Abstract Background: Immunosuppression is a key characteristic of pancreatic ductal adenocarcinoma (PDAC), contributing to metastasis and poor survival. Our studies have identified tumor cell intrinsic Rho-associated coiled-coil containing protein kinase-2 (ROCK2) as a key regulator of extracellular matrix remodeling. In this study, we investigated how ROCK2 regulates immunosuppression in PDAC by modulating Leukemia inhibitory factor (LIF) and its effects on STAT3. Methods: TCGA PDAC patient dataset was used to compare the ROCK2 and LIF expression in normal and PDAC tissues. CIBERSORT analysis of the PDAC dataset estimated the proportion of tumor infiltrating immune cell subsets. Genomic editing using the CRISPR/Cas9-system in LSL-KrasG12D/+; Trp53 R172H/+; Pdx1Cre/+ (KPC) cells was performed to generate KPC Rock2 knockout (Rock2KO) cells. Cytokine array was performed on Rock2EV and Rock2KO conditioned media and ELISA was used to validate the results. Flow cytometry was used to profile LIF receptor (LIFR) expression in different cell types from KPC orthotopic tumors. Bone marrow-derived macrophages from C57BL/6 mice were treated with recombinant LIF (rLIF) and LIFR inhibitor (EC359), then analyzed for polarization by flow cytometry. KPC orthotopic tumors were generated, and Immune cell profiling was performed to evaluate alterations in immune cell subsets following treatment with EC359. Findings from ROCK2 and ROCK2-regulated LIF-STAT3 targeting, both in vitro and in vivo were validated using Immunoblotting and immunohistochemistry. Results: Analysis of the TCGA dataset revealed that Human PDAC tissues have increased expression and correlation of ROCK2 and LIF. Further analysis showed that macrophages constitute a substantial proportion of the immune cell infiltrate. Cytokine array and ELISA-based studies revealed decreased LIF secretion with ROCK2 knockout, providing evidence for ROCK2 dependent regulation of LIF. KPC orthotopic tumors demonstrated higher LIFR expression in tumor-associated macrophages (TAMs) and EC359 treatment reduced ARG1 and PD-L1 expression on these cells. Additionally, EC359 treatment led to a significant increase in the activated effector and effector memory T cell populations. Furthermore, rLIF treatment increased pSTAT3 levels in macrophages, while EC359 lowered its expression, highlighting the role of ROCK2-regulated LIF-LIFR in STAT3 activation. Conclusion: These findings demonstrate that tumor cell-intrinsic ROCK2 regulates LIF-STAT3, which mediates immunosuppression and can serve as a potential therapeutic target for PDAC. Citation Format: Varunkumar Krishnamoorthy, Sudhakar Jinka, Siddharth Mehra, Phuong Hong Ngoc Tao, Daysi Daniela Manrique, Rimpi Khurana, Yuguang Ban, Vineet Kumar Gupta, Austin Dosch, Nagaraj Nagathihalli. ROCK2-regulated LIF-STAT3 drives immunosuppression in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 7011.

