Search

Nitrosamine drug substance-related impurities (NDSRIs) are a sub-category of N-nitrosamine drug impurities that share structural similarity to the corresponding active pharmaceutical ingredient. The mutagenicity of NDSRIs is poorly understood. We previously tested a series of NDSRIs using the Enhanced Ames Test (EAT). In this follow-up study, we further examined the genotoxicity and mutagenicity of 15 of these NDSRIs in human TK6 cells. Seven EAT-positive NDSRIs, including N-nitroso-nortriptyline, N-nitroso-fluoxetine, N-nitroso-desmethyl-diphenhydramine, N-nitroso-duloxetine, N-nitroso-lorcaserin, N-nitroso-varenicline, and N-nitroso-sertraline, induced concentration-dependent increases in micronuclei after bioactivation with hamster liver S9. These NDSRIs were also mutagenic in the TK and HPRT gene mutation assays, consistent with their positive EAT results. In the presence of hamster liver S9, the eight EAT-negative NDSRIs were negative in the micronucleus assay and negative for mutation induction. Using TK6 cells endogenously expressing a single human cytochrome P450 (CYP), we found that CYP2C19, CYP2B6, CYP2A6, and CYP3A4 are key enzymes activating the genotoxicity and mutagenicity of these NDSRIs. Overall, the hamster S9-mediated TK6 cell mutagenicity results agreed with those observed in the EAT, indicating consistency in the mutagenic responses produced by NDSRIs across different testing systems. These data support the use of EAT for hazard identification and safety assessment of NDSRIs., Competing Interests: Declaration of competing interest This article reflects the views of the authors and does not necessarily reflect those of the U.S. Food and Drug Administration (FDA). Any mention of commercial products is for clarification only and is not intended as approval, endorsement, or recommendation. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

We previously reported that certain sub-regions of the thyA gene of Escherichia coli are more mutable than others when many different mutagens and mutators are analyzed (Mashiach et al., Mutation Research Fundamental Molecular Mechansims of Mutagenesis, 821: 111702, 2021). In this study, we focus on a single mutagen, cisplatin and verify that mutations occur preferentially at specific 3 bp sequences, but only when they appear in certain subregions of the gene. Moreover, we show that hotspots for some premutational lesions are camouflaged by the preferential repair effected by the uvrA,B,C-encoded excision repair system, even when they appear on the same strand. We do this by using a novel reporter gene in E. coli, the tdk gene that codes for thymidine deoxykinase, and we describe some of the advantages of utilizing this detection system., (© 2024 The Author(s). Environmental and Molecular Mutagenesis published by Wiley Periodicals LLC on behalf of Environmental Mutagenesis and Genomics Society.)

Regulatory genetic toxicology testing is essential for identifying potentially mutagenic hazards. Duplex Sequencing (DS) is an error-corrected next-generation sequencing technology that provides substantial advantages for mutation analysis over conventional mutagenicity assays including: improved accuracy of mutation detection, ability to measure changes in mutation spectrum, and applicability across diverse biological models. To apply DS for regulatory toxicology testing, power analyses are required to determine suitable sample sizes and study designs. In this study, we explored study designs to achieve sufficient power for various effect sizes in chemical mutagenicity assessment. We collected data from MutaMouse bone marrow and liver samples that were analyzed by DS using TwinStrand's Mouse Mutagenesis Panel. Average duplex reads achieved in two separates studies on liver and bone marrow were 8.4 × 10 8 (± 7.4 × 10 7 ) and 9.5 × 10 8 (± 1.0 × 10 8 ), respectively. Baseline mean mutation frequencies (MF) were 4.6 × 10 -8 (± 6.7 × 10 -9 ) and 4.6 × 10 -8 (± 1.1 × 10 -8 ), with estimated standard deviations for the animal-to-animal random effect of 0.15 and 0.20, for liver and bone marrow, respectively. We conducted simulation analyses based on these empirically derived parameters. We found that a sample size of four animals per group is sufficient to obtain over 80% power to detect a two-fold change in MF relative to baseline. In addition, we estimated the minimal total number of informative duplex bases sequenced with different sample sizes required to retain power for various effect sizes. Our work provides foundational data for establishing suitable study designs for mutagenicity testing using DS., (© 2024 His Majesty the King in Right of Canada and The Author(s). Environmental and Molecular Mutagenesis published by Wiley Periodicals LLC on behalf of Environmental Mutagenesis and Genomics Society. Reproduced with the permission of the Minister of Health Canada.)

Chlorothalonil (CTL) is a pesticide widely used in Brazil, yet its mutagenic potential is not fully determined. Thus, we assessed the mutagenicity of CTL and its bioactivation metabolites using the somatic mutation and recombination test (SMART) in Drosophila melanogaster, by exposing individuals, with basal and high bioactivation capacities (standard and high bioactivation cross offspring, respectively), from third instar larval to early adult fly stages, to CTL-contaminated substrate (0.25, 1, 10 or 20 μM). This substrate served as food and as physical medium. Increased frequency of large single spots in standard cross flies' wings exposed to 0.25 μM indicates that, if CTL is genotoxic, it may affect Drosophila at early life stages. Since the total spot frequency did not change, CTL cannot be considered mutagenic in SMART. The same long-term exposure design was performed to test whether CTL induces oxidative imbalance in flies with basal (wild-type, WT) or high bioactivation (ORR strain) levels. CTL did not alter reactive oxygen species and antioxidant capacity against peroxyl radicals levels in adult flies. However, lipid peroxidation (LPO) levels were increased in WT male flies exposed to 1 μM CTL. SMART and LPO alterations were observed only in flies with basal bioactivation levels, pointing to direct CTL toxicity to DNA and lipids. Survival, emergence and locomotor behavior were not affected, indicating no bias due to lethality, developmental and behavioral impairment. We suggest that, if related to CTL exposure, DNA and lipid damages may be residual damage of earlier life stages of D. melanogaster., (© 2024 Environmental Mutagenesis and Genomics Society.)

