Aberrant Pathways Research Articles

Modulation of nuclear receptor 4A1 expression improves insulin secretion in a mouse model of chronic pancreatitis.

Diabetes secondary to chronic pancreatitis (CP) presents clinical challenges due to insulin secretory defects and associated metabolic alterations. Owing to lack of molecular understanding, no pharmacotherapies to treat insulin secretory defects have been approved to date. We aimed to delineate the molecular mechanism of β-cell dysfunction in CP. Transcriptomic analysis was conducted to identify endocrine specific receptor expression in mice and human CP on microarray. The identified receptor (NR4A1) was overexpressed in MIN6 cells using PEI linear transfection. RNA-Seq analysis on NovaSeq 6000 of NR4A1 overexpressed (OE) MIN6 cells was performed to identify aberrant metabolic pathways. Upstream trigger for NR4A1OE was studied by InBio Discover and cytokine exposure. Downstream effect of NR4A1OE was examined by Fura2 AM based fluorometric and imaging studies of intracellular calcium. Mice with CP were treated with IFN-γ neutralizing monoclonal antibodies to assess NR4A1 expression and insulin secretion. Increased expression of NR4A1 associated with decreased insulin secretion in islets (humans: controls 9 ± 0.2, CP 3.7 ± 0.2, mice: controls 8.5 ± 0.2, CP 2.1 ± 0.1 μg/L). NR4A1OE in MIN6 cells (13.2 ± 0.1) showed reduction in insulin secretion (13 ± 5 to 0.2 ± 0.1 μg/mg protein/minute, p = 0.001) and downregulation of calcium and cAMP signaling pathways. IFN-γ was identified as upstream signal for NR4A1OE in MIN6. Mice treated with IFN-γ neutralizing antibodies showed decreased NR4A1 expression 3.4 ± 0.11-fold (p = 0.03), improved insulin secretion (4.4 ± 0.2-fold, p = 0.01), associated with increased Ca2+ levels (2.39 ± 0.06-fold, p = 0.009). Modulating NR4A1 expression can be a promising therapeutic strategy to improve insulin secretion in CP.

Abstract PO1-06-14: Preliminary results of a phase I dose escalation study of eribulin in combination with copanlisib in patients with metastatic triple negative breast cancer (TNBC)

Abstract Background: Aberrant PI3K pathway signaling is frequently observed in TNBC. Increasing evidence indicate that PI3K pathway activation maintains the stemness and chemoresistance of breast cancer stem cells (CSCs). PI3K inhibition sensitizes CSCs to chemotherapy. Eribulin (E), a non-taxane microtubule dynamics inhibitor, showed overall survival benefit in metastatic HER2 negative BC. Preclinically, the addition of copanlisib (C), a potent pan-class I PI3K inhibitor that is highly selective against α and δ PI3K isoforms, improved anti-tumor effect in both E-sensitive and resistant TNBC patient-derived xenograft models, irrespective of PIK3CA/PTEN alteration status. Here we report the preliminary data of the phase I dose escalation trial of E+C in patients with metastatic TNBC. Methods: This trial includes a phase I study to determine the dose limiting toxicity (DLT) and recommended phase 2 dose (RP2D) of E+C, followed by a randomized phase II study of E+C (at RP2D) versus E (stratified by tumor PIK3CA/PTEN mutation status) with progression-free survival (PFS) as the primary endpoint. Key eligibility included patients with: metastatic TNBC who progressed on ≤5 lines of chemotherapy, prior anthracycline/taxane chemotherapy (unless contraindicated), ECOG 0-1, adequate organ function and known tumor PIK3CA/PTEN/AKT mutation status. Key exclusions include: prior receipt of E or any PI3K/mTOR/AKT inhibitor, grade ≥2 neuropathy, presence of tumor AKT mutation, and uncontrolled diabetes, hypertension or congenital QT prolongation. A 3+3 design was used to determine the DLT and RP2D. Dose level 1 (DL1) started with E at 1.1 mg/m2 mg IV and C at 45 mg IV on days (D) 1/8 of 21-D cycle, with the potential to escalate to 2 additional dose levels – DL2: E 1.4 mg/m2 and C 45 mg on D 1/8; DL3: E 1.4 mg/m2 and C 60 mg on D 1/8. Results: Eight patients were enrolled in the phase I study and received at least 1 dose of E+C. The median age was 48.5 (range, 26-67) years. The majority of patients identified as White (additionally 2 Hispanic, 1 Black) and had an ECOG performance status of 1 (n=6, 75.0%). Tumor PIK3CA and PTEN alterations were each observed in 1 patient. Median number of cycles received were 2 (range, 1-5). All 8 patients had at least 1 treatment-related adverse event (TRAE). The most common TRAEs across all dose levels were: lymphopenia (n=5, 62.5%), neutropenia (n=5, 62.5%), hyperglycemia (n=4, 50.0%), anemia (n=4, 50.0%) and fatigue (n=4, 50.0%); most toxicities were grade (G) 1-2. G3 hyperglycemia and hypertension were observed in 1 patient each, were medically managed and did not lead to treatment interruption. 1 patient was diagnosed with pulmonary embolism, possibly attributed to study therapy and breast cancer. DLTs were reported in 2 of 2 patients treated at DL2 - 1 patient developed G3 rash responsive to oral corticosteroids, and 1 patient developed G3 acute kidney injury in the setting of hypovolemia and discontinued study therapy. No additional patients were enrolled in DL2. No DLTs were observed in all 6 patients treated at DL1. 1 patient treated at DL1 had possible G1 non-infectious pneumonitis that resolved spontaneously on short interval imaging. Stable disease was observed in 2 of 6 response-evaluable patients (33.3%). 1 patient achieved regression of cutaneous disease following 1 cycle of therapy as assessed by the treating investigator. DL1, E at 1.1 mg/m2 plus C at 45 mg IV, was determined to be the RP2D. Tumor tissue biomarker analysis is underway. Conclusion: These data indicate that E+C at DL1 is a well-tolerated regimen warranting further investigation. The study is actively enrolling patients to the phase II study. Clinical trial information: NCT04345913. Funding Source: National Cancer Institute. Citation Format: Nusayba Bagegni, Brittney Haas, Leslie Nehring, Jingqin Luo, Laura Kennedy, Meghna Trivedi, Manali Bhave, Rabih Said, Cynthia Ma. Preliminary results of a phase I dose escalation study of eribulin in combination with copanlisib in patients with metastatic triple negative breast cancer (TNBC) [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 PO1-06-14.