Abstract Background: Patients diagnosed with pancreatic ductal adenocarcinoma (PDAC) face a dismal prognosis, with only about 13% surviving five years. Transcriptional factors play a vital role in PDAC survival and prognosis by defining tumor aggressiveness, therapeutic resistance, and clinical outcome. Key regulators such as CREB1 have previously been linked with tumor progression and shorter survival. While CREB1 signaling driving PDAC is known, the systematic characterization of active Transcription Factor (TF) associated with CREB1, and their clinical relevance is limited. In this study, we aimed to identify critical TF activity associated with CREB1 and assess their combinatorial prognostic impact in PDAC. Methods: We utilized the VIPER (Virtual Inference of Protein-activity by Enriched Regulon) algorithm with high-confidence DoRothEA regulons (A-C) to infer TF activity across 182 TCGA-PAAD RNA-seq samples (178 Primary Tumors [TP] and 4 Normal Tissues [NT]). Differential Activity Analysis (DAA) using limma identified TFs dysregulated between TP and NT. Survival analyses utilized Kaplan-Meier and Cox Proportional Hazards modeling on the TP cohorts, focusing on the interaction between two key dysregulated TFs, CREB1 and ELF3 (ETS transcription factor 3). R version 4.3.0 was used for all analyses, including GSVA (v1.50.5) and VIPER (v1.36.0). Differential expression and pathway analyses were performed using the R packages limma (v3.58.1), GSVA (v1.50.5), msigdbr, gplots, and ggplot2. Results: DAA identified 44 significantly dysregulated TFs (FDR < 0.05). The epithelial lineage regulator ELF3 showed the highest activation in tumors, while the lymphoid regulator PAX5 (paired box 5) was highly repressed. Although single-TF prognostic tests were non-significant, combinatorial analysis revealed a strong context-dependent effect. The simultaneous High CREB1 and High ELF3 activity defined a uniquely aggressive subgroup with a markedly shorter median overall survival (272 days) compared to the lowest-risk combination (492 days). Cox modeling confirmed a significant synergistic interaction between the two TFs (HR =2.31, p = 0.049). Conclusion: This analysis reveals that the highly activated epithelial driver ELF3 acts synergistically with CREB1 to define a high-risk prognostic signature in PDAC. Our findings underscore that TF networks, rather than single factors, are crucial for patient stratification and represent compelling, context-specific therapeutic targets in PDAC. Citation Format: Poorva Poorva, Rimpi Khurana, Varunkumar Krishnamoorthy, Sudhakar Jinka, Yan Guo, Vineet Kumar Gupta, Nagaraj Nagathihalli. Integrated analysis identifies ELF3-CREB1 co-activation as a prognostic driver of high-risk pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2722.

BACKGROUND & AIMS:Chronic alcoholism often leads to pancreatitis, which exacerbates pancreatic damage through acinar cell injury and fibrotic inflammation activating AKT/mTOR/cyclic adenosine monophosphate response element binding protein 1 (CREB) signaling axis. However, the molecular interplay between oncogenic KrasG12D/+(Kras∗) and CREB in promoting pancreatic cancer progression under chronic inflammation remains poorly understood. METHODS:Experimental alcoholic chronic pancreatitis (ACP) induction was established in multiple mouse models, with euthanasia during the recovery stage to evaluate tumor latency. CREB was selectively deleted (Crebfl/fl) in Ptf1aCreERTM/+;LSL-KrasG12D/+(KC) genetic mouse models (KCC-/-). Pancreata from Ptf1aCreERTM/+, KC, and KCC-/- mice were analyzed using histological profiling, Western blotting, phosphokinase array, and quantitative polymerase chain reaction. Single-cell RNA sequencing was performed in ACP-induced KC mice. Lineage tracing analysis using YFP reporter mice and acinar cell explant cultures analysis were also conducted. RESULTS:ACP induction in KC mice significantly impaired pancreas' repair mechanism. Acinar cell-derived ductal lesions demonstrated sustained CREB hyperactivation in acinar-to-ductal metaplasia/pancreatic intraepithelial neoplasia lesions associated with pancreatitis and pancreatic cancer. Persistent CREB activity reprogrammed acinar cells, and increased profibrotic inflammation. Notably, acinar-specific Creb deletion in ACP-induced models suppressed high-grade pancreatic intraepithelial neoplasia development, restrained tumor progression, and improved acinar cell function. CONCLUSIONS:Our findings demonstrate that CREB and Kras∗ promote irreversible acinar-to-ductal metaplasia, accelerating pancreatic cancer progression with ACP. Targeting CREB may present a promising strategy to mitigate inflammation-driven pancreatic tumorigenesis.

