included patients with a relatively high level of circulating leukemic cells ,38,73; eAML cell populations have hierarchical organization and include a minority of clonogenic cells (often <1% of the population) together with a small minority of leukemic stem cells . Even thOur utilized V-ATPase inhibitor concentrations were based on dose–response experiments, and concanamycin A then seemed more potent when testing equimolar doses. We selected concentrations that reflected the variation in antiproliferative effects between patients . StudiesBoth bafilomycin A1 and chloroquine inhibit autophagy in human AML cells ,66,80,81V-ATPase mediates apoptosis resistance/chemoresistance in various malignant cells, but it can also have proapoptotic functions under certain conditions ,18,19 anWe investigated the effect of V-ATPase inhibition on the soluble mediator network in MSC/AML cell co-cultures. Hierarchical clustering analysis then identified two main patient subsets characterized by generally increased and decreased mediator levels, respectively. The high-release profile was associated with secondary AML, an association not seen when AML cells were cultured alone. Thus, the effects of V-ATPase inhibition on the extracellular mediator network of the AML cell microenvironment is further modulated by effects on neighboring non-leukemic cells, including MSCs but possibly also endothelial cells ,86 and oOur studies of AML cell monocultures showed that V-ATPase inhibition had a direct antiproliferative effect that was particularly strong for secondary AML. On the other hand, MSCs mediate AML supporting effects through their release of various soluble mediators , and secSecondary AML is a heterogeneous group  that incPatients with secondary AML  showed several statistically significant differences from patients with de novo AML and were associated with a strong antiproliferative effect of V-ATPase inhibition, a different V-ATPase interactome mRNA profile and increased levels of several extracellular soluble mediators in bafilomycin-containing MSC/AML cell co-cultures. A previous proteomic study also showed that a different patient subset with adverse prognosis  showed low protein levels of certain ATP transporters and strong antiproliferative effects of V-ATPase inhibitors in pretreatment AML cells compared with patients later becoming long-term AML-free survivors after . Thus, bOur conclusion that the effect of V-ATPase inhibition differs between de novo and secondary AML has to be made with care, even though it was observed via various experimental approaches. Our study population included mainly patients with secondary AML following MDS/myeloproliferative neoplasias but only two patients with previous chemotherapy see . FurtherWe conducted a proteomic and phosphoproteomic comparison of pretherapy AML cells showing weak and strong antiproliferative effects of V-ATPase inhibitors. Several molecules that influence the same cellular structures/processes as V-ATPase differed significantly between these two contrasting patient groups. The proteomic comparison identified the differential expression of several proteins involved in molecular (transmembrane) transport, degranulation and cytoskeletal function. On the other hand, the phosphoproteomic profiling identified differences in RNA metabolism/function, cytoskeletal function and growth regulation, and these differences partly reflected increased CK2 activity . The finOur proteomic comparison identified four potential biomarkers with increased levels in AML cells showing strong antiproliferative effects of V-ATPase inhibition. First, transferrin receptor levels possibly reflect differences in iron metabolism that are important for chemosensitivity ,95, extrWe combined V-ATPase inhibition with cytarabine that was tested at concentrations corresponding to the in vivo levels during low-dose cytarabine therapy ; the obs+ cytotoxic T lymphocytes, whereas the levels/functions of CD4+ helper T lymphocytes and B lymphocytes are not affected [+ T lymphopenia after cytotoxic anticancer therapy [We investigated the in vivo toxicity of bafilomycin A1 that can be administered intraperitoneally without severe toxicity ,70,71,72affected ,109. Theaffected . Previou therapy . A pan-T therapy ,113,114.Bafilomycin A1 monotherapy following xenotransplantation of MV4-11 and HL-60 AML cells had limited or no effect on subsequent leukemia progression . AnotherOur present study has limitations that should be addressed in future studies. First, we did not investigate effects of V-ATPase inhibitors on AML stem cells that are thought to be responsible for chemoresistant leukemia relapse , even thOur experimental studies show that V-ATPase inhibition has antiproliferative, proapoptotic and cytokine-modulating effects on primary human AML cells. Both our present study and a small previous study  support


Delamination in reinforced panels is one of the primary challenges facing the safety and reliability of aerospace structures. This article presents a sensitivity analysis of the fatigue behaviour during the compression of a composite aeronautical stiffened panel experiencing delamination. The main objective is to assess the impact of delamination size and depth on the lifecycle and structural integrity of the panel. Different dimensions and positions of delamination are considered to cover a comprehensive range of damage scenarios. The key feature of this sensitivity analysis is the adoption of a numerical procedure that is mesh- and load-step-independent, ensuring reliable results and providing valuable insight into the criticality of delamination and its impact on the fatigue behaviour during the compression of reinforced aeronautical panels. Sensitivity analyses are essential for enhancing the design process of aerospace structures, thereby contributing to the increased safety and reliability of structural components. In this regard, the use of robust and effective numerical procedures is of crucial significance. This may be seen as the real added value of this paper. Fracture has always been a significant issue for the aerospace industry during the fabrication of aerospace structures, and, over the years, it has become more severe due to the extensive use of innovative materials, such as composite materials ,2, whichMany studies have been carried out in the literature to consider the delamination phenomenon due to a variety of causes. The study in  introducThe study of damage propagation in aircraft structures must begin at the preliminary design stage. Indeed, the use of new design concepts and innovative materials, whose damage behaviour is not well understood, often leads to the over-dimensioning of aircraft structures. Furthermore, in a damage-tolerant design philosophy, the loads and stresses that the aircraft is expected to withstand in operation must be considered in detail, and the effects of the damages on the structural integrity of the component must be assessed. The work in  delved iAppropriate experimental testing campaigns should be conducted to identify structural weaknesses. However, the production of aircraft structures, even on a small scale, to identify the best structural configurations can be very expensive as well as time-consuming. Aircraft designers and engineers use a variety of tools and techniques to analyse and predict the behaviour of materials and structures under operating loads, including finite element analyses ,26,27,28Sensitivity analyses are essential in structural design and projects to identify the critical geometrical parameters that have the most significant impact on the structural design or project outcome ,29,30,31The main added value of this article is the use of the FT-SMXB methodology, due to its inherent characteristics, to study, specifically, the influence of the size and depth of typical impact damage on composite panels. The standard virtual