Pharmacological Inhibition Research Articles

Acetylation of TIR domains in the TLR4-Mal-MyD88 complex regulates immune responses in sepsis.

Activation of the Toll-like receptor 4 (TLR4) by bacterial endotoxins in macrophages plays a crucial role in the pathogenesis of sepsis. However, the mechanism underlying TLR4 activation in macrophages is still not fully understood. Here, we reveal that upon lipopolysaccharide (LPS) stimulation, lysine acetyltransferase CBP is recruited to the TLR4 signalosome complex leading to increased acetylation of the TIR domains of the TLR4 signalosome. Acetylation of the TLR4 signalosome TIR domains significantly enhances signaling activation via NF-κB rather than IRF3 pathways. Induction of NF-κB signaling is responsible for gene expression changes leading to M1 macrophage polarization. In sepsis patients, significantly elevated TLR4-TIR acetylation is observed in CD16+ monocytes combined with elevated expression of M1 macrophage markers. Pharmacological inhibition of HDAC1, which deacetylates the TIR domains, or CBP play opposite roles in sepsis. Our findings highlight the important role of TLR4-TIR domain acetylation in the regulation of the immune responses in sepsis, and we propose this reversible acetylation of TLR4 signalosomes as a potential therapeutic target for M1 macrophages during the progression of sepsis.

Targeting ferroptosis with the lipoxygenase inhibitor PTC-041 as a therapeutic strategy for the treatment of Parkinson's disease.

Parkinson's disease is the second most common neurodegenerative disorder, affecting nearly 10 million people worldwide. Ferroptosis, a recently identified form of regulated cell death characterized by 15-lipoxygenase-mediated hydroperoxidation of membrane lipids, has been implicated in neurodegenerative disorders including amyotrophic lateral sclerosis and Parkinson's disease. Pharmacological inhibition of 15 -lipoxygenase to prevent iron- and lipid peroxidation-associated ferroptotic cell death is a rational strategy for the treatment of Parkinson's disease. We report here the characterization of PTC-041 as an anti-ferroptotic reductive lipoxygenase inhibitor developed for the treatment of Parkinson's disease. In these studies, PTC-041 potently protects primary human Parkinson's disease patient-derived fibroblasts from lipid peroxidation and subsequent ferroptotic cell death and prevents ferroptosis-related neuronal loss and astrogliosis in primary rat neuronal cultures. Additionally, PTC-041 prevents ferroptotic-mediated α-synuclein protein aggregation and nitrosylation in vitro, suggesting a potential role for anti-ferroptotic lipoxygenase inhibitors in mitigating pathogenic aspects of synucleinopathies such as Parkinson's disease. We further found that PTC-041 protects against synucleinopathy in vivo, demonstrating that PTC-041 treatment of Line 61 transgenic mice protects against α-synuclein aggregation and phosphorylation as well as prevents associated neuronal and non-neuronal cell death. Finally, we show that. PTC-041 protects against 6-hydroxydopamine-induced motor deficits in a hemiparkinsonian rat model, further validating the potential therapeutic benefits of lipoxygenase inhibitors in the treatment of Parkinson's disease.

Ccrk-Mak/Ick signaling is a ciliary transport regulator essential for retinal photoreceptor survival.

Primary cilia are microtubule-based sensory organelles whose dysfunction causes ciliopathies in humans. The formation, function, and maintenance of primary cilia depend crucially on intraflagellar transport (IFT); however, the regulatory mechanisms of IFT at ciliary tips are poorly understood. Here, we identified that the ciliopathy kinase Mak is a ciliary tip-localized IFT regulator that cooperatively acts with the ciliopathy kinase Ick, an IFT regulator. Simultaneous disruption of Mak and Ick resulted in loss of photoreceptor ciliary axonemes and severe retinal degeneration. Gene delivery of Ick and pharmacological inhibition of FGF receptors, Ick negative regulators, ameliorated retinal degeneration in Mak -/- mice. We also identified that Ccrk kinase is an upstream activator of Mak and Ick in retinal photoreceptor cells. Furthermore, the overexpression of Mak, Ick, and Ccrk and pharmacological inhibition of FGF receptors suppressed ciliopathy-related phenotypes caused by cytoplasmic dynein inhibition in cultured cells. Collectively, our results show that the Ccrk-Mak/Ick axis is an IFT regulator essential for retinal photoreceptor maintenance and present activation of Ick as a potential therapeutic approach for retinitis pigmentosa caused by MAK mutations.

