Biology subjects

Papagiannakopoulos, T.

Publications and source records attributed to Papagiannakopoulos, T..

12 recordsLinked to original sources

bioRxiv2026

Characterization and therapeutic suppression of KEAP1-NRF2-driven resistance to KRAS inhibitors in pancreatic and lung cancer

The recent approval of KRAS inhibitors supports the therapeutic value of targeting mutant KRAS cancers. However, clinical efficacy is hindered by both primary and treatment-associated acquired resistance. We applied a CRISPR-Cas9 loss-of-function screen and identified loss of KEAP1 as a resistance mechanism to the KRASG12D-selective inhibitor MRTX1133 and the RAS(ON) multi-selective inhibitor RMC-7977 in pancreatic cancer models. RNA-sequencing analyses revealed a KEAP1KO transcriptome that is distinct from the ERK-, MYC-, and YAP/TAZ-TEAD-dependent transcriptional programs that drive KRAS inhibitor resistance, demonstrating a distinct mechanism of resistance. We then established a PDAC KEAP1-deficient (PKD) gene signature that was enriched in patients and preclinical models insensitive to KRAS inhibitor treatment. Finally, we observed that KEAP1-deficient cells exhibited elevated glutamine metabolism, and combination treatment with the glutamine antagonist DRP-104 (sirpiglenastat) enhanced KRAS inhibitor suppression of pancreatic and lung tumors. SIGNIFICANCEKEAP1 loss is associated with reduced response to KRAS inhibitor therapy. We demonstrate that KEAP1 loss-associated resistance can be overcome by pharmacologic inhibition of the KEAP1 loss-induced glutamine dependency, establishing a combination to enhance RAS inhibitor clinical efficacy.

cancer biology↗

bioRxiv2025

Targeting FSP1 triggers ferroptosis in lung cancer

Pre-clinical and clinical studies have demonstrated how dietary antioxidants or mutations activating antioxidant metabolism promote cancer, highlighting a central role oxidative stress in tumorigenesis. However, it is unclear if oxidative stress ultimately increases to a point of cell death. Emerging evidence indicates that cancer cells are susceptible to ferroptosis, a form of cell death triggered by uncontrolled lipid peroxidation1-3. Despite broad enthusiasm about harnessing ferroptosis as a novel anti-cancer strategy, whether ferroptosis is a barrier to tumorigenesis and if it can be leveraged therapeutically remains unknown4,5. Using genetically-engineered mouse models (GEMMs) of lung adenocarcinoma (LUAD), we performed tumor specific loss-of-function studies of the two key ferroptosis suppressors, glutathione peroxidase 4 (Gpx4)6,7 and ferroptosis suppressor protein 1 (Fsp1)8,9, and observed increased lipid peroxidation and robust suppression of tumorigenesis, suggesting that lung tumors are highly sensitive to ferroptosis. Furthermore, across multiple pre-clinical models, we found that FSP1 was required for ferroptosis protection in vivo, but not in vitro, underscoring a heightened need to buffer lipid peroxidation under physiological conditions. Lipidomic analyses revealed that Fsp1-knockout (Fsp1KO) tumors had an accumulation of lipid peroxides, and inhibition of ferroptosis with genetic, dietary, or pharmacological approaches effectively restored the growth of Fsp1KO tumors in vivo. Unlike GPX4, FSP1 expression was prognostic for disease progression and poorer survival in LUAD patients, highlighting its potential as a viable therapeutic target. To this end, we demonstrated that pharmacologic inhibition of FSP1 had significant therapeutic benefit in pre-clinical lung cancer models. Our studies highlight the importance of ferroptosis suppression in vivo and pave the way for FSP1 inhibition as a therapeutic strategy in lung cancer patients.

cancer biology↗

bioRxiv2025

FMO4 drives lung adenocarcinoma by stabilizing the MAT2A/MAT2B complex and hindering ferroptosis

Lung cancer is the leading cause of death by cancer in the world and finding new targets is a major medical need to tackle this disease. Here, upon proteomic analysis to identify common players in oncogenic EGFR- and KRAS-driven lung adenocarcinoma mouse models, we uncovered a largely unknown protein in cancer, flavin-containing monooxygenase 4 (FMO4), whose expression was increased in lung tumors compared with adjacent lung tissue. FMO4 expression was strongly increased also in lung cancer samples from patients compared with healthy lung, and its expression level was inversely correlated with overall survival. Remarkably, in vivo deletion of FMO4 greatly decreased tumor burden and increased survival in oncogenic KRAS-driven lung adenocarcinoma mice unveiling its crucial role in tumor biology. Mechanistically, we found that FMO4 loss of function promotes ferroptosis and cooperates with ferroptosis inducers in vitro and in vivo. Moreover, FMO4 facilitates the interaction between MAT2A and MAT2B, promoting the generation of cysteine from methionine, which in turn boosts the generation of glutathione, thus protecting lung adenocarcinoma against ferroptosis. In summary we identified a new target in lung adenocarcinoma with important implications in cancer biology.