Pancreatic ductal adenocarcinoma (PDAC) remains a challenging disease in need of improved treatments. Cyclic adenosine monophosphate response element binding protein 1 (CREB) is an emerging therapeutic target whose oncogenic effects in PDAC have been largely attributed to a key molecular interplay between oncogenic Kras G12D/+ ( Kras* ) and chronic inflammation driving irreversible acinar to ductal reprogramming. Here, we demonstrate that CREB activation fosters tumor associated macrophage (TAM) mediated immunosuppression and promotes PDAC growth in an aggressive LSL-Kras G12D/+ ; Trp53 R172H/+ ;Pdx1 Cre/+ ( KPC ) genetically engineered mouse model. Selective deletion of CREB ( Creb fl/fl ) in KPC ( KPCC -/- ) mice attenuates primary disease burden. Unbiased transcriptomic analysis and validation using diverse molecular, genetic and pharmacological approaches in vitro and in vivo identify CREB-mediated transcriptional regulation of leukemia inhibitory factor ( Lif ) as one of the potential mediators of tumor cell-macrophage crosstalk promoting a pro-tumor polarization of TAMs, thereby attenuating the infiltration of effector T cells. Mechanistically, cancer cell derived LIF facilitates an immunosuppressive, pro-tumorigenic state. Importantly, pharmacological targeting of the CREB-LIF signaling axis between cancer cells and macrophages, using a CREB-specific inhibitor (CREBi), significantly suppresses tumor growth and sensitizes PDAC to immunotherapy, highlighting the therapeutic potential of this treatment combination to improve outcomes in this aggressive disease.

Smoking, a major risk factor for pancreatic cancer, significantly attenuates the survival benefits of standard therapies, contributing to poor clinical outcomes in this malignancy. Previously, we have identified that hyperactivation of cyclic AMP response element-binding protein (CREB) as driver of immunosuppressive tumor immune microenvironment (TME), which limits the therapeutic potential of immune checkpoint blockade (ICB) therapies. The present study explores the therapeutic strategy of targeting CREB to overcome TME associated immunosuppression and to enhance the efficacy ICB in smoking associated preclinical models of pancreatic cancer. An in vivo smoking model was developed using syngeneic orthotopic tumor implantation in C57BL/6 mice and genetically engineered mouse model (GEMM) of pancreatic cancer, including Creb deleted (Crebfl/fl, CREBcKO) LSL-KrasG12D/+; Trp53 R172H/+; Pdx1Cre/+ (KPC) mice. CRISPR/Cas9 technology was used to generate Creb knockout (CrebKO) in KPC tumor cells, which were orthotopically implanted into the pancreata of C57BL/6 mice. Mice with established tumors were randomized and treated with CREB inhibitor (CREBi, 666-15) alone or in combination with anti-PD-1 monoclonal antibody. This combination therapy was further evaluated in Ptf1aCre/+; LSL-KrasG12D/+;Tgfbr2flox/flox (PKT) GEMM. Multiplex immunophenotyping was performed on harvested tumors to assess immune subsets changes, along with histological analyses including H&E, immunohistochemistry (IHC) and Sirius red staining. Smoking-exposed KPC GEMM mice showed accelerated PDAC progression, characterized by increased high-grade PanIN lesions and an enhanced fibroinflammatory TME. CREB deletion (CREBcKO) significantly attenuated these effects, highlighting its role in smoking-induced tumorigenesis. Genetic ablation or pharmacological inhibition of CREB using 666-15 attenuated the immunosuppressive TME, promoted effector CD4+ and CD8+ T cell infiltration with elevated PD-1 expression on T cells. In combination with anti-PD-1 therapy, CREB inhibition enhanced the anti-tumor benefits in both PKT GEMMs and in KPC orthotopic models. Similarly, CrebKO tumor cells implanted in smoke-exposed C57BL/6 mice showed significant tumor suppression and modulates TME, with or without anti-PD-1 therapy. This study demonstrates the therapeutic advantage of targeting CREB to reprogram the stromal immune microenvironment and improve the efficacy of immunotherapy in preclinical models of smoking-associated pancreatic cancer. These findings provide a strong rationale of integrating CREB inhibition into immunotherapeutic strategies for pancreatic cancer, particularly in high-risk smoking populations. Varunkumar Krishnamoorthy, Siddharth Mehra, Sudhakar Jinka, Haleh Amirian, Edmond Box, Chen Zheng, Jashodeep Datta, Nipun Merchant, Nagaraj Nagathihalli. Targeting CREB reprograms the tumor immune microenvironment and enhances immunotherapy efficacy in smoking-associated pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3290.