Incense-burning smoke is a deleterious air pollutant that initiates cytotoxic effects by inducing apoptosis in lung epithelial cells and also acts as a risk factor for lung cancers. Auramine, an ingredient of incense smoke, has been implicated in tumor progression and cellular sensitivity in non-small cell lung cancer (NSCLC) towards anti-cancer agents through unclear mechanisms. Tumor protein p53 (TP53)-activated long intergenic non-coding RNA-p21 (lincRNA-p21) undertakes a pivotal role in regulating cell apoptosis and chemosensitivity. TP53 mutations prevalent in 50% of NSCLC, contribute to diminished therapeutic efficacy. However, the influence of auramine on chemotherapy-induced lincRNA-p21 expression and apoptosis in NSCLC with different TP53 genetic statuses remains unexplored. This study disclosed that both wild-type p53 (wtp53) and mutant p53 (mutp53) mediate lincRNA-p21 expression, albeit through distinct promoter enhancers, p53-response element (p53RE) and non-B DNA structure G-quadruplex (GQ), respectively. Intriguingly, auramine functions as an effective stabilizer of the GQ structure, augmenting mutp53-mediated lincRNA-p21 expression and enhancing apoptosis and cellular sensitivity to chemotherapy in mutp53-expressing NSCLC cells. These findings suggest a mechanism by which mutp53, in the presence of auramine, is endowed with tumor-suppressing function akin to wtp53, thereby aiding in combating chemoresistance in NSCLC cells harboring TP53 mutations., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The finding of N-nitrosodiethylamine (NDEA) and N-nitrosodimethylamine (NDMA) in marketed drugs has led to implementation of risk assessment processes intended to limit exposures to the entire class of N-nitrosamines. A critical component of the risk assessment process is establishing exposure limits that are protective of human health. One approach to establishing exposure limits for novel N-nitrosamines is to conduct an in vivo transgenic rodent (TGR) mutation study. Existing regulatory guidance on N-nitrosamines provides decision making criteria based on interpreting in vivo TGR mutation studies as an overall positive or negative. However, point of departure metrics, such as benchmark dose (BMD), can be used to define potency and provide an opportunity to establish relevant exposure limits. This can be achieved through relative potency comparison of novel N-nitrosamines with model N-nitrosamines possessing robust in vivo mutagenicity and carcinogenicity data. The current work adds to the dataset of model N-nitrosamines by providing in vivo TGR mutation data for N-nitrosopiperidine (NPIP). In vivo TGR mutation data was also generated for a novel N-nitrosamine impurity identified in sitagliptin-containing products, 7-nitroso-3-(trifluoromethyl)-5,6,7,8-tetrahydro-[1,2,4]triazolo-[4,3-a]pyrazine (NTTP). Using the relative potency comparison approach, we have demonstrated the safety of NTTP exposures at or above levels of 1500 ng/day., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

The detection of N-nitrosamines in drug products has raised global regulatory interest in recent years due to the carcinogenic potential of some nitrosamines in animals and a need to identify a testing strategy has emerged. Ideally, methods used would allow for the use of quantitative analysis of dose-response data from in vivo genotoxicity assays to determine a compound-specific acceptable intake for novel nitrosamines without sufficient carcinogenicity data. In a previous study we compared the dose-response relationships of N-nitrosodiethylamine (NDEA) in three in vivo genotoxicity endpoints in rats. Here we report a comparison of NDEA's genotoxicity profile in mice. Big Blue® mice were administered NDEA at doses of 0.001, 0.01, 0.1, 1 and 3 mg/kg/day by oral gavage for 28 days followed by 3 days of expression. Statistically significant increases in the NDEA induced mutations were detected by both the transgenic rodent mutation assay (TGR) using the cII endpoint and by duplex sequencing in the liver but not bone marrow of mice. In addition, administration of NDEA for two consecutive days in male C57BL/6N mice caused elevated DNA damage levels in the liver as measured by % tail DNA in comet assay. The benchmark dose (BMD) analysis shows a BMDL 50 of 0.03, 0.04 and 0.72 mg/kg/day for TGR, duplex sequencing and comet endpoints, respectively. Overall, this study demonstrated a similar genotoxicity profile of NDEA between mice and rats and provides a reference that can be used to compare the potential potency of other novel nitrosamines for the induction of gene mutations., (© 2024 The Author(s). Environmental and Molecular Mutagenesis published by Wiley Periodicals LLC on behalf of Environmental Mutagenesis and Genomics Society.)

The human in vitro organotypic air-liquid-interface (ALI) airway tissue model is structurally and functionally similar to the human large airway epithelium and, as a result, is being used increasingly for studying the toxicity of inhaled substances. Our previous research demonstrated that DNA damage and mutagenesis can be detected in human airway tissue models under conditions used to assess general and respiratory toxicity endpoints. Expanding upon our previous proof-of-principle study, human airway epithelial tissue models were treated with 6.25-100 µg/mL ethyl methanesulfonate (EMS) for 28 days, followed by a 28-day recovery period. Mutagenesis was evaluated by Duplex Sequencing (DS), and clonal expansion of bronchial-cancer-specific cancer-driver mutations (CDMs) was investigated by CarcSeq to determine if both mutation-based endpoints can be assessed in the same system. Additionally, DNA damage and tissue-specific responses were analyzed during the treatment and following the recovery period. EMS exposure led to time-dependent increases in mutagenesis over the 28-day treatment period, without expansion of clones containing CDMs; the mutation frequencies remained elevated following the recovery. EMS also produced an increase in DNA damage measured by the CometChip and MultiFlow assays and the elevated levels of DNA damage were reduced (but not eliminated) following the recovery period. Cytotoxicity and most tissue-function changes induced by EMS treatment recovered to control levels, the exception being reduced proliferating cell frequency. Our results indicate that general, respiratory-tissue-specific and genotoxicity endpoints increased with repeat EMS dosing; expansion of CDM clones, however, was not detected using this repeat treatment protocol. DISCLAIMER: This article reflects the views of its authors and does not necessarily reflect those of the U.S. Food and Drug Administration. Any mention of commercial products is for clarification only and is not intended as approval, endorsement, or recommendation., Competing Interests: Declaration of Competing Interest Authors have no conflict of interest, financial or otherwise., (Copyright © 2024. Published by Elsevier B.V.)

Currently, there is no test system, whether in vitro or in vivo, capable of examining all endpoints required for genotoxicity evaluation used in pre-clinical drug safety assessment. The objective of this study was to develop a model which could assess all the required endpoints and possesses robust human metabolic activity, that could be used in a streamlined, animal-free manner. Liver-on-chip (LOC) models have intrinsic human metabolic activity that mimics the in vivo environment, making it a preferred test system. For our assay, the LOC was assembled using primary human hepatocytes or HepaRG cells, in a MPS-T12 plate, maintained under microfluidic flow conditions using the PhysioMimix® Microphysiological System (MPS), and co-cultured with human lymphoblastoid (TK6) cells in transwells. This system allows for interaction between two compartments and for the analysis of three different genotoxic endpoints, i.e. DNA strand breaks (comet assay) in hepatocytes, chromosome loss or damage (micronucleus assay) and mutation (Duplex Sequencing) in TK6 cells. Both compartments were treated at 0, 24 and 45 h with two direct genotoxicants: methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS), and two genotoxicants requiring metabolic activation: benzo[a]pyrene (B[a]P) and cyclophosphamide (CP). Assessment of cytochrome activity, RNA expression, albumin, urea and lactate dehydrogenase production, demonstrated functional metabolic capacities. Genotoxicity responses were observed for all endpoints with MMS and EMS. Increases in the micronucleus and mutations (MF) frequencies were also observed with CP, and %Tail DNA with B[a]P, indicating the metabolic competency of the test system. CP did not exhibit an increase in the %Tail DNA, which is in line with in vivo data. However, B[a]P did not exhibit an increase in the % micronucleus and MF, which might require an optimization of the test system. In conclusion, this proof-of-principle experiment suggests that LOC-MPS technology is a promising tool for in vitro hazard identification genotoxicants., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: T.B.Z, A.B, P.V, C.C.V and J.J.S, declare that they are employees and equity holders at TwinStrand Biosciences, Inc. and are authors on one or more Duplex Sequencing-related patents., (Copyright © 2024 Elsevier B.V. All rights reserved.)