Abstract PO2-27-04: Unravelling the role of FGFR1 in innate immune sensing and endocrine resistance in breast cancer

Abstract Fibroblast Growth Factor Receptor 1 (FGFR1) has emerged as a critical player in various cancer types, including breast cancer. FGFR1 alterations, including gene amplification, overexpression, and activating mutations, have been identified in subsets of breast cancers, contributing to aberrant signaling pathways that promote tumor growth, angiogenesis, and metastasis. Our current research endeavors are directed towards unravelling the intricate mechanistic underpinnings of FGFR1's dual impact on breast tumor progression, specifically its contribution to endocrine resistance and its regulatory role in innate immune sensing and subsequent induction of Type 1 interferons (IFNs) Our investigative efforts have previously established a direct association between FGFR1 and endocrine resistance in hormone receptor positive T47D breast cancer cells. Moreover, a remarkable and distinct inverse correlation linking FGFR1 expression to the Type 1 IFN response was observed. We noticed that in vitro stimulation of T47D cells with Type 1 interferons induces the expression of FGFR1 and ESR1, thereby suppressing the interferon-stimulated genes. Interestingly, we observed that the FGFR1-mediated suppression of the Type 1 interferon response is associated with intracellular nucleic acid sensors, specifically toll-like receptors TLR7 and TLR9. Furthermore, we found that the inhibition of FGFR kinase enhances the expression of IFN-alpha, IFN-beta, TLR7, and TLR9 in T47D cells when stimulated with the TLR7 agonist loxoribine. These results strongly suggest that FGFR1 functions as an innate checkpoint and may be exploited by tumor cells to dampen Type 1 interferon responses, thereby inhibiting antitumor immunity in hormone receptor-positive breast cancer. Citation Format: Sushil Dhakal, Torbjørn Amundsen Lien, Jens Henrik Norum, Helga Bergholtz, Helene Midtun Flatekvål, Therese Sørlie. Unravelling the role of FGFR1 in innate immune sensing and endocrine resistance in breast cancer [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 PO2-27-04.

Oxidative stress is a shared characteristic of ME/CFS and Long COVID

Abstract More than 65 million individuals worldwide are estimated to have Long COVID (LC), a complex multisystemic condition, wherein patients of all ages report fatigue, post-exertional malaise, and other symptoms resembling myalgic encephalomyelitis / chronic fatigue syndrome (ME/CFS). With no current treatments or reliable diagnostic markers, there is an urgent need to define the molecular underpinnings of these conditions. By studying bioenergetic characteristics of peripheral blood lymphocytes in over 16 healthy controls, 15 ME/CFS, and 15 LC donors, we find both ME/CFS and LC donors exhibit signs of elevated oxidative stress, relative to healthy controls, especially in the memory subset. Using a combination of flow cytometry, bulk RNA-seq analysis, mass spectrometry, and systems chemistry analysis, we observed gender-specific aberrations in ROS clearance and damage pathways. While ME/CFS females exhibit higher total ROS and mitochondrial calcium levels, ME/CFS males have normal ROS levels, but larger changes in lipid oxidative damage. The higher ROS levels correlate with hyperproliferation of T cells and can be attenuated by metformin, suggesting this FDA-approved drug may be an effective treatment. Thus, we report that both ME/CFS and LC are mechanistically related and can be diagnosed with quantitative blood cell measurements. We also suggest that effective, patient tailored drugs might be discovered using standard lymphocyte stimulation assays.

