Acquisition Of Resistance Research Articles

Loss of lncRNA LINC01056 leads to sorafenib resistance in HCC

Background and aimsSorafenib is a major nonsurgical option for patients with advanced hepatocellular carcinoma (HCC); however, its clinical efficacy is largely undermined by the acquisition of resistance. The aim of this study was to identify the key lncRNA involved in the regulation of the sorafenib response in HCC.Materials and methodsA clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) single-guide RNA (sgRNA) synergistic activation mediator (SAM)-pooled lncRNA library was applied to screen for the key lncRNA regulated by sorafenib treatment. The role of the identified lncRNA in mediating the sorafenib response in HCC was examined in vitro and in vivo. The underlying mechanism was delineated by proteomic analysis. The clinical significance of the expression of the identified lncRNA was evaluated by multiplex immunostaining on a human HCC microtissue array.ResultsCRISPR/Cas9 lncRNA library screening revealed that Linc01056 was among the most downregulated lncRNAs in sorafenib-resistant HCC cells. Knockdown of Linc01056 reduced the sensitivity of HCC cells to sorafenib, suppressing apoptosis in vitro and promoting tumour growth in mice in vivo. Proteomic analysis revealed that Linc01056 knockdown in sorafenib-treated HCC cells induced genes related to fatty acid oxidation (FAO) while repressing glycolysis-associated genes, leading to a metabolic switch favouring higher intracellular energy production. FAO inhibition in HCC cells with Linc01056 knockdown significantly restored sensitivity to sorafenib. Mechanistically, we determined that PPARα is the critical molecule governing the metabolic switch upon Linc01056 knockdown in HCC cells and indeed, PPARα inhibition restored the sorafenib response in HCC cells in vitro and HCC tumours in vivo. Clinically, Linc01056 expression predicted optimal overall and progression-free survival outcomes in HCC patients and predicted a better sorafenib response. Linc01056 expression indicated a low FAO level in HCC.ConclusionOur study identified Linc01056 as a critical epigenetic regulator and potential therapeutic target in the regulation of the sorafenib response in HCC.

Nuclear MAST4 Suppresses FOXO3 through Interaction with AKT3 and Induces Chemoresistance in Pancreatic Ductal Carcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is highly malignant, with a 5-year survival rate of less than 10%. Furthermore, the acquisition of anticancer drug resistance makes PDAC treatment difficult. We established MIA-GEM cells, a PDAC cell line resistant to gemcitabine (GEM), a first-line anticancer drug, using the human PDAC cell line-MIA-PaCa-2. Microtubule-associated serine/threonine kinase-4 (MAST4) expression was increased in MIA-GEM cells compared with the parent cell line. Through inhibitor screening, dysregulated AKT signaling was identified in MIA-GEM cells with overexpression of AKT3. MAST4 knockdown effectively suppressed AKT3 overexpression, and both MAST4 and AKT3 translocation into the nucleus, phosphorylating forkhead box O3a (FOXO3) in MIA-GEM cells. Modulating FOXO3 target gene expression in these cells inhibited apoptosis while promoting stemness and proliferation. Notably, nuclear MAST4 demonstrated higher expression in GEM-resistant PDAC cases compared with that in the GEM-sensitive cases. Elevated MAST4 expression correlated with a poorer prognosis in PDAC. Consequently, nuclear MAST4 emerges as a potential marker for GEM resistance and poor prognosis, representing a novel therapeutic target for PDAC.

CD9 shapes glucocorticoid sensitivity in pediatric B-cell precursor acute lymphoblastic leukemia.

Resistance to glucocorticoids (GC), the common agents for remission induction in pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL), poses a significant therapeutic hurdle. Therefore, dissecting the mechanisms shaping GC resistance could lead to new treatment modalities. Here, we showed that CD9- BCP-ALL cells were preferentially resistant to prednisone and dexamethasone over other standard cytotoxic agents. Concordantly, we identified significantly more poor responders to the prednisone prephase among BCP-ALL patients with a CD9- phenotype, especially for those with adverse presenting features including older age, higher white cell count and BCR-ABL1. Furthermore, gain- and loss-offunction experiments dictated a definitive functional linkage between CD9 expression and GC susceptibility, as demonstrated by the reversal and acquisition of relative GC resistance in CD9low and CD9high BCP-ALL cells, respectively. Despite physical binding to the GC receptor NR3C1, CD9 did not alter its expression, phosphorylation or nuclear translocation but potentiated the induction of GC-responsive genes in GC-resistant cells. Importantly, the MEK inhibitor trametinib exhibited higher synergy with GC against CD9- than CD9+ lymphoblasts to reverse drug resistance in vitro and in vivo. Collectively, our results elucidate a previously unrecognized regulatory function of CD9 in GC sensitivity, and inform new strategies for management of children with resistant BCP-ALL.

ABC transporter inhibition by beauvericin partially overcomes drug resistance in Leishmania tropica.