N-Nitrosamines, known as drug impurities and suspected carcinogens, have drawn significant public concern. In response to drug regulatory needs, the European Medicines Agency (EMA) has previously proposed a carcinogenic potency categorization approach based on the N-nitrosamine α-hydroxylation hypothesis, i.e., that N-nitrosamine mutagenicity increases with the number of α-hydrogen atoms. However, this structure-activity relationship has not been fully tested in vivo. NEIPA (N-nitrosoethylisopropylamine) and NDIPA (N-nitrosodiisopropylamine) are small N-Nitrosamines with similar structures, differing in that the former compound has an additional α-hydrogen atom. In this study, NEIPA and NEIPA doses, 25-100 mg/kg, were administered orally to C57BL/6 J mice for seven consecutive days, and their mutation and DNA damage effects were compared. Compared with NDIPA, the mutagenicity and DNA damage potencies of NEIPA (which contains one more α-hydrogen) were much greater. These differences may be related to their distinct metabolic pathways and target organs. This case study confirms the role of α-hydroxyl modification in the mutagenicity of nitrosamines, with oxidation at the α-hydrogen being a crucial step in the formation of mutagens from N-Nitrosamines, and can inform mutagenicity risk assessment and the formulation of regulatory standards for N-nitrosamine impurities., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financialinterestsor personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Molnupiravir, an antiviral medication widely used against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), acts by inducing mutations in the virus genome during replication. Most random mutations are likely to be deleterious to the virus and many will be lethal; thus, molnupiravir-induced elevated mutation rates reduce viral load 1,2 . However, if some patients treated with molnupiravir do not fully clear the SARS-CoV-2 infections, there could be the potential for onward transmission of molnupiravir-mutated viruses. Here we show that SARS-CoV-2 sequencing databases contain extensive evidence of molnupiravir mutagenesis. Using a systematic approach, we find that a specific class of long phylogenetic branches, distinguished by a high proportion of G-to-A and C-to-T mutations, are found almost exclusively in sequences from 2022, after the introduction of molnupiravir treatment, and in countries and age groups with widespread use of the drug. We identify a mutational spectrum, with preferred nucleotide contexts, from viruses in patients known to have been treated with molnupiravir and show that its signature matches that seen in these long branches, in some cases with onward transmission of molnupiravir-derived lineages. Finally, we analyse treatment records to confirm a direct association between these high G-to-A branches and the use of molnupiravir., (© 2023. The Author(s).)

RET fusions occur as a rare mechanism of acquired resistance to osimertinib in patients with EGFR mutation-positive non-small cell lung cancer. Inhibiting RET alongside osimertinib shows promising clinical activity, but innovative approaches are needed to seek regulatory approvals in these rare treatment resistance settings. See related article by Rotow et al., p. 2979., (©2023 American Association for Cancer Research.)

Purpose: Acquired RET fusions have been reported at resistance to treatment with EGFR inhibitors in EGFR-mutant non-small cell lung cancer (NSCLC); however, a multicenter cohort of patients with EGFR-mutant lung cancers treated with osimertinib and selpercatinib for RET fusion-mediated osimertinib resistance has not previously been published., Patients and Methods: Patients who received selpercatinib in combination with osimertinib on a prospective expanded access clinical trial (NCT03906331) and single-patient compassionate use programs across five countries were centrally analyzed. All patients had advanced EGFR-mutant NSCLC with a RET fusion detected from tissue or plasma following osimertinib therapy. Clinicopathologic and outcomes data were collected., Results: Fourteen patients with EGFR-mutant and RET fusion-positive lung cancers who experienced prior progression on osimertinib received osimertinib and selpercatinib. EGFR exon 19 deletions (±T790M, 86%) and non-KIF5B fusions (CCDC6-RET 50%, NCOA4-RET 36%) predominated. Osimertinib 80 mg daily and selpercatinib 80 mg twice daily were the most commonly administered dosages. The response rate, disease control rate, and median treatment duration were 50% [95% confidence interval (CI), 25%-75%, n = 12], 83% (95% CI, 55%-95%), and 7.9 months (range, 0.8-25+), respectively. Resistance was complex, involving EGFR on-target (EGFR C797S), RET on-target (RET G810S), and off-target (EML4-ALK/STRN-ALK, KRAS G12S, BRAF V600E) mechanisms; RET fusion loss; or polyclonal mechanisms., Conclusions: For patients with EGFR-mutant NSCLC with an acquired RET fusion as a mechanism of EGFR inhibitor resistance, the addition of selpercatinib to osimertinib was feasible and safe and offered clinical benefit, supporting the prospective evaluation of this combination. See related commentary by Krebs and Popat, p. 2951., (©2023 American Association for Cancer Research.)

Aims: The purpose of this study was to evaluate the heterogeneities of glutamine metabolism in EGFR-TKI-resistant lung cancer cells and its potential as a therapeutic target., Main Methods: Cell proliferation and cell cycle assays was performed by IncuCyte real-time analysis and flow cytometry, respectively. Tumor growth was assessed in xenografts implanted with HCC827 GR. An isotopologue analysis was conducted by LC-MS/MS using 13 C-(U)-glutamine labeling to determine the amounts of metabolites. Cellular ATP and mitochondrial oxidative phosphorylation were determined by XFp analysis., Key Findings: We found that the cell growth of the two acquired EGFR-TKI-resistant lung cancer cells lines (HCC827 GR and H292 ER) depends on glutamine. In HCC827 GR, glutamine deficiency caused reduced GSH synthesis and, subsequently, enhanced ROS generation relative to their parental cells, HCC827. On the other hand, in H292 ER, glutamine mainly acted as a carbon source for TCA-cycle intermediates, and its depletion led to reduced mitochondrial ATP production. CB-839, a specific GLS inhibitor, inhibited the latter's conversion of glutamine to glutamate and exerted enhanced anti-proliferating effects on the two acquired EGFR-TKI-resistant lung cancer cell lines versus their parental cell lines. Moreover, oral administration of CB-839 significantly suppressed HCC827 GR tumor growth in the xenograft model., Significance: These findings suggest that glutamine dependency in acquired EGFR-TKI-resistant lung cancer is heterogeneous and that inhibition of glutamine metabolism by CB-839 may serve as a therapeutic tool for acquired EGFR-TKI-resistant lung cancer., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The majority of RUNX1 mutations in acute myeloid leukemia (AML) are missense or deletion-truncation and behave as loss-of-function mutations. Following standard therapy, AML patients expressing mtRUNX1 exhibit inferior clinical outcome than those without mutant RUNX1. Studies presented here demonstrate that as compared with AML cells lacking mtRUNX1, their isogenic counterparts harboring mtRUNX1 display impaired ribosomal biogenesis and differentiation, as well as exhibit reduced levels of wild-type RUNX1, PU.1, and c-Myc. Compared with AML cells with only wild-type RUNX1, AML cells expressing mtRUNX1 were also more sensitive to the protein translation inhibitor homoharringtonine (omacetaxine) and BCL2 inhibitor venetoclax. Homoharringtonine treatment repressed enhancers and their BRD4 occupancy and was associated with reduced levels of c-Myc, c-Myb, MCL1, and Bcl-xL. Consistent with this, cotreatment with omacetaxine and venetoclax or BET inhibitor induced synergistic in vitro lethality in AML expressing mtRUNX1. Compared with each agent alone, cotreatment with omacetaxine and venetoclax or BET inhibitor also displayed improved in vivo anti-AML efficacy, associated with improved survival of immune-depleted mice engrafted with AML cells harboring mtRUNX1. These findings highlight superior efficacy of omacetaxine-based combination therapies for AML harboring mtRUNX1., (© 2022 by The American Society of Hematology.)