LKB1 suppresses KSHV reactivation and promotes primary effusion lymphoma progression.

Viruses normally reprogram the host cell metabolic pathways as well as metabolic sensors to facilitate their persistence. The serine-threonine liver kinase B1 (LKB1) is a master upstream kinase of 5'-AMP-activated protein kinase (AMPK) that senses the energy status and therefore regulates the intracellular metabolic homeostasis. Previous studies showed that AMPK restricts Kaposi's sarcoma-associated herpesvirus (KSHV) lytic replication in endothelial cells during primary infection and promotes primary effusion lymphoma (PEL) cell survival. However, the role of LKB1 in KSHV lytic reactivation and KSHV-associated malignancies is unclear. In this study, we found that LKB1 is phosphorylated or activated in KSHV-positive PEL cells. Mechanistically, KSHV-encoded vCyclin mediated LKB1 activation in PEL cells, as vCyclin knockout ablated, while vCyclin overexpression enhanced LKB1 activation. Furthermore, knockdown of LKB1 inactivated AMPK and induced KSHV reactivation, as indicated by the increased expression of viral lytic genes and the increased virions in supernatants. Accordingly, AMPK inhibition by functional knockdown or a pharmacologic inhibitor, Compound C, promoted KSHV reactivation in PEL cells. Furthermore, inhibition of either LKB1 or AMPKα1 efficiently induced cell death by apoptosis of PEL cells both in vitro and in vivo. Together, these results identify LKB1 as a vulnerable target for PEL, which could be potentially exploited for treating other virus-associated diseases.IMPORTANCEKaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic virus associated with several human cancers, such as primary effusion lymphoma (PEL). Here, we showed that serine-threonine liver kinase B1 (LKB1), upstream of 5' AMP-activated protein kinase (AMPK), is activated by KSHV-encoded vCyclin and maintains KSHV latency in PEL cells. Inhibition of either LKB1 or AMPK enhances KSHV lytic replication from latency, which at least partially accounts for PEL cell death by apoptosis. Compound C, a potent AMPK inhibitor, induced KSHV reactivation and efficiently inhibited PEL progression in vivo. Thus, our work revealed that LKB1 is a potential therapeutic target for KSHV-associated cancers.

Arachidonic acid directly activates the human DP2 receptor.

Aberrant type 2 inflammatory responses are the underlying cause of the pathophysiology of allergic asthma, allergic rhinitis and other atopic diseases with an alarming prevalence in relevant parts of the western world. A bulk of evidence points out the important role of the DP2 receptor in this inflammation processes. A screening of different polyunsaturated fatty acids (PUFAs) at a fluorescence resonance energy transfer (FRET)-based DP2 receptor conformation sensor expressed in HEK cells revealed an agonistic effect of the prostaglandin (PG) D2 precursor arachidonic acid (AA) on DP2 receptor activity of about 80% of the effect induced by PGD2 In a combination of experiments at the conformation sensor and using a BRET-based G protein activation sensor expressed together with DP2 receptor-wt in HEK cells, we found that arachidonic acid act as a direct activator of the DP2 receptor but not DP1 receptor, in a concentration range considered physiologically relevant. Pharmacological inhibition of cyclooxygenases and lipoxygenases as well as cytochrome P450 did not lead to a diminished arachidonic acid response on the DP2 receptor, confirming a direct action of arachidonic acid on the receptor. Significance Statement We identified the prostaglandin precursor arachidonic acid to directly activate the DP2 receptor, a G protein-coupled receptor that is known to play an important role in type 2 inflammation.

Discovery of Novel [1,2,4]Triazolo[1,5-a]pyrimidine Derivatives as Novel Potent S-Phase Kinase-Associated Protein 2 (SKP2) Inhibitors for the Treatment of Cancer.