Leishmaniasis is a neglected tropical disease infecting the world's poorest populations. Miltefosine (ML) remains the primary oral drug against the cutaneous form of leishmaniasis. The ATP-binding cassette (ABC) transporters are key players in the xenobiotic efflux, and their inhibition could enhance the therapeutic index. In this study, the ability of beauvericin (BEA) to overcome ABC transporter-mediated resistance of Leishmania tropica to ML was assessed. In addition, the transcription profile of genes involved in resistance acquisition to ML was inspected. Finally, we explored the efflux mechanism of the drug and inhibitor. The efficacy of ML against all developmental stages of L. tropica in the presence or absence of BEA was evaluated using an absolute quantification assay. The expression of resistance genes was evaluated, comparing susceptible and resistant strains. Finally, the mechanisms governing the interaction between the ABC transporter and its ligands were elucidated using molecular docking and dynamic simulation. Relative quantification showed that the expression of the ABCG sub-family is mostly modulated by ML. In this study, we used BEA to impede resistance of Leishmania tropica. The IC50 values, following BEA treatment, were significantly reduced from 30.83, 48.17, and 16.83 µM using ML to 8.14, 11.1, and 7.18 µM when using a combinatorial treatment (ML + BEA) against promastigotes, axenic amastigotes, and intracellular amastigotes, respectively. We also demonstrated a favorable BEA-binding enthalpy to L. tropica ABC transporter compared to ML. Our study revealed that BEA partially reverses the resistance development of L. tropica to ML by blocking the alternate ATP hydrolysis cycle.

Expression of norA, norB and norC efflux pump genes mediating fluoroquinolones resistance in MRSA isolates.

Although fluoroquinolones are used to treat methicillin-resistant Staphylococcus aureus (MRSA)-induced infections, acquisition of antibiotic resistance by bacteria has impaired their clinical relevance. We aimed to evaluate the frequency of norA, norB, and norC efflux pump genes-mediating fluoroquinolones resistance and measure their expression levels in MRSA isolates. 126 S. aureus isolates were collected from different clinical samples of adult hospitalized patients and identified by conventional microbiological methods. MRSA was diagnosed by cefoxitin disc diffusion method and minimum inhibitory concentration (MIC) of ciprofloxacin by broth microdilution method. The expression levels of efflux pump genes were measured by quantitative real-time polymerase chain reaction (qRT-PCR). 80 (63.5%) MRSA isolates were identified and showed high level of resistance to erythromycin (80%), gentamicin (75%), clindamycin (65%) and ciprofloxacin (60 %). norA, norB and norC were detected in 75%, 35% and 55% of the MRSA isolates respectively. norC was the most commonly overexpressed gene measured by qRT-PCR, occurring in 40% of MRSA isolates, followed by norA (35%) and norB (30%). The expression of these genes was significantly higher in ciprofloxacin-resistant than quantitative real-time PCR ciprofloxacin-sensitive MRSA isolates. This study showed high prevalence and overexpression of efflux pump genes among MRSA isolates which indicates the significant role of these genes in the development of multidrug resistance against antibiotics including fluoroquinolones.

Regional Antibiotic Resistance of the Main Uropathogens

The most important reason for the increase in mortality from infectious factors on the planet is the acquisition of resistance to antibacterial agents by microflora. Therefore, modern guidelines for the treatment of human infectious diseases recommend considering the regional sensitivity of the microflora when choosing an antibiotic. The treatment of infectious lesions of the urinary system is no exception, the disease of which is annually included in the list of the most frequent reasons for increasing the «burden of disease» on the planet. Therefore, the study of the microbial landscape of uropathogenic flora and its sensitivity to modern antibiotics in the Ukrainian population will contribute to increasing the effectiveness of treatment, especially empirical treatment, and reducing the antibiotic resistance of local uropathogens. The objective: optimization of the therapy of infectious and inflammatory diseases of the urinary system by considering the sensitivity of the main regional uropathogens to antibacterial agents. Materials and methods. Bacteriological research was carried out on 545 urine samples of symptomatic inpatients and outpatients aged 19 to 95 years, in whom pathological changes in urine sediment were determined during the examination. According to the sex distribution, women predominated in both groups in the ratio of 1.6:1 and 3.8:1, respectively. Identification of causative agents and their properties, as well as sensitivity to antibiotics, was carried out according to the standardized methods of domestic and international guidelines. The growth of microflora with the number of colony-forming units in 1 ml ≥ 104 (CFU/ml) was considered clinically significant. Statistical analysis was carried out with the help of the statistical package EZR v. 1.50. The critical level of significance is 0.05. Results. In urine samples obtained from patients of a multidisciplinary hospital, 81.7% of identified microorganisms were Escherichia coli (31.5%), Enterococcus faecalis (17.2%), Klebsiella spp. (16.2%), Candida albicans (6.6%), Acinetobacter species (5.6%) and Staphylococcus aureus (4.6%). In the outpatient group of patients, 92.1% of uropathogens were Escherichia coli (52.3%), Klebsiella spp. (13.6%), Staphylococcus aureus (13.6%), Enterococcus faecalis (8.0%) and Enterobacter sрp. (4.6%). In the spectrum of pathogens of infectious and inflammatory diseases of the urinary system, the role of microorganisms that form a capsule (Klebsiella spp., Acinetobacter sрp.) has been increasing in recent years: among the uropathogens in examined inpatients in 2013 (n=122) and 2022 (n=108) proportion of Klebsiella spp. increased, respectively, from 14.8% to 37%; and the proportion of Acinetobacter sрp. from 2.8% to 3.3%. The majority of uropathogens identified from the urine of patients of a multidisciplinary somatic hospital, in contrast to the outpatient group, was characterized by polyresistance to the action of antibacterial agents. Escherichia coli and Klebsiella spp. were sensitive only to fosfomycin and carbapenems, and Enterococcus faecalis – to fosfomycin, amoxicillin clavulonate, ampicillin sulbactam, vancomycin, meropenem and linezolid. Acinetobacter sрp. was insensitive to all antibiotics used in the study. The growth of fungi of the genus Candida albicans was inhibited by the main antifungal drugs of the «access» category – fluconazole, itraconazole, and ketoconazole, with a tendency toward a gradual increase in resistance, which was indicated by a significant number of moderately sensitive strains – 35, 25, and 10%, respectively. Conclusions. The spectrum of uropathogenic flora in patients of a multidisciplinary hospital is characterized by greater diversity and a more frequent combination of several types of microorganisms in one patient compared to the outpatient group. The most common causative agents of infectious and inflammatory diseases of the urinary tract in the Kyiv region are polyresistant strains of Escherichia coli, Enterococcus faecalis and Klebsiella spp. In the spectrum of pathogens of infectious and inflammatory diseases of the urinary system, the role of microorganisms that form a capsule (Klebsiella spp., Acinetobacter sрp.) has been increasing in recent years. Therefore, we are witnessing the evolutionary success of the protective mechanism of capsule formation, which is related, among other things, to the phenomenon of Bacterial Quorum Sensing in microorganisms and the formation of biofilms. Implementation of the obtained results of the study of regional sensitivity of uropathogens to antibacterial agents in medical practice will improve the results of treatment of infectious and inflammatory urological pathology and prevent the growth of antibiotic resistance of the local flora.