With the wide clinical use of the third-generation epidermal growth factor receptor (EGFR) inhibitor osimertinib for the treatment of EGFR-mutated non-small cell lung cancer (NSCLC), acquired resistance caused by EGFR C797S tertiary mutation has become a concern. Therefore, fourth-generation EGFR inhibitors that could overcome this mutation have gained increasing attention in recent years. Here, we identified LS-106 as a novel EGFR inhibitor against C797S mutation and evaluated its antitumor activity both in vitro and in vivo. In cell-free assay, LS-106 potently inhibited the kinase activities of EGFR 19del/T790M/C797S and EGFR L858R/T790M/C797S with IC 50 values of 2.4 nmol/L and 3.1 nmol/L, respectively, which was more potent than osimertinib. Meanwhile, LS-106 exhibited comparable kinase inhibitory effect to osimertinib on EGFR L858R/T790M and wild-type EGFR. Results from cellular experiments demonstrated that LS-106 potently blocked the phosphorylation of EGFR C797S triple mutations in the constructed BaF3 cells that highly expressed EGFR 19del/T790M/C797S or EGFR L858R/T790M/C797S , and thus inhibited the proliferation of these cells. We also constructed tumor cells harboring EGFR 19del/T790M/C797S (named PC-9-OR cells) using the CRISPR/Cas9 system and found that LS-106 markedly suppressed the activation of EGFR 19del/T790M/C797S and the proliferation of PC-9-OR cells. Moreover, cells harboring EGFR 19del/T790M/C797S underwent remarkable apoptosis upon LS-106 treatment. In vivo experiments further demonstrated that oral administration of LS-106 caused significant tumor regression in a PC-9-OR xenograft model, with a tumor growth inhibition rate (TGI) of 83.5% and 136.6% at doses of 30 and 60 mg/kg, respectively. Taken together, we identified LS-106 as a novel fourth-generation EGFR inhibitor against C797S mutation and confirmed its preclinical antitumor effects in C797S-triple-mutant tumor models., (© 2021 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

RAF inhibitors unexpectedly induce ERK signaling in normal and tumor cells with elevated RAS activity. Paradoxical activation is believed to be RAS dependent. In this study, we showed that LY3009120, a pan-RAF inhibitor, can unexpectedly cause paradoxical ERK activation in KRAS G12C -dependent lung cancer cell lines, when KRAS is inhibited by ARS1620, a KRAS G12C inhibitor. Using H/N/KRAS-less mouse embryonic fibroblasts, we discovered that classical RAS proteins are not essential for RAF inhibitor-induced paradoxical ERK signaling. In their absence, RAF inhibitors can induce ERK phosphorylation, ERK target gene transcription, and cell proliferation. We further showed that the MRAS/SHOC2 complex is required for this process. This study highlights the complexity of the allosteric RAF regulation by RAF inhibitors, and the importance of other RAS-related proteins in this process., Competing Interests: Competing interest statement: F.M. is a consultant for the following companies: Amgen; Daiichi Ltd., Frontiers Med, Exuma Biotech, Ideaya Biosciences, Kura Oncology, Leidos Biomedical Research, Inc., PellePharm, Pfizer Inc., PMV Pharma and Quanta Therapeutics. F.M. is a consultant for and cofounder of the following companies (with ownership interest including stock options): BridgeBio; DNAtrix Inc., Olema Pharmaceuticals, Inc., and Quartz. F.M. is the scientific director of the National Cancer Institute RAS Initiative at the Frederick National Laboratory for Cancer Research/Leidos Biomedical Research, Inc. F.M. has been a recipient of research grants from Daiichi Sankyo and Gilead Sciences and has a current grant from Boehringer-Ingelheim., (Copyright © 2022 the Author(s). Published by PNAS.)

Hydrogen sulfide (H 2 S) is an endogenous gaseous molecule that plays an important role in the plant life cycle. The multiple transcription factor ABSCISIC ACID INSENSITIVE 4 (ABI4) was precisely regulated to participate in the abscisic acid (ABA) mediated signaling cascade. However, the molecular mechanisms of how H 2 S regulates ABI4 protein level to control seed germination and seedling growth have remained elusive. In this study, we demonstrated that ABI4 controls the expression of L-CYSTEINE DESULFHYDRASE1 (DES1), a critical endogenous H 2 S-producing enzyme, and both ABI4 and DES1-produced H 2 S have inhibitory effects on seed germination. Furthermore, the ABI4 level decreased during seed germination while H 2 S triggered the enhancement of the persulfidation level of ABI4 and alleviated its degradation rate, which in turn inhibited seed germination and seedling establishment. Conversely, the mutation of ABI4 at Cys250 decreased ABI4 protein stability and facilitated seed germination. Moreover, ABI4 degradation is also regulated via the 26S proteasome pathway. Taken together, these findings suggest a molecular link between DES1 and ABI4 through the post-translational modifications of persulfidation during early seedling development.

Response to ritonavir-boosted-protease inhibitors (PI/r)-based regimen is associated with some Gag mutations among HIV-1 B-clade. There is limited data on Gag mutations and their covariation with mutations in protease among HIV-1 non-B-clades at PI/r-based treatment failure. Thus, we characterized Gag mutations present in isolates from HIV-1 infected individuals treated with a PI/r-regimen (n = 143) and compared them with those obtained from individuals not treated with PI/r (ART-naïve [n = 101] or reverse transcriptase inhibitors (RTI) treated [n = 118]). The most frequent HIV-1 subtypes were CRF02&#95;AG (54.69%), A (13.53%), D (6.35%) and G (4.69%). Eighteen Gag mutations showed a significantly higher prevalence in PI/r-treated isolates compared to ART-naïve (p < 0.05): Group 1 (prevalence < 1% in drug-naïve): L449F, D480N, L483Q, Y484P, T487V; group 2 (prevalence 1-5% in drug-naïve): S462L, I479G, I479K, D480E; group 3 (prevalence ≥ 5% in drug-naïve): P453L, E460A, R464G, S465F, V467E, Q474P, I479R, E482G, T487A. Five Gag mutations (L449F, P453L, D480E, S465F, Y484P) positively correlated (Phi ≥ 0.2, p < 0.05) with protease-resistance mutations. At PI/r-failure, no significant difference was observed between patients with and without these associated Gag mutations in term of viremia or CD4 count. This analysis suggests that some Gag mutations show an increased frequency in patients failing PIs among HIV-1 non-B clades., (© 2022. The Author(s).)

