Emergence Of Resistance Mutations Research Articles

Metabolic profiling unveils enhanced antibacterial synergy of polymyxin B and teixobactin against multi-drug resistant Acinetobacter baumannii

This untargeted metabolomics study investigated the synergistic antibacterial activity of polymyxin B and Leu10-teixobactin, a depsipeptide inhibitor of cell wall biosynthesis. Checkerboard microdilution assays revealed a significant synergy against polymyxin-susceptible and -resistant A. baumannii, excluding lipopolysaccharide-deficient variants. Time-kill assays confirmed bactericidal synergy, reducing bacterial burden by approximately 4-6-log10CFU/mL. The combination (2xMIC polymyxin B and 0.5xMIC Leu10-teixobactin) prevented bacterial regrowth after 24 h, indicating sustained efficacy against the emergence of resistant mutants. The analysis of A. baumannii ATCC™ 19606 metabolome demonstrated that the polymyxin B–Leu10-teixobactin combination produced more pronounced perturbation compared to the individual antibiotics across all time points (1, 3 and 6 h). Pathway analysis revealed that lipid metabolism, cell envelope biogenesis, and cellular respiration were predominantly impacted by the combination, and to a lesser extent by polymyxin B monotherapy. Leu10-teixobactin treatment alone had only a minor impact on the metabolome, primarily at the 6 h time point. Peptidoglycan assays confirmed the combination’s concerted deleterious effects on bacterial cell envelope integrity. Electron microscopy further substantiated these findings, revealing pronounced cell envelope damage, membrane blebbing, and vacuole formation. These findings highlight the potential of the polymyxin B–Leu10-teixobactin combination as an effective treatment in preventing resistance in A. baumannii.

Diagnostic utility of ESR1 mutation detection in liquid biopsy of metastatic breast cancer patients.

Molecular analysis of circulating cell-free DNA (cfDNA) extracted from peripheral blood plasma samples of metastatic breast cancer (BC) patients is of rising interest to find optimal therapeutic strategies. Detection of emerging resistance mutations against endocrine therapy is possible with this approach. Here we present the applicability of a laboratory-developed NGS assay in molecular pathology routine diagnostic, covering four genes with therapeutic (ESR1, PIK3CA, ERBB2) and prognostic (TP53) consequences in metastatic BC. We analyzed 162 liquid biopsy samples and 25 corresponding metastases from metastatic BC patients. In the liquid biopsies, we detected ESR1 mutations in 42 cases (25.9%) and ERBB2 mutations in six cases (3.7%), arguing for a change in therapy to fulvestrant, elacestrant, or neratinib. Furthermore, 17 cases had detectable TP53 mutations, associated with resistance against endocrine therapy. We conclude that liquid biopsy testing is a noninvasive, sensitive, and helpful method to optimize therapeutic decisions in metastatic BC.

Clinical Value of Liquid Biopsy in Patients with FGFR2 Fusion-Positive Cholangiocarcinoma During Targeted Therapy.

FGFR2 fusions occur in 10% to 15% of patients with intrahepatic cholangiocarcinoma (iCCA), potentially benefiting from FGFR inhibitors (FGFRi). We aimed to assess the feasibility of detecting FGFR2 fusions in plasma and explore plasma biomarkers for managing FGFRi treatment. We conducted a retrospective study in 18 patients with iCCA and known FGFR2 fusions previously identified in tissue samples from prior FGFRi treatment. Both tissue and synchronous plasma samples were analyzed using a custom hybrid capture gene panel with next-generation sequencing (VHIO-iCCA panel) and validated against commercial vendor results. Longitudinal plasma analysis during FGFRi was performed. Subsequently, we explored the correlation between plasma biomarkers, liver enzymes, tumor volume, and clinical outcomes. Sixteen patients (88.9%) were positive for FGFR2 fusion events in plasma. Remarkably, the analysis of plasma suggests that lower levels of ctDNA are linked to clinical benefits from targeted therapy and result in improved progression-free survival and overall survival. Higher concentrations of cell-free DNA before FGFRi treatment were linked to worse overall survival, correlating with impaired liver function and indicating compromised cell-free DNA removal by the liver. Additionally, increased ctDNA or the emergence of resistance mutations allowed earlier detection of disease progression compared with standard radiologic imaging methods. VHIO-iCCA demonstrated accurate detection of FGFR2 fusions in plasma. The integration of information from various plasma biomarkers holds the potential to predict clinical outcomes and identify treatment failure prior to radiologic progression, offering valuable guidance for the clinical management of patients with iCCA.