Lytic Activity Spectra of Bacteriophages

One of the central topics in bacteriophage research is the host specificity. It depends on the success of completing viral life cycle stages, including adsorption, penetration of the genetic material of the virus into the cell and its replication, assembly of phage particles and cell lysis. Laboratory assessments of the spectrum of lytic activity of phages are inextricably linked to significant methodological biases, and the often used spot test method can be associated with a large percentage of false-positive results. Along with the variety of types of phage specificity, there is temporal variability. The co-evolution of phages and bacteria leads to the acquisition of resistance to viruses by bacteria and the accumulation of mutations in the genomes of bacteriophages aimed at overcoming this resistance. At the same time, the adaptation of bacteriophages to bacteria that are evolutionarily distant from the isolation hosts is barely possible. This barrier is based on the peculiarities of metabolism, cell wall structures and mechanisms for the implementation of matrix processes. The spatial factor of phage specificity is manifested in the greater breadth of the spectra of lytic activity of bacteriophages on local samples of bacteria compared to the spectra assessed on samples of isolates from habitats geographically distant from the place of virus isolation.

FL118 Is a Potent Therapeutic Agent against Chronic Myeloid Leukemia Resistant to BCR-ABL Inhibitors through Targeting RNA Helicase DDX5.

Chronic myeloid leukemia (CML) is induced by the expression of the fused tyrosine kinase BCR-ABL, which is caused by a chromosomal translocation. BCR-ABL inhibitors have been used to treat CML; however, the acquisition of resistance by CML cells during treatment is a serious issue. We herein demonstrated that BCR-ABL induced the expression of the RNA helicase DDX5 in K562 cells derived from CML patients in a manner that was dependent on its kinase activity, which resulted in cell proliferation and survival. The knockout of DDX5 decreased the expression of BIRC5 (survivin) and activated caspase 3, leading to apoptosis in K562 cells. Similar results were obtained in cells treated with FL118, an inhibitor of DDX5 and a derivative compound of camptothecin (CPT). Furthermore, FL118 potently induced apoptosis not only in Ba/F3 cells expressing BCR-ABL, but also in those expressing the BCR-ABL T315I mutant, which is resistant to BCR-ABL inhibitors. Collectively, these results revealed that DDX5 is a critical therapeutic target in CML and that FL118 is an effective candidate compound for the treatment of BCR-ABL inhibitor-resistant CML.

Role of RelA-synthesized (p)ppGpp and ROS-induced mutagenesis in de novo acquisition of antibiotic resistance in E. coli

The stringent response of bacteria to starvation and stress also fulfills a role in addressing the threat of antibiotics. Within this stringent response, (p)ppGpp, synthesized by RelA or SpoT, functions as a global alarmone. However, the effect of this (p)ppGpp on resistance development is poorly understood. Here, we show that knockout of relA or rpoS curtails resistance development against bactericidal antibiotics. The emergence of mutated genes associated with starvation and (p)ppGpp, among others, indicates the activation of stringent responses. The growth rate is decreased in ΔrelA-resistant strains due to the reduced ability to synthesize (p)ppGpp and the persistence of deacylated tRNA impeding protein synthesis. Sluggish cellular activity causes decreased production of reactive oxygen species (ROS), thereby reducing oxidative damage, leading to weakened DNA mismatch repair, potentially reducing the generation of mutations. These findings offer new targets for mitigating antibiotic resistance development, potentially achieved through inhibiting (p)ppGpp or ROS synthesis.

An essential protease, FtsH, influences daptomycin resistance acquisition in Enterococcus faecalis.