Rhinoviruses (RVs) cause recurrent infections of the nasal and pulmonary tracts, life-threatening conditions in chronic respiratory illness patients, predisposition of children to asthmatic exacerbation, and large economic cost. RVs are difficult to treat. They rapidly evolve resistance and are genetically diverse. Here, we provide insight into RV drug resistance mechanisms against chemical compounds neutralizing low pH in endolysosomes. Serial passaging of RV-A16 in the presence of the vacuolar proton ATPase inhibitor bafilomycin A1 (BafA1) or the endolysosomotropic agent ammonium chloride (NH 4 Cl) promoted the emergence of resistant virus populations. We found two reproducible point mutations in viral proteins 1 and 3 (VP1 and VP3), A2526G (serine 66 to asparagine [S66N]), and G2274U (cysteine 220 to phenylalanine [C220F]), respectively. Both mutations conferred cross-resistance to BafA1, NH 4 Cl, and the protonophore niclosamide, as identified by massive parallel sequencing and reverse genetics, but not the double mutation, which we could not rescue. Both VP1-S66 and VP3-C220 locate at the interprotomeric face, and their mutations increase the sensitivity of virions to low pH, elevated temperature, and soluble intercellular adhesion molecule 1 receptor. These results indicate that the ability of RV to uncoat at low endosomal pH confers virion resistance to extracellular stress. The data endorse endosomal acidification inhibitors as a viable strategy against RVs, especially if inhibitors are directly applied to the airways. IMPORTANCE Rhinoviruses (RVs) are the predominant agents causing the common cold. Anti-RV drugs and vaccines are not available, largely due to rapid evolutionary adaptation of RVs giving rise to resistant mutants and an immense diversity of antigens in more than 160 different RV types. In this study, we obtained insight into the cell biology of RVs by harnessing the ability of RVs to evolve resistance against host-targeting small chemical compounds neutralizing endosomal pH, an important cue for uncoating of normal RVs. We show that RVs grown in cells treated with inhibitors of endolysosomal acidification evolved capsid mutations yielding reduced virion stability against elevated temperature, low pH, and incubation with recombinant soluble receptor fragments. This fitness cost makes it unlikely that RV mutants adapted to neutral pH become prevalent in nature. The data support the concept of host-directed drug development against respiratory viruses in general, notably at low risk of gain-of-function mutations.

Preclinically, enasidenib and azacitidine (ENA + AZA) synergistically enhance cell differentiation, and venetoclax (VEN), a small molecule Bcl2 inhibitor (i) is particularly effective in IDH2 mutated acute myeloid leukemia (IDH2 mut AML). This open label phase II trial enrolled patients (pts) with documented IDH2 mut AML. All patients received AZA 75 mg/m 2 /d x 7 d/cycle and ENA 100 mg QD continuously. Concomitant Bcl2i and FLT3i were allowed (NCT03683433).Twenty-six pts received ENA + AZA (median 68 years, range, 24-88); 7 newly diagnosed (ND) and 19 relapsed/refractory (R/R). In R/R AML patients, three had received prior ENA and none had received prior VEN. The composite complete remission rate (CRc) [complete remission (CR) or complete remission with incomplete hematologic recovery (CRi)] was 100% in ND AML, and 58% in R/R AML. Median OS was not reached in ND AML with median follow-up of 13.1 months (mo); Pts treated in first relapse had improved OS than those with ≥2 relapse (median OS not reached vs 5.2 mo; HR 0.24, 95% CI 0.07-0.79, p = 0.04). Two patients received ENA + AZA with a concomitant FLT3i, one responding ND AML patient and one nonresponding R/R AML patient. Seven R/R AML pts received ENA + AZA + VEN triplet, and with median follow up of 11.2 mo, median OS was not reached and 6-mo OS was 70%. The most frequent treatment-emergent adverse events include febrile neutropenia (23%). Adverse events of special interest included all-grade IDH differentiation syndrome (8%) and indirect hyperbilirubinemia (35%). ENA + AZA was a well-tolerated, and effective therapy for elderly pts with IDH2 mut ND AML as well as pts with R/R AML. The addition of VEN to ENA + AZA appears to improve outcomes in R/R IDH2 mut AML.Clinical trial registration information: https://clinicaltrials.gov/.NCT03683433., (© 2022. The Author(s).)

SUMOylation is a post-translational modification of proteins that regulates these proteins' localization, turnover or function. Aberrant SUMOylation is frequently found in cancers but its origin remains elusive. Using a genome-wide transposon mutagenesis screen in a MYC-driven B-cell lymphoma model, we here identify the SUMO isopeptidase (or deconjugase) SENP6 as a tumor suppressor that links unrestricted SUMOylation to tumor development and progression. Notably, SENP6 is recurrently deleted in human lymphomas and SENP6 deficiency results in unrestricted SUMOylation. Mechanistically, SENP6 loss triggers release of DNA repair- and genome maintenance-associated protein complexes from chromatin thereby impairing DNA repair in response to DNA damages and ultimately promoting genomic instability. In line with this hypothesis, SENP6 deficiency drives synthetic lethality to Poly-ADP-Ribose-Polymerase (PARP) inhibition. Together, our results link SENP6 loss to defective genome maintenance and reveal the potential therapeutic application of PARP inhibitors in B-cell lymphoma., (© 2022. The Author(s).)

Treatment with Menin inhibitor (MI) disrupts the interaction between Menin and MLL1 or MLL1-fusion protein (FP), inhibits HOXA9/MEIS1, induces differentiation and loss of survival of AML harboring MLL1 re-arrangement (r) and FP, or expressing mutant (mt)-NPM1. Following MI treatment, although clinical responses are common, the majority of patients with AML with MLL1-r or mt-NPM1 succumb to their disease. Pre-clinical studies presented here demonstrate that genetic knockout or degradation of Menin or treatment with the MI SNDX-50469 reduces MLL1/MLL1-FP targets, associated with MI-induced differentiation and loss of viability. MI treatment also attenuates BCL2 and CDK6 levels. Co-treatment with SNDX-50469 and BCL2 inhibitor (venetoclax), or CDK6 inhibitor (abemaciclib) induces synergistic lethality in cell lines and patient-derived AML cells harboring MLL1-r or mtNPM1. Combined therapy with SNDX-5613 and venetoclax exerts superior in vivo efficacy in a cell line or PD AML cell xenografts harboring MLL1-r or mt-NPM1. Synergy with the MI-based combinations is preserved against MLL1-r AML cells expressing FLT3 mutation, also CRISPR-edited to introduce mtTP53. These findings highlight the promise of clinically testing these MI-based combinations against AML harboring MLL1-r or mtNPM1., (© 2022. The Author(s).)

