Genetic Mechanisms Of Resistance Research Articles

Drug-resistant tuberculosis: a persistent global health concern.

Drug-resistant tuberculosis (TB) is estimated to cause 13% of all antimicrobial resistance-attributable deaths worldwide and is driven by both ongoing resistance acquisition and person-to-person transmission. Poor outcomes are exacerbated by late diagnosis and inadequate access to effective treatment. Advances in rapid molecular testing have recently improved the diagnosis of TB and drug resistance. Next-generation sequencing of Mycobacterium tuberculosis has increased our understanding of genetic resistance mechanisms and can now detect mutations associated with resistance phenotypes. All-oral, shorter drug regimens that can achieve high cure rates of drug-resistant TB within 6-9 months are now available and recommended but have yet to be scaled to global clinical use. Promising regimens for the prevention of drug-resistant TB among high-risk contacts are supported by early clinical trial data but final results are pending. A person-centred approach is crucial in managing drug-resistant TB to reduce the risk of poor treatment outcomes, side effects, stigma and mental health burden associated with the diagnosis. In this Review, we describe current surveillance of drug-resistant TB and the causes, risk factors and determinants of drug resistance as well as the stigma and mental health considerations associated with it. We discuss recent advances in diagnostics and drug-susceptibility testing and outline the progress in developing better treatment and preventive therapies.

Development of New PCR Protocols to Detect Genetic Diversity in the Metronidazole Metabolism Genes in Susceptible and Refractory Clinical Samples of Giardia duodenalis.

Investigating the genetic variation in thioredoxin reductase (TrxR) and nitroreductase (NR) genes in both treatment-resistant and -sensitive Giardia duodenalis isolates can provide valuable information in identifying potential markers of resistance to metronidazole. The rapid increase in metronidazole treatment failures suggests the presence of genetic resistance mechanisms. By analyzing these genes, researchers can gain insights into the efficacy of metronidazole against G. duodenalis and potentially develop alternative treatment strategies. In this regard, four G. duodenalis isolates (two clinically sensitive and two clinically resistant to metronidazole) were collected from various hospitals of Shiraz, southwestern Iran. Parasitological methods including sucrose flotation and microscopy were employed for the primary confirmation of G. duodenalis cysts in stool samples. Microscopy-positive samples were approved by SSU-PCR amplification of the parasite DNA. All four positive G. duodenalis specimens at SSU-PCR were afterward analyzed utilizing designed primers based on important metronidazole metabolism genes including TrxR, NR1, and NR2. Unlike TrxR gene, the results of NR1 and NR2 genes showed that there are non-synonymous variations between sequences of treatment-sensitive and -resistant samples compared to reference sequences. Furthermore, the outcomes of molecular docking revealed that there is an interaction between the protein sequence and spatial shape of treatment-resistant samples and metronidazole in the position of serine amino acid based on the NR1 gene. This issue can be one of the possible factors involved in the resistance of Giardia parasites to metronidazole. To reach more accurate results, a large sample size along with simulation and advanced molecular dynamics investigations are needed.

Genetic Mechanism of Non-Targeted-Site Resistance to Diquat in Spirodela polyrhiza.

Understanding non-target-site resistance (NTSR) to herbicides represents a pressing challenge as NTSR is widespread in many weeds. Using giant duckweed (Spirodela polyrhiza) as a model, we systematically investigated genetic and molecular mechanisms of diquat resistance, which can only be achieved via NTSR. Quantifying the diquat resistance of 138 genotypes, we revealed an 8.5-fold difference in resistance levels between the most resistant and most susceptible genotypes. Further experiments suggested that diquat uptake and antioxidant-related processes jointly contributed to diquat resistance in S. polyrhiza. Using a genome-wide association approach, we identified several candidate genes, including a homolog of dienelactone hydrolase, that are associated with diquat resistance in S. polyrhiza. Together, these results provide new insights into the mechanisms and evolution of NTSR in plants.

The role of genetic variability of the host on the resistance to Neospora caninum infection in cattle.