Daptomycin is a last-line antibiotic commonly used to treat vancomycin-resistant Enterococci, but resistance evolves rapidly and further restricts already limited treatment options. While genetic determinants associated with clinical daptomycin resistance (DAPR) have been described, information on factors affecting the speed of DAPR acquisition is limited. The multiple peptide resistance factor (MprF), a phosphatidylglycerol-modifying enzyme involved in cationic antimicrobial resistance, is linked to DAPR in pathogens such as methicillin-resistant Staphylococcus aureus. Since Enterococcus faecalis encodes two paralogs of mprF and clinical DAPR mutations do not map to mprF, we hypothesized that functional redundancy between the paralogs prevents mprF-mediated resistance and masks other evolutionary pathways to DAPR. Here, we performed invitro evolution to DAPR in mprF mutant background. We discovered that the absence of mprF results in slowed DAPR evolution and is associated with inactivating mutations in ftsH, resulting in the depletion of the chaperone repressor HrcA. We also report that ftsH is essential in the parental, but not in the ΔmprF, strain where FtsH depletion results in growth impairment in the parental strain, a phenotype associated with reduced extracellular acidification and reduced ability for metabolic reduction. This presents FtsH and HrcA as enticing targets for developing anti-resistance strategies.

Abstract 7519: Elucidation of the mechanism of acquisition of immune checkpoint inhibitor resistance by chronic inflammation

Abstract Introduction: Eight molecules that are structurally similar to angiopoietin but do not bind to angiopoietin's specific receptor Tie2 were identified and named angiopoietin-like factors (ANGPTL). ANGPTL2, particularly abundantly expressed in adipose tissue, promotes damage repair through tissue remodeling, and contributes to tissue homeostasis; however, in severe obesity, ANGPTL2 is overexpressed, resulting in irreversible tissue remodeling, which induces chronic inflammation and is involved in systemic insulin resistance and diabetes development. On the other hand, it is unclear how systemic chronic inflammation controlled by ANGPTL2 is involved in cancer progression and acquisition of immune checkpoint inhibitor (ICI) resistance. Therefore, we aimed to elucidate the mechanisms in the acquisition of ICI resistance by systemic chronic inflammation from the perspective of ANGPTL2. Materials and Methods: 1. To induce chronic inflammation in adipose tissue, BALB/c mice were fed high-fat-diet (HFD) for 6 weeks. CT26 cells, a mouse colon cancer cell line, were subcutaneously implanted in normal diet (ND) and HFD mice and anti PD-1/PD-L1 antibody was administered intraperitoneally. Immune cell fractions in tumor tissue and bone marrow were analyzed by flow cytometry. 2. CT26 was subcutaneously implanted in WT and systemic ANGPTL2-deficient (Angptl2−/−) mice, and anti PD-1/PD-L1 antibody was administered intraperitoneally. Tumor size and survival time were analyzed. Bone marrow cells were isolated and the immune cell fractions were analyzed by flow cytometry, and the expression of inflammatory cytokines in adipose tissue were also analyzed. 3. Bone marrow cells were isolated from BALB/c mice, and myeloid-derived suppressor cells (MDSCs) were established in vitro by GM-CSF and IL-6. Recombinant ANGPTL2 protein was added, and MDSC differentiation and function were evaluated by flow cytometry and qRT-PCR. Results: 1. HFD mice had larger tumor size and poorer response to ICI treatment compared to ND mice. In tumor microenvironment, MDSCs were increased in HFD mice. HFD mice had significantly increased MDSCs, especially polymorphonuclear MDSCs (PMN-MDSCs) in bone marrow. 2. ICI-treated Angptl2−/− mice showed higher treatment efficacy and prolonged survival compared to WT mice. In bone marrow, MDSCs were reduced in Angptl2−/− mice under ICI treatment. In adipose tissue, the expression of chronic inflammatory markers was increased. 3. In vitro, addition of recombinant ANGPTL2 protein to MDSCs induced differentiation from mouse bone marrow cells did not significantly increase the cell ratio of MDSCs. No changes in the expression of MDSCs markers were observed in qRT-PCR. Conclusions: Chronic inflammation induced by ANGPTL2 promotes MDSCs differentiation and maturation in the bone marrow, which may suppress anti-tumor immunity and contribute to ICI resistance. Citation Format: Shinsei Yumoto, Haruki Horiguchi, Hideo Baba, Yuichi Oike. Elucidation of the mechanism of acquisition of immune checkpoint inhibitor resistance by chronic inflammation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7519.

Abstract 1986: Acquisition of carboplatin resistance corresponds with reduced sacituzumab govitecan efficacy in triple negative breast cancer patient derived xenografts

Abstract Sacituzumab Govitecan (SG) is an antibody drug conjugate that targets the epithelial glycoprotein Trop-2. SG has been approved for the treatment of patients with triple negative breast cancer (TNBC) or estrogen receptor positive (ER+) breast cancer after they have received at least two previous systemic treatments. The objectives of this study were to identify biomarkers of SG response using breast cancer patient-derived xenografts (PDXs) and determine if SG efficacy changes when cells become carboplatin resistant (CR). Analysis of transcriptomic and proteomic levels of Trop2 using bulk RNAseq, scRNA-seq, and tandem mass tag spectrometry found that overall ER+ PDXs had significantly lower RNA (p-value less than 0.001) and protein (p-value less than 0.001) expression of Trop-2 than TNBC PDXs. Immunohistochemical assessment of 42 TNBC and 27 ER+ patient tumor samples identified variation in Trop-2 expression; within each cohort, expression varied from highly positive to completely negative. To test the association of Trop-2 with drug responsiveness, cells from 19 different PDXs were cultured in vitro and administered increasing doses of SG. TNBC cells were more responsive to SG treatments compared to ER+ cells (p-value equaling 0.005). Correlation analysis identified a positive relationship of drug response with protein abundance which was stronger in TNBC samples. In vivo studies with 7 TNBC PDXs found that over 70% were highly susceptible to SG treatments, with some tumors being completely eradicated or exhibiting a total inhibition of tumor growth which resulted in a long-term durable response after 10 weeks of treatment. In vivo across all the PDXs, Trop-2 expression alone did not strongly correlate with SG treatment success. Interestingly, CR sublines derived from 2 carboplatin sensitive PDXs were significantly less responsive to SG than their parental PDXs (p-value less than 0.001). These two CR sublines were also significantly less sensitive to the majority of 555 other drugs identified by the NCI compared to their parental PDXs. Ongoing studies are defining mechanisms of reduced SG efficacy in CR models. Overall, these studies find that SG is highly effective in many TNBC PDX models and should be utilized earlier in treatment protocols, especially in metastatic patients. Citation Format: Carson J. Walker, Julia E. Altman, Emily K. Zboril, Rachel K. Myrick, Nicole S. Hairr, David C. Boyd, Bin Hu, Mikhail G. Dozmorov, J. Chuck Harrell. Acquisition of carboplatin resistance corresponds with reduced sacituzumab govitecan efficacy in triple negative breast cancer patient derived xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1986.