cancer biology↗

bioRxiv2024

An enhanced Eco1 retron editor enables precision genome engineering in human cells from a single-copy integrated lentivirus

Retrons are a retroelement class found in diverse prokaryotes that can be adapted to augment CRISPR-Cas9 genome engineering technology to efficiently rewrite short stretches of genetic information in bacteria and yeast. However, efficiency in human cells has been limited by unknown factors. We identified non-coding RNA (ncRNA) instability and impaired Cas9 activity due to 5 sgRNA extension as key contributors to low retron editor efficiency in human cells. We re-engineered the Eco1 ncRNA to incorporate an exoribonuclease-resistant RNA (xrRNA) pseudoknot from the Zika virus 3 UTR and devised an RNA processing strategy using Csy4 ribonuclease to minimize 5 sgRNA extension. This strategy increased steady-state ncRNA levels and rescued sgRNA activity, leading to increased templated repair. This work reveals a previously unappreciated role for ncRNA stability in retron editor efficiency in human cells and presents an enhanced Eco1 retron editor capable of precise genome editing in human cells from a single integrated lentivirus and, in the context of the nCas9 H840A nickase, without creating double-strand breaks.

molecular biology↗

bioRxiv2024

Unraveling cysteine deficiency-associated rapid weight loss

Forty percent of the US population and 1 in 6 individuals worldwide are obese, and the incidence of this disease is surging globally1,2. Various dietary interventions, including carbohydrate and fat restriction, and more recently amino acid restriction, have been explored to combat this epidemic3-6. We sought to investigate the impact of removing individual amino acids on the weight profiles of mice. Compared to essential amino acid restriction, induction of conditional cysteine restriction resulted in the most dramatic weight loss, amounting to 20% within 3 days and 30% within one week, which was readily reversed. This weight loss occurred despite the presence of substantial cysteine reserves stored in glutathione (GSH) across various tissues7. Further analysis demonstrated that the weight reduction primarily stemmed from an increase in the utilization of fat mass, while locomotion, circadian rhythm and histological appearance of multiple other tissues remained largely unaffected. Cysteine deficiency activated the integrated stress response (ISR) and NRF2-mediated oxidative stress response (OSR), which amplify each other, leading to the induction of GDF15 and FGF21, hormones associated with increased lipolysis, energy homeostasis and food aversion8-10. We additionally observed rapid tissue coenzyme A (CoA) depletion, resulting in energetically inefficient anaerobic glycolysis and TCA cycle, with sustained urinary excretion of pyruvate, orotate, citrate, -ketoglutarate, nitrogen rich compounds and amino acids. In summary, our investigation highlights that cysteine restriction, by depleting GSH and CoA, exerts a maximal impact on weight loss, metabolism, and stress signaling compared to other amino acid restrictions. These findings may pave the way for innovative strategies for addressing a range of metabolic diseases and the growing obesity crisis.

physiology↗

bioRxiv2023

Tumor-intrinsic LKB1-LIF signaling axis establishes a myeloid niche to promote immune evasion and tumor growth

Tumor mutations can influence the surrounding microenvironment leading to suppression of anti-tumor immune responses and thereby contributing to tumor progression and failure of cancer therapies. Here we use genetically engineered lung cancer mouse models and patient samples to dissect how LKB1 mutations accelerate tumor growth by reshaping the immune microenvironment. Comprehensive immune profiling of LKB1-mutant vs wildtype tumors revealed dramatic changes in myeloid cells, specifically enrichment of Arg1+ interstitial macrophages and SiglecFHi neutrophils. We discovered a novel mechanism whereby autocrine LIF signaling in Lkb1-mutant tumors drives tumorigenesis by reprogramming myeloid cells in the immune microenvironment. Inhibiting LIF signaling in Lkb1-mutant tumors, via gene targeting or with a neutralizing antibody, resulted in a striking reduction in Arg1+ interstitial macrophages and SiglecFHi neutrophils, expansion of antigen specific T cells, and inhibition of tumor progression. Thus, targeting LIF signaling provides a new therapeutic approach to reverse the immunosuppressive microenvironment of LKB1-mutant tumors.