Neospora caninum is one of the most frequently diagnosed abortifacient pathogens in cattle. There is abundant genomic information about the parasite itself, but very little is known about the genetic variability of resistance in the most common intermediate host. The aim of this review was to compile all the available information about the genetic variability associated with the resistance to N. caninum both between and within cattle breeds. We systematically searched for published studies that investigated the influence of genetics of the host on the prevalence of N. caninum and risk of abortion. Beyond the potential confounding effects of feeding systems, management and animal density, some lines of evidence suggest that Holstein, the most popular breed for milk production, has a comparatively higher risk of abortion due to infections by N. caninum, whereas some beef breeds from Continental Europe seem to be more resistant. It is still not clear if different genetic mechanisms of resistance are involved in the two known routes of infection: postnatal ingestion of oocysts or transplacental transmission from the infected dam to the fetus. Genomic information associated with susceptibility to infection and risk of abortion in different cattle breeds is still scarce. The information reported here could be useful to identify new research alternatives and to define novel strategies to deal with this major problem of animal production.

Genetic and non-genetic drug resistance: Darwin or Lamarck?

Drug resistance represents a major limitation to the long-term efficacy of anti-cancer treatments. The commonly accepted view is that the selection of inheritable genetic mechanisms governs the development of secondary resistance. However, compelling evidence suggests an important role for adaptive cell plasticity and non-genetic mechanisms in the development of therapy resistance. The two phenomena are not mutually exclusive and the interplay between genetic and non-genetic mechanisms may affect tumor evolution during treatment. A broader characterization of the genetic and non-genetic mechanisms of drug resistance may pave the way for more precise and effective therapeutic strategies to overcome resistance.

Antifungal Resistance in Cryptococcal Infections.

Antifungal therapy, especially with the azoles, could promote the incidence of less susceptible isolates of Cryptococcus neoformans and C. gattii species complexes (SC), mostly in developing countries. Given that these species affect mostly the immunocompromised host, the infections are severe and difficult to treat. This review encompasses the following topics: 1. infecting species and their virulence, 2. treatment, 3. antifungal susceptibility methods and available categorical endpoints, 4. genetic mechanisms of resistance, 5. clinical resistance, 6. fluconazole minimal inhibitory concentrations (MICs), clinical outcome, 7. environmental influences, and 8. the relevance of host factors, including pharmacokinetic/pharmacodynamic (PK/PD) parameters, in predicting the clinical outcome to therapy. As of now, epidemiologic cutoff endpoints (ECVs/ECOFFs) are the most reliable antifungal resistance detectors for these species, as only one clinical breakpoint (amphotericin B and C. neoformans VNI) is available.

Integrative modeling uncovers p21-driven drug resistance and prioritizes therapies for PIK3CA-mutant breast cancer

Utility of PI3Kα inhibitors like BYL719 is limited by the acquisition of genetic and non-genetic mechanisms of resistance which cause disease recurrence. Several combination therapies based on PI3K inhibition have been proposed, but a way to systematically prioritize them for breast cancer treatment is still missing. By integrating published and in-house studies, we have developed in silico models that quantitatively capture dynamics of PI3K signaling at the network-level under a BYL719-sensitive versus BYL719 resistant-cell state. Computational predictions show that signal rewiring to alternative components of the PI3K pathway promote resistance to BYL719 and identify PDK1 as the most effective co-target with PI3Kα rescuing sensitivity of resistant cells to BYL719. To explore whether PI3K pathway-independent mechanisms further contribute to BYL719 resistance, we performed phosphoproteomics and found that selection of high levels of the cell cycle regulator p21 unexpectedly promoted drug resistance in T47D cells. Functionally, high p21 levels favored repair of BYL719-induced DNA damage and bypass of the associated cellular senescence. Importantly, targeted inhibition of the check-point inhibitor CHK1 with MK-8776 effectively caused death of p21-high T47D cells, thus establishing a new vulnerability of BYL719-resistant breast cancer cells. Together, our integrated studies uncover hidden molecular mediators causing resistance to PI3Kα inhibition and provide a framework to prioritize combination therapies for PI3K-mutant breast cancer.