Pancreatic ductal adenocarcinoma (PDAC) is projected to become a leading cause of cancer-related mortality, driven by its late-stage diagnosis, aggressive progression, and propensity for metastasis. Currently, no approved therapeutic effectively targets PDAC metastasis. Emerging evidence implicates extracellular matrix (ECM), and cytoskeletal dynamics are key to invasion, a pertinent contributor to PDAC metastasis. Our previous studies identified cAMP-response element binding protein 1 (CREB) as a key regulator of ECM alterations in PDAC. This study investigates the role of CREB-ROCK in regulating tumor cell invasion and metastasis at the molecular level. Expression profiles of CREB and invasion-related genes were analyzed using The Cancer Genome Atlas (TCGA) PDAC patient’s dataset. CREB expression and localization were examined in PDAC patient’s tissue microarrays (TMAs) and patient-derived xenograft (PDX) models. A novel conditional Creb knockout (Crebfl/fl) model, in an LSL-KrasG12D/+; Trp53 R172H/+; Pdx1Cre/+ (KPC) background (KPCC-/-), was generated to delineate CREB’s role in PDAC progression and metastasis. Functional studies included lentiviral creb overexpression, CRISPR/Cas9-based Creb knockout, RNA sequencing (RNA-seq), and chromatin immunoprecipitation sequencing (ChIP-seq). Metastatic lesions were assessed in genetically engineered mouse and orthotopic models (GEMMs). Matrigel invasion assays, immunohistochemistry, immunoblotting, and qPCR were used to validate the findings in vitro and in vivo. TCGA analysis revealed elevated CREB and invasive gene expression in PDAC compared to normal pancreatic tissues. Immunostaining of TMAs showed increased CREB expression localized to ductal tumor cells. Creb knockout in KPCC-/- mice reduced metastatic burden, while Creb overexpression induced liver metastasis in orthotopic models. Invasion assays confirmed decreased invasiveness of tumor cells with CREB inhibition or deletion. CREB inhibition showed reduced expression of ROCK1/2, a key mediator of cytoskeletal dynamics and ECM remodeling. RNA-seq, qPCR, and ChIP-Seq analysis identified CREB as a transcriptional regulator of genes essential for invasion and ECM remodeling. CREB deletion or inhibition attenuated the expression of these targets, decreasing tumor cell invasion and metastatic potential. These findings establish CREB-ROCK as a central mediator of PDAC metastasis and targeting CREB-ROCK represents a promising therapeutic strategy for inhibiting invasive PDAC progression and metastasis. Sudhakar Jinka, Siddharth Mehra, Varunkumar Krishnamoorthy, Andrew M. Adams, Anna Bianchi, Luis N. Alberto, Yuguang Ban, Jashodeep Datta, Nipun Merchant, Nagaraj Nagathihalli. CREB-driven tumor cell invasion and ECM remodeling promote metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1311.