Abstract 5611: Unraveling the role of F-actin in resistant GBM: Insights into DNA repair, GSC phenotype, and therapeutic implications

Abstract Glioblastoma (GBM), a highly aggressive brain cancer, poses a formidable challenge marked by its resistance to conventional therapies. F-actin, a crucial cytoskeletal protein, influences DNA repair and impacts genomic stability. F-actin dynamics also shape the glioma stem cell (GSC) phenotype, affecting self-renewal and migration. Altered F-actin organization contributes to GBM resistance to conventional therapies. Understanding F-actin's multifaceted role is vital for developing targeted strategies to overcome treatment resistance and enhance outcomes for GBM patients. This study goes deeper into the intricate interplay between the F-actin cytoskeleton and the acquisition of resistance in GBM cells. Our primary goal was to explore the effects of pharmacological or genetic F-actin depolymerization on the reversal of acquired resistance to ionizing radiation (IR) and temozolomide (TMZ) in GBM cells, particularly focusing on the underlying mechanisms involving DNA repair, the glioma stem cell (GSC) phenotype, and therapeutic implications. To establish resistant sublines, GBM U87-MG cells underwent cycles of IR and TMZ exposure, revealing a significant increase in IC50 values for TMZ and ID50 values for IR through viability assays. Subsequent characterization of these sublines growth was in both adherent and spheroid cultures uncovering a distinct sphere-forming capacity. Notably, alterations in DNA repair and the Rho GTPase pathway were observed, with evident disorganized F-actin polymerization in resistant cells, especially at the periphery of spheroids. Elevated expression of stemness markers (Nestin, CD133, and CD44) highlighted a GSC-like profile in the resistant cells, a characteristic further accentuated in spheroid cultures. To assess the relevance of the actin cytoskeleton in maintaining the resistant phenotype, cells were treated with Rho-GTPase inhibitor (C3) or actin polymerization inhibitor (Cytochalasin D). Both treatments resulted in reduced TMZ IC50 and IR ID50 values, partially attributed to the negative modulation of DNA repair capacity - observed by alkaline comet and host-cell reactivation assays. Additionally, F-actin disassembly was found to impact the expression of stemness markers, further reinforcing the connection between cytoskeletal dynamics and the GSC phenotype. Overall, our data strongly suggest that F-actin cytoskeleton dynamics plays a pivotal role in the development and maintenance of resistance in GBM. Targeting these cytoskeletal components sees a promising pharmacological strategy for resensitizing recurrent and resistant GBM tumors, not only by diminishing DNA repair capabilities but also by influencing the GSC-like phenotype, thus opening new avenues for therapeutic interventions. Citation Format: Yuli Thamires Magalhães, Fábio Luís Forti. Unraveling the role of F-actin in resistant GBM: Insights into DNA repair, GSC phenotype, and therapeutic implications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5611.

Abstract 458: Nanoparticle-mediated delivery of miR-590-3p decreases recurrent GBM tumor growth by inhibits multiple oncogenic nodes