Next-generation sequencing reveals genetic heterogeneity and resistance mechanisms in patients with EGFR-mutated non-small cell lung cancer treated with afatinib.

Afatinib, an irreversible ErbB family inhibitor, is widely used as first-line treatment in advanced lung adenocarcinoma patients harbouring mutant epidermal growth factor receptor (EGFR). With the advancements in next-generation sequencing (NGS), comprehensive research into the clinical impact of co-occurring genetic mutations and the molecular mechanisms of acquired resistance is required for afatinib users. From January 2010 to December 2019, we enrolled patients with advanced lung adenocarcinoma with EGFR mutations using afatinib as first-line treatment, and we retrospectively collected pre- and post-afatinib treatment specimens from these patients for NGS testing. Of the 362 enrolled patients, 73 samples (68.9%) from 56 patients successfully returned complete NGS reports. In pre-afatinib treatment specimens, the most frequent co-occurring alterations were TP53, MUC16, USH2A, SNYE1, RECQL4 and FAT1; however, they were not related to progression-free survival. Small cell lung cancer transformation, EGFR p.T790M, amplification of MET, ERBB2, KRAS, EGFR, cell cycle-regulated genes and MDM2, and PTEN alterations were identified as acquired resistance mechanisms. EGFR p.T790M (p=0.0304) and APC alterations (p=0.0311) in post-afatinib specimens were significantly associated with longer overall survival, while MET amplification was significantly associated with poor overall survival (p=0.0324). The co-occurrence of TP53 alterations was significantly associated with shorter overall survival (p=0.0298). Our results show that the frequent co-occurring alterations in advanced EGFR-mutated lung adenocarcinoma did not influence the effectiveness of afatinib. EGFR p.T790M is not only the major resistance mechanism to afatinib but also related to favourable survival outcomes. MET amplification and TP53 mutations were associated with poorer overall survival.

A Study of the Resistance of Hu Sheep Lambs to Escherichia coli F17 Based on Whole Genome Sequencing.

This study aims to analyze the whole genome sequencing of E. coli F17 in antagonistic and susceptible Hu sheep lambs. The objective is to investigate the critical mutation loci in sheep and understand the genetic mechanism of sheep resistance to E. coli F17 at the genome level. Antagonist and susceptible venous blood samples were collected from Hu sheep lambs for whole genome sequencing and whole genome association analysis. A total of 466 genes with significant SNPs (p < 1.0 × 10-3) were found. GO and KEGG enrichment analysis and protein interaction network analysis were performed on these genes, and preliminary investigations showed that SNPs on CTNNB1, CDH8, APOD, HCLS1, Tet2, MTSS1 and YAP1 genes may be associated with the antagonism and susceptibility of Hu sheep lambs to E. coli F17. There are still some shortcomings that have not been explored via in vivo and in vitro functional experiments of the candidate genes, which will be our next research work. This study provides genetic loci and candidate genes for resistance of Hu sheep lambs to E. coli F17 infection, and provides a genetic basis for breeding disease-resistant sheep.

Longitudinal genomic surveillance of multidrug-resistant Escherichia coli carriage in critical care patients.

