Dose-response Curves Research Articles

CurveCurator: a recalibrated F-statistic to assess, classify, and explore significance of dose–response curves

Dose-response curves are key metrics in pharmacology and biology to assess phenotypic or molecular actions of bioactive compounds in a quantitative fashion. Yet, it is often unclear whether or not a measured response significantly differs from a curve without regulation, particularly in high-throughput applications or unstable assays. Treating potency and effect size estimates from random and true curves with the same level of confidence can lead to incorrect hypotheses and issues in training machine learning models. Here, we present CurveCurator, an open-source software that provides reliable dose-response characteristics by computing p-values and false discovery rates based on a recalibrated F-statistic and a target-decoy procedure that considers dataset-specific effect size distributions. The application of CurveCurator to three large-scale datasets enables a systematic drug mode of action analysis and demonstrates its scalable utility across several application areas, facilitated by a performant, interactive dashboard for fast data exploration.

Selectivity of atrazine to forage grasses through dose-response curve

This research aimed to evaluate the selectivity of atrazine applied in forage pre-emergence: Panicum maximum cv. Massai, Panicum maximum cv. Zuri and Panicum maximum cv. Tamani and Brachiaria brizantha cv. BRS Piatã. A randomized block design was adopted, applying eight doses (8D, 4D, 2D, D, 1/2D, 1/4D, 1/8D and no dose, where D is the dose of atrazine 2,000 g ha- 1) with four replications each, in plastic vases with a capacity of four liters. The evaluation was performed visually, with scores from 0 to 100, where 0 was considered absence of injuries and 100 the death of the plant at 15, 30 and 60 days after application (DAA) and dry mass at 60 DAA. For each experiment, the data were submitted to analysis of variance with application of the F test. When the F test was significant, the atrazine doses were adjusted to log-logistic non-linear regression models. It can be concluded that Panicum maximum cv. Massai shows phytotoxicity at a dose of 2,000 g ha-1 with subsequent growth resumption at 30 and 60 DAA.

Analysis of the short-term noise situation around Bangkok International Airport prior to the second phase of airport expansion begins

Short-term noise measurements carried out around the Bangkok International Airport during 2014-2015 provided baseline datasets of noise levels to illustrate the overview of actual aircraft noise exposure to people living underneath the flight path. The datasets will be useful for social surveys to draw the dose-response relationship curve, validation of noise prediction studies, and noise management plans around the airport before the second phase expansion. This study proposes a post-processing tool for extracting aircraft noise events due to aircraft flyovers from the measurements. The actual noise exposure levels of aircraft fly-over noise events were reported to reflect the existing noise situation around the airport and contribute to the analysis of other studies conducted during the same period.

Pharmacodynamic differences between racemic ibuprofen and dexibuprofen in murine preclinical study

Non-steroidal anti-inflammatory drugs (NSAIDs) are among the best therapeutic options to treat pain, but their use is restricted by their adverse effects. In this study, the pharmacological profile of ibuprofen and dexibuprofen was evaluated using the murine acetic acid writhing test, and the participation of naltrexone, naltrindole, nor-binaltorphimine, L-NAME, risperidone, and tropisetron in the antinociceptive efficacy. Antinociception was assessed by dose–response curves to ibuprofen and dexibuprofen before and after the i.p. administration of 1,0 mg/kg of naltrexone, or naltrindole, or norbinaltorphimine, or 5 mg/kg of L-NAME, 0,5 mg/kg of risperidone, or tropisetron in the murine assay. Results are presented as means ± SEM and differences calculated by one-way ANOVA followed by Tukey's post-test. Ibuprofen and dexibuprofen produced a dose-dependent antinociceptive effect with different potency. After pretreatment of mice with naltrexone, naltrindole, nor-binaltorphimine, L-NAME, risperidone, or tropisetron, a significant increase in ibuprofen efficacy was obtained, however, no effect on dexibuprofen activity was obtained. Data demonstrate that ibuprofen and dexibuprofen induce effective antinociception in the acetic acid writhing test, either directly, through COX inhibition, or indirectly, through the modulatory effects of opioid or serotonin, or nitric oxide receptors. These actions are complex since the effects depending on the chemical structure of the NSAID. Thus, a racemic compound (ibuprofen) induces a significant increase in nociceptive efficacy. However, they have no effect on the dextrorotatory enantiomer (dexibuprofen).

Dose error impacts on a collection of realistic dose-response curves based on a NASA sonic boom community noise survey

The National Aeronautics and Space Administration (NASA) plans to conduct surveys of community response to quiet supersonic flight to collect dose-response data for international regulators. Previous models of noise dose versus annoyance response ignored uncertainty in the noise dose experienced by survey respondents. This dose error causes attenuation bias and results in inaccurate dose-response relationships. The impacts of dose errors can be explored by introducing error into the dose estimates of a simulated community noise survey. The simulated population annoyance response is determined by specific noise response characteristics, including the onset of annoyance intercept, rate of increasing annoyance, participant intercept variability, and response rates. This paper explores the effects of dose errors on notional populations created by perturbing the noise response characteristics observed in participants in a recent NASA flight test, Quiet Supersonic Flights 2018 (QSF18). The QSF18-based population parameters were shifted up to ±20% to create the study populations. For the anticipated dose range of the X-59, changes to the onset of annoyance intercept have the greatest impact on erroneous model results. Quantifying point estimate errors illustrates the impact of dose error, with errors up to 14 dB observed for a fixed high annoyance percentage.

In Vitro Characterization of the Bacteria-derived Hypoxia-selective Cytotoxin BE-43547.