Skp1-CUL1-ROC1-F-box E3 ubiquitin ligases' main component S-phase kinase-associated protein 2 (Skp2) is responsible for specifically recognizing ubiquitination-modified substrates to be degraded such as p27 and p21 in the case of binding with adaptor protein Cks1. Pharmacological inhibition of Skp2 has exhibited promising antitumor activity. Herein, we present the design and optimization of a series of [1,2,4]triazolo[1,5-a]pyrimidine-based small molecules targeting Skp2. Among them, E35 demonstrated excellent inhibitory activities against the binding of Skp2-Cks1. In addition, compound E35 significantly inhibited colony formation and migration, as well as arrested the cell cycle at the S-phase. Mechanistically, compound E35 markedly decreased the expression of Skp2, as well as increased the expression of its substrates p21 and p27. Furthermore, compound E35 showed an obvious inhibitory effect on MGC-803 xenograft mice without obvious toxicity. All of these results suggest that compound E35 might be a valuable lead compound for antitumor agents targeting Skp2.

Hope on the Horizon: A Revolution in KRAS Inhibition Is Creating a New Treatment Paradigm for Patients with Pancreatic Cancer.

KRAS is the most frequently altered oncogene in pancreatic ductal adenocarcinoma, in which the aberrantly activated RAS signaling pathway plays pleiotropic roles in tumor initiation and maintenance. Nearly four decades after the discovery of the RAS oncoprotein, a multitude of pharmacologic inhibitors are now available that directly target mutant KRAS. This In Focus commentary, published simultaneously with the 2024 AACR Special Conference on Pancreatic Cancer, summarizes the current state of this rapidly changing field, including preclinical data and emerging clinical trends with respect to therapeutic efficacy, mechanisms of resistance, and potential combinations to maximize clinical benefit from this promising class of therapies.

Abstract A084: CDK12 inhibition drives mitotic catastrophe, triggering genome instability and innate immune response in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease with limited therapies. In our screening using patient-derived organoids, we identified cyclin-dependent kinase 12 (CDK12) as a promising target in a subset of PDAC. Both pharmacological and genetic inhibition of CDK12 exerted potent antitumor activity in vitro and in vivo, inducing double-strand DNA breaks and apoptosis. CDK12 is a member of cyclin-dependent kinases that is involved in gene expression regulations at multiple levels. Recently, CDK12 has gained attention as a therapeutic target in several types of cancers including triple-negative breast cancer and Ewing sarcoma. However, little is known about the impact and possibility of CDK12 inhibition in pancreatic cancer treatment. As the lack of specific inhibitors that selectively target CDK12, studying CDK12 has long been challenging. To overcome this, we used CRISPR-Cas9 mediated genome editing to generate FKBP12F36V (dTAG) knock-in PDAC cell lines, enabling rapid elimination of endogenous CDK12 using a degrader against dTAG (dTAG-13). Quantitative-image-based cytometry and time lapse imaging showed that CDK12 ablation induces mitotic arrest, DNA damage and apoptosis (mitotic catastrophe). Specifically, loss of CDK12 kinase activity in the S phase was sufficient to slow DNA replication speed, leading to spindle assembly checkpoint activation and mitotic catastrophe. Importantly, cells that escaped mitotic death accumulate micronuclei, triggering immune response signaling that is mediated by the cGAS-STING-type I interferon pathway. Our results suggest that CDK12 is a promising target in a subset of PDAC patients, where CDK12 inhibition exerts antitumor activity both in cell autonomous and non-cell autonomous ways through inducing mitotic catastrophe as well as triggering immune response. Citation Format: Dosuke Iwadate, Keisuke Yamamoto, Hiroyuki Kato, Keisuke Tateishi, Mitsuhiro Fujishiro. CDK12 inhibition drives mitotic catastrophe, triggering genome instability and innate immune response in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A084.