DORAVIR: a French national survey of people with HIV-1 treated with an antiretroviral regimen including doravirine.

Doravirine is the latest NNRTI to be approved for the treatment of HIV-1 and has a different resistance profile from first-generation NNRTIs. Our aim was to investigate the virological efficacy of antiretroviral treatment including doravirine in people living with HIV-1 (PLWHIV), the factors associated with virological failure (VF) and those associated with the emergence of reverse transcriptase (RT) mutations in the case of VF. A retrospective national survey of PLWHIV who were either naive or experienced on antiretroviral treatment including doravirine was conducted. VF was defined as two consecutive plasma viral loads (VLs) of ≥50 copies/mL or one VL of ≥200 copies/mL. Genotypic resistance tests were interpreted using the Stanford (v9.4.1) and ANRS (v33) algorithms. Of the 589 PLWHIV treated with a doravirine-containing regimen, 8.5% were naive and 91.5% had prior antiretroviral experience; 56.9% were infected with HIV-1 B subtype. Overall, 88.3% and 85.1% of participants were virologically controlled at Month (M)3 and M6 of doravirine treatment, respectively. In multivariable analysis, CRF02_AG subtype, higher zenith plasma HIV-1 RNA VL, doravirine initiation in the context of failure and baseline V179D mutation presence were associated with VF. Among 88 PLWHIV who experienced virological failure at M6, 15.9% had a median of 2 (IQR 1-3) HIV RT mutations. In multivariable analysis, the only factor associated with the occurrence of mutations was a genotypic sensitivity score that was not fully sensitive. This study is one of the largest to characterize the virological efficacy of doravirine-containing regimens in clinical practice and to identify factors associated with VF or emergence of resistance mutations that should be considered in clinical management.

Custom-Designed Probes for the Accurate Determination of Epidermal Growth Factor Receptor Mutations and Their Allelic Configuration.

The identification of single nucleotide polymorphisms (SNPs) is of paramount importance for disease diagnosis and clinical prognostication. In the context of nonsmall cell lung cancer (NSCLC), the emergence of resistance mutations, exemplified by the epidermal growth factor receptor (EGFR) T790 M and C797S, is intricately linked to the therapeutic efficacy of EGFR tyrosine kinase inhibitors (EGFR-TKIs). Herein, a highly efficient and specific SNP detection platform for T790 M and C797S mutations has been engineered through the integration of an asymmetric polymerase chain reaction (PCR) and an ingeniously tailored four-way junction (4WJ) probe. Notably, a molecular beacon (MB) probe was judiciously designed to discern the allelic configuration of these mutations. The administration of first- and third-generation EGFR-TKIs demonstrates therapeutic efficacy solely when the mutations are in the trans configuration, characterized by a low fluorescence signal. In contrast, significant fluorescence by the MB probe is indicative of the C797S mutation being in a cis arrangement with T790M, thereby rendering the cells refractory to the therapeutic interventions of both first- and third-generation EGFR-TKIs. The assay is capable of concurrently detecting two point-mutations and ascertaining their allelic positions in a single test within 1.5 h, enhancing both efficiency and simplicity. It also exhibits high accuracy in the identification of clinical samples, offering promising implications for therapeutic guidelines. By enabling tailored treatment plans based on specific genetic profiles, our approach not only advances the precision of NSCLC treatment strategies but also marks a significant contribution to personalized medicine.