Hypoxia-activated pro-drugs, such as TH-302, may kill hypoxic treatment-resistant tumor cells, but have failed in clinical trials. This may be related to variable levels of drug-activating reductases. Compounds such as bacteria-derived BE-43547, which target hypoxic cells independently of reductases, may be beneficial. This study characterized the in vitro potency and hypoxia selectivity of BE-43547 and TH-302. Tumor cells were exposed to different oxygenation levels in the presence/absence of drug, and survival was quantified using total cell number (BE-43547) or clonogenic survival (BE-43547 and TH-302) assays. Half-maximal inhibitory concentration (IC50) values and the hypoxia-cytotoxicity-ratio (HCR: normoxic IC50/hypoxic IC50) were determined from dose-response curves. Finally, both drugs were tested in spheroids exposed to 20% or 0% O2 for 24 h followed by assessment of clonogenic survival. BE-43547 was highly potent and displayed little inter-cell line variability. Strongly enhanced cytotoxicity was observed under oxygen-restricted conditions with HCR's of ~100 and ~20 after 24 h of treatment with 0 or 0.5% O2, respectively. Reducing treatment time somewhat reduced hypoxia selectivity. Hypoxia selectivity was observed regardless of whether the drug was added before or during the hypoxic challenge. TH-302 IC50 values varied 10-fold under oxic conditions, whereas those of the anoxic-to-normoxic HCR varied from 15 to 88. Both BE-43547 and TH-302 were unable to completely sterilize anoxic incubated spheroids. BE-43547 is highly hypoxia-selective, and unlike TH-302, displayed minimal variability between cell lines, suggesting that BE-43547 targets a fundamental feature/target that is only present, or of survival importance, during hypoxia. Spheroid experiments suggested inadequate tissue penetrability, which may be overcome by designing novel drug analogs.

How Much Furosemide Should be Administered to Prevent Transfusion-Associated Circulatory Overload? Results of a Dose-Finding Study

BACKGROUND: Transfusion-associated circulatory overload (TACO) is a common but under-reported complication of transfusion therapy, and results in significant morbidity and mortality. Pre-transfusion furosemide has been proposed as a mitigating strategy but has not received widespread endorsement, in part due to uncertainty regarding safe and effective dosing. An observational study was therefore conducted with the goal of creating a reliable dose-response curve in patients at high risk of TACO, using the statistical technique of Multiple Comparisons Procedure with Modelling (MCP-MOD). METHODS: Eligible patients were identified by screening bolus intravenous (IV) furosemide orders at two large academic hospitals. Eligibility criteria were selected to mimic those of a planned randomized controlled trial (RCT) of pre-transfusion furosemide for the prevention of TACO. Inclusion criteria were inpatients 50 years of age or older. Exclusion criteria were active bleeding, hemodynamic instability, glomerular filtration rate (GFR) < 30mL/min or need for dialysis, diuretic therapy (other than furosemide) administered less than 24 hours, furosemide (either IV or PO) administered less than 12 hours, or exogenous albumin administered less than 8 hours prior to index furosemide therapy. The primary outcome measure was the total volume of urine output in the six hours following IV furosemide administration. MCP-MOD analysis was performed after every 50 patients and continued until a weight-adjusted dose-response curve could be established with 100 mL precision, using the following covariates: age, sex, mean arterial pressure (MAP), serum albumin, GFR and history of chronic furosemide use. RESULTS: A total of 149 participants (53 female and 96 male) were enrolled over 2 years. As too few patients received doses exceeding 0.6 mg/kg to allow for a precise dose-response curve, only 132 patients receiving weight-adjusted doses ranging from 0.1 to 0.6 mg/kg were included in the MCP-MOD analysis. The characteristics of these patients are shown in Table 1. The dose-response curve formula that was derived from these patients was: Urine Output at 6 hours (mL) = 717.21 + 191.33 ln(dose (mg)/weight (kg)) + 0.73 × Age (years) + 103.37 × Sex (M=1/F=0) + 1.63 × GFR (ml/min) + 5.45 × MAP (mmHg) - 9.56 × albumin (g/L) - 62.31 × chronic diuretic use (Y=1/N-0) The curve with accompanying 95% confidence intervals is shown in Figure 1 This formula suggests that in an 80 kg patient with a GFR of 75 mL/min, a MAP of 85 mmHg, albumin of 40 g/L and no history of chronic diuretic use, 10 mg of IV furosemide should produce a diuresis of approximately 600 mL, sufficient to prevent circulatory overload from the transfusion of 1 unit of RBCs. A dose of 20 mg would be required to achieve a similar diuresis in patients with either a MAP < 60 mmHg, or a combination of both a GFR < 45 mL/min and a history of chronic diuretic use. CONCLUSIONS: A statistically precise dose-response curve for IV furosemide is possible with the inclusion of relevant clinical covariates, and can be used to guide dosing decisions in patients at risk of transfusion-associated circulatory overload. This formula derived from this study will inform the dosing recommendations of a planned RCT: Transfusion-Associated Circulatory Overload: Best Eliminated by Lasix® (TACO-BEL).

Drug Response Profiling of Childhood T-Cell Acute Lymphoblastic Leukemia to Inform Functional Precision Medicine