crack closure technique (VCCT) would probably influence the results due to its dependence on the mesh and time step of the finite element analysis. On the contrary, one of the advantages of the cohesive zone model (CZM) methodology is its lower sensitivity to mesh size. However, defining the correct cohesive stiffness is still a challenge , and a rA typical aeronautical stiffened panel was studied under compressive fatigue loading conditions. The panel was 300 mm in length and 400 in width. Two T-shape stringers were tied on the skin, which has a 60 mm foot and 30 mm web. The stacking sequences of the different panel components, which are listed in The elastic and interlaminar properties of the material were evaluated through an experimental characterisation, based on the ASTM standards in ,40,41,42The panel was modelled through the Ansys Parametric Design Language (APDL) to easily modify the value of the variable parameters (radius and depth). The global–local approach was chosen as modelling strategy in order to reduce the computational cost and the analyses’ duration. Indeed, a coarse mesh was considered for the global part, while a more refined discretisation was performed in the local zone, which is the region characterised by the delamination damage and is involved in the propagation. Elements of 12.5 mm were used for the global model, while elements of approximately 2 mm and 5 mm were considered in the local region. According to the fail release approach, typically used in conjunction with the virtual crack closure technique (VCCT), the local zone was built with two overlapping identical solids, as shown in The translational and rotational degrees of freedom on one side of the panel were clamped (interlocking constraint); on the other side, the terminal nodes were connected through rigid connections to a reference point (pilot node) on which the compressive force was applied. The scheme of the boundary conditions is shown in Fracture mechanics relations are the basis of the FT-SMXB numerical method. In particular, the modified virtual crack closure technique (VCCT) equations were implemented to calculate the energy release rate (ERR) values on the nodes of the delamination front. Such values were compared with the critical fracture toughness values to assess the propagation of delamination, as described by Equation (1).The ERR ment see .(2)Gj=FA of the fracture surface, while According to the VCCT, i refers to the nodes of the delamination front.In the FT-SMXB approach, the VCCT limits in terms of mesh and dependence on load step size are avoided through the implementation of an iterative procedure, named SMART TIME, that can iteratively change the load step size until the calculated energy corresponds exactly to the amount needed to propagate an area equal to that of one or more elements. In detail, the equivalence in Equation (3) must be satisfied. The subscript N represents the number of cycles, while C and n are the Paris law constants evaluated experimentally.Cyclic load can be applied on the structure via a succession of static analyses, which can be considered as fatigue cycles. The load applied on the structure in each static analysis is calculated as a percentage of the maximum load that the structure can support when subjected to static loading conditions. Basically, a first static analysis is performed, and the ERR values on the delamination front nodes are calculated and used as input in the Paris law criterion, defined by Equation (4). The number of cycles needed to propagate delamination damage is calculated by reversing the Paris law, as described in Equation (5). Finally, the node with the higher criterion value, which is the node where the energy values are closest to the critical values, is selected and released.Starting from the last converged solution, subsequent analyses are performed until the number of cycles to failure is reached, or until the user-defined limits are encountered. Damage accumulation is considered. A damage variable is considered in each node of the delamination front to account for damage accumulation, and the number of cycles starts from that reached in the previous analysis.Three different radii of circular delamination were studied: 20 mm (R20), 30 mm (R30), and 40 mm (R40). Each of them was positioned at three depths along the thickness see . This plThe propagation initiation load and the overall buckling behaviour of the panel under static loading conditions were needed to proceed with fatigue analyses. Hence, preliminary analyses were conducted considering the panel with one edge fixed  and the other subjected to compressive displacement (free degrees of freedom in the compression direction). Fixing the position along the thickness of the delamination damage, as the radius increases, there are no significant variations in the delaminated area trend with increasing load; as demonstrated in It is worth specifying that the analyses automatically terminate when the delamination  reach the user-defined limit. In this specific case, the limit was set at the outmost nodes of the circular propagation crown considered, as shown in Based on the obtained results, considering the load at which delamination starts propagating, fatigue analyses were conducted, according to The summary of the performed fatigue analysis is reported in The fatigue damage initiation life refers to the duration or number of loading cycles that a material or structure can undergo before the initiation of fatigue damage. Evaluating fatigue damage initiation life involves analytical methods, such as cycle counting methods and the damage accumulation model, which are taken into account in FT-SMXB. The delamination initiation cycle was determined through the Paris law relation, as defined in Equation (5). Increasing the depth of delamination for the same radius can have complex effects on the fatigue behaviour of a composite panel. According to According to The evolution of delamination for different numbers of cycles is shown in In this work, the mesh- and load-step-independent FT-SMXB numerical methodology was employed to assess the compressive fatigue behaviour of a composite material, an aeronautical stiffened panel, affected by typical impact damage in the bay region. Two percentages of the static damage initiation load were considered (80% and 90%). The results revealed that for greater radii of the circular delamination, at a fixed damage depth, delaminated areas experience smooth growth, achieving a higher number of fatigue cycles with a lesser damage extent. Furthermore, increasing the depth of the damage, at a fixed radius, increases the delamination onset cycle but leads to faster and unstable delamination evolution. The overall stiffness of the panel remains almost constant with the fatigue cycles for all the considered radii and depths of delamination. It is important to note that the specific behaviour of composites and the effect of delamination depend on various factors, including the type of composite material, its layup configuration, the loading conditions, and the size and shape of the delamination. However, the use of effective and robust computational methodologies can provide help in designing damage-tolerant composite structures for various applications and loads, thus reducing the waste of money and time derived from manufacturing and experimental tests. The primary contribution of this article lies in the application of the FT-SMXB methodology, which, owing to its intrinsic characteristics, proves exceptionally valuable in examining the impact of size and depth variations in a typical damage scenario on composite panels. Unlike the standard VCCT and CZM methodologies, which are likely to impact the results due to their reliance on the mesh and time step parameters in finite element analysis, the FT-SMXB methodology stands out for its independence from these factors.


