Adaptive Resistance Mechanisms Research Articles

Mechanisms of adaptive resistance to targeted therapy in RET-aberrant cancers.

The success of targeted therapies in oncogene-driven cancer is limited by adaptive or acquired treatment resistance, leading to disease progression. A recent study reports that YAP-dependent HER3 activation constitutes a therapeutic vulnerability of adaptive resistance to RET-targeted therapies in RET-altered cancers, highlighting a promising strategy to improve RET-inhibitor tumor responses.

Moxifloxacin-Loaded Polymeric Nanoparticles for Overcoming Multidrug Resistance in Chronic Pulmonary Infections Caused by Pseudomonas aeruginosa.

Pseudomonas aeruginosa (P. aeruginosa) infections are increasingly challenging due to their propensity to form biofilms and low outer membrane permeability, especially in chronically infected patients with thick mucus. P. aeruginosa exhibits multiple drug resistance mechanisms, making it one of the most significant global public health threats. In this study, we found that moxifloxacin (MXC) and antibacterial peptides (ε-poly-l-lysine, ε-PLL) exhibited a synergistic effect against multidrug-resistant P. aeruginosa (MDR-P. aeruginosa). MXC was combined with ε-PLL to prepare lipase-responsive nanoparticles (MCIP/(PEG-PCL)/PLL NPs) with a weakly negative charge. The weakly negatively charged MCIP/(PEG-PCL)/PLL NPs demonstrated remarkable mucus and biofilm penetration capabilities, thereby overcoming one of the adaptive drug resistance mechanisms. MCIP/(PEG-PCL)/PLL NPs improved the outer and inner membrane permeability and inhibited the expression of the efflux pump MexAB-OprM gene in MDR-P. aeruginosa, thereby overcoming mechanisms of both intrinsic and acquired drug resistance. Meanwhile, the nanoparticles demonstrated an ability to reduce repeated infections with MDR-P. aeruginosa. Additionally, the bacterial burden in the lungs of mice treated with MCIP/(PEG-PCL)/PLL NPs was significantly lower than that in the MXC group, resulting in a 99% clearance rate. Notably, MCIP/(PEG-PCL)/PLL NPs showed no toxicity toward BEAS-2B cells or RAW 267.4 cells, nor did they adversely affect pulmonary function or major organs. This study demonstrated the potential of the nanodrug delivery system composed of the antibiotic moxifloxacin and the antibacterial peptide ε-PLL in addressing the clinical challenges of treating chronic pulmonary infections caused by MDR-P. aeruginosa.

Combined PARP14 inhibition and PD-1 blockade promotes cytotoxic T cell quiescence and modulates macrophage polarization in relapsed melanoma

BackgroundProgrammed cell death 1 (PD-1) signaling blockade by immune checkpoint inhibitors (ICI) effectively restores immune surveillance to treat melanoma. However, chronic interferon-gamma (IFNγ)-induced immune homeostatic responses in melanoma cells contribute...

Exploring Metabolic Approaches for Epithelial Ovarian Cancer Therapy.

Epithelial ovarian cancer (EOC) has the highest mortality rate among malignant tumors of the female reproductive system and the lowest survival rate. This poor prognosis is due to the aggressive nature of EOC, its late-stage diagnosis, and the tumor's ability to adapt to stressors through metabolic reprogramming. EOC cells sustain their rapid proliferation by altering the uptake, utilization, and regulation of carbohydrates, lipids, and amino acids. These metabolic changes support tumor growth and contribute to metastasis, chemotherapy resistance, and immune evasion. Targeting these metabolic vulnerabilities has shown promise in preclinical studies, with some therapies advancing to clinical trials. However, challenges remain due to tumor heterogeneity, adaptive resistance mechanisms, and the influence of the tumor microenvironment. This review provides a comprehensive summary of metabolic targets for EOC treatment and offers an overview of the current landscape of clinical trials focusing on ovarian cancer metabolism. Future efforts should prioritize combination therapies that integrate metabolic inhibitors with immunotherapies or chemotherapy. Advances in precision medicine and multi-omics approaches will be crucial for identifying patient-specific metabolic dependencies and improving outcomes. By addressing these challenges, metabolism-based therapies can significantly transform the treatment of this devastating disease.