Background/Aims: Despite the advancement in risk-directed, chemotherapy-based protocols, the prognosis of children with T-cell acute lymphoblastic leukemia (T-ALL) especially for those who were refractory to existing treatments or relapsed from remissions remains poor. The complexity of mutational landscape and lack of actionable genomic lesions collectively pose significant challenges for implementing targeted therapies based solely on genomics. To address this issue, this study seeks to utilize drug sensitivity and resistance test (DSRT) to directly assess the ex vivo response of primary T-lymphoblasts to a portfolio of targeted agents, aiming to establish its drug sensitivity profile and identify drug candidates of immediate clinical relevance to tailor functional precision medicine. Methods: A serum-free, mesenchymal stem cell (MSC)-based culture system was adopted to perform ex vivo DSRT on primary or patient-derived xenograft (PDX) samples of pediatric T-ALL. The latter was generated by transplanting NSG mice with paucicellular specimens with splenocytes collected at leukemia engraftment as the sources of patient-derived T-lymphoblasts. The 42-drug panel consisted of 35 targeted agents (1 in Phase I trials, 6 in Phase II trials, 6 in Phase III trials, 22 FDA-approved) and 7 standard chemotherapeutics. The DSRT tested six serial drug concentrations ranging from 0.1 to 10,000 nM. After 96 hours of co-culturing with MSCs, T lymphoblasts were identified by CD7 staining, while apoptotic cells were characterized by Annexin V/7-AAD. The half maximal inhibitory concentration (IC50) of individual drugs were curated from the dose-response curves. Unsupervised clustering was performed to map the overall drug response pattern. A case series of relapsed or refractory T-ALL were managed with targeted therapies guided by drug profiling results and evaluated for clinical responses. Results: DSRT was conducted on 19 primary and 14 PDX pediatric T-ALL samples from 20 patients. For primary samples, two had been tested for 42 drugs, ten were tested for 16 drugs and the rest underwent testing with varying numbers of drugs depending on cellularity and sample quality. All PDX samples were subjected to testing for 42 drugs. PDX samples retained the original pattern of drug response in matched patient materials (R 2 = 0.77-0.94, 8 paired samples). Unsupervised clustering identified 5 distinct groups of drug response: (i) highly active compounds with median IC50 <10 nM (4 drugs including the proteasome inhibitors bortezomib and carfizomib, BCL2 inhibitor navitoclax and HDAC inhibitor panobinostat); (ii) generally active compounds (median IC50 <230 nM, 10 drugs), including BCL2 inhibitors, MCL1 inhibitors, JAK2/3 inhibitors and MDM2 inhibitors; (iii) compounds with bimodal activities (wide IC50 ranges, 4 drugs), including dasatinib (tyrosine kinase inhibitor), sirolimus and everolimus (mTOR inhibitors) and standard chemotherapeutics; (iv) generally inactive drugs with sporadic exceptions (12 drugs); and (v) completely inactive compounds (12 drugs). Four children with T-ALL received treatment according to drug profiling results. Two patients were in first relapse, one in third relapse and one in a non-remission state. Three of these patients received bortezomib treatment, while the fourth patient received venetoclax. All patients responded to the treatments with complete or partial remission. One patient was bridged to allogeneic stem cell transplantation, and two were bridged to CD7-directed CAR therapies. Conclusions: Our study unveiled a pediatric-specific drug response profile for T-ALL and highlighted opportunistic drug candidates with clinical relevance. The successful implementation of functional precision medicine in our clinic elucidated its practicality and potential to enable personalized treatment for high-risk pediatric T-ALL.

Exploring the Mechanisms of Venetoclax Resistance Via Drug Screening and Genome-Wide CRISPR Screening

Introduction: Combining Venetoclax, a selective BCL-2 inhibitor, with hypomethylating agents has revolutionized frontline acute myeloid leukemia (AML) treatment. Despite its success, the occurrence of resistance remains a significant concern, limiting the range of available treatment alternatives. Little is known about the mechanism by which the cells escape the treatment. Recent studies have observed MCL1 upregulation in AML cells to promote resistance, and the rationale to use an MCL1 inhibitor became attractive. Although MCL1 inhibitors work exceptionally well in vitro their clinical trials were unsuccessful due to their toxicity. Here, we aim to identify other genes responsible for acquiring resistance and tackle the problem through screening for clinically approved drugs to rapidly benefit the patients with our study. Methods: First, we generated a model of the venetoclax resistance from 3 AML cell lines: MOLM-13, HL-60 and MV4-11. Resistance was achieved through chronic administration of the compound, inhibiting cell viability to 80-90% in multiple rounds by gradually increasing concentrations until the cells developed resistance. We have then characterized these Venetoclax-resistant (VeR) cells using quantitative RT-PCR and RNA sequencing to reveal the mechanisms behind their resistance. VeR cell lines were subjected to drug screening with a library of 859 FDA and EMA-approved compounds with various clinical indications. Cell viability in response to the drug library was assessed after 72h by Cell-Titer Glo and evaluated against the wild-type counterparts. Top-performing compounds were validated in dose-response curves, and synergy scores were identified from combinational treatments. Simultaneously, we performed genome-wide CRISPR/Cas9 knockout screening using Brunello CRISPR knockout library on both MOLM-13 wild-type (WT) and VeR cell lines to identify genes that maintain the resistance to venetoclax and genes that may potentially synergize with targeting BCL-2. Results: Results from the CRISPR screen pointed out multiple genes whose loss of function may contribute to venetoclax resistance in MOLM-13 cells, among which BAX, NOXA, ELAVL1 and TP53 ranked as top hits. On the contrary, we also identified several genes whose loss of function sensitized the cells to venetoclax (e.g., MCL1, OPA1, CDK2, MCAT), suggesting them to be promising therapeutic targets. Among these hits, CDK2 correlates with our drug screening data indicating Flavopiridol as an effective compound in treating VeR cell lines. Moreover, we confirmed the elevated MCL1 gene expression for the MOLM-13 VeR cell line through RNAseq (and qPCR) and detected an altered expression of genes mainly in metabolic pathways and PI3K-Akt signaling pathway via differential gene expression analysis. Our drug screening results revealed overall high efficacy of DNA-damaging agents, proteasome and HDAC inhibitors for both WT and VeR cell lines. We also included an MCL1 inhibitor as a positive control in our drug library and confirmed a synergistic effect when combined with venetoclax. Conclusion: Integrating a range of screening approaches may lead to discovering previously unknown therapeutic targets for venetoclax-resistant AML. Our screens confirmed previously described involvement of the apoptotic pathway genes in venetoclax resistance as well as identified some novel promising targets. Our ongoing efforts involve thorough validations of the identified hits from CRISPR and drug screening to enhance the reliability and translatability of the results. This project was partly supported by grants MUNI/A/1330/2021, MUNI/A/1419/2021 OPRDE (No.CZ.02.2.69/0.0/0.0/19_073/0016943) and project NICR (EU program EXCELES, No. LX22NPO5102).