Abstract IA-08: Syndecan 1 is a therapeutic target for KRAS-driven pancreatic cancer

Abstract KRAS inhibitors targeting G12C mutation have been approved by FDA for the treatment of non-small cell lung cancers and have shown promising clinical responses in pancreatic ductal adenocarcinoma (PDAC). RAS inhibitors targeting additional KRAS mutations or multiple RAS isoforms are under clinical development. Although targeting KRAS exhibits anti-tumor effect in PDAC, majority of tumors inevitably develop resistant disease, as is the case for most targeted therapies. In this new era of targeting KRAS driven cancer, understanding mechanisms driving this resistance to RAS targeted therapies and further devising combination strategies to overcome it have become a necessary, though challenging, task to achieve long term disease control. To tackle this problem, we employed various in vitro and in vivo preclinical model systems to explore the development of resistance to both genetic and pharmacological KRAS blockade. Our previous work identified the membrane proteoglycan Syndecan 1 (SDC1) as a KRAS downstream surrogate whose cell surface localization is tightly controlled by oncogenic KRAS signaling and is critical for KRAS-driven tumor development. Here, our analysis of KRAS driven genetically engineered PDAC mouse model and human pancreatic or colorectal cancer (CRC) cells with KRASG12C mutation discovered that, while cell surface SDC1 is reduced upon acute genetic or pharmacological inhibition of oncogenic KRAS, plasma membrane SDC1 is recovered in tumor cells that become resistant to KRAS inhibition (KRAS-bypass cells). Surface localization of SDC1 leads to the activation of multiple receptor tyrosine kinases (RTKs) and macropinocytosis, resulting in the resistance to KRAS blockade. We further identified that the recovery of plasma membrane SDC1 in KRAS-bypass cells is driven by YAP1 oncogene, which induces the downregulation of ARF6 GAPs, leading to the activation of ARF6, a small GTPase required for SDC1 membrane recycling. To therapeutically targeting SDC1, we have successfully developed a monoclonal antibody (22B mAb), which exhibits remarkable sensitivity and specificity for human SDC1. Our results indicate that 22B mAb exerts substantial inhibition of PDAC cell growth in vitro and desirable tumor inhibitory effects in vivo in murine PDAC tumors and human PDAC cell-derived xenograft tumors. Importantly, our 22B mAb synergizes with KRAS inhibitors, chemotherapy, or immunotherapy, resulting in significantly enhanced therapeutic effects. Taken together, our study identified SDC1 as a functional driver for the bypass of KRAS-dependence and therapeutically targeting SDC1 with a novel mAb will overcome the acquired resistance to KRAS targeted therapy. As such, our research yields important new insights in the fields of cancer biology regarding mechanisms of resistance employed by human cancers, and identifies new actionable therapeutic targets and approaches to overcome resistance to KRAS inhibition. Citation Format: Mitsunobu Takeda, Zecheng Yang, Madeline S Theardy, Alexey Sorokin, Oluwadara Coker, Shuaitong Chen, Long T Vien, Laura Bover, Haoqiang Ying, Scott Kopetz, Wantong Yao. Syndecan 1 is a therapeutic target for KRAS-driven pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr IA-08.

Abstract C063: Inhibition of PRMT5 reduces aggressiveness in basal-like pancreatic ductal adenocarcinoma

Abstract The basal-like pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive type of mesenchymal PDAC that resists treatment and has a poor prognosis. Understanding the mechanisms driving basal-like PDAC biology is crucial for unraveling the intricacies of PDAC plasticity and developing effective treatment strategies for this aggressive tumor. Recent studies from our laboratory have shown that protein arginine methyl transferase 5 (PRMT5) significantly contributes to PDAC plasticity and progression towards the basal-like subtype. Specifically, our data show that patients with high levels of PRMT5 expression have a worse outcome, thus arguing that PRMT5 plays a role in PDAC aggressiveness and outcome. Moreover, our experiments indicate that inhibiting PRMT5, either genetically or chemically, in basal-like PDAC cells induces the expression of epithelial markers like GATA6 and E-CADHERIN, suggesting that PRMT5 promotes mesenchymal features in PDAC. Using wound healing and transwell assays, we have demonstrated that pharmacological inhibition of PRMT5 significantly suppresses cell migration and invasion. Additionally, colony formation and cell proliferation are markedly reduced upon PRMT5 inhibition in basal-like PDAC. Collectively, these findings suggest that inhibiting PRMT5 can effectively constrain the aggressive cellular features characteristic of basal-like PDAC, such as cell proliferation, clonogenicity, and migration. Recent research indicates that PRMT5 preferentially methylates splicing factors in various cell types, implying that RNA splicing regulation is crucial to PRMT5’s role in enhancing tumor plasticity and aggressiveness. We are currently investigating whether PRMT5-mediated RNA splicing alterations contribute to the maintenance of the basal-like PDAC phenotype. These studies provide key evidence that PRMT5 is essential for maintaining the aggressive characteristics of basal-like PDAC, highlighting its potential as a therapeutic target for this challenging cancer subtype. Citation Format: Christina Wang, Zamira Guerra Soares, Dakarai Esgdaille, Stephano Spano Mello. Inhibition of PRMT5 reduces aggressiveness in basal-like pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C063.