Colonization with multidrug-resistant Escherichia coli strains causes a substantial health burden in hospitalized patients. We performed a longitudinal genomics study to investigate the colonization of resistant E. coli strains in critically ill patients and to identify evolutionary changes and strain replacement events within patients. Patients were admitted to the intensive care unit and hematology wards at a major hospital in Lebanon. Perianal swabs were collected from participants on admission and during hospitalization, which were screened for extended-spectrum beta-lactamases and carbapenem-resistant Enterobacterales. We performed whole-genome sequencing and analysis on E. coli strains isolated from patients at multiple time points. The E. coli isolates were genetically diverse, with 11 sequence types (STs) identified among 22 isolates sequenced. Five patients were colonized by E. coli sequence type 131 (ST131)-encoding CTX-M-27, an emerging clone not previously observed in clinical samples from Lebanon. Among the eight patients whose resident E. coli strains were tracked over time, five harbored the same E. coli strain with relatively few mutations over the 5 to 10 days of hospitalization. The other three patients were colonized by different E. coli strains over time. Our study provides evidence of strain diversity within patients during their hospitalization. While strains varied in their antimicrobial resistance profiles, the number of resistance genes did not increase over time. We also show that ST131-encoding CTX-M-27, which appears to be emerging as a globally important multidrug-resistant E. coli strain, is also prevalent among critical care patients and deserves further monitoring.IMPORTANCEUnderstanding the evolution of bacteria over time in hospitalized patients is of utmost significance in the field of infectious diseases. While numerous studies have surveyed genetic diversity and resistance mechanisms in nosocomial infections, time series of within-patient dynamics are rare, and high-income countries are over-represented, leaving low- and middle-income countries understudied. Our study aims to bridge these research gaps by conducting a longitudinal survey of critically ill patients in Lebanon. This allowed us to track Escherichia coli evolution and strain replacements within individual patients over extended periods. Through whole-genome sequencing, we found extensive strain diversity, including the first evidence of the emerging E. coli sequence type 131 clone encoding the CTX-M-27 beta-lactamase in a clinical sample from Lebanon, as well as likely strain replacement events during hospitalization.

ГЕНЕТИЧЕСКИЕ МЕХАНИЗМЫ УСТОЙЧИВОСТИ БАКТЕРИЙ К ЦИПРОФЛОКСАЦИНУ (ЛИТЕРАТУРНЫЙ ОБЗОР)

Ciprofloxacin, as a member of the fluoroquinolone group, is widely used to treat diseases caused by both grampositive and gram-negative bacteria. However, in recent years there has been an increase in resistance to this drug, which may be associated with the overuse of ciprofloxacin due to its broad spectrum of action. In this regard, in order to develop new and effective drugs against drug-resistant pathogens, there is a need to study and analyze such resistance mechanisms to ciprofloxacin as modification of target molecules, changes in drug penetration and plasmid-mediated quinolone resistance. The article presents up-to-date information on genetic mechanisms of bacterial resistance to antimicrobial drugs belonging to the fluoroquinolone group.

Genetic Mechanisms of Crop Disease Resistance: New Advances in GWAS

Genetic Mechanisms of Crop Disease Resistance: New Advances in GWAS

Etiological structure of infections in patients of the surgical intensive care unit in the post-covid era

Objective. To determine etiological structure of infections in the surgical intensive care unit (ICU), identify the most common mechanisms of antibiotic resistance of leading pathogens, and develop recommendations for optimizing antimicrobial therapy in the ICU at the present stage. Materials and Methods. A prospective observational study was conducted in the surgical ICU of the Moscow City Clinical Center «Kommunarka» from September 2022 to February 2024. The subjects of observation were 2120 patients treated in the unit. Microbiological examination of patients’ biological fluids was carried out in accordance with approved methodological guidelines. The results of determining the susceptibility of isolated pathogens were interpreted based on the EUCAST v14.0 criteria. Automatic bacteriological analyzers, MALDI-TOF mass spectrometry for pathogen identification, PCR and immunochromatographic methods for determining resistance mechanisms were used. To determine the etiological structure of infections in hospitalized patients, only original isolates were considered, following the principle of «one patient – one microbe.» Results. A total of 1213 bacterial and 53 fungal pathogens were isolated from 394 patients. The leading pathogens were Klebsiella pneumoniae (18.5%), Acinetobacter baumannii (13.4%), and Pseudomonas aeruginosa (9.5%). Resistance to meropenem was 83.0% for K. pneumoniae, 97.4% for A. baumannii, and 81.5% for P. aeruginosa. The highest in vitro activity against Enterobacterales was exhibited by ceftazidime-avibactam (79.2% of susceptible isolates), while against A. baumannii and P. aeruginosa, it was colistin (98.6% and 90.6% of susceptible isolates, respectively). The genetic mechanisms of carbapenem resistance in Enterobacterales were identified: production of NDM (26.3%), OXA-48 (29.6%), and KPC (44.1%) carbapenemases. Conclusions. The predominant pathogens of infections in surgical ICU patients are gram-negative bacteria with a high level of antibiotic resistance, including carbapenem-resistant Enterobacterales, whose resistance is due to the production of carbapenemases of various molecular classes. An increase in the prevalence of the serine carbapenemase KPC, previously atypical for the Russian Federation, has been noted. Currently, for infections caused by Enterobacterales producing serine carbapenemases, ceftazidime-avibactam can be recommended, and in the case of metallo-beta-lactamase production by Enterobacterales, a combination of ceftazidime-avibactam with aztreonam. For infections caused by A. baumannii and P. aeruginosa, regimens including polymyxins are indicated.