The malaria vector, Anopheles stephensi, which is typically restricted to South Asia and the Middle East, was recently detected in the Horn of Africa. Addressing the spread of this vector could involve integrated vector control that considers the status of insecticide resistance of multiple vector species in the region. Previous reports indicate that the knockdown resistance mutations (kdr) in the voltage-gated sodium channel (vgsc) are absent in both pyrethroid-resistant and pyrethroid-sensitive An. stephensi in eastern Ethiopia; however, similar information about other vector species in the same areas is limited. In this study, kdr and the neighboring intron were analyzed in An. stephensi, An. arabiensis, and Culex pipiens s.l. collected between 2016 and 2017 to determine the evolutionary history of kdr in eastern Ethiopia. A sequence analysis revealed that all of Cx. pipiens s.l. (N = 42) and 71.6% of the An. arabiensis (N = 67) carried kdr L1014F, which is known to confer target-site pyrethroid resistance. Intronic variation was only observed in An. stephensi (six segregating sites, three haplotypes), which was previously shown to have no kdr mutations. In addition, no evidence of non-neutral evolutionary processes was detected at the An. stephensi kdr intron, thereby further supporting the target-site mechanism not being a major resistance mechanism in this An. stephensi population. Overall, these results show key differences in the evolution of target-site pyrethroid/dichlorodiphenyltrichloroethane resistance mutations in populations of vector species from the same region. Variations in insecticide resistance mechanism profiles between eastern Ethiopian mosquito vectors may lead to different responses to insecticides used in integrated vector control.

Purpose: Epidermal growth factor receptor kinase domain duplication (EGFR-KDD) is a rare and poorly understood oncogenic mutation in non-small cell lung cancer (NSCLC). We aimed to investigate the acquired resistance mechanism of EGFR-KDD against EGFR-TKIs., Materials and Methods: We identified EGFR-KDD in tumor tissue obtained from a patient with stage IV lung adenocarcinoma and established the patient-derived cell line SNU-4784. We also established several EGFR-KDD Ba/F3 cell lines: EGFR-KDD wild type (EGFR-KDDWT), EGFR-KDD domain 1 T790M (EGFR-KDDD1T), EGFR-KDD domain 2 T790M (EGFR-KDDD2T), and EGFR-KDD both domain T790M (EGFR-KDDBDT). We treated the cells with EGFR tyrosine kinase inhibitors (TKIs) and performed cell viability assays, immunoblot assays, and ENU (N-ethyl-N-nitrosourea) mutagenesis screening., Results: In cell viability assays, SNU-4784 cells and EGFR-KDDWT Ba/F3 cells were sensitive to 2nd generation and 3rd generation EGFR TKIs. In contrast, the T790M-positive EGFR-KDD Ba/F3 cell lines (EGFR-KDDT790M) were only sensitive to 3rd generation EGFR TKIs. In ENU mutagenesis screening, we identified the C797S mutation in kinase domain 2 of EGFR-KDDBDT Ba/F3 cells. Based on this finding, we established an EGFR-KDD domain 1 T790M/domain 2 cis-T790M+C797S (EGFR-KDDT/T+C) Ba/F3 model, which was resistant to EGFR TKIs and anti-EGFR monoclonal antibody combined with EGFR TKIs., Conclusion: Our study reveals that the T790M mutation in EGFR-KDD confers resistance to 1st and 2nd generation EGFR TKIs, but is sensitive to 3rd generation EGFR TKIs. In addition, we identified that the C797S mutation in kinase domain 2 of EGFR-KDDT790M mediates a resistance mechanism against 3rd generation EGFR TKIs.

The essential metal manganese (Mn) induces incurable neurotoxicity at elevated levels that manifests as parkinsonism in adults and fine motor and executive function deficits in children. Studies on Mn neurotoxicity have largely focused on the role and mechanisms of disease induced by elevated Mn exposure from occupational or environmental sources. In contrast, the critical role of excretion in regulating Mn homeostasis and neurotoxicity has received less attention although 1 ) studies on Mn excretion date back to the 1920s; 2 ) elegant radiotracer Mn excretion assays in the 1940s to 1960s established the routes of Mn excretion; and 3 ) studies on patients with liver cirrhosis in the 1990s to 2000s identified an association between decreased Mn excretion and the risk of developing Mn-induced parkinsonism in the absence of elevated Mn exposure. Notably, the last few years have seen renewed interest in Mn excretion largely driven by the discovery that hereditary Mn neurotoxicity due to mutations in SLC30A10 or SLC39A14 is caused, at least in part, by deficits in Mn excretion. Quite remarkably, some of the recent results on SLC30A10 and SLC39A14 provide explanations for observations made ∼40-50 years ago. The goal of the current review is to integrate the historic studies on Mn excretion with more contemporary recent work and provide a comprehensive state-of-the-art overview of Mn excretion and its role in regulating Mn homeostasis and neurotoxicity. A related goal is to discuss the significance of some of the foundational studies on Mn excretion so that these highly consequential earlier studies remain influential in the field.

The emergence of treatment resistance to targeted agents is currently inevitable and inherently heterogeneous in cancer, presenting significant challenges for improving survival outcomes in patients. This is not an exception for cancers harboring EGFR mutations, one of the most prevalently observed oncogenic alterations in non-small cell lung cancer (NSCLC) targeted clinically. Currently, numerous efforts have attempted to delay or overcome acquired resistance to EGFR-tyrosine kinase inhibitors (TKI), changing the treatment landscape of EGFR -mutant NSCLC. Haikala and colleagues have developed a unique strategy using patritumab deruxtecan, an antibody-drug conjugate targeting human epidermal growth factor receptor 3 (HER3) linked to exatecan derivatives, for treating EGFR -mutant NSCLC. By incorporating EGFR TKIs to upregulate surface HER3 expression, the antitumor efficacy of patritumab deruxtecan was augmented in various preclinical models. In parallel, Jänne and colleagues reported the clinical activity of patrimumab deruxtecan in patients with EGFR -mutant NSCLC with prior EGFR TKI treatment. These two studies provide the grounds for hopeful anticipation for a novel strategy that concurrently targets compensatory feedback loops in addition to oncogenic signaling pathways. See related article by Haikala et al., p. 130 ., (©2022 American Association for Cancer Research.)