Despite the approval of several drugs for AML, cytarabine is still widely used as a therapeutic approach. However, 85% of patients show resistance and only 10% overcome the disease. Using RNA-seq and phosphoproteomics, we show that RNA splicing and serine-arginine-rich (SR) proteins phosphorylation were altered during cytarabine resistance. Moreover, phosphorylation of SR proteins at diagnosis were significantly lower in responder than non-responder patients, pointing to their utility to predict response. These changes correlated with altered transcriptomic profiles of SR protein target genes. Notably, splicing inhibitors were therapeutically effective in treating sensitive and resistant AML cells as monotherapy or combination with other approved drugs. H3B-8800 and venetoclax combination showed the best efficacy in vitro, demonstrating synergistic effects in patient samples and no toxicity in healthy hematopoietic progenitors. Our results establish that RNA splicing inhibition, alone or combined with venetoclax, could be useful for the treatment of newly diagnosed or relapsed/refractory AML. There are now several targeted therapies approved for the treatment of acute myeloid leukemia (AML), including midostaurin, gilteritinib, CPX-351, enasidenib, ivosidenib, gemtuzumab ozogamicin, glasdegib and venetoclax . On stanThe presence of recurrent mutations in the spliceosome machinery, as well as aberrant splicing events, have been described as common alterations in AML disease , 9, and Because functional changes in splicing regulatory proteins can promote oncogenesis through overexpression, alteration-of-function, and mutations, therapeutic targeting of the spliceosome holds promise as a novel cancer therapy . SpliceoIn the present study, we show that phosphorylation patterns of SR proteins are altered during the development of cytarabine resistance in AML, and that the combination of spliceosome inhibitors and other approved drugs, including the BCL2 inhibitor venetoclax, improves the therapeutic response in cells from patients with AML, even in a background of cytarabine resistance.n = 75, median age = 60), myeloproliferative neoplasms  and myelodysplastic syndromes  and from 18 age-matched healthy donors from the same population-based studies were employed for analysis. Demographic and clinical features of the subjects are reported in Supplementary Table Comité Ético de Investigación Clínica of the Instituto de Investigación Biomédica of the Hospital 12 de Octubre, and all patients and donors provided written informed consent in accordance with the Declaration of Helsinki. For patients drug response categorization, we rely on ELN recommendations [Hospital 12 de Octubre. For details see Human OCI-AML3, SKM-1 and THP-1 cell lines were cultured in RPMI-1460  supplemented with fetal bovine serum  and antibiotics . OCI-AML3 cytarabine-resistant cells (OCI-AML3_R) were generated from parental OCI-AML3 cells after sustained and cumulative exposure to 20 µM cytarabine. Samples from patients with AML (n = 173) [n = 70) [We used the Gene Expression Profiling Interactive Analysis (GEPIA2) web-server  to compan = 173)  and heal[n = 70) . For detSRRM2, SRSF12  and β-glucuronidase (GUS)  were measured by qPCR and quantified using the comparative cycle threshold (2ΔCt) method [Gene expression levels of ) method .RNA libraries of paired samples from 25 patients with AML at diagnosis and cytarabine-resistance moments were generated following the KAPA RNA HyperPrep kit with RiboErase (HMR) protocol  and sequenced on the NextSeq 500/550 platform . Differential gene expression (DGE) analysis was performed using DESeq2 ; gene clDifferential exons usage analysis was carried out by Dreamgenics S.L.  using the DEXseq package . Variantn = 3) (diagnosis and resistance) were conducted following the standard filter-aid preparation method and processed by IMAC and liquid chromatography tandem-mass spectrometry (LC-MS/MS) as described [Phosphoproteomic studies of bone marrow mononuclear cells (BMMCs) from paired samples of patients with AML (escribed . Differeescribed . For detn = 64) and at resistance (n = 4) and after cytarabine treatment (n = 3)  or 2 × 104 (primary cells) in 96-microwell plates and exposed to different drug doses (Table 50) values were determined by nonlinear regression using GraphPad Prism 5.01 , and the combination index (CI) and normalized isobolograms were obtained using Compusyn software  [Growth analyses after monotherapy or combination treatments were performed in cells seeded at 3 × 10mus, NJ) . For detCD34+ cells from BMMCs of healthy donors were isolated with the MACs CD34 MicroBead Kit  and cultured in methylcellulose medium  with different drug doses. Colony-forming units (CFU-granulocyte-monocyte and erythroid colonies) were scored after 14 days. For details see t-test or the non-parametric Mann-Whitney U test, for unrelated samples, and the Wilcoxon signed-rank test, for related samples. Differences were considered as statistically significant at P ≤ 0.05. Data are presented as the mean 土 standard error of the mean (SEM).Statistical analyses were performed with GraphPad Prism 5.01 software. Comparisons between two groups were performed using the parametric Student’s SRRM2, SRSF12, and SRSF9  were studied in a cohort of 54 patients from Hospital 12 de Octubre. Results showed that serine–arginine repetitive matrix protein 2 (SRRM2) was significantly overexpressed in the myeloid diseases AML , MPN , and MDS  when compared with controls, with expression being significantly higher in AML than in MDS (P = 0.0382)  in myeloid diseases than in controls, although the differences were not significant  Fig. . We alsoHGF, ATF3 and MYO1B) or refractory   Fig. . SubsequA1) Fig. . To idenA1) Fig. . InteresA1) Fig.  and signA1) Fig. . We concKMT2B, ETS2, SMAD5 and ETV6) among the 20 most variable in activity across samples  by LC-MS/MS. Analysis of the intensity of the phosphoepitopes between the two groups revealed significant differences in the phosphorylation of several SR proteins, with SRRM2 showing the greatest changes after treatment , finding significantly higher levels of phospho-SR proteins at resistance in cytarabine-responder patients that further relapsed  of paired bone marrow smears from patients with AML . In these patients, the phospho-SR protein levels after treatment were similar to patients that relapse, however, they presented remarkable higher ones at diagnosis. , with predominantly nuclear staining . Results showed that non-responders had significantly higher levels of phospho-SR protein staining in positive blasts than responders . Profiling analysis identified differential exon usage between the two groups  with different sensitivity to cytarabine. Results showed that several spliceosome inhibitors inhibited cell growth in vitro in the micromolar range, including madrasin, SPHINX31 and SRPKIN-1; notably, H3B-8800 inhibited cell growth in the low nanomolar range Fig. .Fig. 4Thn = 3). We then used a limiting dilution protocol and isolated 20 independent cytarabine-resistant clones . No differences in the mutational status of most frequent genes mutated in AML , 27 were50 values of each drug in OCI-AML3 and OCI-AML3_R1 cells  Fig. , supportug Table , with thSimilar to the results in cell lines, the combination of H3B-8800 and venetoclax showed enhanced antileukemic activity in ex vivo BMMCs isolated from three patients with AML Fig. . Strong Aberrant mRNA splicing in AML has been previously demonstrated , but itsSRRM2 in a series of patients with myeloid disease, with a particularly significant increase in expression in AML. These findings point to a possible role for gene overexpression in the development of these diseases. SRRM2 overexpression has been observed in nasopharyngeal