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

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

Abstract B002: Tumor-secreted PTHrP facilitates pancreatic cancer cachexia by regulation of de novo lipogenesis pathway

Abstract Pancreatic cancer is associated with one of the highest and most severe forms of cancer- associated cachexia amongst solid tumors. In an effort to further our understanding of the molecular etiology of pancreatic cancer-associated cachexia, we have developed mouse models to study adipose tissue wasting during Pancreatic Ductal Adenocarcinoma (PDAC) progression. In these pursuits, we have identified a pro-cachectic factor, PTHrP, that is produced and secreted by PDAC tumor cells and directly signals to adipocytes to facilitate wasting. Genetic deletion and pharmacological inhibition of PTHrP significantly extended the survival of tumor-bearing animals and dramatically reduced cachectic phenotypes in these animals, manifesting as decreased adipose and muscle tissue wasting. Mechanistic studies have shown crosstalk between tumor-cell derived PTHrP and adipose tissue PTH1R that drives the cachectic phenotype. Finally, bulk RNA sequencing analysis in PTHrP-driven cachectic models point towards a role for reduced de novo lipogenesis and adipogenesis in cachectic adipose depots, suggesting potential rewiring of the metabolic profile of white adipose tissue in tumor-bearing mice with higher PTHrP expression. Citation Format: Nikita Bhalerao, Jessica Peura, Yamini Ogoti, Calvin Johnson, Qingbo Chen, Ekaterina Korobkina, Faith Keller, Maximillan Wengyn, Robert Norgard, Richard Kremmer, Emma Watson, Marcus Ruscetti, David Guertin, Jason R Pitarresi. Tumor-secreted PTHrP facilitates pancreatic cancer cachexia by regulation of de novo lipogenesis pathway [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B002.

Abstract C017: Targeting KRAS selectively induce metabolic reprogramming in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease largely due to the lack of effective treatments that overcome the tumor drug resistance. Oncogenic KRAS mutation contribute to most PDAC and drive multiple metabolic pathways to promote tumor growth. Despite this overarching role of KRAS, inhibition of KRAS/MAPK pathway often lead to resistance by yet unclear mechanism. Here we find that the pharmacological inhibition of KRAS/MAPK pathway induces distinct responses in various pancreatic cancer cells. Specifically, while KRAS/MAPK inhibition suppressed growth in some cells, the same treatment induced or did not change the growth of other cell lines and also selectively impacted cell migration. In addition, metabolic activities were suppressed in cells where growth was suppressed or not changed but was accelerated where KRAS/MAPK inhibition induced growth. Analysis of gene expression profile of human PDAC and KRAS-driven mouse tumors identified nutrient transporters that are upregulated KRAS induction but also upregulated upon KRAS inhibition. Our study reveals context-dependent response to KRAS inhibition in pancreatic cancer and suggest targeting nutrient transporters to overcome therapeutic resistance. Citation Format: Laiba Shiekh, Matthew Cheung, Linyuhui Zheng, Erica Marlette, Chiamaka J. Ezeh, Don-Gerard Conde, Zeribe C. Nwosu. Targeting KRAS selectively induce metabolic reprogramming in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C017.