Genetics of antibiotic resistance in methicillin-resistant Staphylococcus aureus (MRSA).

Methicillin-resistant Staphylococcus aureus (MRSA) strains pose a significant threat as common causes of bacterial infections in hospitals, often resistant to available antibiotics such as daptomycin, vancomycin, and linezolid. The continuous emergence of new MRSA isolates with no effective treatment options underscores a real threat to health among humans and animals, and the number of effective antibiotic therapies decreases with each passing year. In this review, we provide an overview of the most common genetic mechanisms of resistance to a broad spectrum of antibiotics in methicillin-resistant S. aureus.

Small-scale mutations are infrequent as mechanisms of resistance in post-PARP inhibitor tumour samples in high grade serous ovarian cancer

While the introduction of poly-(ADP)-ribose polymerase (PARP) inhibitors in homologous recombination DNA repair (HR) deficient high grade serous ovarian, fallopian tube and primary peritoneal cancers (HGSC) has improved patient survival, resistance to PARP inhibitors frequently occurs. Preclinical and translational studies have identified multiple mechanisms of resistance; here we examined tumour samples collected from 26 women following treatment with PARP inhibitors as part of standard of care or their enrolment in clinical trials. Twenty-one had a germline or somatic BRCA1/2 mutation. We performed targeted sequencing of 63 genes involved in DNA repair processes or implicated in ovarian cancer resistance. We found that just three individuals had a small-scale mutation as a definitive resistance mechanism detected, having reversion mutations, while six had potential mechanisms of resistance detected, with alterations related to BRCA1 function and mutations in SHLD2. This study indicates that mutations in genes related to DNA repair are detected in a minority of HGSC patients as genetic mechanisms of resistance. Future research into resistance in HGSC should focus on copy number, transcriptional and epigenetic aberrations, and the contribution of the tumour microenvironment.

Abstract A086: NST-628 is a novel molecular glue that inhibits signaling and pathway reactivation in oncogenic RAS-MAPK cancers