Abstract Introduction: Alcoholic chronic pancreatitis (ACP) is a progressive fibroinflammatory disease of the pancreas often characterized by extensive acinar cell injury leading to acinar-to-ductal metaplasia (ADM). However, the impact of ADM associated with ACP on the acceleration of oncogenic mutant Kras (Kras*)-driven tumorigenesis remains poorly understood. In this study, we aimed to unravel the underlying mechanism by which ACP-induced dysfunction and with Kras*, leads to the development of irreversible ADM and premalignant lesions that establish a tumor-permissive microenvironment for the emergence of high-grade pancreatic intraepithelial neoplasia (PanINs). Methods: ACP mice model was developed in acinar cell-specific Ptf1aCreERTM/+, Ptf1aCreERTM/+; LSL-KrasG12D/+ (KC), and genetic ablation of cyclic AMP response element-binding protein 1 (CREB) in KC (KCC−/−) mice crossed with or without R26R-EYFP reporter mice, and these mice were fed with alcohol-enriched or regular diet along with repetitive caerulein administration. Lineage tracing studies and acinar explants were employed to investigate the fate of adult acinar cells. ACP-induced mice were subsequently euthanized either 3 days or 21 days after ACP induction, the pancreas was collected for immunohistochemical analysis and examined for ADM, PanINs, fibrosis, and inflammation. To gain insights into the molecular mechanisms; kinase array analysis, acinar cell explant culture, Western blot and qPCR-based studies were conducted. Additionally, single-cell RNA sequencing (scRNA-seq) was employed to investigate cell-specific transcriptomic changes focusing on distinct cellular compartments. Results: Lineage tracing studies showed that ACP-induced ADM stems from direct transdifferentiating adult acinar cells to a ductal phenotype in Kras* mice. ACP induction with Kras* cooperatively provided a sustained Kras - dependent oncogenic program in inflammation-induced metaplastic progenitor cells. The upregulation of CREB accompanied this process of malignant transformation. CREB activation persists even after ACP withdrawal in KC mice compared to Ptf1aCreERTM/+ mice without mutant Kras. Acinar-specific CREB ablation attenuated advanced PanIN lesion development, impeding tumor progression in Kras* mice. Additionally, it reduced inflammation-induced tissue injury and facilitated acinar cell recovery. ScRNA-seq analysis revealed gene enrichment favoring acinar cell homeostasis and downregulation of ductal gene signatures in KCC−/− mice, further emphasizing CREB's pivotal role in modulating these cellular processes. Conclusion: Our findings demonstrate that Kras* sustains an irreversible ADM and PanIN through continuous activation of CREB. Additionally, we have identified that CREB mediates an oncogenic transcriptional program that drives tumor progression in the context of inflammation-induced disease. Importantly, our study reveals that ACP synergistically cooperates with Kras*-driven activated CREB, promoting the progression of ADM toward high-grade PanINs. Citation Format: Siddharth Mehra, Supriya Srinivasan, Anna Bianchi, Austin Dosch, Samara Singh, Vanessa Garrido, Iago De Castro Silva, Varunkumar Krishnamoorthy, Sudhakar Jinka, Jashodeep Datta, Nipun B. Merchant, Nagaraj Nagathihalli. Alcoholic chronic inflammation driven CREB mediates acinar-to-ductal reprogramming and promote neoplastic progression [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B093.

Abstract Background: Pancreatic Ductal Adenocarcinoma (PDAC) is characterized by a heightened oncogenic mutational burden, an immunosuppressive tumor microenvironment, and a dense desmoplastic stroma. During the tumor progression from in situ carcinoma to intraductal neoplasia, cancer cells manage to evade the stromal compartment and infiltrate into the ducts. This invasive tumor progression is often associated with the overexpression of extracellular matrix (ECM) modulating genes. Despite this correlation, there are currently no effective and specific inhibitors of ECM-altering pathways. By understanding the signaling mechanism, the ECM remodeling pathways may be intercepted to prevent invasive tumor progression. This study aims to elucidate the role of cAMP-response element binding protein 1 (CREB1) and its downstream effectors in regulating PDAC ECM remodeling. Methods: The Cancer Genome Atlas (TCGA) was quired for CREB1 expression and other ECM modulators in PDAC patients. In vitro, CRISPR/CAS9-based genomic editing was utilized to knockout CREB1 in the murine KPC cell line, on which RNA-seq analysis was performed. To investigate the effects of CREB in PDAC progression, a novel conditional CREB1 knockout (CREBfl/fl) was created in LSL-KrasG12D/+; Trp53 R172H/+; Pdx1Cre/+ (KPC) mice (KPCC-/-). Tumors were extracted and histologically evaluated. Immunoblotting and staining were conducted on both in vitro and in vivo samples. Chromatin immunoprecipitation sequencing (ChIP-seq) and qPCR were performed on the KPC CREBKO. Eukaryotic Promoter Database (EPD) and ENCODE databases were explored to evaluate the transcriptional role of CREB in regulating ECM genes. Results: Patient PDAC samples data from TCGA revealed elevated levels of CREB1, ROCK1, and ROCK2 compared to normal pancreatic tissues. When comparing high CREB expression to low CREB expression, upregulation of several ECM genes was noted, including ROCK1 and ROCK2 in patients with high CREB expression. In CREBKO, RNA-seq analysis revealed the downregulation of key ECM genes, including Mmp3, Mmp10, Lama3, and Fn1 compared to CREB wildtype. In our novel murine conditional knockout model, a substantial reduction in fibrosis was observed. In both in vitro and in vivo, immunoblot and staining demonstrated a marked reduction in the expression of ECM remodeling proteins upon CREB deletion. Through ChIP-seq, the direct involvement of CREB1 in the transcriptional regulation of Rock1 and Rock2 was established. EPD and ENCODE were utilized to confirm and validate our CREB regulation experimental findings. Conclusion: These findings demonstrate the role of CREB in PDAC progression, which may provide a potential target to intercept ECM remodeling via inhibition of the CREB-ROCK axis to suppress PDAC tumor progression and invasion. Citation Format: Sudhakar Jinka, Siddharth Mehra, Varunkumar Krishnamoorthy, Anna Bianchi, Karthik Rajkumar, Andrew Adams, Haleh Amirian, Samara Singh, Edmond W. Box, Erin Dickey, Nivelo L. Alberto, Ban Yuguang, Jashodeep Datta, Nipun Merchant, Nagaraj Nagathihalli. CREB-ROCK driven extracellular matrix remodeling exasperates pancreatic cancer 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 4277.