Abstract Glioblastoma (GBM) is the most lethal and aggressive form of brain cancer. Despite aggressive therapy consisting of surgery followed by radio/chemotherapy GBM recurs in almost all patients and, currently, there are no proven therapies to treat recurrent GBM (rGBM). Recent developments in nanomedicine provide new and promising opportunities to develop new targeted therapeutics to treat brain tumors. We have recently developed PBAE nanoparticle formulations optimized for in vivo miRNA delivery to brain tumor stem cells, or GSCs. Carefully selecting one miRNA to target several mRNAs dysregulated within a pathway can allow the targeting of multiple nodes using a single agent, mimicking combinatorial therapies. To test this hypothesis, we performed bioinformatic analysis of RNA sequencing from GSCs and clinical rGBM specimens combined with gene set enrichment analysis (GSEA) and identified TGFBR2 signaling as a targetable pathway in rGBM. We show that SMAD2 signaling, a downstream effector of TGFBR2, is enriched in rGBM and that patients with elevated TGFBR2 transcripts respond poorly to current standard of care. Utilizing patient-derived xenograft lines from rGBM, we show that blocking TGFBR2 signaling using siRNAs or ITD1, a selective TGFBR2 inhibitor, decreases the stem cell capacity, cell viability and re-sensitizes clinical rGBM cells to temozolomide (TMZ) in-vitro. In vivo, we show that ITD1 is modestly effective in subcutaneous and ineffective in orthotopic rGBM models. Not surprisingly, these tumors exhibited classical growth patterns associated with acquisition of resistance. To overcome this obstacle, we performed a miRNA-based network analysis in rGBM and identified miR-590-3p as a putative tumor suppressor that targets SMAD2 signaling at multiple nodes in rGBM. In-vitro, transgenic miR-590-3p robustly decreased the expression of 37 putative oncogenes simultaneously concurrent with decreasing stem cell capacity, cell viability and re-sensitizing rGBM PDX cells to TMZ. In vivo delivery of miR-590-3p to established PDX-derived rGBM orthotopic tumors using novel PBAE nanocarriers robustly decreased tumor sizes and increased tumor necrosis. These results show that the promiscuous nature of miRNAs, in combination with cutting-edge nucleic acid delivery vehicles, can be leveraged as advanced targeted therapeutics for rGBM bypasses the resistance that is developed to standard of care and available small molecules. Citation Format: Hernando Lopez-Bertoni, Sophie Sall, Harmon Khela, Jack Korleski, Katherine Luly, Maya Johnson, Bachchu Lal, Amanda Johnson, Stephany Tzeng, Jordan Green, John Laterra. Nanoparticle-mediated delivery of miR-590-3p decreases recurrent GBM tumor growth by inhibits multiple oncogenic nodes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 458.

Abstract 3874: Adaptive heterogeneity enables the survival of residual malignant PDAC cells in response to RAS-GTP inhibition

Abstract More than 90% of human pancreatic ductal adenocarcinoma (PDAC) cases are driven by activating mutations in KRAS. RMC-7977 is a potent inhibitor of GTP-bound RAS proteins (RAS(ON)), including both wild type and mutant variants of KRAS, NRAS, and HRAS. The related investigational agent, RMC-6236, is a first-in-class, potent, orally bioavailable, RASMULTI(ON) inhibitor currently in Phase 1/1b clinical trials (NCT05379985). We performed preclinical studies in a range of models of PDAC, including the highly chemoresistant K-rasLSL.G12D/+, p53LSL.R172H/+, Pdx1Cretg/+ (KPC) genetically engineered mouse. RMC-7977 exhibited broad anti-tumor activity at tolerable doses, provoking radiographic responses in KPC pancreatic tumors and extending overall survival by 3-fold, the largest response observed yet in this model. RMC-7977 dosing produced a metronomic effect on RAS signaling in tumor and normal tissues, with full pathway inhibition at 4 hours post treatment that was restored by 24 hours. This pattern yielded tumor-selective effects on apoptosis and proliferation, consistent with RAS oncogene addition in PDAC. To understand the dynamic response of malignant cells following RAS-GTP inhibition, we did single cell RNA sequencing on KPC pancreatic tumors treated with RMC-7977 or vehicle at multiple timepoints. Using the ARACNe and VIPER algorithms [Maroling., et al. 2006; Alvarez., et al. 2016], we performed regulatory network analysis on the expression profiles of >210,000 cells to calculate the activities of >5000 transcriptional regulatory proteins per cell. Additionally, we assessed the impact of treatment on malignant cell subtypes as previously identified by our team based on their sets of hyperactivated developmental transcription factors [Laise., et al. 2022]. As expected, RAS pathway transcription factors, such as FOS and JUN, were repressed relative to controls in malignant cells by 4 hours after treatment with RMC-7977. However, at 24 hours, responses varied by cellular subtype: well-differentiated Gastrointestinal Lineage State (GLS) cells had restored MAPK activity, while poorly differentiated Morphogenic State (MOS) cells were still inactivated. Strikingly, we found that after one week of treatment (in actively regressing tumors, representing a state of residual disease), nearly all malignant cells had assumed the GLS state. Acquisition of resistance after weeks or months of treatment was associated with a restored diversity of malignant cell states, suggesting that the GLS state serves as a regulatory “safe harbor” in which residual malignant cells can undergo evolution and adaptation. Mechanistically, we found that RAS pathway signaling directly activates transcriptional regulators that drive the MOS state, elucidating an early adaptive response to RAS-GTP inhibition that enables residual cells to survive treatment prior to development of acquired resistance. Citation Format: Lorenzo Tomassoni, Urszula N. Wasko, Alvaro C. Garcia, Tanner C. Dalton, Pasquale Laise, Stephen A. Sastra, Carmine F. Palermo, Andrea Califano, Kenneth P. Olive. Adaptive heterogeneity enables the survival of residual malignant PDAC cells in response to RAS-GTP inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3874.