Abstract C022: Elucidation and exploitation of RAS-inhibitor dependent macropinocytic regulation in pancreatic ductal adenocarcinoma

Abstract Alteration of essential metabolic pathways is a major mechanism by which oncogenic KRAS promotes tumor development and growth in pancreatic ductal adenocarcinoma (PDAC). KRAS- driven PDAC is dependent on nutrient scavenging pathways, including macropinocytosis and autophagy to fuel the metabolic demand of rapid proliferation. Thus, these metabolic processes are attractive targets for the development of treatments for PDAC. Acute KRAS loss results in downregulation of macropinocytosis in PDAC. Additionally, our lab demonstrated that KRAS loss or inhibition of ERK MAPK signaling decreased glucose consumption and glycolysis but increased autophagy, thereby enhancing dependency on autophagy for survival and growth. Accordingly, dual ERK MAPK and autophagy inhibition (via chloroquine) synergistically enhanced anti-tumor efficacy in PDAC. However, early clinical data has demonstrated that resistance to this treatment arises over time through unknown mechanisms. We observed that both autophagy induction and macropinocytosis downregulation are transient following RAS, MEK, or ERK inhibition—with autophagic and macropinocytic activity returning to or surpassing basal levels after within two weeks of treatment. Furthermore, we found that prolonged pharmacological inhibition of RAS, MEK, or ERK pathway consistently resulted in significant upregulation of macropinocytosis. We propose that prolonged MAPK inhibition upregulates macropinocytosis over time, consequently abrogating dependency on autophagy and therefore reducing sensitivity to autophagy inhibition. Furthermore, we found that chloroquine, which is commonly thought of as a lysosomotropic drug, does not prevent degradation of proteins engulfed via macropinocytosis. Together our data suggests that upregulation of macropinocytosis may mediate resistance to the combination of ERK MAPK inhibition and chloroquine. With the recent advance in development RAS inhibitors, the resistance mechanisms by which PDAC can overcome RAS inhibitor treatment is a major focus of the field. Given the dependence of PDAC on macropinocytic uptake for tumorigenic growth, it is imperative to understand how long-term treatment with RAS inhibitors regulate macropinocytosis. We demonstrated that PDAC cells with acquired resistance to RAS inhibition exhibit a 2- to 10-fold increase in macropinocytic uptake. We found that upregulated macropinocytosis following long-term RAS or MAPK inhibition is accompanied by increased albumin uptake and sensitivity to albumin-bound paclitaxel (nab-paclitaxel), a therapeutic that is included in the current standard of care for PDAC patients. These results suggest that RAS inhibitor pretreatment or concurrent treatment with nab-paclitaxel may represent a strategy to improve this current PDAC treatment. We conclude that a more complete understanding of the regulation of essential metabolic pathways following both acute and sustained RAS inhibition will better inform future RAS inhibitor combination therapies. Citation Format: Ryan Robb, Sarah Ackermann, Khalilah Taylor, Runying Yang. Elucidation and exploitation of RAS-inhibitor dependent macropinocytic regulation in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C022.

IĸBζ is a central modulator of inflammatory arthritis pathogenesis.

Current therapies targeting individual factors in inflammatory arthritis (IA) show variable efficacy, often requiring treatment using combinations of drugs and associated with undesirable side effects. NF-ĸB is critical for production and function of most inflammatory cytokines. However, given its essential role in physiologic processes, targeting NF-ĸB is precarious. Hence, identifying pathways downstream of NF-κB that selectively govern expression of inflammatory cytokines in IA would be advantageous. We have previously identified IĸBζ as a unique inflammatory signature of NF-ĸB that controls transcription of inflammatory cytokines only under pathologic conditions while sparing physiologic NF-ĸB signals. We generated mice harboring myeloid, lymphoid and global deletion of Nfkbiz (the gene encoding IĸBζ). These models were subjected to serum transfer-induced arthritis (STIA). Additionally, pharmacologic inhibitors of IĸBζ were injected intraperitonially. Joint swelling, μCT, immunohistochemistry, flow cytometry, and cytokine measurements were carried out using synovial tissues. Global deletion of Nfkbiz or depletion of neutrophils (vastly IĸBζ+ cells) reduced inflammatory synovial cells and increased anti-inflammatory and regenerative synovial cells, plummeted expression of inflammatory factors and ameliorated experimental mouse IA. Further, expression of Irg1, the enzyme responsible for itaconate production, was increased in synovial cells. Accordingly, the itaconate derivative dimethyl itaconate (DI) inhibited IĸBζ-mediated inflammatory factors. Further, in silico screen identified 8-Hydroxyquinoline (HQ) as putative inhibitor of IĸBζ not affecting physiological NF-ĸB activity. Congruently, systemic administration of either DI or HQ inhibited joint swelling and damage. Our study positions IĸBζ as an inflammation-specific target for therapeutic consideration in RA as its inhibition spares the beneficial functions of NF-ĸB.