YAP regulates HER3 signaling-driven adaptive resistance to RET inhibitors in RET-aberrant cancer.

Rearranged during transfection (RET) aberrations represent a targetable oncogene in several tumor types, with RET inhibitors displaying marked efficacy. However, some patients with RET-aberrant cancer are insensitive to RET tyrosine kinase inhibitors (TKIs). Recently, drug-tolerant mechanisms have attracted attention as targets for initial therapies to overcome drug resistance. The underlying mechanisms of drug-tolerant cell emergence treated with RET-TKIs derived from RET-aberrant cancer cells remain unknown. This study investigated the role of YAP-mediated HER3 signaling in the underlying mechanisms of adaptive resistance to RET-TKIs in RET-aberrant cancer cells. Four RET-aberrant cancer cell lines were used to assess sensitivity to the RET-TKIs selpercatinib and pralsetinib and to elucidate molecular mechanisms underlying adaptive resistance using RNA sequencing, phospho-RTK antibody arrays, chromatin immunoprecipitation assay, and luciferase reporter assays. Clinical specimens from patients with RET-fusion-positive lung cancer were analyzed for pre-treatment YAP expression and correlated with treatment outcomes. In high YAP-expressing RET-aberrant cancer cells, YAP-mediated HER3 signaling activation maintained cell survival and induced the emergence of cells tolerant to the RET-TKIs selpercatinib and pralsetinib. The pan-ErBB inhibitor afatinib and YAP/TEAD inhibitors verteporfin and K-975 sensitized YAP-expressing RET-aberrant cancer cells to the RET-TKIs selpercatinib and pralsetinib. Pre-treatment YAP expression in clinical specimens obtained from patients with RET-fusion-positive lung cancer was associated with poor RET-TKI treatment outcomes. The YAP-HER3 axis is crucial for the survival and adaptive resistance of high YAP-expressing RET-aberrant cancer cells treated with RET-TKIs. Combining YAP/HER3 inhibition with RET-TKIs represents a highly potent strategy for initial treatment.

Small cell transformation in EGFR-mutated non-small cell lung cancer: DLL3 expression and efficacy of immune checkpoint inhibitors or tyrosine kinase inhibitors combined with chemotherapy

IntroductionSmall cell transformation (SCT) is a typical mechanism of adaptive resistance to third generation epidermal growth factor receptor inhibitors (EGFRi) which have become the standard of care for EGFR-driven non-small cell lung cancer (EGFR+ NSCLC). Little is known about the optimal management of SCT patients. This study aimed to compare outcomes under platinum/etoposide chemotherapy alone (chemo) or in combination with EGFR inhibitors (EGFRi+chemo) or immune checkpoint inhibitors (ICI+chemo). In addition, DLL3 expression was explored as potential novel therapeutic target. MethodsWe conducted a retrospective study on patients with EGFR+ NSCLC and SCT treated at 19 centers in Europe and the United States. A total of 47 patients were included of whom 17 received chemo, 20 ICI+chemo, and 10 EGFRi+chemo. We analyzed DLL3 expression by immunohistochemistry. ResultsIn the entire cohort, median overall survival (OS) from start of first SCT therapy was 11 months (95 % confidence interval [95 %CI] 9.1–12.9) and median progression-free survival (PFS) was 5 months (95 %CI 4.2–5.8). Median PFS was similar in all three groups (chemo and ICI+chemo 4 months, EGFRi+chemo 6 months), and 12-months PFS was 12 % (95 %CI 2 %−31 %), 13 % (95 %CI 0 %−43 %), and 0 % for ICI+chemo, EGFRi+chemo, and chemo, respectively. Median OS in the ICI+chemo group was 13 months (95 %CI 5.5–20.5) compared to 10 months (95 %CI 7.6–12.4) with chemo and EGFRi+chemo (95 %CI 8.1–11.9), respectively. Before and after SCT, 0 % and 93 % of tumors were DLL3-positive. ConclusionsOur results suggest that ICI+chemo and DLL3-targeting agents are worth further exploration in EGFR+ NSCLC undergoing SCT. Presented elsewherePart of this work has been presented at ESMO annual meeting in Madrid, Spain in October 2023 (Poster 1336 P).