Abstract 6203: An altered profile of ribosomal proteins and ERK-eIF4E translational control in an inflammatory breast cancer model of acquired resistance and reversal is associated with pathological complete response in patients

Abstract Background: Drug resistance is a major challenge in inflammatory breast cancer (IBC), a hyperproliferative subtype characterized by the presence of diffuse tumor cell clusters that show high level of heterogeneity. As the degree of tumor heterogeneity during cancer progression can cause evolution of cell states that can either compete or co-operate resulting in therapeutic failure, the study objective was to identify drug resistance networks that can serve as biomarkers or therapeutic targets. Methods: Integrated transcriptomic and proteomic profiling approach was conducted in a progression model of acquired drug resistance and its reversal comprised of a treatment-naïve patient-derived SUM149 cell line, along with two isogenic derivatives and representing drug-resistant (rSUM149) and resistance-reversal states (rrSUM149). Global gene expression analysis in IBC patients (n=87) recorded responses to neoadjuvant chemotherapy. Based on the data, targeted multikinase inhibitors were evaluated in viability and signaling assays. Results: We identified specific ribosomal proteins associated with resistance acquisition, which correlated with high levels of pERK, CDK1, XIAP, and SOD2. Conversely, the resistance reversal in rrSUM149 showed almost complete normalization to the profile of SUM149. However, VIPER analysis revealed proteins related to ribosomal processes (AGO2, Exportin 1, RPL5) that did not revert in rrSUM149, suggesting this pathway may have pleiotropic effects in governing drug resistance. Notably, genes linked to ribosomal processes were significantly enriched (P<0.001) among the overexpressed genes in IBC patients (n=87) who exhibited pCR to neoadjuvant chemotherapy. This is crucial as IBC tumors, including rSUM149, exhibit MAPK hyperactivation including in the presence of EGFR tyrosine kinase inhibitors. Treatment with Merestinib, a multikinase inhibitor, suppressed pERK, peIF4E, downstream targets like CDK1, and ribosomal protein IMP3 underscoring the impact on protein synthesis signaling crucial for countering translational dependence in cancer cells. Additionally, Merestinib synergized with EGFR-TKI (lapatinib) in increasing cell death in all three states. Conclusion: Overall, our study describes adaptive changes in response to both therapeutic stress and therapy break that have the potential to serve as biomarkers or extended for pharmacological interrogation toward precision medicine for this rare, understudied cancer. Furthermore, the ability of Merestinib, in clinical trials, to target translational dependence is attractive as cancer cells often appropriate these signaling pathways to gain tolerance to various stress stimuli. Support: NIH-R01CA264529; DoD Breast Cancer Breakthrough level 2 Award W81XWH2010153 Citation Format: Gayathri R. Devi, Pritha PAI, Matthew W. Foster, Dorababu S. Sannareddy, Seayoung Lee, Steven V. Laere. An altered profile of ribosomal proteins and ERK-eIF4E translational control in an inflammatory breast cancer model of acquired resistance and reversal is associated with pathological complete response in patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6203.

Abstract 4527: TNG348 is synergistic with PARP inhibitors in tumor models with elevated replication stress

Abstract Defective maintenance of genomic integrity is a hallmark of cancer cells that can result from oncogene-induced replication stress and by loss of DNA repair mechanisms. DNA repair deficiencies and elevated replication stress present targetable vulnerabilities for cancer treatment. Notably, BRCA1/2 mutant and homologous recombination deficient (HRD) tumors cannot repair double-strand breaks by homologous recombination and rely on alternative pathways of DNA repair. PARP inhibitors (PARPi), which are a standard of care in many BRCA1/2 mutant tumors, cause synthetic lethality with BRCA1/2 mutation by inhibiting the DNA base excision repair pathway. Despite the clinical benefit of PARPi, they are not effective in every HRD tumor and the acquisition of PARPi resistance limits long-term response. TNG348, a selective allosteric inhibitor of the deubiquitinating enzyme USP1, was specifically designed to target HRD vulnerabilities through an alternative mechanism. We previously showed that the anti-tumor activity of USP1 inhibition results from disruption of the translesion synthesis DNA damage tolerance pathway, a mechanism of action that is functionally distinct from base excision repair targeted by PARPi. Our preclinical studies show that TNG348 is active in HRD models and strongly synergizes with PARP inhibitors to drive strong anti-tumor responses. We have identified replication stress as a predictive biomarker of TNG348 response using cell line profiling and genome-wide CRISPR screens. For example, overexpression of oncogenes known to induce replication stress sensitized to the TNG348 and PARPi combination both in vitro and in vivo. These data indicate that cancer-specific elevated DNA replication stress could contribute to tumor sensitivity to TNG348 and provide additional patient stratification strategies and opportunities for indication expansion. Citation Format: Antoine Simoneau, Hsin-Jung Wu, Charlotte Pratt, Grace Comer, Shangtao Liu, Samuel Meier, Tenzing Khendu, Ashley Choi, Hongxiang Zhang, Binzhang Shen, Douglas Whittington, Sirimas Sudsakorn, Wenhai Zhang, Yi Yu, Yingnan Chen, Brian Haines, Adam Crystal, Jannik N. Andersen, John Maxwell, Scott Throner. TNG348 is synergistic with PARP inhibitors in tumor models with elevated replication stress [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4527.

Abstract 3026: STK11 negatively regulates NFKB signaling in KRAS-driven lung adenocarcinoma