carcinoma, and its silencing in cellular models of the disease diminishes proliferation, blocks the cell cycle, and enhances apoptosis [Large scale studies such as those of TCGA, which have analyzed the genome of 200 adult patients with de novo AML , have atpoptosis .HGF [MIR12AHG [BLK [KLF6 [RASGEF1B [MYO1B [SPTA1 [KMT2B [ETS2 [SMAD5 [ETV6 [ETV6, involved in different types of leukemia [ETV6 knockout cell lines exhibit an increased gene expression of splicing factors [Mutations in mRNA splicing genes has been linked to drug resistance development in AML , 11, 51.HGF , MIR12AHMIR12AHG , BLK [55LK [KLF6  and RASGRASGEF1B  appear tB [MYO1B  or SPTA1B [SPTA1  can be t1 [KMT2B , ETS2 [32B [ETS2 , SMAD5 [2 [SMAD5  and ETV6D5 [ETV6 . For insleukemia  is downrleukemia , and ETV factors . TherefoWe show by phosphoproteomics and immunohistochemistry that the phosphorylation of SR proteins increases after the development of acquired resistance to cytarabine. This increase was not related to changes in the location or retention of SR proteins, but rather to changes in their activity/functionality , 62, 63.Splicing alterations in cancer can modify the functionality of oncogenes, tumor suppressor proteins, splicing factors, apoptosis proteins, and also cell proliferation , 12, 14.SRRM2 silencing increases the levels of pro-apoptotic proteins such as Bax or caspase 3, while decreasing the levels of anti-apoptotic proteins such as BCL2 or Ki-67 [Preclinical studies with the splicing inhibitor H3B-8800 have revealed its potential in the treatment of myeloid diseases that carry mutations in splicing factors , 37, 69.or Ki-67 . It coulor Ki-67 , and so In sum, we demonstrate that altered phosphorylation of SR proteins is related to primary or secondary resistance to cytarabine, and might be useful to predict response. Moreover, inhibition of the splicing mechanism, alone or in combination with venetoclax, could be a good strategy for the treatment of newly diagnosed or relapse/refractory AML.Supplemental Material (Leukemia - ProvAccepted)


What was the regulatory oversight and evidence generation for idelalisib between accelerated approval and voluntary withdrawal?In this systematic review and meta-analysis, we found 20 trials that investigated idelalisib in indolent non-Hodgkin lymphoma (NHL): 3 were phase 3 trials in indolent NHL, 2 of which were terminated due to increased mortality, and 1 recruited only 96 patients prior to voluntary withdrawal. No efficacy results were published for these trials; Gilead reported cumulative sales revenue of $842 million during market authorization.Periodic assessment of regulatory effectiveness of the accelerated approval pathway is needed. This systematic review with meta-analysis examines the regulatory oversight of the accelerated approval pathway and evidence generation for idelalisib during premarketing, postmarketing, and premarketing withdrawal periods. Idelalisib is a first-in-class phosphatidylinositol 3-kinase inhibitor that received US Food and Drug Administration accelerated approval in July 2014 as a single-agent treatment for relapsed follicular lymphoma (FL) and small lymphocytic lymphoma (SLL). Serious adverse effects were reported in 2016 leading to termination of postmarketing registry trials. However, idelalisib remained on the market until 2022 when Gilead voluntarily withdrew the drug for the accelerated approval indication.Evaluate the regulatory oversight of the accelerated approval pathway and evidence generation for idelalisib during premarketing (2008-2014), postmarketing (2014-2016), and premarketing withdrawal periods (2016-2022).ClinicalTrials.gov, FDA.gov, PubMed database.Clinical trials investigating the safety and effectiveness of idelalisib.Study characteristics and relative risk (RR) of safety outcomes were abstracted. Data were pooled using random effects meta-analysis. The analysis was performed in October of 2022.Trial status, recruitment status, publication status, serious adverse events (SAEs), fatal adverse events (FAEs), and all-cause mortality.Overall, 31 idelalisib trials met selection criteria. In total, 20 of 30 (65%) included SLL and/or FL; 13 (42%) trials were completed, 13 (42%) had published results, and 7 (23%) were randomized clinical trials (RCTs). Overall, 6 RCTs of idelalisib had publicly available data on safety outcomes. By the initial postmarketing period (2016), the cumulative RR for SAEs was 1.86 , for FAEs was 3.30 , and for death was 1.35 . In the premarketing withdrawal period, only a single phase 3 trial was enrolling patients for FL and was terminated. However, idelalisib was not withdrawn from the market until 2022. Gilead reported cumulative sales revenue of $842 million during market authorization (2014-2022) and annual sales had a steady decline from $168 million to $62 million during the premarketing withdrawal period (2016-2021).Findings of this systematic review and meta-analysis show that serious risks of SAE, FAE, and death with idelalisib treatment were evident by 2016. However, idelalisib remained on the market for another 6 years, with minimal evidence generation. It was voluntarily withdrawn for FL and SLL accelerated approval indications coinciding with decreasing revenue generation. Closer attention for safety and effectiveness of drugs reaching market by accelerated approval is needed. Completion of these requirements is often delayed. For example, of 93 accelerated approvals between 1992 and 2017, 37 (40%) had not completed PMR by 2018 and 5 (5%) drugs that received accelerated approval had been withdrawn.2Since the inception of the accelerated approval pathway in 1992, it has been an increasingly used strategy for drug approvals in oncology and hematologic malignant diseases. Between 1992 and 2017, 93 new oncology indications were approved under the accelerated pathway.3 Idelalisib is a first-in-class PI3K inhibitor that received accelerated approval in July 2014 by the US Food and Drug Administration (FDA) as a single-agent treatment for relapsed follicular lymphoma (FL) and small lymphocytic lymphoma (SLL). Subsequently, additional PI3K inhibitors, including copanlisib, duvelisib, and umbralisib, were approved.4 However, during the first 2 years after approval, concerns about safety began to emerge, including risk of drug-related mortality.4 In 2022—7 years after the initial approval and more than 5 years after initial safety concerns were raised—Gilead Sciences, Inc voluntarily withdrew the idelalisib indications for FL and SLL, citing poor enrollment in confirmatory studies.5 This was followed by further withdrawals of next-in-class PI3K agents because further concerns were raised over the safety of the entire class of drugs.6Several drugs in the phosphatidylinositol 3-kinase (PI3K) inhibitor class have received accelerated approval. The PI3K pathway has been of specific interest because of its central role in multiple cellular functions and signaling pathways in tumor development, and consequently, is a target for antitumor drugs.Idelalisib, a first-in-class drug, demonstrated early promising results and was granted accelerated approval, yet longer follow-up resulted in the manifestation of serious risks. Review of the regulatory history of idelalisib is instructive and offers lessons for improving oversight of the accelerated approval pathway. To address questions regarding the overall safety of idelalisib, the timing of its approval, and how decisions by both the FDA and Gilead Sciences coincided with the reporting and dissemination of safety data, we performed a cumulative meta-analysis of the evidence that emerged during market authorization and reviewed the regulatory response by the FDA. We focused on the sequence of events from accelerated approval to voluntary withdrawal of SLL and