Abstract C012: Overcoming Cell Subtype-Specific Adaptive Resistance to KRAS G12D Inhibition in KRAS G12D-Mutant Pancreatic Cancer

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) has a 5-year survival rate of only 12%, with limited treatment options. Oncogenic KRAS mutations drive PDAC tumor progression, which occur in the majority of PDAC tumors. KRAS G12D is the most common KRAS mutation, found in 36% of PDAC cases, which is associated with the worst clinical outcomes. Mutant-selective KRAS G12D inhibitors (KRASi G12D), such as MRTX1133, have recently demonstrated initial responses in KRAS G12D-mutant preclinical models. However, mechanisms of adaptive resistance to KRAS G12D inhibition are largely under-investigated and novel therapeutic strategies to overcome drug resistance are urgently needed. Results: Adaptive RAS signaling reactivation was found to dampen KRAS G12D inhibition in a library of KRAS G12D PDAC cell lines, thereby minimizing drug efficacy. Quantitative downstream analyses of drug combination screening using a KRAS G12D cell line library further identified this adaption to KRAS inhibition was cell subtype specific. Fibroblast growth factor receptor (FGFRs) drove adaptive RAS reactivation specifically in mesenchymal PDAC cells, which are aggressive, and intrinsically resistant to KRASi. In contrast, epithelial growth factor receptor (EGFR) reactivated RAS G12D signaling in epithelial cells, which are sensitive to KRASi G12D. Genetic and pharmacological inhibition of FGFR1 or FGFR2 improved drug efficacy by decreasing FGFR-mediated RAS reactivation specifically in mesenchymal PDAC lines. Pharmacological blockade of EGFR enhanced drug efficacy by inhibiting EGFR-mediated RAS reactivation specifically in epithelial PDAC lines. Furthermore, low-concentration of a novel pan- RAS inhibitor, which inhibits both wild-type and mutant RAS, completely abrogated MAPK signaling and significantly improved efficacy, when combined with KRASi G12D, suggesting a pro-survival dependency on wild-type RAS as a compensatory mechanism in response to KRAS G12D inhibition. Conclusion: Upstream RTK-mediated adaptive RAS signaling re-activation in response to KRAS inhibition in PDAC is cell subtype specific. Complete inhibition of RAS signaling by overcoming adaptive RAS rebound improves efficacy of KRASi G12D. Citation Format: Qingxiang (Nick) Lin, Alvin Morales, Haley Barnes, Ryan Bruce Corcoran. Overcoming Cell Subtype-Specific Adaptive Resistance to KRAS G12D Inhibition in KRAS G12D-Mutant Pancreatic Cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C012.

Mechanisms of adaptive resistance in Phytobacter sp. X4 to antimony stress under anaerobic conditions

Sb(III) oxidation by microorganisms plays a key role in the geochemical cycling of antimony and is effective for bioremediation. A previously discovered novel Sb(III)-oxidizing bacteria, Phytobacter sp. X4, was used to elucidate the response patterns of extracellular polypeptides (EPS), antioxidant system, electron transfer and functional genes to Sb(III) under anaerobic conditions. The toxicity of Sb(III) was mitigated by increasing Sb(III) oxidation capacity, and the EPS regulated the content of each component by sensing the concentration of Sb(III). High Sb(III) concentrations induced significant secretion of proteins and polysaccharides of EPS, and polysaccharides were more important. Functional groups including hydroxyl, carboxyl and amino groups on the cell surface adsorbed Sb(III) to block its entry. Hydroxyl radicals and hydrogen peroxide were involved in anaerobic Sb(III) oxidation, as revealed by changes in the antioxidant system and electron spin resonance (EPR) techniques. qPCR confirmed that proteins concerning nitrate and antimony transfer, antimony resistance and antioxidant system were regulated by Sb(III) concentration, and the synergistic cooperation of multiple proteins conferred high antimony resistance to X4. The adaptive antimony resistance mechanism of Phytobacter sp. X4 under anaerobic conditions was revealed, which also provides a reference value for bioremediation method of antimony contamination in anaerobic environment.