Protein kinase C iota promotes glycolysis via PI3K/AKT/mTOR signalling in high grade serous ovarian cancer.

Epithelial ovarian cancer, especially high grade serous ovarian cancer (HGSOC) is by far, the most lethal gynecological malignancy with poor prognosis and high relapse rate. Despite of availability of several therapeutic interventions including poly-ADP ribose polymerase (PARP) inhibitors, HGSOC remains unmanageable and identification of early detection biomarkers and therapeutic targets for this lethal malady is highly warranted. Aberrant expression of protein kinase C iota (PKCί) is implicated in many cellular and physiological functions involved in tumorigenesis including cell proliferation and cell cycle deregulation. Two high grade serous ovarian cancer cells SKOV3 and COV362 were employed in this study. PKCί was genetically knocked down or pharmacologically inhibited and several functional and biochemical assays were performed. We report that PKCί is overexpressed in HGSOC cells and patient tissue samples with a significant prognostic value. Pharmacological inhibition of PKCί by Na-aurothiomalate or its shRNA-mediated genetic knockdown suppressed HGSOC cell proliferation, EMT and induced apoptosis. Moreover, PKCί positively regulated GLUT1 and several other glycolytic genes including HK1, HK2, PGK1, ENO1 and LDHA to promote elevated glucose uptake and glycolysis in HGSOC cells. Mechanistically, PKCί drove glycolysis via PI3K/AKT/mTOR signalling. Na-aurothiomalate and highly selective, dual PI3K/mTOR inhibitor dactolisib could serve as novel anti-glycolytic drugs in HGSOC. Taken together, our results indicate PKCί/PI3K/AKT/mTOR signalling cascade could be a novel therapeutic target in a lethal pathology like HGSOC.

MTOR signalling controls the formation of smooth muscle cell-derived luminal myofibroblasts during vasculitis.

The accumulation of myofibroblasts within the intimal layer of inflamed blood vessels is a potentially catastrophic complication of vasculitis, which can lead to arterial stenosis and ischaemia. In this study, we have investigated how these luminal myofibroblasts develop during Kawasaki disease (KD), a paediatric vasculitis typically involving the coronary arteries. By performing lineage tracing studies in a murine model of KD, we reveal that luminal myofibroblasts develop independently of adventitial fibroblasts and endothelial cells, and instead derive from smooth muscle cells (SMCs). Notably, the emergence of SMC-derived luminal myofibroblasts-in both mice and patients with KD, Takayasu's arteritis and Giant Cell arteritis-coincided with activation of the mechanistic target of rapamycin (mTOR) signalling pathway. Moreover, SMC-specific deletion of mTOR signalling, or pharmacological inhibition, abrogated the emergence of luminal myofibroblasts. Thus, mTOR is an intrinsic and essential regulator of luminal myofibroblast formation that is activated in vasculitis patients and therapeutically tractable. These findings provide molecular insight into the pathogenesis of coronary artery stenosis and identify mTOR as a therapeutic target in vasculitis.

Oxysterol Sensing Through GPR183 Triggers Endothelial Senescence in Hypertension.