Novel Insights in TKI Resistance in BCR:: ABL1-Positive B-Cell Acute Lymphoblastic Leukemia Beyond Kinase Domain Mutations

Background and Aim Despite the introduction of tyrosine kinase inhibitors (TKIs), approximately 30% of newly diagnosed pediatric and adult BCR:: ABL1-positive B-cell acute lymphoblastic leukemia (B-ALL) patients experience relapsed or refractory disease (Biondi et al, Lancet Haematology 2018; Bassan et al, Journal of Clinical Oncology 2010). ABL1 kinase domain mutations, hindering TKI binding, explain part of the relapses and are acquired during TKI therapy but are rarely detected in newly diagnosed BCR:: ABL1-positive B-ALL patients (Baer et al, Haematologica 2022). Intrinsic TKI resistance mechanisms in BCR:: ABL1-positive B-ALL present prior to TKI treatment are less well documented. Therefore, we aimed to identify potential biomarkers for TKI sensitivity at diagnosis using primary patient samples and newly generated cell lines made from patient-derived xenografts (PDX cell lines). Patients and Methods Leukemic cells from diagnosis of BCR:: ABL1-positive B-ALL patients without ABL1-kinase domain mutations were used to measure cell survival in an ex vivo co-culture assay after imatinib (1.95-125 μM), dasatinib (1.95-125 nM), or bosutinib (0.016-2 μM) exposure. TKI sensitivity was assessed using the area under the dose-response curve (AUC), with <80 arbitrary units (AU) classified as imatinib-sensitive, 80-120 AU as intermediate, and >120 AU as resistant. Multiplex ligation-dependent probe amplification, whole exome sequencing, and RNA sequencing were performed on diagnostic material if available. ABL1 phosphorylation and expression levels were measured using (phospho)flow cytometry in patient-derived xenografts from diagnostic samples and (PDX) cell lines. Results We determined ex vivo sensitivity to imatinib, dasatinib, and bosutinib in 32 pediatric and 19 adult BCR:: ABL1-positive B-ALL patient samples. We observed a substantial variability in ex vivo imatinib response in pediatric and adult patient samples (median AUC: 98 AU; range: 16 to 281 AU). The sensitivity towards imatinib strongly correlated with the sensitivity towards second-generation TKIs dasatinib and bosutinib (rho = 0.84; p < 0.001 and rho= 0.89; p <0.001). Moreover, imatinib's AUC of samples derived from newly diagnosed patients who relapsed after TKI treatment was higher compared with those derived from patients who did not suffer from a relapse (median AUC 123 vs 84 AU; p = 0.03). We observed that an increase in ex vivo imatinib resistance was correlated with a decreased amount of phosphorylated ABL1 protein (rho=-0.92; p=0.0013) and total ABL1 protein (rho=-0.82; p=0.01) in untreated, newly diagnosed samples. Ex vivo exposure to imatinib reduced the ABL1 phosphorylation levels in both sensitive and resistant samples but resistant cells exhibited lower levels of (p)ABL1 protein, indicating reduced dependence on BCR-ABL1 signaling. The ex vivo imatinib resistance in samples carrying deletions in B-cell development genes IKZF1 and/or PAX5 was higher than in samples without these deletions (median AUC: 122 versus 80 AU; p=0.01). In addition, one imatinib-resistant patient had a p.His1038Arg mutation in ZEB2, which is recurrent in B-ALL and this gene has also been described as B-cell development gene (Studd et al, Blood Cancer Journal 2021). Interestingly, another resistant patient had a mutation in SETD2,a gene previously associated with ex vivo TKI resistance in chronic myeloid leukemia cell lines (Sheng et al, Cell Proliferation 2019). Other secondary lesions included SH2B3 inactivation lesions and CRLF2 rearrangements. These lesions were present in intermediately sensitive and resistant BCR:: ABL1-positive samples and are expected to lead to increased JAK/STAT signaling, potentially serving as a resistance mechanism for TKI. Conclusion Our study revealed that the amount of total and phosphorylated ABL1 protein was correlated with ex vivo TKI sensitivity. Newly diagnosed TKI-resistant BCR:: ABL1 samples were further characterized by somatic lesions in B-cell development genes including IKZF1. These results link the known poor prognostic value of IKZF1 deletions in BCR:: ABL1-positive B-ALL patients (van der Veer et al. Blood 2014; Fedullo et al, Haematologica 2019) with intrinsic mechanisms of TKI resistance other than tyrosine kinase domain mutations.

The Novel Profiling Relative Inhibition Simultaneously in Mixtures (PRISM) Platform Identifies Synergistic Activity of Lanraplenib and Ruxolitinib in Hematological Malignancies