Computational Modeling of Drug Response Identifies Mutant-Specific Constraints for Dosing panRAF and MEK Inhibitors in Melanoma.

This study explores the potential of pre-clinical in vitro cell line response data and computational modeling in identifying the optimal dosage requirements of pan-RAF (Belvarafenib) and MEK (Cobimetinib) inhibitors in melanoma treatment. Our research is motivated by the critical role of drug combinations in enhancing anti-cancer responses and the need to close the knowledge gap around selecting effective dosing strategies to maximize their potential. In a drug combination screen of 43 melanoma cell lines, we identified specific dosage landscapes of panRAF and MEK inhibitors for NRAS vs. BRAF mutant melanomas. Both experienced benefits, but with a notably more synergistic and narrow dosage range for NRAS mutant melanoma (mean Bliss score of 0.27 in NRAS vs. 0.1 in BRAF mutants). Computational modeling and follow-up molecular experiments attributed the difference to a mechanism of adaptive resistance by negative feedback. We validated the in vivo translatability of in vitro dose-response maps by predicting tumor growth in xenografts with high accuracy in capturing cytostatic and cytotoxic responses. We analyzed the pharmacokinetic and tumor growth data from Phase 1 clinical trials of Belvarafenib with Cobimetinib to show that the synergy requirement imposes stricter precision dose constraints in NRAS mutant melanoma patients. Leveraging pre-clinical data and computational modeling, our approach proposes dosage strategies that can optimize synergy in drug combinations, while also bringing forth the real-world challenges of staying within a precise dose range. Overall, this work presents a framework to aid dose selection in drug combinations.

Computational modeling of drug response identifies mutant-specific constraints for dosing panRAF and MEK inhibitors in melanoma.

This study explores the potential of preclinical in vitro cell line response data and computational modeling in identifying optimal dosage requirements of pan-RAF (Belvarafenib) and MEK (Cobimetinib) inhibitors in melanoma treatment. Our research is motivated by the critical role of drug combinations in enhancing anti-cancer responses and the need to close the knowledge gap around selecting effective dosing strategies to maximize their potential. In a drug combination screen of 43 melanoma cell lines, we identified unique dosage landscapes of panRAF and MEK inhibitors for NRAS vs BRAF mutant melanomas. Both experienced benefits, but with a notably more synergistic and narrow dosage range for NRAS mutant melanoma. Computational modeling and molecular experiments attributed the difference to a mechanism of adaptive resistance by negative feedback. We validated in vivo translatability of in vitro dose-response maps by accurately predicting tumor growth in xenografts. Then, we analyzed pharmacokinetic and tumor growth data from Phase 1 clinical trials of Belvarafenib with Cobimetinib to show that the synergy requirement imposes stricter precision dose constraints in NRAS mutant melanoma patients. Leveraging pre-clinical data and computational modeling, our approach proposes dosage strategies that can optimize synergy in drug combinations, while also bringing forth the real-world challenges of staying within a precise dose range.

Elacestrant in the treatment landscape of ER-positive, HER2-negative, ESR1-mutated advanced breast cancer: a contemporary narrative review.

Estrogen receptor-positive (ER+), human epidermal growth factor receptor 2-negative (HER2-) breast cancer with ESR1 mutations presents a significant therapeutic challenge due to its adaptive resistance mechanisms to chemotherapy, especially endocrine treatment. Elacestrant, a novel oral selective estrogen receptor degrader (SERD), has emerged as a promising agent in this treatment-resistant era. A comprehensive search was conducted on pivotal clinical trials, including the RAD1901-005 Trial, EMERALD TRIAL, ELIPSE, and ELEVATE, focusing on their methodologies, patient populations, treatment regimens, and outcomes. This narrative review describes the available preclinical and clinical evidence on elacestrant, focusing on its pharmacodynamics, pharmacokinetics, efficacy, and safety within the existing literature. Elacestrant has demonstrated excellent activity against ESR1 mutations associated with resistance to first-line endocrine therapies. Clinical trials have shown improved progression-free survival in patients with advanced ER+/HER2-, ESR1-mutated breast cancer. Safety profiles indicate a tolerable side effect spectrum consistent with other agents. Its oral bioavailability offers a convenient alternative to injectable SERDs, with potential implications for patient adherence and quality of life. The review also discusses the comparative efficacy of elacestrant relative to existing endocrine therapies and its possible use in combination regimens. Ongoing clinical trials assessing elacestrant and other SERDs will yield data that might aid clinicians in determining the optimal selection and order of endocrine treatment drugs for ER+ breast cancer. The integration of targeted and immunotherapeutic agents with traditional chemotherapy represents a pivotal shift in Breast Cancer treatment, moving towards more personalized and effective regimens.