Developing predictive models for HIV Drug resistance: A genomic and AI approach

This paper proposes a novel approach to combating HIV drug resistance through the development of predictive models leveraging genomic data and artificial intelligence (AI). With the increasing prevalence of drug-resistant strains of HIV, there is a critical need for innovative strategies to predict and manage resistance mutations, thereby optimizing treatment outcomes and prolonging the efficacy of antiretroviral therapy (ART). Drawing on advances in genomics and AI, this study outlines a conceptual framework for the development of predictive models that can identify potential drug-resistance mutations in HIV genomes and inform clinical decision-making. The proposed framework integrates genomic data from HIV-infected individuals with AI algorithms capable of learning complex patterns within the data. By analyzing genomic sequences obtained from HIV-positive patients, the models aim to identify genetic variations associated with drug resistance, predict the likelihood of resistance development, and guide the selection of appropriate treatment regimens. This approach holds promise for personalized medicine in HIV care, enabling clinicians to tailor therapy based on an individual's genetic profile and risk of resistance. Key components of the conceptual framework include data preprocessing to extract relevant genomic features, model training using machine learning techniques such as deep learning and ensemble methods, and validation of predictive performance through cross-validation and independent testing. Furthermore, the integration of clinical data, such as treatment history and viral load measurements, enhances the predictive accuracy of the models and provides valuable insights into treatment response dynamics.The development of predictive models for HIV drug resistance represents a paradigm shift in HIV care, offering a proactive approach to treatment management and surveillance. By leveraging genomic and AI technologies, healthcare providers can anticipate and address emerging resistance mutations before they compromise treatment efficacy. Ultimately, the implementation of predictive models holds the potential to improve patient outcomes, reduce the transmission of drug-resistant HIV strains, and advance the global fight against HIV/AIDS. Keywords: Developing, Predictive Models, HIV Drug Resistance, Genomic, AI Approach.

Abstract GS03-10: Allosteric PI3K-alpha inhibition overcomes on-target resistance to orthosteric inhibitors mediated by secondary PIK3CA mutations

Abstract Background: PIK3CA mutations occur in ~40% of HR-positive breast cancers, where alpelisib, an orthosteric PI3Kα inhibitor, is FDA-approved in combination with fulvestrant. Although prior studies have identified potential resistance mechanisms, such as PTEN loss, clinical acquired resistance to orthosteric PI3Kα inhibitors and the role of next-generation allosteric PI3Kα inhibitors remain poorly understood. Methods: To identify on-target and off-target alterations potentially mediating resistance to PI3Kα inhibitors, we used a targeted next-generation sequencing assay (Guardant360; Guardant Health) to analyze ctDNA in serially collected plasma samples from 32 patients with PIK3CA-mutated advanced HR-positive, HER2-negative breast cancer treated with alpelisib and inavolisib. In addition, we performed whole exome sequencing (WES) of 100 tissue samples collected from 8 autopsy series from patients with metastatic, PIK3CA-mutant HR-positive, HER2-negative breast cancer previously treated with PI3Kα inhibitors. Acquired alterations were prioritized through a combination of structural modeling and free-energy perturbation simulation and validated in genomically engineered PIK3CA mutant breast cancer cell lines T47D (PIK3CA H1047R-mutant) or MCF7 (PIK3CA E545K-mutant). Results: We observed that 50% of patients acquire genomic alterations within the PI3K-pathway, including PTEN loss and activating AKT1 mutations. Notably, while secondary PIK3CA mutations were previously reported to increase sensitivity to PI3Ka-inhibitors, we identified emergent secondary resistance mutations in PIK3CA that alter the inhibitor binding pocket including PIK3CA Q859K and PIK3CA W780R. Some mutations had differential effects on PI3Ka-selective vs. pan-PI3K inhibitors, but resistance induced by all mutations could be overcome by the novel allosteric pan-mutant-selective PI3Ka-inhibitor RLY-2608. Conclusion: In one of the largest patient cohorts analyzed to date, this study defines the clinical landscape of acquired resistance to PI3Ka inhibitors. Genomic alterations within the PI3K pathway represent a major mode of resistance and identify a novel class of secondary PIK3CA resistance mutations that can be overcome by an allosteric PI3Ka inhibitor. Together, these findings provide insights to guide strategies to overcome resistance in PIK3CA-mutated cancers. Citation Format: Andreas Varkaris, Ferran Fece de la Cruz, Elizabeth Martin, Bryanna Nordern, Nicholas Chevalier, Ignaty Leshchiner, Anastasia Stavridi, Janice Kim, Alkistis Papatheodoridi, Hakan Gunaydin, Brian Danysh, Laxmi Parida, Ioannis Sanidas, Yongli Ji, Kayao Lau, Gerburg Wulf, Aditya Bardia, Laura Spring, Steven Isakoff, Jochen Lennerz, Levi Pierce, Ermira Pazolli, Gad Getz, Ryan Corcoran, Dejan Juric. Allosteric PI3K-alpha inhibition overcomes on-target resistance to orthosteric inhibitors mediated by secondary PIK3CA mutations [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr GS03-10.