Background: The methylation status of the O 6 -methylguanine-DNA methyltransferase (MGMT) promoter plays a key role in response to temozolomide chemotherapy and disease prognosis in patients with wild-type isocitrate dehydrogenase (IDH) glioblastoma (GBM)., Patients and Methods: The MGMT promoter methylation status and its association with clinicopathological parameters were retrospectively analysed in a cohort of 316 patients with GBM with wild-type IDH., Results: MGMT methylation was significantly associated with ATRX chromatin remodeler (ATRX) loss and completion of the standard Stupp protocol. The median durations of overall and progression-free survival for the unmethylated, low-methylated (10-39%), and hypermethylated (≥40%) groups were 15, 23, and 30 months and 11, 18, and 21 months, respectively. However, the improvement in the survival of the hypermethylated group was not statistically significant., Conclusion: We suggest a possible association between MGMT methylation status and ATRX mutations in GBM with wild-type IDH., (Copyright © 2022 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

New neutralizing agents against SARS-CoV-2 and associated mutant strains are urgently needed for the treatment and prophylaxis of COVID-19. Herein, we develop a spherical cocktail neutralizing aptamer-gold nanoparticle (SNAP) to block the interaction between the receptor-binding domain (RBD) of SARS-CoV-2 and host ACE2. With the multivalent aptamer assembly as well as the steric hindrance effect of the gold scaffold, SNAP exhibits exceptional binding affinity against the RBD with a dissociation constant of 3.90 pM and potent neutralization against authentic SARS-CoV-2 with a half-maximal inhibitory concentration of 142.80 fM, about 2 or 3 orders of magnitude lower than that of the reported neutralizing aptamers and antibodies. More importantly, the synergetic blocking strategy of multivalent multisite binding and steric hindrance ensures broad neutralizing activity of SNAP, almost completely blocking the infection of three mutant pseudoviruses. Overall, the SNAP strategy provides a new direction for the development of antivirus agents against SARS-CoV-2 and other emerging coronaviruses.

Patients with chronic lymphocytic leukemia (CLL) bearing TP53 mutations experience chemorefractory disease and are therefore candidates for targeted therapy. However, the significance of low-burden TP53 mutations with <10% variant allele frequency (VAF) remains a matter for debate. Herein, we describe clonal evolution scenarios of low-burden TP53 mutations, the clinical impact of which we analyzed in a "real-world" CLL cohort. TP53 status was assessed by targeted next-generation sequencing (NGS) in 511 patients entering first-line treatment with chemo- and/or immunotherapy and 159 patients in relapse before treatment with targeted agents. Within the pretherapy cohort, 16% of patients carried low-burden TP53 mutations (0.1% to 10% VAF). Although their presence did not significantly shorten event-free survival after first-line therapy, it affected overall survival (OS). In a subgroup with TP53 mutations of 1% to 10% VAF, the impact on OS was observed only in patients with unmutated IGHV who had not received targeted therapy, as patients benefited from switching to targeted agents, regardless of initial TP53 mutational status. Analysis of the clonal evolution of low-burden TP53 mutations showed that the highest expansion rates were associated with fludarabine, cyclophosphamide, and rituximab regimen in both first- and second-line treatments (median VAF increase, 14.8× and 11.8×, respectively) in contrast to treatment with less intense treatment regimens (1.6×) and no treatment (0.8×). In the relapse cohort, 33% of patients carried low-burden TP53 mutations, which did not expand significantly upon targeted treatment (median VAF change, 1×). Sporadic cases of TP53 mutations' clonal shifts were connected with the development of resistance-associated mutations. Altogether, our data support the incorporation of low-burden TP53 variants in clinical decision making., (© 2021 by The American Society of Hematology.)

The underlying molecular mechanism and their general effect on the replication capacity of HIV 1 drug-resistance-associated mutations is often poorly understood. To elucidate the effect of two such mutations located in a region with a high density of spicing regulatory elements on the HIV-1-splicing outcome, bioinformatic predictions were combined with transfection and infection experiments. Results show that the previously described R263K drug-resistance-associated integrase mutation has additionally a severe effect on the ESE2b splicing regulatory element (SRE) in exon 2b, which causes loss of SD2b recognition. This was confirmed by an R263R silent mutation with a similar predicted effect on the exon 2b SRE. In contrast, a V260I mutation and its silent counterpart with a lower effect on ESS2b did not exhibit any differences in the splicing pattern. Since HIV-1 highly relies on a balanced splicing reaction, changes in the splicing outcome can contribute to changes in viral replication and might add to the effect of escape mutations toward antiviral drugs. Thus, a classification of mutations purely addressing proteins is insufficient.

HIV infection persists in different tissue reservoirs among people with HIV (PWH) despite effective antiretroviral therapy (ART). In the brain, lentiviruses replicate principally in microglia and trafficking macrophages. The impact of ART on this viral reservoir is unknown. We investigated the activity of contemporary ART in various models of lentivirus brain infection. HIV-1 RNA and total and integrated DNA were detected in cerebral cortex from all PWH ( n  = 15), regardless of ART duration or concurrent plasma viral quantity and, interestingly, integrated proviral DNA levels in brain were significantly higher in the aviremic ART-treated group ( P  < 0.005). Most ART drugs tested (dolutegravir, ritonavir, raltegravir, and emtricitabine) displayed significantly lower 50% effective concentration (EC 50 ) values in lymphocytes than in microglia, except tenofovir, which showed 1.5-fold greater activity in microglia ( P  < 0.05). In SIV-infected Chinese rhesus macaques, despite receiving suppressive ( n  = 7) or interrupted ( n  = 8) ART, brain tissues had similar SIV-encoded RNA and total and integrated DNA levels compared to brains from infected animals without ART ( n  = 3). SIV and HIV-1 capsid antigens were immunodetected in brain, principally in microglia/macrophages, regardless of ART duration and outcome. Antiviral immune responses were comparable in the brains of ART-treated and untreated HIV- and SIV-infected hosts. Both HIV-1 and SIV persist in brain tissues despite contemporary ART, with undetectable virus in blood. ART interruption exerted minimal effect on the SIV brain reservoir and did not alter the neuroimmune response profile. These studies underscore the importance of augmenting ART potency in different tissue compartments. IMPORTANCE Antiretroviral therapy (ART) suppresses HIV-1 in plasma and CSF to undetectable levels. However, the impact of contemporary ART on HIV-1 brain reservoirs remains uncertain. An active viral reservoir in the brain during ART could lead to rebound systemic infection after cessation of therapy, development of drug resistance mutations, and neurological disease. ART's impact, including its interruption, on brain proviral DNA remains unclear. The present studies show that in different experimental platforms, contemporary ART did not suppress viral burden in the brain, regardless of ART component regimen, the duration of therapy, and its interruption. Thus, new strategies for effective HIV-1 suppression in the brain are imperative to achieve sustained HIV suppression.

Multivalent membrane disruptors are a relatively new antimicrobial scaffold that are difficult for bacteria to develop resistance to and can act on both Gram-positive and Gram-negative bacteria. Proton Nuclear Magnetic Resonance ( 1 H NMR) metabolomics is an important method for studying resistance development in bacteria, since this is both a quantitative and qualitative method to study and identify phenotypes by changes in metabolic pathways. In this project, the metabolic differences between wild type Bacillus cereus ( B. cereus) samples and B. cereus that was mutated through 33 growth cycles in a nonlethal dose of a multivalent antimicrobial agent were identified. For additional comparison, samples for analysis of the wild type and mutated strains of B. cereus were prepared in both challenged and unchallenged conditions. A C 16 -DABCO (1,4-diazabicyclo-2,2,2-octane) and mannose functionalized poly(amidoamine) dendrimer (DABCOMD) were used as the multivalent quaternary ammonium antimicrobial for this hydrophilic metabolic analysis. Overall, the study reported here indicates that B. cereus likely change their peptidoglycan layer to protect themselves from the highly positively charged DABCOMD. This membrane fortification most likely leads to the slow growth curve of the mutated, and especially the challenged mutant samples. The association of these sample types with metabolites associated with energy expenditure is attributed to the increased energy required for the membrane fortifications to occur as well as to the decreased diffusion of nutrients across the mutated membrane.