FL indications by Gilead Sciences and investigated the historical revenue generated by idelalisib within that time frame. We focused on 3 distinct time periods: the premarketing period , initial postmarketing period , and premarketing withdrawal . We sought to understand what was known regarding the safety of idelalisib by the end of each of these 3 time periods by characterizing study designs, whether studies were completed, and evidence regarding safety, to determine whether its withdrawal could have occurred sooner. We also estimated the trajectory in revenue generated by sales of idelalisib over time.idelalisib and study type interventional, and PubMed with search term idelalisib and lymphoma and study type clinical trials to identify all idelalisib trials. Searches were from database inception through August 2022. All trials were reviewed following the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) reporting guidelines, and only trials with a clinical indication for chronic lymphocytic leukemia (CLL), SLL, or FL were included for analysis.7 For each trial, we abstracted the national clinical trial (NCT) number, title, start and end dates, phase, indication, trial status, and publication status. Studies listed as active, not recruiting were considered complete if results were published or shared on ClinicalTrials.gov, and the date on which the study status was changed from recruiting to not recruiting was noted instead of end date.A systematic review, including a cumulative meta-analysis, was performed by 1 reviewer (T.B.) for all clinical trials investigating the efficacy of idelalisib for indications that received regular or accelerated FDA approval. We searched ClinicalTrials.gov using the search term 8 For all-cause mortality, the hazard ratio for death and confidence intervals (CIs) were obtained in addition to number of deaths.Analysis was performed on phase 3 randomized clinical trials (RCTs), excluding 1 extension study comparing 2 dose levels of idelalisib and 1 study comparing idelalisib to chlorambucil instead of placebo. From this set, we collected safety data reported on ClinicalTrials.gov. Data on the size of treatment and placebo groups, number of deaths, and number of serious adverse events (SAEs) for the placebo and treatment arms were collected. The number of SAEs was found under Study Results on ClinicalTrials.gov. Per their definition, SAEs are adverse events that are life-threatening or result in death, require an extend hospitalization, result in significant incapacity, or cause a congenital anomaly. All-cause mortality and rates of fatal adverse events (FAEs) not available on ClinicalTrials.gov were obtained from publications, if available. For trials that were terminated without reported results, data were collected from the FDA Briefing Document for the April 21, 2022, Oncologic Drugs Advisory Committee meeting discussing PI3K inhibitors.Frequencies of trial characteristics such as phase, indication, status, and availability of published results were calculated. A timeline was created using the trial start and end dates for trials with SLL and FL indications. For the analysis of RCTs, cumulative risk ratios (RRs) of all-cause mortality, SAEs, and FAEs for the placebo and treatment arms were calculated based on the reported deaths, SAEs, and total participants. Using R statistical software , forest plots were then created from the calculated cumulative RRs for all-cause mortality, SAEs, and FAEs. The analysis was performed in October of 2022.Gilead in the company search page of SEC.gov and reviewed the 10-K filings from 2014 to 2021 for reported worldwide idelalisib sales. The US-specific sales data were also collected when available.Worldwide sales revenue from idelalisib, reported by Gilead, was collected from the Securities and Exchange Commission (SEC) Annual Filings. We searched In accordance with 45 CFR §46.102(f), this review was not human participants research and was not submitted to an institutional review board and did not require informed consent procedures.idelalisib and study type interventional. An additional search of PubMed did not reveal additional studies. Of the 62 trials, 31 were excluded because they were for indications other than CLL, SLL, or FL, did not include idelalisib, were withdrawn prior to any patient recruitment, or were extension studies. The remaining 31 clinical trials were included in our analysis. Trial selection was notable for 31 trials in indolent non-Hodgkin lymphoma (NHL), 20 trials including SLL and/or FL, and 6 phase 3 RCTs  were for CLL only but 20 (65%) included SLL and/or FL; the indications that have now been withdrawn. Most trials (23 [74%]) tested idelalisib in combination with other drugs.14 Herein we analyze the characteristics and results of these trials in context of the regulatory history of idelalisib.Less than a quarter of studies were randomized trials (7 [23%]). Six (86%) of the RCTs compared idelalisib to placebo. Less than half (13 [42%]) of trials were completed and 13 (42%) had results published.NCT01539512), which, in July 2014, led to traditional approval of idelalisib in combination with rituximab for relapsed CLL.Between June 2008 and July 2014, 15 trials studying idelalisib in CLL, SLL, and FL treatment were started. Most of these preapproval trials were early phase (phase 1 or 2) (9 of 15 [60%]) and 3 of 15 (20%) were completed . Most 9. The comNCT01282424), which showed a 54% overall response rate  in 72 patients with FL and a 58% ORR in 26 patients with SLL, a surrogate unvalidated end point.12 Continued approval was contingent on further confirmation of clinical benefit through PMR. The safety data of idelalisib monotherapy were based on 146 adults with indolent NHL who received idelalisib in single-arm clinical trials. From initial approval in 2014, idelalisib carried a boxed warning regarding potential for fatal and serious toxic effects including hepatic toxic effects, severe diarrhea, colitis, pneumonitis, infections, and intestinal perforation. In later studies, severe allergic and skin reactions were recorded as well as Pneumocystis jirovecii pneumonia and cytomegalovirus infections.16 Based on the 1 phase 3 trial at this point, there was an RR for SAEs of 1.48  with idelalisib treatment but no increased risk of death , (2) a phase 3 trial comparing rituximab with or without idelalisib (NCT01732913) by December 2017, and (3) a phase 3 trial comparing bendamustine and rituximab with or without idelalisib (NCT01732926) by February 2019. All of these trials were started and ongoing at the time of accelerated approval.The initial PMR to obtain regular approval included completion of (1) a dose optimization trial for stable disease by June 2019  . Thirtee17 Two of these were PMR trials for the SLL and FL indications (NCT01732913 and NCT01732926), and 5 of the 7 ongoing phase 3 RCTs during this time period were terminated. By the end of 2016, there were 4 completed/terminated RCTs comparing idelalisib vs placebo in indolent NHL. Based on data from those 4 studies, the cumulative RR for SAEs was 1.86  and the cumulative RR for FAEs was 3.30 . Moreover, the cumulative RR for death by the end of 2016 was 1.35   .NCT02536300) began in 2016, which compared 3 different doses of idelalisib.The FDA responded to these findings in September 2016 in 3 ways. They issued a new safety alert, updated the boxed warning and safety information on the drug label, and also added a requirement for the sponsor to conduct an additional trial . This new PMR was issued to conduct a trial establishing a safe and effective dosing regimen of idelalisib in patients with relapsed FL by April 2024. As a result, a phase 3 RCT  during this period were still early-phase trials . Of the NCT01659021, NCT01569295). These