A first-in-class selective inhibitor of EGFR and PI3K offers a single-molecule approach to targeting adaptive resistance

Despite tremendous progress in precision oncology, adaptive resistance mechanisms limit the long-term effectiveness of molecularly targeted agents. Here we evaluated the pharmacological profile of MTX-531 that was computationally designed to selectively target two key resistance drivers, epidermal growth factor receptor and phosphatidylinositol 3-OH kinase (PI3K). MTX-531 exhibits low-nanomolar potency against both targets with a high degree of specificity predicted by cocrystal structural analyses. MTX-531 monotherapy uniformly resulted in tumor regressions of squamous head and neck patient-derived xenograft (PDX) models. The combination of MTX-531 with mitogen-activated protein kinase kinase or KRAS-G12C inhibitors led to durable regressions of BRAF-mutant or KRAS-mutant colorectal cancer PDX models, resulting in striking increases in median survival. MTX-531 is exceptionally well tolerated in mice and uniquely does not lead to the hyperglycemia commonly seen with PI3K inhibitors. Here, we show that MTX-531 acts as a weak agonist of peroxisome proliferator-activated receptor-γ, an attribute that likely mitigates hyperglycemia induced by PI3K inhibition. This unique feature of MTX-531 confers a favorable therapeutic index not typically seen with PI3K inhibitors.

Adaptive changes in the fatty acid composition of <i>Oxytropis scheludjakovae</i> and <i>Oxytropis strobilacea</i> under temperature fluctuations during the growth season in Central Yakutia

Although there is growing interest in understanding the mechanisms of plant adaptive resistance, there is still incomplete or missing information for many plant species. In this study, we conducted an analysis of the fatty acid composition in the vegetative and generative organs of two species belonging to the Oxytropis genus, namely O. scheludjakovae and O. strobilacea. Gas-liquid chromatography was used to analyze the qualitative and quantitative changes in fatty acids throughout the growing season. The plants were grown in the natural flora collection nursery at the Yakutsk Botanical Garden. The main fatty acids identified had carbon atoms ranging from 14 to 24. There were no significant differences in fatty acid composition between the endemic species O. scheludjakovae and the more widely distributed species O. strobilacea. The highest total fatty acid content (19.5 mg/g tissue) was observed in the leaves of O. scheludjakovae in June and in the leaves of O. strobilacea (17.0 mg/g tissue) in June and July. Palmitic acid was the primary saturated fatty acid in both species, while linolenic acid was the main unsaturated fatty acid. The adaptation of these plants to temperature changes was reflected in the variation of unsaturated fatty acid content. O. strobilacea showed higher values of fatty acid unsaturation coefficient throughout the growing season compared to O. scheludjakovae.

The evolving role of immune checkpoint inhibitors in cervical and endometrial cancer.

The introduction of immune checkpoint inhibitors (ICIs) has revolutionized the treatment landscape for numerous tumor types, including cervical and endometrial cancers. Multiple ICIs against programmed cell death-1 (PD-1), programmed death-ligand 1 (PD-L1), and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) have demonstrated encouraging outcomes in controlled clinical studies for advanced cervical and endometrial cancers. For advanced cervical cancer, approved ICIs as second-line treatment include cemiplimab, nivolumab, and pembrolizumab as single agents. In the first-line treatment setting, options include pembrolizumab alone or in combination with bevacizumab, as well as atezolizumab combined with a backbone platinum-based chemotherapy plus bevacizumab. Additionally, for locally advanced cervical cancer, pembrolizumab is recommended alongside concurrent chemoradiotherapy. For endometrial cancer, pembrolizumab monotherapy, pembrolizumab in combination with lenvatinib, and dostarlimab are currently approved as second-line treatment options. Moreover, either dostarlimab or pembrolizumab can be added to first-line platinum-based chemotherapy for mismatch repair deficient malignancies. Although the inclusion of these agents in clinical practice has led to improved overall response rates and survival outcomes, many patients still lack benefits, possibly due to multiple intrinsic and adaptive resistance mechanisms to immunotherapy. This review aims to highlight the rationale for utilizing ICIs and their current role, while also delineating the proposed mechanisms of resistance to ICIs in cervical and endometrial cancer.