Mrgprb2-mediated mast cell activation exacerbates Modic changes by regulating immune niches

Modic changes are radiographic features associated with microfracture, low-virulence organism infection and chronic inflammation with inflammatory cell infiltration in the vertebral endplate region. Mast cells, as innate immune cells similar to macrophages, are present in painful degenerated intervertebral discs. However, the involvement and mechanisms of mast cells in the development of Modic changes remain unclear. Herein, we found increased mast cell infiltration in samples from patients with Modic changes and in mouse models of Modic changes. To clarify the role of mast cells in the progression of Modic changes, we used mast cell-deficient (KITW-SH/W-SH) mice to construct a model of Modic changes and found that the severity of Modic changes in KITW-SH/W-SH mice was significantly lower than that in WT mice. These findings were further supported by the use of a mast cell-specific activator (compound 48/80) and a stabilizer (cromolyn). Furthermore, we found that mast cells were not activated via the classic IgE pathway in the Modic change models and that Mrgprb2 is the specific receptor for mast cell activation reported in recent studies. Then, we utilized Mrgprb2 knockout mice to demonstrate that Mrgprb2 knockout inhibited mast cell activation and thus reduced the degree of Modic changes. Transcriptomic sequencing revealed aberrant PI3K-AKT and MAPK pathway activation in the Mrgprb2-deficient mast cells. Additionally, Mrgpbrb2-activated mast cells regulate immune niches by recruiting macrophages, promoting M1 polarization and reducing M2 polarization, thereby promoting the progression of Modic changes. These findings suggest that mast cells may serve as a novel therapeutic target for addressing Modic changes.

Electrocardiographic approach strategies in patients with Parkinson disease treated with deep brain stimulation.

Deep brain stimulation (DBS) is an interdisciplinary and reversible therapy that uses high-frequency electrical stimulation to correct aberrant neural pathways in motor and cognitive neurological disorders. However, the high frequency of the waves used in DBS can interfere with electrical recording devices (e.g., electrocardiogram, electroencephalogram, cardiac monitor), creating artifacts that hinder their interpretation. The compatibility of DBS with these devices varies and depends on factors such as the underlying disease and the configuration of the neurostimulator. In emergencies where obtaining an electrocardiogram is crucial, the need for more consensus on reducing electrical artifacts in patients with DBS becomes a significant challenge. Various strategies have been proposed to attenuate the artifact generated by DBS, such as changing the DBS configuration from monopolar to bipolar, temporarily deactivating DBS during electrocardiographic recording, applying frequency filters both lower and higher than those used by DBS, and using non-standard leads. However, the inexperience of medical personnel, variability in DBS models, or the lack of a controller at the time of approach limit the application of these strategies. Current evidence on their reproducibility and efficacy is limited. Due to the growing elderly population and the rising utilization of DBS, it is imperative to create electrocardiographic methods that are easily accessible and reproducible for general physicians and emergency services.

Lipidomics Analysis Unravels Aberrant Lipid Species and Pathways Induced by Zinc Oxide Nanoparticles in Kidney Cells.

Zinc oxide nanoparticles (ZnO NPs) are widely used in versatile applications, from high technology to household products. While numerous studies have examined the toxic gene profile of ZnO NPs across various tissues, the specific lipid species associated with adverse effects and potential biomarkers remain elusive. In this study, we conducted a liquid chromatography-mass spectrometry based lipidomics analysis to uncover potential lipid biomarkers in human kidney cells following treatment with ZnO NPs. Furthermore, we employed lipid pathway enrichment analysis (LIPEA) to elucidate altered lipid-related signaling pathways. Our results demonstrate that ZnO NPs induce cytotoxicity in renal epithelial cells and modulate lipid species; we identified 64 lipids with a fold change (FC) > 2 and p < 0.01 with corrected p < 0.05 in HK2 cells post-treatment with ZnO NPs. Notably, the altered lipids between control HK2 cells and those treated with ZnO NPs were associated with the sphingolipid, autophagy, and glycerophospholipid pathways. This study unveils novel potential lipid biomarkers of ZnO NP nanotoxicity, representing the first lipidomic profiling of ZnO NPs in human renal epithelial cells.

The DMT1 isoform lacking the iron-response element regulates normal and malignant hematopoiesis via NOTCH pathway activation.

Natural resistance-associated macrophage protein 2 (NRAMP 2; also known as DMT1 and encoded by SLC11A2) is mainly known for its iron transport activity. Recently, the DMT1 isoform lacking the iron-response element (nonIRE) was associated with aberrant NOTCH pathway activity. In this report, we investigated the function of DMT1 nonIRE in normal and malignant hematopoiesis. Knockdown of Dmt1 nonIRE in mice showed that it has non-canonical functions in hematopoietic stem cell differentiation: its knockdown suppressed development along the myeloid and lymphoid lineages, while promoting the production of platelets. These phenotypic effects on the hematopoietic system induced by Dmt1 nonIRE knockdown were linked to suppression of Notch/Myc pathway activity. Conversely, our data indicate a non-canonical function for DMT1 nonIRE overexpression in boosting NOTCH pathway activity in T-cell leukemia homeobox protein 1 (TLX1)-defective leukemia. This work sets the stage for future investigation using a multiple-hit T-cell acute lymphoblastic leukemia (T-ALL) model to further investigate the function of DMT1 nonIRE in T-ALL disease development and progression.

The Role of Fibroblast Growth Factor-Receptor Pathway Aberrations in Pediatric Diseases

The fibroblast growth factor receptors play a crucial role in binding to fibroblast growth factor and are involved in various pathological conditions. These receptors consist of an extracellular ligand domain, a transmembrane helix domain, and an intracellular domain with tyrosine kinase activity. There are over 48 different isoforms of FGFR. Each FGF receptor in ligand-binding properties and kinase domains. The FGF/FGFR signaling pathway is implicated in various pediatric cancers and therefore are both non-selective and selective FGFR inhibitors available. Additionally, there are five distinct membrane FGF receptors dentified in vertebrates. All belonging to the tyrosine kinase superfamily. In this manuscript, the focus is based on an analysis of the role of FGF receptor signaling pathways and aberrations especially in pediatric diseases. Erk 1 and Erk 2 seem to have an important role as a mediator of FGF signaling in different biological and developmental processes. FGF signaling plays an important role in conserved developmental functions in skeletal growth, palate closure, ear development, cranial suture ossification and neuronal development in the child. Aberrant FGF signaling causes different congenital disorders and different forms of cancer in childhood. Modulating FGF signaling is of great importance for the treatment of rare diseases in childhood.