Background: Investigation of drug combinations across different contexts can provide useful insights on the anti-cancer mechanism and can ultimately lead to new treatments. However, conventional drug combination screening methods are limited by throughput. Efforts to systematically identify the most effective active combinations and the optimal molecular contexts in a high throughput screening (HTS) format could greatly accelerate the development of combination treatments. Spleen Tyrosine Kinase, SYK, is a non-receptor tyrosine kinase known to regulate intracellular signaling, including FLT3, AKT/mTOR and STAT5 pathways, via its immunoreceptor tyrosine-based activation motif (ITAM). Deregulated SYK signaling has been reported to play a central role in pathogenesis of allergic and autoimmune diseases as well as hematological malignancies. Lanraplenib (LANRA) is a next-generation SYK inhibitor currently being evaluated in combination with gilteritinib, a FLT3 inhibitor, in patients with relapsed or refractory (R/R) FLT3-mutated acute myeloid leukemia (AML) (NCT05028751). Given its critical role in intracellular signaling and interaction with receptor tyrosine kinases(RTKs), we hypothesized that LANRA could synergize with ruxolitinib, a JAK inhibitor. To address this hypothesis, we performed a high throughput drug combination screen using the Broad Institute's Profiling Relative Inhibition Simultaneously in Mixtures (PRISM) platform, which enables rapid screening of thousands of compounds in a 930-cell line panel across 45 different lineages. Methods: In this study, we performed a combination screen using the PRISM platform with ruxolitinib as the test compound, with a 10 μM top dose (7 dose concentrations with 3-fold dilutions), in combination with LANRA at two anchor doses, 2 μM and 10 μM. PRISM is a pooled, multiplexed cell viability assay that provides 7-point dose response curves, IC 50, AUC values, and relative abundance of unique cell line barcodes. To understand the drug synergy landscape across different lineages, we developed a bioinformatics pipeline which uses PRISM viability data to calculate synergy scores across all the cell lines and drug combinations. Secondary validation studies of the combinations used CTG viability measurements. Phospho-SYK expression was evaluated in archival formalin-fixed paraffin-embedded (FFPE) bone marrow biopsies from patients with myeloid proliferative neoplasm (MPN) by immunohistochemistry (IHC). RNA-seq was performed to evaluate differential changes in gene expression in response to LANRA. Gene set enrichment analysis (GSEA) was performed to evaluate perturbation in leukemogenic signaling pathways. Results: In the PRISM cell line panel, ruxolitinib in combination with LANRA demonstrated synergistic activity in hematological malignancy cell lines. Among AML cell lines in the panel, OCIAML5, OCIM1, HL60, HEL, EOL1, MONOMAC6, NB4, U937, PL21, and TF1 showed the highest synergy scores. The most sensitive cell lines to the combination showed up-regulation of JAK-STAT and inflammatory signaling pathways in a gene set enrichment analysis (GSEA) prior to treatment. Consistent with this, phosphorylated SYK was associated with inflammatory megakaryocytes and fibrosis in primary samples from patients with MPN. LANRA down-regulated JAK-STAT signaling in a reporter cell line and repressed gene expression associated with dysregulated inflammatory pathways in AML cells. Additionally, the combination of LANRA and ruxolitinib showed synergistic antiproliferative activity across a broad range of concentrations in FLT3-ITD primary AML cells and a panel of hematological malignancy cell lines, confirming the PRISM results. Conclusion: These studies demonstrate the utility of PRISM as a platform to rapidly identify rational combination agents. Importantly, LANRA is effective in combination with ruxolitinib in AML and other hematological malignancy preclinical models. This finding is consistent with the observation that SYK can regulate STAT signaling and cooperate with other RTKs like FLT3. Given the central role of SYK in regulation of oncogenic and inflammatory signaling, SYK inhibition with LANRA in combination with ruxolitinib may be a promising strategy for patients with myeloid malignancies.

Computational Discovery and Validation of NAD+ Biosynthesis As Unique Vulnerability in B-Lymphoid Malignancies

Background and Significance: Glucocorticoids have been a mainstay in virtually all therapy regimens for B-lymphoid malignancies since the 1950s. Glucocorticoids selectively kill B-cell leukemia and lymphoma cells but have very limited effects on myeloid and epithelial cells. Likewise, inhibitors of B-cell signaling (e.g. ibrutinib) and selective depletion of B-cells (e.g. rituximab, CD19 CAR-T cells) have achieved important progress and are well tolerated. Drug discovery tool for selective targeting of B-cell malignancies: For these reasons, we developed an interactive computational tool ( lymphoblasts.org) to identify novel B-cell selective vulnerabilities based on integrated genetic and pharmacological compound screens ( Figure 1). Compound screening data from CTD and GDSC databases were reprocessed by fitting 4-parameter log-logistic dose response curves. Differential gene-dependency was also tested using DEMETER2 and Chronos scores, from Project Achilles RNAi and CRISPR datasets, respectively. To prioritize cell-type specific targets, compounds were ranked by average differential rank percentile of both compound-sensitivity and target-gene dependency. NAD+ biosynthesis as unique B-lymphoid vulnerability: Integration of genetic RNAi and CRISPR-based sensitivity data identified NMNAT1 and NAMPT as top-ranking B-cell selective targets that regulate NAD+ salvage and biosynthesis in the nucleus ( Figure 1). NAMPT, was previously identified as “pre-B-cell colony enhancing factor” and functions as rate-limiting enzyme in the salvage biosynthesis pathway of NAD+, which is critical for cellular energy metabolism. These genetic results were further corroborated by the identification of four small molecule inhibitors of NAMPT, with a striking B-cell-selective sensitivity profile. Compared to a panel of 1,078 myeloid leukemia and solid tumor cell lines, B-cell leukemia and lymphoma cell lines (n=98) were dramatically more sensitive to NAMPT inhibitors, including STF31, CAY10618, GMX1778 and Daporinad ( Figure 1). To verify these results based on the DepMap dataset and our drug discovery tool (lymphoblasts.org), we studied the effects of seven NAMPT inhibitors (CAY10618, FK866, GMX1778, OT82, STF118804, STF31 and KPT9274) on a panel of myeloid leukemia and solid tumor cell lines as well as PDX from patients with B-ALL and mantle cell lymphoma. Interestingly, three of these NAMPT inhibitors have demonstrated favorable PK/PD and safety profiles (NCT04281420, NCT04914845, NCT03921879). Genetic modeling of NAMPT-deficiency in B-cell malignancies. To model consequences of Nampt deletion in B-ALL, we transformed pre-B cells from Namptfl/fl mice with BCR-ABL1 and NRAS G12D oncogenes. Deletion of Nampt subverted competitive fitness of B-ALL cells, loss of colony-forming ability and cell cycle arrest. Additionally, we developed a conditional mouse model ( Namptfl/fl x Mb1-Cre) for B-cell-specific Nampt deletion, which resulted near-complete ablation of B-lymphopoiesis. Moreover, in vitro inducible deletion of Nampt caused rapid depletion and reduced colony formation in cultured bone marrow pre-B cells. Deletion of Nampt profoundly affected B-cell energy metabolism, significantly reduced FCCP-stimulated OCR and resulted in cell death owing to energy stress. Metabolomic studies of genetic Nampt-deletion using mass spectrometry revealed depletion of metabolites involved in amino acid metabolism, purine/pyrimidine metabolism, and the TCA cycle. Notably, phosphoserine was among the top-ranked downregulated metabolites, suggesting the involvement of one carbon pathway disruption in mediating energy crisis upon Nampt-deletion in B-ALL cells. Conclusions: These findings highlight that the classical ‘glucocorticoid paradigm’ can be extended to discover novel vulnerabilities, including NAD+ biosynthesis. Computational integration of genetic and pharmacological compound screens was particularly powerful in identifying previously unrecognized opportunities to leverage selective vulnerabilities of B-lymphoid leukemia and lymphoma. Our genetic studies corroborate the unique role of NAMPT and NMNAT1 in B-cell development and metabolic regulation. Further studies will focus on efforts to repurpose three clinically approved NAMPT small molecule inhibitors for the treatment of refractory B-cell malignancies.