were CLL-only studies, 1 completed and 1 terminated, both with results published. Only 1 other phase 3 trial enrolled patients and generated new data after 2016. This was the dose optimization study of idelalisib in FL (NCT02536300), the new PMR established in 2016 following the termination of prior PMR trials.Two phase 3 studies initiated prior to idelalisib approval in 2014 continued enrollment after 2016  were RTCs. Second, 13 of all 31 trials (42%) had published results. Third, the limited phase 3 data available showed increasing RR of SAEs, FAEs, and death, notably including an increased RR for death of 1.35  that was known by 2016 . Fourth,17 The FDA set confirmatory requirements at initial accelerated approval in 2014 and again in 2016, but enforcement of these requirements was delayed. It was not until 2022 that the drug was voluntarily withdrawn for FL and SLL indications.Could the drug have been withdrawn sooner to protect patients from a dangerous drug? Our analysis shows that the bulk of data had been accumulated by the end of the initial postmarketing period in 2016. From 2016 to 2022, patients were exposed to the toxic effects of the product, and physicians did not have access to critical data such as incidence of death or SAEs in terminated trials, while the drugs continued to have marketing authorization. From initial approval, idelalisib carried a boxed warning regarding potential for fatal and serious toxic effects. In March 2016, the FDA issued an additional warning about increased rates of adverse events and deaths in trials combining idelalisib with other cancer treatments after 6 trials were halted due to toxic effects and deaths.18 Gilead purchased Calistoga Pharmaceuticals, which included idelalisib in its portfolio, for $375 million in 2011. Although the drug did not reach the highest blockbuster status, remaining on the market was more lucrative than withdrawal.The withdrawal did not occur due to new data. In fact, there were few ongoing trials after 2016, by which point safety concerns had already been raised. The decision did coincide with a period of decreasing revenue for Gilead (eFigure 2 in The case of idelalisib has implications for the entire class. During the 6 years between the time when safety concerns were raised and the withdrawal of the SLL and FL indications, the FDA approved several next-in-class drugs, including copanlisib, duvelisib, and umbralisib. The FDA decision to approve next-in-class drugs with a known safety signal for the parent is concerning. After the withdrawal of idelalisib, the accelerated approval for duvelisib was withdrawn in April 2022 and the accelerated approvals for umbralisib were withdrawn in June 2022.19The findings of our study suggest that patients would benefit from regulatory reform and increased oversight of the accelerated approval pathway. The Accelerated Approval Integrity Act, introduced in March 2022, would help protect patient safety by including an automatic expiration of accelerated approval status within 1 year if postapproval studies are not completed by the deadline.This study had 2 important limitations. First, these results may not be generalizable to all drugs and disease entities benefiting from the accelerated approval process. Second, we used data that were publicly available and thus were dependent on the accuracy and completeness of these databases.Idelalisib was voluntarily withdrawn for FL and SLL accelerated approval indications at a time of decreasing revenue generation. Six years prior to withdrawal there were already serious toxic effects concerns, and minimal evidence generation to show patient benefits. Evaluation of the effectiveness and integrity of accelerated approval pathway would improve patient safety.


The implementation of small-molecule and immunotherapies in acute myeloid leukemia (AML) has been challenging due to genetic and epigenetic variability amongst patients. There are many potential mechanisms by which immune cells could influence small-molecule or immunotherapy responses, yet, this area remains understudied.Here we performed cell type enrichment analysis from over 560 AML patient bone marrow and peripheral blood samples from the Beat AML dataset to describe the functional immune landscape of AML.ex vivo small-molecule and immunotherapy responses. Additionally, we generated a signature of terminally exhausted T cells (Tex) and identified AML with high monocytic proportions as strongly correlating with increased proportions of these immunosuppressive T cells.We identify multiple cell types that significantly correlate with AML clinical and genetic features, and we also observe significant correlations of immune cell proportions with http://vizome.org/), can be leveraged to investigate potential contributions of different immune cells on many facets of the biology of AML.Our work, which is accessible through a new “Cell Type” module in our visualization platform (Vizome;  FLT3, NPM1, and DNMT3A, only encompassing approximately 30% of all AML patients . As expected, we found bone marrow aspirate samples had decreased proportions of lymphoid populations as compared to the peripheral blood samples . Clinical flow calls used to compare xCell (v1.1.0) proportions were also derived from the Beat AML dataset.Previously deposited data , 12 was Heatmaps were generated by log2 transforming cell proportion estimations for all unique AML patient sample peripheral blood aspirates (n=252). Cell types are clustered by Euclidean distance and patient samples by Pearson’s correlation. The R package “Pheatmap” was used to generate all heatmaps. Clustering of cell types versus specimen was performed using ConsensusClusterPlus (v1.54.0) using partitioning around medoids (PAM)  based onFlow cytometry data was mined from electronic medical records derived from clinical hematology/pathology testing as previously described , 12.Inhibitor-cell type correlations were generated for all inhibitors in the Beat AML database with at least 20 unique patient samples. Details on the drug viability assay and data processing can be found in Tyner et al. 2018 . This reEx vivo aPD1 responses were determined based on Lamble and Kosaka et al.  with gene-sets derived from Jadhav et al.  and Man Unless otherwise stated, * represents P values less than.05, ** less than.01, *** less than.001, and **** less than.0001.https://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/study.cgi?study_id=phs001657.v2.p1). In addition, all data can be accessed and queried through our online, interactive user interface, Vizome, at vizome.org/aml2.Publicly available datasets were analyzed in this study. These data can be found here: All raw and processed sequencing data, along with relevant clinical annotations have been submitted to dbGaP and Genomic Data Commons and are publicly available. The dbGaP study ID is 30641 and accession ID is phs001657.v2.p1 (The studies involving human participants were reviewed and approved by Oregon Health & Science University Institutional Review Board. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.KR: formal analysis, investigation, visualization, methodology, writing–original draft, writing–review and editing. DB: formal analysis, investigation, visualization, methodology, writing-review and editing, resources, data curation. WY: methodology, writing–review and editing. NL: resources. MV: data curation. SM: formal analysis, investigation, visualization, methodology, writing-review and editing, resources, data curation. JT: Conceptualization, resources, formal analysis, supervision, funding acquisition, investigation, visualization, methodology, writing–original draft, project administration, writing–review and editing. All authors contributed to the article and approved the submitted version.