Complex and variable regulation of ΔNp63 and TAp63 by TGFβ has implications for the dynamics of squamous cell epithelial to mesenchymal transition

TGFβ has roles in inflammation, wound healing, epithelial to mesenchymal transition (EMT), and cancer stem cell states, and acts as a tumor suppressor gene for squamous cell carcinoma (SCC). SCCs are also characterized by high levels of ΔNp63, which induces epithelial cell phenotypes and maintains squamous stem cells. Previous studies indicate a complex interplay between ΔNp63 and TGFβ signaling, with contradictory effects reported. We investigated the effects of TGFβ on p63 isoform proteins and mRNAs in non-malignant squamous and SCC cells, and the role of either canonical or non-canonical TGFβ signaling pathways. TGFβ selectively increased ΔNp63 protein levels in non-malignant keratinocytes in association with SMAD3 activation and was prevented by TGFβ receptor inhibition, indicating activation of canonical TGFβ pathway signaling. TP63 isoform mRNAs showed discordance from protein levels, with an initial increase in both TAP63 and ΔNP63 mRNAs followed by a decrease at later times. These data demonstrate complex and heterogeneous effects of TGFβ in squamous cells that depend on the extent of canonical TGFβ pathway aberrations. The interplay between TGFβ and p63 is likely to influence the magnitude of EMT states in SCC, with clinical implications for tumor progression and response to therapy.

Abstract 6284: Multimoics reveal aberrant pathways and regulation of castration resistant and metastatic prostate cancer

Abstract Prostate cancer is a highly heterogeneous disease and in the end, 30% of patients will experience metastatic and fatal castration resistant prostate cancer (mCRPC). In spite of the vigorous efforts to search the causes of metastatic CRPC (mCRPC), the mechanisms are still largely unresolved. To understand the progression of mCRPC, we utilized scRNA-seq to profile global expression patterns in LNCaP, LNCaP-Abl and PC3 representing castration sensitive and resistant states. ScRNA-seq revealed 335 and 2393 differentially expressed (DE) genes in early and advanced castration resistant states enriched in 43 and 163 aberrant pathways. These included focal adhesion, adherens junction, leukocyte transendothelial migration, ECM-receptor interaction, regulation of actins, gap junction, TGFb, WNT, Hippo, Ephrin and other pathways. Upregulation of these pathways predicted worst disease free/overall survival of The Cancer Genomes Atlas (TCGA) PC cohort using Kaplan-Meir estimation. Further scRNA-seq also indicated most of the pathways were also enriched in ex vivo circulating tumor cells derived from biochemical recurrent or mCRPC patients. Small molecule perturbation verified function of 9 pathways involved in cell proliferation, migration, invasion, and organoid formation of mCRPC cell lines and CTCs. We then delved into how these genes are regulated by the global chromatin landscape through Assay for Transposase-Accessible Chromatin and high throughput DNA-seq (ATAC-seq). In castration sensitive LNCaP, active chromatins are associated with genes but most of them are located in distal intergenic regions. Conversely, the majority (51.9-57.6%) of open chromatins in castration resistant PC cells are situated in promoters that lie within 1kb of transcription start sites. KEGG pathway enrichment analysis uncovered Hippo, Wnt, tight junction, adherens junction, circadian and other pathways that corroborate the scRNA-seq findings. GRO-seq data indicated that nascent transcription supports gene body transcription in castration resistant cells but leads to massive pausing in castration sensitive cells. Citation Format: Aaron M. Horning, Chia-Nung Hung, Brandon Liebermann, Chih-Wei Chou, Che-Kuang Lin, Yao Vickie Wang, Victor X. Jin, Michael A. Liss, Chiou-Miin Wang, Chun- Lin, Meizhen Chen, Tim H-M Huang, Chun-Liang Chen. Multimoics reveal aberrant pathways and regulation of castration resistant and metastatic 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 6284.

Abstract 3366: FoRx-06-428 is a novel PARG inhibitor with potent anti-tumor efficacy in preclinical cancer models