Triple Blockade of Oncogenic RAS Signaling Using KRAS and MEK Inhibitors in Combination with Irradiation in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest of human malignancies and carries an exceptionally poor prognosis. It is mostly driven by multiple oncogenic alterations, with the highest mutation frequency being observed in the KRAS gene, which is a key oncogenic driver of tumorogenesis and malignant progression in PDAC. However, KRAS remained undruggable for decades until the emergence of G12C mutation specific KRAS inhibitors. Despite this development, this therapeutic approach to target KRAS directly is not routinely used for PDAC patients, with the reasons being the rare presence of G12C mutation in PDAC with only 1-2% of occurring cases, modest therapeutic efficacy, activation of compensatory pathways leading to cell resistance, and absence of effective KRASG12D or pan-KRAS inhibitors. Additionally, indirect approaches to targeting KRAS through upstream and downstream regulators or effectors were also found to be either ineffective or known to cause major toxicities. For this reason, new and more effective treatment strategies that combine different therapeutic modalities aiming at achieving synergism and minimizing intrinsic or adaptive resistance mechanisms are required. In the current work presented here, pancreatic cancer cell lines with oncogenic KRAS G12C, G12D, or wild-type KRAS were treated with specific KRAS or SOS1/2 inhibitors, and therapeutic synergisms with concomitant MEK inhibition and irradiation were systematically evaluated by means of cell viability, 2D-clonogenic, 3D-anchorage independent soft agar, and bioluminescent ATP assays. Underlying pathophysiological mechanisms were examined by using Western blot analyses, apoptosis assay, and RAS activation assay.

Off-target autophagy inhibition by SHP2 allosteric inhibitors contributes to their antitumor activity in RAS-driven cancers.

Aberrant activation of RAS/MAPK signaling is common in cancer, and efforts to inhibit pathway components have yielded drugs with promising clinical activities. Unfortunately, treatment-provoked adaptive resistance mechanisms inevitably develop, limiting their therapeutic potential. As a central node essential for receptor tyrosine kinase-mediated RAS activation, SHP2 has emerged as an attractive cancer target. Consequently, many SHP2 allosteric inhibitors are now in clinical testing. Here we discovered a previously unrecognized off-target effect associated with SHP2 allosteric inhibitors. We found that these inhibitors accumulate in the lysosome and block autophagic flux in an SHP2-independent manner. We showed that off-target autophagy inhibition by SHP2 allosteric inhibitors contributes to their antitumor activity. We also demonstrated that SHP2 allosteric inhibitors harboring this off-target activity not only suppress oncogenic RAS signaling but also overcome drug resistance such as MAPK rebound and protective autophagy in response to RAS/MAPK pathway blockage. Finally, we exemplified a therapeutic framework that harnesses both the on- and off-target activities of SHP2 allosteric inhibitors for improved treatment of mutant RAS-driven and drug-resistant malignancies such as pancreatic and colorectal cancers.

RND Efflux Pump Induction: A Crucial Network Unveiling Adaptive Antibiotic Resistance Mechanisms of Gram-Negative Bacteria.

The rise of multi-drug-resistant (MDR) pathogenic bacteria presents a grave challenge to global public health, with antimicrobial resistance ranking as the third leading cause of mortality worldwide. Understanding the mechanisms underlying antibiotic resistance is crucial for developing effective treatments. Efflux pumps, particularly those of the resistance-nodulation-cell division (RND) superfamily, play a significant role in expelling molecules from bacterial cells, contributing to the emergence of multi-drug resistance. These are transmembrane transporters naturally produced by Gram-negative bacteria. This review provides comprehensive insights into the modulation of RND efflux pump expression in bacterial pathogens by numerous and common molecules (bile, biocides, pharmaceuticals, additives, plant extracts, etc.). The interplay between these molecules and efflux pump regulators underscores the complexity of antibiotic resistance mechanisms. The clinical implications of efflux pump induction by non-antibiotic compounds highlight the challenges posed to public health and the urgent need for further investigation. By addressing antibiotic resistance from multiple angles, we can mitigate its impact and preserve the efficacy of antimicrobial therapies.