Abstract Alterations in the RAS-MAPK pathway are identified in nearly 30% of cancers and are associated with poor patient prognosis. Although available therapies target various nodes of the RAS-MAPK pathway, clinical anti-tumor activity is limited by paradoxical bypass mechanisms, feedback signaling and pathway reactivation, and genetic resistance mechanisms. NST-628 aims to overcome the limitations of traditional RAS-MAPK pathway inhibitors by stabilizing all RAF-MEK complexes in a conformation that blocks MEK phosphorylation, to sequester these nodes from dynamic signaling processes that promote RAF-mediated bypass signaling. In comparison with clinically approved RAS-MAPK pathway inhibitors, only NST-628 robustly and durably decreases pathway reactivation, as measured by MEK phosphorylation, in KRAS-G13D tumor models in vitro and in vivo. Immunoprecipitation of endogenous ternary complexes demonstrates that NST-628 promotes potent stabilization of CRAF-MEK, BRAF-MEK, and ARAF-MEK complexes with complete shutdown of MEK, ERK, and RSK phosphorylation. Moreover, NST-628 treatment completely prevents RAF paralog heterodimerization in RAS-driven cells. Dose-dependent panRAF-MEK complex stabilization was confirmed using recombinant pre-formed CRAF-MEK, BRAF-MEK, and ARAF-MEK complexes, and MEK and RAF paralog affinity increases in the presence of NST-628 were quantified via surface plasmon resonance (SPR). In this assay, NST-628 significantly increased the affinity of MEK with both inactive monomeric and active dimeric RAF complexes. In parallel, we structurally characterized NST-628 bound to CRAF-MEK, BRAF-MEK, ARAF-MEK complexes using x-ray crystallography and cryogenic electron microscopy (cryo-EM). NST-628 binds the interfacial allosteric pocket along the MEK activation loop, thereby preventing RAF-mediated activation of MEK. We also demonstrate RAF paralog-specific pocket features that NST-628 can engage, helping us rationalize the large increase in affinity observed for MEK and CRAF in the presence of NST-628. The increased stabilization of CRAF-MEK complex by NST-628 may contribute to the decreased pathway reactivation in specific biomarker-driven tumors. Through our robust biophysical and cellular characterization coupled with novel structural insights, we have defined the unique mechanism of action for NST-628 and provide insight into its best-in-class potential in RAS-MAPK activated cancers. Citation Format: Bradley Quade, Meagan Ryan, Brooke Swalm, Steven Cohen, Zhong Fang, Aysegul Ozen, Xin Huang, Arvin C Dar, Yongxin Han, Klaus P Hoeflich, Margit Hagel, Michael Hale. NST-628 is a novel molecular glue that inhibits signaling and pathway reactivation in oncogenic RAS-MAPK cancers [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A086.

Recent Advances in the Genetic and Biochemical Mechanisms of Rice Resistance to Brown Planthoppers (Nilaparvata lugens Stål)

Rice (Oryza sativa L.) is the staple food of more than half of Earth’s population. Brown planthopper (Nilaparvata lugens Stål, BPH) is a host-specific pest of rice responsible for inducing major losses in rice production. Utilizing host resistance to control N. lugens is considered to be the most cost-effective method. Therefore, the exploration of resistance genes and resistance mechanisms has become the focus of breeders’ attention. During the long-term co-evolution process, rice has evolved multiple mechanisms to defend against BPH infection, and BPHs have evolved various mechanisms to overcome the defenses of rice plants. More than 49 BPH-resistance genes/QTLs have been reported to date, and the responses of rice to BPH feeding activity involve various processes, including MAPK activation, plant hormone production, Ca2+ flux, etc. Several secretory proteins of BPHs have been identified and are involved in activating or suppressing a series of defense responses in rice. Here, we review some recent advances in our understanding of rice–BPH interactions. We also discuss research progress in controlling methods of brown planthoppers, including cultural management, trap cropping, and biological control. These studies contribute to the establishment of green integrated management systems for brown planthoppers.

Extended Follow-up and Resistance Mutations in CLL Patients Treated with Acalabrutinib