Abstract The formation of poly(ADP-ribose) (PAR) chains from NAD+ precursors by PARP enzymes is a characteristic posttranslational modification during DNA damage repair. PAR chains function as docking platforms for DNA repair proteins that are required to resolve DNA lesions. Equally important is the subsequent removal of PAR chains from protein substrates to conclude the repair process and restore genomic integrity. Poly(ADP-ribose) glycohydrolase (PARG) is the key enzyme in this process and has recently garnered significant attention as a novel cancer target. FoRx-06-428 is a novel, orally bioavailable and highly potent, low molecular weight PARG inhibitor. It binds to the catalytic domain of human PARG with high affinity, as evidenced by surface plasmon resonance (SPR), and displays potent inhibition of enzymatic activity. FoRx-06-428 shows selective reduction of viability in a panel of cancer cell lines highlighting therapeutic potential in multiple cancer indications. Pharmacological inhibition of PARG leads to accumulation of PAR chains and is indicative of target engagement, accompanied by modulation of DNA damage response markers, such as phosphorylation of replication protein A (pRPA) and phosphorylation of H2AX (γH2AX). Compelling anti-tumor efficacy as single agent was observed in several xenograft models (breast and gastric cancer) with excellent tolerability. Dose-dependent accumulation of PAR chains was demonstrated in vivo and constitutes a tractable target engagement biomarker for clinical exploration. Sensitivity to PARG inhibition is partly driven by distinct genomic instabilities, such as defects in homologous recombination (HR) repair, however the activity of PARG inhibitors is also influenced by aberrations in other cancer-relevant pathways. This report represents the first disclosure of the pharmacological characterization of FoRx-06-428 and highlights the value of PARG inhibition as a novel targeted approach to treat DNA repair-deficient cancers. Citation Format: Olivier Querolle, Ulrich Lücking, Luca Iacovino, Alena Freudenmann, Giacomo Rossetti, Marina Gysin, Serena Bologna, Vasilis Dionellis, Marta Malattia, Alessandro Potenza, Nicolas Bocquet, Irena Konstantinova, Andreas Goutopoulos, Sotirios Sotiriou, Thanos Halazonetis, Tarig Bashir, Frank Zenke. FoRx-06-428 is a novel PARG inhibitor with potent anti-tumor efficacy in preclinical cancer models [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 3366.

Abstract 4737: The combination of the BET inhibitor JQ1 and gemcitabine induces regressions in gemcitabine resistant patient-derived xenograft models of pancreatic cancer

Abstract Efforts to develop targeted therapies for drug resistant tumors have been relatively unsuccessful. The 5-year survival for patients with advanced pancreatic cancer is ~3%. Gemcitabine is usually combined with nab-paclitaxel as a standard of care for treatment of locally advanced or metastatic pancreatic ductal adenocarcinoma (PDAC). However, no chemotherapeutic regimens thus far evaluated have been curative. We developed the first PDAC patient-derived xenograft (PDX) models in which resistance against gemcitabine was acquired in vivo. We used RNA-seq and ingenuity pathway analysis to compare expression profiles of paired parent vs gemcitabine resistant PDX models. This comparison suggested a previously unreported mechanism for gemcitabine resistance, involving aberrations of cholesterol biosynthesis and lipid metabolism pathways. We also made the unexpected observation that gemcitabine-resistant PDAC models derived from primary tumors have high levels of 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2), an enzyme essential to cholesterol biosynthesis and rate limiting in ketogenesis. Notably, the BET bromodomain inhibitor JQ1 decreases HMGCS2 levels; and BET inhibitors + gemcitabine exert synergistic cytotoxicity in gemcitabine-resistant PDAC cells. We determined that the BET bromodomain inhibitor JQ1 + gemcitabine induces regressions of two in vivo gemcitabine-resistant tumor models which we developed directly from primary human tumor tissue. These mechanism-based studies suggest that inhibiting HMGCS2 represents a novel approach to therapy for PDAC. We propose that combining gemcitabine with agents that decrease levels of HMGCS2 comprises effective treatment for patients with drug resistant PDAC. Citation Format: Aubrey L. Miller, Samuel C. Fehling, Eric Josh Brown, Eric O. Heard, Tracy L. Gamblin, Rebecca B. Vance, Karina J. Yoon. The combination of the BET inhibitor JQ1 and gemcitabine induces regressions in gemcitabine resistant patient-derived xenograft models of pancreatic 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 4737.

Abstract 2079: Tumor-derived fibrinogen promotes pancreatic ductal adenocarcinoma progression

Abstract Background: Hypercoagulability is a common clinical manifestation in patients with cancers, especially in pancreatic ductal adenocarcinoma (PDAC). Over 20% of PDAC patients with advanced-stage and worse prognosis undergo a venous thromboembolism (VTE) incidence. Fibrinogen (FG) may contribute to VTE due to its high expression in advanced PDAC tumors. Here, we set to determine the biological roles of tumor-derived FG in PDAC. In addition, our previous study showed that Von Willebrand Factor (VWF) A2 domain can effectively bind to FG and reduce platelet clot formation. Therefore, the functional roles of A2 on FG in PDAC were also studied. Methods: The correlation between survival rate and FG expression in PDAC was analyzed using RNAseq profiles of 149 PDAC patient samples from the TCGA database. PDAC subtyping of Baylor College of Medicine (BCM) PDAC patient-derived xenograft (PDX) cohort was performed using RNAseq data according to Moffitt’s criteria. The FG (three chains FGA, FGB, FGG) overexpression (OE) and knock-out (KO) PDAC cell lines were established. The gene expression and biological functions of FG were performed in PDX and cell lines using IHC and immunoblot assays. The FG effect on PDAC progression in vivo was studied in mouse models. Results: TCGA analysis showed that elevated tumor-derived FG gene expression is significantly correlated with worse overall survival and disease-free survival rates in PDAC patients. PDX lines RNAseq analysis clearly separated BCM PDAC PDXs into two subtypes: basal-like and classical PDACs. Basal-like PDXs showed significantly stronger FG staining than that in classical ones. Compared to the classical subtype, the basal-like subtype showed significantly upregulated genes that are associated with the epithelial-mesenchymal transition (EMT), IL6/JAK/STAT3, and TNFα signaling pathways. Accordingly, basal-like subtype PDAC exposed to IL6 or TNFα significantly increased FG and FG receptor expression with activated STAT3 and AKT pathways and elevated EMT markers, whereas classical subtype cells have minimal changes. In addition, we found that the FG-OE cells exhibit enhanced cell proliferation and migration properties whereas FG-KO cells reduced the effects. Consistent with in vitro data, FG-OE PDAC showed significantly higher tumor burdens than that of control PDAC in both orthotopic and subcutaneous PDAC mouse models. Furthermore, A2 protein treatment can effectively block the FG function and dramatically inactivate the FG-induced EMT process and AKT signaling pathway. Conclusions: Tumor-derived fibrinogen is upregulated in a basal-like subtype of PDAC cells and may contribute to tumor growth and metastasis by activating aberrant signaling pathways and promoting EMT. Our findings imply that elevated fibrinogen in PDAC could contribute to tumor progression and the A2 protein which target to fibrinogen may be a promising therapy for basal-like subtype PDAC. Citation Format: Dongliang Liu, Lisa Brubaker, Yichi Niu, Emily LaPlante, George Van Buren, Aleksandar Milosavljevic, Changyi Chen, Chenghang Zong, Miguel Cruz, Qizhi Yao. Tumor-derived fibrinogen promotes pancreatic ductal adenocarcinoma progression [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 2079.