Molecular Insights into the Impact of Mutations on the Binding Affinity of Targeted Covalent Inhibitors of BTK.

Targeted covalent inhibitors (TCIs) have witnessed a significant resurgence in recent years, particularly in the kinase drug discovery field for treating diverse clinical indications. The inhibition of Bruton's tyrosine kinase (BTK) for treating B-cell cancers is a classic example where TCIs such as ibrutinib have had breakthroughs in targeted therapy. However, selectivity remains challenging, and the emergence of resistance mutations is a critical concern for clinical efficacy. Computational methods that can accurately predict the impact of mutations on inhibitor binding affinity could prove helpful in informing targeted approaches─providing insights into drug resistance mechanisms. In addition, such systems could help guide the systematic evaluation and impact of mutations in disease models for optimal experimental design. Here, we have employed in silico physics-based methods to understand the effects of mutations on the binding affinity and conformational dynamics of select TCIs of BTK. The TCIs studied include ibrutinib, acalabrutinib, and zanubrutinib─all of which are FDA-approved drugs for treating multiple forms of leukemia and lymphoma. Our results offer useful molecular insights into the structural determinants, thermodynamics, and conformational energies that impact ligand binding for this biological target of clinical relevance.

Prevalence of Emergent Dolutegravir Resistance Mutations in People Living with HIV: A Rapid Scoping Review.

Dolutegravir (DTG) is a cornerstone of global antiretroviral (ARV) therapy (ART) due to its high efficacy and favorable tolerability. However, limited data exist regarding the risk of emergent integrase strand transfer inhibitor (INSTI) drug-resistance mutations (DRMs) in individuals receiving DTG-containing ART. We performed a PubMed search using the term "Dolutegravir", last updated 18 December 2023, to estimate the prevalence of VF with emergent INSTI DRMs in people living with HIV (PLWH) without previous VF on an INSTI who received DTG-containing ART. Of 2131 retrieved records, 43 clinical trials, 39 cohorts, and 6 cross-sectional studies provided data across 6 clinical scenarios based on ART history, virological status, and co-administered ARVs: (1) ART-naïve PLWH receiving DTG plus two NRTIs; (2) ART-naïve PLWH receiving DTG plus lamivudine; (3) ART-experienced PLWH with VF on a previous regimen receiving DTG plus two NRTIs; (4) ART-experienced PLWH with virological suppression receiving DTG plus two NRTIs; (5) ART-experienced PLWH with virological suppression receiving DTG and a second ARV; and (6) ART-experienced PLWH with virological suppression receiving DTG monotherapy. The median proportion of PLWH in clinical trials with emergent INSTI DRMs was 1.5% for scenario 3 and 3.4% for scenario 6. In the remaining four trial scenarios, VF prevalence with emergent INSTI DRMs was ≤0.1%. Data from cohort studies minimally influenced prevalence estimates from clinical trials, whereas cross-sectional studies yielded prevalence data lacking denominator details. In clinical trials, the prevalence of VF with emergent INSTI DRMs in PLWH receiving DTG-containing regimens has been low. Novel approaches are required to assess VF prevalence with emergent INSTI DRMs in PLWH receiving DTG in real-world settings.

FGFR-targeted therapeutics: clinical activity, mechanisms of resistance and new directions.