The interaction of pharmacologically active drugs with SC biochemical components is underestimated in pharmaceutical research. The aim of this research was to illustrate that some drugs intended for transdermal delivery could interact with the protein component of SC. Such interactions could be in favor of or opposition to their percutaneous absorption. IR microspectroscopy was used to delineate possible interaction of SC keratin with three losartan salts LOS-K, LOS-DEA and LOS-AML salts in addition to AML-BES salt. The results of PCA, combined with comparisons of average second derivative spectra of SC samples treated with these salts and the control SC, showed that LOS-DEA did not interact with SC, thus providing base line permeation of losartan. AML-BES, LOS-AML and LOS-K salts modified the conformational structure of keratin. The disorganization effect on the α-helical structure and induced formation of parallel β-sheets and random coils were in the order of AML-BES˃LOS-AML˃LOS-K. The order of the impact of treatments which resulted in increased formation of β-turns was AML-BES˃LOS-AML. The formation of antiparallel β-sheets was manifested by LOS-AML. Thus, the overall effect of these salts on the SC protein was AML-BES˃LOS-AML˃LOS-K. The impact of LOS-K was associated with improved permeation whereas the impact of LOS-AML was associated with hindered permeation of both losartan and amlodipine. There is a possibility that losartan and amlodipine when present in combination inside SC, their binding to the protein is enhanced leading to being retained within SC. The layered structure of the skin is well identified with the outermost 10–20 μm thick layer being the stratum corneum (SC) .Some chemical substances are capable to interact with intercellular lipid or intracellular protein components of SC. Taking into consideration the published research in this context, three categories of those substances can be recognized: chemical penetration enhancers, topically applied cosmetic and personal care products, and active ingredients in medicinal products.Chemical penetration enhancers are the most commonly studied ones as they are intended to overcome the barrier properties of SC in order to enhance the percutaneous penetration of drugs thus, facilitating transdermal delivery of therapeutic agents. Many studies utilizes infrared (IR) microspectroscopy to identify the effect of chemical penetration enhancers, such as oleic acid , other fTo a lesser extent, the interaction of topically applied cosmetic products, particularly moisturizers, with SC components is also present in literature. Such interaction may lead to compromised barrier properties of SC or increased susceptibility of skin to irritation, which could be attributed to specific components of the moisturizers . The intVery limited amount of research was devoted to investigate the interaction of pharmacologically active drugs in medicinal products with SC lipids or proteins. Indeed, one can barely find examples. In one study, miltefosine, a drug used to treat visceral leishmaniasis, was found to interact with SC lipid component leading to an increase in its fluidity, an effect that could be utilized for the development of topical treatment of cutaneous leishmaniasis. The study was performed on SC of rats by electron paramagnetic resonance spectroscopy and fluorescence spectroscopy . In a reOn the other hand, we have recently investigated the potential transdermal delivery of a novel salt of an antihypertensive drug losartan, losartan amlodipine (LOS-AML), as an alternative for the conventional available oral delivery of the potassium salt (LOS-K) . LOS-AMLThe scarcity of studies dealing with the interaction of drugs with SC components, and the fact that the limited available research within this context is focused on topically applied drugs, combined with the results obtained from investigating the percutaneous permeation of LOS-AML, have urged us to investigate the interaction of SC with LOS-AML. The occurrence of such interactions is critical since it may have an impact on the drugs retention capability within the SC or their diffusion behavior through it.The investigated drugs were LOS-K, AML-BES, in addition to our novel salts, LOS-AML and losartan diethyl amine (LOS-DEA). The effect of LOS-AML on the protein structure of SC was compared to that of LOS-K and AML-BES. Furthermore, the effect of LOS-DEA was also investigated to reveal whether other losartan salts have the ability to interact with SC or not. IR microspectroscopy combined with multivariate statistical analysis was used to delineate the effect of the applied salts on the protein components of human SC.LOS-K and AML-BES were kindly donated by Jordan Pharmaceutical Manufacturing  and Al Hayat Pharmaceutical Industries Company  respectively. The investigated novel salts prepared in our laboratory were LOS-AML  and losa®, Darmstadt, Germany. Trypsin was obtained from SIGMA-ALDRICH, USA.PG was obtained from SIGMA-AlDRICH2 windows were obtained from Crystran Ltd, UK whereas the diamond compression cell was obtained from Specac, UK.ThermoNicolet 8700 spectrometer coupled with a continuum infrared microscope at IR laboratory of SESAME synchrotron (Jordan) using the globar source was used for IR data measurement. The microscope is equipped with a liquid nitrogen-cooled mercury cadmium telluride detector and OMNIC 9.1 software (Thermo Fisher scientific) for data acquisition for the SC samples. The CaFSixty percent PG in phosphate buffer saline (pH 6.8) was used as a solvent to prepare the treatment solutions of the investigated salts. Solution of each investigated salt of losartan  was prepared at a concentration equivalent to 80% of losartan solubility in each corresponding salt 17, . SimilarThe study was approved by the Ethics Committee for Scientific Research on Humans at Philadelphia University. Human skin of a 44-year-old female donor undergoing abdominal plastic surgery was obtained from a local hospital immediately after the surgery. The research objective was explained to the donor and her approval was verbally granted directly, since she owned the samples after conducting the surgery and had the option to bury them or donate to scientific research. Her approval was granted at two different occasions, before and after conducting the surgery in the presence of her surgeon. The obtained skin was appropriately processed and stored. Three circular discs of 25 mm in diameter for In-vitro permeation experiment in addition to six circular discs of 12 mm in diameter for IR microspectroscopic analysis were punched out from the frozen human skin sheet. The discs were left for few minutes to thaw then soaked in 1% w/v trypsin in distilled water at 32°C for 24 hr to isolate the SC. The isolated SC was soaked for 2 hr in distilled water to wash.24) in μg was obtained from the permeation plots and the results were reported as average ±SD. The obtained values were statistically compared to those of LOS-K and LOS-AML [In-vitro permeation of LOS-DEA was performed as described in reference 18. The total cumulative permeated amount of LOS after 24 hr of permeation  (section 2.2). The fifth piece was swabbed by the same manner with 60% PG to serve as a control. All the solutions were previously equilibrated at 32°C. The sixth piece was left untreated. The SC pieces were then incubated at 32°C for 24 hr. To remove the residual treatment solutions, the pieces were then gently pressed between filter papers and the surfaces were washed by swabbing with a cotton swab impregnated with distilled water and gently pressed between filter papers. The SC pieces were then mounted on CaF2 at step size of 20 μm, and a spectral resolution of 2 cm-1. For each spectrum, 256 co-scans were recorded in the mid-infrared range between 4000 and 700 cm−1 in transmission mode.All IR measurements were carried out at SESAME synchrotron (Jordan). IR microscopic map for each isolated SC sample was acquired using the 15X Schwarzschild objective of the IR microscope. All spectra were obtained using a double path single masking aperture size of 40* 40 μmIn addition, a representative spectrum for each of: LOS-K, LOS-DEA, LOS-AML, AML-BES and PG was obtained after being deposited into diamond compression cell using the same described settings.Splitting of the obtained IR maps was done by Omnic software to yield a data set corresponding to each SC sample. The resultant raw spectra (100–150) of all data sets were preprocessed in the spectral domain corresponding to protein amide I and II vibrations using the Unscrambler 10.3 software (CAMO software). In this domain, the spectra were appropriately smoothed and base line corrected. Savitsky-Golay second derivatives were then calculated using third polynomial order and seven smoothening points, and subsequently range normalized. Principal component analysis (PCA) was performed and the results were represented in the form of score and loadings plots. The unit vector normalized reduced average spectra and their corresponding range normalized Savitsky-Golay second derivative  of the data sets were obtained to compare the effect of the different treatments in accordance with the PCA results. Furthermore, the average Savitsky-Golay second derivative of each data set was range normalized with that of its corresponding treatment  and carefully compared.