Evaluation of the cytotoxicity, antioxidant activity, and molecular docking of biogenic zinc oxide nanoparticles derived from pumpkin seeds.

This study aimed to investigate the characterization of zinc oxide nanoparticles (ZnONPs) produced from Cucurbita pepo L. (pumpkin seeds) and their selective cytotoxic effectiveness on human colon cancer cells (HCT 116) and African Green Monkey Kidney, Vero cells. The study also investigated the antioxidant activity of ZnONPs. The study also examined ZnONPs' antioxidant properties. This was motivated by the limited research on the comparative cytotoxic effects of ZnO NPs on normal and HCT116 cells. The ZnO NPs were characterized using Fourier-transform infrared spectroscopy (FTIR), Thermogravimetric Analysis (TGA), Transmission Electron Microscope/Selected Area Electron Diffraction (TEM/SAED), and Scanning Electron Microscope-Energy Dispersive X-ray (SEM-EDX) for determination of chemical fingerprinting, heat stability, size, and morphology of the elements, respectively. Based on the results, ZnO NPs from pumpkins were found to be less than 5 μm and agglomerates in nature. Furthermore, the ZnO NPs fingerprinting and SEM-EDX element analysis were similar to previous literature, suggesting the sample was proven as ZnO NPs. The ZnO NPs also stable at a temperature of 380°C indicating that the green material is quite robust at 60-400°C. The cell viability of Vero cells and HCT 116 cell line were measured at two different time points (24 and 48 h) to assess the cytotoxicity effects of ZnO NP on these cells using AlamarBlue assay. Cytotoxic results have shown that ZnO NPs did not inhibit Vero cells but were slightly toxic to cancer cells, with a dose-response curve IC50 = ~409.7 μg/mL. This green synthesis of ZnO NPs was found to be non-toxic to normal cells but has a slight cytotoxicity effect on HCT 116 cells. A theoretical study used molecular docking to investigate nanoparticle interaction with cyclin-dependent kinase 2 (CDK2), exploring its mechanism in inhibiting CDK2's role in cancer. Further study should be carried out to determine suitable concentrations for cytotoxicity studies. Additionally, DPPH has a significant antioxidant capacity, with an IC50 of 142.857 μg/mL. RESEARCH HIGHLIGHTS: Pumpkin seed extracts facilitated a rapid, high-yielding, and environmentally friendly synthesis of ZnO nanoparticles. Spectrophotometric analysis was used to investigate the optical properties, scalability, size, shape, dispersity, and stability of ZnO NPs. The cytotoxicity of ZnO NPs on Vero and HCT 116 cells was assessed, showing no inhibition of Vero cells and cytotoxicity of cancer cells. The DPPH assay was also used to investigate the antioxidant potential of biogenic nanoparticles. A molecular docking study was performed to investigate the interaction of ZnO NPs with CDK2 and to explore the mechanism by which they inhibit CDK2's role in cancer.

Enriched Signalling Pathways in Venetoclax-Relapsed Chronic Lymphocytic Leukemia (CLL) Cells and Targeting Using a Protac-Based Bcl-2/Bcl-Xl Degrader