Fibroblast growth factor (FGF) signalling via FGF receptors (FGFR1-4) orchestrates fetal development and contributes to tissue and whole-body homeostasis, but can also promote tumorigenesis. Various agents, including pan-FGFR inhibitors (erdafitinib and futibatinib), FGFR1/2/3 inhibitors (infigratinib and pemigatinib), as well as a range of more-specific agents, have been developed and several have entered clinical use. Erdafitinib is approved for patients with urothelial carcinoma harbouring FGFR2/3 alterations, and futibatinib and pemigatinib are approved for patients with cholangiocarcinoma harbouring FGFR2 fusions and/or rearrangements. Clinical benefit from these agents is in part limited by hyperphosphataemia owing to off-target inhibition of FGFR1 as well as the emergence of resistance mutations in FGFR genes, activation of bypass signalling pathways, concurrent TP53 alterations and possibly epithelial-mesenchymal transition-related isoform switching. The next generation of small-molecule inhibitors, such as lirafugratinib and LOXO-435, and the FGFR2-specific antibody bemarituzumab are expected to have a reduced risk of hyperphosphataemia and the ability to overcome certain resistance mutations. In this Review, we describe the development and current clinical role of FGFR inhibitors and provide perspective on future research directions including expansion of the therapeutic indications for use of FGFR inhibitors, combination of these agents with immune-checkpoint inhibitors and the application of novel technologies, such as artificial intelligence.

Generation and evaluation of protease inhibitor-resistant SARS-CoV-2 strains

Since the start of the SARS-CoV-2 pandemic, the search for antiviral therapies has been at the forefront of medical research. To date, the 3CLpro inhibitor nirmatrelvir (Paxlovid®) has shown the best results in clinical trials and the greatest robustness against variants. A second SARS-CoV-2 protease inhibitor, ensitrelvir (Xocova®), has been developed. Ensitrelvir, currently in Phase 3, was approved in Japan under the emergency regulatory approval procedure in November 2022, and is available since March 31, 2023. One of the limitations for the use of antiviral monotherapies is the emergence of resistance mutations. Here, we experimentally generated mutants resistant to nirmatrelvir and ensitrelvir in vitro following repeating passages of SARS-CoV-2 in the presence of both antivirals. For both molecules, we demonstrated a loss of sensitivity for resistance mutants in vitro. Using a Syrian golden hamster infection model, we showed that the ensitrelvir M49L mutation, in the multi-passage strain, confers a high level of in vivo resistance. Finally, we identified a recent increase in the prevalence of M49L-carrying sequences, which appears to be associated with multiple repeated emergence events in Japan and may be related to the use of Xocova® in the country since November 2022. These results highlight the strategic importance of genetic monitoring of circulating SARS-CoV-2 strains to ensure that treatments administered retain their full effectiveness.

495. De novo emergence of resistance mutations during treatment of SARSCoV-2 infection in immunocompromised patients

Abstract Background Vaccination and therapeutic strategies for COVID-19 are constantly hindered by the rapid emergence of new viral variants less sensitive to antibodies or antivirals. Emerging evidence points to prolonged replication in the immunocompromised host as a key factor contributing to emergence of viral genomic mutations. We analyzed viral evolution and emergence of intra-host variants in response to treatments including antivirals and monoclonal antibodies in immunocompromised subjects with hematological malignancies infected with SARS-CoV2. Methods From April 2020 to August 2022, we collected 245 nasopharyngeal samples from 92 patients including longitudinal samples of 31 subjects with persistent infection (detection of SARS-CoV-2 RNA ≥30 days regardless of symptomatology). The number of samples per subject ranged from 1-14. To identify SARS-CoV-2 mutations, we performed whole genome sequencing using an amplicon-based approach and the Illumina NextSeq platform. Only samples that had 75% genome covered at 5x read depth were included in the analysis. Results After quality control filtering, 21 subjects had at least 2 samples available for longitudinal analyses. Ten of them had no consensus changes during infection (compared to the first sample collected), and 11 had at least 1 change. The spike protein was the most likely location for a consensus change (9 patients), followed by consensus changes in nsp3, nsp12, nsp13, N, ORF8, and nsp4. Mutations tended to appear mostly following treatment, with an increased number of changes apparently associated with worse outcome. After treatment, 3 patients developed mutations in the RNA-dependent RNA polymerase (nsp12) that are known to be associated with remdesivir resistance. One Paxlovid treated patient developed a nsp5 protease mutation potentially associated with resistance. Another patient developed a spike mutation associated with resistance to sotrovimab. Conclusion Our results underscore the need for augmented efforts to study intra-host evolution in the immunocompromised host, identify and isolate viruses resistant to treatment(s) and identify host determinants associated with accelerated resistance. These results are important for implementing optimal treatment strategies and to inform infection control measures. Disclosures All Authors: No reported disclosures

Spike Protein Genetic Evolution in Patients at High Risk of Severe Coronavirus Disease 2019 Treated by Monoclonal Antibodies.