Abstract Therapeutic resistance remains a major obstacle to successful clinical management of diffuse intrinsic pontine glioma (DIPG), a high-grade pediatric tumor of the brain stem. In nearly all patients, available therapies fail to prevent progression. Innovative combinatorial therapies that penetrate the blood–brain barrier and lead to long-term control of tumor growth are desperately needed. We identified mechanisms of resistance to radiotherapy, the standard of care for DIPG. On the basis of these findings, we rationally designed a brain-penetrant small molecule, MTX-241F, that is a highly selective inhibitor of EGFR and PI3 kinase family members, including the DNA repair protein DNA-PK. Preliminary studies demonstrated that micromolar levels of this inhibitor can be achieved in murine brain tissue and that MTX-241F exhibits promising single-agent efficacy and radiosensitizing activity in patient-derived DIPG neurospheres. Its physiochemical properties include high exposure in the brain, indicating excellent brain penetrance. Because radiotherapy results in double-strand breaks that are repaired by homologous recombination (HR) and non-homologous DNA end joining (NHEJ), we have tested the combination of MTX-241F with an inhibitor of Ataxia Telangiectasia Mutated to achieve blockade of HR and NHEJ, respectively, with or without radiotherapy. When HR blockers were combined with MTX-241F and radiotherapy, synthetic lethality was observed, providing impetus to explore this combination in clinically relevant models of DIPG. Our data provide proof-of-concept evidence to support advanced development of MTX-241F for the treatment of DIPG. Future studies will be designed to inform rapid clinical translation to ultimately impact patients diagnosed with this devastating disease.

Supplemental Tables 1, 2, 3

Therapeutic resistance remains a major obstacle to preventing progression of H3K27M-altered Diffuse Midline Glioma (DMG). Resistance is driven in part by ALDH-positive cancer stem cells (CSC), with high ALDH1A3 expression observed in H3K27M-mutant DMG biopsies. We hypothesized that ALDH-mediated stemness and resistance may in part be driven by the oncohistone itself. Upon deletion of H3K27M, ALDH1A3 expression decreased dramatically and was accompanied by a gain in astrocytic marker expression and a loss of neurosphere forming potential, indicative of differentiation. Here we show that the oncohistone regulates histone acetylation through ALDH1A3 in a Wnt-dependent manner and that loss of H3K27M expression results in sensitization of DMGs to radiotherapy. The observed elevated Wnt signaling in H3K27M-altered DMG likely stems from a dramatic suppression of mRNA and protein expression of the Wnt inhibitor EYA4 driven by the oncohistone. Thus, our findings identify EYA4 as a bona fide tumor suppressor in DMG that upon suppression, results in aberrant Wnt signaling to orchestrate stemness and differentiation. Future studies will explore whether overexpression of EYA4 in DMG can impede growth and invasion. In summary, we have gained mechanistic insight into H3K27M-mediated regulation of cancer stemness and differentiation, which provides rationale for exploring new therapeutic targets for DMG.