Abstract 5920: Inhibition of TBL1 and β-catenin interaction sensitizes acute lymphoblastic leukemia to chemotherapy

Abstract BACKGROUND: Relapse of T-cell acute lymphoblastic leukemia (T-ALL) and B-cell acute lymphoblastic leukemia (B-ALL) post-chemotherapy or post-immunotherapy remains a therapeutic problem warranting novel therapeutic approaches. Aberrant Wnt pathway activation is a common feature in ALL as Wnt activation through β-catenin is involved in self- renewal of drug resistant leukemia cells. A therapeutical approach to target Wnt signaling in T-ALL and B-ALL remains elusive. Here, we focus on the effect of transducin β-like protein 1 (TBL1) inhibitor tegavivint in combination with chemotherapy in T-ALL and B-ALL. We hypothesize that interrupting the binding of TBL1 to β-catenin abrogates leukemia cell survival and test if tegavivint sensitizes these cells to chemotherapeutic agents such as VDL (Vincristine, Dexamethasone, L-asparaginase) as part of preclinical evaluation of tegavivint in T- and B-cell ALL. METHODS: Four T-ALL cell lines one patient-derived T-ALL case, B-ALL cell lines and eight patient-derived B-ALL cases were used in vitro. We performed cytotoxicity studies using Annexin V/7AAD stainings and flow cytometry or Trypanblue counts to exclude dead cells, western blots, and co-immunoprecipitation assays to investigate effects of TBL-1 inhibition using tegavivint on β-catenin-mediated signaling. Tegavivint was injected intraperitoneally along with chemotherapeutic drugs. RESULTS: β-catenin and TBL1 were uniformly expressed in all T-ALL and B-ALL cells as determined by western blotting, albeit at different levels. Tegavivint decreases β-catenin protein expression as seen through western blot as well as decreases the binding of TBL1 to β-catenin as seen through co-immunoprecipitation (co-IP) in both the T-ALL and B-ALL samples. We also determined that tegavivint as a monotherapy induced apoptosis in both cell lines and patient samples at as little of a dose of 5nM after 24 hours of incubation using Annexin-V/DAPI staining by flow cytometry. We observed that tegavivint can sensitize all cases to chemotherapy (V: Vincristine, D: Dexamethasone, L:L-Asparaginase or Peg-Asparaginase) within 24 hours of incubation. Lastly, in vivo evaluation of tegavivint showed that tegavivint alone and in combination with VD chemotherapy significantly extends survival of NSG mice engrafted with patient-derived T-ALL and B-ALL compared to control treated mice. CONCLUSION: Our preliminary data shows that T-ALL and B-ALL are sensitive to TBL1-β-catenin inhibition using tegavivint as a monotherapy. Tegavivint can also sensitize T-ALL and B-ALL to chemotherapy (VDL). As our data supports our hypothesis that TBL-1-β-catenin inhibition is a new target in ALL therapy, further studies are in progress to preclinically evaluate this approach for clinical care. Citation Format: Samantha Hurwitz, Rachel Friedmann, Tatiana Fourfouris, Ki Jun Lee, Zesheng Wan, Chintan Parekh, Fariba Navid, Deepa Bhojwani, Kimberly Holloway, Yong-Mi Kim. Inhibition of TBL1 and β-catenin interaction sensitizes acute lymphoblastic leukemia to chemotherapy [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 5920.