Despite endothelial dysfunction being an initial step in the development of hypertension and associated cardiovascular/renal injuries, effective therapeutic strategies to prevent endothelial dysfunction are still lacking. GPR183 (G protein-coupled receptor 183), a recently identified G protein-coupled receptor for oxysterols and hydroxylated metabolites of cholesterol, has pleiotropic roles in lipid metabolism and immune responses. However, the role of GPR183 in the regulation of endothelial function remains unknown. Endothelial-specific GPR183 knockout mice were generated and used to examine the role of GPR183 in endothelial senescence by establishing 2 independent hypertension models: desoxycorticosterone acetate/salt-induced and Ang II (angiotensin II)-induced hypertensive mice. Echocardiography, transmission electron microscopy, blood pressure measurement, vasorelaxation response experiments, flow cytometry analysis, and chromatin immunoprecipitation analysis were performed in this study. Endothelial GPR183 was significantly induced in hypertensive mice, which was further confirmed in renal biopsies from subjects with hypertensive nephropathy. Endothelial-specific deficiency of GPR183 markedly alleviated cardiovascular and renal injuries in hypertensive mice. Moreover, we found that GPR183 regulated endothelial senescence in both hypertensive mice and aged mice. Mechanistically, GPR183 disrupted circadian signaling by inhibiting PER1 (period circadian regulator 1) expression, thereby facilitating endothelial senescence and dysfunction through the cAMP (cyclic adenosine monophosphate)/PKA (protein kinase A)/CREB (cAMP-response element binding protein) signaling pathway. Importantly, pharmacological inhibition of the oxysterol-GPR183 axis by NIBR189 or clotrimazole ameliorated endothelial senescence and cardiovascular/renal injuries in hypertensive mice. This study discovers a previously unrecognized role of GPR183 in promoting endothelial senescence. Pharmacological targeting of GPR183 may be an innovative therapeutic strategy for hypertension and its associated complications.

ERK5 suppression overcomes FAK inhibitor resistance in mutant KRAS-driven non-small cell lung cancer.

Mutated KRAS serves as the oncogenic driver in 30% of non-small cell lung cancers (NSCLCs) and is associated with metastatic and therapy-resistant tumors. Focal Adhesion Kinase (FAK) acts as a mediator in sustaining KRAS-driven lung tumors, and although FAK inhibitors are currently undergoing clinical development, clinical data indicated that their efficacy in producing long-term anti-tumor responses is limited. Here we revealed two FAK interactors, extracellular-signal-regulated kinase 5 (ERK5) and cyclin-dependent kinase 5 (CDK5), as key players underlying FAK-mediated maintenance of KRAS mutant NSCLC. Inhibition of ERK5 and CDK5 synergistically suppressed FAK function, decreased proliferation and induced apoptosis owing to exacerbated ROS-induced DNA damage. Accordingly, concomitant pharmacological inhibition of ERK5 and CDK5 in a mouse model of KrasG12D-driven lung adenocarcinoma suppressed tumor progression and promoted cancer cell death. Cancer cells resistant to FAK inhibitors showed enhanced ERK5-FAK signaling dampening DNA damage. Notably, ERK5 inhibition prevented the development of resistance to FAK inhibitors, significantly enhancing the efficacy of anti-tumor responses. Therefore, we propose ERK5 inhibition as a potential co-targeting strategy to counteract FAK inhibitor resistance in NSCLC.

Inhibition of WNK kinases in NK cells disrupts cellular osmoregulation and control of tumor metastasis.

The serine/threonine With-No-Lysine (WNK) kinase family function in blood pressure control, electrolyte homeostasis, and cellular osmoregulation. These kinases and their downstream effectors are considered promising therapeutic targets in hypertension and stroke. However, the role of WNK kinases in immune cells remains poorly understood. Using the small-molecule WNK kinase inhibitors WNK463 and WNK-IN-11, we investigated how WNK kinase inhibition affects natural killer (NK) cell physiology. WNK kinase inhibition with WNK463 or WNK-IN-11 significantly decreased IL-2-activated NK-cell volume, motility, and cytolytic activity. Treatment of NK cells with these inhibitors induced autophagy by activating AMPK and inhibiting mTOR signaling. Moreover, WNK kinase inhibition increased phosphorylation of Akt and c-Myc by misaligning activity of activating kinases and inhibitory phosphatases. Treatment of tumor-bearing mice with WNK463 impaired tumor metastasis control by adoptively transferred NK cells. The catalytic activity of WNK kinases has a critical role of multiple aspects of NK cell physiology and their pharmacologic inhibition negatively impacts NK cell function.