Network-based elucidation of colon cancer drug resistance mechanisms by phosphoproteomic time-series analysis

Aberrant signaling pathway activity is a hallmark of tumorigenesis and progression, which has guided targeted inhibitor design for over 30 years. Yet, adaptive resistance mechanisms, induced by rapid, context-specific signaling network rewiring, continue to challenge therapeutic efficacy. Leveraging progress in proteomic technologies and network-based methodologies, we introduce Virtual Enrichment-based Signaling Protein-activity Analysis (VESPA)—an algorithm designed to elucidate mechanisms of cell response and adaptation to drug perturbations—and use it to analyze 7-point phosphoproteomic time series from colorectal cancer cells treated with clinically-relevant inhibitors and control media. Interrogating tumor-specific enzyme/substrate interactions accurately infers kinase and phosphatase activity, based on their substrate phosphorylation state, effectively accounting for signal crosstalk and sparse phosphoproteome coverage. The analysis elucidates time-dependent signaling pathway response to each drug perturbation and, more importantly, cell adaptive response and rewiring, experimentally confirmed by CRISPR knock-out assays, suggesting broad applicability to cancer and other diseases.

Abstract PO2-13-07: SMARCA2 compensation of SMARCA4 loss mediates adaptive resistance to neoadjuvant systemic therapy in triple-negative breast cancer: paired transcriptomic analysis of pre- and post-treatment samples from NeoSTOP and NeoPACT

Abstract Objectives/Rationale: Triple-negative breast cancer (TNBC) patients with residual disease (RD) after neoadjuvant systemic therapy (NAST) are at high risk for disease recurrence and death. While multiple prognostic biomarkers associated with response to NAST have been identified, adaptive resistance mechanisms to NAST remain poorly understood. Identifying and characterizing adaptive resistance mechanisms can illuminate novel therapeutic approaches and improve outcomes for TNBC patients with RD. Methods: Total RNA was isolated from pre-treatment and paired surgical specimens (for patients with RD) from TNBC patients treated with chemotherapy on the NeoSTOP (NCT02413320) or chemoimmunotherapy on the NeoPACT (NCT03639948) neoadjuvant trials and was subjected to RNA exome sequencing. Comparative marker selection analysis was performed by computing the two-sided paired samples t-test for each gene followed by pre-ranked gene set enrichment analysis (GSEA) amongst 23,797 annotated gene sets. Gene sets with false-discovery rate (FDR) corrected P values < 0.001 were determined to be significant. Results: Pre-treatment sequencing data were available for N=200 patients and the overall pathologic complete response (pCR) rate was 56.5%. Paired pre- and post-treatment sequencing data were available for N=58 patients with RD (N=27 from NeoSTOP and N=31 from NeoPACT). 165/23,797 gene sets were significantly enriched in post-treatment compared to pre-treatment samples. SHEN_SMARCA2_TARGETS_UP (M29) was among the top five significantly enriched gene set and was selected for further analysis based on the known role of the SWI/SNF complex in cell survival and the availability of clinically viable SMARCA2 degraders. Pre-treatment SMARCA4 expression was associated with pCR, with lower median expression noted in patients with RD compared to patients who achieved pCR (P=0.04). Pre-treatment SMARCA2 expression was not associated with pCR (P=0.48). On paired sample analysis there was pronounced downregulation of SMARCA4 in post-treatment compared to pre-treatment samples in all patients (mean Z=-1.46, P< 0.0001), in patients treated with chemotherapy (mean Z=-1.65, P< 0.0001), and in patients treated with chemoimmunotherapy (mean Z=-1.28, P< 0.0001). Although there was no difference in SMARCA2 expression between paired pre- and post-treatment samples (mean Z=0.03, P=0.83), there was enrichment of SMARCA2 target expression on GSEA analysis (NES=2.63, FDR q< 0.00001) indicating that SMARCA2 activity is compensating for therapy-induced SMARCA4 loss. Conclusions: We identified 165 gene sets that were significantly enriched in paired post-treatment compared to pre-treatment TNBC samples. Altered activity of the SMARCA2 subunit of the SWI/SNF chromatin remodeling complex was identified as one of the top hits. SMARCA2 and SMARCA4 are mutually exclusive subunits of SWI/SNF, and inactivation of both SMARCA2 and SMARCA4 is synthetic lethal. We observed an association of lower pre-treatment SMARCA4 expression with resistance to NAST and pronounced therapy-induced downregulation of SMARCA4 and induction of SMARCA2 target genes in post-treatment compared to pre-treatment samples, suggesting that SMARCA2 compensates for adaptive SMARCA4 loss. SMARCA2 degraders are in early-phase clinical trials and are known to induce synthetic lethality in SMARCA4-deficient cells. Our results identify adaptive loss of SMARCA4 and synthetic lethal targeting of compensatory SMARCA2 as an attractive therapeutic target in TNBC patients with RD after neoadjuvant chemotherapy or chemoimmunotherapy. Citation Format: Shane Stecklein, Rachel Yoder, Joshua Staley, Zachary Schmitt, Anne O'Dea, Lauren Nye, Deepti Satelli, Gregory Crane, Rashna Madan, Maura O'Neil, Andrew Godwin, Harsh Pathak, Qamar Khan, Joyce O'Shaughnessy, Priyanka Sharma. SMARCA2 compensation of SMARCA4 loss mediates adaptive resistance to neoadjuvant systemic therapy in triple-negative breast cancer: paired transcriptomic analysis of pre- and post-treatment samples from NeoSTOP and NeoPACT [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 PO2-13-07.