Abstract 537: Preclinical PD-1 resistance models: Crucial tools for unveiling PD-1 resistance mechanisms and advancing novel treatment strategies

Abstract In the cancer-immunity cycle, the immune checkpoint PD-1 and its ligand PD-L1 collaborate to facilitate tumor immune evasion and promote tumor progression by thwarting immunity-induced apoptosis. Immune checkpoint inhibitors disrupt this alliance by preventing PD-1 from binding to PD-L1, thereby reactivating the patient's immune system. While anti-PD-1/PD-L1 therapy has demonstrated remarkable efficacy in treating solid tumors, durable responses are observed in only a minority of patients. The majority of patients do not experience the full benefits of anti-PD-1/PD-L1 therapy, and a significant proportion of initial responders eventually develop acquired resistance. Thus, the establishment of reliable preclinical PD-1 resistance models is of paramount importance for uncovering resistance mechanisms and devising novel treatment strategies. Common mechanisms contributing to PD-1/PD-L1 resistance include the absence of appropriate tumor antigens, inactivation of tumor surface MHC molecules, aberrant IFNγ signaling pathways, and the presence of an immunosuppressive tumor microenvironment. We have successfully generated multiple drug resistance models at both cellular and animal levels, offering valuable tools for the investigation of diverse drug resistance mechanisms. For instance, our animal model of drug resistance, induced by long-term treatment with the PD-1 antibody (Keytruda), simulates the clinical scenario of acquired resistance due to prolonged medication. Through gene editing techniques, we have created a drug-resistant cell line (CT26-B2M KO) by disrupting the B2M gene in the CT26 cell line, thereby mimicking a tumor model with B2M functional deficiency in mice. Additionally, we have compiled efficacy data from various naturally occurring resistant models, which serve as valuable resources for studying primary resistance or intricate resistance mechanisms. In summary, preclinical PD-1 resistance models serve as crucial tools for researchers to dissect the intricacies of PD-1 resistance mechanisms and offer essential guidance for the development of innovative treatment strategies. Citation Format: Yini Zhu, Yuan Fang, Huiyi Wang, Jun Xing, Lu Yang, Jing Zhao, Xiang Gao, Cunxiang Ju. Preclinical PD-1 resistance models: Crucial tools for unveiling PD-1 resistance mechanisms and advancing novel treatment strategies [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 537.

Abstract 7577: A new cancer therapy strategy: Synthetic lethality induced by inhibition of mTOR and cMYC and JNK activation

Abstract Cancer cells are characterized by aberrant signal transduction and cell cycle control pathways that lead to unchecked cell proliferation. Uncontrolled cancer cell proliferation, as well as immune suppressive macrophage proliferation, contribute to tumor growth and spread. Effort to suppress tumor cell growth using Inhibitors of PI3K or MEK signaling pathways can initially inhibit tumor growth, but resistance develops due to upregulation of the alternative pathway. We identified a novel series of allosteric tyrosine kinase receptor inhibitors that induce synthetic lethality in AML, lung carcinoma, pancreatic carcinoma, and other tumor cells and in proliferating macrophages in vitro and in vivo by simultaneously inhibiting cMyc and Tor and activating the stress activated kinase JNK, leading to p53 mediated cell cycle arrest. Select kinase inhibitors with a unique positioning of a single side chain inhibited CDK8/19 and thereby Myc while stimulating phosphorylation and activation of JNK and p38. JNK activation promoted sustained ATF-2 and cJun-mediated p53 and Chk1/Chk2 activation, thereby promoting cell cycle arrest in G2/M and ultimately, apoptosis. These inhibitors potently suppressed tumor growth in vivo by halting both tumor cell and macrophage proliferation in G2/M. They synergized with anti-PD-1 and anti-CTLA-4 and led to tumor eradication and immunological memory formation. These studies identify novel allosteric inhibitors with potent synthetically lethal anti-tumor activity. Citation Format: Hui Chen, Jason Rivera, Marc Paradise, Zihan Xu, Giuliana Mognol, Judith Varner. A new cancer therapy strategy: Synthetic lethality induced by inhibition of mTOR and cMYC and JNK activation [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 7577.

Abstract 5047: Identifying genomic features associated with pathologic lymph node metastasis in lung adenocarcinoma patients

Abstract Introduction: Lymph node metastasis evaluation in solid tumors is mainly complicated by the diagnostic yield of standard imaging modalities and tumor size contradictions. Exploring clinical/molecular features that complement the use of radiologic and bronchoscopy findings may provide significant values for a more accurate pathologic determination of nodal metastasis. Methods: We analyzed surgery-resected tumor samples from 72 treatment-naïve lung adenocarcinoma (LUAD) patients via whole-exome sequencing (WES). Mutational profiles of patients categorized based on their pathologic lymph node metastasis (pN) status were assessed, and the association between metastatic-driving features and overall survival (OS) was then explored in both the study cohort and the Cancer Genome Atlas Program (TCGA) external cohort. Results: Among all 72 patients, 41 (56.9%) were pathologically confirmed as positive for lymph node metastasis (pN-positive), while 31 (43.1%) were categorized as pN-negative. The most frequently mutated gene was TP53 in both subgroups (54.8% and 53.7%, respectively), and the aberrant KEAP1-NRF2 signaling pathway was found significantly more enriched in pN-negative patients. Copy-number variant analysis showed that chromosome 7p amplifications (X7p_amp) at the arm level and EGFR amplifications at the focal gene level were more frequently identified in pN-positive patients (P=0.008 and P=0.004, respectively). Importantly, these amplifications were associated with worse OS in both the study cohort and the TCGA external dataset. Consistent with the unfavorable prognosis, pN-positive patients demonstrated a higher chromosome instability score (CIS) (median: 0.32 vs. 0.14, P=0.14) and a lower tumor mutation burden (TMB) (median: 1.83 vs. 3.85 muts/Mb, P=0.048) compared to pN-negative patients. Conclusion: Our findings suggest that X7p_amp, particularly EGFR amplification, might be associated with pathologic involvement of lymph nodes and an unfavorable prognosis in LUAD patients. Our study highlights the importance of integrating clinical and genomic data into conventional radiologic modalities for a more accurate determination of pathologic nodal involvement. Citation Format: Shijie Wang, Zhiwei Zhou, Song Wang, Rongyun Guo, Zeming Ma, Dachuan Zhao, Liang Wang, Yinan Liu, Yuanyuan Ma, Jianzhi Zhang, Sha Wang, Yedan Chen, Haimeng Tang, Qiuxiang Ou, Jinfeng Chen. Identifying genomic features associated with pathologic lymph node metastasis in lung adenocarcinoma 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 5047.