Venetoclax is a specific inhibitor of Bcl-2, the key protein which protects CLL cells from intrinsic apoptosis, whereas the Bruton's Tyrosine Kinase (BTK) inhibitor ibrutinib kills CLL cells via blockade of B-cell receptor (BCR) signalling. Unfortunately, some patients develop treatment resistance after receiving these agents in combination or sequentially. We aimed to evaluate whether CLL cells collected upon venetoclax and ibrutinib relapse retain survival dependency on alternative targets of the BCR pathway and Bcl-2 family, and to propose new vulnerabilities based on gene expression changes. Using single cell RNA sequencing (sc-RNA-seq), we analyzed paired samples from a CLL patient collected at baseline and at relapse, which occurred after cessation of fixed-duration treatment with venetoclax and ibrutinib. The proportion of cells in each CLL cluster was vastly altered at relapse. Cluster 3 represented 4% of CLL cells at baseline versus 36% at relapse and had a high BCR score, including high expression of MAP3K8 FOSB8 and NFKB1 genes. Moreover, at relapse, it contained more CLL cells expressing transcripts from the Bcl-2 family of anti-apoptotic proteins, including Bcl-2 and Bcl-xL. We also integrated sc-RNA-seq data from three primary CLL samples collected at relapse after venetoclax and ibrutinib treatment. The four clusters of CLL cells identified were enriched for BCR, interleukin-mediated, ErbB and the Fc epsilon receptor I signalling pathways. Importantly, most of the relapsed CLL cells which expressed high levels of Mcl-1 or Bcl-xL also expressed Bcl-2, suggesting that these resistant CLL cells became dependent on various anti-apoptotic proteins for survival. These observations led us to test Bcl-2/Bcl-xL targeting PROTACs PZ18753b and WH25244 in venetoclax-resistant CLL. These PROTACs were derived from navitoclax (Bcl-2/Bcl-xL dual inhibitor) linked to a VHL E3 ligase ligand to target Bcl-2 and Bcl-xL for degradation, with improved specificity to cancer cells while sparing platelets. We used three models of venetoclax-resistant CLL: (1) OSU-CLL cells expressing mutant Bcl-2 (G101V, F104L, R107-110dup or A113G), (2) treatment-naïve primary CLL cells co-cultured with NK.Tert stromal cells for 24h (CLL+stroma), and (3) primary CLL cells collected at venetoclax relapse. CLL cells were treated with venetoclax, navitoclax, PZ18753b, WH25244, or negative controls (NC) lacking VHL recruitment, PZ18753b-NC or WH25244-NC. Protein degradation was evaluated by Western Blot; viability, by Cell Titer Glo; and apoptosis, by flow cytometric detection of active BAX and BAK, mitochondrial cytochrome C, and AnnexinV binding. WH25244 was superior to venetoclax at targeting OSU-CLL cells expressing either wildtype or mutant Bcl-2, upon 72h treatment. Cell death by WH25244 was preceded by degradation of Bcl-xL and partial degradation of Bcl-2, despite presence of Bcl-2 mutations. In treatment-naïve primary CLL cells, Bcl-xL degradation by PZ18753b and WH25244 proceeded in a dose-, time- and VHL-dependent manner. The potency of these PROTACs fell in between that of venetoclax and navitoclax, with mitochondrial apoptosis occurring at low nanomolar concentrations known to spare human platelets. Stepwise, activation of BAK and BAX, and cytochrome C release were evident at 14h of treatment, followed by phosphatidyl serine exposure at 18h of treatment. A rightward shift in the dose-response curves of PZ18753b (16-fold) and WH25244 (8-fold) was noted for CLL+stroma cells relative to paired CLL cells treated in suspension. This reduction in potency was associated with (1) uptake of the PROTACs by NK.Tert cells, since Bcl-xL degradation was noted in stromal cells yet without evident toxicity, and (2) up to 2.5-fold increased levels of Bcl-xL in CLL+stroma, which shifted the dose-dependent degradation of Bcl-xL. Finally, 2 out of 3 venetoclax-relapsed primary CLL samples showed more active BAK and cytochrome C release upon treatment with WH25244 than venetoclax, evaluated at 10 nM. In conclusion, WH25244 is a PROTAC-based Bcl-2/Bcl-xL degrader with the potential to overcome venetoclax-resistant CLL dependent on Bcl-xL and mutant Bcl-2. Relative to its precursor, navitoclax, it shows increased potency against CLL cells and decreased toxicity against platelets in vitro, due to its VHL-dependent activity and minimal expression of VHL in platelets.

Sensitivity of dominant UKPhytophthora infestans genotypes to a range of fungicide active ingredients

AbstractLate blight, caused by Phytophthora infestans, is a serious disease of potatoes worldwide and is predominantly controlled by repeated prophylactic use of fungicides throughout the growing season. Effective blight management and integrated pest management strategies rely on knowledge of the efficacy of available fungicides to control contemporary genotypes of P. infestans. Between 2019 and 2022, representative isolates of the newer dominant genotypes EU36, EU37 and the older dominant genotype EU6 were sampled from GB crops and tested for sensitivity to seven commonly used fungicide active ingredients (cyazofamid, fluopicolide, mandipropamid, propamocarb, oxathiapiprolin, amisulbrom and mancozeb) used preventatively in detached leaf tests and zoospore motility assays where appropriate. Dose–response curves based on lesion area (mm2) were constructed, and EC50 values calculated. Isolates of P. infestans genotype EU37 were insensitive to fluazinam as previously reported. No insensitivity, or progression towards resistance over time, to any of the other fungicides was observed in any isolates of the genotypes tested, and EC50 values were generally in line with previous testing. Zoospore motility tests with fluopicolide revealed significantly higher MIC values for isolates of genotype EU36 compared with EU37 and EU6, which, whilst not indicative of resistance at the low concentrations of active ingredient tested, may be a factor contributing to the overall dominance of that genotype in the wider population of P. infestans. The evidence suggests that the fungicides tested, with the exception of fluazinam and EU37, are effective for the control of P. infestans genotypes dominant in the GB population up until 2022.

Acetylcholinesterase Inhibition in Rats and Humans Following Acute Fenitrothion Exposure Predicted by Physiologically Based Kinetic Modeling-Facilitated Quantitative In Vitro to In Vivo Extrapolation.

Worldwide use of organophosphate pesticides as agricultural chemicals aims to maintain a stable food supply, while their toxicity remains a major public health concern. A common mechanism of acute neurotoxicity following organophosphate pesticide exposure is the inhibition of acetylcholinesterase (AChE). To support Next Generation Risk Assessment for public health upon acute neurotoxicity induced by organophosphate pesticides, physiologically based kinetic (PBK) modeling-facilitated quantitative in vitro to in vivo extrapolation (QIVIVE) approach was employed in this study, with fenitrothion (FNT) as an exemplary organophosphate pesticide. Rat and human PBK models were parametrized with data derived from in silico predictions and in vitro incubations. Then, PBK model-based QIVIVE was performed to convert species-specific concentration-dependent AChE inhibition obtained from in vitro blood assays to corresponding in vivo dose-response curves, from which points of departure (PODs) were derived. The obtained values for rats and humans were comparable with reported no-observed-adverse-effect levels (NOAELs). Humans were found to be more susceptible than rats toward erythrocyte AChE inhibition induced by acute FNT exposure due to interspecies differences in toxicokinetics and toxicodynamics. The described approach adequately predicts toxicokinetics and acute toxicity of FNT, providing a proof-of-principle for applying this approach in a 3R-based chemical risk assessment paradigm.