Background: Covalent Bruton tyrosine kinase inhibitor (cBTKi) is a standard treatment for chronic lymphocytic leukemia (CLL). Understanding of acquired resistance to cBTKi therapy has largely come from data on patients (pts) treated with the first-in-class cBTKi ibrutinib. Mutations in BTK C481 and to a lesser extent PLCG2 are frequently detected in ibrutinib-treated pts at the time of progressive disease (PD). In contrast, much less is known about genetic mechanisms of drug resistance in pts treated with next generation cBTKi acalabrutinib and zanubrutinib. A recent study identified 3 pts with BTK C481 mutations and no pts with PLCG2 mutations among 8 acalabrutinib-treated pts with progressive disease (PD) (Black, et al. Blood 2022). A second study found a kinase-dead BTK L528W mutation in 7/13 zanubrutinib-treated pts (Blombery, et al. Blood Adv 2022). Although cBTKi share the same mechanism of action by binding to BTK C481, the latter report discloses differences in clonal selection and underscores the need for further investigation in pts treated with next generation cBTKi. In a phase 2 study of acalabrutinib in pts with treatment naïve (TN) CLL or small lymphocytic lymphoma (SLL) with 17p deletion, or TP53 or NOTCH1 mutation, or relapsed/refractory (R/R) CLL/SLL regardless of cytogenetics or somatic mutations (Sun, et al. Blood 2020), we sought to understand clonal evolution during treatment. Methods: Peripheral blood mononuclear cells (PBMCs) were collected from 48 pts at baseline, after 6 months, then annually on treatment until PD. Current analysis includes 14 PD pts [excluding 3 Richter transformation (RT) pts] comprising 10 R/R and 4 TN CLL pts. Additionally, paired baseline and PD bone marrow (BM) from 6 pts and lymph node (LN) samples from 2 pts were included. Targeted next-generation sequencing of ~700 recurrently mutated genes in hematological malignancies (Collins, et al. Leuk Lymphoma. 2021) was performed using DNA isolated from CD19 + enriched cryopreserved PBMCs and BM aspirates, as well as fresh-frozen core LN biopsies. Filtered variants in putative CLL driver genes (Knisbacher, et al. Nat Genet. 2022) with variant allele frequencies (VAF) ≥1% for BTK and PLCG2, ≥5% for variants with known oncological significance, and ≥7.5% for all other variants were included in downstream analysis. Results: After a median follow-up of 6.5 years (IQR 2.9-6.3), 23/48 (48%) pts developed PD (20 CLL, 3 RT). PD occurred in 17/32 (53%) R/R pts and 6/16 (38%) TN pts. Median progression-free survival was reached at 6.0 years. Best response was complete remission in 2/20 (10%) pts and partial remission in 18/20 (90%) pts with CLL PD. IGHV was unmutated in all but one PD patient. At baseline, 34 CLL driver genes were mutated in 41/45 pts (91%; excluding 3 RT pts) with most mutations in NOTCH1, TP53, ATM, SF3B1, BIRC3, and NFKBIE. During acalabrutinib treatment but before PD, 14/45 pts (31%; 8 R/R and 6 TN) acquired mutations in 11 driver genes, most commonly in BRAF (n=4), CREBBP (n=4), and KMT2D (n=2). At the time of PD, 11/14 pts (79%) had newly acquired mutations in 4 genes, predominantly BTK (n=6) and PLCG2 (n=4). BTK C481 mutations (VAF 2-42%) were detected in 6/14 (43%; 4 R/R and 2 TN) pts and T474I mutation (VAF 4-46%) in 3/14 (21%; 1 R/R and 2 TN) pts. Paired BM samples in 2 pts and LN samples in 2 pts showed the same BTK mutations as PBMCs with similar VAFs. PLCG2 mutations (VAF 1-8%) were found in 4/14 (29%; 2 R/R and 2 TN) pts. BTK C481 co-occurred with BTK T474I in 3 pts, but on different sequencing reads suggesting independent subclones, and with a PLCG2 mutation in 1 pt. There have been no pts with BTK L528 mutations. In conclusion, mutations in BTK and PLCG2 are the predominant resistance mechanism to acalabrutinib. Among BTK mutations, C481 was the most common. All T474I mutations co-occurred with C481.

Single-Cell Proteogenomics Analysis of AML Samples Treated with Decitabine and Venetoclax Reveals Divergent Treatment Escape Mechanisms