High-risk patients, often immunocompromised and not responding to vaccine, continue to experience severe coronavirus disease 2019 (COVID-19) and death. Monoclonal antibodies (mAbs) were shown to be effective to prevent severe COVID-19 for these patients. Nevertheless, concerns about the emergence of resistance mutations were raised. We conducted a multicentric prospective cohort study, including 264 patients with mild to moderate COVID-19 at high risk for progression to severe COVID-19 and treated early with casirivimab/imdevimab, sotrovimab, or tixagevimab/cilgavimab. We sequenced the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome during follow-up and searched for emerging spike mutations. Immunocompromised patients have a 6-fold increased risk of developing mutations, which are associated with a prolonged duration of viral clearance but no clinical worsening. Emerging P337S/R/L/H, E340D/K/A/Q/V/G, and K356T/R substitutions in patients treated with sotrovimab are associated with higher viral RNA loads for up to 14 days post-treatment initiation. Tixagevimab/cilgavimab is associated with a 5-fold increased risk of developing mutations. R346K/I/T/S and K444R/N/M substitutions associated with tixagevimab/cilgavimab have been identified in multiple SARS-CoV-2 lineages, including BQ.1 and XBB. The probability of emerging mutations arising in response to mAbs is significant, emphasizing the crucial need to investigate these mutations thoroughly and assess their impact on patients and the evolutionary trajectory of SARS-CoV-2.

Exploring novel HIV-1 reverse transcriptase inhibitors with drug-resistant mutants: A double mutant surprise.

HIV-1 reverse transcriptase (RT) remains a key target for HIV drug development. As successful management of the disease requires lifelong treatment, the emergence of resistance mutations is inevitable, making development of new RT inhibitors, which remain effective against resistant variants crucial. To this end, previous computationally guided drug design efforts have resulted in catechol diether compounds, which inhibit wildtype RT with picomolar affinities and appear to be promising preclinical candidates. To confirm that these compounds remain potent against Y181C, a widespread mutation conferring resistance to first generation inhibitors, they were screened against the HIV-1 N119 clinical isolate, reported as a Y181C single mutant. In comparison to a molecular clone with the same mutation, N119 appears less susceptible to inhibition by our preclinical candidate compounds. A more detailed sequencing effort determined that N119 was misidentified and carries V106A in combination with Y181C. While both indolizine and naphthalene substituted catechol diethers are potent against the classical Y181C single mutant, the addition of V106A confers more resistance against the indolizine derivatives than the naphthalene derivatives. Crystal structures presented in this study highlight key features of the naphthyl group, which allow these compounds to remain potent in the double mutant, including stronger interactions with F227 and less reliance on V106 for stabilization of the ethoxy-uracil ring, which makes critical hydrogen bonds with other residues in the binding pocket.

Allosteric PI3Kα Inhibition Overcomes On-target Resistance to Orthosteric Inhibitors Mediated by Secondary PIK3CA Mutations.

In one of the largest patient cohorts analyzed to date, this study defines the clinical landscape of acquired resistance to PI3Kα inhibitors. Genomic alterations within the PI3K pathway represent a major mode of resistance and identify a novel class of secondary PIK3CA resistance mutations that can be overcome by an allosteric PI3Kα inhibitor. See related commentary by Gong and Vanhaesebroeck, p. 204 . See related article by Varkaris et al., p. 240 . This article is featured in Selected Articles from This Issue, p. 201.

A biophysical framework for double-drugging kinases

Selective orthosteric inhibition of kinases has been challenging due to the conserved active site architecture of kinases and emergence of resistance mutants. Simultaneous inhibition of distant orthosteric and allosteric sites, which we refer to as "double-drugging", has recently been shown to be effective in overcoming drug resistance. However, detailed biophysical characterization of the cooperative nature between orthosteric and allosteric modulators has not been undertaken. Here, we provide a quantitative framework for double-drugging of kinases employing isothermal titration calorimetry, Förster resonance energy transfer, coupled-enzyme assays, and X-ray crystallography. We discern positive and negative cooperativity for Aurora A kinase (AurA) and Abelson kinase (Abl) with different combinations of orthosteric and allosteric modulators. We find that a conformational equilibrium shift is the main principle governing cooperativity. Notably, for both kinases, we find a synergistic decrease of the required orthosteric and allosteric drug dosages when used in combination to inhibit kinase activities to clinically relevant inhibition levels. X-ray crystal structures of the double-drugged kinase complexes reveal the molecular principles underlying the cooperative nature of double-drugging AurA and Abl with orthosteric and allosteric inhibitors. Finally, we observe a fully closed conformation of Abl when bound to a pair of positively cooperative orthosteric and allosteric modulators, shedding light on the puzzling abnormality of previously solved closed Abl structures. Collectively, our data provide mechanistic and structural insights into rational design and evaluation of double-drugging strategies.