Abstract 3576: CRISPR Cas9 mediated MADD deletion can induce autophagy via downregulation of MAPK and PI3K/AKT/mTOR signaling in anaplastic thyroid cancer

Abstract Thyroid cancer is a highly prevalent endocrine malignancy with one of the fastest growing incidences of all cancers in the United States. Anaplastic thyroid cancer (ATC) has an incidence of about 1.7% of all thyroid cancers, but it causes over 40% of thyroid cancer-related deaths with a median survival period of less than one year. This is why agents that inhibit the aberrant signaling pathways which drive ATC aggressiveness are of significant interest. MAPK-activating Death Domain-activating protein (MADD) plays a key role in cancer cell survival and proliferation. MADD is constitutively expressed at higher levels in most tumor tissues and cancer cells relative to normal tissues. Studies using shRNA suggest that MADD prevents apoptosis in ATC cells. For a more definitive and mechanistic analysis of MADD in ATC, we used conditional CRISPR-Cas9 gene editing to target MADD. We observed that MADD deletion in ATC cells induced spontaneous apoptosis and also activated the autophagy pathway in human ATC cells through downregulation of ERK and mTOR signals involving the MAPK and PI3K/Akt/mTOR signaling pathways. Transcriptomic analysis of publicly available TCGA data showed several PI3K/AKT/mTOR pathway genes were positively correlated with MADD levels. RNA sequencing data of ATC lines demonstrated that enhanced activity of the PI3K/AKT/mTOR signaling pathway was correlated with a worse prognosis in ATC. Targeted MADD deletion in ATC cells combined with treatment using ERK/PI3K/Akt/mTOR/autophagy inhibitors resulted in enhanced cell death compared with MADD deletion alone. Further, MADD deleted 8505C-ATC cells showed significantly delayed tumor formation in an orthotopic nude mouse model. These findings suggest that combining MADD deletion with inhibitors of PI3K/Akt/mTOR pathway could provide an attractive new therapeutic option for ATC. Citation Format: Velavan Bakthavachalam, Manikannan Madhayan, Mark Sanborn, Jalees Rehman, Prabhakar Bellur. CRISPR Cas9 mediated MADD deletion can induce autophagy via downregulation of MAPK and PI3K/AKT/mTOR signaling in anaplastic thyroid 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 3576.

Abstract 7575: BPI-460372, a covalent, irreversible TEAD inhibitor in Phase I clinical development

Abstract The Hippo signaling pathway is critical for the regulation of organ development, tissue homeostasis, and tumorigenesis by controlling the activation status of yes-associated protein (YAP) or its homolog PDZ-binding motif (TAZ). As a major downstream effector, the transcription factor TEAD is activated by forming a complex with YAP/TAZ. Hippo pathway aberrations leading to TEAD activation have been reported in many cancers. Inhibiting TEAD function by directly blocking the palmitoylation pocket of TEAD is a potential therapeutic approach for cancer treatment. Betta Pharma has discovered a selective, covalent and irreversible TEAD inhibitor known as BPI-460372. The discovery of BPI-460372 was first reported in AACR 2023. Here we present additional preclinical and clinical data characterizing BPI-460372 as a promising candidate drug. In the dose-escalating part of the Phase Ι study, the exposure of BPI-460372 increased proportionally over 10 to 80 mg dose range after oral administration and exhibited linear pharmacokinetics, with a clearance rate suitable for once-daily dosing. The compound is generally well tolerated, with no drug-related Grade ≥3 adverse event observed. Remarkably, we have not seen renal toxicities that was perceived as a potential risk associated with certain TEAD inhibitors. Preliminary efficacy was observed in a few subjects. In preclinical studies, we found that intermittent dosing schedule of 3 days on/4 days off, compared with continuous dosing at equivalent weekly exposure, can ameliorate kidney damage and improve early renal injury biomarkers while maintaining anti- tumor activity. Thus, alternative dosing schedule could further improve the therapeutic window and enable combination studies in clinical development. Collectively, these results support further development of BPI-460372 for the treatment of cancers harboring Hippo pathway alterations. Citation Format: Xiaohong Han, Jing Guo, Xiangyu Jin, Lingyin Zhu, Hongling Shen, Xiaofeng Xu, Mengjie Zhang, Xiaoyun Liu, Yanju Liu, Hongke Cheng, Shunna Guo, Lieming Ding, Jiabing Wang, Hong Lan, Mengzhao Wang. BPI-460372, a covalent, irreversible TEAD inhibitor in Phase I clinical development [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 7575.