Background: The combination therapy of hypomethylating agents and venetoclax (HMA+VEN) has emerged as a standard of care for acute myeloid leukemia (AML) patients unfit for intensive chemotherapy. Nonetheless, significant clinical challenges persist, including relapse and treatment resistance. Previous studies have revealed genetic ( RAS/RTK pathway mutations and TP53 mutations) and phenotypic (monocytic differentiation) resistance mechanisms in the context of HMA+VEN therapy. Despite these advancements, our comprehensive understanding of the HMA+VEN resistance mechanisms remains incomplete. To gain further insights into the resistance mechanism in HMA+VEN therapy, we implemented single-cell proteogenomics analysis on longitudinally collected samples from AML patients treated with decitabine+venetoclax (DAC+VEN) trial. Methods: We performed a high-throughput, single-cell targeted DNA-antibody sequencing (DAb-seq) using Tapestri platform from Mission Bio Inc. Sixty-three AML samples sequentially collected from 33 unique patients who participated in DAC+VEN clinical trial (NCT03404193) were analyzed. Our custom DNA panel covered 37 recurrently mutated genes in AML, whereas the custom antibody panel included either 15 cell surface proteins (N = 20 patients) or 48 proteins (N = 13 patients). Cellular phenotypes were inferred based on the patterns of cell surface protein expression. Median 2,767 cells were sequenced per sample, with total 196,719 cells sequenced for this study. Results: The median age of the cohort was 70 years (range 21-84). Twenty-four (73%) patients had primary AML and 9 (27%) had secondary or therapy-related AML. Based on the FAB classification, 13 patients had M0/M1/M2, 6 had M4/M5, one had M7, and the rest had unknown subtypes. The most frequent gene mutation at baseline was DNMT3A (45%), followed by NPM1 (39%), TET2 (30%), and TP53 (24%). Among the 33 patients, 32 had evaluable treatment response; 27 (82%) were defined as responders by achieving CR or CRi, whereas 5 (15%) were non-responders. Six patients experienced relapse after responding to the therapy, and their baseline-relapse pairs were analyzed by the single-cell platform. Using the composition of cellular phenotype data, AML samples were classified into either stem-like (N = 5), progenitor-like (N = 1), predominantly monocytic (N = 4), predominantly erythroid (N = 2) and mixed population AML (mixed population of stem, progenitor, monocyte or erythroid-like population, N = 21). Genotype-phenotype analysis showed a significant correlation between the KRAS mutation and the mixed phenotype (p= 0.0403), as well as between the U2AF1 mutation and the mixed phenotype (p= 0.0471). There was no significant association between treatment response and AML phenotype. Analysis of six baseline-relapse pairs showed that relapse was associated with phenotypic shift of AML cells. Stem-like cells were largely eradicated at relapse and replaced by more differentiated cells. Among the six baseline-relapse pairs, three showed monocytic shift, one showed erythroid shift and two showed mixed monocytic/erythroid shift. Phenotypic shifts at relapse were not always associated with expansion of new genetic mutations, except for in two cases: one had monocytic shift accompanied with expansion of KRAS mutation, and another had erythroid shift accompanied with expansion of NRAS mutation. Conclusions: Phenotypic transition from stem-like cells to more differentiated cells (either monocytic or erythroid) was frequently observed in AML relapse following DAC+VEN therapy. These findings are consistent with the known dependency of these cells on non-BCL-2 anti-apoptotic proteins such as MCL-1 and BCL-xL. Close monitoring of these phenotypic alterations throughout the course of HMA+VEN treatment may potentially facilitate the precise identification of cellular populations predisposed to relapse.

Contrasting roles of cytochrome P450s in amitraz and chlorfenapyr resistance in the crop pest Tetranychus urticae

The molecular mechanisms of amitraz and chlorfenapyr resistance remain only poorly understood for major agricultural pests and vectors of human diseases. This study focusses on a multi-resistant field strain of the crop pest Tetranychus urticae, which could be readily selected in the laboratory to high levels of amitraz and chlorfenapyr resistance. Toxicity experiments using tralopyril, the active toxophore of chlorfenapyr, suggested decreased activation as a likely mechanism underlying resistance. Starting from the same parental strain, transcriptome profiling revealed that a cluster of detoxifying genes was upregulated after amitraz selection, but unexpectedly downregulated after chlorfenapyr selection. Further functional validation associated the upregulation of CYP392A16 with amitraz metabolism and the downregulation of CYP392D8 with reduced activation of chlorfenapyr to tralopyril. Genetic mapping (QTL analysis by BSA) was conducted in an attempt to unravel the genetic mechanisms of expression variation and resistance. This revealed that chlorfenapyr resistance was associated with a single QTL, while 3 QTLs were uncovered for amitraz resistance. Together with the observed contrasting gene expression patterns, we argue that transcriptional regulators most likely underly the distinct expression profiles associated with resistance, but these await further functional validation.