cancer biology↗

bioRxiv2023

Glutamine antagonist DRP-104 suppresses tumor growth and enhances response to checkpoint blockade in KEAP1 mutant lung cancer

Loss-of-function mutations in KEAP1 frequently occur in lung cancer and are associated with resistance to standard of care treatment, highlighting the need for the development of targeted therapies. We have previously shown that KEAP1 mutant tumors have increased glutamine consumption to support the metabolic rewiring associated with NRF2 activation. Here, using patient-derived xenograft models and antigenic orthotopic lung cancer models, we show that the novel glutamine antagonist DRP-104 impairs the growth of KEAP1 mutant tumors. We find that DRP-104 suppresses KEAP1 mutant tumor growth by inhibiting glutamine-dependent nucleotide synthesis and promoting anti-tumor CD4 and CD8 T cell responses. Using multimodal single-cell sequencing and ex vivo functional assays, we discover that DRP-104 reverses T cell exhaustion and enhances the function of CD4 and CD8 T cells culminating in an improved response to anti-PD1 therapy. Our pre-clinical findings provide compelling evidence that DRP-104, currently in phase 1 clinical trials, offers a promising therapeutic approach for treating patients with KEAP1 mutant lung cancer. Furthermore, we demonstrate that by combining DRP-104 with checkpoint inhibition, we can achieve suppression of tumor intrinsic metabolism and augmentation of anti-tumor T cell responses.

cancer biology↗

bioRxiv2023

Metabolic Reprogramming by Histone Deacetylase Inhibition Selectively Targets NRF2-activated tumors.

Interplay between metabolism and chromatin signaling have been implicated in cancer initiation and progression. However, whether and how metabolic reprogramming in tumors generates specific epigenetic vulnerabilities remain unclear. Lung adenocarcinoma (LUAD) tumors frequently harbor mutations that cause aberrant activation of the NRF2 antioxidant pathway and drive aggressive and chemo-resistant disease. We performed a chromatin-focused CRISPR screen and report that NRF2 activation sensitized LUAD cells to genetic and chemical inhibition of class I histone deacetylases (HDAC). This association was consistently observed across cultured cells, syngeneic mouse models and patient-derived xenografts. HDAC inhibition causes widespread increases in histone H4 acetylation (H4ac) at intergenic regions, but also drives re-targeting of H4ac reader protein BRD4 away from promoters with high H4ac levels and transcriptional downregulation of corresponding genes. Integrative epigenomic, transcriptomic and metabolomic analysis demonstrates that these chromatin changes are associated with reduced flux into amino acid metabolism and de novo nucleotide synthesis pathways that are preferentially required for the survival of NRF2-active cancer cells. Together, our findings suggest that metabolic alterations such as NRF2 activation could serve as biomarkers for effective repurposing of HDAC inhibitors to treat solid tumors.

cancer biology↗

bioRxiv2023

In vivo metabolomics identifies CD38 as an emergent vulnerability in LKB1-mutant lung cancer

LKB1/STK11 is a serine/threonine kinase that plays a major role in controlling cell metabolism, resulting in potential therapeutic vulnerabilities in LKB1-mutant cancers. Here, we identify the NAD+ degrading ectoenzyme, CD38, as a new target in LKB1-mutant NSCLC. Metabolic profiling of genetically engineered mouse models (GEMMs) revealed that LKB1 mutant lung cancers have a striking increase in ADP-ribose, a breakdown product of the critical redox co-factor, NAD+. Surprisingly, compared with other genetic subsets, murine and human LKB1-mutant NSCLC show marked overexpression of the NAD+-catabolizing ectoenzyme, CD38 on the surface of tumor cells. Loss of LKB1 or inactivation of Salt-Inducible Kinases (SIKs)--key downstream effectors of LKB1-- induces CD38 transcription induction via a CREB binding site in the CD38 promoter. Treatment with the FDA-approved anti-CD38 antibody, daratumumab, inhibited growth of LKB1-mutant NSCLC xenografts. Together, these results reveal CD38 as a promising therapeutic target in patients with LKB1 mutant lung cancer. SIGNIFICANCELoss-of-function mutations in the LKB1 tumor suppressor of lung adenocarcinoma patients and are associated with resistance to current treatments. Our study identified CD38 as a potential therapeutic target that is highly overexpressed in this specific subtype of cancer, associated with a shift in NAD homeostasis.