The present study aims to investigate the effect and the molecular mechanism of N-(2-hydroxyphenyl)-2-phenazinamine (NHP) isolated from Nocardiopsis exhalans against the proliferation of human lung cancer cells. The cytotoxic activity of NHP against A549 and H520 cells was determined using MTT assay. The cytotoxic activity of NHP against A549 and H520 lung cancer cells showed excellent activity at 75 μg/mL and damage the mitochondrial membrane and nucleus by generating oxidative stress. NHP causes nuclear condensation and induces apoptosis which was confirmed using AO/EB and PI/DAPI dual staining assay. Moreover, the NHP downregulates the oncogenic genes such as IL-8, TNFα, MMPs and BcL2 and also upregulates the expression of apoptosis marker genes such as Cyto C, p53, p21, caspase 9/3 in A549 and H520 human lung cancer cells. Considering the strong anticancer activity of NHP against lung cancer, NHP may be further evaluated as a potential anticancer drug for the treatment of lung cancer.

With plenteous accessible therapeutics, lung cancer endures a preeminent cause of the worldwide fatality. Apart from medical advancements, various plant parts are still used to treat cancer based on proven tradition. The present study focuses on analysing the anticancer efficacy of silver nanoparticles coupled with the aqueous leaf extract of Annona muricata. Nanoparticles play a momentous role in drug delivery due to their size and high surface to volume ratio and are with fewer side effect when phytofabricated. Annona muricata aqueous leaf extract mediated silver nanoparticles were characterized using UV visible spectrophotometer, Fourier Transform Infrared Spectroscopy (FTIR), X-ray Diffraction and Zeta-sizer. Their antiproliferative potency was analysed by studying the mRNA and protein expressions of various apoptotic, anti-apoptotic and cell cycle regulatory genes. In addition, the cell cycle regulation was further confirmed using flow cytometry. The nanoparticles were found to be spherical shaped crystals with 80 +/- 6.3 nm as average size and 6 mu g/ml as inhibitory concentration 50 (IC50) on A549 human lung cancer cell line. It was observed that the nanoparticles efficiently induced apoptotic protein expression with a simultaneous suppression of anti-apoptotic protein. The results demonstrate activation of an intertwined intrinsic apoptotic pathway via caspases and the death receptors. The observations infer that the nanoparticles show excellent anticancer efficacy than the crude extract of Annona muricata leaves. Hence these nanoparticles would be a promising adjuvant for treating non-small cell lung cancer.

Sirtuins (SIRT1-7), are NAD-dependent deacetylases and ADP-ribosyl transferases, plays a major part in carcinogenesis. The previous report suggests that in cancer, sirtuins gained tremendous interest and critical regulators of the unusual processes. In carcinogenesis, sirtuins possess either tumor suppressor or promoter. However, in lung cancer condition the studies of sirtuins are less studied. Hence, this designed study investigates the impact of multifaceted sirtuins in NSCLC cells. We evaluated the mRNA and protein expressions of sirtuins by RTPCR and western blot. We found SIRT6 significantly overexpressed in NCI-H520, A549, and NCI-H460 compared with the normal BEAS-2B cell line. Silencing of SIRT6 by siRNA in NSCLC cells caused activation of p53/p21 mediated inhibition of cell proliferation leading to arrest in cell cycle and apoptosis induction. Our results implied that SIRT6 is a tumor promoter in NSCLC development, progression, and regulation. The silencing of SIRT6 to be a novel therapy for lung cancer.

The biogenic engineered silver nanoparticles (AgNPs) were synthesized using aqueous extract of marine mangrove Avicennia marina leaves and its anticancer activity was checked in lung cancer cell line. Initially, the UV-vis spectra exhibited the characteristics SPR absorption peak for AgNPs at 425 nm and further characterized using TEM, SAED, XRD and FT-IR analysis. The TEM pictures displayed the spherical crystalline and monodispersed nature of AgNPs and the size range observed between 25-30 nm. The SAED showed the AgNPs are face-centered cubic pattern which is further confirmed with XRD analysis. The FTIR spectral analysis exposed the presence of necessary biomolecules for the reduction and stabilization of silver ions. Synthesized AgNPs showed dose-dependent cytotoxic activity in A549 cell line. The fluorescence studies showed that AgNPs induces apoptosis by increasing the generation of ROS in mitochondria and cleaving the mitochondrial membrane of A549 cells. Further, the molecular studies were conducted using RT-PCR and western blotting analysis and the results confirmed that the AgNPs induce apoptosis through both p53-dependent and -independent caspase intermediated signaling pathway. Together, the present study concludes that the bioengineered AgNPs can act as a potential therapeutic agent against lung cancer.