Treatment-emergent reverse transcriptase resistance during antiretroviral therapy with bictegravir, tenofovir alafenamide, and emtricitabine: A case series.

Bictegravir/tenofovir alafenamide/emtricitabine (BIC/TAF/FTC) is a complete regimen for the treatment of HIV with a high barrier to resistance and few reported cases of treatment failure. We present three cases of treatment-emergent resistance to nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs) in patients with suboptimal treatment adherence and assess whether the resistance-associated mutations were present before BIC/TAF/FTC initiation or emerged during therapy. We used genotypic drug resistance testing by Sanger sequencing to identify emergent resistance mutations in plasma viral load specimens collected after combination antiretroviral therapy initiation in all participants. Additionally, we performed ultra-deep sequencing by Illumina MiSeq on the earliest available plasma HIV-1 viral load specimen and on any available specimens closest in time to the initiation of BIC/TAF/FTC therapy to identify low-abundance resistance mutations present in the viral quasispecies. All three participants developed NRTI resistance after prolonged exposure and incomplete adherence to BIC/TAF/FTC. The T69N, K70E, M184I, and/or T215I mutations identified in clinical samples at the time of virological failure were not present on deep sequencing of either baseline samples or samples collected before BIC/TAF/FTC initiation. Despite a generally high genetic barrier to resistance, NRTI resistance-associated mutations may emerge during therapy with BIC/TAF/FTC in the setting of suboptimal adherence.

Abstract 4022: Preclinical intracranial activity of NVL-655 in an alectinib-resistant patient-derived model harboring EML4-ALK fusion with G1202R mutation

Abstract Introduction: NVL-655 is a brain-penetrant, ALK-selective tyrosine kinase inhibitor (TKI) designed to maintain activity against ALK and ALK mutations that confer resistance to currently approved therapies, and to avoid neurological adverse events and dose-limiting toxicities associated with TRK inhibition. Brain metastases are common in ALK-positive non-small cell lung cancer (NSCLC), with an incidence of ~40% at diagnosis and ~60% after progressive disease on the first-generation ALK TKI (crizotinib) due to its limited brain penetration. Second- (ceritinib, alectinib, brigatinib, and ensartinib) and third-generation (lorlatinib) ALK TKIs have higher intracranial responses than crizotinib but are still limited by emergence of resistance mutations, most commonly G1202R-containing single and compound mutations. We previously reported that NVL-655 has intracranial activity in a preclinical model of Ba/F3 EML4-ALK v1 G1202R/L1196M. Here we further demonstrate the intracranial activity of NVL-655 in a preclinical patient-derived model harboring EML4-ALK v3 G1202R. Methods: The YU-1077 cell line was established from a patient with ALK-positive NSCLC after relapse on alectinib and was confirmed to harbor EML4-ALK v3 G1202R alteration. ALK TKIs (crizotinib, ceritinib, alectinib, brigatinib, ensartinib, lorlatinib, and NVL-655) were profiled against YU-1077 cells in 3-day viability assays and signaling assays. YU-1077 cells were intracranially implanted in Balb/c nude mice and treated with NVL-655 orally twice daily. Brain tumor burden was monitored weekly using magnetic resonance imaging (MRI). Results: NVL-655 suppressed the viability of YU-1077 cells bearing EML4-ALK v3 G1202R (IC50 < 1 nM) with potency >10-fold that of lorlatinib. NVL-655 at ≤10 nM fully inhibited ALK signaling as measured by ALK, ERK, and S6 phosphorylation, compared to ≥100 nM of lorlatinib required to achieve similar inhibition. Other ALK TKIs tested had limited activity against YU-1077 cells in viability and signaling assays, consistent with the G1202R mutation conferring resistance to first- and second-generation ALK TKIs. In mice intracranially implanted with YU-1077 cells, MRI scans confirmed rapid tumor regression upon treatment with 0.5 and 1.5 mg/kg NVL-655. Conclusion: Patient-derived models are widely viewed as among the most disease-relevant models for evaluating new therapies and drug resistance. Using a xenograft model derived from an alectinib-relapsed patient, we showed that NVL-655 had high intracranial activity against brain tumors bearing the ALK G1202R mutation that confers resistance to multiple ALK TKIs. NVL-655 is being evaluated in a Phase 1/2 clinical trial for patients with advanced NSCLC and other solid tumors harboring ALK rearrangement or activating ALK mutation (ALKOVE-1): NCT05384626. [J. Lee and M. Yu contributed equally.] Citation Format: Jii Bum Lee, Mi Ra Yu, Mi Ran Yun, You Won Lee, Seung Yeon Oh, Eun Ji Lee, Anupong Tangpeerachaikul, Henry E. Pelish, Byoung Chul Cho. Preclinical intracranial activity of NVL-655 in an alectinib-resistant patient-derived model harboring EML4-ALK fusion with G1202R mutation. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4022.