cancer biology↗

bioRxiv2022

Inflammation in the tumor-adjacent lung as a predictor of clinical outcome in lung adenocarcinoma

Early-stage lung adenocarcinoma is typically treated by surgical resection of the tumor. While in the majority of cases surgery can lead to cure, approximately 30% of patients progress. Despite intense efforts to map the genetic landscape of early-stage lung tumors, there has been limited success in discovering accurate biomarkers that can predict clinical outcomes. Meanwhile, the role of the tumor-adjacent tissue in cancer progression has been largely ignored. To test whether tumor-adjacent tissue can be informative of progression-free survival and to probe the underlying molecular pathways involved, we designed a multi-omic study in both tumor and matched tumor-adjacent histologically normal lung tissue from the same patient. Our study includes 143 treatment naive stage I cases with long-term patient follow-up and is, to our knowledge, the largest such study with the longest follow-up. We performed a comprehensive histologic characterization of all tumors, mapped the mutational landscape and probed the transcriptome of both tumor and adjacent normal tissue. We evaluated the predictive power of each data modality and showed that the transcriptome of tumor-adjacent histologically normal lung tissue is the only reliable predictor of clinical outcome. Unbiased discovery of co-expressed gene modules revealed that inflammatory pathways are upregulated in the tumor-adjacent tissue of patients at high risk for disease progression. Furthermore, single-cell transcriptome analysis in the tumor-adjacent lung demonstrated that progression-associated inflammatory signatures were broadly expressed by both immune and non-immune cells including mesothelial cells, alveolar type 2 cells and fibroblasts, CD1 dendritic cells and MAST cells. Collectively, our studies suggest that molecular profiling of tumor-adjacent tissue can identify patients that are at high risk for disease progression.

bioinformatics↗

bioRxiv2021

KEAP1 mutation in lung adenocarcinoma promotes immune evasion and immunotherapy resistance

Lung cancer treatment has benefited greatly from the development of effective immune-based therapies. However, these strategies still fail in a large subset of patients. Tumor-intrinsic mutations can drive immune evasion via recruiting immunosuppressive populations or suppressing anti-tumor immune responses. KEAP1 is one of the most frequently mutated genes in lung adenocarcinoma patients and is associated with poor prognosis and inferior response to all therapies, including checkpoint blockade. Here, we established a novel antigenic lung cancer model and showed that Keap1-mutant tumors promote dramatic remodeling of the tumor immune microenvironment. Combining single-cell technology and depletion studies, we demonstrate that Keap1-mutant tumors diminish dendritic cell and T cell responses driving immunotherapy resistance. Importantly, analysis of KEAP1 mutant patient tumors revealed analogous decrease in dendritic cell and T cell infiltration. Our study provides new insight into the role of KEAP1 mutations in promoting immune evasion and suggests a path to novel immune-based therapeutic strategies for KEAP1 mutant lung cancer. Statement of significanceThis study establishes that tumor-intrinsic KEAP1 mutations contribute to immune evasion through suppression of dendritic cell and T cell responses, explaining the observed resistance to immunotherapy of KEAP1 mutant tumors. These results highlight the importance of stratifying patients based on KEAP1 status and paves the way for novel therapeutic strategies.

cancer biology↗

bioRxiv2020

Improving oligo-conjugated antibody signal in multimodal single-cell analysis

Simultaneous measurement of surface proteins and gene expression within single cells using oligo-conjugated antibodies offers high resolution snapshots of complex cell populations. Signal from oligo-conjugated antibodies is quantified by high-throughput sequencing and is highly scalable and sensitive. In this study, we investigated the response of oligo-conjugated antibodies towards four variables: Concentration, staining volume, cell number at staining, and tissue. We find that staining with recommended antibody concentrations cause unnecessarily high background and that concentrations can be drastically reduced without loss of biological information. Reducing volume only affects antibodies targeting abundant epitopes used at low concentrations and is counteracted by reducing cell numbers. Adjusting concentrations increases signal, lowers background and reduces costs. Background signal can account for a major fraction of the total sequencing and is primarily derived from antibodies used at high concentrations. Together, this study provides new insight into the titration response and background of oligo-conjugated antibodies and offers concrete guidelines on how such panels can be improved. Impact statementOligo-conjugated antibodies are a powerful tool but require thorough optimization to reduce background signal, increase sensitivity, and reduce sequencing costs.

genomics↗