Broad-spectrum antiviral therapies hold promise as a first-line defense against emerging viruses by blunting illness severity and spread until vaccines and virus-specific antivirals are developed. The nucleobase favipiravir, often discussed as a broad-spectrum inhibitor, was not effective in recent clinical trials involving patients infected with Ebola virus or SARS-CoV-2. A drawback of favipiravir use is its rapid clearance before conversion to its active nucleoside-5'-triphosphate form. In this work, we report a synergistic reduction of flavivirus (dengue, Zika), orthomyxovirus (influenza A), and coronavirus (HCoV-OC43 and SARS-CoV-2) replication when the nucleobases favipiravir or T-1105 were combined with the antimetabolite 6-methylmercaptopurine riboside (6MMPr). The 6MMPr/T-1105 combination increased the C-U and G-A mutation frequency compared to treatment with T-1105 or 6MMPr alone. A further analysis revealed that the 6MMPr/T-1105 co-treatment reduced cellular purine nucleotide triphosphate synthesis and increased conversion of the antiviral nucleobase to its nucleoside-5'-monophosphate, -diphosphate, and -triphosphate forms. The 6MMPr co-treatment specifically increased production of the active antiviral form of the nucleobases (but not corresponding nucleosides) while also reducing levels of competing cellular NTPs to produce the synergistic effect. This in-depth work establishes a foundation for development of small molecules as possible co-treatments with nucleobases like favipiravir in response to emerging RNA virus infections.

Amino acid deprivation from reduced hemoglobin degradation in Pfkelch13 artemisinin-resistant parasites reduces fitness. In this issue of Cell Host & Microbe, Mesén-Ramírez et al. decipher the role of nutrient permeable channel activity within the parasitophorous vacuolar membrane to compensate for this fitness cost in asexual blood-stage Plasmodium falciparum parasites., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Blocked cellular differentiation is a central pathologic feature of the myeloid malignancies, myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Treatment regimens promoting differentiation have resulted in incredible cure rates in certain AML subtypes, such as acute promyelocytic leukemia. Over the past several years, we have seen many new therapies for MDS/AML enter clinical practice, including epigenetic therapies (e.g., 5-azacitidine), isocitrate dehydrogenase (IDH) inhibitors, fms-like kinase 3 (FLT3) inhibitors, and lenalidomide for deletion 5q (del5q) MDS. Despite not being developed with the intent of manipulating differentiation, induction of differentiation is a major mechanism by which several of these novel agents function. In this review, we examine the new therapeutic landscape for these diseases, focusing on the role of hematopoietic differentiation and the impact of inflammation and aging. We review how current therapies in MDS/AML promote differentiation as a part of their therapeutic effect, and the cellular mechanisms by which this occurs. We then outline potential novel avenues to achieve differentiation in the myeloid malignancies for therapeutic purposes. This emerging body of knowledge about the importance of relieving differentiation blockade with anti-neoplastic therapies is important to understand how current novel agents function and may open avenues to developing new treatments that explicitly target cellular differentiation. Moving beyond cytotoxic agents has the potential to open new and unexpected avenues in the treatment of myeloid malignancies, hopefully providing more efficacy with reduced toxicity., (© 2021. The Author(s).)

Classical BCR-ABL-negative myeloproliferative neoplasms (MPNs) are clonal disorders of hematopoietic stem cells (HSCs) caused mainly by recurrent mutations in genes encoding JAK2 (JAK2), calreticulin (CALR), or the thrombopoietin receptor (MPL). Interferon α (IFNα) has demonstrated some efficacy in inducing molecular remission in MPNs. To determine factors that influence molecular response rate, we evaluated the long-term molecular efficacy of IFNα in patients with MPN by monitoring the fate of cells carrying driver mutations in a prospective observational and longitudinal study of 48 patients over more than 5 years. We measured the clonal architecture of early and late hematopoietic progenitors (84 845 measurements) and the global variant allele frequency in mature cells (409 measurements) several times per year. Using mathematical modeling and hierarchical Bayesian inference, we further inferred the dynamics of IFNα-targeted mutated HSCs. Our data support the hypothesis that IFNα targets JAK2V617F HSCs by inducing their exit from quiescence and differentiation into progenitors. Our observations indicate that treatment efficacy is higher in homozygous than heterozygous JAK2V617F HSCs and increases with high IFNα dose in heterozygous JAK2V617F HSCs. We also found that the molecular responses of CALRm HSCs to IFNα were heterogeneous, varying between type 1 and type 2 CALRm, and a high dose of IFNα correlates with worse outcomes. Our work indicates that the long-term molecular efficacy of IFNα implies an HSC exhaustion mechanism and depends on both the driver mutation type and IFNα dose., (© 2021 by The American Society of Hematology.)

Three families with multiple gastrointestinal stromal tumors (GISTs) caused by a germline Asp820Tyr mutation at exon 17 of the c-kit gene (KIT-Asp820Tyr) have been reported. We previously generated a knock-in mouse model of the family, and the mice with KIT-Asp818Tyr corresponding to human KIT-Asp820Tyr showed a cecal tumor equivalent to human GIST. In the model mice, we reported that tyrosine kinase inhibitor, imatinib, could stabilize but not decrease the cecal tumor volume. In this report, we examined whether a heat shock protein 90 inhibitor, pimitespib (TAS-116), has an inhibitory effect on phosphorylation of KIT-Asp818Tyr and can decrease the cecal tumor volume in the model mice. First, we showed that pimitespib inhibited KIT phosphorylation both dose- and time-dependently in KIT-Asp818Tyr transfected murine Ba/F3 cells. Then, four 1-week courses of pimitespib were orally administered to heterozygous (KIT-Asp818Tyr/+) model mice. Each course consisted of once-daily administration for consecutive 5 days followed by 2 days-off. Cecal tumors were dissected, and tumor volume was histologically analyzed, Ki-67 labeling index was immunohistochemically examined, and apoptotic figures were counted. Compared to the vehicle treated mice, pimitespib administered mice showed statistically significantly smaller cecal tumor volume, lower Ki-67 labeling index, and higher number of apoptotic figures in 10 high power fields (P = 0.0344, P = 0.0019 and P = 0.0269, respectively). Western blotting revealed that activation of KIT signaling molecules was strongly inhibited in the tumor tissues of pimitespib-administered mice compared to control mice. Thus, pimitespib seemed to inhibit in vivo tumor progression effectively in the model mice. These results suggest that the progression of multiple GISTs in patients with germline KIT-Asp820Tyr might be controllable by pimitespib., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)