Urinary biomarkers of drinking-water disinfection byproducts in relation to diminished ovarian reserve risk: A case-control study from the TREE cohort

BackgroundDisinfection byproducts (DBPs) as ovarian toxicants have been documented in toxicological studies. However, no human studies have explored the effects of exposure to DBPs on diminished ovarian reserve (DOR). ObjectiveTo assess whether urinary biomarkers of exposure to drinking-water DBPs were associated with DOR risk. MethodsA total of 311 women undergoing assisted reproductive technology were diagnosed with DOR in the Tongji Reproductive and Environmental (TREE) cohort from December 2018 to August 2021. The cases were matched to the controls with normal ovarian reserve function by age in a ratio of 1:1. Urinary trichloroacetic acid (TCAA) and dichloroacetic acid (DCAA) were quantified as biomarkers of drinking-water DBP exposures. The conditional logistic regression and restricted cubic spline (RCS) were used to explore urinary biomarkers of drinking-water DBP exposures in associations with the risk of DOR. ResultsElevated urinary DCAA levels were associated with higher DOR risk [adjusted odds ratio (OR) = 1.87; 95 % confidence interval (CI): 1.16, 3.03 for the highest vs. lowest quartiles; P for trend = 0.016]. The association was confirmed in the RCS model, with a linear dose-response curve (P for overall association = 0.029 and P for non-linear association = 0.708). The subgroup analysis by age and body mass index (BMI) showed that urinary DCAA in association with DOR risk was observed among women ≥35 years old and leaner women (BMI < 24 kg/m2), but the group differences were not statistically significant. Moreover, a U-shaped dose-response curve between urinary TCAA and DOR risk was estimated in the RCS model (P for overall association = 0.011 and P for non-linear association = 0.004). ConclusionsExposure to drinking-water DBPs may contribute to the risk of DOR among women undergoing assisted reproductive technology.

Assessment of the performance of the Ames MPF™ assay: A multicenter collaborative study with six coded chemicals

The Ames MPF™ is a miniaturized, microplate fluctuation format of the Ames test. It is a standardized, commercially available product which can be used to assess mutagenicity in Salmonella and E. coli strains in 384-well plates using a color change-based readout. Several peer-reviewed comparisons of the Ames MPF™ to the Ames test in Petri dishes confirmed its suitability to evaluate the mutagenic potential of a variety of test items. An international multicenter study involving seven laboratories tested six coded chemicals with this assay using five bacterial strains, as recommended by the OECD test guideline 471. The data generated by the participating laboratories was in excellent agreement (93%), and the similarity of their dose response curves, as analyzed with sophisticated statistical approaches further confirmed the suitability of the Ames MPF™ assay as an alternative to the Ames test on agar plates, but with advantages with respect to significantly reduced amount of test substance and S9 requirements, speed, hands-on time and, potentially automation.

A Photo-transfer Thermoluminescence (PTTL) Study of TLD-100 over a Wide Dose Range

Photo-transferred thermoluminescence (PTTL) is defined as the transfer of electrons from deep traps into shallow traps via optical stimulation. The importance of PTTL is that it allows for a second measurement of dose assessments for accuracy in cases such as an erroneous dose evaluation. In this study, the PTTL signal of TLD-100 was investigated in detail for a wide dose range from mGy to Gy. The investigation of PTTL signals in the order of Gy is the main innovation of this study. Based on the results of the low dose measurement (mGy), the PTTL dose-response curve has a significant sublinear characteristic in the order of mGy for the total area condition. Additionally, PTTL signals could not be distinguished from the background signal up to 5mGy. Therefore, the PTTL method can be used by taking into account the sublinear function obtained after 5mGy for the total area. On the other hand, it can be applied to TLD-100 between 0.5mGy and 50mGy using ROI. Based on the high dose measurement results (Gy), the PTTL method can be applied up to 10Gy regardless of the total area and ROI. Therefore, the dose reassessment can be performed with PTTL signal in high dose measurements (Gy) such as in the radiotherapy field. Furthermore, in future studies, heating the dosimeters during UV exposure, predose effect, or subjecting the dosimeters to fast cooling following the annealing process may provide important outputs to obtaining higher PTTL intensity, thus, it may allow measuring lower radiation doses.

Cancer organoid-based diagnosis reactivity prediction (CODRP) index-based anticancer drug sensitivity test in ALK-rearrangement positive non-small cell lung cancer (NSCLC)

BackgroundRecently, cancer organoid-based drug sensitivity tests have been studied to predict patient responses to anticancer drugs. The area under curve (AUC) or IC50 value of the dose-response curve (DRC) is used to differentiate between sensitive and resistant patient‘s groups. This study proposes a multi-parameter analysis method (cancer organoid-based diagnosis reactivity prediction, CODRP) that considers the cancer stage and cancer cell growth rate, which represent the severity of cancer patients, in the sensitivity test.MethodsOn the CODRP platform, patient-derived organoids (PDOs) that recapitulate patients with lung cancer were implemented by applying a mechanical dissociation method capable of high yields and proliferation rates. A disposable nozzle-type cell spotter with efficient high-throughput screening (HTS) has also been developed to dispense a very small number of cells due to limited patient cells. A drug sensitivity test was performed using PDO from the patient tissue and the primary cancer characteristics of PDOs were confirmed by pathological comparision with tissue slides.ResultsThe conventional index of drug sensitivity is the AUC of the DRC. In this study, the CODRP index for drug sensitivity test was proposed through multi-parameter analyses considering cancer cell proliferation rate, the cancer diagnosis stage, and AUC values. We tested PDOs from eight patients with lung cancer to verify the CODRP index. According to the anaplastic lymphoma kinase (ALK) rearrangement status, the conventional AUC index for the three ALK-targeted drugs (crizotinib, alectinib, and brigatinib) did not classify into sensitive and resistant groups. The proposed CODRP index-based drug sensitivity test classified ALK-targeted drug responses according to ALK rearrangement status and was verified to be consistent with the clinical drug treatment response.ConclusionsTherefore, the PDO-based HTS and CODRP index drug sensitivity tests described in this paper may be useful for predicting and analyzing promising anticancer drug efficacy for patients with lung cancer and can be applied to a precision medicine platform.