Abstract In the US, more people die each year from lung cancer than breast, colorectal, and prostate cancers combined. Despite advances in lung cancer detection and treatment, the overall prognosis for patients remains poor. Patients whose lung tumors harbor druggable mutations often experience initial responses with targeted therapies, but acquisition of therapy resistance results in 5-year survival rates that have remained near ~20%. Immune check-point inhibitors (ICI) promise to improve outcomes in patients with lung cancer by targeting mechanisms of immune evasion. Drugs that disrupt PD-L1-mediated immune evasion, including pembrolizumab and nivolumab, have revolutionized treatment for some cancers. Unfortunately, only ~30% of lung adenocarcinomas (LUADs) respond to anti-PD-1 therapy and we cannot reliably identify this group prior to treatment. This underscores the need to discriminate which patients will respond to ICI therapies, while simultaneously highlighting the ~70% non-responder rate. To that end, recent clinical studies have linked anti-PD-1 therapy resistance with Serine/Threonine Kinase 11 (STK11) loss of function (LoF). STK11 operates in a heterotrimeric complex with the pseudo-kinase STRADα and the scaffolding protein MO25 where it regulates numerous intracellular signaling networks impacting metabolism, proliferation, transcription, and cell morphology. Why STK11 LoF correlates with anti-PD-1 resistance in the context of KRAS-driven LUAD remains unknown and represents a critical question in lung oncology. As an initial approach to understand this phenomenon, we knocked-out STK11 in multiple KRAS-driven, STK11-competent human LUAD cell lines and performed whole transcriptome analyses to identify STK11-loss-dependent differential gene expression profiles. We assert the transcriptional mediators downstream of STK11 governing the changes we report represent therapeutic targets whose antagonism may restore anti-PD-1 efficacy. Supporting this rationale, our data has identified STK11-loss-dependent activation of the NFκ-B transcriptional network. NFκB is a master transcription factor that regulates numerous cytokines, but whether and/or how STK11 directs NFκB activity remains largely unaddressed. We highlight NFκB activation as a potential mechanistic link between STK11 loss and tumor-intrinsic cytokine upregulation. We speculate that STK11 normally acts as a key player in the negative feedback loop limiting NFκB activity. When STK11 is lost, that activity continues unabated. In summary, we propose STK11 loss drives anti-PD-1 therapy resistance by generating an altered tumor immune microenvironment via constitutive activation of NFκB-mediated cytokine production. In future we intend to assess strategies for reversing anti-PD-1 therapy resistance via disruption of the NFκB transcriptional axis in KRAS-driven LUAD lacking STK11. Citation Format: David Joseph Seward, Sean Lenahan, Allison Racela, Israel Odekunle. STK11 negatively regulates NFKB signaling in KRAS-driven lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3026.

Abstract 3266: Plk1 promotes Pdcd4 degradation to enhance enzalutamide resistance in prostate cancer

Abstract The androgen receptor (AR) signaling pathway plays critical role in the development of prostate cancer (PCa), including castration-resistant prostate cancer (CRPC). As a result, androgen signaling inhibitors (ASIs) like abiraterone and enzalutamide (ENZ) have emerged as the first-line treatment for CRPC. Nevertheless, the limited success of ASIs underscores the urgency of developing new approaches for managing CRPC patients who have become unresponsive to ASIs. Programmed cell death 4 (Pdcd4) is a tumor suppressor, which is frequently down-regulated during PCa progression. It has been shown that reduced Pdcd4 expression promotes both AR-dependent and AR-independent growth in PCa. In this study, we found that down-regulation of Pdcd4 led to acquisition of ENZ resistance in PCa. We also observed that down-regulation of Pdcd4 resulted in activation of mTORC2 activity. Additionally, knockdown of Rictor, a component of mTORC2 complex, enhanced sensitivity to ENZ treatment in PCa. These findings suggested that activation of mTORC2 due to Pdcd4 loss contributes to ASI resistance in PCa. To understand how Pdcd4 is down-regulated in the PCa, we identified that polo-like kinase (Plk1) phosphorylated Pdcd4 at Ser239. Phosphorylation of Pdcd4 by Plk1 led to ubiquitin-mediated proteasomal degradation of Pdcd4. Over-expression of the phosphomimic Pdcd4(S239D) in 22Rv1 cells promoted tumor cell growth in cultured cells and mice. Taken together, our data suggest that targeting Plk1/Pdcd4/mTORC2 signaling axis holds promise as a strategy to overcome ENZ resistance in PCa. Citation Format: Qing Wang, Qiongsi Zhang, Yanquan Zhang, Xiaoqi Liu, Hsin-Sheng Yang. Plk1 promotes Pdcd4 degradation to enhance enzalutamide resistance in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3266.

Abstract 548: Exploiting pronounced selection pressure from therapeutic bottlenecks to identify orthogonal immunotherapy targets

Abstract Evolutionary processes play a crucial role in the development of cancer, where tumor cells continuously adapt, leading to the dominance of cell populations over their non-tumor counterparts, evading treatment and ultimately resulting in death. Effective cancer therapies induce significant and targeted selection pressures. During and in response to the induced bottleneck by therapy, the heterogeneous cancer cell population either diminishes or evolves, causing a subsequent selective sweep and the emergence of cells with resistance traits. However, this bottleneck phase presents a unique opportunity to predictably reduce the diversity of tumor cells. In this context, we propose a framework to quantify the selection on variants before, during, and after the therapeutic bottleneck, enhancing our understanding of the mechanisms underlying the acquisition of therapeutic resistance. Furthermore, we combine measures of selection intensity with the prediction of neoantigens in a patient cohort to identify potential alternative targets for immunotherapy. The outcome is a strategy that exploits the predictability introduced by the primary therapeutic bottleneck to eliminate the remaining tumor cell population. Finally, we illustrate the efficacy of this strategy in the context of several cancer types and empirically identify potential cancer type specific and pan-cancer immunotherapy targets orthogonal to primary, first-line therapies. Citation Format: J. Nic Fisk, Jeffrey P. Townsend. Exploiting pronounced selection pressure from therapeutic bottlenecks to identify orthogonal immunotherapy targets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 548.