Genomic Sequencing from Sputum for Tuberculosis Disease Diagnosis, Lineage Determination, and Drug Susceptibility Prediction.

Universal access to drug susceptibility testing for newly diagnosed tuberculosis patients is recommended. Access to culture-based diagnostics remains limited, and targeted molecular assays are vulnerable to emerging resistance mutations. Improved protocols for direct-from-sputum Mycobacterium tuberculosis sequencing would accelerate access to comprehensive drug susceptibility testing and molecular typing. We assessed a thermo-protection buffer-based direct-from-sample M. tuberculosis whole-genome sequencing protocol. We prospectively analyzed 60 acid-fast bacilli smear-positive clinical sputum samples in India and Madagascar. A diversity of semiquantitative smear positivity-level samples were included. Sequencing was performed using Illumina and MinION (monoplex and multiplex) technologies. We measured the impact of bacterial inoculum and sequencing platforms on genomic read depth, drug susceptibility prediction performance, and typing accuracy. M. tuberculosis was identified by direct sputum sequencing in 45/51 samples using Illumina, 34/38 were identified using MinION-monoplex sequencing, and 20/24 were identified using MinION-multiplex sequencing. The fraction of M. tuberculosis reads from MinION sequencing was lower than from Illumina, but monoplexing grade 3+ samples on MinION produced higher read depth than Illumina (P < 0.05) and MinION multiplexing (P < 0.01). No significant differences in sensitivity and specificity of drug susceptibility predictions were seen across sequencing modalities or within each technology when stratified by smear grade. Illumina sequencing from sputum accurately identified 1/8 (rifampin) and 6/12 (isoniazid) resistant samples, compared to 2/3 (rifampin) and 3/6 (isoniazid) accurately identified with Nanopore monoplex. Lineage agreement levels between direct and culture-based sequencing were 85% (MinION-monoplex), 88% (Illumina), and 100% (MinION-multiplex). M. tuberculosis direct-from-sample whole-genome sequencing remains challenging. Improved and affordable sample treatment protocols are needed prior to clinical deployment.

RET Proto-Oncogene-Not Such an Obvious Starting Point in Cancer Therapy.

Mutations and fusions of RET (rearranged during transfection) gene are detected in a few common types of tumors including thyroid or non-small cells lung cancers. Multiple kinase inhibitors (MKIs) do not show spectacular effectiveness in patients with RET-altered tumors. Hence, recently, two novel RET-specific inhibitors were registered in the US and in Europe. Selpercatinib and pralsetinib showed high efficacy in clinical trials, with fewer adverse effects, in comparison to previously used MKIs. However, the effectiveness of these new drugs may be reduced by the emergence of resistance mutations in RET gene and activation of different activating signaling pathways. This review presents the function of the normal RET receptor, types of molecular disturbances of the RET gene in patients with various cancers, methods of detecting these abnormalities, and the effectiveness of modern anticancer therapies (ranging from immunotherapies, through MKIs, to RET-specific inhibitors).