Intraspecific variation in antibiotic resistance potential within E. coli.

Intraspecific genomic diversity brings the potential for an unreported and diverse reservoir of cryptic antibiotic resistance genes in pathogens, as cryptic resistance can occur without major mutations and horizontal transmission. Here, we predicted the differences in the types of antibiotics and genes that induce cryptic and latent resistance between micro-diverse Escherichia coli strains. For example, we hypothesize that known resistance genes will be the culprit of latent resistance within clinical strains. We used a modified functional metagenomics method to induce expression in eight E. coli strains. We found a total of 66 individual genes conferring phenotypic resistance to 11 out of 16 antibiotics. A total of 14 known antibiotic resistance genes comprised 21% of total identified genes, whereas the majority (52 genes) were unclassified cryptic resistance genes. Between the eight strains, 1.2% of core orthologous genes were positive (conferred resistance in at least one strain). Sixty-four percent of positive orthologous genes conferred resistance to only one strain, demonstrating high intraspecific variability of latent resistance genes. Cryptic resistance genes comprised most resistance genes among laboratory and clinical strains as well as natural, semisynthetic, and synthetic antibiotics. Known antibiotic resistance genes primarily conferred resistance to multiple antibiotics from varying origins and within multiple strains. Hence, it is uncommon for E. coli to develop cross-cryptic resistance to antibiotics from multiple origins or within multiple strains. We have uncovered prospective and previously unknown resistance genes as well as antibiotics that have the potential to trigger latent antibiotic resistance in E. coli strains from varying origins.IMPORTANCEIntraspecific genomic diversity may be a driving force in the emergence of adaptive antibiotic resistance. Adaptive antibiotic resistance enables sensitive bacterial cells to acquire temporary antibiotic resistance, creating an optimal window for the development of permanent mutational resistance. In this study, we investigate cryptic resistance, an adaptive resistance mechanism, and unveil novel (cryptic) antibiotic resistance genes that confer resistance when amplified within eight E. coli strains derived from clinical and laboratory origins. We identify the potential of cryptic resistance genes to confer cross-resistance to antibiotics from varying origins and within multiple strains. We discern antibiotic characteristics that promote latent resistance in multiple strains, considering intraspecific diversity. This study may help detect novel resistance genes and functional genes that could become responsible for cryptic resistance among diverse strains and antibiotics, thus also identifying potential novel antibiotic targets and mechanisms.