The potent biosurfactant-producing marine actinomycete Micromonospora marina was isolated from the mucus of the scleractinia coral Acropora formosa. The strain was screened for biosurfactant production and showed positive signs for hemolytic activity, drop-collapse test, oil displacement, emulsification activity and lipase production. The yield of biosurfactant was found to be high when the medium containing sucrose and peptone were used as carbon and nitrogen sources, respectively. The chemical characterization of the biosurfactant confirmed the presence of proteins, lipids and carbohydrates, thus indicating that the biosurfactant was lipopeptide in nature. Further, the presence of palmitoleic acid in GC-MS analysis also confirmed that the produced biosurfactant was a surfactin derivative. The structural characterization of surfactin was confirmed by H-1 NMR and C-13 NMR, and the results showed the presence of mystric fatty acid (-CH3CH2-), palmitoleic acid, lipopeptide monoesters and aromatic protons and carbons. Further, the surfactin exhibited potential cytotoxic activity against human breast cancer cells by inducing apoptosis through the increase of ROS generation and cleaving mitochondrial membrane potential. Taken together, the surfactin produced by M. marina could act as an excellent anticancer agent against breast cancer.

Distinct morphological silver nanoparticles were synthesized using Dodonaea viscosa leaves, extracted using different polar and non-polar solvents. Petroleum ether, methanol, acetone, acetonitrile and water were used for the extraction of active ingredients from the leaves of Dodonaea viscosa which attributed to obtaining nanoparticles with different physical, chemical, antibacterial and cytotoxic properties. The synthesized nanoparticles were characterized by UV-vis, FT-IR, XRD, HR-SEM with EDX and HR-TEM with SAED patterns. The XRD, HR-SEM and HR-TEM results reveal different nano sizes (15, 18, 12 and 20 nm) with different surface morphology (worm-like, irregular flower, spherical and dendritic structures) of the nanoparticles prepared using different solvent extracts (methanol, acetone, acetonitrile and water). The antibacterial results show significant zone of inhibition (20, 16, 13, 18 mm) against the test bacterium Streptococcus pyogenes for AgNPs synthesized by methanol, acetone, acetonitrile and water extracts, respectively. The cytotoxicity of synthesized silver nanoparticles in A549 NSCLC cells using the MTT assay were found to be 14, 3, 80, and 4 mu g/mL for AgNPs synthesized using leaf extracts obtained from methanol, acetone, acetonitrile and water, respectively. The results revealed that the synthesized AgNPs were effective in inhibiting the growth of A549 NSCLC cells.

Development of drug delivery system conjugated with doxorubicin (dox) on the surface of AuNPs with polyvinylpyrrolidone (Dox@PVP-AuNPs), we have demonstrated that human lung cancer cells can significantly overcome by the combination of highly effective cellular entry and responsive intracellular release of doxorubicin from Dox@PVP-AuNPs complex. Previously drug release from doxorubicin-conjugated AuNPs was confirmed by the recovered fluorescence of doxorubicin from quenching due to the nanosurface energy transfer between doxorubicinyl groups and AuNPs. Dox@PVP-AuNPs achieved enhanced inhibition of lung cancer cells growth than free Doxorubicin and PVP-AuNPs. The in vitro cytotoxic effect of PVP-AuNPs, free Dox and Dox@PVP-AuNPs inhibited the proliferation of human lung cancer cells with IC50 concentration. Compared with control cells, PVP-AuNPs and free Dox, Dox@PVP-AuNPs can increases ROS generation, sensitize mitochondrial membrane potential and induces both early and late apoptosis in lung cancer cells. Moreover, Dox@PVP-AuNPs highly upregulates the expression of tumor suppressor genes than free Dox and PVP-AuNPs and induces intrinsic apoptosis in lung cancer cells. From the results, Dox@PVP-AuNPs can be considered as an potential drug delivery system for effective treatment of human lung cancer.