-1, whereas amide II feature arises mainly from N–H bending and C-N stretching vibrations of amide groups of the peptide backbone in proteins and is located at around 1550 cm-1. Both bands, particularly the amide I band, are sensitive to protein secondary structure [Amide I and Amide II bands constitute the main IR spectral features of SC that correspond to its protein content. Amide I feature arises mainly from C = O stretching of amide groups of the peptide backbone in proteins and is located at around 1650 cmtructure –25, thusKeratin is the main protein constituent of human SC, which is predominantly present in α-helix secondary structure and is known to significantly contribute to the Amide I band. Ceramides and other proteins provide minor contribution to this band, consequently, it is indicative to keratin structural changes , 27.-1 except for AML-BES treated sample in which a shift to higher frequency (1652.7 cm-1) occurred. A shift of the amide I band to a slightly higher frequency is consistent with a decrease in hydrogen bond strength of the peptide bond [-1. In addition, emergence of a shoulder at lower frequency is evident for PG SC and LOS-DEA SC. equency 152.7 cm-1The above discussion highlights possible alterations in the secondary structure of keratin, however, it is difficult to manifest and analyze these alterations by analysis of the raw spectra. To identify the overlapping IR bands in the protein spectral region, that correspond to different protein secondary structures, second derivative spectrum analysis provides one of the most popular approaches , 28. AccPG SC and not SC was taken as the control sample in order to exclude the spectral differences arising from the presence of PG since PG exhibits IR features within the investigated spectral range . This prConcerning the score plot , it is cThe loadings of PC1 and PC2  shows thIn agreement with the score plot, PG and LOS-DEA treated SC samples have noticeable high degree of similarity in the amide I and II regions. On the other hand, -1 usually arising from intermolecular parallel β-sheets.Within amide I range, Peak 1 is attributed to parallel β-sheets –28. LOS--1. The former shoulder could be attributed to formation of random coil structure of the protein [Peak 2 occurs in the range of the α-helical structure of protein –28. LOS- protein –28 where protein . The low protein .A small increase in peak 3 intensity was exhibited in LOS-AML and AML-BES, the latter was also associated with peak shifting to higher frequency. Peaks within this range are usually attributed to β turns –28.AccorAll of the noticed shifts, shoulders and intensity changes in peaks 1–3 were attributed to be induced by the interaction of the corresponding salt with of the corresponding protein structure since the IR signal of the said salts does not overlap with that of PG SC at these peak positons .Peaks 4 and 5 arise from antiparallel β-sheets –28. A smSimilar to amide I, amide II region showed several changes in peaks positions and intensity. Peaks 1, 2 and 3 within amide II are generally assigned to random coil. Peaks positioned at the higher frequency side of this range can be also assigned to antiparallel β-sheets –31. RefeDepending on the above detailed discussion on the impact of the tested salts on the secondary structure of SC protein component, it can be concluded that the disorganization effect on the α-helical structure and induced formation of parallel β-sheets and random coils were in the order of AML-BES˃LOS-AML˃LOS-K. In addition, the order of the impact of treatments that resulted in increased formation of β-turns was AML-BES˃LOS-AML. The formation of antiparallel β-sheets was manifested by LOS-AML. Thus, the overall effect of these salts on the SC protein was AML-BES˃LOS-AML˃LOS-K. Finally, LOS-DEA had no effect on the protein structure and the α-helical structure of keratin was preserved. These conclusions reflect the trends of inter-sample separation seen in the score plot . LOS-DEA24 values of LOS were statistically significant. By not being able to modify SC protein, LOS-DEA exhibited the base line permeation of LOS, which was enhanced by the interaction of LOS-K with keratin, leading to disorganized structure of the protein and subsequent compromised barrier properties of SC. On the other hand, the more drastic interaction of LOS-AML with keratin could be associated with strong binding of these species with protein component because of their simultaneous presence, consequently hindering their permeation despite their effect on disrupting the protein structural conformation. Pyatski et al. (2020) pointed to a similar observation with terbinafine. The interaction of terbinafine with SC keratin, which was evident by induced formation of β-sheet, could regulate its bioavailability in deeper skin layers. The presence of a reservoir of the drug in SC as a result of this interaction was suggested which might allow the controlled released of the drug into the surrounding tissues [In relation to our recent results concerning the retarded percutaneous permeation of losartan and amlodipine from LOS-AML solution compared to LOS-K and AML-BES respectively, the evident interaction of these salts with SC protein could provide the basis for such effect. Possibly, the interaction of losartan and amlodipine with SC when applied individually and not combined helps to modify the barrier properties of SC, thus the interaction is in favor of permeability to some extent. Contrarily, the two drugs when present in combination inside SC, their binding to the protein is enhanced leading to being retained within SC and their permeation hindered. This conclusion is supported by the fact that LOS-DEA provided a permeation that is intermediate between those provided by LOS-K and LOS-AML as shown in  tissues . On the  tissues .Indeed, our observations hypothesize the potential use of IR microscopy in predicting the permeation of different forms of a drug through SC, thus providing a rapid screening tool prior to conduction of permeation studies saving time, effort and the valuable skin samples.The interaction of pharmacologically active drugs with SC biochemical components is underestimated in pharmaceutical research. The nature of this interaction could be crucial for enhancing/retarding the percutaneous permeation of the drug through SC. It could also provide basis for controlled release of the drug through skin. LOS-K was found to enhance the permeation of losartan in a mechanism similar to permeation enhancers that affect the α-helical structure of keratin component of SC. On the other hand, LOS-DEA failed to modify keratin conformation and no signs of interaction with protein were observed by the employed technique. LOS-AML salt impact on the secondary structure of keratin was more drastic than the other two salts of losartan, an effect similar to that of AML-BES. The overall permeation effect of LOS-AML was a retardant for both LOS and AML. Possible binding of these two species to keratin was suggested. Finally, the obtained observation highlight potential use of IR microscopy technique in screening the effect of variable drug forms on the physical barrier properties of SC, and this could be of high value for investigating the percutaneous permeation of substances intended for transdermal/topical delivery.S1 File(DOCX)Click here for additional data file.